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SECTION III
NEUROSURGERY
CHAPTER XIII
EXPERIMENTAL OBSERVATIONS ON PERIPHERAL NERVE REPAIR a
INTRODUCTION
In no field of medicine, perhaps, is the interdependence of experimental and clinical work so clearly demonstrated as in peripheral nerve surgery.
Questions which concern the structure, development, growth, degeneration, and regeneration, after injury, of the peripheral nerves have engaged the attention of observers for more than a century, and there exists an extensive literature, dealing with these and relative problems, to which both the experimenter and the clinician have contributed. A study of this literature, while showing constant advance in knowledge, as concerns all phases of the questions involved, will reveal also wide and fundamental divergence of opinions which have influenced and retarded progress as regards structural interpretations and their clinical applications.
The nervous system consists of independent, anatomic units, the neurons, related to each other by contiguity and not by continuity. The peripheral nervous system is, therefore, both on anatomic and functional considerations, ia part of the central nervous system, and in its surgical treatment should be regarded as such. There are 31 pairs of spinal nerves, quite symmetrically arranged, which course singly or join to form plexuses and which throughout the thoracic and upper lumbar regions and for certain sacral nerves are connected with the ganglia of the sympathetic system through the white rami or preganglionic branches. Considered structurally, each spinal nerve consists of bundles of nerve fibers, certain of which are processes of neurons, known as neuraxes or axons, the cell bodies of which are situated in the ventral gray of the central nervous system or in the sympathetic ganglia and carry nerve impulses from the central nervous system to the periphery, and are thus known is efferent nerve fibers, while other nerve fibers are processes of neurons known as dendrites, the cell bodies of which are found in the spinal ganglia and conduct nerve impulses toward the central nervous system and constitute the neuraxes of afferent nerve fibers. The neuraxes and neuraxes dendrites, forming the nerve fibers of the spinal nerve, may been sheathed in a layer of myelin, known as myelinated or medullated nerve fibers, or they may be naked, and are then known as nonmyelinated or nonmedullated nerve fibers. Considered functionally, we recognize in each spinal nerve nerve fibers belonging to one of four
a Report of the work of the Neurosurgical Laboratory, Department of Anatomy, University of Michigan, Ann Arborr, Mich. The following medical officers, on special assignments, collaborated in the experimental work for stated periods Lieut. Col. Dean Lewis, Maj. J. F. Corbitt, Maj. Byron Stookey, and Maj. T. Roberg. During the latter portion of this investigation technical laboratory assistance was made available through a grant received from the committee on research of the American Medical Association, for which acknowledgment is here made.
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functional systems, designating a functional system "as the sum of all the neurons in the body which possesses certain physiological and anatomical characters in common so that they may react in a common mode." 1 The functional systems of the spinal nerves are designated: 1, Somatic efferent; 2.visceral efferent; 3, somatic afferent; 4, visceral afferent. The nerve fiber composition of a spinal nerve may be represented graphically as shown in Figure 211.
FIG. 211.- Diagrammatic cross section of the Spinal cord, showing on the right side the nerve roots and type nerve fibers. A, Somatic efferent neuron; B, preganglionic neuron of the visceral efferent system; C, postganglionic neuron of the visceral efferent system ; D, somatic afferent neuron; D, somatic afferent neuron with nonmedullated processes, E, visceral afferent neuron (interoceptive impulses); PR. G., preganglionic bundle or white ramus; PO. G., postganglionic bundle or gray ramus; S Y. G., sympathetic ganglion of the sympathetic chain
The somatic efferent fibers (fig. 211-A) are the neuraxes of neurons the cell bodies of which are located in the ventral portion of the spinal gray. They leave the cord through the ventral roots and terminate in skeletal muscle in sublammellar end plates or motor nerve endings. The nerve fibers belonging to this functional system are relatively large, myelinated fibers.
The visceral efferent fibers (figs. 211-B and C), or the sympathetic fibers of the spinal nerves, are neuraxes of sympathetic neurons, the cell bodies of which are situated in the sympathetic ganglia of the symipathetie trunks. They constitute the postganglionic nerve fibers. They enter the spinal nerves through
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the gray rami communicants, each spinal nerve having such connection with the sympathetic trunks. The sympathetic ganglion cells of the sympathetic trunks are in synaptic relation with the preganglionic neurons, the cell bodies of which are situated in the visceral efferent column of the spinal gray, their neuraxes leaving the spinal cord by way of the ventral roots of the successive thoracic and four upper lumbar nerves and by way of the white rami, branches reaching the sympathetic ganglia where they form synaptic relations with the sympathetic nerve cells, it being well known, through the fundamental experimental researches of Langley, that there are always two neurons concerned in carrying an impulse from the central nervous system, through the spinal nerves to involuntary muscle and glandular tissue. The investigations of Boeke make it probable that the visceral efferent fibers may play a part in the innervation of certain skeletal muscle fibers.
The somatic afferent fibers (figs. 211-D and D') of the spinal nerves are the dendritic branches of sensory neurons, the cell bodies of which are situated in the spinal ganglia and convey impulses from the periphery to the central nervous system. A certain per cent of these nerve fibers are relatively large myelinated fibers, which have origin in peripheral nerve endings. both encapsuled and nonencapsuled. They are connected with the larger and more complex ganglion cells. A certain per cent of somatic afferent nerve fibers are nonmyelinated (Ranson) or are very fine myelinated fibers (Langley) which are connected with the smaller and simpler ganglion cells of the spinal ganglia. They are distributed in large part to the skin but also to the muscular branches.
The visceral afferent fibers (fig. 211-E), strictly speaking, are not primarily distributed to the peripheral nerves of the body wall and extremities. but to the thoracic and abdominal viscera which they reach by way of the white rami. The cell bodies of these fibers are in the spinal ganglia. their neuraxes pass through the respective dorsal roots to the dorsal column of the spinal cord.
According to function, the somatic afferent neurons are classified either as exteroceptive fibers, which carry impulses from sense organs and from the surface of the body, or as proprioceptive fibers, carrying impulses arising from within the body, from joint, tendon, and deep connective tissue and from muscular tissue, and also from the semicircular apparatus of the ears. The visceral afferent nerve fibers are said to carry interoceptive nerve impulses. There is no recognizable structural differentiation in afferent nerve fibers to be correlated with the type of impulses carried, though each group of afferent fibers is connected with special receptors or sensory nerve endings in the periphery and with distinctive neuron paths in the central nervous system. The cranial nerves, structurally considered, are very similar to the spinal nerves. but collectively considered contain additional functional systems having restricted distribution and specialized function; considerations which need not receive special discussion here.
STRUCTURE OF A NERVE
The efferent and afferent fibers of a nerve trunk are intermingled and run together in small bundles known as funiculi. A funiculus may approach a millimeter in size. Each funiculus is surrounded by a connective tissue sheath known as the perineurium, composed of several lamellae. of flattened collagenous
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connective tissue bundles; anastomosing here and there, between these flattened connective bundles, spread out fibroblasts are found which, on the inner surface of the perineurium, form a fairly distinct layer. The writer has not been able to demonstrate the fairly definite layer of endothelioid cells described by Key and Retzius. A few wandering cells and a few clasmatocytes are found between the lamellae of the perineurium. Relatively few elastic fibers, arranged in network, are found in the perineurium. Flattened trabeculae of collagenous connective tissue pass from the perineural sheaths to the interior of the funiculi and there is found a loose connective tissue, consisting of fibrils and fine fibers of collagenous connective tissue and a few elastic fibrils, disposed as a network and found between the fibers or small bundles of such, and forming more or less distinct tubular sheaths for the fibers of the funiculi. This constitutes the endoneurium of the funiculus. In it are found a few fibroblasts and a few clasmatocytes and wandering cells. The spinal nerves generally consist of more than one funiculus, and certain of the larger ones have many funiculi. Surrounding the whole nerve trunk and extending between the funiculi, there is found an areolar connective tissue, continuous with the surrounding Connective tissue, which is known as epineurium. The name would suggest that this sheath is upon the nerve. It should be understood that the epineurium extends between and surrounds the several funiculi of a nerve trunk; therefore. an epineurial stitch or suture may pass through a nerve trunk, conceivably between the funiculi. The epineurium consists of looser and denser areolar connective tissue with often, especially in the larger nerves, an appreciable amount of adipose tissue, disposed in small groups of fat cells or in scattered cells. In the epineurium are found the larger blood vessels and the lymph vessels of a nerve trunk, also sensory nerve endings for the nerve itself. The cells of the epineurium are largely of the type of the fixed fibroblasts; wandering cells and clasmatocytes are also found, but in variable numbers: mast cells have be es described. The details of the ultimate distribution of the blood and lymph vessels of a nerve trunk require further study and should be given special consideration for each of the several larger nerve trunks, subject to injury. The larger blood vessels, both arteries and veins, course in the epineurium. Terminal arterioles pass through the perineural sheaths of the several funiculi and break up into capillaries which course in the endoneurium, between the nerve fibers, forming long-meshed anastomoses. The capillaries of the funiculi are relatively scanty. Definite lymph vessels and lymph capillaries have not been shown to exist within the funiculi and perineural sheath. They have been demonstrated by injection in the epineural sheath.
Special interest was drawn to the funicular structure of peripheral nerves through the studies and publications of Stoffel 2 and his followers, who claimed that the several peripheral nerves presented a definite funicular morphology which extended throughout the nerve trunk and was fairly constant, so that definite sensory and motor paths could be demarked in the internal topography of the nerve. This laboratory was not especially concerned with this problem. realizing that during healing or regeneration of a cut nerve, in the field of the scar, even with the best of suture, the funicular pattern is to a large extent lot. Extended researches bearing on this problem were undertaken by Heinemann, 3
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Borchardt and Wyasmenski,4 Langley and Hashimoto, 5 Coinpton,6 Dustin,7 and Kunzel.8 These observers have shown, either by careful dissections of macerated nerve trunks, or by means of carefully oriented serial sections of nerve trunks, that Stoffel 's views can not be maintained. It has been shown that an extensive anastomosis and exchange of nerve fiber bundles exists between funiculi and that the funicular pattern is not the same even at relatively short intervals and not necessarily alike in the same anatomic nerve in different individuals. This question has been well summarized by Stookey, 9 who has reviewed the pertinent literature.
STRUCTURE OF NERVE FIBERS
Nerve fibers are either myelinated-medullated, or nonmyelinated-non-medullated. A myelinated nerve fiber consists of the neuraxis or axon, the myelin or the medullary sheath, and the neurolemma with its neurolemma nuclei or sheath cells. The neuraxis is the direct continuation of the respective nerve cell or neuron, the essential and conducting part of a nerve fiber, and passes uninterruptedly from the cell body to its destination. It must be regarded as in protoplasmic continuity with the cell body of the neuron, its trophic center. The neuraxis is devoid of any sheath in the immediate vicinity of the nerve cell and very generally loses all sheaths before termination. It consists of neurofibrils, continuous with the neurofibrils of the cell body of the peripheral neuron, embedded in a homogeneous neuroplasm. A delicate, peripheral protoplasmic sheath may be present, known as the axolemma, but this difficult to establish conclusively. The myelin sheath in the living and structurally unaltered nerve fibers appears as a homogeneous and structure less sheath which is interrupted from place to place at stated intervals, at the nodes or constrictions of Ranvier. These nodes occur at regular intervals of a length approximately one hundred times the diameter of the fiber. The segment of a nerve intervening between two nodes is known as an internodal segment. The structure of the myelin has not been fully determined, nor is it clear whether the myelin layer is to be regarded as a part of the neuron or iasispecial sheath quite distinct from the neuraxis. The myelin sheath presents quite distinctive structural appearances depending on the mode of fixation and staining of the nerve fibers. In segments of the same nerve, treated with different reagents in fixation and staining, quite dissimilar pictures of myelin structure may be obtained. It would seem that from the complex material which forms the myelin, largely made up of lecithin, there separates out a coagulable substance which under certain treatment forms a reticulum, keratin-like in nature, and known as the neurokeratin net, the arrangement of which varies with different fixations. The majority of the special structural characteristics described for myelin are regarded as fixation artifacts. It is stained black in osmic acid and is differentially stainable by a variety of methods. It presents special manifestations in degenerating nerves. In early stages of development of nerve fibers or in early stages of regeneration of peripheral nerves, the myelin seems to appear as a continuous, delicate sheath, a differentiation of the peripheral part of the neuraxis; in further development the nodes of Ranvier appear. There is at hand evidence, though not conclusive, to
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warrant considering the myelin sheath as a part of the respective neuro. The suggestion that the myelin is in its histogenesis closely related to the neurolemma sheath is not without its supporters. In a comprehensive histologic and histopathologic study of peripheral nerves by Donikow,10 in which extended consideration is given to the structure of the myelin sheath, the conclusion is reached that the plasma cells of Schwann consist of a dense nuclear zone and a looser meshwork which pervades the myelin sheath of the entire internodal segment, in the meshes of which is contained myelin substance. Not unlike this conclusion is that of Nemiloff, 11 who regards the nuclei of the neurolemma sheath as related to a protoplasmic reticulum which pervades the myelin sheath. According to these observations, the myelin sheath is not a part of the neuron but an ensheathing structure. The neurolemma forms the outermost layer of the myelinated nerve fiber of peripheral nerves. It is very thin, apparently homogeneous layer closely applied to the myelin sheath. There is at hand evidence of a delicate fibrillar structure of the neurolemma sheath. The flattened, oval nuclei found lying on the inner surface of the neurolemma sheath, one for each internodal segment, are considered as part of the neurolemma sheath and are known as neurolemma cells or sheath cells Histogenetically, considered, they are of ectodermal origin. It has not bees possible to obtain conclusive evidence as to whether the neurolemma forms a continuous sheath or is interrupted and cemented end to end at the nodes of Ranvier. In degenerating nerve fibers the neurolemma sheaths form a delicate tubular structure which does not fragment with the neuraxis and myelin and the sheath cells proliferate and separate from its inner surface. The neurolemma sheath is absent from nerve fibers of central nervous system; it’s place is there very probably taken by the neuroglia tissue. A nonmedullated fiber consists of a neuraxis which is made up of neurofibrils and neuroplasm with a delicate outer protoplasmic layer or axolemma. They present nuclei at relatively frequent intervals; nuclei which have the appearance of neurolemma or sheath nuclei, although it is difficult to demonstrate clearly a definite neurolemma sheath. It is quite possible that in the nonmedullated fibers the sheath cells do not form a continuous neurolemma sheath.
DEVELOPMENT OF PERIPHERAL NERVE FIBERS
Since histogenesis and experimental embryology of the nervous system has done much to clarify the problems of degeneration and regeneration of peripheral nerves, a brief consideration may here be given to the question of development of peripheral nerves. It is now generally believed that the nerve fibers of the entire nervous system are derived from the neurosensory ectoderm through the neuroblasts; the afferent fibers and the visceral or sympathetic efferent fibers largely from the neural crest, the anlage of the spinal and indirectly of the sympathetic ganglia; the somatic efferent fibers from the neural tube. The "outgrowth theory" of nerve developments first formulated by His 12 is now very generally accepted. According to this theory the neuraxis (and the dendrites) of a neuron are regarded as the outgrowth from a single cell. the neuroblast, no matter what the length of these processes. The growing tip of the neuraxis shows an expansion, known as the end-disc or the incremental
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cone; this is thought to have ameboid properties. The theory of His does not admit of demonstration in adult tissue, but admits of "near proof" in early embryonic stages, especially in tissues stained in differential neuron stains. Harrison's13 experimental observations of growing in coagulated lymph ganglion cells from the spinal cord of amphibian embryos, in which growing and budding neuraxes could be observed under the microscope, very substantially confirmed the outgrowth theory of nerve development. Growing neuraxes of the ventral and dorsal roots of suitably early embryonic stages are from the beginning accompanied by cellular elements which are in very close apposition with the growing nerve fibers. These cells, which were regarded as of mesodermal origin by earlier observers, are now known to be of ectodermal origin and are variously thought of as contributing to the formation of sheath cells or as participating in the formation of the neuraxis itself. The constant presence of these sheath cells in the early stages of growing nerve roots led to the formulation of the " chain theory " of peripheral nerve development, according to which, in essential, each internodal segment is thought to be derived from a cell, the neurons thus constituting a colony of cells in chain, or a syncytiun. With this theory the names of Balfour, 14 Dohrn, 15 and Bethe, 16, are especially associated. Modifications and interpretation of these two theories of peripheral nerve origin and growth are extant. Hensen 17 early contended for a primary connection between the nerve cells and the muscular and other tissues and thought that out of this primary syncytial net the nerve fibers were developed. Held 18 has more recently amplified this view. Such questions are not determined by a study of sections alone, although such study has contributed largely to the solution of the problem; experimental embryology has been of material assistance. Harrison 19 was able to ablate the neural crest in very young amphibian embryos, thus removing the anlage of the dorsal spinal ganglia without injuring the ventral part of the spinal cord from which the ventral root fibers have origin. It was found on development of the ventral root fibers that these were devoid of sheath cells. Further experiments by the same observer and others, including limb transplantation in young amphibian embryos, which on attachment and outgrowth in new positions became neurotized, indicate that there does not exist a primary connection between nerve cells and the peripheral tissues, a sine qua non to nerve growth. Observations made and deductions drawn from experimental embryology bearing on peripheral nerve development have been summarized as follows by Streeter: 20 It was shown that no peripheral nerve fibers would develop in an embryo from which the nerve center had been removed, thus establishing the fact that the ganglion cells are an essential element of the fibers. It was shown that the sheath cells of Schwann, upon the influence of which in the formation of the fibers many histologists had placed much emphasis, were not essential to the growth of the nerve fiber, and that the axis cylinders will develop and extend out in the surrounding tissues in the normal way and reach their normal length in specimens where the sheath cells have been eliminated. It was shown by modifying the environment of the developing nerves that fibers will form in surroundings entirely different from their natural path and establish completely foreign connections." Histogenetic studies have shown quite
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conclusively that the neuraxis is a protoplasmic outgrowth from a single neuroblast. The prevailing opinion is that the neurolemma sheath and its sheath nuclei are of ectodermal origin, derived directly or indirectly from the neuro-sensory ectoderm, as also the capsule cells of spinal and sympathetic ganglia. These neurolemma sheath cells, however, are not to be regarded as potential neuroblasts. There exists less certainty as to the development of the myelin sheath. This sheath seems a part of the neuraxis, which in its deposition is influenced by the sheath cells. Very careful studies of the histogenesis of the myelin are required before the structure of the myelin sheath and its relation to the neuraxis can be fully determined; in such studies experimental embryology must play its rǒle.
DEGENERATION AND REGENERATION OF PERIPHERAL NERVES
There exists a very extensive literature dealing with the problem of peripheral nerve degeneration and regeneration, far too extensive to receive even cursory review here; certain main phases of the development may be noted and briefly considered, since such treatment will obviate repeated restatement in discussing the experimental work. Arnemann, 21 as early as 1787, recognized the fact that a severed nerve lost its conductivity, and Cruikshank 22 and Haighton 23 believed themselves to have demonstrated experimentally regeneration of a severed peripheral nerve. However, it was not until 1852 that Waller 24 clearly demonstrated that the portion of a nerve fiber separated from a "ganglion cell," when a nerve is severed, undergoes degeneration and is regenerated through down growth from the central part. Ranvier 25 and Vanlair 26 mate-rially extended our knowledge more particularly as concerns the down growth of central fibers in regeneration. Their views were controverted by Schiff, Erb, and Wolberg, so that about 1890 there existed three main views concerning the mode of regeneration of severed peripheral nerves: 1, The view of Waller, that after degeneration of the peripheral stump regeneration was through downgrowth of neuraxes derived from the central stump; 2, that after secondary degeneration neuraxes developed in the peripheral stump which were secondarily united to the central fibers; 3, that the neuraxes of the peripheral stump did not degenerate. In the few years following 1890 there appeared a series of mono-graphic contributions dealing with this problem: Bu ngner,27 Howell and Huber 28 Stroebe, 29 Huber. 30 Bűngner, in his frequently quoted communication, paid especial attention to sheath-cell proliferation, and defined clearly the nucleated protoplasmic strands, derived from the proliferating sheath cells and designated by him as "bandfasern," which appear during the second and third week after injury, and were interpreted by him as new nerve fibers or potential nerve fibers. Stroebe, in his experimental studies, believes himself to have shown the down growth of neuraxes from the central stump. Huber, using Stroebe's method of neuraxis staining in an experimental study of bridging nerve defects, believed he had demonstrated the downgrowth of central neuraxes, the division of neuraxes in regeneration and the incremental cone at the growing ends of the neuraxes, the same as found on the growing tips of neuraxes in development, and in neurons grown in tissue cultures. These observations were followed by studies of Galeoti and Levi,31 Kennedy,32 and Wieting, 33 each
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of whom favored the view of peripheral autoregeneration in degenerated nerve. At about 1900 two main and opposing views as to the regeneration of degenerated nerve were held: 1, What is known as the monogenetic conception of nerve regeneration, according to which regeneration of a degenerated severed nerve is through downgrowth of neuraxes derived from the neuraxes of the nerve fibers of the central stump, at all times connected with central nerve cells;2, a polygenetic conception, according to which regeneration is obtained through cells derived from both the central and peripheral stump. The technical staining methods then at the disposal of observers were inadequate to admit of clear and differential staining of neuraxes, leading often to differences in the interpretation of observations made. Bethe 16 hoped to bring solution to the problem through a series of especially devised experiments in which downgrowth of central fibers was thought to be obviated. Bethe believed he had demonstrated new nerve fibers in a distal stump completely separated from the central connection. The experiments of Bethe seemed conclusive, and received wide consideration; they were refuted by the experimental observations of Langley and Anderson,34 Lugaro,35 and others. Several lines of investigation in correlated fields did much to bring solution to the problem: The experimental embryonic observations of Harrison and others contributed largely to the confirmation of the neuron doctrine and the outgrowth theory of nerve development, as above stated; the histogenesis of neurons was much more carefully studied; marked improvement in technical histologic methods was effected, especially as concerns the silver precipitation methods of Golgi, Cajal, Bielsehowski, and Ranson, and the intra vitam methylene blue method of Ehrlich. A series of experimental studies on nerve degenerations and regenerations was undertaken, controlled by careful histologic studies, in which the downgrowth of the neuraxis in regeneration could be followed step by step. This more recent literature includes contributions by Perroncito, 36 Poscharissky, 37 Cajal, 38 Ranson, 39 Boeke, 40 41 Dustin, 42 Ingebrigsten,43 and others, in all of which the modern silver precipitation methods for staining neuraxes have been used to control experimental results, the consensus of their work confirming the monogenetic or downgrowth theory of neuraxis development.
An injury to a peripheral nerve, producing severance of continuity induced be crush, sharp instrument, bullet wound or laceration, calls forth a series of structural changes in the distal segment, known as secondary degeneration or Wallerian degeneration, involving at about the same time the entire distal stump. except for a narrow zone in the immediate vicinity of the wound, a zone of traumatic injury the width of which rarely exceeds 0.5 cm. These structural changes are influenced by the presence or absence of the myelin and will be described separately for the two types of nerve fibers.
DEGENERATION OF MYELINATED NERVE FIBERS
For a period of three to four days in dog and man, two to three days in the rabbit and guinea pig, the nerve fibers distal to the line of injury, except in the zone of traumatism, show no demonstrable structural change and respond to mechanical and electrical stimulation. Structural change is first demonstrable in the neurofibrils of the neuraxis; they show varicosities and hypertrophy and
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granular change (Mönkeberg and Bethe),44 and in pyridine-silver preparations, irregularities of contour and staining (Ranson). 39 Beginning with the third or fourth day (man and dog; second or third day, rabbit and guinea pig) changes in the myelin sheaths in certain of the myelinated fibers is noted, consisting of irregularly placed enlargements and constrictions, followed by fragmentation or segmentation of the myelin and soon after of the neuraxis, resulting in the formation of segments, found within the continuous neurolemma sheath, known as myelin ellipsoids, presenting rounded ends inclosed by a laver of myelin and containing fragments of the neuraxis. This fragmentation of the myelin sheath and neuraxis is preceded or accompanied by the hypertrophy of the protoplasm of the sheath cells, their nuclei showing richer chromatin content. These, in their growth, first compress the myelin and neuraxis and, as these fragment, hypertrophy to occupy the lumen of the neurolemma sheath. These primary changes in the myelin and neuraxis are followed by progressive fragmentation of the myelin ellipsoids, resulting in the formation of larger or smaller masses of oval or spheric shape, many still containing fragments of the neuraxis differentially stainable with silver methods. This is accompanied by growth of the protoplasm of the sheath cells and proliferation of their nuclei in part at least by mitosis (Büngner,27 Huber 45). The hypertrophied protoplasm of the sheath cells begins to surround the myelin remaining. By the end of the first week after injury there are still to be found some few myelinated fibers which do not show fragmentation of the myelin; however, the majority of them show evidence of degeneration but to a variable degree. During the second week after injury there is noted a growth of the sheath cell protoplasm and a proliferation of their nuclei and a progressive fragmentation of the myelin remains. The sheath cells exert a phagocytic action on the myelin remains, so that during the second and third weeks after the injury the myelin globules become progressively less numerous and smaller and the protoplasm and nuclei of the sheath cells form distinct nucleated syncytial bands, still containing a variable number of myelin globules. The nucleated syncytial bands were first fully described by Bu ngner 27 as the "bandfasern" and, regarded as new nerve fibers, they were designated as "embryonic fibers" by Howell and Huber 28. These syncytial, protoplasmic, nucleated bands, developed through hypertrophv of the sheath cells, constitute a stage in the degeneration of peripheral nerve fibers which extends through weeks and months. They represent an undif-ferentiated, perhaps embryonic, protoplasm, and are of ectodermal origin. Büngner 27 and Bethe 16 and other adherents of the school of polygenetic nerve regeneration, have described a delicate, longitudinal striation within the proto-plasm of the "bandfasern," indicative of neurax is development and of peripheral autoregeneration. Faint longitudinal striation is now and again seen in the"bandfasern" in silver preparations, but transition stages between the syncytial protoplasmic bands and developed neuraxes have not been demonstrated. In considering regeneration of nerves it will be noted that neuraxes appear in the distal degenerated stump before the "bandfasern" are fully developed. The question of the mode of removal of the myelin in degenerating nerve fibers has received extensive consideration; both fixed and wandering cells of the
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peripheral trunk and its vessels have been brought in causal relation with this process. Donikow, 10 using various fixing and staining methods, has made a critical study of this question in degenerating nerves of the rabbit and finds that with the beginning of fragmentation of the myelin, fat droplets are found in the protoplasm of the sheath cells which are thought to pass to the tissue lymph spaces of the endoneurium probably in colloidal solution. The fourth day after injury, fat droplets are found in the endoneural cells and by the end of the first week in the cellular elements of the perineurium.
DEGENERATION OF THE NONMYELINATED FIBERS
The account here given is based on Ranson's 39 observations in preparations stained with pyridine-silver methods. Two types of nonmyelinated fibers are recognized; such as degenerated during the first week, thought to be afferent fibers; such as degenerated during the second week, thought to be visceral efferent fibers. Degeneration of the former begins soon after injury, the neuraxis becoming granular and during the second and third day showing alternate darker and lighter segments, the former representing neuraxis remains, the latter perhaps exudate. By the fourth day the darker segments disintegrate and disappear, so that by the end of the first week after injury the degenerated fibers are difficult to see. A proliferation of the sheath cells takes place and fine, nucleated protoplasmic bands develop and are found, usually, grouped in small bundles. The more slowly degenerating non-myelinated fibers may present uniform staining nearly two weeks after injury and be mistaken for newly formed neuraxes; their mode of degeneration is the same as that of the more rapidly degenerating nonmyelinated fibers. "We have, therefore, as the terminal stage of the degeneration of the non-medullated fibers, nucleated protoplasmic bands which differ from similar bands formed from the medullated fibers only in size and in absence of myelin droplets." (Ranson.) 39
DEGENERATION OF NERVE ENDINGS
Tello 46 and Boeke 40 have used differential silver staining methods in the study of motor nerve endings in degeneration. The account of Boeke is here followed. This investigator found that changes are observed in the neuro fibrillar net of the end plate during the first day after injury in that these fibrils stain only lightly. This stage lasts only a short time and is followed by one in which the fibrils hypertrophy and agglutinate and stain more deeply. This agglutination of the neurofibrils proceeds until darkly staining irregular strands are formed; these clump, run together, fragment and form irregular stainable masses which ultimately disappear. The telolemma nuclei are said to disappear and the sole plate nuclei to proliferate; the sole plate hypertrophies. The degeneration of the neuromuscular and neurotendinous and other pro- prioceptive nerve endings awaits special study with the use of modern differential neuraxis staining methods. It can be stated that the neuraxes disappear completely from the endings with the degeneration of peripheral nerves.
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THE NEUROLEMMA SHEATH
The ultimate fate of the neurolemma sheath of degenerating peripheral nerve fibers has not been conclusively determined. As has been stated, during the early stages of degeneration, while the protoplasm of the sheath cells shows hypertrophy and their nuclei proliferation, the neurolemma sheath as such appears to show no structural changes. In cross or in longitudinal sections of the peripheral stump of a degenerating nerve, one to three months after the beginning of degeneration, it seems possible to differentiate membranous sheaths, surrounding the nucleated syncytial bands, quite evident in regions where myelin globules are still present, though it is not possible to stain the neurolemma sheaths differentially. In experimental observations on degenerate nerves, extending 12 to 15 months after injury to the nerve, what appear as collapsed neurolemma sheaths, surrounding nucleated protoplasmic bands are thought to be present. According to the observations of Cajal, the neurolemma sheaths are said to disappear several weeks after the formation of the nucleated protoplasmic bands, these bands remaining surrounded by a fibrillar sheath of connective tissue origin, the fibrillar sheath of Retzius or of Henle. That a delicate sheath, either of ectodermal origin, neuroleinma sheath, or of mesodermal origin, the fibrillar sheath of Retzius or of Henle surrounds the nucleated, syncytial protoplasmic bands, the product of sheath cell proliferation, months after the degeneration of a peripheral nerve is under way, seems well established.
DEGENERATION IN THE PERIPHERAL AND CENTRAL ZONE OF TRAUTMATISM
There are observed certain structural changes in the immediate vicinity of the wound, differing from the changes noted for secondary nerve degeneration, involving both myelinated and unmyelinated nerve fibers. In sections of an injured nerve trunk, including that portion of the distal stump adjacent to the wound, fixed in chrom-acetic-osmic mixture, about 24 hours after the injury it will be observed that the myelin sheaths of the myelinated fibers do not stain black in osmic acid, as do the nerve fibers more distal, but present a granular appearance and indistinctive coloring. This appears to be the result of the traumatic injury. The neurolemma sheaths appear distended, leucocytes appear in appreciable numbers, both between and, now and again, within the neurolemma sheaths. These changes are noted near the wound at a time when 1 cm. distal, the nerve fibers show no structural change nor alteration in conductivity. In preparations of the distal stump of the wound region stained after differential neuraxis staining methods, Perroncito,36 Cajal, 38, Ranson,39 and others have noted, both for myelinated and nonmyelinated nerve, changes in the neuraxis regarded as an abortive auto regeneration, consisting of side branches terminating in bulbous ends. These may be observed in certain nonmedullated fibers by the end of the first day and may be quite numerous; they do not show much growth and the majority disappear within a few days. Certain of the myelinated nerve fibers show a similar phenomenon. The peripheral end of the central stump of a divided or injured nerve degenerates for a certain distance centrally; the distance varying with the type of injury, though it is usually not more than 0.5 cm.
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to 1 cm. The degenerative changes noted here are essentially the same as described for peripheral secondary nerve degeneration. Kirk and Lewis 47 believe that there is present an early hpertrophic reaction of the neurolemma sheaths adjacent to the line of section, first noticed as an increase in the protoplasm surrounding the sheath cell nuclei followed by mitosis of these nuclei, so that between the fourth and fifth day syncytial nucleated protoplasmic bands have developed. Ranson 39 has described certain changes in the nonmyelinated fibers of the central stump. An early abortive regeneration analogous to that described for similar fibers in the wound region of the distal stump was noted the fine side branches terminating in end-discs which develop only slightly after their first appearance and disappear with the cellulipetal degeneration of nonmyelinated fibers, which begins about the third day after injury and extends about 2 cm. centralward, degenerating as do the nonmyelinated fibers of the distal stump. In preparations stained in differential neuraxis stains, certain neuraxis phenomena are observed in the myelinated fibers of the distal end of the central stump which are regarded as degenerative in character. In certain of these myelinated fibers, 0.2 mm. to 0.5 mm. from the wound, a zone of reaction is found in which the neuraxis is greatly increased in diameter, with neurofibrils staining more deeply and apparently hypertrophied, with increase in neuroplasm; distal to this zone of neuraxis reaction, in a respective fiber, the neuraxis disintegrates. Very early branching of the neuraxes in the immediate vicinity of the wound is noted. The newly formed branches may grow into the exudate of the wound, ending in small discs, or remain within the old neurolemma sheaths, where they become entangled and in further growth often form quite complex skeins. These phenomena appear to be abortive attempts at regenerative and are rather to be associated with degenerative processes.
REGENERATION OF PERIPHERAL NERVES
The process of regeneration of a peripheral nerve degenerated after injury, is initiated before the formation of the nucleated syncytial protoplasmic bands is well under way; a topical and timely separation of the two processes call not well be made since they are concurrent; they deserve separate discussion since they are independent. Certain of the main phases in the interpretation of the process of nerve regeneration were considered in the brief review of the literature preceding this section and it was noted that with the introduction of differential neuraxis staining methods and their use in experimental studies of nerve degeneration and regeneration that the downgrowth or monogenetic theory of neuraxis development has become quite generally accepted. The studies of Perroncito,36 Cajal,38 Ranson,39 Boeke40, 41 Kirk and Lewis,47 Dustin,42 Ingebritsen,43 and others. all of whom have used these special silver methods to control the results of experimental observations, have been especially helpful in bringing clarity to this subject. Boeke, one of these more recent workers, in an excellent study expresses himself as follows: "I place myself without question as adopting the viewpoint that as in development of embryonic nerve fibers, the regenerating nerve fibers arise exclusively through outgrowth from divided nerves of the central stump, which enter the peripheral path and in
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this reach their peripheral destination." While unanimity has not been reached in all points there is quite general agreement that the down-growing neuraxes, derived from the central nerve fibers, are a sine qua non of the regeneration of the peripheral degenerated portion of a divided or injured nerve. These
FIG. 212.- Microphotograph of a pyridine-silver preparation from a longitudinal section of the distal end of the central stump of the sciatic of a dog, 11 days after the operation of cable-auto-nerve transplant, showing division of down-growing central neuraxes
more modern observations have extended but have confirmed fully conclusions reached by a group of observers, using less satisfactory methods and working nearly 25 years ago. One quotation may be permitted. Huber, 30 at the conclusion of an extended study on the repair of nerves after loss of substance
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expresses himself as follows: “The regeneration of the peripheral end (which always degenerates so that only the old sheaths of Schwann containing band of nucleated protoplasm, developed from the hyperthro pied protoplasm and proliferated nuclei of its fibers, are met with) is the result of all out-growth of new axis cylinders from undegenerated axes of the central stump, the budding axes following paths of least resistance."
Evidences of regeneration are noted in the distal end of the central stump of a divided nerve within the first day after injury (Perroncito36 and others) in the form of fine branches derived from central neuraxes, which grow into the exudate of the wound or remain within the neurolemma sheaths. These
FIG. 213.- From longitudinal section of a regenerating distal segment of a severed nerve several weeks after operation. Note the single neuraxis deflected upon itself and terminating in a prominent end-disc or incremental cone. Pyridine-silver preparation
early branches of the central neuraxes, very likely, in part degenerate again; at least there is not marked progress until toward the end of the first. week after injury, evinced now by numerous side branches and end divisions of central neuraxes, usually several tenths of millimeters central to the cut ends of the fibers. The central neuraxis of a myelinated fiber may here give off numerous new branches, the number estimated as high as 50 by Ranson, 39 clearly evident in cross sections of suitable stages in which, in the crosscut regenerating fibers of the central stump, a variable though appreciable number of new neuraxes may be found within one neurolemma sheath, often found arranged in a circle surrounding the old neuraxis of the respective fiber. These newly formed
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neuraxes grow toward the periphery of the central stump, within the old neurolemma sheaths, having a more or less parallel course, or for reasons not satisfactorily explained may assume a very complex coiled or spiral arrangement, forming longer or shorter skeins which were first clearly defined by
FIG. 214.- Taken from the distal half of a neurorna, 21 days after severance of the sciatic nerve of a dog; pyridine-silver preparation. The figure presents a number of spiral formations (Perroncito spirals), several end-discs or incremental cones, the ends of down-growing neuraxes, crisscrossing of down-growing neuraxes, typical of their growth through embryonic connective tissue
Perroncito 36 and are often referred to as the spirals of Perroncito. On the ends of the down-growing neuraxes smaller or larger end-discs or incremental cones, similar to those found on developing, embryonic nerve fibers are easily demonstrated. They often are directed toward the periphery but are also
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found directed centralward and are conspicuous in the Perroncito spirals.The end-discs on the neuraxes are often many times the diameter of the respective neuraxis, and it is conjectured that their relative size is proportionate to the resistance met with by the downgrowing neuraxis. The nonmedullated fibers of the central stump begin regeneration about the fourteenth day after injury; these downgrowing neuraxes having small end-discs and arc often found arranged in small compact bundles. The downgrowing branches of the neuraxes of both the myelinated and the nonmyelinated nerve fibers of the central stump are first nonmyelinated. The very marked increase in the number of the central neuraxes was not appreciated until these fine nonmedullated nerve fibers were brought to view with silver staining, the evidence contributing largely to the substantiation of the theory of the downgrowth of central neuraxes in the regeneration of a peripheral degenerated nerve. From the beginning of the time of budding and downgrowth of central neuraxes, certain of them reach the exudate of the central wound; their number is appreciable toward the end of the first week after injury. As the down-growing neuraxes reach the exudate and the organizing embryonic connective tissue of the wound region, whether suture has taken place or not, the small bundles of down-growing neuraxes lose their regular arrangement and direction parallel to the long axis of the nerve and assume an irregular crisscross course and either as single fibers or small bundles of such they course through the wound region. Immediate suture of a severed nerve does not seem to accentuate the rate of downgrowth of the central neuraxes, nor is the forming scar tissue of the wound region negligible, even under the most favorable suture conditions, in influencing the downgrowing neuraxes and in dispersing them sufficiently to influence the funicular structure. Now and again central neuraxes may be traced through several divisions in the same or successive sections passing through the wound region. Neuraxes terminating in end-discs within the organizing scar tissue of the wound after suture or in the central stump when no suture has taken place, are to be observed in nearly every section in suitably stained sections of requisite stages; they are found extending in every direction. In cross and longitudinal serial sections of the wound region, including approximately one centimeter of the distal and central stumps adjacent to the wound and taken at intervals of three to five days during the first three weeks after severance or injury of the nerve, and prepared after the differential silver neuraxis staining methods, a progressive neurotization of the scar tissue maybe observed, proceeding from the center to the periphery, and easily demontrated in the region at a time when no neuraxes are found in the more distal segments of the degenerated nerve. There is noted a gradual decrease in the number of neuraxes as one passes from the more central to the more distal portion of the wound. In cases of primary suture of a severed nerve, new neuraxes of central origin are found in the central end of the distal segment of the divided nerve only a few days after they appear in the wound region, at first in small numbers and scattered here and there and then in progressively larger numbers as time advances. After the down-growing neuraxes have reached and penetrated the central end of the distal stump, the course of the majority of them becomes again quite regular and parallel to the long axis of
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the nerve. Under favorable conditions new neuraxes may penetrate the central end of the distal stump before its nerves have reached the end stages of nerve degeneration, evidenced by the presence of nucleated syncytial bands developed from the hypertrophied and proliferated sheath cells. In their downgrowth into the central end of the distal stump many of the down-growing central neuraxes penetrate the neurolemma sheaths of the degenerating peripheral fibers, others are found in endoneural spaces between the nerve fibers, others pass along the inner or outer surface of the perineural sheath others again are found in the epineurium. It is not unusual to observe several new neuraxes within one neurolemma sheath of a degenerating nerve fiber of the distal stump and, in longitudinal sections of this region, neuraxes with end
FIG. 215.- From a longitudinal section of the proximal zone of a neurorna on the sciatic of a dog, 31 days after section; pyridine-silver preparation. The figure presents the central down-growing neuraxes, in approximately parallel arrangement, so long as the down-growing neuraxes are in the main within the old neurolemma sheaths and the dispersed and irregular arrangement of the neuraxes as found in the scar tissue of the more distal zone of the neuroma
discs are often noted indicating the distal termination of a new neuraxis within the limits of the section. In cross or in longitudinal serial sections it can be readily determined that neurotization of the peripheral degenerated portion of a nerve progresses gradually from the region of the wound and suture toward the periphery and this at a rate which is estimated at from 1 mm. to 2 mm. in 24 hours. The progressive neurotization is not only in the distance of the penetration of the downgrowing central neuraxes but also in the increase in the number of neuraxes which reach the distal stump; this number gradually decreasing toward the periphery.
Attention has been called to the very great increase in the number of the neuraxes, formed by division and budding in the distal end of the central stumps
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Only a variable percentage of these reach the distal stump through the wound region, proportionately more, the more favorable and shorter the path. The downgrowing central neuraxes are diverted from their course toward the periphery in the organizing scar tissue in various ways. Certain of them are deflected centralward, ending in the endoneural tissue of the central stump. Others grow toward the edges of the wound and are lost in the surrounding connective tissue, while others pass to the fibrous layers of the peripheral stump outside of the funiculi. In experimental work, it has been observed, that the more successful the suture the earlier do downgrowing neuraxes reach the distal stump and the greater the number of downgrowing neuraxes which penetrate it. In case a primary suture is not made and the severed nerve ends are not in close
FIG. 216.- From a longitudinal section of a neuroma on the sciatic of a dog, 31 days after nerve section; pyridine-silver preparation. The figure makes evident the great increase in the number of down-growing neuraxes as found in the central zone of the neuroma, or proximal to the wound after severance and suture
approximation, even though there be not extensive separation, the down-growing central neuraxes become dispersed and deflected in their course in the organizing scar tissue forming the central end of a cut nerve resulting in the formation of an amputation neuroma, even in cases in which the central down-growing neuraxes ultimately reach the distal stump and bring about its partial neurotization. The down-growing neuraxes of the central stump, whether the branches of myelinated or of nonmyelinated fibers, in the wound region and in the distal stump are all at first nonmyelinated fibers. Whether these down-growing neuraxes are preceded or accompanied by sheath cells derived from the central fibers has not been determined conclusively. The modern silver methods which give such clear differentiation of neuraxes and terminal end-discs do not
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differentiate equally clearly between cells derived from the ectodermal sheath cells and the cells derived from the mesodermal fibroblasts and both types of cells appear to form syncytial structures in the earlier stages of nerve repair. It is difficult to conceive of the relatively large end-discs of growing neuraxes, thought to have ameboid properties, as coursing within the nucleated protoplasmic bands. The same may be thought of the complex spiral structures or the numerous neuraxis buds growing from a single central neuraxis. Experimental embryologic evidence is at hand to show that neuraxes may grow without the presence of sheath cells; resulting in the formation of naked neuraxes
FIG. 217.- From a longitudinal section of a neuroma, removed-three-weeks after section of the sciatic of a dog; pyridine-silver preparation. Note- the branching of neuraxes; the crisscrossing of neuraxes; prominent spiral formations and a number of typical though relatively large end-discs or incremental cones denoting the distal ends of downgrowing neuraxes
What appear to be naked neuraxes are now and then followed for quite a long distance in the connective tissue or between the muscle fibers and this some distance from the wound. The statement has been made that the nu-cleated syncytial protoplasmic bands of the distal stump exert a chemotactic influence on the downgrowing neuraxes, drawing them toward the neurolemma sheath of the peripheral stump and influencing their downgrowth toward the periphery. This has not been experimentally demonstrated. There are often found several down-growing neuraxes in one neurolemma sheath of the distal stump, but they are also found between the nerve fibers of the funiculi and in the connective tissue outside of the perineural sheaths. The relation of the
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down-growing neuraxes found within the old neurolemma sheath to the nu-cleated syncytial bands also found within the sheaths has been interpreted differently by observers. They have been regarded either as playing a passive r le or as forming conduits for the down-growing neuraxes. Ranson39 thinks that all the new axons lie in protoplasmic bands and Boeke,40 as a result of a study in which he made use of various technical methods, reached the conclusion that neurofibril strands are always intraprotoplasmic. Kirk and Lewis 47 believe that they have shown that the nucleated, syncytial, protoplasmic bands constitute conduits in the substance of which the nonmedullated nerve fibers regenerating from the proximal stump rapidly grow down, the bands forming first, the fibers following along them though they state that they do not wish to imply "that the axis cylinders always and necessarily track along the protoplasmic bands." The prevailing opinion at the present time regarding the down-growing neuraxes found in the peripheral neurolemma sheaths is that they have an intraprotoplasmic position. So far as has been determined there is at hand no evidence to warrant the conclusion that down-growing neuraxes show any selectivity on reaching the peripheral stump. Thus branches from the central motor neurons may and do, no doubt, enter the sheath of degenerating afferent or sensory nerve fibers and vice versa; and that nonmedullated central fibers may enter the sheaths of medullated peripheral fibers and vice versa. It has long been known that central motor fibers may grow into distal sensory fibers, and vice versa. This has recently been confirmed by Boeke.41 Functional regeneration has not been attained although neuraxes are seen in the periplheral segments and a beginning in the formation of nerve terminations has been noted. It is assumed that chance brings as many down-growing central neuraxes of motor neuron origin to degenerated peripheral motor nerve fibers as of central fibers of other functional types and so for other types of nerve fibers. It has seemed to the writer that a simple mechanical explanation is perhaps the correct one. It may accordingly be assumed that developing neuraxes of the central stump, which reach degenerating homologous nerve fibers of the peripheral stump, develop the functional activity after the formation of the requisite nerve terminations; while down-growing central neuraxes which reach degenerating heterogeneous nerve fibers of the peripheral stump ultimately degenerate. The enormous increase in the number of central nerve fibers, through division and formation of side branches, permits many nerve fiber branches to go astray and ultimately to undergo degeneration by reason of want of functional activity and still leave a sufficient number to admit of structural and functional regeneration of a nerve going to a given part. The conclusion is, as a result of many experimental observations on nerve repair, that there is obtained at best only partial regeneration of the distal segment of a peripheral divided or injured nerve.
Huber,48 Tello,46 and Boeke40 have studied the regeneration of the motor and sensory nerve ending in striated muscle tissue after experimental degeneration of nerves, with the aid of the intra vitam methylene blue method and certain silver precipitation neuraxis stains. Experimental observations show that a muscle responds to electrical stimulation of its nerve which had previously been degenerated, so soon as newly formed motor nerve endings can be demonstrated in said muscle by suitable staining methods. Down-growing neuraxes,
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now and then terminating in end-discs can often be demonstrated in the interfascicular muscular branches when stimulation of the motor branches fails to incite muscular contraction. Developing motor endings in regeneration have been demonstrated as forming as the result of the branching of end discs, or as branches of collaterals led to the muscle fibers within the old neurolemma sheaths or along syncytial strands of sheath cells and perhaps along cells of connective tissue origin. There may be formed more than one nerve termination on a single fiber. Motor endings regenerate before sensory endings; possibly explaining a voluntary control of a muscle before there is synergic action of said muscle. Coarser or finer nerve fibers are often found within the capsule of neuromuscular and neurotendinous nerve endings before the endings show full development. Complete regeneration of the complex endings has been observed.
THE MYELIN SHEATH AND NEUROLEMMA SHEATH OF REGENERATING NERVE FIBERS
The silver precipitation methods used as differential staining for neuraxes, and used by nearly all observers in the more recent studies of nerve degeneration and regeneration are not suitable methods for the study of the histogenesis of the myelin or neurolemma sheath, so that these more recent observation have on the whole not contributed materially to the solution of this question. As has been stated, all downgrowing neuraxes derived from the central stump are at first nonmyelinated. It seems quite clear that such of these fibers as are destined to become myelinated acquire their myelin sheath beginning with the proximal end, and proceeding from here distalward. New myelinated nerve fibers can be recognized first in the distal end of the proximal segment of a divided nerve toward the end of the first month after injury in the formof very delicate and apparently continuous sheaths demonstrable in chromatized tissue treated by differential myelin staining, methods. The structural appearances presented suggest the differentiation of the myelin sheath as a peripheral layer of the neuraxis, though this can not be stated without reservation. Myelin sheath formation proceeds distalward and does not involve all of the nerve fibers at the same time. This process of myelinization begins in the proximal stump before functional connection of the nerve fibers with formation of nerve terminations has been effected.
The neurolemma sheaths of regenerating nerve fibers appear to be new formations, developed from the sheath cells, which migrate with budding neuraxes or are found in situ by the down-growing neuraxes and derived through, sheath cell proliferation during the process of degeneration of the nerve. The detail of the development and formation of the neurolemma sheath of regenerating nerve fibers has not been definitely determined.
EXPERIMENTAL OBSERVATIONS
The experimental observations to be recorded and discussed in the following pages deal in a large measure with the question of bridging nerve defects peripheral nerves, due to loss of substance. These defects were bridged by nerve transplants or otherwise. No experimental observations on simple, primary, or secondary end-to-end suture of a severed nerve were made. The
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end-to-end suture of a divided peripheral nerve, either as a primary or a secondary operation, is a recognized procedure in surgery, even though there may not be full agreement as to technical details of the operation. The differences of opinion as to the technique of end-to-end suture of a divided nerve were not such as seemed to warrant extended experimentation at a time when other questions seemed to be more pressing. The same can not be said for operative procedure suggested and used for the repair of a divided nerve after loss of substance. The various methods which have been suggested to bridge the gap between the severed ends of a peripheral nerve in case there is loss of tissue to the extent that the severed ends can not be brought together without undue tension were reviewed by Huber 4 and critically considered in the light of experimental observations. Such operative procedures include the formation of a nerve flap from either the central or distal stump or both; operations of nerve implantations; operations of nerve crossing, either complete or incomplete; suture a distance; tubular sutures and nerve grafting or nerve transplantation. The value of the nerve flap in the repair of peripheral nerves was tested experimentally by Huber 30 and discredited and shown to be of no value by Stookey 50 as a result of a critical review of all the reported cases. Nerve implantation consists of the insertion of the severed end of one nerve, either the central or distal end or both, into the interfunicular tissue of an adjacent nerve. By multiple or serial implantation is meant where one or several parallel nerves are injured, with loss of substance and are then serially implanted (Hofmeister).51 Neither of these operations can be approved since on implantation of the central stump of a divided nerve into the interfunicular connective tissue of a sound nerve there is not furnished a suitable path for the downgrowth of central neuraxes; the less so in serial implantation. By nerve crossing is here meant the suture of the central end, complete crossing, or a flap from a normal nerve, incomplete nerve crossing, to the distal end of another nerve. It has been shown experimentally that complete and incomplete nerve crossing are feasible and logical procedures, whether made as a primary or a secondary operation. In selecting this as the operation of choice it needs to be decided whether other operative procedure may not offer more favorable end results when considering both of the nerves involved. It should be understood that in the operation of complete crossing, in forming the central flap, the sound nerve is injured to the extent of the flap and paralysis will ensue in the field of the cut fibers, not relieved or only partially relieved by the accidental downgrowth of central neuraxes from the parent nerve. In suture a distance a bundle of catgut strands has been used to bridge a defect in a divided nerve with loss of substance by Huber 30, with some success as concerns neurotization of the distal stump. However, other methods offer greater opportunity for bridging nerve defects. Muscle and tendon fibers, woolen, silk, and other fibers have been used for suture a distance with unfavorable results and need receive no further consideration here. The operation of uniting the separated ends of a divided nerve by means of a tube through the lumen of which the central neuraxes were thought to be directed to the distal stump-tubular nerve suture-is an old operation and has received more than incidental attention by surgeons even in relatively recent years. A variety of materials have been
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used, not all of which have been tested experimentally. Of materials used at operation or in experiments, mention is here made of decalcified bone tube, iodoform gauze, epidermis of man, magnesium tubes, hardened-gelatine tubes, rubber tubes and sheet rubber wrapped to form a tube, galalith tubes, hardened arteries and fresh arteries and veins, fascia lata and other connective tissue membranes, peritoneum, Cargile membrane, celloid in tubes, arteries filled with agar, as suggested by Edinger and extensively used for a brief period in the German army. Certain of these materials for tubular nerve suture were given consideration in our experimental work and will be discussed in connection with the experimental observations. Other materials used are really more of academic interest than of practical use and their consideration need not occupy space here. Nerve grafts or nerve transplants were given extensive consideration in our experimental work, new methods were devised which, judging from experimental results, should receive favorable consideration at the hands of surgeons in dealing with the repair of injured nerves after loss of substance. We have followed surgical usage and have designated a nerve segment from another nerve of the same individual as an auto-nerve-trans-plant; a nerve segment taken from another individual but of the same species as a homo-nerve transplant; a nerve segment taken from another individual but of a different species, a hetero-nerve transplant.
The literature dealing with both the clinical and the experimental phases of the question of repair of peripheral nerves after loss of substance is an extensive one, too extensive to be reviewed here. Certain of the contributions will be considered in connection with the discussion of the experimental observations. Many citations are given by Huber 49 and by Stookey 9, who give critical reviews of clinical and experimental observations.
The experimental observations to be discussed here will be considered under the following heads: a
Series No. 1. Injection of absolute alcohol into a normal nerve, without subsequent cutting of the nerve; 12 experiments.
Series No. 2. Injection of full-strength acetone into a normal nerve, without subsequent cutting of the nerve; 3 experiments.
Series No. 3. Injection of absolute alcohol into the central end of a divided nerve to obviate the formation of an amputation neuroma; 37 experiments.
Series No. 4. Amputation neuroma formation in aseptic wounds; 12experiments.
Series No. 5. Auto-nerve transplants, including cable auto-nerve transplants; 17 experiments.
Series No. 6. Homo-nerve transplants; 6 experiments.
Series No. 7. Hetero-nerve transplants, mainly guinea pig's nerves to rabbits, also nerves of dogs to rabbits; 39 experiments.
Series No. 8. Degenerated auto-nerve transplants; 3 experiments.
Series No. 9. Degenerated homo-nerve transplants; 5 experiments.
Series No. 10. Degenerated hetero-nerve transplants; 16 experiments.
a Brief preliminary reports have been given Of these experimental observations before the Chicago Neurological and Pathological Societies (Huber) and before the Chicago Surgical and Neurological Societies (Huber), 53 Reports of progress were given by Huber 54 on special detail before the Neurosurgical School in New York City.
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Series No. 11. Homo-nerve transplants which had been stored in sterile vaseline at 3° C.; 8 experiments.
Series No. 12. Homo-nerve transplants which had been stored in sterile liquid petrolatum at 3? C; 40 experiments.
Series No. 13. Homo-nerve transplants which had been stored in 50 percent alcohol at room temperature; 19 experiments.
Series No. 14. Hetero-nerve transplants, dog's nerve to rabbit; stored in sterile liquid petrolatum at 3° C.; 6 experiments.
Series No. 15. Hetero-nerve transplant, dog's nerve to rabbit, stored in 50 percent alcohol; 3 experiments.
Series No. 16. Auto-nerve transplants, wrapped in Cargile membrane; 14experiments.
Series No. 17. Auto-nerve transplants, wrapped in auto-fascial sheaths of fascia lata; 14experiments.
Series No. 18. Auto-nerve transplants, wrapped in formalized arterial sheaths; 6 experiments.
Series No. 19. Auto-nerve transplants, wrapped in auto-fat sheaths; 2experiments. Series No. 20. Tubular sutures with use of formalized arteries; 13 experiments.
Series No. 21. Tension sutures; resected nerve sutured under extreme tension, with or without wrapping in Cargile membrane or formalized arterial sheaths; 13 experiments.
All of the operative work was carried out under strict asepsis and by surgeons on special detail for experimental nerve surgery. This work was very carefully done, especially as pertains to the technique of the operative work on the nerves. Special care was exercised in placing sutures so as to obtain accurate coaptation of the severed nerve ends, to stay all hemorrhage, and to obviate clot formation between the severed nerve ends. In practically all of the experimental operations healing of the wound was attained by primary intention; when otherwise, notation is made in the respective protocol. In the several series of experiments undertaken the respective animals were kept under observation for periods which varied in the several experiments from one day to about a year. Very few animals were lost as a result of operation. There was evidently great difference in resistance to disease. Now and again epidemics would interfere with the schedule of experiments, though not always with complete loss of the experiment. At fixed times the animals operated upon were killed and the operated nerves exposed and gross observations recorded. Functional tests were made in nearly all cases in which return of function was crucial to the experiment. After exposure of the nerve and the making of the necessary functional tests the operated nerve was removed for histologic study. In cases in which the operated animals died during the night, especially if this occurred in an experiment in which some time had elapsed since the initial operation, equally careful exposure of the operated nerve was made and a histologic study undertaken. In nearly all of the experiments a segment of the nerve central to the wound region, the wound region, and the portion of the nerve distal to the wound were removed for
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histologic study, and sectioned serially in alternate longitudinal and crosscut series. Considering the primary purpose of these series of studies, namely the opportunity of applying in practical surgery the conclusions reached in the work of repair of peripheral nerves, our attention was especially directed toward the behavior of the central neuraxes in the process of neurotization of the peripheral segment, the sine qua non of peripheral nerve repair. It was necessary to determine on a reasonably reliable differential neuraxis stain, one that could be used in staining en masse and permit of paraffin embedding to facilitate, so far as possible, the making of serial sections, so essential to the adequate study of the problem in question. After brief trial of several methods the pyridine-silver method of neuraxis staining (Ranson) was selected and the great majority of the histologic sections made were stained after this method. The essential steps of the pyridine-silver method of neuraxis staining are as follows: 1. Fixation of tissue in ammoniated absolute alcohol for 2 to 3 days. The ammoniated alcohol is prepared by adding 1 c. c. of strong ammonia water to 100 c. c. of absolute alcohol. A relatively large quantity of the fixative is essential. 2. Wash for about 2 minutes in distilled water. 3. Place for 24 hours in pyridine. 4. Wash in distilled water for 24 hours, changing the water frequently. 5. Place in 2 per cent aqueous solution of nitrate of silver, in which the tissue remains for 3 to 5 days, in the dark and at a temperature of 32? C. to 35° C. 6. Rinse in distilled water and place for 1 to 2 days in a pyrogallic acid-formalin solution prepared by dissolving 4 gms. of pyrogallic acid in 100 c. c. of 5 percent formalin. Keep in the dark and at a temperature of 32? C. to 35? C. 7. After washing in distilled water for several minutes dehydrate in graded alcohol, beginning with 80 percent alcohol. The dehydration needs to be thoroughly done; several changes of absolute alcohol are required. 8. Clear in xylol and embed in paraffin. A necessary stay in the warm oven even for 48 hours, to insure thorough penetration of the paraffin, does not deleteriously affect the stain.
The great bulk of the sections stained by the pyridine-silver method were cut serially by Huber's “water on the knife" method with the knife in the oblique position and on the sliding microtome. The sections as cut were arranged serially on the slide and fixed to the slide by the water-albumin fixation method and warmed to attain flattening of the sections. After removal of the paraffin thorough dehydration and clearing in xylol, the sections were mounted under cover glass in damar. Attempts at contrast staining were made but not generally used. In successfully stained preparations after use of the pyridine-silver method the nonmyelinated neuraxes stain a nearly black or brown-black or dark brown color, which usually contrasts quite distinctly with the yellow-brown color of the connective tissue. The neuraxes of myelinated fibers of the central stump, especially of the larger nerve fibers, are of a light brown or yellowish-brown color. The Perroncito spirals and the incremental cones or end-discs are very clearly differentiated. The myelin sheaths are not differentially stained. The nuclei of the neurolemma sheaths and of the fibrous connective tissue cells and fibroblasts of growing fibrous tissue. may or may not be differentiated, usually not clearly nor certainly enough to determine definitely questions concerning their participation
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in the downgrowth of central neuraxes. The Bielschowsky method and this method as modified by Boeke in his studies of degeneration and regeneration of nerves, as also Cajal's methods, were used; but they did not give as reliable and constant results as the pyridine-silver method. The pyridine-silver method does not give as satisfactory results in cases where the tissues are removed several hours after death, though in certain of the experiments in which the animal died during the night, very successful staining of the neuraxes was attained. It should be understood that with all silver precipitation methods of neuraxis staining, as with intra vitam methylene blue method, positive results can be received with much greater assurance than negative results. The fact that no neuraxes are found stained in such preparations is not proof positive of their non existence. Certain of the operated nerves were fixed in formalin and after chromatization stained in differential myelinstains, in iron-lac-hematoxylin or double stained in safranine and licht gr u n. In other cases the nerve was fixed in chrom-acetic-osmic acid mixture and stained in safranin and licht grün. The experiments in which these latter histologic methods were used in place of the pyridine-silver method are relatively few and mostly in experiments of short duration.
INJECTION INTO LIVING UNCUT NERVE
SERIES NO. 1
INJECTION OF ABSOLUTE ALCOHOL INTO A LIVING NERVE WITHOUT CUTTING THE NERVE
Schlösser 54, 55 introduced the use of alcohol injection into a nerve trunk or the tissue surrounding the nerve trunk for the relief of neuralgia or other peripheral nerve irritations. Brissaud, Sicard, and Tanon 56 record the use of alcohol injection for facial spasms. Since then the method has been extensively used in the operative relief for neuralgic conditions. Experimental observations on alcohol injection into a living nerve trunk are first recorded by Finkelnburgs 57 who, taking part in a general discussion on the treatment of neuralgia, reports briefly on experimental observations in which 0.6 c. c. to 1.5 c. c. of 60 per cent to 80 per cent alcohol was injected into the sciatic of dogs after exposure of the nerve or into the tissues surrounding the nerve. A complete paralysis was produced on injecting the nerve, lasting for months, with complete degeneration of the nerve in the wound region and the peripheral segment of the nerve. A much more extended and careful study of the question was undertaken by May.58 He injected the infraorbital as a pure sensory nerve, the sciatic and anterior crural as mixed nerves, and also the Gasserian ganglion. General histologic methods, as also Cajal's and Bielschowsky's silver impregnation methods for neuraxis staining, were used. The protocols of the experiments made are given, as also figures illustrative of the changes resulting in the nerve on injection of alcohol. The following are certain of the conclusions reached by May:58
1. Alcohol injected into the trunk of a peripheral nerve produces more or less complete local necrosis of the nerve at the point of injection.
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2. The change is not an ascending one; the nerve above the point of injection remains normal; the cells of origin of the fibers flay show some degree of chromatolysis, but do not exhibit signs of permanent injury.
3. The conditions produced by such injection are more favorable to regeneration than those resulting from simple section without suture. The anatomical continuity of the nerve trunk favors rapid regeneration, though this is to some extent retarded by the fibrosis which occurs to a greater or less extent in every ease of alcohol injection.
Harris 59, 60 and Patrick 61 have reported extensive clinical observations, dealing, mainly with the injection of the trigeminal ganglion for the relief of trifacial neuralgia, an operation experimentally studied by May. 58 The injection of a nerve trunk with alcohol in causalgia was first recommended by Sicarde 62 and has since received consideration by a number of other observers, who have used the method with success.
The experimental observations in this series were undertaken with a primary view of gaining experimental data for comparison and correlation with the experiments reported upon under Series No. 3, dealing with the amputation neuroma. The histologic findings in the several experiments are of interest per se, especially as concerns the more immediate effects of the alcohol on the living nerve fibers. Our observations were made on the sciatic nerve of the rabbit. This nerve, in the rabbit, can be exposed from the popliteal space to the sciatic notch with very little bleeding. The exposed nerve was then freed from its bed at about the middle of its course and raised slightly on a blunt hook and injected through a fine hypodermic needle inserted if possible beneath the perineural sheath of the large funiculus and into the epineural sheath surrounding the smaller funiculi and in a direction nearly parallel to the long axis of the nerve. The injection of the alcohol was made slowly and with the nerve exposed, so as to enable the observer to follow the immediate result of the injection. The nerve for the length of 1.5 cm. to 2 cm. presents, after successful injection, a "milky white" appearance. The few drops of alcohol which might escape into the wound were taken up with cotton. The wound was then closed with deep catgut or silk stitches and the skin wound with interrupted silk stitches.
PROTOCOLS
EXPERIMENT No. 1.- Rabbit No. 24a. Large; full grown; 1 hour. March 12, 1918.4 p. m., right sciatic exposed and injected while in place, with absolute alcohol. Nerve not cut. Wound closed. March 12, 5 p. m., wound opened, one hour after alcohol injection A slight hemorrhage is found at the point of injection. The part of the nerve affected by the alcohol, a little over 1 cm. in length, presents a dull white appearance. The sciatic removed and fixed in neutral formalin. Bielsehowsky's differential staining method used.
Microscopic findings.- In series of longitudinal sections, including the field of alcohol injection and the nerve trunk adjacent, central and distal thereto, it may be observed that in the nerve fibers found in the field of alcohol injection the neuraxes are not interrupted and present the staining reaction of normal neuraxes. The myelin of many of the fibers presents a distinctly granular appearance; the granules appear to have been derived from the neurokeratin net. In other fibers the "Golgi funnels" are clearly differentiated. The neurolemma sheaths in the injected field are well maintained; of regular contour and found deeply stained. Not as yet distinct structural changes noted as the result of alcohol injection.
EXPERIMENT No. 2.- Rabbit No. 40a; full grown; 3 hours. March 23, 1918, 11 a. m., left sciatic exposed and injected while in place with absolute alcohol. Nerve not cut. Wound closed. March 23, 2 p. m., three hours after alcohol injection. Wound opened and let
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sciatic exposed. The area of alcohol injection recognized by tile dull white appearance presented by the nerve trunk in this region. This area extends along the length of the nerve trunk for a distance a little over 1 cm. Calf muscles do not contract when nerve is cut central to field of alcohol injection. The nerve removed and fixed in neutral formalin. Bielschowsky's silver stain used.
Microscopic findings.- Of the several series of longitudinal sections, in those including the injected area, it may be observed that the neuraxes of the nerve fibers found in the alcohol injected area are as yet unfragmented and show normal differential staining. In one small area a few segmented neuraxes noted. The myelin sheaths present a distinctly grani lar appearance; neurolemma sheaths have normal appearance. The nerve fibers of this area present structurally no distinct departure from that presented by the normal fibers.
EXPERIMENT No. 3.- Rabbit No. 61a; large; full grown; abscess on neck; 6 hours. March 23, 1918, 11.20 a. m., the right sciatic exposed and injected while in place with absolute alcohol. Nerve not cut. Wound closed. March 23, 5.20 p. m., wound opened and sciatic exposed. Area of alcohol injection easily recognized by its dull white color. Muscles do not contract when nerve is cut central to field of injection. Nerve fixed in neutral formalin. Bielschowsky's silver staining method used.
Microscopic findings.- In several series of longitudinal sections including the area of alcohol injection, in the immediate alcoholized field and especially in the larger internal popliteal bundle, many of the nerve fibers are found to contain fragmented neuraxes. These fragments of neuraxes are of longer or shorter length; certain of the fragments present a wavy course; others are coiled. Certain other neuraxes present a granular disintegration. In the external popliteal funiculus, fragmentation of neuraxes not so distinct. The myelin of the nerve fibers presents a granular appearance; the neurolemma sheaths are found well maintained.
EXPERIMENT No. 4.- Rabbit No. 6a; large; full grown; 1 day. February 21, 1918, right sciatic exposed and injected while in place with absolute alcohol. Nerve not cut. Wound closed. February 22, killed, 24 hours after injection; wound opened and nerve exposed. Area of alcohol injection recognized by dull white color of nerve in the region of alcohol injections. Muscles do not contract on cutting central sciatic. Nerve removed and fixed in neutral formalin. Bielschowsky's silver staining method used.
Microscopic findings.-Several series of longitudinal sections made. In the series including the field of alcohol injection numerous fragmented neuraxes are found. These neuraxes fragments stain differentially in the silver stain; they vary in length; many are distinctly coiled, like a spiral spring; others present a wavy course; others show alternate enlargements and constrictions. Many of the nerve fibers more peripherally placed in the sections do not show this neuraxis fragmentation. Presumably such fibers were not affected by the alcohol. In the affected nerve fibers the myelin presents a granular appearance; the neurolemma sheaths do not show a distinct structural change.
EXPERIMENT No. 5.- Rabbit No. 6; large; full grown; 2 days. February 20, 1918, left sciatic exposed and injected in place with absolute alcohol. Nerve not cut. Wound Whose. February 22, killed, two days after alcohol injection. On exposing the nerve the area of alcohol injection is recognized by its dull white color. The nerve removed and fixed in neutral formalin. Bielschowsky's silver staining method used.
Microscopic findings.-Several series of longitudinal sections made. In such of the sections as include the field of alcohol injection, the majority of the neuraxes are found fragmented into longer and shorter segments, which take the differential silver stain. Many of these neuraxis segments appear to be breaking up into granules, which granules are differentially stained .A certain number of nerve fibers having unfragmented neuraxes are found here and there in the sections. Nerve fibers with fragmented and unfragmented neuraxes are often found in close proximity. The myelin of the nerve fibers distinctly granular; the neurolemma sheaths found of normal contour and appear structurally well preserved. The sheath cells not clearly differentiated.
EXPERIMENT No. 6.- Rabbit No. 7a; full grown; 3 days. February 22,1918, right sciatic exposed and while in place injected with absolute alcohol. Nerve not cut. Wound closed.
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February 25, killed. On exposing the nerve the field of alcohol injection recognized by the dull white color assumed by the nerve in this region. Nerve removed and fixed in neutral formalin. Bielschowskv's silver staining method used.
Microscopic findings.-Several series of longitudinal sections made. In such as include the field of alcohol injection, it is observed that nearly all of the neuraxes of the nerve fibers show a fragmentation. The majority of these neuraxes segments are of relatively short length, many of which appear to be breaking down into granules. In certain of the nerve fibers neuraxes segments are no longer evident. Here and there in the field, nerve fibers with unbroken neuraxes are to be seen. The rayelin of the nerve fibers presents a granular appearance; neurolemma sheaths well maintained.
EXPERIMENT No. 7.- Rabbit No. 7; full grown; 4 days. February 21, 1918, left sciatic exposed and while in place injected with absolute alcohol. Nerve not cut. Wound closed. February 25, killed. Wound nearly healed. On exposing the sciatic, area of injection with absolute alcohol no longer dull white color, but appears slightly congested; has not the glistening appearance of normal nerve trunk. Sciatic removed and fixed in neutral formalin. Bielschowsky's silver staining method used.
Microscopic findings.- Several series of longitudinal sections made. In such as include the area of alcohol injection, the neuraxes of nearly all of the nerve fibers found broken in segments of very variable length; relatively few unfragmented neuraxes observed. In many of the neurolemma sheaths, no longer any fragments of neuraxes found; in others again the neuraxes segments are quite long and of wavy or spiral course. The myelin of the nerve fibers granular; neurolemma sheaths of the great majority of the fibers seem well preserved.
EXPERIMENT No. 8.- Rabbit No. 61; large; full grown; abscess on back; 11 days. March 12, 1918, left sciatic exposed and while in place injected with absolute alcohol. Nerve not cut. Wound closed. March 23, killed, wound well healed. The area of alcohol injection not clearly defined. For a short segment the nerve seems congested. Sciatic removed and fixed in neutral formalin. Bielschowsky's silver staining method used.
Microscopic findings.-Three series of longitudinal sections made, of a little over 3 cm.of the nerve trunk including area of alcohol injection. In such sections as include the area of alcohol injection, the neurolemma sheaths of the nerve fibers clearly made out; within the great majority of these a granular detritus with only here and there a fragment of neuranis remaining. In other neurolemma sheaths deeply stained globular masses; the histogenesis of which is not clearly made out. Evidence of in-wandering of leucocytes is noted; though the stain used does not clearly define these. In longitudinal sections of the nerve distal to the field of alcohol injection, the microscopic field is quite different. In such segments longer and shorter segments of neuraxes are found, differentially stained. A few fibers in which unbroken neuraxes are present are seen. The distal nerve segment sectioned presents the appearance of degenerating peripheral nerve fibers.
EXPERIMENT No. 9.- Rabbit No. 26a; full grown; 62 days. March 19, 1918. Right sciatic exposed and while in place injected with absolute alcohol. Nerve not cut. Wound closed. May 23, killed. Severe neurotrophic changes right hind foot. On exposing the right sciatic the region of alcohol injection is detected by reason of a slight discoloration if nerve; in this region it does not present the white glistening appearance of a normal nerve trunk. No twitching nor contraction of the calf muscles is observed on cutting the nerve either central or distal to field of alcohol injection. Nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings.- Several series of longitudinal sections made. In such sections as include the nerve just central to the place of injection, numerous smaller and larger bundles of neuraxes, having in the main a longitudinal course, but here and there exchanging fibers are found, separated by long, spindle-shaped areas containing granular detritus and large vesicular cells, mutually compressed. In cross sections of the nerve in approximately the middle of the field of alcohol injection, it may be observed that the fibrous sheaths of the nerve are thickened, and that the funicular structure of the nerve is lost. Numerous neuraxes arranged in smaller and larger groups are observed in cross sections. Areas of vesicular cells and granular detritus are here and there evident; some few neuraxes are found in such areas. In cross section of the nerve trunk, approximately 2 cm. distal to the place of alcohol
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injection, the funicular structure of the nerve is again observed, with new down-growing neuraxes observed in all of the funisiculi.
EXPERIMENT No. 10.- Rabbit No. 65; full grown; 66 days. M1arch 21, 1918, the right seistic exposed and while in place injected with absolute alcohol. Nerve not cut. Wound closed. May 25, killed; much emaciated; for three weeks posterior extremities partially paralyzed. Cause not ascertained. On exposing the right sciatic, in what appears as the region of alcohol injection the nerve trunk shows distinct spindle-shaped enlargement; nerve is here somewhat adherent to the surrounding connective tissue. No contraction of calf muscles on cutting nerve central or distal to place of alcohol injection was observed .Nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. No wholly success fill silver differentiation of neuraxes attained.
Microscopic findings.-Three series of longitudinal sections made, including the field of alcohol injection. In these series it can be observed that numerous neuraxes growing from the central portion of the nerve have reached the portion distal to the field of alcohol injection. In longitudinal sections including the region of the spindle-shaped enlargement bundles of neuraxes are found to cross and crisscross in all directions. These microscopic fields give the impression that many of the nerve fibers were torn at the time of alcohol injection, the appearance being that of neuraxes passing through a fibrinous wound region. Distal to this spindle-shaped enlargement, they have a more regular longitudinal course much as seen in regenerating peripheral stump. The differential staining in this series is not wholly satisfactory; neuraxes in sufficient numbers were found differentially stained so that their course could be determined at different levels.
EXPERIMENT No. l1.- Rabbit No. 46a; full grown; 71 days. March 20, 1918, right sciatic exposed and injected while in place with absolute alcohol. Nerve not cut. Wound closed. May 31, rabbit found (lead in the morning; severe neurotrophic changes in the right hind foot. On exposing the right sciatic in the region of alcohol injection, the nerve trunk presents a slightly smaller diameter and appears slightly congested; there is further a light Yellow color. The distal segment presents the appearance of a degenerated nerve. Sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.- Three series of longitudinal and one of cross sections made. In series of longitudinal sections including the field of alcohol injection, numerous larger and smaller bundles of neuraxes having in the main a longitudinal course are to be observed; between these bundles of neuraxes long spindle-shaped areas or columns composed of granular detritus and mutually compressed vesicular cells occur. In the series of cross sections made through the field of alcohol injections, the main funiculi (internal and external popliteal) are found demarked. Neuraxes arranged in smaller or larger groups and seen in cross section are found; these are separated by irregularly round or oval areas composed of granular detritus and vesicular cells. In the longitudinal sections made distal to the field of alcohol injection the nerve presents the appearance of a regenerating peripheral nerve.
EXPERIMENT No. 12.- Rabbit No. 64; full grown; 137 days. March 21, 1918, right sciatic exposed and while in place injected with absolute alcohol. Nerve not cut. Wound closed. August 5, killed. Rabbit in good condition; small neurotrophic ulcer on right heel. In exposing the sciatic, this in the middle of the thigh, the region of the alcohol injection presents for a length of about 1.5 cm. a somewhat smaller diameter than the nerve central and distal thereto, and presents the appearance of a normal nerve though the funicular structure can not be made out and is in this region moderately adherent to the underlying muscle. On cutting the sciatic central and then distal to the field of alcohol injection, after exposure of the calf muscles, these muscles were seen to contract and twitch. Sciatic removed and fixed in ammoniated alcohol. Fair silver differentiation attained.
Microscopic findings.- Series of longitudinal and cross sections were made at successive levels. Nerve fibers, certain of which are myelinated, can be traced from the central nerve through the field of alcohol injection to the distal nerve. In cross sections of the nerve through the field of alcohol injection, it can be seen that the funicular structure of the nerve is lost in this region. The neuraxes are found arranged in smaller and larger bundles, separated by strands of endoneural connective tissue, which is very materially increased in this region.
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Very little remains of the old nerve fibers observed. The nerve distal to the field of alcohol injections contains numerous both myelinated and nonmyelinated nerve fibers. The muscle tissue was not studied in sections.
SUMMARY AND CONCLUSIONS
In Experiment No. 1, in which the nerve was removed for study one hour after the injection of absolute alcohol, the gross changes observed in the nerve in the field of injection were more evident than the microscopic changes. In the region of the spread of the alcohol, the nerve is coagulated, appears "milky white" or "dull white" and there is evidence of capillary hemorrhage. Unfortunately the record of this case does not state whether the nerve responded to mechanical stimulation on being cut central to the region of alcohol injection. There is only very slight evidence of structural change in the region of alcohol injection as seen under the microscope when the nerve is removed soon after the injection; the neuraxes of the nerve fibers presenting, on the whole, a normal appearance and showing normal staining reaction on silver impregnation.
In Experiments No. 2, No. 3, and No. 4, in which the nerve was removed for study at a progressively longer period after the injection of alcohol, beginning with three hours after the operation, the nerve did not respond to mechanical stimulus on being sectioned central to the field of injection, while the nerve distal to this field presented normal structure and function. In the field immediately influenced by the injected alcohol, there is evident a fragmentation of neuraxes and myelin sheaths of nerve fibers not comparable to that observed in secondary degeneration, in that the change is not accompanied by proliferation of sheath cells and comes on soon after injury. Not all of the nerve fibers of an injected nerve trunk are equally affected. A certain number of nerve fibers, the number varying in the several experiments, appears not to be affected by the alcohol. The number of nerve fibers not affected, it would seem, is greater in case the alcohol is injected into the surrounding tissue rather than into the nerve trunk. Beginning with the third day after the alcohol injection, the neuraxes fragments, many of which may still stain differentially, begin to show evidence of further breaking down and, by the eleventh day after alcohol injection, the neurolemma sheaths (the sheaths of Henle?) are found filled with a granular detritus in which neuraxis fragments may or may not be found. The breaking down nerve fibers in the region of alcohol injection do not present the successive stages of secondary nerve degeneration. leading to the formation of nucleated, syncytial, protoplasmic bands, but present a microscopic picture which resembles more that of a nerve transplant removed some10 to 15 days after transplantation. The segment of the nerve peripheral to the field of alcohol injection, on the other hand, presents the histologic changes characteristic of secondary nerve degeneration, as also the region immediately central to the field of alcohol injection.
In a nerve removed approximately two months after alcohol injection, Experiments No. 9 and No. 10, there is found abundant evidence of regeneration, although this has not extended far into the distal segment. In the region of alcohol injection, readily recognized in section, remains of the old nerve fibers are found in the form of granular detritus and also there are found large vesicular cells with relatively small nuclei, presumably with phagocytic function,
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arranged in irregular columns or spindle-shaped areas; these may be found within or between old neurolemma sheaths and endoneural connective tissue septa. In this region there are found new neuraxes grouped in smaller and larger bundles, traceable to the central segment, having in the main a longitudinal course but here and there exchanging neuraxes. The new neuraxes did not appear in the distal regions of the peripheral segment. In only one experiment, No. 12, was the animal kept for a period long enough to admit are generation of the distal segment. In this experiment the nerve was removed somewhat over four months after the operation, at which time there was functional evidence of nerve regeneration. The region of alcohol injection is recognized by the absence of funicular structure, which structure is evident central and distal to the region. Bundles of nerve fibers, both myelinated and non-myelinated, are found in the "wound" region, separated by relatively large areas of connective tissue. These bundles have in the main a longitudinal course but are serpentine as they wind through the connective tissue. The appearance of a wound region after severance and suture of a nerve trunk with not especially good approximation of ends is not unlike that of the region of alcohol injection followed by regeneration, except that this special area in and old alcohol injected nerve extends over a longer distance in the course of the nerve. Peripheral to the region of alcohol injection the process of regeneration is as after nerve section. The conditions resulting from injection of alcohol into a nerve trunk May "I speaks of as a "chemical section" of the nerve and thinks that it is probable that regeneration would follow more quickly than after mechanical section. This, it would seem, depends entirely on the thoroughness and extent of the alcohol injection. If a number of point injections are made extending over several centimeters of nerve the resulting fibrosis would be quite extensive.
So far as can be determined there is no selective action as regards afferent and efferent nerves as a result of alcohol injection. In cases of causalgia in which 60 percent alcohol was injected, it was thought by certain observers that motor functions might persist even though reaction of degeneration were present. It is difficult to explain such selective action, except on the possible ground that the larger myelinated motor nerve fibers are more resistant to the weaker solutions of alcohol than the smaller myelinated or nonmyelinated fibers of the exteroceptive pain and temperature functional systems. In cases of causalgia treated by the injection of alcohol into the respective nerve, ultimate regeneration of the injected nerve may be anticipated with reasonable assurance.
SERIES NO. 2
INJECTION OF FULL STRENGTH ACETONE INTO LIVING NERVE WITHOUT CUTTING THE NERVE
In this series of three experiments full strength acetone was injected into the sciatics of rabbits precisely as was described for Series No. 1, except that acetone solution was used instead of absolute alcohol.
PROTOCOLS
EXPERIMENT No. 13.- Rabbit No. 32a; full grown; 65 days. March 19, 1918, right sciatic exposed and injected with about 0.5 c. c. of full strength acetone. Nerve not cut.
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Appearance of portion of nerve injected resembles closely that obtained when absolute alcohol is injected; dull white color. Wound closed. May 23, killed. Rabbit presents severe neurotrophic changes right hind foot. On exposing the right sciatic, increase of connective tissue about nerve in region of acetone injection; when nerve is dissected free, nerve trunk presents slight enlargement in this region. Calf muscles exposed; atrophic. Muscles did not contract on cutting nerve central and distal to field of acetone injection. Sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.- Four series of longitudinal sections were cut taken at successive levels and including the field of acetone injection and the nerve just proximal and distal thereto. In the series of sections including the nerve about 1.5 cm. proximal to the point of acetone injection, practically normal nerve structure is observed, the stained neuraxes, both myelinated and nonmyelinated, having a longitudinal direction. Here and there "end discs," the distal ends of down-growing neuraxes, are encountered. In longitudinal sections of the immediate field of acetone injection, numerous neuraxes in the form of larger and smaller bundles, having in the main a longitudinal course, but here and there interchanging fibers, are encountered. These bundles of neuraxes are separated by areas and columns of vesicular cells with small nuclei as well as granular detritus. Areas of crisscrossing of neuraxes are here and there encountered. In more distally placed sections many neuraxes are found growing distalward on the inner surface of the perineural sheath; such neuraxes interlace and have a plexus form of arrangement. In sections placed distal to the field of injection, new nerve fibers are found in large numbers, having again a longitudinal course. The nerve trunk in this region presents the appearance of a regenerating peripheral nerve after severance of continuity.
EXPERIMENT No. 14.- Rabbit No. 35a; full grown; 78 days. March 19, 1918, right sciatic exposed and injected while in place with full strength acetone. Nerve not Cut. Wound closed. June 5, rabbit found dead in the morning. On exposing the right sciatic this is found nonadherent to the surrounding connective tissue. In the middle of the thigh. the region of acetone injection, the sciatic presents a short segment having a light yellow color. The nerve of this region is not thickened nor adherent. Sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Only in part successful silver differentiation attained.
Microscopic findings.- In the several series of longitudinal sections made, sufficiently good silver differentiation obtained to determine the fact that central down-growing neuraxes, in large numbers, pass through the field of acetone injection into the nerve trunk distal to this field. In the field of acetone injection are observed areas and columns of granular detritus and large vesicular cells, separating bundles of neuraxes. At the point of injection, distinct increase of endoneural and perineural connective tissue is noted.
EXPERIMENT No. 15.- Rabbit No. 53a; nearly full grown; 323 days. March 20, l918, right sciatic exposed and injected while in place with full strength acetone. Nerve not cut, wound closed. January 17, 1919, rabbit found Lead in the morning; seemed in fairly good condition the day before, though somewhat emaciated. On exposing the right sciatic there is found no material increase in the connective tissue surrounding the nerve; central portion of the nerve adherent to underlying muscle. The main funiculi of the nerve evident practically the entire length. A slight increase in the diameter of the nerve is noted in the region of acetone injection. The calf muscles presented normal size and color; owing to death of animal could not be tested functionally. Sciatic removed and fixed in ammoniated alcohol or pyridine-silver staining. Good but faint differentiation attained.
Microscopic findings.- In three series of longitudinal sections, numerous both myelinated and nonmyelinated neuraxes, having in the main a longitudinal course, can be traced through the field of acetone injection. Scarcely any of the remains of the degenerated portions of nerve fibers found. In cross sections of the nerve, made just central to the place of acetone injection but in the region affected by the acetone, the funicular arrangement of the nerve trunk is found to be maintained. Within the several funiculi there is found a distinct increase of the endoneural connective tissue. So far as can be determined from histologic findings, very complete regeneration of the nerve distal to the place of acetone injection has taken place. Pieces of calf muscle were not removed for histologic study.
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CONCLUSIONS
So far as can be determined from the limited number of experiments in which full strength acetone was injected into the nerve instead of absolute alcohol as in Series No. 1, this may be regarded as a safe procedure and affects the nerve in the region of the injection very much as does absolute alcohol. There is a "chemical section" of the nerve and the operation is followed by loss of function in the peripheral field of the respective nerve. In due time regeneration, through the region immediately affected by the acetone, takes place in a manner as after alcohol injection.
INJECTION INTO DIVIDED NERVE TO PREVENT AMPUTATION NEUROMA
SERIES NO. 3
INJECTION OF ABSOLUTE ALCOHOL INTO THE CENTRAL END OF A DIVIDED NERVE TO OBVIATE THE FORMATION OF AMPUTATION NEUROMA
SERIES NO. 4
AMPUTATION NEUROMA FORMATION IN ASEPTIC WOUNDS
The experimental observations here reported under Series No. 3 and No.4 were undertaken with a view of studying the factors which cause and govern neuroma formation and if possible to devise a safe and practical method to prevent their formation, and to determine if possible the general principle according to which methods suggested to prevent neuroma formation might be judged critically on the basis of experimental observations. There has been abundant opportunity to study the neuroma formation in experimental operations other than those recorded under Series 4, since in many of the operations listed under other series, nerves were cut and resected incidental to the respective operation; this in experiments made on dogs as well as on rabbits. The work here reported was supplemented by further experiments, in which several of the methods suggested for the prevention of neuroma formation were tested experimentally. In these supplementary experiments, made post bellum, the operative work was done by Dean Lewis, in the animal laboratory of Rush Medical College, in affiliation with the University of Chicago, while the histologic study was under-taken by Huber at the University of Michigan. Their joint work formed the basis of a communication 03 dealing with the question of amputation neuromas, their development and their prevention, in which many of the experiments here listed under Series No. 3 and No. 4 were given consideration.
PROTOCOLS
EXPERIMENT No. 16.- Rabbit No. 24; large; full grown; 11 days. March 1, 1918 left sciatic exposed; large nerve. Absolute alcohol injected in several point injections; approximately 2.5 cm. of nerve well injected. Nerve cut distal to field of injection and resected about 1 cm. Wound closed. March 12, killed. Sciatic exposed. Distal end of central sciatic stump surrounded by a small amount of pus. Nerve ends in slight enlargement, having light yellow color. Hemorrhage into nerve trunk, extending for a distance of about 3 cm. from end. Central stump removed and fixed in neutral formalin. Tissue stained after the Bielschowsky silver staining method.
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Microscopic findings.-In several series of longitudinal sections, of successive levels of the distal end of the central sciatic stump, in the region affected by the injected alcohol the neuraxes of the nerve fibers are found in the form of short irregular segments. The myelin of the fibers is present in the form of a granular detritus and smaller and larger globules. The neurolemma sheaths appear well preserved. Here and there irregularly formed cellula elements are found within the neurolemma sheaths, the histogenesis of which is not clearly determined.
EXPERIMENT No. 17.-Rabbit No. 40; full grown; 18 days. March 5, 1918, left sciatic exposed and absolute alcohol injected; in the larger internal popliteal bundle several point injections. Quite a little absolute alcohol escaped into the wound. The nerve cut distal to the injection and resected. The wound closed. March 23, rabbit killed. Wound well healed. On exposing the sciatic no material increase of connective tissue about nerve. Distal end of central sciatic stump tapers to a fine point; slightly adherent to underlying muscle. About 2.5 cm. of end of central stump of light yellow color. Central sciatic stump removed and fixed in neutral formalin for Bielschowsky silver staining.
Microscopic findings.-In two series of longitudinal sections of successive levels of the distal end of the central sciatic stump it may be clearly ascertained that both of the main bundles of the sciatic were well injected, in that in practically all of the nerve fibers only scattered neuraxis fragments are to be found. The myelin remains found in the form of granular detritus. The neurolemma sheaths present, many showing spindle-shaped enlargement at irregular intervals. Small nuclei of doubtful source found scattered through the granular myelin detritus. The fibrous tissue sheaths of the distal end of the central sciatic stump thickened; fibrous tissue at the cut end of the nerve.
EXPERIMENT No. 18.-Rabbit No. 14; full grown; 20 days. February 26, 1918, left sciatic exposed and injected with absolute alcohol, making several point injections. Very little alcohol escaped to wound. Nerve cut distal to place of injection and resected 1 cm. Wound closed. March 18, rabbit found dead in the morning. Wound well healed. On exposing the sciatic, the distal end of the central stump found tapering to a fine line adherent to the underlying muscle. A small blood clot found surrounding the distal end of the central sciatic stump. Central sciatic removed and fixed in neutral formalin. Sections stained in iron hematoxylin and picro-fuchsin.
Microscopic findings.-In the several series of longitudinal sections made at successive levels, the structural appearance presented is such that the sections would not be recognized as sections of peripheral nerve tissue, endoneural connective tissue strands and neurolemma sheaths being the only portion of nerve structure recognized within the funiculi of the larger internal popliteal bundle. Within these sheaths, the neuraxes of the fibers have completely disappeared. The myelin remains are found in the form of a granular detritus or as inclusions in large vesicular cells having very small nuclei. In the external popliteal bundle, not so fully injected, certain normal fibers are to be found; other fibers showing degeneration phenomena, resembling those found in a peripheral nerve after section, are observed. The perineural sheaths of both the internal and external popliteal bundles present a structural appearance which is not unlike that of a normal nerve.
EXPERIMENT No. 19.-Rabbit No. 28; small; half grown; 21 days. March 1, 1918, left sciatic exposed and injected with absolute alcohol; very successfully injected; hardly any alcohol escaped. Nerve cut distal to injection and resected. Wound closed. March 22, rabbit found dead in the morning; seemed well nourished; wound well healed. On exposing the left sciatic this found only slightly adherent to the muscle bed. Distal end of central stump, for about 1.5 cm. tapers to fine strand and is of light yellow color. Distal end of central stump removed and fixed in neutral formalin. Sections stained in iron hematoxylin and picro-fuchsin.
Microscopic findings.-In several series of longitudinal sections, including the area of alcohol injection, the perineural sheaths of time funiculi appear slightly thickened. Of the old nerve fibers, the neurolemma sheaths only in part present; areas in which they have disappeared. In such areas, and within distended neuroleninia sheaths in other parts, there are found large vesicular cells, mutually compressed, having granular and globular inclusions. The cells have small nuclei. In the most distal part of the central stump such cells are less numerous, with a consequent reduction in the size of the nerve.
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EXPERIMENT No. 20.- Rabbit No. 11; full grown; 24 days. February 26, 1918, left sciatic exposed and injected with absolute alcohol; cut distal to place of injection and resected. Wound closed. March 22, rabbit found dead in the morning; seemed well nourished; wound well healed. On exposing the nerve this was found only slightly adherent to the underlying muscle. Distal end of central stump presents tapering end. The central stump removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin.
Microscopic findings.- In series of longitudinal sections of the distal end of the central stump, including the area injected with absolute alcohol and 2 cm. central, perineural sheath found well maintained. In the injected area, practically only the old neurolema sheaths observed; many of these greatly distended, and here and there small areas where these have disappeared. In such areas and within certain of the neurolemma sheaths large vesicular cells with small nuclei are observed. Both of the main bundles equally involved.
EXPERIMENT No. 21.- Rabbit No. 1; large; full grown; 36 days. February 18, 1918, left sciatic exposed and raised from bed for several centimeters. Lifted on hook and injected with absolute alcohol. Area well injected presents a milky white appearance. Cut distal to injection and resected 5 mm. Wound closed. March 26, rabbit died during morning; still warm when found. Very much emaciated. Abscesses filled with "cheesy" pus in various parts of body. The wound was well healed. On exposing the nerve it was found that the external popliteal was not cut at the operation and apparently was not injected. The internal popliteal stump found with tapering end. Nerve fixed in neutral formalin. Bielschowsky silver staining; good differentiation of neuraxes.
Microscopic findings.- From a study of several series of longitudinal sections, it is evident that the external popliteal was insufficiently injected in that a large portion of this nerve bundle seems not to have been affected by the alcohol; showing normal nerve fibers. In the part of the nerve affected by the alcohol the neuraxes and myelin sheaths have disappeared and are replaced by a granular detritus and large vesicular cells with protoplasmic inclusions. Perineural sheaths not materially thickened.
EXPERIMENT No. 22.- Rabbit No. 21; full grown; 36l days. February 28, 1918, left sciatic exposed and injected with absolute alcohol; injected in two regions about 8 mm. apart. Nerve cut distal to field of injection and resected. Wound closed. April 5, rabbit found dead in the morning; emaciated; wound well healed. On exposing the nerve a discoloration about distal end of central stump noted (probably due to hemorrhage). The distal end of the central stump tapers to nearly a point anil presents a light yellow color. About 1.5 cm. proximal to the distal end of central stump nerve presents a normal appearance. Nerve removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and piero- fuchsin.
Microscopic findings.- In three series of longitudinal section:; taken at successive levels, including about 3 cm. of the distal end of the central stump, the following observations are permitted. Approximately 2 cm. central to the place of injection normal nerve tissue is reached. Distal thereto in progressive degree, neuraxes and myelin are replaced by granular detritus, globules and phagocytic cells, in part within neurolemma sheaths, in part in areas in which the neuroleninna sheaths have disappeared, only strands of endonicural connective tissue remaining. Down-growing nerve fibers, in part With very thin myelin sheaths, can be traced from the central undegenerated portion into the degenerated area. These fibers are found singly or in small bundles; present a very regular course, with direction in the main parallel to the long axis of the nerve, and reach to within 1 cm. of the distal end of the central stump. Here and there strands of nucleated bands of syncytial protoplasm are noted in the degenerated portion of the nerve.
EXPERIMENT No. 23.- Rabbit No. 2; large; full grown; 49 days. February 18, 1918,right sciatic exposed; partly freed and injected with absolute alcohol; cut distal to injection and resected 5 mm. Wound closed. March 18, wound completely healed; hair growing over shaved area. April 8, found dead in the morning. On exposing nerve it was found that the external popliteal bundle was cut but not the internal bundle. External popliteal presents slight enlargement of distal end of central stump; end tapers to fine strand and is of light yellow color. Nerve removed and fixed in neutral formalin. Section stained in iron-hematoxylin.
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Microscopic findings.-Only the cut external popliteal nerve sectioned. Evident from series of longitudinal sections that this branch was only partly injected, since in it only a small area in which neuraxes and invelin sheath disintegration is observed. The remainder of the stump resembles in structure closely amputation neurorna, with proliferation of connective tissue and down-growing neuraxes. This experiment can not be regarded as successful.
EXPERIMENT No. 24.-Rabbit No. 43; half grown; 52 days. March 5, 1918, left sciatic exposed and injected with absolute alcohol; one injection. Quite a little alcohol escaped to wound. Nerve cut and resected. Wound closed. April 25, killed. Rabbit in good condition. On exposing nerve, this presents normal appearance to about 2.5 cm. from distal end of the central stump. Distal end shows a slight spindle-shaped enlargement central-ward, then tapers to a fine strand. Streaks of yellow-white color, parallel to long axis oh-served. The distal end of the central stump removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.-In three series of longitudinal sections, taken at different levels ,it may be observed, that in the distal part of the central stump to the extent of about 2 cm. the neuraxes and myelin and in part the neurolemma sheaths of the nerves have been re-placed by granular and globular detritus and vesicular cells, arranged in columns or groups separated by strands of endoneural connective tissue and neurolernma remains. Single neuraxes or small groups of such, growing from the central undegenerated portion of the nerve can be traced into degenerated area. These neuraxes have a regular course, in the main parallel to the long axis of the nerve. Neucleated protoplasmic strands accompany these neuraxes. In the distal most part of the central stump as vet no new neuraxes are found; from this part also the granular detritus and vesicular cells have disappeared.
EXPERIMENT No. 25.-Rabbit No. 22; full grown; 56 days. February 28, 1918.left sciatic exposed and injected with absolute alcohol; well injected. Nerve cut distal to field of injection and resected. Wound closed. April 25, killed. Rabbit in good condition; slight neurotrophic ulcer left foot. Wound well healed. On exposing nerve it is found that it presents a normal appearance to about 1.5 cm. from distal end of the central stump, which presents only very slight enlargement, is of yellow-white color, and tapers to fine strand, which seems continuous with surrounding connective tissue. Nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. Successful silver differentiation attained.
Microscopic findings.-In series of longitudinal sections taken at successive levels it may be observed that numerous new neuraxes growing from the central undegenerated portion of the nerve have grown distally into the portion injected with absolute alcohol. These neuraxes course singly or in small bundles, having in the main a longitudinal course, the small bundles of neuraxes showing here and there interchange of fibers. Between these neuraxes are found columns or areas of granular detritus and vesicular cells. The down-growing neuraxes have practically reached the distal end of the central stump. There is observed no tangling or crisscrossing of neuraxes as seen in a neuroma, nor in the intergrowth of neuraxes and connective tissue as observed at the end of a neuroma.
EXPERIMENT No. 26-Rabbit No. 41; large; full grown; 58 days. March 5, 1918, left sciatic exposed and internal and external popliteal bundles injected separately; the former two injections; well injected. Nerve cut distal to injection and resected Wound closed. May 3, rabbit found dead in the morning. Neurotrophic changes left heel; popliteal lymph gland enlarged. On exposing the nerve it is found that the central stump tapers to fine strand, and presents light-yellow color. About 2 cm. central to distal end of central stump nerve presents normal appearance, with funiculi distinct. Nerve remove and fixed in Flemming chrom-osmic-acetic solution. Sections stained in safranine and licht
grün.
Microscopic findings.-In three series of longitudinal sections taken at successive level sit may be observed, beginning with the most distally placed series, that the neuraxes and myelin of the nerve fibers of both of the main funiculi have entirely disappeared, with fine strands of endoneural connective tissue and neurolemma sheath remains forming a very open meshed network, surrounding areas of granular and globular detritus, through which are scattered small round or oval nuclei. In the series of the next higher level the same
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general structure is found for the greater part of the section. In the more central portion of the sections small strands of syncytial nucleated bands of protoplasm are observed, which become more numerous in the centrally placed of the three series. These nucleated proto-plasmic bands have grown into the degenerated portion of the nerve from the central undegenerated portion.
EXPERIMENT No. 27.-Rabbit No. 38; nearly full grown; 63 days. March 5, 1918,left sciatic exposed; quite a little bleeding; controlled. Two injections of absolute alcohol made; both bundles injected. Nerve cut and 5 mm. resected. Wound closed. May 13, rabbit found dead in the morning; in fairly good condition. On exposing the nerve, distal end of central stump found tapering to fine strand slightly adherent to muscle bed. The distal end for a distance of about 2 cm. presents a light-yellow color; central to this nerve normal appearance. Nerve removed and fixed in neutral formalin. Sections stained iniron-hematoxylin and picro-fuchsin.
Microscopic findings.-In several series of longitudinal sections taken at successive levels, it is observed that in the distal end of the central stump, in the area of the alcohol injection, neuraxes and myelin of the nerve fibers have entirely disappeared, fine strands of endoneural connective tissue and remnants of neurolemma sheaths remaining. This portion of the nerve consisting almost wholly of granular and globular detritus, surrounded by the perineural sheaths. More centralward in the series of sections, nucleated protoplasmic bands growing distally from the undegenerated central nerve are to be observed, to one side, near the perineural sheath, these protoplasmic bands extend distally to near the distal end of the central stump.
EXPERIMENT No. 28.-Rabbit No. 18; full grown; 65 days. February 27, 1918, left sciatic exposed; large vein cut; clamped. Absolute alcohol injected and nerve cut just distal to injected field and resected. Wound closed. May 5, found dead in the morning. Protocol incomplete, simple statement, " No neuroma."
EXPERIMENT No. 29.-Rabbit No. 10; full grown; 71 days. February 26, 1918, left sciatic exposed and injected with absolute alcohol. Nerve cut distal to field of injection; resected 1 cm. Wound closed. May 8, killed. Rabbit not in good condition; emaciated;"fungus" ears. On exposing, the left sciatic central stump is found ending in fine tapering strand, not especially adherent to the muscle bed; of light-yellow color. About 2 cm. central to distal end nerve presents the appearance of normal nerve. The nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good differential silver staining attained.
Microscopic findings.-From the microscopic appearances presented in the several series of longitudinal sections taken at successive levels, it is evident that the injection of alcohol was not wholly successful in this experiment. Numerous neuraxes may be traced from the central portion of the nerve, toward the end of the central stump, numerous large end-discs found at various levels. Especially to one side of the nerve, and about 2 mm. from its distal end, numerous complex spirals of neuraxes are to be observed. At the distal end of the central stump, crossing and recrossing of neuraxes is noted, though there is not observed that intergrowth of neuraxes and connective tissue as is generally seen in a neuroma. In the entire series of sections, few remains of myelin and neuraxes of the old nerve fibers observed. The conclusion seems warranted that at the time of operation the nerve trunk was partially injected with absolute alcohol, and that after section of the nerve a partial neuroma developed.
EXPERIMENT No. 30.- Rabbit No. 32; full grown; 80 days. March 4, 1918, left sciatic exposed. Several injections of absolute alcohol made, spaced at intervals of about 5 mm. Well injected. Nerve resected 1.3 cm. just distal to field of injection. Wound closed. May 23, killed. Rabbit in good condition; severe neurotrophic changes left hind foot. On exposing left sciatic, this presents a normal appearance to within 2 cm. of distal end of central stump. The end presents first a slight enlargement, then tapers to a fine strand only loosely adherent to the surrounding connective tissue. Distal nerve segment completely degenerated. Central sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
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Microscopic findings.- In three series of longitudinal sections and one series of cross sections approximately 3 cm. of the distal end of the central sciatic stump was sectioned. The most centrally placed sections include a portion of the normal nerve. Numerous downgrowing neuraxes may be traced from this portion of the nerve into that portion immediately influenced by the absolute alcohol. In this latter portion areas and columns of globular and granular detritus are found, coursing between which there may le observed smaller and larger bundles of neuraxes, which have in the main a regular course, with here and there interchange of fibers. These down-growing neuraxes may be traced to the distal end of the central stump, but present no tangling or intertwining as observed in a neuroma. The perineural sheath surrounds these down-growing neuraxes.
EXPERIMENT No. 31.- Rabbit No. 26; full grown; 83 davs. March 1. 1918, left sciatic exposed and injected with absolute alcohol. Well injected. Nerve cut distal to field of injection and resected 8 mm. Wound closed. May 23, killed. Rabbit in goon condition. On exposing the left sciatic, this presents normal appearance to within 2 cm. of distal end of central stump. Distal end of central stump presents slight enlargement. then tapers to fine strand, adherent to underlying muscles. Nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings.- In two series of longitudinal sections in which approximately 4 cm. of nerve is sectioned, it is observed that numerous neuraxes growing distalward from the central uninjured portion of the nerve extend into the portion affected by the absolute alcohol. These neuraxes are inclosed within the thickened perineural and epineural sheaths and have in the main a longitudinal course, except those found in close proximity to the fibrinous sheaths; many of them cross and recross and intertwine oil the inner surface of the perineural sheath. These down-growing neuraxes can be traced to the attenuated end of the central stump. The remains of the fibers affected by the absolute alcohol found in areas of granular detritus, interspersed with large vesicular cells and fat cells, between which course the neuraxes.
EXPERIMENT No. 32.-Rabbit No. 29; full grown; 83 days. March 1, 1918, left sciatic exposed and injected with absolute alcohol; well injected. Nerve cut distal to field of injection and resected. Wound closed. May 23, rabbit found dead in the morning. Left femur found broken; apparently some days before death. On exposing the sciatic, tissues about nerve found much congested and containing extravasated blood, owing to fracture. The distal end of the central sciatic found to taper to fine strand; relations not clear owing to extravasated blood. Nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings.-In two series of longitudinal sections including approximately 4 cm. of the distal end of the promixal stump), large number of down-growing neuraxes maybe traced from the uninjured central portion of the nerve to its distal end. These nueraxes are inclosed in the thickened fibrous tissue sheaths of the nerve and have in the main a regular course. Within the area injected with absolute alcohol a few columns and areas of granular detritus, certain large vesicular cells and many fat cells are found. Such columns and areas are separated by bundles of down-growing Neuraxis, a few of which cross such fields either as single fibers or as small bundles of such.
EXPERIMENT No. 33.-Rabbit No. 17; nearly fully grown; 84 days. February 27, 1918. left sciatic exposed and injected with absolute alcohol. First injection not successful; nerve slightly torn. Second attempt at a higher level, was successful; well injected. Nerve cut and resected. Wound closed. May 22, rabbit found dead in the morning; seemed in good condition; severe neurotrophic changes left hind foot. On exposing the left sciatic. the distal end of the central stump is found to taper to fine strand; adherent to the underlying muscle. Several delicate nerve bundles appear to extend on the muscle bed for a distance of about 1 cm. beyond the cut end of the nerve. No evidence of a neuroma noted. Nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained. A portion of the nerve removed in this experiment was lost; the portion at hand represents the most distal portion of the central stump for the length of a little over 1 cm.
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Microscopic findings.-In a series of longitudinal sections, small bundles of fine neuraxes inclosed within the thickened fibrous sheath are observed. These bundles of neuraxes are found separated by areas of granular detritus and fat cells.
EXPERIMENT No. 34.- Rabbit No. 42; half grown; 84 days. March 5, 191S, left sciatic exposed and injected with absolute alcohol; larger bundle in two stages; smaller bundle, one injection. Well injected. Nerve cut and resected 1 cm. Wound closed. May 29, rabbit found dead in the morning. Protocol incomplete. Nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings.- In three series of longitudinal and cross sections in which approximately 4 cm. of nerve were cut, central neuraxes are found passing distalward through the area injected by absolute alcohol, and have reached the distal end of the central stump, and as scattered neuraxes or as small bundles of such can be traced into the connective tissue overlying the muscle bed for a distance of about 1 cm. beyond the cut end of the nerve. In the main these neuraxes have a very regular longitudinal course. Very little detritus, the remains of the injured nerve fibers found in the area injected with absolute alcohol.
EXPERIMENT No. 35.- Rabbit No. 8; full grown; 90 days. February 23, 1918, left sciatic exposed and injected with absolute alcohol; well injected. Nerve cut just distal to injection; resected 1 cm. Wound closed. May 23, killed. Rabbit very much emaciated; severe neurotrophic changes foot, two-toes missing; large ulcer on heel. On exposing the sciatic, this is found of normal appearance to about 1.5 cm. from end of central stump. End of stump presents slight enlargement then tapers to a fine strand. The nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. Faint but differential neuraxis staining attained.
Microscopic findings.- In three series of longitudinal and one of cross sections, taking in a little over 4 cm. of the distal end of the nerve the following may be observed: Neuraxes in large numbers can be traced from the central practically uninjured portion of the nerve, through the area affected by the alcohol to the distal end of the central stump. In a series of cross sections, taken about 2 cm. above the point of puncture for alcohol injection, the funicular structure of the nerve is not lost; the perineural sheaths are distinctly thickened. Within the funiculi, numerous neuraxes seen in cross section, four to ten within one neurolemma sheath. Not all of the funiculi found equally affected. In the more distal portion of the nerve, in two series of longitudinal sections, numerous neuraxes, having in the main a longitudinal course, and arranged in larger or smaller bundles, and separated by elongated areas and columns of granular detritus, vesicular cells and fat cells, are to be observed. inclosed within the thickened fibrous sheaths.
EXPERIMENT No. 36.- Rabbit No. 37; full grown; 93 days. March 5, 1918, left sciatic exposed and injected with absolute alcohol; both bundles well injected. Nerve cut about .5 cm. distal to place of injection and resected 1 cm. A small amount of alcohol escaped to wound. Wound closed. June 6, killed. Rabbit in fair condition; neurotrophic ulcer on left heel. On exposing the sciatic nerve is found to present normal appearance to near end of distal stump which tapers to a fine strand. Nerve removed and fixed in Flemming's chrom-osmic-acetic mixture. Sections stained in safranine and licht grün.
Microscopic findings.-In several series of cross and longitudinal sections made from the distal 4 cm. of the central stump the following observations are made: In the series of longitudinal sections small bundles composed of nucleated protoplasmic bands and fine myelinated nerve fibers may be traced from the central normal portion of the nerve to the end of the distal stump. Between these there are found broader or narrower columns composed of, in the main, large vesicular cells with small nuclei, having globular and granular protoplasmic inclusions. These cells would appear to have phagocytized the remains of the nerve fibers affected by the absolute alcohol. The fibrous sheaths of the distal end of the central stump are found materially thickened. In the distal 1.5 cm. the funicular structure of the nerve is lost.
EXPERIMENT No. 37.- Rabbit No. 34; full grown; 94 days. March 4, 1918, the left sciatic exposed and injected with absolute alcohol; well injected. Nerve cut 5 mm. distal to place of injection and resected 1 cm. Wound closed. June 5, killed. Much emaciated; severe neurotrophic ulcer, left heel. On exposing the left sciatic nerve found normal to within1.5 cm. of end of the central stump; presents slight enlargement, then tapers to fine strand;
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adherent to the underlying muscle. Nerve removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin. Tissue not well embedded, sections torn.
Microscopic findings.-In the sections remaining it can be determined that nucleated protoplasmic bands extend from the central undegenerated portion of the nerve to the distal end of the central stump. These bands of nucleated protoplasm regarded as nonmyelinated fibers. Between such bands or bundles are found columns or long spindle-shaped areas of large closely arranged vesicular cells with globular or granular inclusions.
EXPERIMENT No. 38.- Rabbit No. 39; small rabbit; not full grown; 95 days. March 5, 1918, left sciatic exposed; free venous bleeding. Absolute alcohol injected; larger bundle in several places; smaller bundle one injection. Well injected. A small amount of alcohol escaped to wound. Wound closed. June 8, rabbit found dead in the morning. On exposing the left sciatic this presents a normal appearance to within a short distance of the distal end of the central stump, which tapers to a fine strand. Nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation is not successful.
Microscopic findings.-Several series of longitudinal sections, though not showing differentiation of neuraxes, are sufficiently stained to admit of making the interpretation that there was no neuroma formation; the arrangement of the connective tissue warrants this conclusion.
EXPERIMENT No. 39.-Rabbit No. 27; full grown; 97 days. March 1, 1918, left sciatic exposed and injected with absolute alcohol. On first attempt, movement of animal prevents successful injection; on second trial successful injection made. Nerve cut distal to injection and resected. June 6, killed. Rabbit in good condition. On exposing the left sciatic the nerve presents normal appearance to near distal end of the central stump, which appears to end in fine tapering strand. The relations of distal end of central stump not clearly made out owing to presence of dense cicatricial tissue at end of the fine tapering strand. Nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation attained; sheath nuclei as well as neuraxes stained.
Microscopic findings.-Several series of longitudinal sections made. In these it is possible to trace numerous neuraxes from the more centrally placed sections, through the field affected by the alcohol injection to the distal end of the central stump. At the distal end of the central stump the neuraxes are found to cross and recross, especially those found in close relation to the outer fibrous sheath. In the more central portion the neuraxes present a more regular longitudinal course. In this portion of the nerve, between small bundles of neuraxes, large spindle-shaped areas composed of vesicular cells and granular detritus are to be found. In relation with the distal end of the central stump there was noted at the time the nerve was removed a small irregular mass about 5 mm. in diameter which appeared to consist of dense fibrous tissue. In sections this mass was found to contain a nucleus of osseous tissue surrounded by dense fibrous tissue. In this fibrous layer, mainly to one side, several small bundles of neuraxes were found. A study of this series of sections suggests imperfect alcohol injection, as a result partial neuroma formation, with proliferation of fibrous tissue consequent to escape of alcohol into the wound.
EXPERIMENT No. 40.-Rabbit No. 15; full grown; 102 days. February 26, 1918, left sciatic exposed and injected with absolute alcohol; nerve cut distal to injection and resected1 cm. Wound closed. June 4, killed. Animal not in good condition; severe neurotrophic ulcer on left heel; popliteal lymph gland greatly enlarged. On exposing the left sciatic, the central stump found tapering to fine strand; distal end of light-yellow color. Connective tissue in proximal part of popliteal space quite dense. Central sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained; especially more central portion of nerve.
Microscopic findings.-Two series of longitudinal sections, including the 2 cm. of the distal end of the central stump, made. In the more distally placed series, within the thickened fibrous sheath, a granular detritus and vesicular cells occupy nearly the entire area. No down-growing neuraxes appear to have reached this portion of the central stump. In the more centrally placed series, numerous new neuraxes are found; those more axially placed have a regular course; those more peripherally placed crisscross on the inner surface of the fibrous tissue but do not present the intergrowth of fibrous tissue and neuraxes as noted at the distal end of a neuroma.
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EXPERIMENT No. 41.-Rabbit No. 3; large; full grown; 108 days. February 18, 1918, left sciatic exposed and injected with absolute alcohol. Nerve cut about 5 mm. distal to place of injection; not resected. Wound closed. June 6, killed. Left hind foot slight neurotrophic changes on heel. On exposing the sciatic it is found that the external popliteal was not cut, and probably not injected. Internal popliteal central stump presents a tapering end. Some delicate fine strands seem to extend beyond the cut end; on cutting of these “fibers," no twitching of calf muscles observed. The nerve removed and fixed in an ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings.-The noncut and noninjected external popliteal sectioned with the cut and injected internal popliteal, cut together in series of longitudinal sections. In the sections, the external popliteal presents the appearance of a normal nerve; here and there a few degenerated fibers are noted. In the distal end of central stump of the internal popliteal, central down-growing neuraxes can be traced to the distal end, having in the main a regular longitudinal course, and separated into smaller and larger bundles by long spindle-shaped areas, occupied by granular detritus and large vesicular cells. A few neuraxes can be traced into the connective tissue surrounding the distal end of the central stump of the internal popliteal.
EXPERIMENT No. 42.-Rabbit No. 25; half grown rabbit; 150 days. March 1, 1918, left sciatic exposed and injected with absolute alcohol. Well injected; practically no alcohol escaped to the wound. Sciatic cut 5 mm. distal to place of injection and resected 1 cm. Wound closed. July 30, killed. Rabbit not in good condition. On exposing the left sciatic the central stump is found to end in a fine tapering strand. No bulb. No nerve fibers could be traced beyond the cut end of the nerve. Central sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained. During embedding the end of the central sciatic stump became bent, so that it was not possible to cut longitudinal sections including the entire length of the piece.
Microscopic findings.-It is evident on study of the entire series, that down-growing neuraxes coming from the central uninjected portion of the nerve, have passed through the area injected with absolute alcohol and have reached the distal end of the central sciatic stump. These neuraxes have in the main a longitudinal course. Toward the distal end some crisscrossing of neuraxes is observed; not to the extent found in a neuroma, and such crisscrossing of neuraxes as is observed occurs within the fibrous tissue sheath and mainly on its inner surface.
EXPERIMENT No. 43.-Rabbit No. 23; nearly full grown; 157 days. March 1, 1918,left sciatic exposed and injected with absolute alcohol. Well injected. A small quantity of alcohol escaped to the wound. Nerve cut just distal to place of injection and resected. Wound closed. August 5, killed. Rabbit in good condition; foot missing; stump completely healed. On exposing the sciatic the central stump is found to end in fine tapering strand from the distal end of which a fine filament can be traced toward the distal sciatic stump, but does not reach it. Calf and foot flexor muscles completely degenerated. Central sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Not wholly successful differentiation attained; patchy.
Microscopic findings.-In fairly complete series of longitudinal sections, taking in the distal 2 cm. of the central sciatic stump, it may be observed that numerous neuraxes, both myelinated and nonmyelinated, grow distal through the field of alcohol injection to the extreme distal end of the central stump. Here the connective tissue sheath of the nerve is found very materially thickened, the connective tissue extending into the interior of the nerve end and separating the nerves into small intertwining bundles. More centrally the neuraxes have a more regular longitudinal course, except those found in close relation to the fibrous tissue sheath, which course along the inner surface without definite arrangement. The structural appearances presented are not those of a neuroma.
The following eight experiments are briefly listed but not numbered:
Rabbit No. 30. Small rabbit; full grown; 1 day. March 1, 1918, left sciatic exposed; injected with absolute alcohol; cut; resected. Wound closed. March 2, found dead in the morning. On opening wound, evidence of hemorrhage along line of incision. Portion of
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sciatic injected with alcohol of soft consistence; no gross hemorrhage into nerve. Tissue not sectioned.
Rabbit No. 45. Full grown; 7 days. March 5, 1918, left sciatic exposed; injected with absolute alcohol; large bundle in three stages. Nerve cut and resected. Wound closed. March 12, found dead. Reported too late to use tissue for microscopic study.
Rabbit No. 44. Nearly full grown; 8 days. March 5, 1918, left sciatic exposed; absolute alcohol injected. A small amount escaped to wound. Nerve cut and resected. Wound closed. March 13, a second operation attempted on right sciatic. Rabbit did not recover from second operation. Left sciatic wound found well healed. The distal end of the central sciatic stump found loosely adherent to the underlying muscle and presenting a slightly tapering end. Central sciatic removed and fixed in neutral formalin. Tissue lost in washing after fixation.
Rabbit No. 49. Half grown; 9 days. March 6, 1918, left sciatic exposed; injected with absolute alcohol. Nerve cut and resected. Wound closed. March 15, found dead in the morning. Reported too late to be used for microscopic study. Central end of sciatic found slightly tapering.
Rabbit No. 31. Half grown; 10 days. March 4, 1918, left sciatic exposed and injected with absolute alcohol. Nerve cut and resected. Wound closed. March 14, found dead in the morning. Must have been dead many hours. Wound well healed. Central sciatic stump found slightly tapering. Tissue not studied microscopically.
Rabbit No. 36. Full grown; 11 days. March 5, 1918, left sciatic exposed and injected with absolute alcohol. Nerve cut and resected. Wound closed. March 16, found dead in the morning. Rabbit dead many hours. Tissue not used for study. Central sciatic stump found slightly tapering.
Rabbit No. 19. Full grown; 93 days. February 27, left sciatic exposed and injected with absolute alcohol. Nerve cut and resected. Wound closed. June 1, rabbit found dead in the morning. Severe neurotrophic changes of left hind foot; secondary injection. On exposing the sciatic this is found to taper to fine strand. No neuroma. The tissue not studied.
Rabbit No. 30- Full grown; 98 days. February 28, 1918, left sciatic exposed and absolute alcohol injected. Wound closed. June 6, rabbit found dead in the morning. Reported too late to be of use in study of the tissue. On exposing the nerve this is found to taper to a fine strand. No neuroma.
EXPERIMENT No. 44.- Rabbit No. 28a; small; half grown; 9 days. March 13, 1918,right sciatic exposed; cut and resected 1.2 cm. Wound closed. March 22, rabbit found dead in the morning. Wound 'well healed. On exposing the sciatic, a small swelling on the distal end of central sciatic stump noted. Beginning of neuroma. Central sciatic stump removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin.
Microscopic findings.-In longitudinal sections of the distal end of the central stump early stages of amputation neuroma formation noted, evidenced structurally by fragmentation of the myelin to the extent of about 8 mm. of the distal end of the central nerve fibers; proliferation of the sheath cells in this region; proliferation of the connective tissue.
EXPERIMENT No. 45.- Rabbit No. 12; full grown; 25 days. February 26, 1918, left sciatic exposed; cut and resected. Wound closed. March 23, killed. Wound healed. On exposing nerve a distinct bulb on distal end of the central sciatic stump found. Removed and fixed in ammoniated alcohol for pyridine-silver staining. Tissue misplaced; not sectioned.
EXPERIMENT No. 46a.- Rabbit No. 16; small rabbit; not full grown; 28 days. February 26, 1918, left sciatic exposed; cut and resected. Wound closed. March 26, killed. Rabbit in good condition. On exposing the left sciatic distinct bulb found on central end of the distal stump. A delicate filament, having the appearance of a small nerve, traced a short distance beyond the distal end of the nerve bulb. Bulb removed and fixed in neutral formalin for silver staining. Bielschowsky silver method used.
Microscopic findings.-In longitudinal section it is noted that the neuraxes were not differentially stained, but that the fibrous tissue is very clearly differentiated. This enables
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the observation that in a neuroma the endoneural connective tissue as well as the perineural sheaths show distinct proliferation.
EXPERIMENT No. 46b.-Rabbit No. 4; full grown; 34 days. February 19, 1918, left sciatic exposed; cut and resected. Wound closed. March 26, killed. Wound well healed. On exposing the left sciatic a long spindle-shaped enlargement is found on the distal end of the central sciatic stump, from which is seen to pass a fine nerve bundle, lost in the connective tissue a short distance distal to the bulb. Central sciatic and bulb removed and fixed in neutral formalin for Bielschowskv's silver staining method.
Microscopic findings.-In longitudinal sections of the distal end of the central stump amputation neuroma evidenced structurally; branching of down-growing neuraxes, many ending in terminal disks; neuraxes with irregular serrated borders; neuraxes showing spiral arrangement are observed; endoneural connective tissue proliferated.
EXPERIMENT No. 47.- Rabbit No. 47; full grown; 35 days. March 6, 1918, left sciatic exposed; cut and resected 2 cm. Wound closed. April 10, rabbit found dead in the morning; severe neurotrophic changes of heel; wound well healed. Nerve not exposed until about 18 hours after death. Central sciatic stump found to end in distal spindle-shaped bulb. Central sciatic and bulb removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin.
Microscopic findings.- In a series of longitudinal sections of the distal end of the central sciatic stump, neuraxes conning from the distal end of the bulbous enlargement can be traced into the connective tissue distal, in the form of small myelinated fibers, either singly or in small bundles. These have a very irregular course in the connective tissue and extend for a distance of about 3 mm. beyond the end of the bulb. A distinct mass of connective tissue, blending the internal and external popliteal bundles and extending through the bulb region centralward, is found to contain many small bundles of nerve fibers. There is found a distinct increase in the thickness of the connective tissue sheaths in the bulb region, and also of the endoneural connective tissue.
EXPERIMENT No. 48.- Rabbit No. 22a; full grown; 44 days. March 13, 1918, right sciatic exposed; cut and resected 1.5 cm. Wound closed. April 25, killed. Wound well healed. On exposing the right sciatic its central stump found to end in a long spindle-shaped bulb adhering to the underlying muscle. Nerve and bulb removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good differential silver staining attained.
Microscopic findings.-In a removal of the distal end of this nerve there was removed with it a portion of underlying fascia and muscle; in the serial longitudinal sections these tissues are included in normal relation. In study of the series of sections it is found that down-growing neuraxes have grown distally beyond the limits of the bulbous enlargement, and after passing a tangled irregular course in the connective tissue penetrate the underlying fascia, and in smaller and larger bundles extend distally between muscle fibers in quite regular longitudinal course for a distance of at least 1 cm., the distal limits of the section. Certain of these neuraxes are found to end abruptly in terminal discs, these often showing branching, so that two or three discs are found at the distal end of one neuraxis. Within the bulbous enlargement marked increase in number of neuraxes is noted.
EXPERIMENT No. 49.- Rabbit No. 10a; full grown; 57 days. March 12, 1918, right sciatic exposed; cut and resected 1.8 cm. Wound closed. May 8, killed. Rabbit much emaciated; "fungus" ears. On exposing the right sciatic its central stump is found to end in a distinct bulbous enlargement, from the distal end of which several fine nerve bundles, spreading out fan-shaped, can be traced for a short distance on the fascia overlying the muscle. Nerve and bulb removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.-In a series of longitudinal sections neuroma structure is evidenced by the great increase in the number of down-growing neuraxes, and their irregular crisscross course at the level of nerve section. Numerous small bundles of neuraxes traced into the connective tissue distal to the bulbous enlargement; these are lost in the connective tissue.
EXPERIMENT No. 50.- Rabbit No. 17a; nearly full grown; 71 days. March 12, 1918, right sciatic exposed; cut without lifting from bed; resected 1.5 cm. Wound closed. May
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22, rabbit found dead in the morning. On exposing the right sciatic its central stump found to end in a distinct bulbous enlargement, not adherent to the underlying fascia and muscle. Nerve and bulb removed and fixed in ammoniated alcohol for pyridine-silver staining. Only fair differential silver staining attained.
Microscopic findings.- In section, sufficient neuraxes staining found to determine neuromastructure; few neuraxes have grow in distally beyond the limits of the neuroma.
EXPERIMENT No. 51.- Rabbit No. 60; full grown; 79 days. March 12, 1918, left sciatic exposed; nerve cut and resected 0.6 cm. Not resected. Wound closed. May 31, rabbit found dead in the morning; severe neurotrophic changes in left hind foot. On exposing the left sciatic, no distinct bulbous end noted on distal end of central stump, in place of bulb a long spindle-shaped enlargement from the distal end of which several fine nerve bundles can be traced to the central end of the distal sciatic stump, which end is slightly enlarged. Sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings.- In several series of longitudinal sections including the central and distal resected nerve ends and the intervening connective tissue, it can be observed that when structurally considered there is present a well-developed amputation neuroma. Many neuraxes spirals arc found, with evidence of great increase in the number of neuraxes. In the region representing the central cut end of the nerve, neuraxes in smaller and larger bundles intertwine with bundles of fibrous tissue; certain of the neuraxes bundles extend distally in the connective tissue, and may be traced to the central end of the distal stump; others can be traced to the underlying muscle, between muscle fibers of which they course.
EXPERIMENT No. 52.- Rabbit No. 34a; full grown; 84 days. March 13, 1918, right sciatic exposed; cut and resected 1.3 cm. Wound closed. June 6, killed. Severe neurotrophic changes right foot. On exposing the right sciatic, its distal end is found to end in a distinct, long, spindle-shaped bull), from the distal end of which several small nerve bundles can be traced to the central end of the distal sciatic stump. The central bulb and these nerve strands not adherent to the underlying muscle. No contraction of the calf muscle observed on cutting nerve central and then distal to place of sciatic resection. Sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation good for the more centrally placed series of sections; for more distal series, incomplete.
Microscopic findings.- In longitudinal sections of the central bulb region amputation neuroma evidenced structurally from the distal end of which many small bundles of neuraxes can be traced into the connective tissue intervening between the resected nerve ends. In cross sections of this connective tissue area numerous small bundles of neuraxes, cut in cross, oblique or, for a distance, in longitudinal section, are found separated by connective tissue. In longitudinal sections of the central end of the distal sciatic stump the silver differentiation not wholly successful, sufficient neuraxes staining observed to warrant the conclusion that certain of the central neuraxes have reached the distal sciatic stump through the connective tissue intervening between the resected nerve ends.
EXPERIMENT No. 53.- Rabbit No. 46; full grown; 85 clays. .March 6, 1918, left sciatic exposed; cut and resected 2 cm. Wound closed. May 31, rabbit found dead in the morning; slight neurotrophic changes in the left foot. On exposing the left sciatic very distinct bulbous ends on both the main branches noted; these bulbs taper distalward into fine nerve strands which can be traced a short distance beyond the nerve bulbs. Central sciatic and bulbous enlargement fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.- In a series of longitudinal sections of the central bulbous enlargement amputation neuroma evidenced structurally; many spirals of neuraxes found, numerous branching neuraxes and terminal end-discs noted. Relatively few neuraxes can be traced into the connective tissue distal to the bulb.
EXPERIMENT No. 54.-Rabbit No. 15a; large; full grown; 87 days. March 13, 1918,right sciatic exposed; cut and resected 1.5 cm. Wound closed. June 8, killed. Severe neurotrophic changes of right foot. On exposing the right sciatic distinct bulbous enlargement on distal end of central sciatic stump found. A fine strand of nerve fibers can he traced from the distal end of the central bulb to the central end of the distal sciatic stump.
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Sciatic removed and fixed in ammoniated alcohol for pyridine silver staining. Good silver differentiation attained.
Microscopic findings.- In sections a large amputation neuroma evidenced structurally; in this noted spirals of neuraxes, end-discs, crisscrossing of neuraxes, especially at its distal end. Relatively few neuraxes can be traced to the connective tissue distal to the neuroma. The distal sciatic degenerated; no evidence of regeneration.
EXPERIMENT No. 55.- Rabbit No. 35; full grown; 93 days. March 4, 1918, left sciatic exposed; cut and resected 1 cm. Wound closed. June 5, rabbit found dead in the morning. On exposing the left sciatic the distal end of the central stump presents a well-formed, relatively large bulbous end. Several fine nerve strands traced a distance beyond the bulb; lost in the connective tissue. Sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Unfortunately tissue lost; not sectioned.
EXPERIMENT No. 56.- Rabbit No. 25a; half grown; 129 days. March 20, 1918, right sciatic exposed; cut and resected 1.2 cm. Ends of cut sciatic placed in alignment, and muscles sutured over them. Wound closed. July 30, killed. Rabbit not in good condition. On exposing the right sciatic the central stump is found to end in a long spindle shaped bulbous end, the distal end of which reaches the central end of the distal sciatic stump; the nerve bundles uniting the resected nerve ends is adherent to the underlying muscle. The distal sciatic stump presents a spindle-shaped enlargement nearly as large as that found on the central sciatic stump. Sciatic nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. Only partial differential staining attained.
Microscopic findings.- In series of longitudinal sections, well-developed amputation neuroma evidenced structurally, from the distal end of which larger and smaller bundles of neuraxes can be traced to and through the connective tissue intervening between the resected nerve ends. In the central end of the distal sciatic stump relatively large numbers of neuraxes observed in such portions of the series of sections in which the silver differentiation is sufficiently good to determine them.
EXPERIMENT No. 57.- Rabbit No. 13; full grown; 160 days. February 26, 1918, left sciatic exposed; cut and resected 1 cm. Wound closed. August 5, killed. Much emaciated; left foot missing; healed over. On exposing the left sciatic a distinct bulbous end on the distal end of the central sciatic stump noted, from the distal end of which fine nerve strands can be traced toward the central end of the distal sciatic stump, but do not appear to reach it. Calf muscles found atrophic. Central sciatic and bulb fixed in ammoniated alcohol for pyridine-silver staining; distal internal popliteal fixed in neutral formalin. Only fair differential silver staining attained.
Microscopic findings.- Well-developed amputation neuroma evidenced structurally, from the distal end of which only a few neuraxes can be traced into the connective tissue distal to the neuroma. The nerve fibers of the distal internal popliteal found completely degenerated; relatively few nuclei observed; neurolemma sheaths found thickened and collapsed.
EXPERIMENT No. 58.- Rabbit No. 58; full grown; 179 days. March 12, 1918, left; sciatic exposed and cut high in thigh; the cut ends retracted so as to be separated 8 mm. Wound closed. September 8, killed. Rabbit in fairly good condition; somewhat emaciated; “fungus" ears. On exposing the left sciatic there is observed a spindle-shaped enlargement in the central sciatic stump, the distal end of which continues to the central end of the distal, sciatic stump, the intervening bundle being of nearly the same size as the sciatic. The distal sciatic presents the appearance of a normal nerve. The distal end of the central bulb, as also the intervening nerve bundle, presents a light red color as though more vascular than the remainder of the nerve. The sciatic and a segment of the internal popliteal and posterior tibial fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin.
Microscopic findings.- In longitudinal sections of the central bulb it is seen that numerous nerve fibers, both myelinated and nonmyelinated, pass from the distal end of the bulb into the connective tissue. Of these, those coming from the more axial portion of the bulb have in the main a longitudinal direction, while those which come from the more peripheral portion of the bulb present a very irregular course. In the central end of the distal stump and
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at levels to the middle of the leg in the posterior tibial, in cross sections, numerous myelinated nerve fibers are to be seen.
EXPERIMENT No. 59.- Rabbit No. 57; full grown; 180 days. March 12, 1918. left sciatic exposed and cut high in thigh; ends retracted so as to be separated 6 mm. Muscle, stitched over cut nerve ends. Wound closed. September 9, rabbit found dead in the morning; left foot missing; stump healed. On exposing the sciatic a nerve bundle of a diameter nearly as large as the sciatic extends from a central sciatic bulb to the distal sciatic stump. The distal sciatic presents the appearance of a normal nerve. Sciatic removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht-grün.
Microscopic findings.- In longitudinal sections of the central bulb region numerous nerve fibers arranged in small bundles can, be traced from the distal end of the central bulb into the connective tissue distal to the bulb. In cross sections of the nerve bundle intervening between the resected nerve ends, it is observed that the nerve fibers are arranged in numerous small funiculi, having no definite Perineural sheaths and separated by intervening connective tissue. In the distal sciatic stump, in both cross and longitudinal sections, numerous myelinated fibers are observed among fibers not yet regenerated.
EXPERIMENT No. 60.- Rabbit No. 51; full grown-; 10 months. March 11, 1918, left sciatic exposed, cut high in thigh; ends retracted 7 mm. Wound closed. January 9, 1919, rabbit seemed normal 4 p. in.; found dead 9 p. m.; still warm. Toes of left foot found missing; healed. On exposing the left sciatic, except for slight central enlargement and loss of the funicular structure in the region of the nerve section, sciatic nerve presented the appearance of a normal nerve. Calf muscles and foot exteriors seemed fully regenerated. Sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Faint but differential silver staining attained.
Microscopic findings.-In longitudinal sections of the bulbous enlargement on the central sciatic stump the neuroma structure evidenced by the crisscrossing of the neuraxes and loss of the funicular structure of the nerve. In cross sections of the tissue intervening between the resected nerve ends, numerous small Funiculi of both myelinated and non-myelinated nerve fibers, separated by bands of fibrous tissue are observed. In a series of longitudinal sections embracing the central end of the distal stump, both myelinated and nonmyelinated nerve fibers, with regular order of direction arc found in the connective tissue over the end of the distal stump and can be traced into the distal nerve in which they assume a definite longitudinal course.
EXPERIMENT No. 61.-Rabbit No. 53; nearly full grown; 11 mouths. March 11. 1911.left sciatic exposed and cut; ends retracted 5 mm. Wound closed. January 17, 1918, rabbit found dead in the morning; somewhat emaciated. On exposing the left sciatic the nerve is observed to present two spindle-shaped enlargements about 2 cm. apart, in the region of the nerve section. These are found united by a nerve bundle of about the same size as the sciatic. In the region of the two enlargements and the intervening bundle the nerve adherent to the underlying muscle. Calf and extensor foot muscle seem regenerated; presenting normal size and color. The animal was found dead too long after death to make tissue of much value for special differential neuraxes staining. Fixed in ammoniated alcohol for pyridine-silver staining. Imperfect differentiation attained.
Microscopic findings.-Sufficient silver differentiation of neuraxes obtained to determine in several series of longitudinal sections that both myelinated and nonmyelinated nerve; coming from the central bulb, passing through the intervening connective tissue, have reached the distal sciatic.
EXPERIMENT No. 62.-Rabbit No. 55; large; full grown; 1 year. March 11, 1918, left sciatic exposed and cut high in thigh, end retracted 8 mm. Very little bleeding. Muscle stitched over nerve. Wound closed. March 11, 1919, killed. Active, in good condition: left foot missing; healed. On exposing the left sciatic two slight, spindle-shaped swellings are observed about 1 cm. apart, the distance bridged by a nerve bundle nearly the size of the sciatic; this in the region of nerve section. In this region nerve is adherent to the underlying muscle. On exposing calf and foot extensor muscles, these present a normal appearance cutting nerve near sciatic notch, vigorous contractions of calf and foot extensor muscles noted; the same on cutting nerve in the popliteal space. Sciatic fixed in ammoniated alcohol
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for pyridine-silver staining; portions of calf muscles stained in gold chloride. Very good silver, differentiation attained.
Microscopic findings.-In longitudinal sections of the central bulb numerous myelinated and nonmyelinated nerve fibers are seen to cross and recross in the distal end of the central bulb and enter the connective tissue intervening between the resected nerve ends. In cross sections of the field a large number of small nerve funiculi, without special fibrous sheaths but separated by bands of fibrous tissue, can be observed. In longitudinal sections of the distal end of the central sciatic small nerve bundles with very sinuous course can be traced from the connective tissue into the distal sciatic in which the neuraxes assume a regular longitudinal course. Cross and longitudinal sections of the distal sciatic at several levels present an appearance which resembles closely that of a normal nerve. Differentiation of nerve and endings in the muscle not successful.
EXPERIMENT No. 63.- Rabbit No. 56; full grown; 1 year. March 11, 1918, left sciatic exposed; cut high in thigh; cut ends retracted 8 mm. Very little bleeding. Muscle stitched over nerve. Wound closed. March 11, 1919, killed. Rabbit in good condition. On exposing the sciatic the two resected ends found united, without appreciable enlargement of the central or distal resected ends. Calf and foot extensor muscles present normal appearance and contract vigorously when nerve is cut central and distal to the region of section. Sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.-In series of cross and longitudinal sections, including the field of operation and the nerve distal, it can be observed that numerous neuraxes coming from the distal end of the central bulbous enlargement pass through the connective tissue intervening between the resected nerve ends and enter the distal sciatic, in which they are found in large numbers in all of the funiculi. In cross section of the connective tissue found between the resected nerve ends the neuraxes, both myelinated and nonmyelinated, are found in the form, numerous small nerve funiculi, separated by bands of connective tissue.
For more than a century consideration has been given to the swellings which form on the distal end of the proximal stump of a completely or partially severed nerve and known as neuroma or neuromata. The literature dealing with the structure of neuroma is quite extensive and the surgical literature dealing with the operative means for the prevention of neuroma formation covers a period of many years. The histologic description of neuroma, presented by various writers, differs widely, especially for the period preceding the introduction of specific neuraxis stains. It is not thought necessary to review here at length this extensive literature; certain of the more pertinent references will be given incidental consideration.
The operations reported upon under Series No. 4 were made under strictly aseptic precautions and with as little bleeding as possible, hemorrhage and suppuration having been looked upon as important causative factors in neuroma formation. The structural changes observed in the distal end of the proximal stump of a divided nerve, severed by means of a sharp instrument placed in an aseptic wound, are, during the first few days after the operation, precisely the structural changes observed in the distal end of the proximal stump of a severed nerve, immediately sutured under aseptic conditions. For a distance of from5 mm. to 1 cm. from the cut surface, both myelinated and nonmyelinated nerve fibers show degenerative changes accompanied by proliferation of sheath cells, comparable in every way to the degenerative changes noted in the peripheral stump. The abortive regenerative changes, considered in the general introduction, may also be observed. The connective tissue of the nerve trunk early shows reaction to the injury, evidenced by cell proliferation
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in the region of the cut surface and exudate covering it, so that the cut end of the nerve is early covered by a connective tissue cap which becomes continuous with the epineurium of the nerve and less with the surrounding tissue. This connective tissue cap occurs quite regularly in neuromas. It is found organiz-
FIG. 218.- A longitudinal section of a typical neuroma removed from the sciatic of a dog 31 days after section; pyridine-silver preparation. The relations of the epineurium and the connective tissue cap found on the end of the neuroma are clearly seen. Note the regular arrangement of the nerve fibers and neuraxes in the upper half of the figure and the crisscrossing and otherwise irregular arrangement of the neuraxes as evident in the lower half of the figure
ing when the central neuraxes show the early evidences of regeneration and downgrowth. These evidences of regeneration, as concerns the neuraxes, are best seen and studied in silver preparations, and consist of end and side branches of neuraxes often found terminating in end- discs, very much as observed in regeneration of a severed nerve and subsequent suture. There is distinct
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evidence of sheath cell proliferation, but their relation to the budding and growing neuraxes is not quite clear in silver preparations nor can they be definitely differentiated from the proliferating connective tissue cells. During the second week after resection of the nerve, neuraxis budding and down-growth is clearly demonstrable. The down-growing neuraxes approach the region of neuroma formation with fairly regular and approximately parallel course; as they approach the region of the fibrous cap, single neuraxes or small bundles of such begin to intertwine; many are deflected from their course, even to the extent of turning centralward, and many terminal end-discs are observed. There is fairly distinct interlacement of organizing connective tissue bundles. Very characteristic of the earlier stages of neuroma formation are peculiar spiral complexes, first described by Perroncito, and formed of a single relatively large neuraxis or several neuraxes in axial position, about which are wound in spiral form a variable number of neuraxes and their branches, many ending in end-discs and all found within a neurolemma sheath. These Perroncito spirals may be scattered singly here and there or be found in larger or smaller groups. As such a spiral grows in diameter it would seem that the old neurolemma sheath disappears so that the spirals come to lie in the endoneural connective tissue. The connective tissue of a neuroma is deserving of consideration. It is composed of loosely woven, wavy, connective tissue bundles, is quite cellular and differentiates quite slowly into a compact tissue. One gains the impression that the growing neuraxes, as they reach the region of the connective tissue cap and the sides of the growing neuroma, for sometime stimulate connective tissue to growth. The formation of the central budding and growing neuraxes and the connective tissue of the neuroma progress simultaneously and there develops an intergrowth of connective tissue and neuraxes which characterize the terminal part of the neuroma. A neuroma is to be regarded as a thwarted attempt at regeneration of the nerve, the down-growing neuraxes being blocked by scar tissue. In many instances a well developed neuroma has formed in a strictly aseptic wound by the end of the third or the beginning of the fourth week after operation. As time progresses, in many instances, neuraxes singly or in small bundles, penetrate the cap over the end of the neuroma and penetrate the surrounding connective tissue, pass into intermuscular septa and may penetrate adjacent muscle and course between muscle fibers. In course of time, the down-growing neuraxes may reach the central end of the distal stump and bring about at least partial neurotization of the distal segment. The importance of the participation of growing and budding central neuraxes in neuroma formation, is clearly seen in pyridine-silver preparations of suitable stages. Cone 34 has stressed the fact that three-fourths of each painful bulb consists of nerve fibers and we are led to think that their proliferation against resistance is a cause of pain. All neuroma in the earlier stages of development consist, to a large extent, of nerve fibers or of neuraxes and in all neuromata there is to be noted proliferation of neuraxes against resistance. This fact led us to feel that any measure employed with a view of preventing neuroma formation, in order to attain success, must be directed primarily toward the neuraxes and not the connective tissue of the severed nerve end. Huber and Lewis 33 tested experimentally
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several methods recommended in clinical surgery and directed more particularly toward the connective tissue at the end of the severed nerve, such as "swing door operation" and “crush and tie," and under most favorable conditions of asepsis, neuroma formation was accentuated rather than obviated. In the experiments in which absolute alcohol was injected in the nerve, the procedure was directed toward the neuraxes and it was hoped distinct delay in neuraxis downgrowth would be attained. It may be stated that the escape of a few drops of alcohol, an accomplishment more likely to occur while injecting a nerve the size of that of the sciatic of rabbit, than the larger nerves of the extremities of man, is not to be regarded as of serious consequence, since the records show that the escape of absolute alcohol into the wound was not followed
FIG. 220.- Spiral formations of Neuraxis from neuroma shown in Figure 219. The figure presents a number of end-discs, certain of which are the terminations of neuraxes participating in the spiral structures
by excessive scar tissue formation. One can not agree, therefore, with Corner 65 who states that "injection of alcohol, quinine, and urea should not be used, as about three or four fifths of an injection flows out of a nerve into the surrounding tissue, causing later large formation of scar tissue round the nerve and subsequent strangulation."
The procedure employed in the experiments listed under Series No. 3 was to inject the central stump of a resected nerve approximately 1.5 cm. from the cut surface with absolute alcohol, through a hypodermic needle, inserting the needle very obliquely and centralward. Enough alcohol was slowly injected to give the nerve in the region of the injection and for a distance of approximately 2 cm. a milk white appearance or the appearance of cooked white of egg. If alcohol escaped into the wound, this was taken up with sterile cotton
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and the wound closed. At the end of stated periods the operated nerves were removed and studied macroscopically and microscopically as detailed in the protocols of the several experiments of Series No. 3.
SUMMARY
From a study of the protocols of the series it may be noted that as a consequence of alcohol injection there ensues a fragmentation of the neuraxes and a granular breaking down of the myelin sheaths, and a destruction of the sheath cells in the region of the alcohol injection while the fibrous tissue sheath and the endoneural connective tissue are not affected to the extent of losing their fibrillar structure. These changes affect a region of approximately 2 cm. in the nerves experimented with, namely, the sciatic of rabbits. There is then in the distal portion of the proximal stump thus treated no neuraxis regeneration nor fibrous tissue proliferation of the tissue under consideration. By the end of the third week and the beginning of the fourth week, the fragments of the old neuraxes have disappeared as also much of the myelin detritus. The old neurolemma sheaths seem to persist, and large vesicular cells, many with relatively small nuclei, and having lipoid granules and globules in their protoplasm, the histogenesis of which it is difficult to determine in the pyridine-silver preparations, make their appearance. They are wholly unlike the hypertrophied sheath cells of degenerating nerves. A resected nerve without alcohol injection, or with "swing door" or "crush and tie" operation after resection, shows by the end of the first month after operation a well-developed neuroma. Beginning with the fifth or sixth week after operation, a down-growth of central neuraxes into the region affected by the alcohol begins to be noted. The down-growing neuraxes, as observed in pyridine-silver preparations, in longitudinal section present a fairly regular course in the main parallel to the long axis of the nerve. They appear to course, in part at least, within old neurolemma sheath remains (or sheath of Henle) and gradually reach the distal end of the proximal stump by the end of the second or the middle of the third month after operation. The down-growing neuraxes are accompanied by sheath cells, it is thought derived from central sheath cells. Even in nerve ends seen four to five months after resection and injection of alcohol there is no evidence of neuroma formation, although central neuraxes. both myelinated and nonmyelinated, have passed through the injected portion of the nerve to the extreme distal end of the proximal segment. The connective tissue sheaths of this region are distinctly thickened and the endoneural tissue increased so that the nerve fibers found in the distal end are seen in small interlacing and intertwining bundles, separated by connective tissue. In none of the nerves studied in this series was there any distinct evidence of neuromas formation except in cases in which alcohol injection was not successful. As a result of observations on experiments of Series No. 3 and No. 4.evidence at hand warrants the statement that a neuroma indicates an attempt at nerve regeneration which is thwarted by the formation of scar tissue found at the end of the neuroma; that they form in aseptic wounds and their formation is in no sense dependent on the presence of blood clot or infection resulting in suppuration. We believe ourselves to have demonstrated that absolute
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alcohol injected into the nerve, 2 cm. to 2.5 cm. from its cut surface in several point injections arranged so as to involve all parts of the nerve trunk, is a procedure which is successful in preventing neuroma formation.
NERVE TRANSPLANTS
The great majority of the experimental observations listed in the following series deal with cases in which, owing to loss of nerve substance at the time of injury, the severed nerve ends were separated to such extents that they could not be brought together for suture. The question of bridging defects in nerve resulting from loss of substance at the time of injury is one that has received consideration for a time nearly coincident with that of the use of suture in uniting severed nerves. It is not proposed to enter on a general discussion of the methods used or suggested for the purpose of bridging nerve defects nor to consider critically the extensive literature bearing on this question; incidentally, certain pertinent references will be considered. Certain of the methods suggested as of service in bridging nerve defects which have received general recognition, such as suture a distance, tubular suture, nerve implantation and nerve flaps, were tested experimentally by Huber 30 several decades ago and discarded as not justified on experimental grounds. A few of these methods, such as the operation of nerve flap made from the central or distal stump, or from both stumps are still in use by surgeons. A critical review of all of the cases in which the operation of nerve flap was used to bridge a nerve defect was made by Stookey 50, who found that in not a single case was there conclusive evidence of regeneration. There has been a revival of the operation of nerve implantation, in case of loss of nerve substance in peripheral nerves, as a result of the advocacy of this method by Hofmeister 51, but there is no warrant for this method if properly done. It is only when nerve fibers are cut in the sound nerve at the seat of implantation, in which case the operation becomes one of nerve crossing, that there is any justification for attempting the method. Consideration is given to tubular suture in Series No. 20.
The use of a segment of nerve to bridge a defect due to loss of substance in a nerve has long been advocated, by both the experimenter and the clinician. This procedure was first tried experimentally by Philipeaux and Vulpian 66 and was first used by Albert 67 in human surgery. A segment of nerve used to bridge a defect in a peripheral nerve, taken from another nerve from the same individual, is designated an autogenous transplant or graft-an auto-nerve trans-plant; a segment of nerve taken from another individual but of the same species is known as a homogenous transplant or graft-a homo-nerve transplant; a nerve segment taken from another individual but of a different species is called a heterogenous transplant or graft-a hetero-nerve transplant. Having in mind the practical application in human surgery, various types of nerve transplants were tested experimentally, such as normal or degenerated nerves; fresh and preserved or stored in aseptic state; as single nerves or a bundle of nerves, the latter known as "cable-nerve transplant" or "multi-nerve transplant"; and wrapped or unwrapped in protecting membrane or sheath. These series constitute a very comprehensive experimental study of the question of nerve transplant.
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SERIES NO. 5
AUTO-NERVE TRANSPLANTS, INCLUDING CABLE-AUTO-NERVE TRANSPLANTS
General surgical experience regarding the question of tissue graft or transplants, and the favor with which autogenous tissue grafts arc regarded, made it imperative that auto-nerve transplants be given special consideration, even though it was recognized that in using an autogenous nerve transplant a normal functioning nerve was of necessity resected to the extent necessary to bridge the existing defect, with consequent loss of function in the nerve resected throughout its entire field of distribution. Consideration should be given the fact that a nerve transplant should have a cross-section area approximately that of the nerve to be repaired, in order to admit of ready downgrowth of central neuraxes. The mere question of determining the relative value of an auto-nerve transplant as over against other types of nerve transplants did not appear to have sufficient value, since it could hardly be regarded as good surgery to resect one major nerve of an extremity to repair another even though an auto-nerve transplant should on experimentation prove to have special merit. In our endeavor to make auto-nerve transplants of practical use in surgery, the operation here known as cable-auto-nerve transplant was developed and tested experimentally. This operation consists in using several segments of a nerve which could be resected without serious inconvenience as a result of loss of function. Certain cutaneous nerves were selected for this purpose and a sufficient number of segments placed side by side so that their combined cross-section area approximated that of the nerve to be repaired. These operations were made with great care, especially those in which cable transplants were made. It is so essential to obtain good end-to-end approximation in nerve suture and it was our special endeavor to obtain this. In valuating these and other experiments in the light of possible application to human surgery. it is recognized that a direct transfer of results is not permissible. The aseptic wounds in normal tissue and the nerve resected by means of sharp instruments present a condition not found in a severed nerve torn or crushed by high explosive or otherwise, perhaps wound infected, and perhaps not seen until months after injury with abundant formation of scar tissue. It was felt, however, that certain general deductions could be made and certain general principles formulated. In suturing the nerve transplants very fine silk threads, waxed with sterile wax, were used. It is essential and necessary to have good approximation of the cut surface. This is more readily attained with waxed silk sutures passed through the nerve transplant and the resected ends of the nerve than when catgut is used. Stookey 68 and Elsberg 69 have described special technical methods, recommended for use in cable-auto-nerve transplants in human surgery. This operation is not an easy one and is very time consuming. It generally necessitates the making of a second wound, which even though it is quite superficial, is of necessity of some length. It is suggested that for purpose of cable-auto-nerve transplant the internal saphenous nerve, the anterior femoral cutaneous, and the sural nerve from the lower extremity and the cutaneous branch of the musculocutaneous (lateral antibrachial cutaneous)
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and dorsal antibrachial cutaneous from the upper extremity are, of the larger cutaneous nerves, available.
The protocols of experiments under Series No. 5, auto-nerve transplants and cable-auto-nerve transplants follow:
PROTOCOLS
EXPERIMENT NO. 64.- Dog No. 6; large; full grown; somewhat emaciated; 119 days. April 11, 1918, left sciatic exposed for a distance of 6 cm. Incision made through skin and muscle; free bleeding. Superficial radial of right forearm exposed and freed of connective tissue. Using No. 60 linen thread and fine, straight, round needles, two sutures passed through nerve about 3 cm. apart. Radial cut with scissors 2 mm. proximal and distal to suture lines and nerve segment transferred to sciatic wound. One needle and suture passed through sciatic centrally and sciatic cut 2 mm. distal to suture line; central suture tied. Distal suture passed through sciatic and the nerve cut 2 mm. proximal; distal suture tied. Only fair central and distal approximation of nerve ends attained. Radial segment has much smaller diameter than the sciatic nerve. Wounds closed. August 8, killed. Dog much emaciated, has not been well for several days. Left hind foot, small neurotrophic ulcer of heel; does not stand on ball of foot. Sear tissue found in line of sciatic wound; extends to deeper tissues. Left sciatic found surrounded by dense fibrous tissue. On dissecting free, a large central bulb noted. A small nerve bundle can be traced from this to distal sciatic stump. Distal sciatic presents the appearance of a partially regenerated nerve. Calf muscles exposed and sciatic and the transplant freed from the bed. On slowly cutting with scissors, sciatic central to the transplant, distinct twitching of calf muscles noted; same when sciatic is cut distal to the transplant. Sciatic and transplant and internal and external popliteal branches removed and fixed in ammoniated alcohol for pyridine-silver staining. After silver staining this material was by accident overheated in oven; section series unsatisfactory.
Microscopic findings.-In very broken and irregular sections, sufficient evidence of down-growth of neuraxes from the central bulb to warrant the conclusion that neuraxes coming from the central bulbous enlargement had grown through and outside of transplant to the distal sciatic stump. Details could not be determined.
EXPERIMENT NO. 65.-Dog No. 7; large; full grown; well fed; 120 days. April 12, 1918, left superficial radial exposed and freed from connective tissue. Two silk sutures passed 2.8 cm. apart and nerve cut central and distal to sutured lines with sharp razor blade. Right sciatic exposed and freed. Sciatic resected distal and proximal to suture line and sutures tied. Good approximation of nerve ends attained. Wound not quite dry; wounds closed. August 10, killed. Dog emaciated, but seems in good condition. Walks well; uses right hind foot quite normally; standing on ball of foot. On exposing the right sciatic a relatively large bulbous enlargement is found on the distal end of the central sciatic stump. A small bundle of nerves leads from this to the distal stump, the central end of which is not materially enlarged. Central sciatic bulb and the transplant not especially adherent to underlying muscle. Calf muscles exposed. After completely freeing the sciatic from notch to popliteal space, on slowly cutting the nerve central to the transplant distinct twitching and contraction of the calf muscles noted. Sciatic and transplant, external and internal popliteal and pieces of calf muscles and extensor leg muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findinqs.-Large oval, central bulb evidenced structurally, from the distal end of which numerous down-growing neuraxes can be traced to the central end of the transplant and also into the connective tissue surrounding the central wound. In cross sections of the transplant its funicular structure is found retained, with the fibrous sheath thickened. Numerous neuraxis found in each of the funiculi of the transplant, also in small bundles in the connective tissue surrounding the transplant. In successive cross and longitudinal series of sections, these neuraxes can be traced to and through the distal wound into the distal sciatic branches; in the internal popliteal nearly to the level of the heel; in the external popliteal to the region of the head of the fibula. In sections of the calf muscles new neuraxes are found
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in the larger and smaller muscular branches, and as single nerve fibers, between and on the muscle fibers. Here and there quite well developed motor nerve endings were noted.
EXPERIMENT No. 66.- Dog No. 3; small dog; full grown; 132 days. March 8, 1918, left sciatic cut, high up; wound closed. March 27, left sciatic exposed, nineteen days after section. Difficult to find central stump; much bleeding; muscles somewhat torn. Ends of cut sciatic stumps resected. Right ulnar exposed and a segment transplanted to the resected sciatic. Ulnar segment somewhat short; central suture gave way; difficult to suture again. One central and distal catgut suture used. Not good approximation attained; transplant ultimately 1.5 cm. in length. Wounds closed. August 6, killed. Dog in good condition; not materially emaciated. Uses left hind leg well; now and then steps on dorsun of foot; small ulcer on dorsum of foot. On exposing left sciatic large bulbous enlargement is found on central sciatic stump; from this a small bundle of nerves leads to distal sciatic stump. Distal sciatic, especially internal popliteal branch presents the appearance of a regenerated nerve. Central bulb and region of transplant surrounded by quite dense fibrous tissue; adherent to underlying muscle. After exposing calf muscles and freeing the sciatic from bed on slowly cutting sciatic central to transplant, feeble twitching of calf muscles noted. Muscle not fully recovered, pale red color, streaked with yellow white. Sciatic nerve and transplant, internal popliteal and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Fair differential silver staining attained.
Microscopic findings.-From the distal end of a long spindle-shaped bulbous enlargement, down-growing neuraxes may be traced through a long fibrous union, extending several millimeters, to the central end of the transplant. In cross sections of the transplant, about 1 cm. distal to the central wound, the funicular structure of the ulnar can only be partially made out; there is observed material increase of its fibrous sheaths. New neuraxes are observed within the funiculi of the transplant and also in the surrounding connective tissue, especially to one side. These new neuraxes can be traced in sections, to the distal wound and through it to the distal internal popliteal, the external popliteal not having been united to the transplant at the distal wound. In sections of the calf muscles, new neuraxes are noted in the muscular branches entering the several calf muscles, and in many of the inter- fascicular nerve branches.
EXPERIMENT No. 67.- Dog No. 8; large dog; full grown; 282 days. August 12, 1918,the left superficial radial exposed and freed from connective tissue. The right sciatic exposed by cutting through muscle; good direction of incision; free bleeding. Sutures passed through radial branch, 3 cm. apart before cutting the same, and transplanted to the resected right sciatic; good approximation attained. Muscle stitched over sciatic and transplant and the wounds closed. May 21, 1919, killed. Dog in good condition. Uses right foot well, though has slight limp in walking. On exposing right sciatic this is found embedded in loose connective tissue, interspersed with small fat globules. Large central bulbous enlargement noted, which extends in a fine nerve strand to the distal sciatic. In the region of the transplant diameter not quite half that of the sciatic. Calf and plantar muscles exposed. After freeing sciatic and the transplant from the bed, on slowly cutting the nerve with scissors, central to the transplant, distinct contraction of the calf and interossei muscles observed. On cutting the internal popliteal distal to the transplant the same observation noted. Sciatic transplant and the internal popliteal and several interossei muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.- Long spindle-shaped central bulb evidenced structurally, from the distal end of which numerous neuraxes pass into the transplant and into the connective tissue surrounding it. In cross sections taken about 1.5 cm. distal to the central wound, it may be observed that the funicular structure of the transplant is still retained, surrounded by a distinct though not dense layer of fibrous tissue. Numerous new neuraxes are found within the surrounding connective tissue. In sections these new neuraxes may be traced to and through the distal wound into the distal popliteals. In the posterior tibial, both myelinated and nonmyelinated fibers may be traced to the level of the heel. In sections of several interossei muscles new neuraxes are to be observed in the muscular branches and here an there motor end organs are noted. Regeneration of distal popliteal to foot muscles, through the transplant.
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EXPERIMENT NO. 68.- Dog No. 2; medium size; full grown; 439 days. March 7, 1918, left sciatic exposed and cut high in thigh. March 26, 19 days later, severe neurotrophic changes left foot; foot in part missing. Left sciatic exposed; large central bulb note. Central and distal sciatic stump resected. A segment of the right ulna of 2 cm. length transplanted. One central and distal No. 000 catgut suture used. Distal external popliteal resected for another operation; transplant sutured distally only to internal popliteal branch. Wounds closed. June 12, left foot completely healed; dog in good condition. May 20,1919, killed. Dog in good condition; very active. Left foot to metacarpals missing. On exposing left sciatic this is found surrounded by loose connective tissue. Large bulb on central sciatic stump; this continuous distally with a nerve bundle about the size of ulnar traced to distal internal popliteal; external popliteal attached loosely to internal popliteal; no organic union. Calf muscle fully exposed; these have the appearance of normal muscle tissue. After completely freeing the sciatic from its bed, on slowly cutting the nerve with scissors central to the transplant, distinct contraction of the calf muscles observed. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation attained.
Microscopic findings.-Very large bulbous enlargement on central sciatic stump evidenced structurally, from the distal end of which numerous new neuraxes can be traced distally through a wide, central, fibrous wound, into the transplant and into the connective tissue surrounding the same. In cross sections of the transplant, about 1 cm. distal to the central wound, one large funiculus of the transplanted nerve segment clearly outlined by perineural sheath. Within this numerous myelinated and nonmyelinated nerve fibers, separated into small bundles by endoneural connective tissue, are observed. In the connective tissue surrounding the transplant numerous small bundles of nerves are found. These neuraxes can be traced through the distal wound into the distal internal popliteal, in which they are present in all of its funiculi, both as myelinated and nonmyelinated nerve fibers. Regeneration to lower level of popliteal space (the extent of nerve removed).
EXPERIMENT No. 69.-Dog No. 1; medium size; full grown; 81 days. May 16, 1918, right sciatic exposed and freed. Left ulnar exposed. Two segments of the left ulnar of approximately 3 cm. length transplanted to the resected right sciatic. Each ulnar segment sutured centrally and distally, separately to resected sciatic ends. Waxed, fine, silk thread sutures used; good approximation of the nerve ends attained. Wounds closed. August 5, killed. Dog in good condition; slight foot-drop right hind leg; very little neurotrophic change right hind foot. On exposing the right sciatic, transplants found well in place; no distinct central bulb. Transplant surrounded by loose connective tissue, not adherent to underlying muscle. Calf muscles exposed. After freeing transplant from bed on slowly cutting nerve central to the transplant, no contraction of calf muscles observed. Sciatic removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation only partially successful.
Microscopic findings.-In longitudinal sections through the central wound a central bulbous enlargement is clearly made out structurally, from the distal end of which numerous neuraxes traced into the transplant. In cross sections of the transplant the two ulnar segments clearly made out, each retaining its funicular structure. The two nerves are found surrounded by a common fibrous tissue sheath. Within the transplants new neuraxes found about equally distributed. The down-growing neuraxes can be traced into the distal wound and through this into the distal sciatic in which they may be traced in lessening numbers, approximately 3 cm. distal to the distal wound.
EXPERIMENT No. 70.-Dog No. 4; large dog; full grown; 152 days. March 8,1918, left sciatic exposed and cut high in thigh. Wound closed. April 15, slight neurotrophic changes left heel; left sciatic again exposed, 38 days after section. Large neuroma on central sciatic sump; central end of distal sciatic only slightly enlarged. Central bulb removed and distal sciatic stump resected 5 mm. Two superficial radial branches, having parallel course, exposed and freed from connective tissue, brought together and clamped with artery forceps. Two No. 110 silk thread sutures formed 4 cm. apart and nerve cut with safety razor blade 2 mm. beyond sutures. The two nerve segments transferred to sciatic wound and sutured to resected sciatic ends; good approximation attained. Diameter of the two radial branches
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not as great as the resected sciatic. Muscle stitched over nerve and transplant and wounds closed. April 23, superficial sciatic wound open to the extent 2.5 cm.; deeper wound seemed healed; no infection. August 7, killed. Dog very much emanciated; has not been feeding well for several days; left foot in part missing; nearly healed. Had had very severe neurotroplic changes of the foot. On exposing the left sciatic, contiguous muscles found to have yellow red color and much reduced in size. Large central sciatic bulb noted .A small nerve bundle extends from this to the distal sciatic stump. No material increase of fibrous tissue about the nerve. Calf muscles exposed; these appear very atrophic and not of normal color. After freeing nerve and slowly cutting the same central to the transplant no distinct contractions of calf muscles noted. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. In part good silver differentiation attained; not uniformly stained.
Microscopic findings.-Large central bulbous enlargement evidenced structurally, from the distal end of which numerous neuraxes can be traced to the central ends of the transplanted nerve segments and the connective tissue surrounding the same. In cross sections of the transplant, about 1.5 cm. distal to the central Wound, both of the transplanted nerve segments clearly made out, with the funicular structure retained. The transplanted nerve segments surrounded by a common, fairly dense, fibrous sheath, which extends between the nerves. Within the funilculi of the transplant many new neuraxes observed; there appear to be more of these in one nerve than in the other, though this can not be definitely determined since the silver differentiation is not uniform. In the connective tissue surrounding the transplant many small bundles of nerve fibers encountered; this mainly to one side. New neuraxes in large numbers may be traced to and through the distal wound into the distal popliteal stump, for a distance of about 4 cm., the extent of the nerve removed for sections.
EXPERIMENT No. 71.-Dog No. 15; large, black bound; full grown; 376 days. May 8, 1918, superficial radial exposed and freed from connective tissue cut to segments about 4 cm. length, placed side by side and clamped at each end with artery forceps. Thus suspended, two fine, silk sutures passed through the two nerve segments 2.2 cm. apart, cut beyond suture lines and transplanted to the resected left sciatic, in suturing only the internal popliteal bundle used, external popliteal disregarded. Fairly good approximation of nerve end attained. Wounds closed. May 20, 1919, killed. Dog in very good condition; still favors the left hind foot; no distinct foot or toe drop note. Shape and size of bulb in part due to large bulbous end on external popliteal. From the distal end of the bulb there may be traced a nerve bundle to the internal popliteal. Calf muscles and foot muscles exposed. After freeing sciatic and the transplant from the bed, on slowly cutting with scissors central to the transplant, good contraction of calf muscles noted; foot muscles contraction feeble and uncertain. External popliteal cut, but not included in the sutures, is distally found closely united to internal popliteal. Cutting of external popliteal near head of fibula calls forth good contraction of leg flexors. Sciatic and transplant and two interossei muscles removed an fixed in ammoniated alcohol for pyridine-silver staining Good differential silver staining attained.
Microscopic findings.-Very large bulbous end on central sciatic evidenced structurally. In longitudinal sections of this bulb, the unabsorbed central suture observed. Down-growing neuraxes, crisscrossing in every direction, reach the distal part of the bulb and can be traced to central ends of the transplants and the surrounding connective tissue. In cross sections of the transplant at two levels, near central and distal wounds, only one nerve bundle is clearly demarked. Whether there exists an error of record and only one nerve bundle was transplanted and not two as recorded, or whether one of the bundles pulled free centrally and disappeared, can not now1 be determined. The single nerve present is surrounded by a dense, felted layer of fibrous tissue. Within its funiculi numerous, both myelinated and nonmyelinated, neuraxes found; largely arranged in small bundles separated by endoneural connective tissue. In the connective tissue surrounding the transplant in centrally and distally placed cross sections, many small bundles of nerve fibers found. Neuraxes found within and without the transplant may be traced through the distal wound to the distal popliteal, in which in successive levels they are observed in cross and longitudinal sections to the level of the heel. In sections of interossei muscles new neuraxes are observed in the small interfascicular nerve branches and in one instance in a neuromuscular spindle.
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Complete distal regeneration of the internal popliteal and branches, through the transplant, attained.
EXPERIMENT No. 72.- Dog No. 14; relatively large dog; full grown; 91 days. May 2, 1918, left sciatic exposed and freed. Two branches of the cutaneous radial exposed and freed from connective tissue. Two segments of the larger nerve of 2.5 cm. length transplanted to the resected internal popliteal branch of sciatic; one central and distal silk suture and one segment of the smaller nerve of 2.5 length transplanted to the resected external popliteal; one central and distal silk suture; good central and distal approximation attained in all three segments of cutaneous nerves used as transplants. Muscles stitched over nerve and transplant and wounds closed. May 14, severe neurotrophic changes left foot; possible infection of foot. Sciatic and forearm wounds healed. August 2, found dead in the morning; had not been eating well for several days; very much emaciated; still neurotrophic changes of foot. On exposing the left sciatic indistinct central bulb noted. Several bundles of nerves lead from this to the distal sciatic; can not determine definitely whether these nerve bundles are within the transplanted nerve segments. Distal sciatic does not present the appearance of normal nerve. Sciatic and internal popliteal removed and fixed in ammoniate alcohol for pyridine-silver staining. Silver differentiation only partially successful.
Microscopic findings.- In longitudinal sections of the central wound, scarcely any evidence of central bulb noted oil microscopic inspection, under the microscope crisscrossing of central neuraxes as they pass to transplant is noted. In cross sections of the transplant, the three transplanted nerve segments quite clearly made out; found surrounded by a common fibrous tissue sheath. Within the three transplanted nerve segments the funicular arrangement quite clearly retained. In each of these funiculi are found new neuraxes, quite evenly distributed. Very few neuraxes found in the connective tissue outside of the funiculi. The new neuraxes may be traced into the distal stump in which they are found in good numbers 3 mi. beyond the distal wound, the extent of the nerve sectioned.
EXPERIMENT No. 73.- Dog. No. 9; large; full grown; 323 days. June 29, 1918, right sciatic exposed and freed from bed. Two branches of the left cutaneous radial exposed and freed from connective tissue. Three segments of these nerves having a length of 2 cm. each sutured separately between the resected ends of the sciatic, using three No. 00 catgut sutures softened for a short time in sterile distilled -water. Only fair approximation of the nerve ends attained. Dry field obtained by use of adrenalin. Wounds closed. May 18, 1919, killed. Dog in good condition; walks well; severe skin disease; nails on two inner toes long and curved so as to form complete circle. On exposing the right sciatic no material increase of connective tissue about the nerve noted; quite large central bulb, with 1ransplant well in place. Calf muscles exposed; sciatic a transplant freed the entire length. On slowly cutting with scissors the nerve central to the transplant, good contraction of calf and flexor leg muscles noted. Foot muscles exposed, on cutting posterior tibial near heel, good contraction of the plantar interossei muscles observed. Sciatic, transplant, posterior tibial, pieces rf calf and interossei muscles removed and fixed in an ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings- In longitudinal sections of central wound region a large central bulb evidenced structurally from the distal end of which numerous neuraxes extend to the central end of the transplant. in cross section of the transplant at two levels, near central and distal wounds, only two of the transplanted nerves made out, both of these have retained their funicular structure. The fate of the third nerve segment can only be conjectured; it is thought that it pulled free centrally and completely degenerated. Within the funiculi of the two nerve segments present there are found numerous new neuraxes. In the more centrally, placed cross sections numerous small nerve bundles are found in the connective tissue surrounding the transplant; scarcely any of the extra funicular nerve bundles are found in the cross sections of the lower level. New neuraxes were traced in the distal internal popliteal, the level of the heel, and in sections of the interossei muscles into the smaller interfascicular muscular branches; motor and sensory muscle nerve endings were observed in sections of the interossei muscles. Complete peripheral regeneration attained, so far as distribution of peripheral motor branches is concerned.
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EXPERIMENT No. 74. Dog No. 12; large; full grown; 11 days. April 26, 1918, left sciatic exposed and freed. Two superficial cutaneous radial branches exposed and freed from connective tissue. Free venous oozing in the wound; after freeing nerves they were bathed in partly clotted blood for about fifteen minutes. Four segments were made of the cutaneos radial branches, placed side by side, and clamped together at the ends with artery forceps. A single silk thread suture passed centrally and distally through the four nerve segments 2.5
FIG. 221.-Cross section through the middle of a cable-auto-nerve transplant, Experiment No. 74, 11 days after operation; pyridine-silver preparation. Note the new epineural sheath, seemingly uniting the four separate nerve segments into one compact nerve with many funiculi. The funicular structure of each nerve segment is well maintained
cm. apart, and the nerve segments were cut beyond the sutures and the sutures tied, the forming a complete bundle. This bundle of nerve was transferred to sciatic wound and fixed between the resected ends of the sciatic by suturing the same to the underlying muscles. The resected nerve ends and the bundle of four nerve segments were united by one central and distal epineural suture. Only fair approximation of nerve ends attained. Muscle stitched over nerve and transplant. Wound closed. Oozing in radial wound not full
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controlled. Closed. May 7, killed. Forearm wound open; superficial sciatic wound open to the extent of 3 cm.; deep wound healed. Left sciatic exposed; transplants found well in place; surrounded by newly forming connective tissue. A small amount of sanguineous exudate surrounds nerve and transplants. Sciatic and the transplants removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good differential silver staining attained.
Microscopic findings.-In longitudinal sections of the central wound, taking in I cm. each of central end of transplant and distal end of proximal stump; early stages of the downgrowth of central neuraxes very beautifully shown; the ends of the down-growing central neuraxes have reached the fibrous wound, which the more advanced are in the act of penetrating. Many of the central neuraxes terminate in relatively large end-discs, others show division, others still early stages of spiral formation. As yet no new neuraxes can be traced to the central end of the transplant. In cross sections of the transplant the four
FIG. 222.- Cross section through the middle of a cable-auto-nerve transplant, Experiment No. 75, 26 days after the operation; pyridine-silver preparation. The funicular structure of the four segments of the nerve used is well maintained. Newly formed epineural tissue has united the nerve segments so as to form a single nerve trunk
segments of nerve transplanted can be clearly made out, with the funicular structure of each stained and perineural sheaths not thickened. The four nerves surrounded by a com-mon connective tissue layer, forming a new epineural sheath common to the four nerves. In both cross and longitudinal sections of the transplant the neuraxes of the transplanted nerves appear fragmented into irregular segments, still staining differentially in silver. Sheath nuclei only here and there noted; not well stained in silver. Distal sciatic in early stages of degeneration.
EXPERIMENT No. 75.- Dog No. 10; large dog; full grown; 26 days. April 24, 1918,the left sciatic exposed and freed. Two cutaneous radial branches exposed and freed from connective tissue. These nerves cut into four segments of a little over 4 cm. length, placed side by side in two groups and together clamped at the ends with artery forceps. Two fine silk thread sutures passed 2 cm. apart in each group of two nerves, and the nerves cut a little beyond the suture. These segments of the cutaneous radial, with sutures in place, were transferred to the sciatic wound, and each pair sutured separately to resected ends of
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the sciatic. Good approximation of nerve ends attained. Muscle stitched over nerve and transplant; wound closed. May 20, dog died during the afternoon; nerve removed about two hours after death; left hind foot, severe neurotrotphic changes, slightly infected; sciatic wound well healed; forearm wound partly opened. On exposing the left sciatic no evidence of infection is noted; transplant found well in place and firmly united to resected nerve ends. Quite distinct bulbous enlargement on central sciatic noted; central end of distal stump not materially enlarged. The four transplanted nerve segments surrounded by newly formed connective tissue so as to form one bundle. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good silver differential attained.
Microscopic findings.-In longitudinal sections through the central wound and about 1 cm. each of sciatic and transplants, the central bulbous enlargement is seen to include the central wound and central sutures. Numerous neuraxes coming from the central sciatic can he traced through the central wound, in which they are found to crisscross in all directions
FIG. 223.- Longitudinal section through the central wound region, cable-auto-nerve transplant, Experiment No..75 20 days after operation; pyridine-silver preparation . The central end is found directed toward the left. The actual wound region found between the central and distal loops of the respective sutures is clearly seen in the figure
tions, into the central ends of th transplanted nerve segment. In cross section of the transplant, 1 cm. distal to the central wound, the four nerve segments transplanted can be clearly made out, each retaining its funicular structure, and are surrounded by a common fibrous tissue sheath serving as an epineural sheath, the whole transplant presenting the appearance of a relatively large nerve trunk with 10 larger and smaller funiculi; in about equal distribution there are found large numbers of new neuraxes, many found to be within old neurolemma sheaths, others in the endoneural tissue between these sheaths. Very few neuraxes observed in the connective tissue surrounding the several transplanted nerve segments. In longitudinal sections of the distal wound and adjacent nerve trunk it may be observed that certain of the down-growing neuraxes have reached the distal wound (transplant only 2 cm. in length), having thus nearly reached the central end of the distal sciatic. In this series of sections there was obtained very successful differential staining of neuraxes. Very often when this is the case the cellular elements of the tissue are not clearly stained; therefore this series of sections is not satisfactory for determining the behavior of the sheath cells of the transplanted nerve segments.
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EXPERIMENT No. 76.- Dog No. 11; large log; full grown; 109 days. April 25, 1918 left sciatic exposed and freed. The cutaneous radial branches exposed and freed from connective tissue. Quite a little venous oozing in the radial wound, so that the isolated cutaneous radial branches were bathed in partly clotted blood for 15 to 20 minutes. The cutaneous radial branches cut into segments of about 4 cm. lengths and placed side by side and together clamped at each end with an artery forceps. Two fine silk thread sutures passed 2.5 cm. apart, and nerves cut 2 mm. beyond the sutures. The sutures were then tied loosely and cut short, thus forming a compact nerve bundle of 2.5 cm. length, consisting of four segments of cutaneous radial. This bundle was transplanted to the resected left sciatic, and sutured centrally and distally by means of two fine silk thread epineural stitches. Fairly good approximation of cut nerve ends attained. Muscle stitch
FIG. 225.- From a cross section of a cable-auto-nerve transplant, Experiment No. 75, 26 days after operation; pyridine- silver preparation. The figure presents a portion of one of the larger funiculi of a transplanted nerve seen to seen in Figure 222. The black dots represent cross sections of a single neuraxis or small bundles of such which grew through the central wound and into the neurolemma sheaths of the transplanted nerves
over nerve and transplant and wound closed. Forearm wound open a long time; not well protected; in part dry; bleeding not fully controlled. Wound closed. Both wounds healed well. August 12, killed. Slight foot- and toe-drop of left hind foot; walks quite well. On exposing the left sciatic no material increase of connective tissue about nerve and transplant is found. Quite distinct bulbous enlargement of the central sciatic is noted; slightly adherent to underlying muscle. From distal end of the central bulb several small nerve bundles can be traced to the distal sciatic stump, which presents the appearance of a regenerated nerve of nearly normal size. On exposing the calf muscles, these appear of nearly normal size and color and manifest quite rhythmic twitching. After freeing sciatic and transplant from bed, on slowly cutting sciatic with scissors, central to the transplant, distinct contractions of the calf muscles observed; the same on cutting distal to transplant. The sciatic
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and transplant, internal and external popliteal and posterior tibial, portions of calf and leg flexor muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. In part very good differential silver staining attained.
Microscopic findings.- In longitudinal sections of the central wound and adjacent nerve ends, central bulbous end is seen to include central wound and sutures and an area of small cell infiltration near one of the central sutures. Many new neuraxes can be traced from the central sciatic through the central wound into the central end of the transplant; others in the form of small nerve funiculi, having very tortuous course, are traced distally in the connective tissue outside of the transplant. In cross sections of the transplant, made about 1.5 cm. distal to the central wound, the four transplanted nerve segments with their respective funiculi can be clearly made out, and are found surrounded by a common fibrous tissue sheath, consisting of quite densely felted fibrous tissue. Numerous new neuraxes are found in each of the funiculi of the several transplanted nerve segments, also in the form of small nerve bundles in the connective tissue surrounding the nerve transplants. Neuraxes in large numbers can, in sections of successive levels, be traced through the transplanted nerve segments and distal wound into the distal sciatic stump. In cross sections of the internal popliteal at the lower level of the popliteal space, numerous new neuraxes are found in all of the several funiculi. In alternate cross and longitudinal sections of the distal internal popliteal, posterior tibial, and internal plantar new neuraxes were observed, these becoming progressively less numerous distalward, so that in the internal plantar only a few scattered neuraxes were found in an otherwise degenerated nerve. In sections of the calf muscles, numerous new neuraxes are found in the larger and smaller intramuscular nerve branches and as single nerve fibers on and between muscle fibers; here and there motor nerve endings are to be seen. Regeneration of the distal sciatic through a cable autonerve transplant attained. Partial regeneration of the distal nerve to the level of the internal plantar.
EXPERIMENT No. 77.- Dog No. 5; medium size; full grown; 152 days. March 8,1918, left sciatic exposed and cut quite high in thigh; nerve ends retracted 8 mm. Wound closed. April 16, left sciatic exposed, 39 days after section. Dog in good condition; only slight limp noted. Large bulb found on the central sciatic stump, from which extends a fine nerve thread, which appears to reach the distal sciatic stump, which presents slight central enlargement; central and distal sciatic stumps resected; end 3 cm. apart. Two branches of the cutaneous radial exposed and freed of connective tissue, and cut in segments about 4.5 cm. long; placed side by side and clamped at each end with an artery forceps. Two No. 110 linen thread sutures passed through the four nerve segments 3 cm. apart. Nerves cut beyond suture lines and sutures tied loosely so as to form compact bundle. The same suture used to unite this nerve bundle to the ends of the resected sciatic; one extra epineural stitch central and distal; fairly good approximation of cut nerve ends attained. The four nerves together are only about two-thirds the diameter of the resected sciatic. Dry field; muscle stitched over nerve and transplant; wounds closed. Wounds healed well. August 7, killed. Dog emaciated; skin diseases; toe-drop on left hind foot; small ulcer on dorsum of foot. On exposing the left sciatic, there is noted an increase of connective tissue about nerve and transplant, especially in region of central and distal wound. No distinct central bulb observed and central end of distal sciatic only slightly enlarged. The four transplanted nerve segments surrounded by a common connective tissue sheath so as to form one nerve bundle about two-thirds as large as the distal sciatic. After exposing the calf muscles and freeing the sciatic and the transplant from its bed, on slowly cutting the sciatic with scissors, central to the transplant, indistinct, feeble twitching of calf muscles observed; this observed somewhat more clearly on cutting internal popliteal distal to the transplant. Calf muscles do not present normal color; pale red with narrow, light yellow streaks. Sciatic and the transplant with internal and external popliteal and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.- In longitudinal sections of the central wound and the adjacent nerve ends, the central bulbous enlargement is found to embrace the central wound and central sutures. Numerous new neuraxes coming from the central stump can be traced through the central wound, in which they are seen to crisscross in all directions into the central ends of
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the transplanted nerve segments. Many neuraxes are seen to pass distally in the connective tissue surrounding the transplanted nerve segments. In cross sections of the transplant, the four nerve segments transplanted are seen clearly demarked; each retaining its funicula structure. They arc found surrounded by a common connective tissue sheath which serves as an epineural sheath. Numerous new neuraxes found in each of the funiculi of the four transplanted nerve segments. In the surrounding connective tissue, especially to one side, some 15 to 20 small nerve bundles are to be observed. New neuraxes can be traced in successive sections through the distal wound into the distal sciatic in which they are found in large numbers in all of the several funiculi. In sections of the calf muscles, new neuraxes are to be observed in the larger and smaller intramuscular nerve branches, and as single nerve fibers between and on the muscle fibers; a few motor nerve endings observed. Only partial regeneration of nerves in the calf muscles attained.
FIG. 227.- Cross section of cable-auto-nerve transplant, Experiment No. 77, 152 days after operation; pyridine-silver preparation. The four nerve segments used as a nerve bridge in this operation are clearly made out nearly four months after the operation
EXPERIMENT No. 78.- Dog No. 9; large dog; full grown; 389 days. April 24, 1918,left sciatic exposed and resected; quite free bleeding from the central stump; small artery ligatured. Two branches of the right superficial radial exposed and freed from connective tissue; cut into segments about 4 cm. long, placed side by side, and clamped at each end with an artery forceps. Two fine silk thread sutures passed through the four nerve segments 2 cm. apart and nerve cut beyond sutures. The sutures tied loosely, so as to form one compact bundle. This bundle transferred to sciatic wound and with one continuous suture each of the four nerve segments was sutured separately, centrally and distally, to the resected nerve ends; fine vessel silk was used for this suture. Muscle stitched over nerve and transplant; wounds closed. May 18, 1919, killed. Dog not in good condition, severe skin disease, active; walks well. Nails on two of the toes very long and curved. On exposing the left sciatic quite a large bulb found on the central sciatic; no distinct enlargement of central end of distal sciatic; transplant found well in place. Distal sciatic presents the appearance of a normal nerve. Calf muscles exposed; these have the appearance of normal muscle. After freeing sciatic from the bed, on slowly cutting nerve with scissors, central to transplant, calf muscles found to contract well, even though this functional test was made nearly forty minutes after the dog was killed. Contraction of the foot muscles not so clearly made out; they had not been exposed. Sciatic and transplant, distal popliteal and portions of interossei muscles removed, fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
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Microscopic findings.- In longitudinal sections of the central wound and adjacent nerve ends, the central bulbous enlargement is found to include the central nerve wound and central sutures. Numerous neuraxes can be traced from the central sciatic through the central wound, in which they crisscross in all directions into the central end of the transplant. In cross sections of the transplant, though more than one year after operation, the four transplanted nerve segments can be clearly demarked, each with distinctive funicular structure. These four nerves are surrounded by a common fibrous tissue sheath which serves as an epineural sheath. Numerous neuraxes, many of which are myelinated, pass through the transplant to the distal sciatic; these are found about equally distributed through the several funiculi of the four transplanted nerve segments. Only very few neuraxes, in the form of several small nerve bundles, are found in the surrounding connective tissue. In longitudinal sections of the distal wound and adjacent nerve ends, there is also observed a crisscrossing of neuraxes; the neuraxes having here a very irregular course. In sections of the popliteal branches of the sciatic, to the level of the posterior tibial at the heel, new neuraxes in large numbers are to be observed. Unfortunately the pieces of interossei muscles removed at post mortem were lost in process of staining and can not be reported upon. In so far as Microscopic evidence of nerves is concerned, nearly complete regeneration of distal branches of the sciatic was obtained.
EXPERIMENT No. 79.- Dog No. 13; large dog; full grown; 385 days. April 30, 1918, left sciatic exposed; resected. Two cutaneous radial branches exposed and freed from connective tissue. Each branch cut centrally and distally and pinned out, side by side, on piece of smooth wood, which had been sterilized with the instruments. With the nerves thus pinned out, sutures were passed so as to form two bundles of nerves, each with two nerve segments with sutures at each end; nerve bundles of 2.5 cm. length. Each of the two bundles, each composed of two nerve segments, was sutured separately between the resected nerve ends. One bundle was cut longer than the other, so that when they were sutured in place, one of the bundles presented a wavy course. Fairly good central and distal approximation of the nerve ends attained. Muscles stitched over nerve and the transplant; wounds closed. May 20, 1919, killed. Dog in very good condition; well fed and active; still seems to favor left hind leg a little; small ulcer on dorsum of the foot; two nails on this foot of large and irregular form. On exposing the left sciatic a distinct bulbous enlargement on central sciatic is found, from the distal end of which several nerve bundles are seen to pass to the distal sciatic stump. Distal sciatic and the popliteal branches present the appearance of normal nerves. Calf and leg flexor muscles fully exposed; after freeing sciatic and transplant from bed, on slowly cutting sciatic central to the transplant, vigorous contractions of the calf and leg flexor muscles observed. Cutting of the posterior tibial at heel does not call forth distinct contraction of the interossei muscles. Sciatic and the trans- plant, internal and external popliteal branches, portions of calf and interossei muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.-In longitudinal sections of the central wound and the adjacent nerve ends, it is quite clearly to be made out that good approximation of the severed ends was not obtained at the time of operation. Down-growing neuraxes coming from the central stump, on reaching the central wound, present a felt-work arrangement, crisscrossing in all directions; many can be traced into the connective tissue surrounding the transplants. In cross sections of the transplant, about 1 cm. distal to the central wound, the field is made up of large numbers of small nerve funiculi separated by fibrous tissue and three quite distinct funiculi, surrounded by perineural sheath. The latter appear to be the only funiculi surviving out of about ten as found in other experiments in which four segments of the cutaneous radial nerves were transplanted. Numerous neuraxes may be traced through the distal wound into the distal sciatic, in which they are found in sections taken at successive levels to the posterior tibial at the level of the heel. In both cross and longitudinal sections, made of pieces taken from the interossei muscles, new neuraxes may be observed in the intramuscular nerve branches, and nerve endings in at least one neuromuscular spindle. No fully formed motor endings were observed; however, this may be due to imperfect silver differentiation. Regeneration of the distal popliteal to the level of the interossei muscles attained.
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EXPERIMENT No. 80.-Rabbit No. 2a; large; full grown; 21 days. March 18, 1918, left and right sciatic exposed. A segment of the right sciatic having a length of 1.5 cm. trans-planted to the resected left sciatic. One through-and-through Chinese silk suture placed centrally and distally; wounds closed. April 8, rabbit found dead in the morning. Wounds well healed. On exposing the left sciatic transplant is found well in place. Distinct bulb on distal end of central sciatic stump noted. Sciatic and the transplant removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin.
Microscopic findings.-In longitudinal sections taken from the transplanted nerve segment, numerous small round cells, found mostly in the looser connective tissue outside of the perineural sheath, observed. In the transplanted nerve fibers, the myelin is found in the form of irregular globules, separated by a granular detritus. Neuraxis remains not clearly made out. No distinct proliferation of sheath cells can be observed in such broken-down fibers. The picture is not that of a Wallerian degeneration. Strands of what appear to be syncytial protoplasmic bands, with short, rod-shaped nuclei, are noted. These bands resemble those found in a peripheral nerve trunk after section. Small bundles of such protoplasmic bands are separated by areas having neurolemma sheaths in which myelin remains are found.
In all of the experiments of auto-nerve transplants, kept for a time sufficiently long to admit of nerve regeneration, the results obtained were very satisfactory. In five of these experiments (No. 64 to No. 68), only one small nerve was used to bridge a defect in a resected sciatic, in three (No. 69 to No.71), two segments of ulnar or superficial radial nerves were used to bridge such a defect, and in two experiments (No. 72 and No. 73) three pieces taken from the cutaneous radial were sutured between the resected ends. In all of the several experiments there was noted a nerve bulb on the distal end of the central sciatic stump, in general the more prominent the less adequately the nerve defect was bridged. On the whole there appeared to be little difference in the results attained in a primary operation in which the nerve transplant was sutured in place at the time of resection of the sciatic or in a secondary operation (No. 66 and No. 88) in which the sciatic was resected, the wound closed to be reopened some weeks later, and a nerve transplant made. A study of the protocols of the respective experiment will show that the central neuraxes were found to grow through the transplant, and to a variable extent in the connective tissue surrounding the nerve transplanted, to reach the distal segment of the resected nerve. It is not the purpose to discuss at this time the relative merits of the auto-nerve transplants. This will be undertaken after considering the observations considered in Series No. 6 and No. 7. A somewhat further consideration may here be given to the six experiments (No. 74to No. 79) in which four segments of the radial cutaneous nerves were used to bridge a defect in sciatic nerves the result of resection. This operation we have designated a "cable-auto-nerve transplant" or a multiple nerve transplant. The radial cutaneous branches of the dog are relatively small nerves consisting of three or four major funiculi and presenting the form of a flattened oval in cross section. Four segments of these nerves arranged in parallel bundles were sutured singly or in pairs or as one bundle between the resected ends of the sciatic, by both central and distal sutures. In the dog killed 11days after the operation (No. 74), before regeneration could have taken place, attention is called to the fact that on exposing the nerve, the four nerve segments transplanted were found united in one compact bundle, having the appearance of a single nerve trunk, by newly formed connective tissue. This
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newly formed connective tissue forming an epineural sheath surrounding and enclosing the four segments of nerve transplanted, giving in cross section the appearance of a nerve trunk with many funiculi. A little study of the cross section reveals the fact that each of the four nerve segments has in reality maintained its own identity, each showing its respective funiculi surrounded by perineural sheaths. In this experiment there is distinct evidence of an active downgrowth of central neuraxes, which have reached the central wound region but have not as yet penetrated the transplants. In the experiment (No. 75),which terminated 26 days after the operation, the transplanted nerve segments were found united in one compact bundle by a newly formed epineural sheath. However, in each nerve segment there can easily be determined the several funiculi, each surrounded by a distinct perineural sheath. The transplanted nerve segments were found firmly united to the central and distal stumps of the resected nerve. The nerve segment removed for study included the distal end of the central stump to the extent of about 2 cm., the transplant, and some3 cm. of the distal sciatic. This segment was divided into pieces so as to admit of longitudinal sections of the central and distal wound regions and contiguous transplant and sciatic segments and cross sections of the middle of the transplant. Nearly complete serial sections of the several pieces were made after the tissue had been stained by the pyridine-silver method. In the series of longitudinal sections of the central wound region, central neuraxes may be traced in large numbers through the central wound and into the central part of the nerve transplant. The picture presented is very much that of a section through the wound region after primary suture, except that in the cable auto-transplant the approximation of nerve ends is not so good; there is much more crisscrossing of the down-growing neuraxes. The cross-section series is very instructive. In the sections, taken from about the middle of the transplant, new neuraxes are found in all of the funiculi of the several transplanted nerve segments and in approximately equal distribution. Many of the neurolemma sheaths of the transplanted nerve fibers contain more than one neuraxis, others only one, and again others none. Very few neuraxes are found in the connective tissue outside of the funiculi. In the longitudinal sections, including the distal wound, it may be noted that the down-growing neuraxes in lessening number have penetrated the transplant to the region of the distal wound. The distal sciatic presents the picture of a degenerated nerve, the majority of the neurolemma sheaths containing syncytial strands.
In the remaining four of the cable auto-nerve transplants (No. 76 to No.79) ranging in length of observation from nearly 4 months to a little over12 months, even in those of long duration, could there be made out readily the four segments of nerve transplanted each with its several funiculi showing conclusively that the severed nerve segments formed definite paths along which the down-growing central neuraxes proceeded to reach the distal segment. In each of these four experiments was there a return of motor function as tested on the exposed muscles. This is corroborated by the presence of new motor endings in the calf muscles of certain experiments and even in the foot muscles of other experiments. The morphologic evidence of regeneration through several nerve segments as used in the operation of auto-nerve transplants, it seems, is conclusive. It is here shown that it is possible to use several segments of a relatively small cutaneous nerve to bridge a defect in a major nerve, such as the sciatic, thus making available in human surgery the operation of auto-nerve transplant.
SERIES NO. 6
HOMO-NERVE TRANSPLANTS
Under Series No. 6 are presented observations on six experiments, all but one on the sciatic nerve of the rabbit, in which the sciatic was resected in one animal and a segment of requisite length removed from the sciatic of another animal used as a nerve bridge. The operation of homo-nerve transplant in human surgery has limited opportunity for application since it is to a large extent chance that would make available normal, fresh human nerve tissue to be used for purpose of transplant. As a secondary operation, where the operation of nerve repair may be timed, nerves from amputations may be made use of. We present an extended series of observations on the use of stored homo-nerve transplant which will be considered under a separate heading; in this series only operations in which fresh homo-nerve transplants were used are included.
PROTOCOLS
EXPERIMENT NO. 81.- Rabbit No. 1l a; full-grown rabbit; 8 days. March 14, 1918, right sciatic exposed and resected to the extent of 1 cm. A segment taken from the right sciatic of another rabbit, of 1.2 cm. length, used as a transplant. One central and distal Chinese silk suture placed. Fairly good approximation of nerve ends attained. Wounds closed. March 22, rabbit found dead in the morning. Sciatic wound healed. On exposing the sciatic, transplant found in place and united to the resected nerve ends. Sutures still show clearly. No distinct central bulb observed. Sciatic and transplant removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin.
Microscopic findings.-In series of longitudinal sections of the transplant, the myelin fthe transplanted nerve fibers seen to be fragmented in many of the fibers. The neurokeratinnet is clearly made out even in the fragments of myelin. Neurolemma sheaths very distinct. Sheath cells evident but do not manifest proliferation.
EXPERIMENT No. 82.- Rabbit No. la; large rabbit; full grown; 8 days. March 18,1918, the right sciatic exposed and resected 1.3 cm. The right sciatic of another rabbit exposed and a segment of 1.5 cm. length transplanted. One central and distal Chinese silk suture placed. Good approximation attained; a little clotted blood between nerve ends in distal wound. Wound closed. March 26, rabbit found dead in the morning. On exposing the right sciatic it is found that the central suture had given away a little; distal suture good. Sciatic and the transplant removed and fixed in neutral formalin for Bielschowsky silver staining.
Microscopic findings.-In a series of longitudinal sections of the transplant it is found that the epineural sheath has been invaded by numerous small round cells. In the transplanted nerve fibers the neuraxes are found to be fragmented; these fragments, differentially stained, are either bent upon themselves, are coiled, or have a wavy course. The myelin is not clearly defined; appears granular. The neurolemma sheaths intact and appear thickened. Sheath nuclei not differentiated.
EXPERIMENT No. 83.- Dog No. 36a; medium size; full grown; 17 days. June 4, 1918, right sciatic exposed and internal popliteal freed, and resected. A segment of 3 cm. length taken from the right internal popliteal of another dog, under the anesthetic at the same time, used as a transplant. One central and distal fine silk thread suture placed; very good approximation. The uncut external popliteal funilculi lies at the side of the operated internal popliteal
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Clean field; wound closed. June 21, dog found dead in the morning; very slight neurotrophic changes right hind foot. Wound well healed. On exposing the right sciatic the transplant is found well in place; appears as if slightly swollen; united to the resected nerve ends, though distally the wound had separated a little. No distinct central bulb noted. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good silver differentiation for the central segment attained.
Microscopic findings.- In longitudinal sections of the central wound and the adjacent nerve ends, central wound is clearly demarked by the presence of the central suture; good fibrous union. Only indistinct central bulb evidenced structurally. Numerous downgrowing neuraxes of- the central stump, end, often after branching, in large end-discs at the point of the fibrous union. Others have penetrated the fibrous tissue of this region and are found in the central end of the transplant in which they may be traced distally, becoming gradually less numerous, for a distance of nearly 1.5 cm. Now and again more than one neuraxis may be observed in old neurolemma sheath of the transplant. In cross section of the transplant about 1 cm. distal to the central wound relatively few neuraxes are to be found, scattered fairly evenly through the several funiculi of the transplanted nerve. In the distal popliteal stump early nerve degeneration stages observed. No new neuraxes traced to the distal nerve.
EXPERIMENT No. 84.- Rabbit No 62; full grown; 23 days. March 21, 1918, right sciatic exposed and resected 1.2 cm. Right sciatic of another rabbit exposed, while nerve was being exposed rabbit died under anesthetic; operation completed and nerve used in transplant. One central and one distal Chinese silk suture placed; fair approximation. Wound closed. April 13, killed. Caudal half of rabbit paralyzed; cause not determined. Sciatic wound well healed. On exposing nerve, transplant was found well in place; slightly adherent to muscle bed. Central and distal sutures clearly made out; no distinct central bulb. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good silver differentiation attained.
Microscopic findings.-In longitudinal sections through the central wound region and adjacent nerve ends, numerous down-growing neuraxes, crisscrossing through the fibrous wound, can be traced into the central end of the transplant. In cross sections of the transplant, about its middle, the funicular arrangement of the transplanted nerve segment is found well retained. The fibrous sheaths of the funiculi found thickened, within them and between the funiculi are found numerous small round cells. Within the funiculi numerous down- growing neuraxes are to be seen. In many instances four to six or eight new neuraxes found in one old neurolemma sheath. Certain of these down-growing neuraxes have reached the distal wound and can be traced to the distal sciatic in which they extend for a distance of about 1 cm.
EXPERIMENT NO. 85.- Rabbit No. 8a; full grown; 68 days. March 16, 1918, right sciatic exposed and resected a little over 1 cm. A segment of equal length taken from the right sciatic of another rabbit used as transplant. One central and distal Chinese silk suture placed; good approximation. Wound closed. May 23, killed. Severe neurotrophic change right foot; rabbit in good condition. On exposing the right sciatic, transplant is found well in place; sutures clearly seen. Small spindle-shaped central bulb. After exposing the calf muscles and freeing nerve from bed, no contraction of muscles observed on cutting nerve central to the transplant. Calf muscles appear atrophic and of yellow-red color. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings.-In longitudinal sections of the central wound and the adjacent nerve ends, it may be observed that numerous neuraxes pass from the central stump through the central wound to the central end of the transplant. In cross sections of the transplant it is to be observed that its fibrous sheaths are materially thickened and that its funicular structure is not fully maintained. Numerous new neuraxes are observed both within the funiculi of the transplant and especially to one side in the surrounding connective tissue. In sections taken at successive levels, the down-growing neuraxes may be traced into and through the distal wound into the distal sciatic in which they are found in good number 2 cm. beyond the distal found; the extent of the sections.
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EXPERIMENT No. 86.-Rabbit No. 27a; full grown; 83 days. March 15, 1918, right sciatic exposed and resected about 1 cm. A segment of equal length taken from the right sciatic of another rabbit and used as transplant. One central and distal Chinese silk suture placed. The muscle stitched over the nerve and transplant and the wound closed. June 6, killed. Wound well healed; rabbit in good condition. On exposing the right sciatic transplant was found well in place, with nerve sutures still evident. Small central bulb observed. Transplant of smaller diameter than resected nerve and slightly adherent to the underlying muscle. After freeing nerve from bed and exposing the calf muscles, on slowly cutting central sciatic no contraction of calf muscles noted. Sciatic and the transplant and the internal popliteal removed and fixed in ammoniated alcohol for pyridine silver staining. Fairly good silver differentiation attained.
Microscopic findings.-In longitudinal sections of the central wound, the central suture appears in the sections, clearly indicating the region of the central wound. Numerous down-growing neuraxes coming from the central stump penetrate the central end of the transplant. In cross sections of the transplant the funicular structure is clearly demarked. In the funiculi numerous new neuraxes, evenly distributed, are to be observed. Several groups of small nerve bundles are found in the connective tissue surrounding the transplant. In successive series of sections these neuraxes can be traced into the distal sciatic stump in which they are present in large numbers to the extent of the sections, 2 cm. beyond the distal wound.
The experiments in which fresh homo-nerve transplants were used, though relatively few in number and of relatively short duration, nevertheless show conclusively the feasibility of using fresh homo-nerve transplants to bridge a nerve defect. In the last three of the experiments listed (No. 84 to No. 86), ranging in duration from a little over one month to nearly three months, the down-growing central neuraxes were traceable through the transplant and a variable distance into the distal sciatic stump. As seen best in cross sections of the transplant, the down-growing neuraxes are found within the neurolemma sheath of the transplanted fibers, in the endoneural tissue between the nerve fibers and in the connective tissue surrounding the perineural sheaths of the funiculi of the transplant. In Experiment No. 83, in which the sciatic of a dog was operated upon and the experiment terminated at the end of approximately three weeks, the series of longitudinal sections of the central wound region presents a typical picture of regeneration from the central nerve stump with many branching central neuraxes and many down-growing neurax esterminating at various levels in end-discs. These down-growing neuraxes are traceable through the wound region and into the central end of the transplant. No neuraxes are found in the distal wound region; the distal sciatic presenting a typical picture of a degenerated peripheral nerve of about three weeks standing.
SERIES NO. 7
HETERO-NERVE TRANSPLANTS
There can be no question that should hetero-nerve transplants prove to be a feasible operation it would become the operation of choice in cases in which a nerve bridge was found necessary. Therefore, it seemed to us worth while to reinvestigate the merits of a hetero-nerve transplant. In the majority of the experiments of this series (No. 87 to No. 125) one or two sciatic nerves, taken from the guinea pig, were used to bridge defects in the sciatic of rabbits, the result of resection. In a few experiments a nerve taken from a dog was used
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to bridge a resected sciatic of a rabbit. The operated animals were killed at stated intervals, ranging in the several experiments from three days to nearly a year. Only fresh nerves were used for purposes of transplants. While one animal was under an anesthetic having one of the sciatic nerves exposed, resected, and sutures placed, a guinea pig (or dog) was placed under anesthesia, the region of the operation shaved and made aseptic, so that at the proper time the desired nerve could be exposed, a segment of requisite length taken and transferred to the host and sutured in place. In a number of instances two sciatics of a guinea pig were used to bring the diameter of the transplant to approximate that of the nerve to be bridged. Especial care was taken to suture the transplant to the resected nerve ends with as good end-to-end approximation as was possible so as to make the conditions favorable for regeneration.
PROTOCOLS
EXPERIMENT No. 87.- Rabbit No. 73; old; large rabbit; 3 days. April 22, 1918, left sciatic exposed; resected about 1 cm. The two sciatics of a half-grown guinea pig exposed, exsected and placed side by side and clamped at ends with artery forceps. Two fine silk threads passed a little over 1 cm. apart through both nerves, and nerves cut beyond sutures. The two nerves together used as a transplant and sutured to the resected ends of the sciatic. Wound closed. April 25, killed. Superficial wound found healed; deep wound easily separated. On exposing the sciatic it was observed that one of the nerves used as a trans- plant had separated from the central stump; the other from the distal stump; the respective ends lying free in the wound. The sciatic and the transplanted nerves removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation of central sciatic stump attained.
Microscopic findings.-In sections it is evident that the ends of the transplanted nerve segment were not well sutured to the resected ends. In longitudinal sections of the distal end of the central sciatic stump, many of the central neuraxes present distinctly swollen ends, many of irregular shape; no distinct fibrillar differentiation is made out. No clear evidence of the downgrowth of central neuraxes is observed. Transplanted nerve segments and the distal sciatic not clearly differentiated in this series.
EXPERIMENT No. 88.- Rabbit No. 73a; old; large rabbit; 3 days. April 22, 1918, right sciatic exposed and resected about 1 cm. Both of the sciatics of a half-grown guinea pig exsected and together used as transplant. One central and distal silk thread suture used. Wound closed. April 25, killed. Superficial wound found healed; deep wound easily separated. On exposing nerve, transplanted nerve segments found well in place. Sciatic and transplants removed and fixed in Flemming's chromo-osmic-acetic mixture. Sections stained in safranine and licht grün.
Microscopic findings.- In longitudinal sections of the central and distal wound regions, it is evident that there was good end-to-end approximation of the ends-of the transplanted nerve segments and the resected sciatic. Central and distal wound region consists of a loose fibrocellular tissue. In cross section of the transplanted nerve segments, the funicular structure of the respective nerves well maintained. The two nerves found surrounded by a common layer of exudate with beginning fibrous tissue formation. Evidence of in wandering of leucocytes; these found between the nerve fibers. Very little fragmentation of the myelin of the transplanted nerve fibers observed; their sheath cells only indistinctly stained. In the distal sciatic beginning fragmentation of the myelin and the beginning of proliferation of the sheath cells noted; here and there a mitotic figure in these.
EXPERIMENT No. 89.- Rabbit No. 74; full grown; 5 days. April 22, 1918, the left sciatic exposed and resected 1.3 cm. The two sciatics of a half-grown guinea pig together used as a transplant. One central and distal silk suture placed. Wound closed. April 27, killed. Superficial wound healed; deeper wound not fully united. On exposing nerve, transplants found well in place, though only one of the nerves is clearly made out; not adherent
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to the underlying muscle. Nerve and transplant surrounded by sanguineous exudate. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation in the central stump attained.
Microscopic findings.-In longitudinal sections of the central wound region, beginning stages of the down growth of central neuraxes noted. Certain of these have reached the central wound region. Not good alignment of central end of the transplants and distal end of sciatic found. Silver staining not satisfactory for detail study of transplant. Distal sciatic beginning degeneration of peripheral fibers made out.
EXPERIMENT No. 90.- Rabbit No. 74a; full grown; 3 days. April 22, 1918, the right sciatic exposed and resected 1.5 cm. The two sciatics of a half-grown guinea pig used as a transplant. One central and distal silk suture placed. On suturing distally one nerve was twisted over other; half spiral turn. Wound closed. April 25, killed. Superficial wound healed; deep wound not completely healed. On exposing sciatic it is found that the external popliteal bundle was not cut, the transplant found sutured to internal popliteal; transplants well in place, easily demarked by presence of sutures. Internal popliteal and the transplant removal and fixed in Flemming's chromo-osmic-acetic mixture. Sections stained in safranine and licht grün.
Microscopic findings.- In longitudinal sections of central and distal wound regions, it may be observed that the ends of the transplanted nerves are bent over hook-shaped; thus not found in alignment with the resected nerve ends. In longitudinal sections of the distal end of the central stump, for a distance of about 8 mm., fragmentation of myelin of central fibers and proliferation of the sheath cells noted. In cross sections of the transplant, the funicular structure of the two nerves is well maintained, with exudate and newly forming fibrous tissue inclosing the two nerves. The perineural sheaths of the funiculi not thickened. Beginning of in wandering of cells through the perineural sheaths observed. Only in a few of the larger funiculi, and in these in the more peripherally placed fibers, is fragmentation of the myelin noted. The great majority of the nerve fibers of the transplanted nerves show as vet no distinct fragmentation of the myelin and no proliferation of sheath cells. In the distal sciatic the peripheral nerve fibers show fragmentation of myelin and proliferation of sheath cells.
EXPERIMENT No. 91.-Rabbit No. 69; large; full grown; 9 days. April 18, 1918, left sciatic exposed and resected 1.2 cm. The two sciatics of a half-grown guinea pig together used as a transplant. One central and one distal suture placed; good approximation. Wound closed. April 27, killed. Superficial wound healed; deep wound healing. On exposing the left sciatic transplant found well in place, surrounded by exudate and newly forming connective tissue, which unites the two nerves in one bundle. The transplant appears congested, giving it a pink-red color. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings.-In a series of longitudinal sections of the central wound region, numerous neuraxes growing from the neuraxes of the central sciatic can be traced to the central wound region, which they have invaded. Certain of the neuraxes are seen to branch others to terminate in end-discs; the beginning of spiral formation seems evident. Fine naked neuraxes found in the fibrocellular central wound. In the transplant, especially near the central wound, the neuraxes of the transplanted nerve, found in the form of long segments, having a regular course. Further distalward shorter neuraxis segments are found. Distal sciatic presents early stages of degeneration.
EXPERIMENT No. 92.-Rabbit No. 69a; large; full grown; 9 days. April 18, 1918,right sciatic exposed and resected 1.2 cm. The two sciatics of a half-grown guinea pig together used as a transplant. One central and distal silk suture placed; good approximation. Wound closed. April 27, killed. Superficial wound healed; deeper wound healing. On exposing the right sciatic the transplants found well in place and surrounded by newly forming connective tissue. Sciatic and the transplant removed and fixed in Flemming's chromo-osmic-acetic mixture. Sections stained in safranine and licht grün.
Microscopic findings.-Transplant found well united to the resected nerve ends. In cross sections of the transplant, through its middle region, the two nerves transplanted clearly made out, each showing typical funicular structure. The two nerves united by a
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thick, common fibrous tissue sheath, in which are seen numerous small round cells; these also in large numbers between the two nerves. Wandering cells have invaded certain of the nerve funictuli. In certain of the funiculi the nerve fibers more peripherally placed show fragmentation of the myelin, not distinctly evident in the more centrally placed fibers. Not all of the funiculi react in the same way. Certain of the sheath nuclei of the transplanted nerve fibers are found to stain deeply; others more faintly. No proliferation of these cells is noted. In the distal end of the central sciatic stump and in the distal sciatic there is observed a distinct increase in the number of the sheath cell nuclei; here and there mitotic division is noted. Fragmentation of the myelin, which varies in degree in different nerve fibers, is observed in the distal sciatic.
EXPERIMENT No. 93.- Rabbit No. 67; large; full grown; 9 days. April 18, 1918, left sciatic exposed and resected 1.4 cm. The-two sciatics of a nearly grown guinea pig used as transplants. One central and distal suture placed; centrally good approximation, distally"fair." Wound closed. April 27, killed. Wound healed. On exposing the left sciatic, transplants found well in place; surrounded by newly formed connective tissue and adherent to the underlying muscle. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. For central portion of nerve good silver differentiation attained.
Microscopic findings.-In longitudinal sections of the central wound region down-growing neuraxes in relatively small number are found to have reached the central wound region; branching of these neuraxes is observed. In longitudinal sections of the transplanted nerves, the neuraxes of the nerve fibers are found fragmented into longer or shorter segments, staining differentially in the silver. A distinct layer of fibrous tissue surrounds the transplant his tissue incloses many small cells. Distal sciatic shows early stages of nerve degeneration.
EXPERIMENT No. 94.- Rabbit No. 67a; large; full grown; 9 days. April 18, 1918, right sciatic exposed and resected 1.5 cm. The two sciatics of a nearly grown guinea pig used as transplant. One central and distal silk suture placed. Clean, dry wound. Wound closed. April 27, killed. Wound healed. On exposing the right sciatic, transplant found well in place; surrounded by exudate and newly formed fibrous tissue, not adherent to under-lying muscle. A dead space nearly surrounds transplant; in this a small amount of sanguineous exudate. Sciatic and transplant removed and fixed in Flemming's chrom-osmic-acetic mixture. Sections stained in safranine and licht grün.
Microscopic findings.-In longitudinal sections of the central and distal wound regions, the ends of the transplants are found united to the resected nerve ends by means of fibro- cellular tissue. The wound regions surrounded by numerous small cells; these have penetrated the distal end of the central sciatic stump for a distance of about 2 mm. In cross sections of the transplant the two nerves clearly demarked, with funicular structure well maintained. These nerves as seen in cross sections present an appearance which resembles closely that of normal nerves. At the periphery of the funiculi, beginning breaking down of the myelin is noted. In wandered cells are found here and there between the nerve fibers; not to equal extent in all of the funiculi. The distal sciatic presents early stages of nerve degeneration, with great increase in the number of the sheath cells, and fragmentation of the myelin.
EXPERIMENT No. 95.- Rabbit No. 71; large; full grown: 15 days. April 19, 1918, left sciatic exposed and resected 1.4 cm. The two sciatics of a nearly grown guinea pig used as transplants. One central and distal No. 110 linen thread suture placed; only "fair" central and distal approximation of the nerve ends attained. Wound closed. May 4, killed. Rabbit not well; emaciated; wound well healed. On exposing the left sciatic, transplant found well in place; seems of smaller diameter than when used; not adherent to muscle. Distinct central sciatic bulb noted. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. For central portion of nerve fair silver differentiation attained.
Microscopic findings.- In longitudinal sections of the central wound region a distinct central bulb evidenced structurally, from the distal end of which a few down-growing central neuraxes have reached and penetrated the central wound region; numerous bulbous end-discs on such neuraxes noted. Within the transplant the old neuraxes found in short segments still showing the differential silver staining. The distal sciatic presents early stages of degeneration.
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EXPERIMENT No. 96.- Rabbit No. 71a; large; full grown; 15 days. April 19, 1918, right sciatic exposed and resected 1.2 cm. The two sciatics of a half-grown guinea pig used as transplants. One central and distal No. 110 linen thread suture placed; good central approximation, "fair'" distal. Wound closed. May 4, killed. Rabbit not well; emaciated; wound was healed. On exposing the right sciatic transplant is found well in place and of dull white color. The two nerves are held together by scant connective tissue. Well developed central bulb noted. Sciatic and transplant removed and fixed in Flemming's chrom-osmic-acetic mixture. Sections stained in safranine and licht grün.
Microscopic findings.- Transplant found well united to resected nerve ends; fibrous unio In cross sections of the transplant, the two nerves transplanted are clearly demarked with funicular structure well retained. The two nerves found surrounded by common connective tissue sheath infiltrated with small round cells; found in large numbers between the two nerves. Here and there cells which have penetrated the perineural sheaths are found between the nerve fibers. It may be observed that the peripheral fibers of the funiculi have their myelin and neuraxes more fragmented than those more centrally placed. Wandering cells found within certain of the neurolemma sheaths. In the distal sciatic the nerve fibers found in early stages of degeneration.
EXPERIMENT No. 97.- Rabbit No. 88; large; full grown; 42 days. August 20, 1918, the left sciatic exposed; internal popliteal bundle freed; resected 2.8 cm. A segment of equal length taken from the left sciatic of a full-grown guinea pig used as transplant. One central and distal suture of waxed fine silk thread used; good approximation. Wound closed. October 1, rabbit found dead in the morning; neurotrophic ulcer left heel. On exposing the left sciatic, it was found that the transplant remained united to the central sciatic stump, the suture showing, but had pulled free from the distal stump; its end lying free in the wound; the portion of the transplant remaining having a yellow-white color. Large bulbous end on central sciatic stump noted. Central sciatic and remains of transplant removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchin; safranine and licht grün.
Microscopic findings.-In cross sections of the remains of the transplant the funicular structure of the nerve found well maintained; the perineural sheaths invaded by small round cells. Within the perineural sheaths, large and in fact multinucleated masses of protoplasm, indefinitely bounded, are to be seen. In the nerve fibers, the neurolemma sheaths found containing globular masses, varying in size. This more particularly in the perineural fibers of the funiculi. The more centrally placed fibers found better preserved; certain ones still showing the neurokeratin net of the myelin.
EXPERIMENT No. 98.- Rabbit No. 88a; large; full grown; 33 days. August 29, 1918, right sciatic exposed; internal popliteal freed; resected 2.4 cm. The right sciatic of a full-grown guinea pig used as transplant. One central and distal waxed, fine silk-thread suture placed; good approximation. Wound closed. October 1, rabbit found dead in the morning; neurotrophic ulcer on right heel. On exposing the right sciatic, transplant was found well in place; no material increase of connective tissue. Transplant is of dull white color. No distinct bulb on the central sciatic stump noted. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. For central part of nerve good silver differentiation attained; for distal nerve not so good.
Microscopic findings.- In longitudinal sections of the central wound region a long spindle-shaped bulb evidenced structurally, including the central end of the transplanted nerve segment. Down-growing neuraxes coming from the central nerve are found to enter several of the funiculi of the transplant in which they can be traced several millimeters. In cross sections of the transplant taken about 1 cm. distal to the central wound, the funicular structure of the transplanted nerve is found well preserved, with the fibrous sheaths of the funiculi thickened. The old neuraxes of the transplanted nerve fibers evident within many of the neurolemma sheaths. No new neuraxes made out at this level. In longitudinal sections of the transplant, nearly all of the myelinated nerve fibers show remains of the neuraxes, as short segments of spiral form or looped, showing the characteristic silver reaction. The nerves of the distal popliteal found degenerated.
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EXPERIMENT No. 99.-Rabbit No. 70; full grown; 34 days. April 19, 1918, left sciatic exposed and resected 1.2 cm. The two sciatics of a half-grown guinea pig used as transplants. One central and distal silk thread suture placed. Good central approximation attained; distal not good; an additional epineural stitch improves somewhat. Wound closed. May23, killed. Wound well healed; beginning neurotrophic ulcer on the left heel. On exposing the left sciatic, transplant is found well in place, surrounded by fibrous tissue and found adherent to underlying muscle. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good differential silver staining attained.
Microscopic findings.- A large distinct central bulb evidenced structurally, from the distal end of which numerous neuraxes can be traced distally. Certain of the neuraxes can be traced into the central end of the transplanted nerves; others pass to the connective tissue surrounding the transplanted nerves or found between them. In cross sections of the middle of the transplant the two nerves are clearly made out. The two nerves are found surrounded by a common fibrous tissue sheath. At this level no new or down-growing neuraxes made out within the perineural sheaths of the nerve funiculi. In many of the nerve fibers fragments of the old neuraxes may be seen. In the fibrous tissue surrounding the transplanted nerves, mainly to one side, there are to be found numerous neuraxes, singly or in small bundles, separated by fibrous tissue. In longitudinal sections of the distal wound region it is evident certain of the down-growing neuraxes have reached this region and may here be traced into the central end of the distal popliteal. These new neuraxes appear to reach this level mainly by way of the connective tissue found surrounding the transplant.
EXPERIMENT NO. 100.- Rabbit No. 70a; full grown; 34 days. April 19, 1918, right sciatic exposed and resected 1.4 cm. The two sciatics of a half-grown guinea pig used as a transplant. One central and distal silk suture placed. After suture, the two nerve segments transplanted found twisted one spiral turn; the central and distal approximation "fair."Wound closed. May 23, killed. Wound appeared well healed. On removing skin over wound area, a small focus of suppuration found in wound line; does not appear to extend to deeper wound. On exposing sciatic, tissue about nerve presents no evidence of infection. The transplant found well in place, surrounded by connective tissue and adherent to underlying muscle. No distinct bulbous enlargement of central sciatic noted. Sciatic and trans-plant removed and fixed in Flemming's chrom-osmic-acetic mixture. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-In longitudinal sections including the central and distal wounds, the transplant appears well united to resected nerve ends; fibrous tissue union. An indistinct central bulbous enlargement is found, from the distal end of which down-growing neuraxes in the form of nonmyelinated nerve fibers may be observed; these approach the central ends of the transplanted nerve segments. In cross sections of the transplant, the funicular arrangement of the transplanted nerves found well retained; surrounded by a common fibrous sheath, showing much round cell infiltration. The nerve fibers of the transplant appear to be appreciably enlarged, with neurolemma sheath distinct. Within these sheaths here and there globular remains of myelin. Phagocytic cells found within the neurolemma sheaths. The nerve fibers of the distal popliteal found degenerated.
EXPERIMENT NO. 101.- Rabbit No. 66; full grown; 50 days. April 17, 1918, left sciatic exposed and resected 1.3 cm. The two sciatics of a half-grown guinea pig used as a transplant. One central and distal No. 110 linen-thread suture placed. Fairly good approximation attained. Wound closed. June 6, killed. Rabbit good condition; no distinct neurotrophic changes of left hind foot. On exposing the left sciatic, transplants found well in place; surrounded by a common fibrous sheath; adherent to underlying muscle. A distinct bulbous enlargement on the central sciatic stump noted. Calf muscles fully exposed; these appear degenerated. On cutting nerve central to transplant, no contraction of calf muscles noted. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good differential silver staining attained.
Microscopic findings.-In longitudinal sections of the central wound region, a long spindle-shaped central bulb is recognized; line of union with central end of transplants clearly recognized by presence in the sections of central sutures. Numerous down-growing central neuraxes can be traced to the central end of the transplant, within which they are traced distalward for
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a distance of nearly 1 cm. Certain of these neuraxes are found in the remains of the old neurolemma sheaths, found in the detritus derived from the transplanted nerve fibers. In cross sections of the transplant, taken about 1 cm. distal to the central wound, new neuraxes are to be observed within several of the funiculi of the transplanted nerve segments, even in the most necrotic portions. In longitudinal sections of the distal wound region, a few of the down-growing neuraxis can be traced from the distal end of the transplant into the distal wound and from this a few scattered neuraxes to the central end of the distal internal popliteal.
EXPERIMENT NO. 102.- Rabbit No. 66a; full grown; 50 days. April 17, 1918, right sciatic exposed and resected 1.5 cm. The two sciatics of a half-grown guinea pig used as transplants. One central and distal No. 110 linen-thread suture placed; fairly good approximation. Wound closed. June 6, killed. Rabbit in good condition; scarcely any neurotrophic changes in right hind foot. On exposing the right sciatic, transplant found well in place; surrounded by connective tissue; only moderately adherent to the underlying muscle. Relatively dense fibrous tissue surrounds distal wound. Distinct bulbous enlargement noted on central sciatic stump. Calf muscles exposed; these have the appearance of degenerated muscle; do not contract on cutting nerve central to the transplant. Sciatic and transplant removed and fixed in Flemming's chromo-osmic-acetic mixture. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-In longitudinal sections of the central wound region a very large central bull is evidenced structurally, from the distal end of which numerous young nerve fibers, the majority of which are as yet nonmyelinated, are found to extend to the central end of the transplant, in which larger and smaller syncytial masses, irregular multinucleated giant cells, occupy the regions of the transplanted nerve fibers. In cross sections of the transplant the two nerve segments are recognized by their funicular arrangement and are surrounded by a common connective-tissue sheath. Within the perineural sheaths of the several funiculi, masses of large vesicular cells and irregular masses of syncytial protoplasm and granular detritus occupy the greater part of the cross-section area of each funiculus. In the fibrous tissue surrounding the transplanted nerve segments, especially to one side, there is observed an area in which some fifteen small funiculi of nerve fibers arc to be found. No new nerve fibers were traced to the distal sciatic. The nerves of the distal sciatic found degenerated.
EXPERIMENT No. 103.-Rabbit No. 68; large; full grown; 61 days. April 18, 1918, left sciatic exposed and resected 1.4 cm. The two sciatics of a nearly-grown guinea pig used as transplants. One central and distal No. 110 linen-thread suture placed; good approximation. Wound closed. June 18, rabbit found dead in the morning; severe neuro-trophic changes left hind foot. On exposing the left sciatic a large bulbous enlargement on the central sciatic stump is noted, from the distal end of which a fine strand, not quite the size of one of the sciatics used as transplant, leads to the distal sciatic stump. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Only in part good silver differentiation attained.
Microscopic findings.-In longitudinal sections of the central wound region a large spindle-shaped bulbous enlargement of end of central sciatic is evidenced structurally, from the distal end of which down-growing neuraxes may be traced to the central end of the transplant and the connective tissue surrounding the transplant. III cross sections distant about 1 cm. from central wound, the funicular structure of one of the transplanted nerves can be made out, with epineural sheaths thickened. Within the fibrous sheath areas of granular detritus and faintly outlined vesicular cells are noted. This mass occupies nearly the entire cross area of each funiculus. No down-growing neuraxes recognized in this detritus nor at this level in the surrounding fibrous tissue. The distal popliteal found completely degenerated.
EXPERIMENT No. 104.-Rabbit No. 68a; large; full grown; 61 days. April 18, 1918, right sciatic exposed and resected 1.5 cm. The two sciatics of a nearly-grown guinea pig used as transplants. One central and distal No. 110 linen-thread suture placed; good approximation. Wound closed. June 18, rabbit found dead in the morning; very severe neuro-trophic changes right heel. On exposing the right sciatic, transplant found well in place; the two nerves distinctly evident; of dull white color. No distinct central sciatic bulb
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noted. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Not well differentiated- not well embedded; sections torn.
Microscopic findings.-From well-developed central bulbous enlargement down-growing neuraxes can be traced in part into the central end of the transplant, the majority to the side of the transplant into the surrounding connective tissue. The remainder of the series of sections, especially those of the transplant, so badly torn, owing to faulty embedding, the resulting sections could not be used for critical study. The nerves of the distal sciatic stump found completely degenerated.
EXPERIMENT No. 105.- Rabbit No. 96; full grown; 69 days. September 6, 1918, left sciatic exposed and the internal popliteal freed and resected 2.5 cm. A segment of equal length taken from the left sciatic of a large guinea pig used as transplant. One central and distal waxed fine silk-thread suture placed; good approximation. Slight hemorrhage from central sciatic stump, not fully controlled. Wound closed. November 15, rabbit found dead in the morning; not well for several days; slight neurotrophic changes left heel. On exposing the left sciatic, the external popliteal found in close approximation to operated internal popliteal. Transplant found well in place and easily recognized by reason of its yellow-white color. Small spindle-shaped bulbous end central internal popliteal. Internal popliteal and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining, Only in part good silver staining attained; sections torn.
Microscopic findings.-In longitudinal sections of the central wound region certain down-growing neuraxes are found to enter the central end of the transplant; the majority are seen to pass to the side of the transplant into the surrounding connective tissue. In cross sections of the transplant it is seen that the funicular structure of the transplanted nerve is well retained, with epineural sheath thickened. In many of the old nerve fibers of the transplant remnants of the old neuraxes seen, both in cross and longitudinal sections. The neurolemma sheaths seem thickened and contain granular detritus. No new neuraxes traced to the distal wound. The nerves of the distal popliteal found degenerated.
EXPERIMENT No. 106.- Rabbit No. 96a; full grown; 69 days. September 6, 1918, right sciatic exposed, internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the right sciatic of a large full-grown guinea pig used as transplant. One central and distal waxed fine silk-thread suture placed; good approximation. Wound closed. November 15, rabbit found dead in the morning; not well for several days; severe neurotrophic changes right heel. On exposing the right sciatic, the central internal popliteal is found to end in a large bull to which the central end of the transplant is adherent. The transplant had pulled free from the distal popliteal; the distal suture is found in the free distal end of the transplant. Central and distal internal popliteal and the transplant removed and fixed in neutral formalin. The sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht-grün.
Microscopic findings.-In longitudinal sections of the central wound region, active downgrowth of small myelinated and nonmyelinated neuraxes pass to the side of the transplant and are lost in the surrounding connective tissue. In cross sections of the transplant, just distal to central wound, it is observed that the funicular structure of the nerve is well retained; with each funiculus surrounded by a perineural sheath. The nerve fibers within the perineural sheaths of larger diameter; remnants of neuraxes noted. Under low magnification the cross sections of the transplanted nerve resemble closely in general structure a normal nerve. The nerve fibers of the distal popliteal completely degenerated.
EXPERIMENT No. 107.- Rabbit No. 89; full grown; 87 days. August 30, 1918, left sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length, taken from the left sciatic of a large, full-grown guinea pig, used as a transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed November 26, rabbit found dead in the morning; much emaciated, on left heel neurotrophic ulcer. On exposing the left sciatic the transplant is found well in place, demarked by its light yellow color; no material increase of connective tissue about it. Distinct bulbous enlargement on central internal popliteal. Internal popliteal removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-In longitudinal sections of the central wound region, from the large central bulb, there may be traced many small myelinated and nonmyelinated nerve
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fibers to the central end of the nerve transplant. In cross and longitudinal sections of the transplanted nerve segment, it may be observed that the perineural sheath of the nerve is not materially thickened. Within this sheath large areas in which are found closely arranged large vesicular cells, with globular and granular inclusions, not Clearly defined. Other areas in which similar cells and granular detritus are found in what appear to be distended neurolemma sheaths. Within the perineural sheath, mainly to one side, many small funiculi of nerve fibers may be observed. No new nerve fibers traced to and through the distal wound. The nerve fibers of the distal popliteal stump found completely degenerated.
EXPERIMENT No. 108.-Rabbit No. 89a; full grown; 87 days. August 30, 1918, right sciatic exposed; internal popliteal freed and resected 3 cm. A segment of equal length, taken from the right sciatic of a large, full-grown guinea pig, used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. November 26, rabbit found dead in the morning; much emaciated; severe neutrophic ulcer right heel. On exposing the right sciatic, transplant is found well in place; no material increase of connective tissue and only moderately adherent to underlying muscle. Distinct central bulbous enlargement found. Internal popliteal and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings.-In longitudinal sections of the central wound region, and series of sections at successive levels, the transplant is clearly demarked by reason of its jet-black staining. Down-growing central neuraxes can be seen to penetrate the central end of the transplant, in which they can be traced distally until the jet black, nontransparent colorations reached; here they are lost to view. Certain of the central neuraxes pass to the side of the transplant, coursing distalward in the surrounding connective tissue. In cross sections of the transplant, the perineural sheaths are not found materially thickened. Within the sheaths the remains of the transplanted nerves so deeply stained-jet black-that no structural details can be determined, and it can not be ascertained whether central neuraxes have reached this level. fn longitudinal sections of the distal wound region, a few neuraxes can be traced from the distal end of the transplant to the distal wound; others appear to reach the wound region from the surrounding connective tissue. Certain few neuraxes have reached the central end of the distal popliteal in which they have grown for a distance approximating 1 cm.
EXPERIMENT No. 109.-Rabbit No. 93; full grown; 96 days. September 4, 1918, left sciatic exposed; internal popliteal bundle freed; resected 2.5 cm. A segment of equal length, taken from the left sciatic of a full-grown guinea pig, used as a transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. December 10, killed. Rabbit moribund; breathing when killed; much emaciated, severe neurotrophic ulcer left heel. On exposing the left sciatic, the external popliteal is found closely adherent to operated internal popliteal; dissected free without cutting perineural sheath. Large spindle-shaped bulb on internal popliteal noted. The transplant is found well in place, but of small size; only about one-half the size as when used. Transplant presents several short stretches of yellow-white color. Calf muscles exposed; these are atrophic and of yellow-red color and do not contract nor show twitching on cutting nerve. Internal popliteal and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fair silver differentiation attained.
Microscopic findings.- In longitudinal sections of the central wound region, a distinct central bulbous enlargement is evidenced structurally, this included the central end of the transplant, recognized as a granular mass of detritus and faintly stained vesicular cells. Central neuraxes may be traced mainly to the side of this mass into the connective tissue surrounding the transplant. In cross sections of the transplant, about its mid region, a very material thickening of its fibrous sheath is noted. Within this fibrous sheath there are found numerous small bundles of neuraxes, separated by bands of fibrous tissue. The necrotic remains of the transplanted nerve found to one side. In longitudinal sections of the distal wound region, certain neuraxes coming from the distal end of the transplant, more numerous from the connective tissue surrounding the transplant, can be traced through the distal wound into the distal popliteal nerve, in which they are found, scattered through the several fulliculi to the lower level of the popliteal space.
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EXPERIMENT NO. 110.- Rabbit No. 93a; full grown; 96 days. September 4, 1918, right sciatic exposed; internal popliteal freed; resected 2.2 emu . A segment of equal length, taken from the right sciatic of a full-grown guinea pig, used as a transplant. One central and distal waxed, fine silk thread suture passed; good approximation. Wound closed. December 10, killed. Moribund; just breathing; much emaciated; severe neurotrophic ulcer right heel. On exposing the right sciatic, the external popliteal found free. Transplanted nerve segment found well in place, of small diameter, and shows several short stretch of yellow-white color. Large central bulb noted. Calf muscles exposed; these are atrophic and of pale yellow-red color. No contraction or twitching of muscles observed on cutting the nerve. Internal popliteal and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Only in part good differential silver staining attained.
Microscopic findings.-In longitudinal sections of the central wound region, a distinct central bulb evidenced structurally from the distal end of which down-growing neuraxes maybe traced to the central end of the transplant and to the connective tissue by the side of the transplant. The series of cross sections of the transplant, torn and found not well differentiated. To one side there may be made out the necrotic remains of the transplanted nerves, occupying about one-half of the cross section area of the transplant, the remaining half consists largely of dense fibrous tissue in which small bundles of neuraxes are observed this portion of the section is fragmented, so that relations are difficult to make out. In longitudinal sections of the distal wound region, new neuraxes in small numbers are seen to pass through the distal wound and to enter the central end of the distal popliteal, in which they extend for a distance of about 1 cm. beyond the distal wound.
EXPERIMENT No. 111.-Rabbit No. 87; not quite full grown; 99 days. August 28,1918, left sciatic exposed; internal popliteal bundle freed; resected 2.8 cm. A segment of equal length taken from the left sciatic of a large, full-grown guinea pig, used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. December 6, rabbit found dead in the morning; mulch emaciated; severe neuro-trophic ulcer left heel; an encapsuled, sausage-shaped, cold abscess over left tendo Achillis. On exposing the left sciatic, external popliteal found free. Transplant found well in place, throughout of light yellow color, which clearly demarks it; small diameter. Large spindle-shaped central bulb. Calf muscles are atrophic and present the appearance of degenerated muscle. Internal popliteal and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation attained.
Microscopic findings.-In longitudinal sections of the central wound region, a large central bulb is recognized structurally, in the distal end of this a necrotic area, stained jet-black, interpreted as the central end of the transplant, by the side of this area numerous neuraxes grow distalward. In cross sections of the transplant in its mid region, the perineural sheath found materially thickened; to one side and within the fibrous sheath, a deeply stained black mass is found occupying about one-half of the cross area of the transplant and representing the necrotic remains of the transplanted nerves. To the other side, also within the fibrous sheath, there are found small groups of neuraxes, separated by strands of fibrous tissue; a few new neuraxes are to be observed in the region of the distal wound. These may be traced to the distal popliteal in which they are followed to the lower level of the popliteal space; the remainder of the distal popliteal found completely degenerated.
EXPERIMENT No. 112.-Rabbit No. 87a; not quite full grown; 99 days. August 28,1918, right sciatic exposed; internal popliteal freed; resected 2.0 cm. A segment of equal length taken from the right sciatic of a full-grown guinea pig, used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. December 6, found dead in the morning; much emaciated; severe neurotrophic ulcer right heel. On exposing the right sciatic, external popliteal bundle found free. The transplant found well in place, of light yellow color; On it or in it there may be traced a fine nerve bundle. Large spindle-shaped central bulb noted. Both central and distal sutures clearly evident. Calf muscles atrophic. The internal popliteal and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fair silver differentiation attained.
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Microscopic findings.- In longitudinal sections of the central wound region, a large central bulb evidenced structurally, from the distal end of which new neuraxes may be traced to the central end of the transplant. In cross sections of the transplant, the perineural sheath found very materially thickened and blended with the surrounding connective tissue. Within this connective tissue sheath small bundles of neuraxes, separated by strands of fibrous tissue, arc to be found; certain small funiculi of nerve fibers seen in the surrounding connective tissue. New neuraxes in relatively small numbers found in the region of the distal wound, and in the distal internal popliteal just distal. In the posterior tibial continuation only degenerated nerve fibers found.
EXPERIMENT No. 113.-Rabbit No. 92; large; full grown; 104 days. September 3. 1918, left sciatic exposed; internal popliteal freed and resected 2.2 cm. A segment of equal length taken from the left sciatic of a full-grown guinea pig, used as transplant; good approximation. Wound closed. December 17, killed. Rabbit much emaciated; snuffles; neurotrophic ulcer on left heel. On exposing the left sciatic, external popliteal found only loosely adherent to the operated internal popliteal. On internal popliteal large central bulb is noted. Transplant found well in place, distal two-thirds of light yellow color, with several glistening white streaks. Central end of distal popliteal found distinctly enlarged. Calf muscles found atrophic, do not respond on cutting nerve centrally. Internal popliteal and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Imperfect silver differentiation attained.
Microscopic findings.-In series of longitudinal and cross sections taken at successive levels, the transplant clearly demarked by reason of its jet-black, nontransparent staining. The neuraxes of the central stump not clearly differentiated and for the remainder of the series not differentiated.
EXPERIMENT No. 114.-Rabbit No. 92a; large; full grown; 104 days. September 3, 1918, right sciatic exposed and internal popliteal freed; resected 2.0 cm. A segment of equal length taken from the right sciatic of a full-grown guinea pig, used as a transplant. One central and distal waxed, silk thread suture placed; central approximation good; distal suture pulled out; an epineural stitch made. Wound closed. December 17, killed. Rabbit much emaciated; snuffles; neurotrophic ulcer on right heel. On exposing right sciatic, external popliteal bound only loosely to operated internal popliteal. The internal popliteal presents a large central bull. The transplant found well in place; of small diameter and in the main of light yellow color. Several small bundles of nerve appear to run on or in the trans- plant to reach the distal popliteal. Calf muscles atrophic; do not respond on cutting the nerve centrally. Internal popliteal and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Imperfect silver differentiation attained; sections light yellow color, with neuraxes not differentiated.
Microscopic findings.-The results of this experiment can not be clearly determined from study of sections. In cross sections of the transplant, small areas, which are quite certainly small funiculi of nerve fibers, found in the fibrous tissue to one side of the transplant; the region of the nerve fibers of the transplant only necrotic tissue made out. No ne1uraxesdifferentiated in the distal popliteal.
EXPERIMENT No. 115.-Rabbit No. 91; large; full grown; 191 days. September 2, 191s,left sciatic exposed; internal popliteal freed; resected 2.6 cm. A segment of equal length take from a large full-grown guinea pig, 12 minutes after it stopped breathing, used as trans-plant. One central and distal waxed, fine silk thread suture passed; good approximation. Wound closed. March 12, 1919, killed. Rabbit in good condition; slightly emaciated; old neurotrophic ulcer on left heel nearly healed. A large, encapsuled, cold abscess found over tendo Achillis; this does not involve deeper tissues. On exposing the left sciatic the external popliteal found free; cutting of the nerve does not cause contraction of the muscles supplied by it. The operated internal popliteal bundle presents a large central bulb; transplant found well in place but of small diameter. Calf muscles found atrophic and do not respond on cutting nerve central to the transplant. Internal popliteal removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation attained.
Microscopic findings.-In longitudinal sections of the central wound region, long spindle-shaped bulb evidenced structurally from the distal end of which numerous down-growing
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neuraxes are found to enter the central end of the transplant. In cross sections of the transplant the fibrous tissue sheaths of the nerves transplanted are found materially thickened. Numerous new neuraxes are found within the transplant, in the form of very small bundles separated by fibrous tissue. Only relatively few nerve fibers or neuraxes found in the surrounding fibrous tissue. Within the transplant only here and there necrotic remains of the transplanted nerve fibers found. Down-growing neuraxes traced to the distal wound and into the distal popliteal, in which they are found in good numbers at the lower level of the popliteal, the extent of the sections cut. The calf muscles were not studied in this experiment.
EXPERIMENT No. 116.- Rabbit No. 91a; large; fill grown; 191 days. September 2,1918, right sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length taken from the right sciatic of a large guinea pig, which had stopped breathing 45 minutes previous, was used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. March 12, 1919, killed. Rabbit in good condition; old neurotrophic ulcer on right heel nearly healed. On exposing right sciatic, external popliteal found free. Large central bulb noted on the operated internal popliteal. Transplant found well in place, of good size, and presenting the appearance of a small nerve bundle. The distal internal popliteal presents the appearance of a normal nerve. External popliteal cut and resected and internal popliteal freed from bed. Calf muscles exposed. On slowly cutting with scissors operated nerve central to the transplant, vigorous contraction of the calf muscles observed; same on cutting distal to the transplant. The internal popliteal and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining; portions of the calf muscles removed for gold chloride staining. Fair differential silver staining attained.
Microscopic findings.- In longitudinal sections of the central wound region numerous neuraxes are observed to extend from the distal end of a large central bulb to the central end of the transplant. In cross sections of the transplant, in its mid region, the fibrous tissue sheaths of the nerve transplanted are found very materially thickened. Within the fibrous tissue sheaths numerous new neuraxes are found in the form of small bundles, separated by connective tissue. In the distal end of the central bulb and at several levels in the transplant areas of granular detritus, vesicular cells with globular and granular inclusions are to be found; remains of the transplanted nerves. New neuraxes traced to and through the distal wound into the central end of the distal popliteal. In gold chloride stained pieces of calf muscles numerous new neuraxes are found in the larger nerve bundles of the muscle and followed into the smaller interfascicular branches, but motor end plates were not found differentiated; teased muscle fibers presented normal appearance.
EXPERIMENT No. 117.- Rabbit No. 90; full grown; 194 days. August 30, 1918, left sciatic exposed; internal popliteal freed; resected 2.5 cm. A segment of equal length, taken from the left sciatic of a large guinea pig, which had stopped breathing 12 minutes previously, used as a transplant. One central and distal waxed, fine silk thread suture placed; centrally good approximation; distally "fair." Wound closed. March 12, 1919, killed. Rabbit in good condition; severe neurotrophic ulcer left heel, which appears to be healing. On exposing the left sciatic, external popliteal found free. Operated internal popliteal presents a large central bulb. Transplant in place as a fine strand extending from central bulb to the distal popliteal. Distal popliteal does not appear degenerated. On exposing the calf muscles these are found atrophic and of light yellow-red color. External popliteal removed and internal popliteal and transplant freed from bed. On slowly cutting with scissors nerve central to the transplant feeble to distinct contraction of the calf muscles observed. Internal popliteal and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained; sections somewhat torn.
Microscopic findings.- In longitudinal sections of the central wound region including the central end of the transplant numerous new neuraxes can be traced from the central bulb into the transplant and into the surrounding tissue. In cross sections of the transplant the transplanted nerve segment is recognized by its distinctly thickened fibrous sheaths, within which numerous new neuraxes may be seen. Scarcely any necrotic remains of the transplanted nerve observed. In the fibrous tissue surrounding the transplanted nerve segment there are to be seen many small funiculi of nerve fibers. Down-growing neuraxes can be traced through
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the central wound into the central end of the distal popliteal, in which they are found in relatively large numbers in its several funiculi.
EXPERIMENT No. 118.- Rabbit No. 90a; full grown; 194 days. August 30, 1918, right sciatic exposed; the internal popliteal freed; resected 2.3 cm. A segment of equal l length taken from the right sciatic of a full grown guinea pig, which stopped breathing 32 minutes previous, used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. March 12, 1919, killed. Rabbit in good condition; severe neurotrophic ulcer right heel. On exposing the right sciatic external popliteal found free. The operated internal popliteal presents a large central bulb. The transplant found in place; its distal half of light brown color. Calf muscles atrophic. Cutting of nerve central to transplant causes no contraction of the calf muscles. Internal popliteal and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good silver differentiation attained.
Microscopic findings.- In longitudinal sections of the central wound region, including the central end of the transplant, numerous neuraxes can be traced from the central bulb to the central end of the transplant and surrounding connective tissue. In the distal end of the central bulb, the neuraxes are found to cross and intercross central wound region. In cross sections of the transplant, about 1 cm. distal to central wound, the perineural sheaths of the transplanted nerve segments evident and not materially thickened; within these sheaths numerous neuraxes are observed. Outside of the perineural sheaths an area of connective tissue is observed in which there are found numerous small funiculi of nerve fibers. Down- growing neuraxes can be traced through the transplant and from the surrounding connective tissue to the distal wound and through this to the distal popliteal stump in which, in the several funiculi, they are found in relatively large numbers.
EXPERIMENT No. 119.- Rabbit No. 95; full grown; 277 days. September 6, 1918, the left sciatic exposed; internal popliteal freed; resected 2.7 cm. A segment of equal length taken from the left sciatic of a full-grown guinea pig, used as a transplant. One central and distal waxed, fine silk thread suture placed; central approximation good; distal approximation, nerve ends not in good alignment. Wound closed. June 10, 1919, rabbit found dead in the morning; very much emaciated; severe neurotrophic ulcer left heel. On exposing the left sciatic the operated internal popliteal is found to end in a large central bulb, from the distal end of which no transplant nor nerve bundles can be traced to the distal popliteal, the central end of which ends free and presents an S-shaped curve. It would appear that the central suture gave way soon after the operation and that the transplanted nerve segment had completely disappeared. Calf muscles found atrophic and distal internal popliteal completely degenerated. Central bulb and distal internal popliteal removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver staining attained.
Microscopic findings.- In longitudinal sections of the central bulb this is found to include the central end of the transplant, consisting of necrotic detritus. Active down-growth of central neuraxes is evident; these are lost in the surrounding connective tissue In the distal popliteal the nerve fibers found completely degenerated.
EXPERIMENT No. 120.-Rabbit No. 95a; full grown; 277 days. September 6, 1918, p right sciatic exposed; internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the sciatic of a large, full-grown guinea pig, used as transplant. One central and distal waxed fine silk thread suture placed; very good approximation. Wound closed. June 10, 1919, rabbit found dead in the morning; very much emaciated; severe neurotrophic ulcer right heel. On exposing the right sciatic, the operated internal popliteal found to end in a large spindle-shaped bulb; the transplant well in place and of good size. Conditions of calf muscles not recorded. Internal popliteal and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fair silver differentiation attained.
Microscopic findings.-In longitudinal sections of the central wound region a large central bulb, with characteristic structure of neuroma, including spiral neuraxes, evidenced structurally. In distal end of bulb, the necrotic remains of the central end of the transplant are found. Numerous down-growing neuraxes pass by the side of this necrotic area and extend distalward in the connective tissue. In the several successive series of sections the greater part of the transplanted nerve fibers, or the remains of the same, stained jet-
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black in the silver. In cross sections of the transplant taken from its mild region small fuiniculi of nerve fibers are found in the connective tissue surrounding the transplant. In the region of the distal wound and in sections of the central end of the distal popliteal only a few neuraxes are to be observed; the greater part of the distal popliteal showing degenerated nerve fibers.
EXPERIMENT NO. 121.- Rabbit No. 94; large Belgian hare; 358 days. September 5, 1918, left sciatic exposed; internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the left sciatic of a large guinea pig and used as transplant. One central and distal waxed fine silk thread suture passed. Centrally "fair" approximation attained; distally good alignment, but cut nerve ends not quite end to end. Wound closed. August 28, 1919, rabbit in good condition; still large neurotrophic ulcer left heel; appears to be healing; spreads toes of left hind foot when held up by ears. On exposing the left sciatic external popliteal is found in close apposition to the operated internal popliteal; adherent to it. Large spindle-shaped central bulb on the operated internal popliteal. Transplant found well in place and presents the appearance of a normal nerve. Calf muscles exposed and external popliteal cut at the level of head of fibula; internal popliteal and transplant freed. On slowly cutting with scissors, central sciatic, good contraction of calf muscles and less vigorous contraction of the plantar foot muscles observed. Calf muscles found of nearly normal size and of pale red color streaked with yellow. The internal popliteal and transplant, portions of calf and foot muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Quite good differential silver staining attained.
Microscopic findings.-In longitudinal sections of the central wound region, from the distal end of the large central bulb, certain down-growing neuraxes can be traced into the central end of the nerve transplant, the majority into the connective tissue surrounding the transplant. In the connective tissue the small bundles of nerve fibers present a very serpentine course. In cross sections of the transplant, taken about 1 cm. distal to the central wound, numerous small funiculi of nerve fibers are found in the connective tissue surrounding the transplant, outside of the perineural sheaths. Within the transplant crosscut neuraxes, differentially stained, are found in good numbers, separated by strands of connective tissue. Only small remnants of the necrotic remains of the transplanted nerve fibers found within the transplant. Down-growing neuraxes can be traced to and through the distal wound, into the distal popliteal, in which they are found in good numbers in all of the funiculi. In sections of the calf muscles new neuraxes are found in the larger and smaller intramuscular nerve branches and as single fibers between and on the muscle fibers.
EXPERIMENT No. 122.- Rabbit No. 94a; large Belgian hare; 358 days. September 5, 1918, right sciatic exposed; internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the right sciatic of a large, full-grown guinea pig, used as transplant. One central and distal waxed silk thread suture placed; good approximation. Wound closed August 28, 1919, killed. Rabbit in very good condition; still large neurotrophic ulcer right heel, which appears to be healing. On exposing the right sciatic, the external popliteal is found free. A long spindle-shaped bulb found on the central internal popliteal. Transplant found well in place, of small diameter but presents the appearance of a normal nerve. Distal popliteal; looks like a normal nerve. Calf muscles exposed; very nearly of normal size and of pale red color streaked with light yellow. After cutting and resecting external popliteal and freeing the operated internal popliteal from bed, on slowly cutting nerve with scissors, central to the transplant, distinct but feeble contractions in the calf muscles noted; foot muscles uncertain. Functional test 20 minutes after the animal was killed. Internal popliteal and transplant and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.-Only in part good silver differentiation attained; in part fine granular silver deposit in sections. In longitudinal sections of the central wound region large numbers of down-growing neuraxes can be traced from the distal end of the central bulb to the central end of the transplant and the connective tissue surrounding the transplant; the latter in the form of numerous small funiculi, very much coiled and twisted; those entering the transplant follow a more regular longitudinal course after having passed the central
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wound region. In cross sections of the transplant, about 1 cm. distal to the central wound, it may be observed that new neuraxes are found both within and without the perineural sheaths of the nerve segment transplanted. New neuraxes can be traced through the distal wound into the distal popliteal, in which they arc found in good numbers in all of the fulliculi. In the sections made from the calf muscles neuraxes are found in the larger fasictilar nerve bundles and here and there as single nerve fibers on the muscle fibers. The muscle fibers so far as can be determined in silver stained preparations appear to present normal structure; here and there areas or columns of fat cells within the muscle.
EXPERIMENT NO. 123.- Rabbit No. 78a; large; full grown; 7 days. June 3, 1918, right sciatic exposed; fascial plane not readily found, consequence muscle torn; sciatic resected 2.5 cm. A segment of equal length taken from the right external popliteal of a full-grown dog; nerve resected two hours previous; nerve segment lying in the wound used as transplant. One central and distal waxed fine silk thread suture placed; good approximation. Ultimately dry field; wound closed. June 10, rabbit found dead in the morning; superficial wound healed; deep wound congested. In deep wound near distal suture a small hematoma in the connective tissue. Transplant found well in place; united to the resected nerve ends; central and distal sutures distinct. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Faint but differential silver staining attained.
Microscopic findings.- Transplant united to central sciatic only at one border; for the remainder of the cross section separated by an appreciable distance. Distally good fibrous union obtained. In longitudinal sections of the transplant the neuraxes of the transplanted nerve fibers found fragmented into relatively long segments, having a wavy or spiral course, and stained differentially in the silver stain. The neurolemma sheaths seem well maintained, the perineural sheaths not appreciably thickened. In the distal end of the central sciatic stump early stages of the downgrowth of the central neuraxes evident. The nerve fibers of the distal sciatic present early stages of degeneration.
EXPERIMENT No. 124.- Rabbit No. 75a; full grown, 13 days. May 10, 1918, right sciatic exposed and resected 2.5 cm. A segment of equal length, taken from the left internal popliteal of a dog, used as transplant. One central and distal silk-thread suture placed; good approximation. Muscle stitched over nerve. Wound closed. May 23, rabbit found dead in the morning; "snuffles;" wound well healed. On exposing the right sciatic, transplant found well in place; appears of slightly greater diameter than when used; found well united to the resected nerve ends. Transplant surrounded by newly formed connective tissue. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential neuraxis staining attained.
Microscopic findings.-In longitudinal sections of the central and distal wound regions, ends of transplant found well united to the resected nerve ends. In longitudinal sections of the transplanted nerve segment, the neuraxes of the old nerve fibers are found segmented into longer and shorter segments, staining differentially in the silver stain; the neurolemma sheaths found well preserved; no distinct evidence of the proliferation of the sheath cells. Structural evidence of the beginning of a central bulb, from the distal end of which many down-growing neuraxes, terminating in bulbous end-discs, can be traced through the central wound for a short distance into the central end of the transplant. In certain of the neurolemma sheaths, near the central wound, remnants of old neuraxes and down-grown new neuraxes are to be found side by side. The fibers of the distal sciatic found in process of degeneration; many nucleated syncytial protoplasmic bands are seen, with proliferation of sheath cells.
EXPERIMENT No. 125.- Rabbit No. 76a; large; full grown; 52 days. May 17, 1918,right sciatic exposed and resected 2 cm. A segment of equal length, taken from the left ulnar of a dog, used as a transplant. One central and distal silk-thread suture passed; good approximation. Wound closed. July 8, rabbit found dead in the morning; wound well healed. On exposing the right sciatic, transplant was found to be well in place, firmly united to the resected sciatic stumps; adherent to the underlying muscle. Transplant of yellow-white color but seems of good consistency. No well-marked central bulb noted. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
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Microscopic findings.-In the successive series of longitudinal and cross sections, the transplanted nerve segment is clearly demarked by reason of a jet-black nontransparent coloring, the connective tissue sheaths being stained a yellow-brown color. Within the transplant, here and there fragments of neuraxes are found within the neurolemma sheaths. In cross sections of the transplant, wherever the dark coloring admits of the observation, in nearly every neurolemma sheath there may be observed the cut end of the old neuraxis. In longitudinal sections of the central wound region, clown-growing neuraxes, traced from the distal end of the central bulb, may be seen passing by the side of the nerve transplant into the surrounding connective tissue, and in longitudinal sections of the transplant, new neuraxes are found in the fibrous tissue between the funiculi. No new neuraxes are found in the region of the distal wound. The nerve fibers of the distal sciatic are found completely degenerated.
It is evident from a study of the protocols of the experiments on hetero-nerve transplant, that on the face of the results attained the statement iswarranted that a hetero-nerve transplant may be used to bridge a nerve defect with probability of success, to the extent that certain of the down-growing central neuraxes will penetrate the central end of the transplant and through it reach the distal segment. In all of the experiments of this series, kept for more than three months (No. 109 to No. 122) after the operation, the down-growing neuraxes derived from the distal end of the central stump could be traced into the central end of the transplant, to the distal wound region, and thence to the distal sciatic stump. There seems no question that a certain number of down-growing neuraxes, the number varying in the different experiments, reach the distal sciatic segment through the hetero-nerve transplant. Having established this general conclusion, the protocols of the experiments of long duration may be studied more critically, and it will be found that in nearly every record of microscopic findings it is noted that the down-growing neuraxes derived from the central stump not only penetrate the central end of the trans-plant but at the central wound region pass into the connective tissue surrounding the transplant and in close contiguity to it reach the distal wound and, perchance, enter the distal sciatic stump. These extrafunicular nerve bundles are most easily determined in cross sections of the transplant and surrounding connective tissue. In such sections, the funicular structure of the nerve transplanted is usually readily made out, even months after the operation, and the perineural sheaths surrounding the funiculi are evident. In properly stained sections, with neuraxis differentiation, extra funicular nerve fibers, singly or in small bundles, are easily determined. Such extrafunicular fiber bundles usually have an irregular serpentine course, as though winding their way through the interstices of the connective tissue. It may also be noted that these extrafunicular nerve fiber bundles are on the whole much more numerous than is the case when auto- or homo-nerve transplants are used, and mav include a relatively large per cent of the central nerve fibers reaching the distal wound and the distal nerve segment. It can also be shown that the rate of regeneration appears much slower when a hetero-nerve transplant is used than when using an auto- or homo-nerve transplant. Further, that the results are not so satisfactory, taking as an index the number of neuraxes which reach the distal stump through the hetero-nerve transplant, as when auto-or homo-nerve transplants are used. However, the hetero-nerve transplant does not become necrotic as is stated by certain observers. If properly sutured to the resected
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nerve ends, the ends of the hetero-nerve transplant quickly form fibrous union with the resected nerve ends and become surrounded by newly formed connective tissue. In experiments terminated from six to twelve months after the operation, there is usually present a prominent central nerve bulb which includes the central wound region. In the region of the transplant the nerve bundle has the appearance of a living nerve, though of smaller diameter than when the nerve was transplanted. The hetero-nerve transplant has not disappeared, since months after the transplant was placed its funicular structure can be made out.
In the earlier stages of experimental operative work on nerve bridging nospecial consideration was given to the relative value of auto-, homo-, and hetero-nerve transplants; the need of making such differentiation was not recognized. As early as 1869, Philippeaux and Fulpian used a lingual nerve to bridge the resected hypoglossal nerve of dogs; auto-nerve transplant. These experiments were followed by others in which auto-, homo-, and hetero-nerve transplants were used, generally with indifferent or unfavorable results. Huber, in 1895, reported on a series of 26 experiments of nerve transplantation. Of this number in 10 of the experiments the animals were kept for a period of four months or more before the operated nerve was tested functionally and the nerve removed for examination. In five of these, all hetero-nerve transplants (cat's sciatic to resected ulnar of dog), the results were very satisfactory; in four others the down-growing central neuraxes had passed the region of the transplant and entered the distal nerve segment. With the microscopic methods available then, such precise neuraxis differentiation could not be had as now and it was not determined whether all of the down-growing neuraxes passed to the distal stump through the funiculi of the transplant or extra-funicular in the surrounding connective tissue. In an unsigned statement, found in the "Medical Supplement, Daily Review of Foreign Press," London, October 1, 1918, giving a review of the treatment of gun-shot injuries of nerves in Germany to the middle of 1917, the following statement appears:
When a gap between the two divided nerve ends can not be obliterated, the unsettled question as to the regeneration of nerves has to be taken into consideration. The dominant view before the war was that regeneration in the peripheral segment was due entirely to the down growth of axis-cylinders from the proximal segment. But this theory does not seem to receive much confirmation. The regeneration of potential nerve fibers in the still separated distal segment, as described by Ballance and Purves Stewart, has found supporters, the potential nerve fibers becoming linked up with axis cylinders in the proximal segment when the two ends are brought together * * *. The second view that the distal segment regenerates so far affords encouragement to the method of inserting into the gap a nerve graft, which shall serve, as it were, to prolong the peripheral segment to meet the proximal end. Then the axis cylinders in the proximal segment, without growing out more than is seen incase of nerve end bulb, can become connected by a series of links with the potentially regenerated nerve fibers, and then these become fully developed. This nerve grafting to fill gaps gains support from animal experimentation and appears to be the plan which should be adopted in surgery.
The experimental observations on nerve transplantation furnish the most conclusive evidence for the monogenetic ol downgrowth theory of nerve regeneration. In none of the experiments recorded under Series No. 5, No. 6,and No. 7 is there found any evidence in support of auto-regeneration of the
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peripheral stump. In suitably stained pyridine-silver preparations of a nerve bridged by a nerve transplant and removed for study at the right time, in a serial order at progressive stated intervals, it can be seen that the budding central neuraxes grow to the region of the central wound and step for step into the transplant, through the transplant into and through the distal wound and into the distal segment, and in this progressively until the end organs are reached. As a result of the observations accumulated in this series of experiments, there is found abundant warrant for stating that the nerve bridge or nerve transplant offers a suitable path for down-growing central neuraxes and that regeneration of the distal segment after nerve bridging is only through down-growing central neuraxes.
In more recent experimental observations and in the more modern surgical work more precise recognition has been given to the relative merits of the auto-, homo-, and hetero-nerve transplants as with other tissue grafts. As a result of experimental observations, Forssman 70 and a little later Morzbacher,71 whose results were confirmed by Segale,72 were the first to suggest that there were important differences between homo- and hetero-nerve transplants. As a result of their observations it was concluded that in auto- and homo-nerve transplants the transplanted nerves survived and were capable of undergoing degenerative changes while a hetero-nerve transplant was subject to a necrobiotic process owing to the fact that it did not survive in the host. On the other hand, Maccabruni 73 working in the laboratory of Golgi, found that there was little difference in the behavior of auto-, homo-, or heterogenous nerve segments transplanted into connective tissue or intermuscular septa, the axial portion of each becoming necrotic while the more peripheral portions, subject to better nutrition, presented the phenomena of nerve degeneration, even proliferation of sheath cells. Ingebrigtsen has considered this question in a number of contributions. In the account of 1915, 74 the following statement appears: "In the problem of transplantation of nerves the question of the fate and survival and multiplication of the cells of Schwann is of importance. The solution of this point, which is the only reliable sign of the survival of the transplanted piece, gives the key to the problem and will influence the procedure of surgeons in cases of nerve defects. If the grafts die and become necrotic they are no more suitable for bridges than strands of catgut." Writing in 1916, Ingebrigtsen 75 states that Wallerian degeneration occurs in auto- and homo-nerve transplants in the same manner as in the peripheral end of a cut nerve, except that the various changes take place somewhat slowly, while in hetero-transplants there is no Wallerian degeneration and no proliferation of sheath cells and 12 to 15 days after transplantation the nerve becomes necrotic and on histologic examination of the later stages no new neuraxes were found in the heterogenous transplant. After more extended study and in a comprehensive monograph (1918) 43 Ingebrigtsen had broadened his viewpoint, as may be noticed from the following quotation which is presented in this quite literal translation:
And we come then to the conclusion that the cells of the sheath of Schwann of the auto-and homo-nerve transplants are without biological significance whatever for the regeneration of the new neuro-fibrils of the transplant, which grow into the transplant from the central stump whether the transplant is living or dead.
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In the extended series of operations on nerve transplants, included in Series No. 5, No. 6, and No. 7, auto-, homo-, and lietero-nerve transplants, primary consideration was given to the downgrowth of central neuraxes in regeneration and their relation to the transplanted nerve fibers, and for this purpose the pyridine-silver neuraxis differentiation method was largely used. This method is not suitable for a detailed study of the myelin fragmentation nor the behavior of the sheath cells of the transplanted nerves. The evidence at hand warrants the conclusion that none of the transplanted nerve fibers, whether of auto-, homo-, or heterogenous source, undergo typical Wallerian degeneration, if sheath cell proliferation is to be considered a sine qua non of Wallerian degeneration. Further, the conviction has been gained that the sheath cells of the transplant play a very subsidiary and a negligible r ole as concerns regeneration through a nerve transplant. (Series No. I1, No. 12,and No. 13 seem to demonstrate this conclusively.) That there is a (difference in the behavior of auto-and homo-nerve transplants on the one hand, and hetero-nerve transplant on the other there can be no question. However, one can not accept the statement that heteroplastic nerve transplants become necrotic. Months after such a transplant has been placed, can its funicular structure be determined, with funiculi surrounded by perineural sheaths? That regeneration may take place through a hetero-nerve transplant the earlier observations of Huber (1895) 30 may serve to show, as also certain of the experiments of longer duration of Series No. 7. In preparations made from this series the neuraxes were differentially stained by the pyridine-silver method and in successful preparations stained by this method there is no difficulty in determining neuraxes. The results obtained as regards regeneration of the peripheral segment are not nearly so favorable on use of the heterogenous transplant as when auto- and homo-nerve transplants are used. However, this would seem to be due not so much to a difference in the mode of fragmentation of the myelin and a want of sheath cell proliferation but to a relatively retarded and at times imperfect phagocytosis of the products of myelin fragmentation, leaving the neurolemma tubes less suitable for neuraxis downgrowth than when homo- or auto-nerve transplants are used. The answer to the question of chemotactic or want of chemotactic action of the products of nerve degeneration and sheath cell proliferation can not now be given, since sufficient, and conclusive experimental evidence is not now at hand. Conditions being approximately equal as concerns operation, relative size of nerve and sutures, the extrafunicular nerve fibers coming from the central stump and passing into the connective tissue surrounding the transplant are much more numerous when heterogenous transplants are used than with autogenous or homogenous transplants. This is interpreted as an index that the latter are more favorable than the former for downgrowth of neuraxes. Thus, while the regeneration of the distal segment of a resected nerve can be obtained through a heterogenous nerve bridge in experimental work, the outcome is less certain and less satisfactory and it requires a longer time than when auto-or homo-nerve transplants are used. Thus, hetero-nerve transplantation is not recommended as an operation in the repair of human nerves after loss of nerve substance.
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DEGENERATED NERVE TRANSPLANTS
SERIES NO. 8, NO. 9, AND NO. 10
DEGENERATED AUTO-, HOMO-, AND HETERO-NERVE TRANSPLANTS
In Series No. 8, No. 9, and No. 10, including degnerated auto-, homo-,and hetero-nerve transplants, the nerve segment selected for the transplant was taken from a nerve which had been caused to undergo Wallerian degeneration as a result of nerve section some weeks before the nerve segment was used as a nerve bridge. This series of experiments was undertaken to test a number of hypotheses relative to nerve transplants. It was conjectured that since a transplanted nerve segment degenerates after transplantation, the process of regeneration through a nerve transplant might be facilitated by using a nerve segment already degenerated to the extent of presentingthe nucleated syncytial strands (" band fasern ") in the neurolemina sheaths. In a measure one may regard the nucleated syncytial strands, the product of sheath cell proliferation, as less differentiated protoplasm than developed sheath cells, conceivably a protoplasm more favorable to downgrowth of central neuraxes. Especially was it conjectured that a degenerated hetero-nerve transplant might for this reason prove more satisfactory than a hetero-nerve transplant taken from a normal nerve. It was further felt, consequent to the suggestion of certain observers who have regarded degenerating nerve fibers and proliferating sheath cells as capable of exerting a chemotactic in-fluence on down-growing neuraxes in nerve regeneration, a degenerated nerve transplant might serve to attract, from the beginning of transplantation, central neuraxes in early stages of regeneration. None of these suppositions were well founded. These three series of experiments are jointly presented and considered. The protocols of experiments 126 to 149 (Series No. 8, No. 9,and No. 10) are as follows:
PROTOCOLS
EXPERIMENT No. 126.- Dog No. 1; medium size; full grown; 133 clays. March 25,1918, right ulnar exposed and resected 1.2 cm. As a transplant, used 1.2 cm. of the distal segment of the left sciatic of the same clog, cut March 7, 18 days previous. One central and one distal Chinese silk suture placed; good approximation. Quite a little bleeding, which was not folly controlled. Fascia stitched over the nerve and transplant. Wound closed. August 5, killed. Dog in good condition. On exposing the ulnar, the transplant was found well in place; easily demarked, since central and distal sutures are still clearly evident. Transplant has diameter slightly larger than ulnar. Ulnar distal to transplant presents the appearance of normal nerve. Forearm muscles supplied by ulnar do not contract when ulnar is cut central to the transplant. Ulnar and the transplant and distal ulnar removed and fixed in ammoniated alcohol. Only in part good differential staining attained. Tissues not well embedded, sections torn.
Microscopic findings.- In longitudinal sections of the central wound region, distinct central bulb evidenced structurally, from the distal end of which down-growing neuraxes can be traced to the transplant. In cross sections of the transplant, the funicular structure of the degenerated nerve segment transplanted, is in part retained. New neuraxes observed in the funiculi, in which they are arranged in small bundles separated by fibrous tissue; also in the connective tissue surrounding the transplant; especially to one side. In longitudinal sections of the distal wound region, new neuraxes can be traced to the distal ulnar and in this, in good numbers to the level of the elbow; the extent of the distal ulnar segment sectioned.
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EXPERIMENT No. 127.- Dog No. 3; medium size; full grown; 134 days. March 27,1918, right ulnar exposed and resected 1.5 Cm. A segment of equal length, taken from the external popliteal bundle of the left sciatic of the same dog, cut March 8,18 days previously, used as transplant. One central and distal Chinese silk suture placed; fair approximation attained. Free venous bleeding, not fully controlled. Fascia stitched over nerve; wound closed. August 8, killed. Dog in very good condition. On exposing the right ulnar, a large bulb is observed on end of central ulnar stump, from which a small nerve bundle lead to the distal ulnar segment. On cutting ulnar central to the transplant, no distinct contraction of the forearm muscles supplied by the ulnar is observed. 1lnar and transplant and segment of distal ulnar removed and fixed in ammoniated alcohol for pyridine-silver staining. Not entirely successful silver differentiation attained.
Microscopic findings.- In alternate longitudinal and cross sections, new neuraxes call be traced from the central ulnar stump to the distal ulnar. In the cross sections taken from the middle of the transplant, the funicular structure of the transplanted nerve segment not clearly made out. Small funiculi of nerve fibers are observed. Their relation to the transplanted nerve segment is uncertain.
EXPERIMENT No. 128.- Dog No. 2; medium size; full grown; 420 days. March 26,1918, right ulnar exposed and resected to the extent of 1.5 cm. A segment of equal length taken from the left external popliteal of the same dog, the left sciatic of which was cut March7, 19 days previous, used as a transplant. One central and distal Chinese silk suture placed; good approximation. Fascia stitched over nerve; wound closed. May 20, 1919, killed. Dog in good condition. On exposing the right ulnar, distinct central ulnar bulb is found, from the distal end of which a well-formed bundle of nerves, of slightly smaller diameter than the ulnar, leads to the distal ulnar stump, the central end of which is only slightly enlarged. In the region of the transplant nerve firmly adherent to the surrounding tissue, the distal ulnar has the appearance of a normal nerve. After exposing the forearm muscles, and freeing the ulnar from its bed, on slowly cutting the ulnar central to the transplant, distinct and vigorous contraction of the forearm muscles supplied by the ulnar. Ulnar and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.-In series of alternate longitudinal and cross sections, through transplant and distal ulnar, neuraxes coming from the central ulnar can be traced through the transplant into the distal ulnar. In cross section of the transplant, taken at its middle, it may be clearly seen that the connective tissue of the transplant is very materially increased; this blending with the perineural sheaths. Certain of the down-growing neuraxes, both myelinated and nonmyelinated, appear within a large funiculus of the transplanted nerve segment. Others are found in small bundles in the connective tissue outside of the transplant. In the distal ulnar, cut in cross sections 2 cm. below the elbow, both myelinated and nonmyelinated neuraxes are found scattered through all of the funicili, in large numbers.
EXPERIMENT No. 129.- Dog No. 36; medium size; full grown; 17 days. June 4, 1918, left sciatic exposed; internal popliteal bundle freed and resected 3 cm. A segment of equal length, taken from the left internal popliteal of dog No. 24, the sciatic of which was cut May 18, 17 days previous, used as a transplant. One central and distal waxed, fine silk thread suture placed; very good central and distal approximation attained. Wound closed. June 21, dog found dead in the morning; no neurotrophic changes of left hind foot. On exposing the left sciatic, transplant is found well in place, and appears of slightly larger diameter than when transplanted; no distant central bulb observed. The internal popliteal and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.- In longitudinal sections of the central and distal wound regions, transplant found well united to resected nerve ends with only narrow fibrous tissue union intervening. Scarcely any evidence of central bulb noted. In the distal end of the central stump, active outgrowth of central neuraxes observed. These have reached the scar tissue of the central wound, which many have penetrated and which they traverse by crisscrossing in all directions. Many end-discs and evidence of branching of neuraxes seen. A certain few of the central neuraxes have passed through the central wound into the central end of
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the nerve transplant. These are much more nunerous near the central wound than a little more distally, but can be traced to nearly the middle of the transplant. Within the transplant there arc observed the thickened neurolemma sheaths of the degenerated, transplanted nerve fibers, and remnants of myelin. The distal internal popliteal is found in early stage of degeneration.
EXPERIMENT No. 130.-Dog No. 7; large; full grown; 36 days. July 5, 1918, left sciatic exposed; internal popliteal bundle freed; resected 4 cm. A segment of equal length, taken from the left internal popliteal of dog No. 28, the sciatic of which was cut 28 days previously, used as a transplant. One central and distal waxed, fine silk thread suture placed; very good approximation attained. Wound closed. August 10, killed. Dog seemed well, though emaciated; no neurotrophic changes left hind foot; wound well healed. On exposing the left sciatic, the external popliteal bundle found free; the transplant in the internal popliteal well in place, and clearly demarked by its light yellow color; no material increase of connective tissue about the transplant. No distinct central bulb noted. The internal popliteal removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and piero-fuchsin; safranine and light-grün.
Microscopic findings.-In longitudinal sections of the central and distal wound regions, transplant found well united to the resected nerve ends; fibrous tissue union at the wounds. In the sections from the central wound region, new nerve fibers, in part with fine myelin sheaths, can be traced from the central stump, through the central wound into the transplant. In cross and longitudinal sections of the transplant, the neurolemma sheaths found give the impression of being thickened. Many of these sheaths contain large vesicular cells, having globular, myelin remains in their protoplasm; further, granular detritus; not many sheath nuclei evident. The distal nerve presents degeneration phenomena.
EXPERIMENT No. 131.- Dog No. 5a; large; full grown; 37 days. July 3, 1918, the right sciatic exposed; internal popliteal freed; resected 3.4 cm. A segment of equal length taken from the internal popliteal of the right sciatic of dog No. 27, cut June 6, 27 days previous and used as a transplant. One central and distal waxed fine silk thread suture placed; good alignment attained, but distal end of transplant rotated; good approximation of nerve ends. Wound closed. August 9, killed. Dog emaciated; small neurotrophic ulcer right hind foot. Wound well healed. On exposing the right sciatic, it is found that the external popliteal is closely adherent to the operated internal popliteal; was not dissected free. Distinct increase of connective tissue in the region of operation, about the sciatic. The transplant found well ii place; demarked by its light yellow color. No distinct central bulb noted. External and operated internal popliteal bundle removed together and fixed in ammoniated alcohol for pyridine-silver staining. Good differential neuraxis staining attained.
Microscopic findings.- In longitudinal sections of the central wound region neuraxes from the central stump in large numbers can be traced into the central end of the transplant. In cross sections of the transplant, 1 cm. distal to the central wound, numerous neuraxes are found within the transplant, in the form of small bundles, separated by strands of fibrous tissue. In longitudinal sections of the transplant, it may be observed, that while these small bundles have in the main a longitudinal direction, contiguous bundles frequently are found anastomosing. Only a small number of old myelin remnants are found in the transplant. A few of the down-growing neuraxes have reached the distal wound and can be traced for a short distance into the central end of the distal popliteal; by far the greater portion of the distal popliteal showing only degenerated nerve fibers.
EXPERIMENT No. 132.- Dog No. 37; medium size; full grown; 146 days. June 18. 1918, left sciatic exposed; internal popliteal freed, resected 2.7 cm. A segment of equal length, taken from the internal popliteal bundle of the left sciatic of dog No. 23, cut May 31, 18 days previous, used as transplant. One central and distal waxed fine silk thread suture placed; good approximation. Wound closed. November 11, killed. On this day participated in fight with another dog and nearly killed; was still breathing when found. Dog in good condition. No neurotrophic changes left hind foot noted. On exposing the left sciatic, external popliteal found free; no distinct bulb on central internal popliteal. Transplant found well in place, has the appearance of normal nerve, except that a light pink color is evident. Distal nerve has the appearance of normal nerve. Calf and the plantar muscles exposed and external popliteal resected and removed. After freeing the internal popliteal and
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transplant from the bed, on slowly cutting the nerve central to transplant, good contraction of calf and interossei muscles observed. On cutting posterior tibial at heel, interossei muscles seen to contract. Internal popliteal and the transplant, posterior tibial, internal plantar and portions of several interossei muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential neuraxis staining attained.
Microscopic findings.- In longitudinal sections of the central wound region, a long spindle-shaped bulb evidenced structurally from the distal end of which numerous down-growing neuraxes can be traced to the central end of the transplant. In cross sections of the transplant 1 cm. distal to the central wound, the perineural sheaths of the transplant found thickened. Within these sheaths are found, within the funiculi, numerous small bundles of neuraxes, certain of which are myelinated, separated by strands of fibrous tissue. Large numbers of the neuraxes in these small bundles may be traced to and through the distal wound into the distal internal popliteal, in which new neuraxes are found in all of the funiculi. New neuraxes can he traced to the interfascicular nerve branches in the interossei muscles; a few motor ending observed. Good regeneration of the distal popliteal attained.
EXPERIMENT No. 133.-Dog No. 8a; large dog; full grown; 316 days. July 9, 1918.left sciatic exposed; internal popliteal freed; resected 3.8 cm. A segment of equal length, taken from the internal popliteal of the sciatic of dog No. 26, cut June 7, 32 days previous, used as transplant. One central and distal waxed fine silk thread suture placed. Good distal approximation; central good alignment, but after tying distal suture, central suture gave way slightly, so that nerve ends were nearly 2 mm. apart. Wound closed. May 21,1919, killed. Dog in very good condition; uses left hind foot well; no neurotrophic changes. On exposing the left sciatic, the external popliteal is found free. No distinct enlargement on central internal popliteal noted. The transplant found well in place and presents the appearance of a normal nerve, though somewhat spread out and of flattened form. Distal popliteal has the appearance of a normal nerve. Calf and plantar muscles exposed; external popliteal resected and removed. After separating nerve and transplant from bed, on slowly cutting nerve with scissors central to the transplant, distinct and vigorous contraction of calf and foot muscles noted. Internal popliteal and transplant, posterior tibial, portions of calf and foot muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential neuraxis staining attained.
Microscopic findings.-In longitudinal sections of the central wound region, quite distinct spindle-shaped bulb evidenced structurally, from the distal end of which numerous neuraxes enter the transplant. In cross sections of the transplant, taken at levels near the central and distal wounds, the transplanted nerve segment found to be well outlined with thickened fibrous sheaths. Within the transplant are found numerous small bundles of neuraxes separated by strands of fibrous tissue. Numerous small bundles of neuraxes also found in the connective tissue surrounding the nerve transplant. Many of the neuraxes within and without the transplant are found to be myelinated. New neuraxes call, in sections made at successive levels, be traced through the transplant into and through the distal would into the distal popliteal, in which they are followed to the interossei muscles. In sections of portions of the interossei muscles, new nerve fibers may be observed in inter-fascicular nerve branches and as single nerve fibers, on and between muscle fibers. Motor endings found not well differentiated. Nearly complete regeneration of the distal popliteal observed.
EXPERIMENT No. 134.-Rabbit No. 78; large; full grown; 7 days. June 3, 191, left sciatic exposed and resected 2.5 cm. A segment of equal length, taken from the external popliteal of dog No. 21, the left sciatic of which was cut 16 days previous, used as transplant. One central and distal waxed fine silk thread suture placed; good approximation. Wound closed. June 10, rabbit found dead in the morning. Wound well healed. On exposing the sciatic, tissues about the nerve found congested. Transplant found well in place, though distal suture had drawn out a little. Transplant united to resected nerve ends; good color. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation of neuraxes attained.
Microscopic findings.-In longitudinal sections of the central wound region the very early stages of a bulbous end on central sciatic stump noted. Certain of the larger neuraxes
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of the central stump present large, bulbous ends; growing end-discs noted just central to central wound. Central wound consists of loose fibrocellular tissue, coagulum, and tissue detritus. In longitudinal sections of the transplant, remnants of old neuraxes, in the formof short segments, twisted, coiled, or bent, are to be observed. Faintly stained nuclei are found within the old neurolemma sheaths. In the distal sciatic beginning of nerve degeneration observed.
EXPERIMENT No. 135.- Rabbit No. 75; full grown; emaciated; 13 days. May 10, 1918, left sciatic exposed and resected 2.5 cm. A segment of equal length, taken from the internal popliteal bundle of the left sciatic of dog No. 2, cut April 23, 17 days previous, used as transplant. One central and distal waxed, fine silk thread suture passed; good approximation. Wound closed. May 23, rabbit found dead in the morning; snuffles; wound well healed. On exposing the left sciatic, transplant found well in place; demarked by sutures. Transplant seems of slightly larger diameter than when used, and found surrounded by newly formed connective tissue. Beginning of bulbous enlargement on the distal end of the central stump. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good differential staining, especially central stump, attained.
Microscopic findings.-In a series of longitudinal sections of the central wound region, distinct bulbous end of central stump evidenced structurally, at the distal end of which is found the wound line, consisting of fibrocellular tissue; suture found in sections. Many down-growing neuraxes of the central stump have reached the wound line. Many of these show bulbous end-discs; certain of them are directed centralward, others toward the periphery. In longitudinal sections of the transplant, the fibrous tissue sheaths are found very materially thickened by means of newly formed connective tissue containing many leucocytes. The neurolemma sheaths very materially thickened. These in longitudinal sections present a wavy zigzag course. Globules of myelin and remnants of neuraxes observed. Many wandering leucocytes are found between the old nerve fibers. The distal sciatic presents early stages of degeneration.
EXPERIMENT No. 136.- Rabbit No. 85; nearly full grown; Belgian hare; 42 days. July 9, 1918, left sciatic exposed and resected 2.2 cm. A segment of equal length, taken from the right median of dog No. 26, cut June 7, 32 days pervious, used as transplant. One central and distal suture of waxed, fine silk thread placed; approximation good, though the transplant has slightly greater diameter than the sciatic resected. Wound closed. August 20, rabbit found dead in the morning; very much emaciated; large neurotrophic ulcer on left heel. On exposing the left sciatic, the transplant is found well in place; of larger diameter than the resected nerve and of yellow-white color. No distinct central bulb noted. Transplant and suture lines surrounded by relatively dense fibrous tissue and adherent to underlying muscle. Sciatic and the transplant removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht-grün.
Microscopic findings.- In longitudinal sections of the wound regions, the resected nerve ends and ends of transplant found firmly united. There is noted a marked increase of fibrous tissue about the transplant. In longitudinal sections of the transplant, the neurolemma sheaths of the transplanted nerve fibers appear thickened and as if consisting of a delicate fibrillar structure. Near the central and distal wound leucocytes found within the neurolemma sheaths and here and there are found to contain globules. In cross sections of the transplant, about 1 cm. distal to the central wound, numerous small funiculi consisting of nonmyelinated fibers found in the connective tissue surrounding the transplant, outside of its perineural sheaths. Distal popliteal found in advance stages of nerve degeneration. Near the distal wound leucocytes found within the neurolemma sheaths of the distal popliteal.
EXPERIMENT No. 137.- Rabbit No. 77; full grown; 48 days. May 20, 1918, left sciatic exposed and resected 2.2 cm. A segment of equal length taken from the right ulnar of dog No. 22, cut April 29, 21 days previous, used as transplant. One central and distal fine Chinese silk thread suture placed; good approximation. Wound closed. July 7, rabbit found dead in the morning; neurotrophic changes left heel. On exposing the left sciatic, transplant is found well in place, of light yellow color, of slightly smaller diameter distally and surrounded
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by a relatively firm layer of fibrous tissue. Only slight evidence of bulbous enlargement of central sciatic noted. Distal sciatic presents the appearance of a normal nerve. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.-In series of longitudinal and cross sections taken at successive levels, nearly the entire nerve transplant is very clearly demarked by reason of a peculiar silver reaction. Exclusive of the perineural sheaths, and the nerve fibers immediately adjacent, the entire transplant is stained a jet-black, making it nontransparent even in sections of 5 microns thickness, so that no structure can be made out in the parts thus stained. In longitudinal sections of the central wound region, numerous neuraxes growing from the central nerve can be traced through the wound tissue to the beginning of the transplant, but appear to pass no distance into the transplant. Certain of these neuraxes deviated to one side and may be traced for a distance of several millimeters in the connective tissue sheath surrounding the transplant. Distal sciatic degenerated.
EXPERIMENT No. 138.- Rabbit No. 77a; full grown; 48 days. May 20, 1918, right sciatic exposed and resected 2.2 cm. A segment of equal length, taken from the right median of dog No. 22, cut April 29, 21 days previous, used as transplant. One central and distal fine Chinese silk thread suture placed; good approximation central; distal "fair." Wound closed. July 7, rabbit found dead in the morning; neurotrophic changes right heel. On exposing the right sciatic, transplant is found well in place, of light yellow color, of firm consistency; united to the resected nerve ends. No material increase of connective tissue found surrounding the transplant. Distinct central bulb noted. Distal sciatic presents the appearance of a degenerated nerve. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.- In longitudinal and cross sections taken at successive levels, trans-plant clearly demarked by reason of its jet-black color after silver staining. Neuraxes from the central sciatic may be observed to grow toward the central end of the transplant, but not to penetrate it. Certain of these neuraxes deviate to one side and can be traced distal ward into the connective tissue sheath of the transplant. These relatively few neuraxes can be traced in the connective tissue sheath, in cross and longitudinal sections of the transplant tonear the distal wound, where they escape the plane of section. Relatively few neulraxes are again recognized in the distal wound and for a distance of about 2 mm. in the central end of the distal sciatic stump, in longitudinal sections of which only ten to fifteen neuraxes are recognized in one section, all other nerve fibers included in the section degenerated.
EXPERIMENT No. 139.- Rabbit No. 76; large; full grown; 52 days. May 17, 1918, right sciatic exposed and resected 2 cm. A segment of equal length taken from the right ulnar of dog No. 21, cut April 29, 18 days previous, used as transplant. One central and distal fine Chinese silk thread suture placed; good approximation. Wound closed. July 8, rabbit found dead in the morning; severe neurotrophic changes right heel. On exposing the right sciatic an encapsuled abscess in the region of the transplant found. The transplant appears to have pulled free from distal stump and has almost completely disappeared; only a short segment, of light yellow color, adhering to central sciatic stump. Large bulbous end found on the distal end of central sciatic. Central sciatic and bulbous end removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good silver differentiation attained.
Microscopic findings.-In two series of sections, the segment of the transplant remaining, clearly demarked by reason of its jet-black color. In longitudinal sections through the central bulb and contiguous central end of transplant, it is evident that there was not obtained as end-to-end suture of resected nerve end and transplant, the central end of the transplant having slipped to one side, so that the distal end of the central sciatic stump rests against the perineural sheath of the transplant. A well-developed central bulb is evidenced structurally from the end of which numerous neuraxes grow distalward; meeting the perineural sheath of the transplant, they are diverted from their course and form small convoluted bundles of nerve found in the surrounding connective tissue. Many of the central neuraxes end dis-tally in large end-discs. In the bulb itself, just central to the wound region, numerous spirals composed of neuraxes may be observed. Central neuraxes can be traced distally for only a short distance. In cross sections of the transplant, I cm. distal to the central wound, no neuraxes are found in the connective tissue surrounding the transplant. The distal sciatic was found completely degenerated.
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EXPERIMENT No. 140- Rabbit No. X2; full grown; 62 days, July 8, 1918, right sciatic exposed anil resected 2.6 cm. A segment of equal length taken from the external popliteal bundle of the left sciatic of dog No. 25, cut June 7, 31 days previous, used as a transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. September 8, rabbit found dead in the morning; very severe neurotrophic changes right hind foot; foot in part missing; “fungus'' cars. On exposing the right sciatic, transplant is found well in place, of distinct yellow color, thus clearly demarked from resected sciatic ends. Transplant found of larger diameter than the sciatic. Well-developed central sciatic bulb noted. Sciatic and nerve transplant removed and fixed in neutral formalin. Sections stained in iron-hemmatoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-In a series of longitudinal sections through the distal end of the central stump and the central end of the transplant, it may be observed that nucleated syncytial strands of protoplasm extend for a short distance into the central end of the transplant; beyond this region the thickened neurolemma sheaths of the transplanted nerve fibers are found and seem to contain granular and globular detritus, and inwandered cells. There is observed a distinct small cell infiltration in this region. In the connective tissue to one side of the transplant, protoplasmic syncytial strands, grouped in small bundles, are observed in cross sections. These can not be definitely traced to the distal wound. The distal sciatic found completely degenerated.
EXPERIMENT No. 141.- Rabbit No. 86; only about one-half grown; 65 days. July 9, 1918, left sciatic exposed and resected 2.5 cm. A segment of equal length taken from the external popliteal bundle of the left sciatic of dog No. 26, cut June 7, 32 days previous, used as transplant. One central and distal waxed, fine silk thread suture placed; distal approximation good; central, good alignment but nerve ends about 2 mm. apart. Wound closed. September 12, found dead in the morning; much emaciated; slight neurotrophic changes left heel; posterior half of body paralyzed for past few days. On exposing the left sciatic, the transplant is found well in place; clearly demarked by its light yellow color; of good size and consistence and adherent to the underlying muscle. Quite distinct central sciatic bulb noted. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Not satisfactory silver differentiation attained.
Microscopic findings.- In sections, transplant clearly demarked by reason of its jet-black coloring. Neuraxes from the central nerve are seen to approach the central end of the transplant but do not penetrate it. Certain few neuraxes pass into the connective tissue to one side of the transplant, passing distally in the connective tissue. No new neuraxes are observed as having reached the distal wound. Distal sciatic degenerated.
EXPERIMENT No. 142.- Rabbit No. 80; full grown; 61 days. July 5, 1918, left sciatic exposed and resected 3 cm. A segment of equal length, taken from the right median of dog No. 26, cut June 7, 27 days previous, used as a transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Adrenal in used to control oozing. Wound closed. September 4, found dead in the morning; seemed in good Condition; slight neurotrophic ulcer left heel. On exposing the left sciatic, transplant is found well in place, of small diameter and of light yellow color; adherent to the underlying muscle. Distinct bulbous enlargement of the distal end of the central stump noted. The sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fair silver differentiation attained.
Microscopic findings.-In longitudinal sections of the central wound. region, down-growing neuraxes from the central stump in small number are seen to pass distally to one side of the transplant and enter the connective tissue in which they may be traced not quite to the distal wound region. The region of the transplanted nerve fibers stained jet black; no structural details can be ascertained.
EXPERIMENT No. 143.- Rabbit No. 80a; full grown; 63 days. July 3, 1918, the right sciatic exposed and repete 1 3 cm. A segment of the right ulnar of dog No. 26, cut June 6,27 days previous, used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Adrenalin used to stop oozing; wound closed. September 4, rabbit found dead in the morning; seemed in good condition; neurotrophic ulcer right heel. On exposing the right sciatic, transplant is found well in place; clearly demarked
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by its light yellow color; is of good size and consistency and is found adherent to the underlying muscle. Distinct central bulb noted. Sciatic and transplant removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-In longitudinal sections through the central wound region, distinct central bulb evidenced structurally, from the distal end of which nucleated, syncytial, protoplasmic strands can be traced to the connective tissue found to one side of the transplant. In longitudinal sections of the transplant, the of neurolemma sheaths of certain of the transplanted nerve fibers seen, these appear thickened and present a wavy or zigzag course, containing a granular and globular detritus. Here and there inwandered leucoctes may be observed. The perineural sheaths present small cell infiltration. The distal nerve completely degenerated. No new nerve fibers found in the connective tissue surrounding the greater length of the transplant.
EXPERIMENT No. 144.-Rabbit No. 79; full grown; 84 days. June 19, 1918, the left sciatic exposed; internal popliteal freed and resected 3.1 cm. .A segment of equal length, taken from the distal ulnar of dog No. 23, cut May 31, 19 days previous, used as transplant. One central and distal waxed, fine silk thread suture placed. Good approximation of nerve ends attained centrally; distally "fair.” Wound closed. September 11, rabbit found dead in the morning; scarcely any neurotrophic changes in left hind foot. On exposing the left sciatic, external popliteal found free. The operated internal popliteal presents large central bulb. The transplant found well in place; is of light yellow color, of small diameter, especially in its middle portion; and found adherent to underlying muscle. The nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. The tissues removed in this experiment were lost in process of fixing and staining; no sections made.
EXPERIMENT No. 145.-Rabbit No. 79a; full grown; 84 lays. June 19, 1918, right sciatic exposed; internal popliteal freed and resected 3 cm. A segment of equal length taken from the right median of dog No. 23, cut May 31, 19 days previous, used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. September 11. Rabbit found dead in the morning; very slight neurotrophic changes right hind foot. On exposing the right sciatic, operated internal popliteal is found to present large bulb along side of which external popliteal is found closely adherent. Transplant found well in place; clearly demarked by reason of light yellow color, and is adherent to underlying muscle. Nerve and transplant removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and light grün.
Microscopic findings.- In longitudinal sections of the central wound region, a large, distinct bulb is evidenced structurally, from the distal end of which nerve fibers, in part myelinated, pass to the connective tissue to one side of the transplant. In cross sections of the middle transplant region, the perineural sheaths found distinctly thickened and penetrated by inwandered cells. Within this sheath necrotic remains of the transplanted nerve fibers are found. To one side of the transplant, and outside of the perineural sheath, numerous small funiculi of nerves with certain fibers myelinated are to be observed. In cross and longitudinal sections taken at successive levels, these small bundles of nerves may be traced to the distal wound region, coursing in the connective tissue outside of the transplant; certain few are found to have reached the central end of the distal popliteal, here clearly recognized as small myelinated fibers.
EXPERIMENT No. 146.- Rabbit No. 81; full grown; 93 days. July 5, 1918, left sciatic exposed and resected 2.4 cm. A segment of equal length taken from the right median of dog No. 28, cut June 7, 28 days previous, used as a transplant. One central and distal waxed, fine silk thread suture placed. Good central approximation attained; distal suture does not include the external popliteal branch. Wound closed. October 6, killed. Rabbit found dying; much emaciated; severe neurotrophic ulcer left heel. On exposing, the left sciatic, transplant is found well in place; is of good size and light yellow color, and only moderately adherent to underlying muscle. Only slight spindle-shaped enlargement on central sciatic noted. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
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Microscopic findings.-In series of longitudinal and cross sections taken at successive levels, the transplanted nerve segment is clearly demarked by reason of the jet-black color assumed in the silver stain. For a distance of approximately 2 mm., at the central and distal end of the transplant and in the peripheral part, adjacent to the perineural sheath the substance responsible for the peculiar jet-black silver reaction noted, has apparently disappeared, in that in these regions the transplant is colored a yellow brown. In longitudinal sections of the central wound region, certain neuraxes may be traced into central end of the transplant, to the region of the jet-black coloring; here they can no longer be differentiated. Other neuraxes pass to one side of the transplant, to the connective tissue. In cross sections of the transplant, mainly to one side, numerous small bundles of neuraxes are found in the connective tissue sheath. New neuraxes are observed in longitudinal sections of the distal wound region; certain of these appear to enter the distal wound through the transplant, others from the surrounding connective tissue. New neuraxes are found in good number in all of the funiculi of the distal popliteal in the region of the distal wound.
EXPERIMENT No. 147.-Rabbit No. 81a; full grown; 93 days. July 5, 1918, right sciatic exposed and resected 2.2 cm. A segment of equal length taken from the right ulnar of dog No. 28, cut June 7, 28 days previous, used as a transplant. One central and distal waxed, fine silk thread suture placed; good central and distal approximation. Wound closed. October 6, killed. Rabbit found dying; much emaciated; severe neurotrophic ulcer right heel. On exposing the right sciatic, transplant is found well in place; demarked by its light yellow color. At distal suture transplant appears to have pulled away slightly from distal sciatic stump. Transplant found only moderately adherent to underlying muscle. Quite distinct central bulb noted. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation attained.
Microscopic findings.-In series of cross and longitudinal sections, taken at successive levels, it may be observed that certain neuraxes coming from the central sciatic stump, enter the central end of the transplant and may be traced in it for a short distance. However, the majority of the central neuraxes are found to pass into the connective tissue to one side of the transplant and in cross sections of the transplant these are found in the form of small nerve funiculi outside of the perineural sheaths. Neuraxes can be traced through the distal wound into the distal popliteal stump. Certain of these appear to take exit from the distal end of the transplant; these can not be identified more centrally by reason of the jet-black coloring of the greater part of the transplant. The neuraxes found in the distal popliteal appear to be about equally distributed through the several fuiniculi and can be traced distally to the end of the series of sections approximately 3 em. beyond the distal wound.
EXPERIMENT No. 148.-Rabbit No. 83; not quite full grown; 217 days. July 8, 1918,left sciatic exposed and resected 2.0 cm. A segment of equal length taken from the external popliteal of dog No. 25, the sciatic of which was cut June 7, 31 days previous, was used as a transplant. Dog No. 25 stopped breathing while under ether anesthesia, 45 minutes before the nerve was removed. One central and distal waxed, fine silk thread suture passed. Central suture not good; removed and another made; slight trauma of nerve ends; finally central and distal approximation good. Wound closed. February 10, 1919, killed. Rabbit in good condition; part of left hind foot missing; practically healed over; scarcely any evidence of long-standing neurotrophic ulcer left heel. On exposing the left sciatic, a large central bulb is found. The nerve in region of transplant adherent to underlying muscle. The distal sciatic presents the appearance of a normal nerve. After exposing the calf muscles and the leg flexors, and freeing nerve and transplant from the bed, on slowly cutting the sciatic with scissors, central to the transplant, good contraction of the calf and leg flexor muscles noted; the same on cutting nerve distal to the transplant. The calf muscles have nearly recovered size, but are of a pale red color. The sciatic and the transplant and portions of the calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good silver differentiation attained.
Microscopic findings.-In series of longitudinal and cross sections, taken at successive levels, it is observed that except for two small regions, of about 1.5 mm. in length, near the distal and central ends of the transplant, which are colored jet-black, the remainder ofthe nerve transplant is colored a light yellow-brown, as is the remainder of the nerve. In longitudinal sections of the central wound region numerous new neuraxes can be traced from
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the distal end of the large central bulb into the transplant as well as into the connective tissue surrounding the transplant. In cross sections of the transplant in its middle region many small nerve funiculi, separated by endoneural fibrous tissue, are seen with the perineural sheath of the transplant. There are also found many small nerve bundles in the connective tissue outside of the perineural sheaths of the transplant. New neuraxes can be traced to and through the distal wound into the distal sciatic. In cross sections of the internal popliteal, taken at the lower level of the popliteal space, new neuraxes in large numbers are found in all of its several funiculi. In sections of the calf muscles new neuraxes in good relative numbers are found in the interfascicular muscle nerves and as single fibers on and between muscle fibers; motor endings and nerve endings in neuromuscular spindles are observed.
EXPERIMENT No. 149.-Rabbit No. 84; Belgian hare; not quite full grown; 240 days. July 9, 1918, right sciatic exposed and resected 2.2 cm. A segment of equal length, taken from the right ulnar of dog No. 26, cut June 7, 32 days previous, used as a transplant. One central and distal waxed, fine silk thread suture passed; good approximation. Diameter of the degenerated ulnar segment slightly greater than that of the resected sciatic. Adrenalin used to obtain dry field; wound closed. March 6, 1919, killed. Rabbit in good condition. No neurotrophic ulcer on right heel. On exposing the right sciatic a large central bulb is found on the central sciatic. Transplant is found of light yellow color, of good size and adherent to underlying muscles. The distal sciatic presents the appearance of a normal nerve. After exposing the calf muscles and freeing the sciatic from bed, on slowly cutting with scissors, sciatic central to the transplant, good contraction of calf muscles observed. Calf muscles are found to have nearly recovered size but are of pale red color. Sciatic and the transplant and portions of the calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good differential silver staining attained.
Microscopic findings.-In series of longitudinal and cross sections taken at successive levels the transplanted nerve segment is clearly demarked by reason of the jet-black coloring assumed by the transplanted nerve tissue. In longitudinal sections of the central wound region numerous central neuraxes can be traced to the periphery, mainly in the connective tissue outside of the transplant, as clearly seen in cross sections, taken about midway between central and distal wounds, in which two areas, situated at opposing sides of the transplant, there are found in the connective tissue numerous small bundles of nerve fibers situated outside of the perineural sheaths. Whether any neuraxes pass through the transplant cannot be determined by reason of the jet-black coloring of the nerve bundle remains of the transplant. New neuraxes are observed in the distal wound and in the central end of the distal sciatic, in which they are found evenly distributed through the several fulliculi. In sections of the calf muscle new neuraxes are found in the interfascicular nerve bundles, and here and there as single nerve fibers on and between muscle fibers.
As concerns degenerated auto-nerve transplant, Experiments No. 126 to No. 128, while only three in number, they may serve to show that a degenerated nerve can serve the purpose of an auto-nerve transplant though there is no indication that regeneration from the central stump through such a transplant takes place more readily than when an undegenerated auto-nerve transplant is used. Indeed the results attained are, on the whole, less satisfactory, if one may judge from the relatively few experiments. There are relatively more extrafunicular nerve fibers in the experiments in which a degenerated auto-nerve transplant was used than in the experiments in which cable-auto-nerve transplants were made.
In the series of five experiments (No. 129 to No. 133) in which degenerated homo-nerve transplants were used, the end results were on the whole very satisfactory and may compare very favorably with the end results in experiments in which a homogenous transplant was made, using a fresh nerve. Attention is especially called to Experiment No. 131, terminated 37 days after the operation. A nerve segment 3.4 cm. in length. taken from the distal portion of
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a nerve cut 27 days previously, was used as a nerve bridge. In this experiment, numerous down-growing neuraxes are readily determined within the funiculi of the transplant; relatively few new nerve fibers are extra-funicular. In Experiment No. 132, a little over four months' duration, it was possible to trace new-neuraxes to the foot interossei muscles, with good return of function. It is notlikely that a degenerated nerve would be available for an auto-nerve transplant, in human surgery. It is quite within the bounds of possibility that a degenerated nerve from another individual, one to several months after injury, may be available for bridging a nerve defect, in which event it may be stated that experimental evidence warrants the use of a degenerated homo-nerve transplant. In nerve defect bridged by degenerated auto- and homo- nerve transplants, the down-growing central neuraxes make use of the patent or semipatent neurolemma sheath of the transplanted nerve fibers. There is no evidence that the transplanted syncytial nucleated strands stand in any definite relation to tie down-growing neuraxes, since these strands undergo change, degenerate, after transplantation.
The experiments on degenerated hetero-nerve transplants (No. 134 to No.149) did not substantiate the conjecture that the less specifically differentiated protoplasm of the "bandfasern ", which may be regarded as in a measure representing a reversion to embryonic structure, offered a better avenue for the down growth of central neuraxes than would an undegenerated nerve trans-plant of the heterogenous origin. It may be noted that in the majority of the experiment protocols the notation is made that the transplant presents a light yellow color when the nerve is exposed some time after the operation. This enables ready demarkation of the transplant and is indicative of a necrobiotic change, involving not only the transplanted nerve fibers but the fibrous tissue sheaths of the nerve funiculi. The products of this necrobiotic change show a peculiar reaction to the pyridine-silver stain in that they assume a jet-black color, in which no tissue elements can be made out, and would mask any neuraxes in case they were present. This series of sixteen experiments need not be discussed seriatim; they range in time after operation from 7 days to 244 days. The results may be summarized by stating that a degenerated hetero-nerve transplant was found less serviceable than a nondegenerated nerve transplantof heterogenous origin owing to the fact that a degenerated hetero-transplant undergoes further change of necrobiotic nature, resulting in the formation of tissue detritus which appears to offer an effective block to the down-growing neuraxes. Whether this block is largely of a mechanical or to a large part of a chemical nature has not been determined. In all of the experiments under observation after the initial operation for more than 45 days, down-growing neuraxes could be traced into the central wound, but no distance into the transplant. Numerous neuraxes could be traced into the connective tissue surrounding the transplant, thus having extra funicular position. Only in a few experiments were nerve-fibers in number found within the nerve funiculi of the transplant. Cross sections of the transplant region with good differential neuraxis staining are necessary to determine the relative position of the neuraxes, whether extrafunicular or intrafunicular, the latter indicating the efficacy of the trans-plant.
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STORED HOMO-NERVE TRANSPLANTS
SERIES NO. 11
HOMO-NERVE TRANSPLANTS, STORED IN STERILE VASELINE SERIES NO. 12
HOMO-NERVE TRANSPLANTS, STORED IN LIQUID PETROLATUM SERIES NO. 13
HOMO-NERVE TRANSPLANTS, STORED IN 50 PERCENT ALCOHOL
In these series of stored homogenous transplants, totaling 67 experiments, we present a body of experimental observations, which we regard as of crucial importance in determining the true function of a nerve transplant. In none of these experiments can the transplant he regarded as being in the state of a living tissue.
The experiments under Series No. 11 were suggested to us through the publication of Dujarier and Francois,76 who reported briefly a series of 20 cases in which homogenous nerve transplants. stored in vaseline, were used to bridge nerve defects. Dujarier and Francois recommended that nerves removed from amputated limbs under aseptic precautions be placed in sterile vaseline and kept at nearly 00 temperature. In their work as reported, nerves were kept in this way for 41 davs. Before use the vaseline was warmed to melting, the nerve segment removed and rinsed in warmed serum, then sutured between the severed nerve ends. In the cases reported, healing took place by primary intention. Not enough time had elapsed from the time of operation to the time of the publication to make a report on the ultimate results. At the time our observations were undertaken we were not aware of any experimental observations in which stored nerve transplants had been used. We followed as closely as possible the method as briefly outlined by Dujarier and Francois. The sciatics of large and full grown rabbits were removed under aseptic precautions, placed in large tube vials containing sterile melted vaseline, after which the tube vials were plugged with sterile cotton plugs. The tube vials were then placed in a small ice chest regulated to 30 C.. in which they remained for periods varying in the several experiments from 9 days to13 days. The nerves thus stored were used to bridge defects in the sciatics of rabbits caused by resection. Just before use the vial containing the nerve to be selected for the experiment was carefully warmed to an extent sufficient to melt the vaseline. The contained nerve segment was then removed and rinsed in warmed sterile serum, and a segment of proper length cut and sutured to the resected stumps of a rabbit's sciatic nerve. One fine, waxed silk suture was placed centrally and distally. The experimental operations are relatively simple. A nerve segment stored in vaseline is readily manipulated: the small amount of vaseline clinging to the nerve was disregarded at the operation, since it seemed to play no special part in the healing of the wound. The necessary warming of the vaseline, so that the nerve segment may be readily removed, with possibility of overheating the nerve segment, more particularly the washing of the nerve segment in serum, seemed to he objections to the
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method as suggested by the French observers. It occurred to us that the same ends might be attained by using liquid petrolatum as a medium for storing nerve segments. This method of procedure was tested in Series No. 12,40 experiments, in which homogenous nerve transplants stored in liquid petrolatum were used. In our experiments we used Squibb's liquid petrolatum, which is a clear, bland fluid. The required quantity was placed in large tube vials, corked with cotton plugs anti autoclaved on successive days. After cooling to room temperature the tube vials were placed in a small ice chest regulated to 3VC. The sciatics of rabbits were removed under asepsis, placed in sterile cooled liquid petrolatum and stored in the ice chest until required for operation, for periods varying from 7 days to 39 days in the several experiments. Before an experiment the tube vial containing the nerve selected was taken from the ice chest and placed in the operating room, and when required the nerve segment was taken from the tube vial by means of forceps and, grasping the nerve segment at one end, was allowed to drain for a few minutes. The sutures were then placed at requisite distance and the nerve cut about 2 mm.distal to the suture and the nerve transplant sutured proximally and distally to the resected nerve ends. Nerve segments stored in liquid petrolatum have good consistency four to five weeks after removal from the animal and have nearly the same appearance as a normal nerve. The excess of liquid petrolatum drains off very readily; the thin coating clinging to the nerve transplant plays no part in the healing of the wound, so far as can be determined. It was our experience that storing of nerves in liquid petrolatum, as used by us, was much to be preferred to storing in vaseline as suggested by Dujarier and Francois.
Homogenous nerves transplants stored in 50 percent alcohol were used in a further series of experiments (Series No. 13). The suggestion for this series of experiments came quite indirectly from observations published by Nageotte. 77 Nageotte had determined as a result of bilateral experimental operations on the sciatics of six dogs, in which on one side direct suture of the severed sciatic was made, while on the other side a 5 mm. long hetero-nerve transplant which had been stored in 50 percent alcohol for some time was interposed between the severed ends of the cut sciatics and sutured in place, that better results could be reported in certain experiments for the side in which the short heterogenous nerve segment stored in alcohol was used. In Nageotte's experiments the heterogenous nerve was obtained from the slaughter house, placed in 50 percent alcohol in sealed tubes, some of which were kept as long as 15 months. In our experiments the sciatics of full grown rabbits were removed under aseptic precautions and placed at once in 50 percent alcohol, in sterile, wide-mouthed glass-stopped bottles, in which they were kept for periods varying from 7 days to 29 days. In the 50 percent alcohol the nerve trunk becomes hardened, though not brittle, and of course can not be regarded as tissue retaining latent viability. Just before use as nerve transplants the nerve segments were taken from the alcohol and placed for 10 to 20 minutes in warmed, sterile, normal salt solution, in which, after a short stay, the nerve again becomes quite pliable. The nerve segments were taken from the normal salt solution; the sutures placed at requisite distance, the ends freshened by cutting with sharp scissors about 2 mm. beyond the suture lines, and
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the operation completed by placing the alcoholized transplant between the resected sciatic stumps and sutured in place by making one central anal one distal suture with fine silk thread waxed with sterile wax. The nerve segments stored in alcohol, after a short stay in the saline solution, are of good consistency and lend themselves readily to operative technique; sutures pass easily; end-to-end approximation is easily made. In our experimental work, nerve segments were stored in 50 percent alcohol for about four weeks and at room temperature; they were kept in a dark cabinet. We have no observations indicating that nerve segments might not be stored in 50 per cent alcohol for a period of four months or more and then used as nerve transplants. This method of storing nerve transplants is so simple, the necessary precautions so easily met, that this method should commend itself as at least worthy of further experimental test.
Protocols of experiments under Series No. 11, No. 12, and No. 13, homo-nerve transplants stored in vaseline, liquid petrolatum, and 50 percent alcohol follow:
PROTOCOLS
EXPERIMENT No. 150.- Rabbit No. 97; large; full grown; 66 days. October 4, 1918,left sciatic exposed, internal popliteal bundle freed and resected 3 cm. For nerve transplant there was used the internal popliteal bundle of another rabbit, removed nine days previous and stored in sterile vaseline at a temperature of 30 C. Just before use as transplant, the nerve segment washed for some minutes in sterile rabbit's serum. One central and one distal waxed fine silk suture placed. Good central and distal approximation of nerve end attained. Dry field. Wound closed. December 8, rabbit found dead in the morning. Wound well healed. On exposing left sciatic no material increase of connective tissue about nerve and transplant noted. Transplant found well in place. Indistinct central bulbous enlargement; no material enlargement of central end of distal internal popliteal stump. Nerve and transplant not adherent to muscle bed. Central and distal popliteal and trans-plant removed and fixed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.-Numerous down-growing neuraxes can be traced from the distal end of the central stump, through central wound to proximal end of transplant. Through this neuraxes can be traced in good number to and through distal wound to proximal end of the distal internal popliteal. In cross sections of the transplant neuraxes are found in small groups, separated by small areas containing vesicular cells and tissue detritus.
EXPERIMENT No. 151.- Rabbit No. 97a; large; full grown; 66 days. October 4, 1918,right sciatic exposed, internal popliteal bundle freed and resected 3 cm. A nerve segment of equal length,-taken from the sciatic of another rabbit, stored in sterile vaseline nine days at 30° C. temperature, washed in sterile rabbit's serum several minutes, used as transplant. One central and one distal waxed, fine silk suture placed; good nerve-end approximation attained Dry field. Wound closed. December 8, rabbit found dead in the morning. Wound well healed. Small neurotrophic ulcer right heel. Right sciatic exposed. Transplant found well in place. Only indistinct central bulbous enlargement. No material increase of connective tissue about nerve and transplant. Nerve and transplant removed and fixed in 5 percent neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.- Structurally considered, quite well defined central bulbous enlargement from the distal end of which many small nerve fibers nucleated, syncytial strands of protoplasm can be traced into the proximal end of the transplant and through this to the distal wound. Certain of the nerve fibers within the nerve transplants present thin myelin sheaths. The great majority of the new nerve fibers found within the transplant are arranged in the form of small bundles of narrow bands, here and there anastomosing, and separated by small areas or columns of vesicular cells and tissue detritus. The endoneural and perineural connective tissue not materially increased.
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EXPERIMENT No. 152.- Rabbit No. 99; medium size; full grown; 89 days. October 7,1918, left sciatic exposed, internal popliteal bundle freed and resected 3.0 cm. A nerve segment of equal length taken from the sciatic of another rabbit, stored 13 days in sterile vaseline, temperature 30 C., used as transplant. One central and one distal suture of waxed, fine silk thread placed. Good central and distal nerve-end approximation attained. Dry field. Wound closed. January 4, 1919, rabbit found dead 1 p. m.; living in the morning. Moderate emaciation; neurotrophic ulcer left heel. Wound well healed. Left sciatic exposed full length. External popliteal bundle found adherent along central wound. Transplant found well in place; small spindle-shaped central bulbous enlargement. Transplant found slightly adherent to underlying muscle; of light gray color and not quite as glistening as normal nerve. Distal wound not distinctly made out. Central and distal sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.-Good differential neuraxis staining attained. From the distal end of central bulbous enlargement which embraces central end of the transplant, numerous necuraxes are traced through the transplant, to and through the distal wound and into the proximal end of the distal popliteal stump in which for several centimeters well differentiate neuraxes are found in large numbers. In cross sections of the transplant, the new neuraxes present are found in the form of small funiculi separated by strands of endoneural connective tissue. Here and there small groups of vesicular cells, inclosing what appear to be lipoid globules, are noted. Distal internal popliteal well regenerated.
EXPERIMENT No. 153.- Rabbit No. 99a; medium size; full grown; 89 days. October7, 1918, right sciatic exposed, internal popliteal bundle freed; resected 3.2 cm. A segment of equal length taken from the sciatic of another rabbit, stored in sterile vaseline, at a temperature of 30° C. for 13 days, used as transplant. One central and one distal waxed, fine silk thread suture placed. Good central and distal approximation attained. Dry field; wound closed. January 4, 1919, rabbit found dead 1 p. m.; nerve removed at once. Moderately emaciated; slight neurotrophic ulcer right heel. Wound well healed. Right sciatic exposed full length. Transplant found well in place; clearly demarked by presence of central and distal suture. Small spindle-shaped, central, bulbous enlargement. External popliteal bundle not adherent to underlying muscle. No appreciable distal enlargement. Central and distal sciatic and transplant removed and fixed in 5 percent neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-Quite distinct central bulbous enlargement evidenced structurally, from the distal end of which many fine, myelinated nerve fibers and nucleated, proto-plasimic, syncytial strands pass through the transplant to the distal wound. In cross sections of the transplant about 1 cm. distal to central wound, one large funiculus and two small funiculi of the transplant almost completely filled with new nerve fibers with only here and there small areas of tissue detritus and vesicular cells evident. The perineural sheaths of the transplanted nerve funniculi well maintained and only slightly thickened. Apparent regeneration through transplant; used histologic methods do not enable full determination of distal growth of new neuraxes.
EXPERIMENT No. 154.-Rabbit No. 98; large; full grown; Belgian hare; 96 days. October 5, 1918, left sciatic exposed, internal popliteal freed; resected 2.5 cm. A segment of equal length, taken from the sciatic of another rabbit, stored in sterile vaseline, temperature 3?C., 11 days, used as nerve transplant. One central and one distal suture of waxed, fine silk thread placed. Only fair central and distal nerve-end approximation attained. Dry field; wound closed. January 9, 1919, killed. Rabbit much emaciated;"fungus" ears; severe neurotrophic ulcer left heel; on the whole quite active. Wound well healed. Left sciatic exposed full length. External popliteal bundle found quite free. Large spindle-shaped central bulbous enlargement noted on central internal popliteal stump; slight enlargement of the central end of distal stump. Transplant well in place; light gray color, not adherent to underlying muscle. Unoperated external popliteal bundle resected and removed. Calf muscles fully exposed; operated internal popliteal and transplant completely freed from bed. On slowly cutting with scissors sciatic central to transplant doubtful, feeble contractions of calf muscles. On cutting internal popliteal lower level of popliteal space, feeble contraction of calf muscles; uncertain. Central and distal sciatic and transplant
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and pieces of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.- Oily fair silver differentiation attained; calf muscles silver staining good. Distinct central bulbous enlargement evidenced structurally from the distal end of which numerous new neuraxes pass through the transplant to the distal popliteal nerve. In sections of the calf muscles, numerous new neuraxes found in the larger muscular branches, relatively fewer in the smaller interfasicular branches and here and there single nerve fibers seem to pass to muscle fibers. Muscle fibers of small diameter, but show distinct cross striations. Muscle capillaries very numerous and very tortuous. Regeneration of distal popliteal to muscular branches of the calf muscle, beginning recovery of in motor function of these muscles.
EXPERIMENT No. 155-Rabbit No. 98a; large; full grown; Belgian hare; 96 days. October 5, 1918, right sciatic exposed; internal popliteal bundle freed; resection 2.5 cm. A segment of equal length taken from the sciatic of another rabbit, stored 11 days in sterile vaseline at 3? C., used as transplant. One central and one distal waxed, fine silk thread suture placed; quite good approximation of nerve ends attained. Dry field; wound closed. January 9, 1919, killed. Rabbit much emaciated; 'fungus' ears; severe neurotrophic ulcer right heel. Wound well healed. Right sciatic exposed full length. External polpliteal bundle quite free; resected and removed. Operated internal popliteal bundle shows the transplant well in place, of good size and light gray color. Large, spindle-shaped central bulbous enlargement noted . After fully exposing the calf muscles and freeing the internal popliteal bundle from bed, on slowly cutting with scissors, the nerve central to transplant, no distinct twitching of calf muscles; the same on cutting distal to transplant. Operated nerve with transplant and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation fair; calf muscles good.
Microscopic findings.- Large central bulb evidenced structurally, from the distal end of which numerous new neuraxes may be traced through the transplant to the distal segment. In sections of calf muscles, numerous new neuraxes noted in the muscular nerves; a few of these may be traced between muscle fibers. Distal regeneration to and into the calf muscles.
EXPERIMENT No. 156- Rabbit No. 100; large; full grown; 155 days. October 8, 1918, left sciatic exposed; internal popliteal bundle freed and resected 3.0 cm. A segment of equal length taken from the sciatic of. another rabbit, stored 12 days in sterile vaseline temperature 3?C., used as transplant. One central and one distal -waxed, fine silk thread suture placed. Good central and distal nerve-end approximation attained. Dry field; wound closed. March 12, 1919, killed. Rabbit in very good condition; neurotrophic ulcer healed; left foot appears normal; walks well, except now and then toe-drop; spreads toes of left foot on holding up by ears. Left sciatic exposed the whole length; external popliteal bundle free; resected and removed. Internal popliteal in operated region has the appearance of a normal nerve; scarcely any evidence of central bulb; no enlargement at the distal wound. Transplant not adherent; no material increase of connective tissue surrounding operated nerve. After exposing the calf muscles and removing skin to heel and completely freeing the operated nerve, on slowly cutting the nerve central to the transplant, vigorous and contraction of calf muscles and apparently foot muscles; the same on cutting nerve distal to transplant. Operated nerve removed and fixed in ammoniated alcohol for pyridine-silver staining; portions of calf muscles removed for gold chloride method of staining nerve terminations. Differential silver staining only of a portion of the nerve good
Microscopic findings.-Transplant well united to resected nerve ends; scarcely any evi-dence of central and distal wounds; these demarked by retained silk sutures. New neuraxes in great numbers pass through transplant to the distal nerve. For the transplanted nerve segment, the perineural sheaths of the funiculi well maintained. Within the funiculi the new neuraxes arranged in small groups, separated by endoneural connective tissue, much more extensive than in normal nerve. An attempt was made to endeavor to stain the motor nerve ending in gold chloride. This attempt not successful. In certain of the large muscular nerve bundles the neuraxes beautifully differentiated even into the smaller branches, but motor ending not differentiated. It seems clear that this is due to faults in the method,
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perhaps impurity of chemical used. Very complete regeneration of distal nerve through the transplanted nerve segment.
EXPERIMENT No. 157.-Rabbit No. 10a; full grown; 155 days. October 8, 1918,right sciatic exposed; internal popliteal bundle freed and resected 3.0 cm. A segment of equal length taken from the sciatic of another rabbit, stored 12 days in sterile vaseline at3° C. used as transplant. One central and distal suture of waxed, fine silk thread placed. Central suture nerve-end approximation good; distal not good. Slight manipulation caused this distal suture to give way. Another suture placed; slight traumatism of nerve end, otherwise approximation good. Fairly dry field; wound closed. March 12, 1919, killed. Rabbit very good condition; small neurotrophic ulcer right heel. Right foot otherwise normal; spreads toes on holding up by ears. Left sciatic exposed the full length. External popliteal free full length; resected; removed. Operated internal popliteal presents scarcely any evidence of central bulb; transplant well in place; good color; good size. After exposing fully the calf muscles and freeing internal popliteal, on slowly cutting nerve central to transplant, good contraction of calf muscles. Operated nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining; calf muscles removed for gold chloride staining. Silver differentiation of neuraxes fairly good.
Microscopic findings.-Transplant well united to resected nerve ends; only slight struc-tural evidence of central bulbous enlargement. New neuraxes in large numbers traced through transplant to distal nerve. In the transplant these new neuraxes arranged in the form of small bundles separated by endoneural connective tissue. An attempt made to stain the motor endings in gold chloride not successful. Numerous neuraxes found in the larger and smaller nerve branches clearly differentiated, but not motor endings. The muscle fibers present the size and structure of normal muscle fibers. Very good regeneration of distal nerve through the transplant.
EXPERIMENT No. 158.-Rabbit No. 108; large; full grown; 1 hour. December 17, 1918, left sciatic exposed; internal popliteal bundle freed; resected 3.0 cm. A segment of equal length taken from the sciatic of another rabbit, stored seven days in sterile liquid petrolatum at 3? C., used as transplant. One central and one distal waxed fine silk thread suture placed; good approximation attained. Wound closed. As soon as wound was closed rabbit stopped breathing and could not be revived. Wound was reopened and the sciatic and transplant removed, about one hour after the operation was completed, and fixed in ammoniated alcohol for pyridine-silver staining. Experiment is recorded, since it enabled examining histologically a nerve stored in liquid petrolatum immediately after it had been placed in the wound. Good differential silver staining.
Microscopic findings.-The appearance presented in cross and longitudinal sections of the transplant, embedded in living tissue only about one hour, may be regarded as essentially the same as that of a nerve stored in liquid petrolatum for a period and examined before transplantation. The sections obtained present in essentials the appearance presented by a fresh nerve fixed and stained after the pyridine-silver method. Especially is this true of cross sections. In longitudinal sections the neuraxes are seen as unsegmented strands of regular contour. The "Golgi-funnels" of the myelin, are distinctly evident though not quite so regular as in a fresh, normal nerve. The sheath cells were not differentiated.
EXPERIMENT No. 159.-Rabbit No. 108a; large; full grown; 1 hour. December 17, 1918, right sciatic exposed; internal popliteal freed; resected 3.0 cm. A segment of equal length removed from the sciatic of another rabbit, stored seven days in sterile liquid petrolatum at 30° C., used as transplant. One central and one distal waxed, fine silk thread suture placed. Good approximation attained. Wound closed. Rabbit stopped breathing while this operation was being completed and could not be revived. About one hour after the operation was begun, sciatic and the transplant removed and fixed in neutral formalin. Stained in iron-hematoxylin and picro-fuchsin and in safranine and licht grün.
Microscopic findings.-Cross and longitudinal sections of the transplant present appear-ances which resemble very closely that of a normal nerve fixed and stained as above indicated In cross sections of the nerve transplant, the nerve fibers have not so compact an arrangement as in a normal nerve, though the fibers themselves have the appearance of normal nerve fibers. In longitudinal sections, the fibers present a regular contour, the neuraxes even
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borders and are not shrunken; the neurokeratin net of the myelin, very regular and distinct. The sheath nuclei present normal form and size and reaction to stains.
EXPERIMENT No. 160.-Rabbit No. 105; full grown; Belgian hare; 2 days. November S.1918, left sciatic exposed; internal popliteal freed; resected 3.0 cm. A segment of equal length taken from the sciatic of another rabbit, stored 38 days in liquid petrolatum at 3? C.,used as transplant. One central and one distal waxed, fine silk thread suture placed; good approximation attained. Wound closed. November 10, rabbit found dead in the morning. Wound clean; healing. Left sciatic exposed. Quite a large blood clot over nerve region of central wound. Nerve sutures in place. Ends of transplant and resected nerve ends not as yet united. Sciatic and transplant removed and fixed in neutral formalin. Stained iniron-hematoxylin, picro-fuchsin and safranine and licht grün.
Microscopic findings.-In both cross and longitudinal sections of the transplant, it may be observed that the transplanted nerve fibers retain their form and structure very well. The neuraxes are not segmented, the myeline sheaths show clearly a neurokeratin net, the sheath nuclei distinctly evident and stain readily, though of more uniform color than normal nuclei.
EXPERIMENT No. 161.-Rabbit No. 105a; full grown; Belgian hare; 2 days. November 8, 1918, right sciatic exposed; internal popliteal freed; resected 2.8 cm. A segment of equal length taken from the sciatic of another rabbit, stored in liquid petrolatum 38 days at 30 C., used as transplant. One central and one distal waxed, fine silk thread suture placed; good approximation. Wound closed. November 10, rabbit found dead in the morning. Wound clean; healing. Right sciatic exposed. Nerve transplant found well in place, not adherent to resected nerve ends and surrounding tissues. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.-Silver staining not successful; no differentiation of neuraxes in normal central stump. In the transplanted nerve the neurolemma sheaths differentiated. These appear slightly thickened; other structures not clearly differentiated.
EXPERIMENT No. 162.-Rabbit No. 106; full grown; 2 days. November 8, 1918, left sciatic exposed; internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the sciatic of another rabbit, stored 38 days in liquid petrolatum at 30 C., used as transplant. Central and distal waxed, fine silk suture placed; good approximation. Wound closed. November 10, rabbit found dead in the morning. Sciatic and transplant removed and fixed in neutral formalin. Stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-Nerve fibers of the transplant very well preserved. No segmentation of the neuraxes of nerve fibers of the transplant noted. In many of the nerve fibers neurokeratin net of the myelin well stained, in others no longer evident. Sheath nuclei evident and well stained. Transplant loosely adherent to resected nerve ends. Centrally very little inwandering of leucocytes into the nerve transplant. Centrally and distally, hemorrhage into resected nerve ends. As yet no evidence of degeneration of the nerve fibers of the distal nerves.
EXPERIMENT No. 163.-Rabbit No. 106a; full grown; 2 days. November 8, 1918, right sciatic exposed; internal popliteal bundle freed; resected 2.5 cm. A segment of equal length taken from the sciatic of another rabbit, stored in liquid petrolatum 38 days at 3? C., used as transplant. One central and one distal waxed, fine silk thread suture placed; approximation good. Wound closed. November 10, rabbit found dead in the morning. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.-In cross sections of the nerve transplant, neuraxes differentially stained, though of paler color than in normal nerve; neurolemma sheaths stand out clearly and appear as if slightly thickened.
EXPERIMENT No. 164.-Rabbit No. 102; full grown; 4 days. November 4, 1918, left sciatic exposed; internal popliteal freed; resected 2.8 cm. A segment of equal length taken from the sciatic of another rabbit, stored in sterile petrolatum 35 days at 3VC., used as transplant. One central and one distal waxed, fine silk thread suture placed; good approximation. Wound closed. November 8, rabbit found dead in the morning. Superficial wound clean and dry; nearly healed. Left sciatic exposed. Transplant found well in place; sutures show
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clearly. Transplant presents a light yellow-white color; not adherent to underlying muscle; loosely united to the resected nerve ends. Resected nerve ends appear congested. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fair differential silver staining attained.
Microscopic findings.-In the transplanted nerve segment , both in cross and longitudinal sections, the neuraxes still evident, though staining very lightly, In the majority not as vet frag- mented; certain ones showing a granular change. Neurolemma sheaths well maintained, and appear slightly thickened. Neurokeratin net and Golgi funnels not clearly seen. In the central end of the transplant are seen a number of distended capillaries, grown into the transplant from the central nerve stump. These capillaries have only endothelial walls and are distended with blood cells and have grown toward the periphery between the nerve fibers of the transplant. The distal segment presents evidence of early stages of nerve degeneration.
EXPERIMENT No. 165.-Rabbit No. 102a; full grown; 4 days. November 4, 1918, right sciatic exposed; internal popliteal freed; resected 2.8 cm. A segment of equal length taken from the sciatic of another rabbit and stored in sterile liquid petrolatum 35 days at 30 C., used as transplant. One central and one distal waxed, fine silk thread suture placed; good approxi- mation. Wound closed. November 8, rabbit found dead in the morning. Superficial wound found clean and dry and nearly healed. Right sciatic exposed. Transplant found well in place, of yellow-white color, nonadherent and loosely united to the resected nerve ends. Sciatic and transplant removed and fixed in neutral formalin. Stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-In cross and longitudinal sections of the nerve transplant, the nerve fibers found to be very well maintained; neuraxes present and not fragmented. The neurokeratin net of myelin only here and there clearly brought out. The neurolemma sheaths not collapsed, and of regular contour. Neuraxes of the transplant seen best preserved in the immediate vicinity of the central and distal wounds; in these regions stain much more clearly than in the body of the transplant. Nerves of the distal segment show beginning stages of degeneration evidenced in fragmentation of the myelin.
EXPERIMENT No. 166.- Rabbit No. 117; full grown; 4 days. December 27, 1918, left sciatic exposed; internal popliteal freed; resected 3.1 cm. A segment of equal length taken from the sciatic of another rabbit, stored 13 days in sterile liquid petrolatum at 3° C., used as transplant. One central and one distal waxed, fine silk thread suture placed; good approximation. Wound closed. December 31, rabbit found dead in the morning. Superficial wound healed. On removing skin over operated field, bloody exudate in subcutaneous tissue about wound and in deeper wound about transplant noted. Transplant found well in place; not adherent to surrounding tissue, loosely united to resected nerve ends. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. In part very good silver differentiation attained. Microscopic findings.-In longitudinal sections of the transplanted nerve segment, it may be observed that the neuraxes are undergoing changes; many appear fragmented into longer or shorter segments, of distinctly granular structure. The neurolemma sheaths well maintained, though in many fibers showing alternate slight distensions or constriction Capillaries coming from the central nerve stump extend nearly the whole length of the nerve transplant. Distal nerve shows early stages of degeneration.
EXPERIMENT No. 167.- Rabbit No. 117a; full grown; 4 days. December 27, 1918, right sciatic exposed; internal popliteal freed; resected 3.2 cm. A segment of equal length taken from the sciatic of another rabbit, stored 13 days in sterile liquid petrolatum at 3? C., used as transplant. One central and one distal waxed, fine silk suture placed; good approximation. Diameter of transplant smaller than that of the resected nerve. Wound closed. December 31, rabbit found dead in the morning. Superficial wound found healed. Right sciatic exposed. Transplant found well in place; but surrounded by a blood clot. Sciatic and transplant removed and fixed in neutral formalin. Stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.- In cross and longitudinal sections of the transplanted nerve segment, it is observed that the neuraxes are beginning to show a fragmentation. These fragments of neuraxes are found inclosed in nmyelin segments in which the neurokeratin net ;
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is still evident, and are found within neurolemma sheaths. No evidence of proliferation of the sheath cells of the transplanted nerves ascertained. Capillaries containing blood cells, and extravasated blood cells found between the nerve fibers of the transplant. In the distal segment, beginning fragmentation of neuraxes and myelin; hypertrophy of the sheath cells noted; here and there these fill the neurolemma sheaths; as yet no distinct proliferation of the sheath cells observed.
EXPERIMENT No. 168.-Rabbit No. 127; full grown; 5 days. March 4, 1919, left sciatic exposed; internal popliteal freed; resected 3.1 cm. A segment of equal length taken from the sciatic of another rabbit, stored twenty-one days in liquid petrolatum at 3° C., used as transplant. One central and one distal waxed, fine silk thread suture placed. Good central approximation attained; distal only fair. Wound closed. March 9, rabbit found dead in the morning. Transplant found well in place, easily demarked by sutures; united to resected nerve ends; not adherent to surrounding tissue. Sciatic and transplant removed and fixed in neutral formalin. Stained in safranine and licht grün.
Microscopic findings.-In longitudinal sections of the transplanted nerve segments fragmentation of neuraxes noted; these fragments have a granular structure. The neurolemma sheaths well maintained but of irregular contour. Long, rod-shaped nuclei are found in relation with the nerve fibers. It is difficult to determine whether these nuclei are within it he neurolemma sheaths or situated on their outer surface. The appearances presented in cross sections of the transplant enable the determination that the majority of these nuclei are situated between the nerve fibers, thus outside of the neurolemma sheaths and of connective tissue derivation. Numerous wandering leucocytes found between the nerve fibers of transplant in the immediate vicinity of both the central and distal wounds.
EXPERIMENT No. 169.-Rabbit No. 112; full grown; 6 days. December 21, 1918, left sciatic exposed; internal popliteal freed; resected 2.3 cm. A segment of equal length taken from the sciatic of another rabbit, stored nine days in sterile liquid petrolatum at 3° C.,used as transplant. One central and one distal waxed, fine silk thread suture placed. Central suture not good; removed and resutured; approximation fair; distal good. Wound closed. December 27, rabbit found dead in the morning. Superficial wound healed; deep wound, blood clot about transplant. Central suture not good. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. It part good silver differentiation attained.
Microscopic findings.-New neuraxes budding from central stump; neuraxes can be traced into the central wound; these not as yet numerous. In the transplant, the neuraxes of the transplanted nerves found for the greater part in the form of short segments having wavy or spiral course. Neurolemma sheaths well maintained and of regular contour. Beginning stages of nerve degeneration noted in the distal segment.
EXPERIMENT No. 170.-Rabbit No. 112a; full grown; 6 days. December 21. 1918, right sciatic exposed; internal popliteal freed; resected 2.6 cm. A segment of equal length taken from the sciatic of another rabbit, stored nine days in sterile liquid petrolatum at 3? C., used as transplant. One central and one distal waxed, fine silk suture placed; good approximation; small hematoma under fascial tissue in popliteal space. Wound closed. December 27, rabbit found dead in the morning. Wound well healed. On exposing sciatic, transplant found well in place and united to the resected nerve ends. Sciatic and transplant removed and fixed in neutral formalin. Stained in iron-hematoxylin and picro-fuchin, safranine and licht grün.
Microscopic findings.-In longitudinal sections of the transplanted nerve segment, the neuraxes of the transplanted nerves appear fragmented in short segments. These segments are here and there swollen and globular and for the most part inclosed in myelin in which a neurokeratin net is still evident. The neurolemma sheaths are well maintained. Within these sheaths are found the myelin segments and neuraxes segments shrunken in longitudinal direction, leaving spaces in which a granular precipitate is seen. Sheath cells not evident. Capillaries course between the nerve fibers of the transplant. Distal segment found degenerating.
EXPERIMENT No. 171.-Rabbit No. 109; full grown; Belgian hare; 12 days. December18, 1918, left sciatic exposed and internal popliteal bundle freed; resected 2.4 cm. A segment
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of equal length taken from the sciatic of another rabbit, stored eight days in liquid petrolatum at 3? C., used as transplant. One central and one distal suture placed; approximation, both central and distal, only fair. Wound closed. December 30, rabbit found dead in the morning. Wound well healed. Left sciatic exposed. Transplant found well in place and adherent to surrounding tissue which is discolored; bloody exudate. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation good; nuclei in part stained.
Microscopic findings.-Early stages of central bulbous enlargement evidenced struc-turally. Central end of transplant well united to central stump; fibrous and cellular tissue constitutes central wound. Down-growing neuraxes from the central stump noted in passage through central wound. Certain of these have reached the central end of the transplant in which they have passed distally to the extent of approximately 2 mm. In the greater part of the nerve transplant no new neuraxes seen. Many remnants of old neuraxes evident,
FIG. 228- Cross section of homo-nerve transplant, stored in liquid petrolatum, at 3°C., 8 days before use as transplant, Experiment No. 171, 12 days after operation; pyridine-silver prepara- tion. Note the distinct funicular structure of the nerve transplanted and the want of fibro blastic differentiation about the nerve transplant as a whole either deeply stained fragments or of lighter color and granular structure. Sheath cells here and there evident. Myelin not clearly differentiated. In the distal nerve segment, nerve fibers in degeneration, sheath cells proliferated; many nucleated, syncytial protoplasmic bands; myelin ovoids and fragments of old neuraxes present.
EXPERIMENT No. 172.-Rabbit No. 109a; full grown; Belgian hare; 12 days. December 18, 1918, right sciatic exposed; internal popliteal bundle freed; resected 2.5 cm. A segment of equal length taken from the sciatic of another rabbit, stored eight days in sterile liquid petrolatum at 3° C., used as transplant. One central and one distal waxed, fine silk-thread suture placed; good approximation attained. Dry wound; wound closed. December 30, rabbit found dead in the morning. Superficial wound well healed. Oil removing skin over operated area, small area of focal infection noted; deep wound healed, no evidence of infection. Transplant found well in place; only moderately adherent to surrounding tissue. The sciatic and transplant removed and fixed in neutral formalin. Stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
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Microscopic findings.-Transplant firmly united to central and distal ends of resectednerve by means of cellular fibrous tissue layer. Distinct central bulbous enlargement and slight distal enlargement. In longitudinal sections of the transplant, taken from its middle third, neurolemma sheaths of the transplanted nerve fibers well maintained and appear as slightly thickened. Myelin observed in the form of smaller and larger globules or segments, the larger of which inclose fragments of old neuraxes. Fibrous sheaths of the transplant invaded by wandering cells. Within the transplant many long nuclei with rounded ends. The majority of these appear to be situated between the nerve fibers; others appear to be within the neurolemma sheaths; their histogenesis uncertain. Distal segment of nerve found in degeneration.
EXPERIMENT No. 173.-Rabbit No. 107; large; full grown; Belgian hare; 23 days. November 10, 1918, left sciatic exposed; internal popliteal bundle freed; resected 3.2 cm. A segment of equal length taken from the sciatic of another rabbit, stored thirty-nine days in sterile liquid petrolatum at 3º C., used as transplant. One central and one distal waxed, fine silk-thread suture placed; very good approximation attained. Dry field; wound closed. December 2, rabbit found dead in the morning. Wound well healed. Left sciatic exposed full length. External popliteal free and runs normal course. The nerve transplant found well in place. Material increase of connective tissue about the central and distal wounds. Transplant of yellow-white color and appears as if slightly congested. Transplant firmly united to resected nerve ends. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation centrally very good, distally not uniform.
Microscopic findings.-A distinct central bulb evidenced structurally. From the distal end of this bulb numerous neuraxes pass to and through the central wound into central end of transplant, in which they may be traced to the neighborhood of the distal wound. The distal wound appears not to have been penetrated by down-growing neuraxes nor area any new neuraxes to be found in the central end of the distal nerve. In cross sections of the nerve transplant, about 1 cm. distal to the central wound, new neuraxes found in all parts of the transplanted nerve. In many regions small groups of new neuraxes appear to pass distally within one old neurolomma sheath; certain neuraxes observed between or out-side of neurolemma sheaths. Perineural sheaths of the transplanted nerve funiculi not materially thickened.
EXPERIMENT No. 174.-Rabbit No. 107a; large, full grown; Belgian hare; 23 days. November 9, 1918, right sciatic exposed; internal popliteal freed; resected 3.2 cm. A segment of equal length taken from the sciatic of another rabbit, stored 39 days in sterile liquid petrolatum at 30 C., used as transplant. One central and one distal waxed, fine silk-thread suture placed; good approximation; dry field; wound closed. December 2, rabbit found dead in the morning. Wound healed. Cold abscess over right gluteal region; not related to wound. The right sciatic exposed full length. Transplant found well in place and firmly united to the resected nerve ends; quite adherent to surrounding tissue; of yellow-white color. Quite well developed central bulbous enlargement. Sciatic and transplant removed and fixed in neutral formalin. Stained in iron-hematoxvlin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-Fibrocellular central wound into which extend nucleated prot plasmic bands. In longitudinal sections of the transplant, from about its middle third, myelin of the transplanted nerve segments in the form of larger and smaller globules, certain of which are found to contain fragments of the old neuraxes. Numerous, relatively large round or oval nuclei found within the old neurolemma sheaths. Histogenesis of these is uncertain. The neurolemma sheaths well maintained and appear slightly thickened. Distal nerve not sectioned.
EXPERIMENT No. 175.-Rabbit No. 133; large; old; Belgain hare; 82 days. March 15,1919, left sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length taken from the sciatic of another rabbit, stored seven days in sterile liquid petrolatum at 3ºC., used as transplant. One central and one distal waxed, fine silk-thread suture placed; good approximation attained. Wound not quite dry; wound closed. June 5, killed. For several days had not eaten well; much emaciated; moribund when killed. Severe neurotrophic
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ulcer left heel. Wound well healed. Left, sciatic exposed full length. External popliteal found free from operated internal popliteal. The transplant found well in place; no material increase of connective tissue about transplant. Large, spindle-shaped central bulbous enlargement. Distal suture clearly seen. Calf muscles exposed; these are atrophic and of pale-red color; appearance of degenerated muscles. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation not uniform throughout.
FIG. 229.- Cross section of homo-nerve transplant, stored in liquid petrolatum at 30 C. for 39 days before use, Experiment No. 174, removed 23 days after operation; pyridine-silver preparation. Attention is called to the distinct funicular structure presented by this nerve and the small amount of fibrous tissue development surrounding the nerve
Microscopic findings:-New down-growing neuraxes can he traced from distal end of central bulbous enlargement to and into transplant and in this to and through the distal wound into the central end of the distal nerve segment. Within the transplant these new neuraxes have a very regular, longitudinal course and appear to pass distally within and outside of old neurolemma sheaths. Areas of degenerated or fragmented myelin of the transplanted nerves observed, between which and around which the down-growing neuraxes pass.
EXPERIMENT No. 176.-Rabbit No. 131; large; full grown; 82 days. March 12, 1919.left sciatic exposed; internal popliteal bundle freed; resected 2.9 cm. A segment of equal
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length taken from the sciatic of another rabbit, stored in sterile liquid petrolatum 23 days at 3ºC., used as transplant. One central and one distal suture of waxed, fine silk thread placed. Central suture pulled out; resutured, a little traumatism to central resected stump resulted; central approximation only fair; distal good. Wound closed. June 2, rabbit found dead in the morning. Slightly emaciated; neurotrophic ulcer left heel. Wound well healed. Left sciatic exposed full length. No material increase of connective tissue about nerve. External adherent to side of operated internal popliteal. Transplant well in place; of light reddish-brown color; not adherent to surrounding tissue. Sciatic and transplant
FIG. 230.- Cross section of homo-nerve transplant stored in liquid petrolatum 39 days at 3º C. before use; Experiment No. 174. Experiment terminated 23 days after operation. Higher magnification of portion of the larger funiculus shown in Figure 229. Note the new neuraxes seen in cross section as fine black dots, many of which are found within neurolemma sheaths of the transplanted nerve fibers
removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation only partially successful; tissue not well embedded and difficult to cut; sections torn.
Microscopic findings.-Numerous new neuraxes can be traced from the central stump and through the distal wound. The differential staining of the distal nerve not successful, thus can not clearly determine whether new neuraxes have reached the distal popliteal.
EXPERIMENT No. 177.-Rabbit No. 129; full grown; 112 days. March 7, 1919, left sciatic exposed; internal popliteal freed; resected 2.9 cm. A segment of equal length taken from the sciatic of another rabbit, stored in sterile liquid petrolatum 17 days at 3° C., used as
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transplant. One central and one distal waxed, fine silk thread suture placed; good approximation. Wound closed. June 27, killed. Rabbit much emaciated; not well; severe neurotrophic ulcer left heel. Wound well healed. Left sciatic exposed full length. External popliteal adherent to operated internal popliteal; both bundles surrounded by a dense fibrous tissue sheath and adherent to underlying muscle. Transplant found well in place and of good size. Calf muscles fully exposed; are atrophic and of pale red color. Sciatic completely freed from bed. On slowly cutting with scissors, sciatic central to transplant, vigorous contraction of the foot flexors supplied by the external popliteal, unoperated, but only very feeble and doubtful twitching of calf muscles supplied by operated internal popliteal. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fair differential silver staining throughout the series of sections attained.
Microscopic findings.-Large, spindle-shaped central bulbous enlargement evidenced structurally, from the distal end of which new, down-growing neuraxes may be traced through the transplant, to and through the distal wound and in good numbers into the central end of the distal popliteal, in which they may be traced to the lower level of the popliteal space. Unfortunately the calf muscles were not removed for microscopic examination; it is thus not possible to report on the presence or absence of new neuraxes in the calf muscles. Microscopic findings indicate partial regeneration of the distal nerve segment through the nerve transplant.
EXPERIMENT No. 178.-Rabbit No. 104; full grown; 117 days. November 7, 1918, left sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length taken from the sciatic of another rabbit, stored 38 days in sterile liquid petrolatum at 3º C., used as transplant. One central and one distal waxed, fine silk thread suture placed; good central approximation, distal fair. Wound closed. March 4, 1919, killed. Rabbit found nearly moribund; "fungus" ears; eyes infected; much emaciated; wound well healed; neurotrophic ulcer left heel. On exposing the left sciatic, transplant found well in place, of small diameter, adherent to underlying muscle. Large, spindle-shaped central bulb. Calf muscles fully exposed; these appear atrophic and of pale red color and do not respond distinctly on cutting the sciatic central to the transplant. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Not complete and uniform silver differentiation.
Microscopic findings.-Sections show sufficient silver differentiation of neuraxes to determine the observation that neuraxes growing from the central bulb enter the transplant and pass through this to and through distal wound into the central end of distal popliteal, in which region the staining is better; here numerous new neuraxes are differentiated. Regeneration through transplant of central end of distal popliteal.
EXPERIMENT No. 179.-Rabbit No. 104a; full grown; 117 days. November 7, 1918,right sciatic exposed; internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the sciatic of another rabbit, stored 38 days in sterile liquid petrolatum at 3º C.,used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. March 4, 1919, killed. Rabbit found nearly moribund; only slight neurotrophic ulcer right heel. On exposing right sciatic, transplant is found well in place, of good size and only slightly adherent to the underlying muscle. Large central bulb. No record of test of muscles. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation very unsatisfactory and imperfect.
Microscopic findings.-Transplant well united to resected nerve end. New neuraxes appear to be present in the transplant; this can not be determined definitely since silver differential staining is unsuccessful. Experiment not conclusive.
EXPERIMENT No. 180.-Rabbit No. 130; full grown; 120 days. March 10, 1919, left sciatic exposed; internal popliteal bundle freed; resected 3.4 cm. A segment of equal length taken from another rabbit, stored 20 days in sterile liquid petrolatum at 3º C., used as transplant. One central and one distal waxed, fine silk thread suture placed; centrally, good approximation; distally only fair. Wound closed. July 8, rabbit found dead in the morning; examined soon after death. Neurotrophic ulcer left heel noted. On exposing left sciatic, the nerve transplant found well in place, of good size and dull-white color and only moderately adherent to underlying muscle. Small, spindle-shaped central bulb, and slight enlargement
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of central end of distal stump noted. Calf muscles fully exposed; these appear as if partly recovered; good color though not full size. Muscles could not be tested as regards functional return, because animal had been dead some time before it was examined. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation attained.
Microscopic findings.-In alternate cross and longitudinal sections of the operated nerve, numerous new neuraxes coming from the central stump may be traced through the trans-plant to the distal popliteal. In cross sections of the transplant near the central and distal wounds, there may be observed an increase of the endoneural connective tissue but no material thickening of the perineural sheaths of the funiculi. The new neuraxes are found in small groups, separated by endoneural connective tissue and are equally distributed through all parts of the transplant.
EXPERIMENT No. 181.-Rabbit No. 114; full grown; 145 days. December 24, 1918,left sciatic exposed; internal popliteal freed; resected 2.1 cm. A segment of equal length taken from the sciatic of another rabbit, stored 11 days in sterile liquid petrolatum at 3 º C.,used as transplant. Only fair central and distal approximation attained. Wound closed. May 18, 1919, rabbit found dead in the morning. Emaciated; "fungus" ears, neurotrophic ulcer left heel, which seemed to be healing; wound well healed. On exposing left sciatic, transplant found well in place, no material increase of connective tissue about operated nerve not adherent to underlying muscle. Quite distinct central bulb noted. Distal nerve presents the appearance of a normal nerve. Calf muscles fully exposed, have the appearance of regenerating muscle, though not fully recovered. Could not be tested as to functional return-animal dead. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining; silver staining faint but differential.
Microscopic findings.-Neuraxes in very good numbers pass from the distal end of the central stump, through the transplant to the distal nerve, in which they are distributed in good numbers in all of the funiculi. In cross sections of the transplant, the new neuraxes found mostly in small groups, separated by strands of endoneural tissue; quite evenly distributed over the entire transplant. Here and there small areas or columns of m yeling lobules and detritus, derived from the myelin sheaths of the transplanted nerve fibers, are to be found. Such areas not generally traversed by neuraxes. Very complete regeneration of the distal nerve, through the transplant, evidenced structurally.
EXPERIMENT No. 182.-Rabbit No. 114a; full grown; 145 days. December 24, 1918, right sciatic exposed; internal popliteal bundle freed; resected 2.9 cm. A segment of equal length taken from the sciatic of another rabbit, stored 11 days in sterile liquid petrolatum at 3 º C., used as transplant. One central and one distal waxed, silk thread suture placed. Good central, only fair distal approximation attained. Wound closed. May 18, 1919,rabbit found dead in the morning. Large neurotrophic ulcer right heel; apparently healing. On exposing right sciatic, transplant found well in place; external popliteal looped over it, though not adherent; no material increase of connective tissue about nerves. Distinct spindle-shaped central bulb, only indistinct enlargement of central end of distal stump. Distal nerve has the appearance of normal nerve. Calf muscles exposed; not fully recovered, though present the appearance of regenerating muscle. Could not be tested as to functional return. Sciatic and transplant removed and fixed in neutral formalin. While tissues were being prepared for embedding and serial section, preparatory to staining iniron-hematoxylin and picro-fuchsin, it was accidentally thrown out, thus not available for report as to microscopic findings.
EXPERIMENT No. 183.-Rabbit No. 128; full grown; Belgian hare; 138 days. March 5, 1919, left sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length taken from the sciatic of another rabbit, stored 22 days in sterile liquid petrolatum at 3 º C., used as transplant. One central and distal waxed, fine silk-thread suture placed. Good approximation attained. Wound closed. July 21, rabbit found dead in the morning; much emaciated. Severe neurotrophic ulcer left heel. On exposing the left sciatic, transplant found well in place, of good size and of an appearance similar to resected nerve. Moderately large central bulb noted. Calf muscles still appear somewhat atrophic and of pale red color. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
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Microscopic findings.-Large numbers of neluraxes pass from distal end of central stump, through central wound and transplant, through distal wound into distal popliteal. Certain of the neuraxes passing through the transplant have acquired a myelin sheath. Spindle-shaped areas of myelin globules and granular detritus noted ill longitudinal sections of the transplant. These lie between bundles of descending neuraxes. Endoneural connective tissue not materially increased; while the perineural sheaths are materially thick Numerous new neuraxes in the distal popliteal in all of the funniculi. Regeneration of the distal nerve evidenced structurally.
EXPERIMENT No. 184.-Rabbit No. 101; full grown; 155 days. October 8, 1918, left sciatic exposed; the internal popliteal freed; resected 3.5 cm. External popliteal bundle accidently cut while separating it from the internal popliteal; disregarded .A segment of equal length taken from the sciatic of another rabbit, stored nine days in sterile liquid petrolatum at 3 º C., used as transplant. One central and one distal waxed, silk thread suture placed. Approximation of central ends fair; distally good direction, but suture not well placed, not good approximation attained. Muscle torn in exposing nerve; oozing, not fully controlled. Wound closed. March 12, 1919, killed. Rabbit in very good condition. One toe left foot missing; neurotrophic ulcer of left heel practically healed; does not spread toes of left foot on lifting up rabbit by ears. On exposing the left sciatic, transplant found well in place, of good size; quite material increase of connective tissue about operated nerve. Only indistinct central bulb. External popliteal accidently cut and not sutured, found united, slight bulbous enlargement at the place of cutting. Calf muscles and other leg muscles fully exposed, and internal popliteal and transplant completely freed from bed. On slowly cutting internal popliteal bundle central to transplant, vigorous contraction of calf muscles; same on cutting distal to transplant; no distinct response from plantar foot muscles. Plantar foot muscles have nearly recovered normal size and are of pale red color. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining; portions of calf muscles removed for gold chloride staining. Good differential silver staining attained.
Microscopic findings.-Numerous new fibers, both myolinated and nonmyelinated, pass through transplant to the distal stump. Scarcely any myelin globules, remains of myelin of transplanted nerve, to be found in the transplant. In cross sections of the transplant near both central and distal wounds, numerous neuraxes, evenly distributed over transplant, to be seen. Very little increase of endoneural connective tissue to be noted; perineural tissue not materially increased. Numerous new neuraxes in distal popliteal in all of the funiculi. In the calf muscles stained after the gold chloride method, the neuraxes of the nerve fibers of the larger muscular bundles well stained, as also in certain smaller muscular branches. Motor endings nowhere stained. This is regarded as inconclusive, and appears to be due to imperfect differentiation, since the teased muscle fibers appear to present normal structure. Regeneration and return of motor function in calf muscle, though this latter is not fully checked by histologic findings.
EXPERIMENT No. 185.-Rabbit No. 101a; full grown; 154 days. October 9, 1918, right sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length taken from the sciatic of another rabbit, stored nine days in sterile liquid petrolatum, at 3° C..used as transplant. One central and one distal waxed, fine silk thread suture placed; good approximation attained. Wound closed. March 12, 1919, killed. Rabbit in very good condition, healed neurotrophic ulcer right heel; spread toes of right foot on elevating rabbit by ears. On fully exposing right sciatic and calf muscles, transplant found well in place with only slight increase of connective tissue about operated nerve. Transplant of good size and good color. Only slight bulbous enlargement of central stump noted. After completely freeing the operated internal popliteal and transplant, cutting the same central to the transplant, good contraction of calf muscles, contraction of foot muscles somewhat uncertain. Operated nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining; portions of calf muscles for gold chloride staining. Good silver differentiation attained.
Microscopic findings.-Numerous, both myelinated and nonmyelinated neuraxes to be traced from the distal end of the central stump to and through transplant, to distal popliteal.
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In cross sections of the transplant, neuraxes seem to be evenly distributed over transplant. Narrow streaks of myelin remains, with these certain large vesicular cells, found between bundles of new neuraxes; these remains not numerous. Endoneural connective tissue not materially increased. In the gold chloride stained pieces of muscle, neuraxes well stained in the larger and smaller muscular nerves. The motor endings not differentiated; apparently due to faulty differential staining; muscle fibers present a normal structure. Regeneration of distal popliteal through transplant.
EXPERIMENT No. 186.-Rabbit No. 111; full grown; 196 days. December 20, 1918, left sciatic exposed; internal popliteal bundle freed; resected 3.0 cm. A segment of equal length taken from the sciatic of another rabbit, stored 10 days in sterile liquid petrolatum at 3º C., used as transplant. One central and one distal suture placed, waxed, fine silk thread used. Good approximation attained. Dry field; wound closed. July 4, 1919, rabbit found dead in the morning; seemed in fairly good condition the day before; severe neurotrophic ulcer left heel; healing. On exposing the left sciatic, transplant found well in place and presents the appearance of a normal nerve; no material increase of connective tissue about it. Large oval-shaped central bulb; central end of distal stump not materially increased. Distal nerve presents the appearance of a normal nerve trunk. The calf muscles exposed; they present a pale red color and are still slightly atrophic. Could not be tested as to functional return. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation not wholly satisfactory.
Microscopic findings.-In cross and longitudinal sections, sufficient differential silver staining of neuraxes found to determine the fact that numerous, both myelinated and non-myelinated neuraxes, extend through the transplant from the central to the distal stump. Columns and spindle-shaped areas of myelin globules and detritus, are here and there noted in the longitudinal sections of the transplant; these show as round or oval areas in cross sections of the transplant. Apparent regeneration of the distal nerve through the transplant.
EXPERIMENT No. 187.-Rabbit No. 111a; full grown; 196 days. December 20, 1918, right sciatic exposed; internal popliteal freed; resected 2.6 cm. A segment of equal length taken from the sciatic of another rabbit, stored 10 days in sterile liquid petrolatum at 3º C used as transplant. One central and one distal waxed, fine silk thread suture placed; good approximation attained. Dry field; wound closed. July 4, 1919, rabbit found dead in the morning. Seemed in fairly good condition the day before; healing neurotrophic ulcer right heel. On exposing the right sciatic, it is found that the transplant is well in place of good size and color and not adherent to underlying muscle. Small, oval-shaped central bulb noted. Distal nerve presents the appearance of normal nerve trunk. Notes do not record the appearance and condition of the calf muscles. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation not wholly satisfactory.
Microscopic findings.-In cross and longitudinal sections, sufficient differentiation of neuraxes to enable determining the fact that numerous neuraxes, both myelinated and non-myelinated, pass from the central stump through the transplant to the distal internal popliteal. Scarcely any remains of the myelin of the transplanted nerve noted. Apparent regeneration of the distal popliteal through the transplant.
EXPERIMENT No. 188.-Rabbit No. 116; full grown; 223 days. December 27, 1918, left sciatic exposed; internal popliteal freed; resected 3.1 cm. A segment of equal length taken from the sciatic of another rabbit, stored 13 days in sterile liquid petrolatum at 3º C.,used as transplant. One central and distal suture placed; good approximation attained. Slight oozing of blood from distal stump into distal wound; not fully controlled. Wound closed. August 7, 1919, rabbit found dead in the morning; seemed in good condition, "fungus" ears; neurotrophic ulcer left heel, seems to be healing. On exposing the left sciatic, transplant found well in place; of good size and color; presents the appearance of normal nerve; not materially adherent to underlying muscle. Calf muscle exposed, of nearly normal size, of yellow-red color, streaked with narrow yellow stripes. Sciatic and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation attained throughout whole length of nerve.
Microscopic findings.-Well-developed central bulbous enlargement evidenced structur-ally, from the distal end of which numerous myelinated and nonmyelinated neuraxes can be
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traced through the transplant to the distal popliteal. Scarcely any neuraxes in the connective tissue, surrounding the transplant, found. In cross sections of the transplant, it may be observed that the down-growing neuraxes pass through and between the old neurolemma sheaths. Endoneural and perineural connective tissue not found materially increased. Numerous new neuraxes found in all the funiculi of the distal pop)liteal cut at the lower level of the popliteal space. In longitudinal and cross sections of pieces of calf muscle, silver stained, new neuraxes observed in the larger muscular nerve branches, in the smaller inter-fascicular nerve branches and as single fibers between the muscle fibers; here and there evidence of motor nerve-endings noted. Regeneration of distal popliteal through the transplant, recovery of motor function in calf muscles, as evidenced structurally, obtained.
EXPERIMENT No. 189.-Rabbit No. 116a; full grown; 223 days. December 27, 1918, right sciatic exposed; internal popliteal freed; resected 3 cm. A segment taken from the sciatic of another rabbit, stored 13 days in sterile liquid petrolatum at 3º C., used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Oozing in deep popliteal space, not fully controlled. Wound closed. August 7, 1919, rabbit found dead in the morning; seemed in good condition, "fungus" ears; severe neuro-trophic ulcer right heel; apparently healing. On exposing right sciatic, transplant found well in place; of good size and color; surrounded by relatively dense connective tissue, adherent to underlying muscle. Calf muscles fully exposed; are of good size, yellow-red color, with narrow yellow streaks evident. Sciatic and transplant, with portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining throughout.
Microscopic findings.-Distinct, large spindle-shaped central bulb, from the distal end of which numerous new neuraxes pass through the transplant to the distal popliteal. Scarcely any neuraxes course in the connective tissue outside of the perineural sheaths of the transplanted nerve. In cross sections of the transplanted nerve it may be observed that new neuraxes are distributed quite evenly over the entire transplant. In the distal popliteal lower level of the popliteal space, numerous neuraxes, both myelinated and nonmyelinated, found in all of the funiculi. In sections of the calf muscles, both in cross and longitudinal sections, new down-growing neuraxes found in the larger and smaller muscular branches and as single terminal nerve fibers on and between the muscle fibers. Regeneration of the distal popliteal through transplant and recovery of the motor fibers in the calf muscle.
EXPERIMENT No. 190.-Rabbit No. 115; full grown; 235 days. December 26, 1918,left sciatic exposed; internal popliteal freed; resected 2.9 cm. A segment of equal length taken from the sciatic of another rabbit, stored 13 days in sterile liquid petrolatum at 3º C.,used as transplant. One central and one distal suture placed, waxed, fine silk thread suture; approximation good. Dry field; wound healed. August 18, 1919, killed. Rabbit in good condition; holds head to left side; when attempting to walk forward rolls over; some semi-circular canal condition not pertinent to experiment. On exposing the left sciatic, transplant found well in place; presents appearance of normal nerve, though not as distinctly bounded; only moderate increase of connective tissue about transplant. Distinct central bulb; only slight enlargement of central end of distal stump. External popliteal adherent to operated internal popliteal; cut and in part resected. Calf muscles fully exposed; these present nearly normal size and color. After completely freeing operated internal popliteal and transplant from bed and slowly cutting with scissors internal popliteal central to the transplant, vigorous contraction of the muscles and movement of toes; the same on cutting distal to the transplant. Sciatic nerve and transplant and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Only in part good silver differentiation attained.
Microscopic findings.-Large spindle-shaped bulb, to which is adherent the external popliteal. From the distal end of the central bulbous enlargement on the internal popliteal, numerous myelinated and nonmyelinated neuraxes can be traced through the transplant to the distal popliteal. A few small funiculi of nerve fibers found in the connective tissue outside of the perineural sheaths of the transplanted nerves, on the side toward the adherent external popliteal. In the distal popliteal, new neuraxes in large numbers in all of the funiculi. In sections of the calf muscles, neuraxes noted in the larger and smaller intramuscular branches -
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Regeneration of the distal popliteal through the transplant with regeneration of motor nerves to the calf muscles. Interossei not removed and studied.
EXPERIMENT No. 1919.-Rabbit No. 115a; full grown; 235 days. December 26, 1918, right sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length taken from the sciatic of another rabbit, stored 13 days in sterile liquid petrolatum at 3º C.,used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Dry field; wound closed. August 18, 1919, killed. Good general condition; holds head to left side; when attempting to walk, falls to side. On exposing the right sciatic, transplant found well in place; presents the appearance of a normal nerve; external popliteal adherent. Large central bulb; central end of distal segment distinctly enlarged. On exposing calf muscles, which are nearly of normal size and color, and completely freeing the internal popliteal and transplant from its bed, slowly cutting with scissors the internal popliteal central to the transplant, causes feeble contraction of calf muscles; more vigorous contraction on cutting distal to the transplant; contraction and movement of toes doubtful. Sciatic and transplant and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Only in part good differential silver staining attained.
Microscopic findings.-Large central bulb, which tapers into transplant. From the distal end of bulb, numerous myelinated and nonmyelinated neuraxes traced through the transplant to the distal popliteal. Scarcely any neuraxes found in the connective tissue outside of the perineural sheaths of the transplanted nerve. These perineural sheaths found quite distinctly thickened. All of the funiculi of the distal popliteal nerve possess numerous new neuraxes, many of which are myelinated. In sections of the calf muscles, new neuraxes found in the intramuscular branches and as single terminal branches on the muscular fibers. Regeneration of distal popliteal through the transplant, also motor nerves in the calf muscles.
EXPERIMENT No. 192.-Rabbit No. 113; full grown; 238 days. December 23, 1918,left sciatic exposed; internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the sciatic of another rabbit, stored 11 days in sterile liquid petrolatum at 3º C., used as transplant. One central and distal waxed, silk thread suture placed; approximation central good; distal good direction,, but transplant twisted one-half spiral. Field not quite dry; wound closed. August 18, 1918, killed. Rabbit not in good condition;"fungus" ears; emaciated; neurotrophic ulcer left heel, not completely healed. On exposing left sciatic, it is noted that muscles of thigh look pale and flabby. Transplant found well in place; good size and color; no material increase of connective tissue about it; well-developed central bulb; quite distinct enlargement of central end of distal stump. Calf muscles fully exposed; these are small and of pale color. After freeing nerve and transplant from bed and on cutting slowly with scissors nerve central to transplant, feeble but distinct contraction of calf muscles and movement of toes noted; also on cutting distal to transplant. Sciatic and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Differential silver staining very good in part; granular deposit obscures in part.
Microscopic findings.-Large central bulb from the distal end of which numerous new neuraxes can be traced to distal popliteal, in which to the level of entrance of branches into the calf muscles new neuraxes are found in large numbers in all of the funiculi. Scarcely any neuraxes found in the connective tissue outside of the perineural sheaths of the transplanted nerves. Portions of calf muscle, in this experiment, were accidently lost. Regellera-tion of distal popliteal to level of calf muscles through the transplant.
EXPERIMENT No. 193.-Rabbit No. 113a; full grown; 238 days. December 23, 1918, right sciatic exposed; internal popliteal bundle freed; resected 2.4 cm. A segment of equal length taken from the sciatic of another rabbit, stored 11 days in sterile liquid petrolatum at 3º C.,used as transplant. One central and one distal suture of waxed, fine silk thread placed; good approximation. Dry field; wound closed. August 18, 1919, killed. Rabbit not in good condition; much emaciated; neurotrophic ulcer right heel; not completely healed. On exposing the right sciatic, external popliteal found free; transplant well in place; good size and color; connective tissue not materially increased about it. Calf muscles fully exposed; these appear small and of pale red color. After freeing nerve and transplant completely, on slowly cutting nerve with scissors central to the transplant, good contraction of the calf
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muscles and distinct movement of the toes noted, the same on cutting distal to the transplant. Sciatic and transplant and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation only in part successful.
Microscopic findings.-From the distal end of a long, spindle-shaped central enlargement embracing the central wound, numerous myelinated and nonmyelinated neuraxes traced through the transplant to the distal popliteal, in which new neuraxes are found in large numbers in all of the funiculi. Scarcely any neuraxes found in the connective tissue surrounding the transplant. Pieces of calf muscle accidently lost. Regeneration of distal popliteal through transplant.
EXPERIMENT NO. 194.-Rabbit No. 110; full grown; 242 days. December 19, 1918, left sciatic exposed; internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the sciatic of another rabbit, stored eight days in sterile liquid petrolatum at 3º C..used as transplant. One central and one distal suture of waxed, fine silk thread used; good approximation; dry field; wound closed. August 18, 1919, killed. Rabbit in good condition; walks well; neurotrophic ulcer left heel very nearly healed. On exposing the left sciatic, the external popliteal found in very close approximation to operated internal popliteal. The transplant found well in place; good size and color, no material increase of connective tissue about the operated nerve. Distinct central bulb and distinct enlargement of central end of distal popliteal. Calf muscles and plantar foot muscles fully exposed; these of normal size and color. After completely freeing the operated nerve from bed, slowly cutting the nerve central to transplant, continued and vigorous contraction of calf and plantar foot muscles; the latter exposed so that contraction was directly observed. Sciatic and transplant and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Fair silver differentiation attained.
Microscopic findings.-From distinct bulbous enlargement of the central stump, numerous myelinated and nonmyelinated neuraxes traced through the transplant to the distal popliteal, through which they are traced to the level of entrance to calf muscles. Scarcely any neuraxes in the connective tissue surrounding the transplanted nerve segments. Numerous neuraxes noted in the intramuscular branches in the sections of the calf muscles. Regeneration of distal popliteal through the transplant, recovery of calf muscles.
EXPERIMENT NO. 195.-Rabbit No. 110a; full grown; 242 days. December 19, 1918, right sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length taken from the sciatic of another rabbit, stored in liquid petrolatum in eight days at 3º C., used as transplant. One central and distal suture of waxed, fine silk thread used; good approximation; field not quite dry; wound closed. August 18, 1919, killed. Rabbit in very good condition; uses right hind foot well; neurotrophic ulcer on right heel very nearly healed. On exposing right sciatic, external popliteal quite free, resected. The transplant found well in place; good size and color, no material increase of connective tissue about it. Distinct central bulb and quite distinct enlargement of central end of distal popliteal. Calf muscles and plantar foot muscles fully exposed; these of normal size and color. After freeing transplant and nerve from bed, on slowly cutting with scissors central to the transplant, good contraction of foot and calf muscles noted. Sciatic and transplant and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Only partially successful silver differentiation attained. This owing to fact that while this and several other series were being embedded in paraffin, steam escaped into chamber containing paraffin dishes with tissues. Sufficient silver differentiation obtained to enable determination of the neuraxes, though these are not distinctly stained.
Microscopic findings.-Large spindle-shaped central bulb, from the distal end of which numerous neuraxes, myelinated and nonmyelinated, can be traced through the transplant to the distal popliteal, in which neuraxes in large numbers are found in all of the funiculi. In sections of the calf muscles, neuraxes noted in the larger and smaller calf muscles and as single terminal nerve branches between and on the muscle fibers. Regeneration of distal popliteal through the transplant, and recovery of motor nerves in calf muscles.
EXPERIMENT No. 196.-Rabbit No. 103; large; full grown; Belgian hare; 286 days. November 5, 1918, left sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length taken from the sciatic of another rabbit, stored 36 days in sterile liquid petrolatum
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at 3 ? C., used as transplant. One central and one distal waxed, fine silk-thread suture placed. Centrally one funiculus of central stump not in good approximation; distally good approximation attained. Dry field; wound closed. August 18, 1919, killed. Rabbit in good condition; walks well; neurotrophic ulcer left heel very nearly healed. On exposing left sciatic, external popliteal found in close apposition to operated internal popliteal. Transplant found well in place; good size and color, very little connective tissue increase about it. Only small, indistinct central bulbous enlargement noted, distally scarcely any enlargement. Calf muscles fully exposed; these have the appearance of normal muscle. After completely freeing transplant and nerve, on slowly cutting nerve with scissors central to transplant, vigorous contraction of calf muscles and movement of toes noted. Sciatic and transplant and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Only partially successful silver differentiation attained.
Microscopic findings.-Not well marked central bulbous enlargement noted, in the center of which an accumulation of small cells, not clearly defined in silver stain, but presenting the appearance of a small localized pus pocket, is noted. From the distal end of this bulbous enlargement, numerous neuraxes, both myelinated and nonmyelinated, can be traced through the transplant to the distal popliteal. In cross sections of the transplant, approximately 1 cm. from the central wound, an accumulation of small cells under the perineural sheath is noted; possibly a small focus of pus cells. Perineural and endoneural connective tissue of the transplant found materially increased. Numerous small nerve bundles in the connective tissue outside of perineural sheaths observed. Muscle pieces came in contact with water while in paraffin; silver differentiation unsuccessful. Regeneration through transplant, although this gives evidence of having been slightly infected, of the distal popliteal to the level of muscular branches to calf muscles, more distally not controlled histologically.
EXPERIMENT No. 197.-Rabbit No. 103a; large; full grown; 286 days. November 5, 1918, right sciatic exposed; internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the sciatic of another rabbit, stored 36 days in liquid petrolatum at 3º C., used as transplant. One central and distal waxed, fine silk-thread suture placed; good approximation; dry field; wound closed. August 18, 1919, killed. Rabbit in very good condition; small healing neurotrophic ulcer left heel. On exposing the right sciatic, external popliteal found in close apposition to operated internal popliteal. Transplant found well in place, good size and color; very little increase of connective tissue about it. Only small central bulbous enlargement noted. Observations necessarily and unavoidably interrupted at this point; before they could be resumed sufficient time had elapsed to make it impossible to obtain contraction of muscles supplied by unoperated external popliteal. Calf muscles presented the appearance of normal muscles, both as to size and color; return of function could not be tested. Sciatic and portions of calf muscles removed, fixed in ammoniated alcohol for pyridine-silver staining. Only partial silver differentiation attained.
Microscopic .findings.-Silver differentiation sufficient to determine the fact that neuraxes, both myelinated and nonmyelinated, pass through the transplant to the distal popliteal, where they were traced to and into the muscular branches of the calf muscles. In cross sections of the transplant it is observed that very few neuraxes pass distally in the connective tissue surrounding the transplanted nerve segment. In cross sections of the distal popliteal, numerous neuraxes to be observed in all of its funiculi. Regeneration of distal popliteal through the transplant to the calf muscles.
EXPERIMENT NO. 198.-Rabbit No. 120; full grown; 2 days. January 10, 1919, left sciatic exposed; internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the sciatic of another rabbit, and stored 11 days at room temperature in sterile 50 percent alcohol, used as transplant. Before use, nerve was taken from alcohol and placed for15 minutes in warm, sterile saline solution. One central and distal waxed fine silk thread suture placed; good approximation; slight oozing from central stump, not fully controlled; wound closed. January 12, rabbit found dead in the morning. On exposing sciatic, dry field noted. Transplant found well in place, of dull gray-green color; sutures in place. Ends of resected nerve found congested. Sciatic with transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver staining very pale.
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Microscopic findings.-In longitudinal sections of the transplanted nerve segment the neurolemma sheaths of the nerve fibers distinctly made out; in in myelin in many places traces of Golgi funnels; neuraxes not found segmented but staining very lightly, having even borders and of approximately normal size. In cross sections outline of nerve fibers well maintained, distinctly bounded by neurolemma sheaths; myelin scarcely stained; neuraxes centrally placed in fibers and staining very lightly.
EXPERIMENT No. 199.-Rabbit No. 120a; full grown; 2 days. January 10, 1919, right sciatic exposed; internal popliteal freed; resected 3.3 cm. A segment of equal length taken from the sciatic of another rabbit, stored 11 days at room temperature in sterile 50 percent alcohol, used as transplant. Before use, placed in warm, sterile saline solution for one hour. One central and distal waxed fine silk thread suture placed; good approximation. Wound closed. January 12, rabbit found dead in the morning. On exposing right sciatic, transplant found well in place, sutures evident. Transplant of dull gray-green color, and found not adherent to surrounding tissues. Nerve and transplant removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-In longitudinal sections of the transplant, the neurolemma sheaths of the nerve fibers found well maintained; neurokeratin net of myelin only faintly stained. Neuraxes found not segmented. In longitudinal sections embracing the central and distal wounds, respectively, inwandered leucocytes in ends of the transplant distinctly observed. These inwandered cells extend for a distance of about 2 mm., both at the central and distal ends of the transplant, mainly between the nerve fibers, certain few within the neurolemma sheaths of the transplanted nerve fibers. No neurolemma sheath cells of transplanted nerves clearly made out.
EXPERIMENT No. 200.-Rabbit No. 123; full grown; 23 days. January 18, 1919,left sciatic exposed; internal popliteal freed; resected 2.8 cm. A segment of equal length taken from the sciatic of another rabbit, stored 17 days at room temperature in sterile 50 per cent alcohol, used as transplant. Before use, placed in sterile, warm saline solution 10 minutes. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. February 10. Died, nerve taken out just after death of rabbit; still warm; beginning neurotrophic ulcer left heel. Wound well healed. On exposing the left sciatic, transplant found well in place, united to resected nerve ends; of slightly smaller diameter in middle portion than at ends; no material increase of connective tissue about transplant. Distinct, central bulbous enlargement noted. The nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver staining differential but faint.
Microscopic findings.-Numerous neuraxes extend from distal end of the central stump, through central wound into the central end of the nerve transplant, in which they may be traced distally for a distance of approximately 2 cm. In cross sections of the middle of the transplant small groups of neuraxes found within as well as outside of the neurolemma sheaths are to be observed. Neurolemma sheaths in certain regions widely distended or broken down. In such regions myelin globules, granular detritus, and large vesicular cells enclosing what appears to be lipoid globules encountered. Perineural sheaths of transplant well maintained, not materially thickened. No neuraxes found in the connective tissue out-side of this sheath. New neuraxes have not reached the distal stump; this shows nerve fibers in process of degeneration.
EXPERIMENT No. 201.-Rabbit No. 123a; full grown; 23 days. January 18, 1919,right sciatic exposed; internal popliteal freed; resected approximately 3 cm. A segment of equal length taken from the sciatic of another rabbit, stored 17 days in 50 percent alcohol at room temperature, used as transplant. One central and one distal suture placed. While manipulating transplant, central suture pulled out; transplant resected and resutured, final length approximately 2 cm.; good approximation. Wound closed. February 10, died. Nerve taken out just after death; still warm. Wound well healed. On exposing the right sciatic, transplant found well in place; united to resected nerve ends, of good size and dull gray-green color. Distinct central bulbous enlargement noted. Nerve and transplant removed and fixed in neutral formalin. Sections stained in safranine and licht grün.
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Microscopic findings.-Transplant found well united to central and distal resected ends of internal popliteal; only narrow connective tissue wounds evident. In longitudinal sectionsof the transplanted nerve segment, strands of syncytial nucleated protoplasmic bands, apparently extending from the distal end of the central stump into the transplant, are found separated by narrow areas or columns of myelin globules, granular detritus, and vesicular cells with lipoid globules, the remains of the myelin of the transplanted nerve fibers. This structure extends to the distal end of the transplant, where, near the distal wound, the protoplasmic bands are more widely separated and less numerous, the intervening spaces wider. The central end of the distal stump presents the appearance of a nerve in degeneration.
EXPERIMENT No. 202.-Rabbit No. 118; full grown; 42 days. December 28, 1918,left sciatic exposed; the internal popliteal bundle freed; resected 2.4 cm. A segment of equal length taken from the sciatic of another rabbit, stored seven days at room temperature in sterile 50 percent alcohol, used as transplant. Before use, nerve placed in sterile, warmed saline solution ten minutes. One central and one distal waxed, fine silk thread suture placed. Good approximation attained, though the transplanted nerve segment is of distinctly smaller diameter than the resected nerve. Wound closed. February 8, 1919, killed. Rabbit not in good condition; "fungus" ears; emaciated; neurotrophic ulcer left heel. Wound well healed. On exposing the left sciatic, the external popliteal found adherent to operated internal popliteal. Transplant found well in place; of yellow-white color; adherent to underlying muscle. Large central bulbous enlargement. Distal nerve presents the appearance of a degenerated nerve. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining throughout.
Microscopic findings.-In longitudinal sections of central and distal wounds, the transplant well united to resected nerve ends, little connective tissue intervening. From the distal end of the central bulb, numerous neuraxes may be traced into the central end of the transplant, in which they end distally, grouped mainly to one side in the transplant, along the inner surface of its perineural sheath; only a few scattered bundles of neuraxes in the substance of the transplant. In the transplant areas and columns of myelin globules, granular detritus and masses of large vesicular cells, with lipoid globules. The downgrowing neuraxes traced to and through the distal wound, only a few having reached the central end of the distal popliteal, in which they may be traced distally for a distance of about 1 cm.
EXPERIMENT No. 203.-Rabbit No. 118a; full grown; 42 days. December 28, 1918, right sciatic exposed; the internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the sciatic of another rabbit, stored seven days in sterile 50 per cent alcohol at room temperature, used as transplant. One central and distal waxed, fine silk thread suture placed. Good central approximation attained, distal good direction, end of transplant twisted one half turn. Wound closed. February 8, 1919, killed. Rabbit not in good condition; "fungus" ears; emaciated; neurotrophic ulcer right heel. Wound well healed. Right sciatic exposed. External popliteal found adherent. Transplant found well in place; of yellow-white color, clearly demarked; adherent to underlying muscle. Large central bulb. Nerve and transplant removed and fixed in neutral formalin. Sections stained in safranine and licht grün.
Microscopic findings.-In longitudinal sections of central and distal wounds, the transplant found well united to resected nerve ends. New nerve fibers and nucleated, syncytial protoplasmic bands extend from distal end of the central stump into the transplant. In longitudinal sections of the transplant, larger and smaller bundles of syncytial protoplasmic bands, having in the main a longitudinal direction, but here and there anastomosing, and separated by areas and columns of large vesicular cells, are to be observed. In cross sections of the transplant about 1 cm. distal to central wound, one relatively large bundle of syncytial protoplasmic bands, placed largely to one side, but extending into the middle of the transplant, is recognized. Fewer of these nucleated protoplasmic bands seen in the distal part of the transplant, but may be traced to and into the distal wound. The distal nerve presents the appearance of a degenerated nerve.
EXPERIMENT No. 204.-Rabbit No. 121; full grown; 70 days. January 14, 1919, left sciatic exposed; internal popliteal freed; resected 3.2 cm. A segment of equal length taken
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from the sciatic of another rabbit, stored 14 clays in sterile 50 per cent alcohol at room tempera-ture, used as transplant. Before use, the nerve placed for eight minutes in sterile, warmed saline solution. One central and distal waxed, fine silk thread suture placed. Central approximation recorded as good, distal "fair." Wound closed. March 25, rabbit found dead in the morning. Had had convulsions previous day; much emaciated; severe neurotrophic ulcer left heel. Wound well healed. On exposing the left sciatic, transplant found well in place, of light yellow-white color, of smaller size than when used; no material increase of connective tissue about it. Relatively large central bulb noted. The nerve and the transplant removed and fixed in neutral formalin. Sections stained in safranine and licht grün.
Microscopic findings.-No distinct central bulb evidenced structurally. New nerve fibers and syncytial protoplasmic strands extend from the distal end of the central stump into the nerve transplant, in the proximal half of which these are arranged in small bundles, within the perineural sheaths of the nerve transplant, with relatively few myelin globules and vesicular cells separating such bundles. In the distal half of the transplant these small bundles of nucleated protoplasmic bands are separated by larger and smaller areas or columns of vesicular cells and myelin globules. Many of the nucleated protoplasmic bands reach and penetrate the distal wound. The distal popliteal found degenerated; numerous nucleated, syncytial protoplasmic strands noted; relatively few myelin globules are evident.
EXPERIMENT No. 205.-Rabbit No. 121a; full grown; 70 days. January 14, 1919, right sciatic exposed; internal popliteal freed; resected 3.3 cm. A segment of equal length taken from the sciatic of another rabbit, stored 14 days in sterile 50 per cent alcohol at room temperature, used as transplant. Nerve placed in sterile, warmed saline solution forty-five minutes before use. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. March 25, rabbit found dead in the morning; much emaciated; severe neurotrophic ulcer right heel. Wound well healed. On exposing the right sciatic, transplant found well in place, of light yellow-white color, much smaller diameter than when use; not adherent. Large central bulb. Nerve and transplant removed and fixed in neutral formalin. Sections stained in safranine and licht grün.
Microscopic findings.-Transplant well united to resected nerve ends. Distinct central bulb evidenced structurally, from the distal end of which numerous new nerve fibers and nucleated, syncytial strands extend into the transplant, extending to and into the distal wound. In the distal end of the transplant, areas and columns of large vesicular cells found between protoplasmic strands. The distal nerve presents the appearance of a degenerated nerve.
EXPERIMENT No. 206.-Rabbit No. 119; full grown; 62 days. January 9, 1919, left sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length taken from the sciatic of another rabbit, stored 10 days at room temperature in sterile 50 percent alcohol, used as transplant. Nerve, before use, placed fifteen minutes in sterile, warmed saline solution. One central and distal suture of waxed, fine silk thread placed; good approximation. Field not quite dry; wound closed. March 12, rabbit found dead in the morning. Not much emaciated; severe neurotrophic ulcer left heel. Wound well healed. On exposing the left sciatic, external popliteal found closely adherent to operated internal popliteal. Transplant found well in place, in part of light pink color in part dull white color; no material increase in connective tissue surrounding operated nerve. Large central bulbous enlargement, adherent to underlying muscle. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Throughout, good differential neuraxis staining attained.
Microscopic findings.-The transplant found well united to resected nerve ends. From the distal end of the central bulb, numerous down-growing neuraxes found growing distally through central wound to central end of transplant. In cross sections of the transplant about1 cm. distal to central wound, numerous small bundles of neuraxes, separated by endoneural connective tissue are to be found. In cross sections taken near the distal wound, essentially the same structural appearance observed for the greater part of the transplant; to one side, within perineural sheath, a relatively large area, containing large vesicular cells and granular detritus noted. In this field no neuraxes observed. New neuraxes traced through the distal wound into the distal nerve segment, in which they may be traced in good numbers for a
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distance of about 2 cm. Regeneration of proximal end of distal nerve through the nerve transplant.
EXPERIMENT No. 207.-Rabbit No. 119a; full grown; 62 days. January 9, 1919, right sciatic exposed; internal popliteal bundle freed; resected 2.2 cm. A segment of equal length taken from the sciatic of another rabbit, stored at room temperature in sterile 50 percent
FIG. 231.- From a longitudinal section of homo-nerve transplant, stored in 50 per cent alcohol for 10 days before use as transplant; Experiment No. 200. Nerve removed 62 days after operation. Note the regular course of the new neuraxes, evident as black lines, as they pass distally within the neurolemma sheaths of the transplanted nerve fibers
alcohol for a period of 10 days, used as transplant. Nerve placed in sterile, warmed saline solution one hour and ten minutes before use. One central and distal waxed, fine silk thread suture placed. Central suture torn out; resected and resutured; approximation good. Field not quite dry; wound closed. March 12, rabbit found dead in the morning; not much emaciated; severe neurotrophic ulcer right heel. Wound well healed. On exposing the right
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sciatic, external popliteal found closely adherent to operated internal popliteal. The transplant found well in place; relatively small diameter; not adherent to underlying muscle. Central end of transplant of dull white color; distal of light pink color. Large, oval-shaped central bulb. Nerve and transplant removed and fixed in neutral formalin. Sections stained in safranine and licht grün.
Microscopic findings.-Transplant found well united to the resected nerve ends; narrow connective tissue wounds. From the distinct central bulb, new small nerve fibers and
FIG. 232.-From a cross section of homo-nerve transplant, stored in 50 percent alcohol for 10 days before use as transplant; Experiment No. 206. Nerve removed 62 days after operation. Section made approximately 15 mm. distal to the central wound. Note the numerous central neuraxes seen in cross section, showing as fine darkly stained points, very evenly distributed through the field. There is here evident excellent neurotization of the transplanted nerve segment
nucleated, protoplasmic bands extend into the transplant in which they may be traced to and through the distal wound. Especially in the longitudinal sections taken from the distal half of the transplant, irregular columns and areas of large vesicular cells and cell detritus, separating bundles of new nerve fibers, are to be noted.
EXPERIMENT No. 208.-Rabbit No. 135; large; old; Belgian hare; 67 days. March18, 1919, left sciatic exposed; internal popliteal freed; resected 3.5 cm. A segment of equal length taken from the sciatic of another rabbit, stored at room temperature for 28 days in sterile 50 percent alcohol, used as transplant. Nerve placed in sterile saline solution 15
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minutes before use. One central and distal waxed, fine silk thread suture placed; good approximation. Dry field; wound closed. May 24, rabbit found dead in the morning; not much emaciated; severe neurotrophic ulcer left heel. Wound well healed. On exposing the left sciatic, external popliteal found free. Transplant well in place, found only moderately adherent to underlying muscle; of yellow-white color, tinged here and there a brown color. Distinct central bulb; central end of distal nerve enlarged. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation not successful throughout.
Microscopic findings.- Transplant well united to resected nerve ends. The silver differ-entiation of sufficient clearness to determine the fact that many new neuraxes coining from The distal end of the central bulb pass to and through the transplant. These are seen to descend mainly along the inner surface of the perineural sheath and peripheral part of the main funiculus, the core of which is occupied by a relatively large area containing large vesicular cells and granular detritus. Few, if any, of the down-growing neuraxes appear to have reached the distal segment of the resected nerve, which presents the appearance of a degenerated nerve.
EXPERIMENT No. 209.- Rabbit No. 122; full grown; 144 days. January 16, 1919, left sciatic exposed; internal popliteal bundle freed; resected 3.0 cm. A segment of equal length taken from the sciatic of another rabbit, stored at room temperature for a period of 15 daysin sterile 50 percent alcohol, used as transplant. Before use placed in warmed, sterile saline solution 15 minutes. One central and distal waxed, fine silk thread suture placed; good approximation attained. Wound closed. June 9, rabbit found dead in the morning; not much emaciated; small neurotrophic ulcer left heel. Nerve and transplant removed, fixed in ammoniated alcohol for pyridine-silver staining. Fair silver differentiation attained; not well embedded, sections torn.
Microscopic findings.- Transplant found well united to the resected nerve ends. From the distal end of the central bulb, numerous neuraxes are found to pass to the transplant in which they are arranged in small bundles separated by endoneural connective tissue, presenting much greater amount than in a normal nerve trunk. To one side of transplant, remains of the transplanted nerve fibers noted particularly in cross sections. Neuraxes of the transplant pass to and through the distal wound and are found in good numbers in the distal nerve. Regeneration of the central end of the distal segment through the transplant.
EXPERIMENT No. 210.- Rabbit No. 122a; full grown; 144 days. January 16, 1919, right sciatic exposed; internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the sciatic of another rabbit, stored at room temperature for 15 days in sterile 50 per cent alcohol, used as transplant. Before use, nerve placed for one hour in warmed, sterile saline solution. One central and distal waxed, fine silk thread suture placed; distal resutured, first very unsatisfactory; good approximation attained. Small blood clot in the connective tissue near distal wound. Wound closed. June 9, rabbit found dead in the morning; not much emaciated; moderately large neurotrophic ulcer right heel. On exposing the right sciatic, external popliteal found free. Transplant found well in place; of good size; only moderately adherent to underlying muscle. Large central bulb. The nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fair silver differentiation attained; tissue not well embedded, sections torn.
Microscopic findings.-Neuraxes coming from the distal end of the central bulbous enlargement can be traced through the transplant into the distal segment of the resected nerve in which they are present in large numbers. Within the transplant, these down-growing neuraxes are arranged in small bundles, separated by endoneural connective tissue, present in larger amount than in a normal nerve trunk. Regeneration of the distal nerve segment through the nerve transplant attained.
EXPERIMENT No. 211.- Rabbit No. 137; small rabbit; seemed full grown; 152 days. March 19, 1919, left sciatic exposed; internal popliteal freed; resected 2.5 cm. A segment of equal length taken from the sciatic of another rabbit, stored for a period of 29 days at room temperature in sterile 50 percent alcohol, used as transplant. Before use, nerve placed for 10 minutes in warmed, sterile saline solution. One central and distal waxed, fine silk thread suture placed. Good central approximation attained; a small amount of
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clotted blood in central wound; distally, good direction, but distal end of transplant twisted one half turn. Wound closed. August 18, killed. Rabbit in good condition; not large, but appears well fed; neurotrophic ulcer left heel, which appears to be healing. On exposing the left sciatic, external popliteal found in close approximation to the operated internal popliteal. Transplant found well in place, of good size; has the appearance of a normal nerve, though of slightly brown color. No distinct central bulbous enlargement noted. Calf muscles fully exposed, these present a pale red color but have not fully recovered their normal size. The external popliteal cut central and distal to the region of the transplant and the internal popliteal and transplant completely freed from bed. On slowly cutting the sciatic central to the transplant good contraction of the calf muscles and slight movement of the toes noted; the same cutting distal to the transplant. The nerve and the transplant and portions of the calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation not good for distal portion of nerve.
Microscopic findings.- Numerous new neuraxes traced through the transplant into the distal portion of resected nerve to level of calf muscles. In the transplant these neuraxes in the form of small bundles separated by endoneural connective tissue. Very little detritus derived from the transplanted nerves noted. In sections of the calf muscles, numerous neuraxes observed in the intrafascicular nerve bundles and as single nerve fibers, between and on muscle fibers; a few motor nerve endings noted. In sections of the posterior tibial nerve, not successful silver differentiation. Regeneration of distal popliteal including motor branches and endings in the calf muscles attained.
EXPERIMENT No. 212.- Rabbit No. 134; full grown; not large; 154 days. March 17, 1919, left sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length taken from the sciatic of another rabbit, stored at room temperature in sterile 50 per cent alcohol for a period of 27 days, used as transplant. Before use, nerve placed for 15 minutes in warmed, sterile saline solution. One central and distal nerve suture placed; good approximation. Wound closed. August 18; killed. Rabbit in good condition; neurotrophic ulcer left heel nearly healed. On exposing the left sciatic, the external popliteal found in close apposition to the operated internal popliteal. Transplant found well in place; of small diameter; but presents the appearance of a normal nerve; only moderately adherent to the underlying muscle. Distinct central bulb noted. Distal nerve presents the appearance of a normal nerve. Calf muscles fully exposed; these still somewhat atrophic and of pale-red color. Nerve and transplant completely freed from the bed. On cutting slowly with scissors central to the transplant, indistinct contractions-"feeble contractions"-of calf muscles noted. Nerve and transplant and portions of the calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining for central part of nerve, but not for distal part, attained.
Microscopic findings.- Numerous neuraxes can be traced from the distal end of the central stump through the transplant to the distal nerve. Silver staining of pieces of calf muscles not satisfactory-no neuraxes stained. Regeneration of distal nerve through transplant, recovery of calf muscles not confirmed by microscopic findings.
EXPERIMENT No. 213.- Rabbit No. 125; full grown; 208 days. January 22, 1919,left sciatic exposed; internal popliteal freed; resected 2.9 cm. A segment of equal length taken from the sciatic of another rabbit, stored at room temperature for 21 days in sterile 50 percent alcohol, used as transplant. Nerve placed in sterile saline solution for 15 minutes before use. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. August 18, killed. Rabbit in fairly good condition; healing neurotrophic ulcer left heel; walks on heel and does not bring foot down to floor. On exposing left sciatic external popliteal found free; transplant well in place; of small diameter; only slightly adherent to underlying muscle. Large and distinct central bulb noted; evident enlargement of the central end of distal nerve. Calf muscles fully exposed; these still slightly atrophic, of pale red color, streaked with light yellow bands. External popliteal resected, operated internal popliteal and transplant completely freed from bed. On slowly cutting nerve with scissors central to the transplant, feeble but distinct contraction of calf muscles noted; the same on cutting distal to transplant. No distinct movement of the toes observed. Nerve and transplant and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good differential silver staining attained.
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Microscopic findings.-The distinct central bulb evidenced structurally, from the distal end of which numerous new neuraxes can be traced through the transplant to the distal nerve. Within the transplant the neuraxes arranged in the form of small funiculi separated by endoneural connective tissue. New neuraxes traced into the calf muscles in which they are found in the intrafascicular nerves and as separate nerve fibers between and on the muscle fibers. Regeneration of distal nerve through transplant, partial return of motor function in calf muscles.
EXPERIMENT No. 214.-Rabbit No. 125a; full grown; 203 days. January 27, 1919, right sciatic exposed; the internal popliteal bundle freed; resected 2.9 cm. A segment of equal length taken from the sciatic of another rabbit, stored at room temperature in sterile 50 percent alcohol for 21 days, used as transplant. Before use, nerve kept in sterile saline solution for 50 minutes. One central and distal waxed, fine silk suture placed; good approximation. Wound closed. August 18, killed. Rabbit in fairly good condition; healing neuro-trophic ulcer right heel. On exposing the right sciatic, it is found that the external popliteal is closely adherent to operated internal popliteal. The transplant found well in place and about one-half the diameter as when used; only moderately adherent to underlying muscle. Relatively large central bulb; central end of distal nerve found only slightly enlarged. Calf muscles fully exposed; these have not fully recovered size; pale red color. The nerve and the transplant completely freed and external popliteal cut in popliteal space; on slowly cutting with scissors, the nerve central to the transplant, distinct but feeble contractions of the calf muscles noted; the same on cutting distal to the transplant; no toe movement noted. Nerve and transplant and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.-From the large central bulb, numerous new neuraxes can be traced through the transplant to the distal nerve and through the muscular branches to the calf muscles. Very good regeneration of motor fibers in calf muscles noted. Regeneration of distal nerve through the transplant including muscular branches to calf muscles.
EXPERIMENT No. 215.-Rabbit No. 126; full grown; 208 days. January 22, 1919, left sciatic exposed; internal popliteal freed; resected 2.6 cm. A segment of equal length taken from the sciatic of another rabbit, stored at room temperature in sterile 50 percent alcohol for 22 days, used as transplant. Before use, nerve placed in warmed, sterile saline solution for 15 minutes. One central and distal waxed, fine silk-thread suture placed; central approximation not quite end to end, distal good. Wound closed. August 18, killed. Rabbit in good condition; small healing neurotrophic ulcer left heel; not full use of foot. On exposing the left sciatic, it is found that external popliteal is in close approximation to operated internal popliteal. Transplant found well in place; nearly of same size as when used. Distinct central bulb noted; moderate enlargement of central end of distal nerve. Calf muscles fully exposed; these have not fully recovered size. Nerve and the transplant completely freed from bed. On slowly cutting nerve central to transplant, contraction of the calf muscles noted, as also slight movement of the toes; same on cutting distal to the transplant. Nerve and transplant and portions of calf muscles removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good differential silver staining attained; pale.
Microscopic findings.- Transplant firmly united to resected nerve ends. Well-developed central bulb evidenced structurally, from the distal end of which numerous myelinated nerve fibers can be traced through the transplant to the distal nerve. In cross sections of the transplant made about 1.5 cm. distal to the central wound, it may be observed that the perineural sheaths of the transplanted nerve segment are intact and only moderately thickened, while the endoneural tissue is distinctly increased in amount. The neuraxes are found in small groups separated by endoneural tissue. In sections of the calf muscles new neuraxes are found in the interfascicular nerve bundles, and here and there as single fibers passing to the muscle fibers. Good regeneration of distal internal popliteal through the nerve trans-plant attained.
EXPERIMENT No. 216.-Rabbit No. 126a; full grown; 208 days. January 22, 1919, right sciatic exposed; internal popliteal freed; resected 2.9 cm. A segment of equal length taken from the sciatic of another rabbit, stored at room temperature in sterile 50 percent cent alcohol for 22 days, used as a transplant. Before use, nerve placed in warmed, sterile
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saline solution for one hour. One central and distal waxed, fine silk-thread suture placed; good approximation. Wound closed. August 18, killed. Rabbit in good condition; healing neurotrophic ulcer right heel. On exposing the right sciatic, external popliteal found closely adherent to operated internal popliteal. Transplant found well in place; of good size; of dull white color and only moderately adherent to underlying muscle. Large central bulb noted; central end of distal nerve only slightly enlarged. Calf muscles fully exposed; these appear to have nearly recovered size and color. The nerve and the transplant completely freed from bed and external popliteal cut in popliteal space. On slowly cutting with scissors the nerve central to the transplant, good contraction of the calf muscles observed. Nerve and transplant and portions of the calf muscles removed for pyridine-silver staining. Pale, but fairly good differential silver staining attained.
Microscopic findings.-Transplant found well in place and firmly united to the resected nerve ends; line of suture hardly evident. From the central bulb numerous neuraxes traced into proximal end of the transplant, relatively few traced into the connective tissue surrounding the transplant. In cross sections of the transplant taken about 1.5 cm. distal to the central wound, the epineural sheaths found materially thickened and there is noted a marked increase of the endoneural connective tissue. Within the transplant, nerve fibers, both myelinated and nonmyelinated, observed in small bundles, separated by varying amounts of endoneural connective tissue. These neuraxes can be traced through the transplant to the distal nerve within which they are present in large numbers, both myelinated and nonmyelinated fibers, and are found quite evenly distributed in the several funiculi. In sections of the calf muscles, neuraxes observed in the interfascicular nerve branches and as single nerve fibers on and between muscle fibers. Structurally considered, the muscles appear as regenerated. Fairly complete regeneration of the distal nerve through the transplant, including nerves to calf muscles, attained.
The end results of the experimental observations on stored, homo-nerve transplants are on the whole very satisfactory. Functional return is recorded for all of the experiments of longer duration. Stored homo-nerve transplants seem to serve the purpose of nerve bridge quite as well as fresh homo-nerve transplants, thus obviating in a large measure certain difficulties connected with the use of homo-nerve transplants in human surgery. If nerves obtained at amputations can be stored for weeks, to be at hand when required, with promise of favorable results on use, stored homo-nerve transplant deserves consideration in human surgery. It is our belief that smaller nerve bundles, used if necessary as cable or multiple nerve transplants, should give promise of more favorable end results than the use of one large nerve, such as the sciatic or its main branches used as a nerve bridge.
In all of the experiments (No. 150 to No. 157) in which a homogenous nerve bridge of nerve stored in vaseline was made there was noted relatively little increase in the connective tissue surrounding the nerve transplant at the time when the nerve was exposed for study of functional return. The transplant presented the appearance of a normal nerve. In all of these experiments down-growing neuraxes derived from the central stump neuraxes were traced through the central wound into the transplant, and through the transplant to and through the distal wound into the distal stump. In the cross sections of the transplants the down-growing neuraxes encountered are manv of them found within what appear to be neurolemma sheaths remains of transplanted nerve fibers. Tissue detritus and large vesicular phagocytic cells, unlike the end results of Wallerian degeneration, are met with within the nerve funiculi. In the experiments of about two months' duration (No. 154and No. 155) feeble muscle contraction was noted and interfascicular, muscular
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nerve bundles with new neuraxes were noted in the sections of the calf muscles; and in experiments of nearly five months' duration (No. 156 and No. 157) regeneration of the distal segment of the nerve with down-growing neuraxes was quite complete. On the whole, very satisfactory neurotization of the degenerated distal segment was obtained in experiments dealing with homo-nerve transplants, stored in vaseline, following in the main the method of procedure suggested by Dujarier and Francois. 76 The much larger series of homogenous-nerve transplants stored in liquid petrolatum (No. 158 to No.197) included 14 experiments of relatively short duration (1 hour to 12 days) in which the behavior of the transplant soon after it was placed as a nerve bridge could be studied. A nerve stored in liquid petrolatum at 3º C. retains its microscopic structure quite completely and will stain differentially by the pyridine-silver method. Two days and even four days after such a transplant is placed the neuraxes of the nerve fibers as seen in the pyridine-silver preparations are not fragmented. From 6 days to 12 days after transplantation the neuraxes are found segmented and show a granular change. The myelin shows fragmentation, but there is no evidence of proliferation of sheath cells. At the end of 12 days there are still found fragments of old neuraxes in the transplanted nerve segment. In the central wound region down-growing central neuraxes have penetrated the wound region and certain ones have extended into the transplant for a distance of about 2 mm. Experiments Nos. 173 and 174 deserve special consideration. A homogenous-nerve transplant stored 39 days in liquid petrolatum was used to bridge a nerve defect. The animal died 23 days after the operation but was used for histologic study. In cross sections of the transplants, made about 1 cm. distal to the central wound, new neuraxes were found in all parts of the transplant. In many parts of the field more than one neuraxis was found in one neurolemma sheath, while other neuraxes are found outside of the neurolemma sheaths. Extensive neurotization of the transplant had taken place, by downward growth of central neuraxes, at the end of 23 days after the operation. Of the experiments of this series 20 were carried on for a period of 3 months or longer; the longest for a period of nearly 7 months. In all of these experiments, where functional tests could be made, return of functions in the calf muscles is recorded, and incertain of the longer time experiments return of function in the foot interossei was observed. All of the experiments were controlled by histologic study of practically the whole sciatic nerve, and in all of the experiments could central neuraxes be traced through the transplant into the distal popliteal nerve and thence into the calf muscles. In cross sections of the transplanted nerves in the respective experiments, stained by the pyridine-silver method, it could be determined in nearly every experiment that the down-growing central neuraxes made use of the neurolemma sheaths of the transplanted nerve fibers in their course through the transplant. In all of these experiments relatively few nerve fibers are found in the connective tissue surrounding the perineural sheaths of the funiculi of the nerve transplant, interpreted as meaning that in this series the nerve transplant is the main avenue along which the down-growing neuraxes reach the distal stump. The experimental observations dealing with homogenous-nerve transplants stored in liquid petrolatum seem
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to us to warrant the deduction that human nerves obtained from amputated members and stored in liquid petrolatum as here directed and, on need, used for bridging nerve defects, deserve serious consideration as a surgical procedure. We were agreeably surprised at the favorable results attained on use of homogenous-nerve transplants stored in 50 percent alcohol for purpose of nerve bridge. In this series of 18 experiments (No. 198 to No. 216) relatively few were of short duration. In Experiments No. 198 and No. 199 the rabbit was found dead 2 days after operation. In sections of the transplant stained by the pyridine-silver method the neuraxes were found to stain very lightly, but were found unsegmented. Inwandered leucocytes were found in theends ot the transplant at the central and distal wounds, both within the neurolemma sheaths and between the nerve fibers. In Experiments No. 200 and No. 201 (compare Experiments No. 173 and No. 174) the rabbit had died 23 days after the operation but the tissue was used for histologic study. In preparations stained after the pyridine-silver method down-growing central neuraxes can be traced through the central wound and for a distance of about 2 cm. into the transplant. In cross sections of the transplant the down-growing neuraxes are found within as well as without the neurolemma sheaths, but practically no nerve fibers are found in the connective tissue surrounding the transplant, outside of the perineural sheaths. By the end of 42 days, more clearly 2 months after the operation, down-growing neuraxes were traced through the transplant to the distal wound and through this into the central end of the distal popliteal, the down-growing neuraxes decreasing in number the farther distal the observation is made. In the experiments of longer duration, 8 in number, in which the observations were carried on to from 4 months to nearly 6 months after the operation, functional return was noted in the experiments in which this could be tested and histologically new neuraxes were found in the distal nerve, conveyed there through the transplant. In several experiments new neuraxes were found in the interfascicular and intrafascicular nerve bundles of the calf muscles.
By way of summary it may here be added that very good neurotization was attained through homogenous nerve bridges which had been stored in 50 percent alcohol. The supposition is permissible that in nerves stored in sterile vaseline and liquid petrolatum at a temperature of 3º C. there maybe some degree of viability of certain tissue elements-sheath cells or
connective tissue cells-even though there is no satisfactory evidence of the proliferation of the sheath cells of transplanted nerve fibers, nor of the participation of the sheath cells of the nerve transplant, direct or indirect, in the down growth of the central neuraxes. In case of nerves stored in alcohol, it can not be supposed that any viability is retained by the tissue elements or cells of the nerves transplanted. There is no evidence of sheath cell participation and no evidence that they proliferate. The fragmentation of the myelin and neuraxes of the transplanted nerves after storage in vaseline, liquid petrolatum, and alcohol is a necrobiotic change and not a secondary degeneration-Wallerian degeneration---as observed in the distal segment.
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STORED HETERO-NERVE TRANSPLANTS
SERIES NO. 14
HETERO-NERVE TRANSPLANTS STORED IN LIQUID PETROLATUM
SERIES NO. 15
HETERO-NERVE TRANSPLANTS STORED IN 50 PERCENT ALCOHOL
In the discussion of Series No. 11, No. 12 and No. 13, stored homogenous transplants, consideration was given to the fact that little if any viability is retained by any of the tissue elements of nerves stored for stated periods before use as a transplant, especially so when stored in 50 percent alcohol. Therefore, it was thought that the sheath cells of the stored, transplanted nerves take no active part in the fragmentation of the neuraxes and myelin sheaths of the transplanted nerves and, so far as can be determined, are not causally related to the downgrowth of the central neuraxes, in their passage through the transplant to reach the distal segment of the resected nerve. The conviction seems warranted that the neurolemma sheaths of the stored, transplanted nerve fibers, which do not fragment with the neuraxes and myelin sheaths, act in a purely mechanical way in serving as conduits through which the down-growing neuraxes are conveyed through the transplant to the distal nerve segment. Therefore, the supposition seemed justified that a hetero-nerve transplant, stored in liquid petrolatum and especially in alcohol, would prove more satisfactory as a nerve- bridge than a fresh heterogenous nerve transplant. Series No. 14 and No. 15 were undertaken to test this hypothesis. In Series No. 14, the internal popliteal or ulnar nerve of dogs was removed under aseptic precautions and stored in liquid petrolatum, as described under Series No. 12, for periods varying for from 12 days to 25 days and were then used to bridge nerve defects caused by resecting the sciatic nerve of rabbits. One central and one distal fine waxed silk suture was placed to fix the transplant to the resected nerve ends. In Series No. 15, segments, taken from the internal popliteal and ulnar nerves of dogs and stored for periods varying from 5 days to 7 days in 50 per cent alcohol, were used to bridge nerve defects in the sciatic of rabbits caused by resection and sutured in place by fine waxed silk sutures.
The protocols of the experiments of Series No. 14 and No. 15, stored heterogenous nerve-transplants, are as follows:
PROTOCOLS
EXPERIMENT No. 217.- Rabbit No. 127a; full grown; 5 days. March 4, 1919, the right sciatic exposed; internal popliteal freed; resected 3.1 cm. A segment of equal length taken from the internal popliteal of a dog, stored for 10 days in sterile liquid petrolatum at 3º C, used as transplant. The transplant of dull white color and of larger diameter than the resected nerve. One central and distal suture of waxed, fine silk thread placed; good approximation. Dry field; wound closed. March 9, rabbit found dead in the morning. Superficial wound healed. On exposing the right sciatic, transplant is found well in place; loosely united to resected nerve ends; not adherent to surrounding muscle. Nerve and transplant removed and fixed in neutral formalin. Sections stained in safranine and licht grün.
Microscopic findings.-Longitudinal sections, embracing central and distal wounds, show very good approximation; a few extravasated blood cells found in the intervening connective
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tissue. Leucocytes noted in the central and distal ends of the transplant for a distance of about 8 mm. The great majority of these found between the nerve fibers. Leucocytes have wandered in less number and for a shorter distance into the resected nerve ends. In cross and longitudinal sections of the transplant taken from its middle third, the contained nerve fibers appear as very well preserved; the neuraxes staining pale, though readily made out, and not fragmented; the myelin sheaths not fragmented; the neurolemma sheaths clearly seen. The few sheath cell nuclei here and there seen, appear in form and staining reaction much as do similar nuclei in normal nerves. The perineural sheaths present the characteristic appearance of this structure.
EXPERIMENT No. 218.- Rabbit No. 133a; large; old; Belgian hare; 82 days. March15, 1919, right sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length taken from the left ulnar of a dog, stored 25 days in sterile liquid petrolatum at 3º C.,used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. June 5, killed. For several days rabbit had not been well; emaciated; nearly moribund, when killed; right heel swollen and red, no ulcer. On exposing the right sciatic, external popliteal found free. Transplant found well in place and firmly united to resected nerve ends; seemed of smaller diameter than when used; of distinct light yellow color; only moderately adherent to the underlying muscle. Large central bulb, which tapers toward the transplant; slight enlargement of central end of distal nerve noted. Calf muscles very atrophic; no response on cutting nerves. Nerve and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings.- In longitudinal sections, the large central bulb clearly demarked from the transplant. In the portion of the bulb developed from the distal end of the central stump the light yellow coloring, differential staining of neuraxes characteristic for pyridine silver methods, is to be observed. The sections of the transplant throughout, present a jet-black, nontransparent coloration, by reason of which structural differentiation can not be made out. By reason of this silver reaction, it is not possible to determine whether neuraxes coming from the central stump penetrate the transplant. In cross sections of the transplant, its connective tissue sheaths present a brown-yellow color. In close relation with this sheath new neuraxes are observed. Within the sheaths, the portions containing the nerve fibers of the funiculi colored jet-black. The distal internal popliteal stump completely degenerated.
EXPERIMENT No. 219.-Rabbit No. 131a; large; full grown; 82 days. March 12, 1919, right sciatic exposed; internal popliteal freed; resected 2.8 cm. A segment of equal length taken from the right ulnar of a dog, stored in sterile liquid petrolatum at 3º C. for a period of 20 days, used as transplant. One central and distal waxed, fine silk thread suture placed. Good central approximation attained; at the distal wound, thread through transplant very nearly cut through. Wound closed. June 2, rabbit found dead in the morning; moderate emaciation; severe neurotrophic ulcer right heel. On exposing the right sciatic, it is noted that the transplant is united to central end of resected nerve, but pulled free from the distal segment, the transplant ending free 1.5 cm. distal to central wound; remaining transplant segment yellow-white color; large central bulb. The central and distal segments of the resected nerve and remains of transplant removed and placed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.-Numerous neuraxes can be traced from the distal end of the central bulb along the side of the transplanted nerve segment, but do not appear to have penetrated the same. Transplanted nerve segment clearly demarked by reason of its jet-black coloration. Distal nerve completely degenerated.
EXPERIMENT No. 220.-Rabbit No. 129a; full grown; 112 days. March 7, 1919, right sciatic exposed; internal popliteal freed; resected 2.8 cm. A segment of equal length taken from the left internal popliteal bundle of a dog, stored 14 days in sterile liquid petrolatum at 3º C., used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. The diameter of the transplant somewhat greater than that of the resected nerve. Dry field; wound closed. June 27, killed. Rabbit not well for several days; neurotrophic ulcer right heel. On exposing right sciatic. external popliteal found free
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Transplant found well in place and firmly united to resected nerve ends; seems of slightly larger diameter than when used; distinct yellow-white color, which enables demarking it clearly. Moderately large central bulb. Calf muscles atrophic. No response on cutting nerve central and distal to the transplant. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.-Distinct central bulb evidenced structurally, from the distal endof which new neuraxes pass to the side of the transplant. The transplant itself is stained a deep black color, admitting no determination of structural details. In cross and longitudinal sections of the transplant, it is not possible to differentiate any neuraxes within he perineural sheaths of the transplant; this by reason of the dark silver reaction. In cross nd longitudinal sections of the internal popliteal distal to the transplant a goodly number of new neuraxes are to be observed. In a series of longitudinal sections embracing the distal wound and adjacent nerve ends, neuraxes are to be observed entering the field of the distal ound to one side of the distal end of the transplant. It would appear, though by reason f the peculiar staining of the transplant this can not be determined conclusively, regeneration in the distal stump is attained through neuraxes that pass distally outside of the transplant.
EXPERIMENT No. 221.- Rabbit No. 130a; full grown; 121 days. March 10, 1919,right sciatic exposed; internal popliteal freed; resected 3.2 cm. A segment of equal length taken from the right ulnar of a dog, stored in sterile liquid petrolatum 17 days at 3º C., used as transplant. One central and distal suture waxed, fine silk thread placed; good approximation. The diameter of the transplant greater than that of the resected nerve. Wound closed. July 8, rabbit found dead in the morning; moderately emaciated; severe neuro-trophic ulcer right heel. On exposing the right sciatic, external popliteal found free; trans-plant well in place; clearly demarked by reason of its light yellow color; no material increase of connective tissue about it. Large central bulb, adherent to underlying muscle, noted. Calf muscles still atrophic, present the appearance of degenerated muscle. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings.- From the distal end of the central bulb new neuraxes call be traced mainly to one side of the transplant; some few appear to enter the transplant, but by reason of the dark, nontransparent coloration they can not be traced any distance in the transplant. No neuraxes appear to have reached the distal internal popliteal, which structurally considered has the appearance of a completely degenerated nerve.
EXPERIMENT No. 222.- Rabbit No. 129a; full grown; Belgian hare; 138 days. March5, 1919, right sciatic exposed; internal popliteal freed; resected 3.2 cm. A segment of equal length taken from the internal popliteal of the right sciatic of a dog, stored in sterile liquid petrolatum for 11 days at 3º C., used as transplant. One central and distal waxed, fine silk thread suture placed; good approximation. Wound closed. July 21, rabbit found dead in the morning; much emaciated; severe neurotrophic ulcer right heel. On exposing the right sciatic, external popliteal found closely adherent to operated internal popliteal. Transplant found well in place; of distinct yellow-white color, thus clearly demarked. Large spindle-shaped central bulb. Calf muscles atrophic; present the appearance of degenerated muscle. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation not good in all parts of the series.
Microscopic findings.- From the distal end of the very well developed central bulb, numerous neuraxes passing mainly to one side of the transplanted nerve segment noted. In cross and longitudinal sections of the nerve transplant, the appearance presented in sections warrants conjecture that the chemical state of the transplanted nerve segment, which may be correlated with the peculiar jet-black coloration noted on staining with the pyridine-silver method, is undergoing a change in that the outer portion of the transplant no longer presents this peculiar coloration, only the core or central portion being thus colored. No neuraxes are to be observed within the transplant nor in the distal stump, which presents the appearance of a completely degenerated nerve.
EXPERIMENT No. 223.-Rabbit No. 135a; old; Belgian hare; 67 days. March 18,1919, right sciatic exposed; the internal popliteal freed; resected 3.3 cm. A segment of equal
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length taken from the right ulnar of a dog, stored at room temperature in sterile 50 per cent alcohol for 6 days, used as transplant. Before use, the nerve was placed in warm, sterile saline solution for 15 minutes. One central and distal waxed, fine silk thread suture placed. Wound closed. May 24, rabbit found dead in the morning; moderate emaciation; severe neurotrophic ulcer right heel. On exposing the right sciatic, the external popliteal found free. Transplant found well in place, united to resected nerve ends; clearly demarked by its yellow color; no material increase of connective tissue about it. Large spindle-shaped bulb noted. Calf muscles atrophic and flabby. Nerve transplant and nerve removed and fixed in ammoniated alcohol for pyridine-silver staining. Good neuraxes differentiation attained; tissue blocks not well embedded, sections in part torn.
Microscopic findings.-In longitudinal sections embracing the central wound, scattered neuraxes traced from the central nerve stump into central end of the transplant. In cross sections of the transplant, approximately 1.5 cm. distal to the central wound, the perineural sheath of the transplanted nerve segment is found very materially thickened; within this there is found a detritus, the remains of the transplanted nerves. In it no definite tissue can be recognized; even the neurolemma sheaths of the transplanted nerves have disappeared. No new neuraxes are to be recognized. In longitudinal sections of the transplant the same general appearances are presented, except that here and there short fragments of old neuraxes, having no definite arrangement, are found scattered through the detritus. Distal nerve completely degenerated.
EXPERIMENT No. 224.- Rabbit No. 137a; small; full grown; 152 days. March 19,1919, right sciatic exposed; internal popliteal freed; resected 3 cm. A segment of equal length taken from the left ulnar of a dog, stored at room temperature in sterile 50 percent alcohol for 7 days, used as a transplant. Before use, the nerve was placed for 15 minutes in a sterile saline solution. One central and distal waxed, fine silk thread suture placed; very good approximation of nerve ends. Wound closed. August 18, killed. Rabbit in good condition; small neurotrophic ulcer of the left heel. On exposing the right sciatic, external popliteal found free. Transplant found well in place; clearly demarked by its yellow-white color; good size and firmly united to resected nerve ends. Calf muscles ex-posed; atrophic and of a pale yellow-red color. Nerve and transplant freed from bed, on slowly cutting the nerve central to the transplant, no response of calf muscles noted; no evidence of contraction. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation only in part successful. Resulting sections somewhat torn.
Microscopic findings.-In longitudinal sections of the central wound region, it is to be observed that neuraxes passing from the distal end of the central bulb, pass to the region of the central wound, which they do not penetrate for any distance; certain of them turning centralward. In the central end of the transplant, near the central wound and for several millimeters distal, quite long fragments of the old neuraxes of the transplanted nerves, differentially stained in silver, may be observed; distal to this region, such neuraxes remains no longer observed. In cross and longitudinal section of the transplant 1.5 cm. to 2 cm. and 3 cm. distal to the central wound, no new neuraxes observed within the transplanted nerve segment. The nerve fibers of the transplant in part completely broken down, in part the old neurolemma sheaths found persisting, filled with detritus and leucocytes filled with lipoid globules. Just central to the distal wound, new neuraxes recognized in the transplant, passing through the distal wound into the distal popliteal in which they maybe traced to the level of the calf muscles. Apparent regeneration of the distal nerve, down-growing neuraxes appearing to pass distally mainly outside of the transplant is concluded.
EXPERIMENT No. 225.-Rabbit No. 134a; full grown; 154 days. March 17, 1919, right sciatic exposed; internal popliteal freed; resected 3.0 cm. A segment of equal length taken from the external popliteal bundle of the left sciatic of a dog, stored in sterile 50 percent alcohol at room temperature for 5 days, used as a transplant. Before use, nerve placed15 minutes in warmed, sterile saline solution. One central and distal waxed, fine silk thread suture placed; good approximation. Clean, dry field; wound closed. August 18, killed. Rabbit in good condition; neurotrophic ulcer on right heel nearly healed; does not use right hind leg and foot normally. On exposing the right sciatic, the external popliteal found
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quite free. Transplant found well in place; demarked by light yellow color; united to resected nerve ends. Spindle-shaped central bulb noted. Calf muscles atrophic and of pale red color. Section of nerve causes no contraction of the calf muscles. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good silver differentiation attained.
Microscopic findings.- In longitudinal sections embracing the central wound, new neuraxes can be traced from the distal end of the central stump into the central end of the transplant, either as single neuraxes or as small groups of such, which course distally in collapsed neurolemma sheaths. In cross sections of the transplant approximately 1.5 cm. distal to the central wound, it may be observed that mainly to one side many new neuraxes are found within the perineural sheath of the transplant, in part within this sheath, as also in the detritus, derived from the transplanted nerve fibers. Within and between the persisting neurolemma sheaths, numerous leucocytes greatly distended with lipoid globules are to be observed. Certain of the neuraxes which are found in the transplant and its sheath are to be traced to and through the distal wound into the distal popliteal through the transplant attained.
The end results of the experiments on stored hetero-nerve transplants are, on the whole, unsatisfactory. As concerns Series No. 14, heterogenous nerve transplants stored in liquid petrolatum, in none of the experiments of longer duration was regeneration of the distal segment of the resected and bridged nerve attained through the heterogenous transplant. The nerve segment was found firmly united to the central and distal nerve stump. On staining after the pyridine-silver method, the stored, heterogenous nerve transplant presented a peculiar reaction toward the silver nitrate in that the silver appeared to be reduced en masse, so that no differentiation of elements was possible within the perineural sheath of the funiculi transplanted. This made a close histologic study of the behavior of the transplanted nerve segments in these experiments difficult and in the main unsatisfactory, in that it could not be determined with certainty whether down-growing neuraxes of central origin passed through the transplant to reach the distal segment of the nerve. Down-growing neuraxes were found in the central nerve bulb and from this could be traced in the general direction of the central end of the nerve transplant, but also into the connective tissue surrounding the transplant and in this connective tissue to the level of the distal wound. In none of the experiments was neurotization of the distal segment attained. In the experiments of Series No. 15, in which heterogenous nerve transplants stored in 50 percent alcohol were used, the end results attained are much less satisfactory than in the series in which homogenous nerve transplants were used (Series No. 13). The several animals were under observation for from 64 days to 154 days, thus for a time of sufficient length to admit of regeneration through the transplant, under favorable conditions. It may be noted on study of the protocols that to a limited extent downgrowth of central neuraxes through the transplants was observed; our results thus con- firming Nageotte.77 The long persistence of fragments of the neuraxes of the transplanted nerves is to be noted, especially in Experiment No. 224, terminated 152 days after the operation. In the central portion of the transplant near the central wound and for several millimeters distal, quite long fragments of old neuraxes stained differentially by the pyridine-silver method are to be found. Distal to this region they have disappeared from the remains of the transplanted
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nerve fibers. Judging from the limited number of experiments here presented (3), testing the value of heterogenous nerve transplants stored in 50 per cent alcohol, it seems clear that this form of nerve bridge is not to be advocated as worthy of consideration in human surgery. The experiments of this series in so far as they can be compared with the series in which homogenous nerve transplants stored in 50 per cent alcohol (Series No. 13) were tested, indicate that there is distinct difference as regards serviceability between homogenous and heterogenous nerve transplants stored in 50 per cent alcohol and in favor of the alcoholized homogenous nerve transplants.
AUTO-NERVE TRANSPLANTS WRAPPED IN PROTECTIVE MATERIAL
SERIES NO. 16
AUTO-NERVE TRANSPLANTS WRAPPED IN CARGILE MEMBRANE
In this and the following several series (Series No. 16, No. 17, No. 18, and No. 19) we have attempted to test the merits of certain membranous structures which had been recommended for use in surgical practice, as a covering for suture lines in operations of nerve suture or as a wrapping about a nerve transplant and the suture lines or in other operative procedures in peripheral nerve repair. Incidental references are found in surgical literature to a number of membranous structures used for wrapping nerves or tendons after operative procedure. Our list of experiments might have been extended had cognizance been given to all of the materials used for this purpose. References to the use of Cargile membrane are not infrequent. It seemed to be used sufficiently frequently to warrant renewed experimental inquiry. Morris 78 states that he had received from Dr. Charles H. Cargile, of Arkansas,"sterilized animal membrane" (dried and sterilized peritoneum of the ox) with the request that he test its use and value in surgical practice, especially as a means of preventing adhesions in certain cases of abdominal surgery. Twelve experiments on rabbits were made. Morris found that the membrane resisted absorption for more than 10 days but less than 30 days when placed in the peritoneal space. Craig and Ellis 79 undertook a series of experiments on dogs using both chromatized and unchromatized Cargile membrane to wrap tendons and also nerves. They reached the conclusion that both the chromatized and the unchromatized membrane are of value in preventing adhesions of wounded nerves and tendons when such structures lie in tissue subjected to trauma. It was their observation that especially unchromatized Cargile membrane is absorbed relatively quickly in the tissues; macroscopically within 5days, microscopically within 14 days. The membrane appears to be destroyed by a lytic substance contained in the body fluids, phagocytes being regarded as of less importance in this process of disintegration and absorption. Sherren, 80 in several places, refers to the use of Cargile membrane or foil in connection with nerve suture and nerve transplantation. Other writers refer to the use of other animal membranes such as hernial sac, peritoneum, omentum, etc., used as fresh or as dried sterilized membranes. Meuriot and Platon 81 used strips some 20 cm. long and 5 cm. wide, cut from rubber gloves and wrapped spirally
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about the nerve, at the seat of injury. This technique was used in 100 cases and in 9:3 without unfavorable reaction.
In experiments of Series No. 16, the sciatic nerve of a dog was resected to the extent of approximately 4 cm. and the defect bridged by a segment of necessary length removed from the ulnar of the opposite side of the same dog. After the nerve transplant had been completed with the necessary sutures placed, one or several layers of Cargile membrane of sufficient length to extend about 1 cm. beyond the central and distal suture lines were wrapped about the nerve transplant and the ends of the central and distal stumps as closely and as evenly as could be. The Cargile membrane used was that made by Johnston and Johnston and was designated as "medium hard chromic." Pieces of requisite size, either of one laver or of several layers, were cut from pieces found within the several envelopes, as found in the market, and used at once as wrapping for the operated nerve, after which the wound was closed. An attempt was made to sterilize the portion of the Cargile membrane not used at any one operation, until it occurred to us to place the unused portion of any membrane in 70 per cent or 95 per cent alcohol, in which the membrane fragments were kept until further used. Before actual use, the membrane piece to be used was placed in absolute alcohol for several hours or perhaps a day. From the absolute alcohol the membrane was placed, just before use, on a dry, sterile towel so as to enable the absolute alcohol to evaporate. In this dry state, after the evaporation of the alcohol, the membrane was used as a wrapping for an operated nerve. As will be noted on reading the protocols, Cargile membrane thus stored in absolute alcohol reacts very differently toward tissues than Cargile membrane not stored in alcohol. In our discussion we shall use the term "alcoholized Cargile membrane" meaning thereby Cargile membrane, either chromatized or unchromatized, which had for a time been kept in alcohol. The characteristics of an alcoholized Cargile membrane were discovered quite by accident. Our own observations pertain to the use of such a membrane as a wrapping for an operated nerve; its wider application in surgery has not been considered.
The protocols of experiments under Series No. 16, auto-nerve transplants with a wrapping of " Cargile membrane " and " alcoholized Cargile membrane,"are as follows:
PROTOCOLS
EXPERIMENT No. 226.-Dog No. 34; medium size; full grown; 20 hours. May 28,1918, left sciatic exposed and internal popliteal freed. Right ulnar exposed. A segment 4.6 cm. length of right ulnar transplanted to the resected internal popliteal; one central and distal fine silk thread suture used; distally a second epineural suture placed. Good approximation attained. A single layer of Cargile membrane, cut long enough to overlap suture lines 5 mm., wrapped about nerve; well applied, forms close-fitting tube. Both wounds closed. May 29, dog found dead next morning; distemper. Wound reopened, and sciatic exposed. Resected nerve ends appear slightly congested. Transplant and Cargile membrane found well in place; a small amount of fluid noted within membrane; membrane loosely adherent to surrounding tissues; adhesions easily broken down. Nerve and transplant surrounded by Cargile membrane removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin.
Microscopic findings.-In cross and longitudinal sections, Cargile membrane presents the appearance of a thin layer of dense collagenous connective tissue. Numerous leucocytes
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between membrane and epineural sheath of the transplant. Coagulum and numerous leucocytes on outer surface of membrane. At the central wound, beginning degeneration of distal end of central nerve fibers noted; leucocytes and extravasated red blood cells between these fibers. In central end of transplant, for a distance of about 1 mm., myelin degeneration noted; here leucocytes in and between neurolemma sheaths of fibers. More distal in the transplant, few leucocytes observed.
EXPERIMENT No. 227.- Dog No. 33; half grown; medium size; 3 days. May 27, 1918, left sciatic exposed and the internal popliteal freed. Right ulnar exposed and freed. A segment of 5 cm. length of right ulnar transplanted to left internal popliteal. One central and distal silk suture placed. Distal suture gave way; in resuturing this central suture gave way; central resutured. Fair approximation attained. A single layer of Cargile membrane wrapped about transplant and suture lines; well applied. Wounds closed. May 30, dog found dead in the morning; distemper. Superficial wounds healed. On exposing sciatic, slight infection of deep wound noted. The Cargile membrane evident; small amount of exudate within the membrane. Resected nerve and transplant with Cargile membrane removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin.
Microscopic findings.-In sections, Cargile membrane is found well in place. Coagulum leucocytes, extravasated blood observed within membrane and about its outer surface. Beginning degeneration of distal end of central stump and central end of the transplant to be observed. Central end of transplant and distal end of central stump united; fibrin, coagulum, leucocytes, extravasated red blood cells intervening. Leucocytes observed in and between neurolemma sheaths of the transplanted nerves.
EXPERIMENT No. 228.- Dog No. 3; medium size; full grown; 56 days. June 11, 1918, right sciatic exposed and the internal popliteal freed. The left ulnar exposed. A segment 2 cm. length of left ulnar transplanted to the right internal popliteal. One central and distal fine silk thread suture placed; good approximation. Two layers of Cargile membrane wrapped about transplant and the resected nerve ends; well applied, forming close-fitting tube. Slight oozing from resected nerve ends, not controlled. Both wounds closed. August 6, killed. Dog in good condition; active; no neurotrophic ulcer right foot. Wound well healed. On exposing the right sciatic, transplant is found well in place; no trace of Cargile membranes evident. Quite a little increase of connective tissue about the transplant and suture lines noted; adherent to underlying muscles. No distinct central bulb noted. No contraction of the calf muscles on cutting the nerve. Resected nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Differential neuraxis silver staining not entirely satisfactory.
Microscopic findings.-In both cross and longitudinal sections of the region of the transplanted nerve segment no trace of the Cargile membranes observed. Distinct thickening of the epineural sheath of the transplant observed. New neuraxes can be traced from the distal end of the central stump, through the central wound into the transplant, and through the transplant into the central end of the distal popliteal; relatively few neuraxes have passed the distal wound. Remains of degenerated myelin observed in the transplant. Many small myelin ovoids seen in the distal popliteal, as also numerous nucleated syncytial bands in which no neuraxes are differentiated observed.
EXPERIMENT No. 229.- Dog No. 30; large; full grown; 129 days. May 22, 1918, two segments of the right ulnar of 2.3 cm. length transplanted to the resected left sciatic. Each segment sutured separately, centrally, and distally, using fine Chinese silk sutures. Fair approximation attained. The transplants and the resected nerve ends, for a distance of 8 mm., wrapped in a single layer of Cargile membrane well applied. Both wounds closed. September 28, dog found dead in the morning. Seemed fairly well day previous; moderate emaciation; skin disease; no neurotrophic changes left foot. On removing skin over operated nerve a small encapsuled stitch abscess noted; does not extend to deeper tissues. On exposing the left sciatic, no material increase of connective tissue noted. Transplants found well in place, demarked by presence of sutures. Transplant presents the appearance of a normal nerve. No trace of Cargile membrane observed. No distinct central bulb noted. Calf muscles of good size and color. Left sciatic and transplant, posterior tibial and external popliteal removed and fixed in ammoniated alcohol for pyridine-silver staining.
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Microscopic findings.-No trace of Cargile membrane observed in sections. In cross sections of the transplant about 1 cm. distal to the central wound, the funicular structure of both of the transplanted ulnar segments preserved, and are surrounded by common fibrous tissue sheath. All of the funiculi on the transplanted ulnar segments contain new neuraxes, certain of these are myelinated, the majority not. Numerous small bundles of nerves observed in the connective tissue intervening between the two nerve segments transplanted. Numerous neuraxes can be traced from the transplants through the distal wound into the distal sciatic segment, all of the funiculi containing them. New neuraxes in good number observed in the posterior tibial and the external popliteal.
EXPERIMENT No. 230.- Dog No. 2; medium size; full grown; 342 days. June 12, 1918, right sciatic exposed and the internal popliteal bundle freed. The left ulnar exposed and freed. A segment of 2 cm. length of the left ulnar transplanted to the right internal popliteal. One central and one distal fine silk thread suture placed; good approximation. A double layer of Cargile membrane wrapped about transplant and resected nerve ends; well applied. Both wounds closed. May 20, 1919, killed. Dog in very good condition; uses right foot well as normal dog. On exposing the right sciatic, external popliteal bundle found free. Distinct central bulb, which tapers toward the transplant, noted. Transplant has the appearance of normal nerve, though surrounded by quite dense connective tissue and adherent to underlying muscle. No trace of Cargile membrane. Calf muscles exposed; these have the appearance of normal muscle. Nerve and transplant completely freed. On slowly cutting nerve with scissors central to the transplant, vigorous contraction of calf and plantar foot muscles. Nerve and the transplant removed and fixed in ammoniated alcohol for pyridine- silver staining. Neuraxes only very lightly stained.
Microscopic findings.- Numerous myelinated and nonmyelinated nerves traced from central bulbous enlargement to the distal popliteal. In cross sections of the transplant funiculi are found to be well maintained, with only moderate increase of connective tissue about the transplant. Calf and plantar muscle not studied in this experiment.
EXPERIMENT No. 231.- Dog No. 35; small dog; full grown; 350 days. June 1, 1918, left sciatic exposed; internal popliteal bundle freed. Right ulnar exposed. A segment. 3.4 cm. length taken from the right ulnar and transplanted to the resected left internal popliteal. One central suture of vessel silk; vessel silk suture attempted for distal suture, broken twice; finally Chinese silk suture used. Good central approximation attained, distal “fair.” Double layer of Cargile membrane wrapped about transplant and the resected nerve ends; well applied; formed close fitting tube. Both wounds closed. May 16, 1919, killed. Dog in very good condition; active; no neurotrophic changes on left hind foot. On exposing the left sciatic, external popliteal bundle found free. Operated internal popliteal presents appearance of a normal nerve, except that region of the transplanted nerve appears slightly smaller than resected nerve. No trace of Cargile membrane, only very moderate increase of connective tissue about the transplant. Small spindle-shaped central bulb noted. Calf muscles exposed; these have the appearance of normal muscle. External popliteal resected and internal popliteal freed from bed. On cutting nerve slowly with scissors central to the transplant, vigorous contraction of calf and plantar muscles observed. Resected nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Silver differentiation faint.
Microscopic findings.- In longitudinal sections embracing central wound, line of central wound is indistinct. Numerous myelinated and nonmyelinated nerve fibers can be traced from central stump through the transplant to the distal nerve. In cross sections made about middle of the transplant, many myelinated and nonmyelinated fibers observed within the transplant, the funiculi of which are well maintained. Outside of perineural sheath, but within the denser connective tissue surrounding the transplant, many small funiculi of nerve fibers observed. Many new nerve fibers, both myelinated and nonmyelinated, noted in the distal popliteal. No trace of Cargile membrane observed in any of the sections.
EXPERIMENT No. 232.- Dog No. 32; medium size dog; full grown; 358 days. May 24, 1918, left sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment of 3 cm. length taken from the right ulnar transplanted to the left internal popliteal. Three epineural sutures placed distally; good approximation. A single layer of Cargile
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membrane wrapped about transplant and resected nerve ends; well applied. Both wounds closed. May 19, 1919, killed. Dog in very good condition; walks well. No neurotrophic changes left hind foot. On exposing the left sciatic external popliteal found free. Operated internal popliteal presents a small spindle-shaped central bulb; otherwise the appearance of a normal nerve. No trace of Cargile membrane. No material increase of connective tissue about the transplant noted. Calf muscles exposed; these of normal size and appearance. The internal popliteal and transplant freed from the bed. On slowly cutting the nerve with scissors central to the transplant, good contraction of the calf and plantar muscles observed. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good differential silver staining attained.
Microscopic findings.- In longitudinal sections of the central wound region, the central wound not easily located. Numerous myelinated and nonmyelinated nerves pass from distal end of the central bulb to and through the transplant to the distal nerve. In cross sections of the transplant taken near the central wound, many new nerve fibers found within the transplant. Funiculi and perineural sheaths maintained. Many small nerve funiculi in the connective tissue surrounding the transplant. In cross sections through the distal part of the transplant, perineural sheaths of transplant not so distinct. Numerous smaller and larger funiculi in the connective tissue outside of the transplant, in distribution more or less clearly bounded by an outer fairly dense connective tissue sheath, probably connective tissue replacing the Cargile membrane. Many new nerve fibers, nearly as many as seen in a normal nerve, traced through the distal wound into the distal popliteal. Very complete regeneration through the transplanted nerve segment.
EXPERIMENT No. 233.- Dog No. 31; large; full grown; 359 days. May 23, 1918, left sciatic exposed and freed. Right ulnar exposed and freed. A segment of 2.9 cm. length taken from the right ulnar transplanted to the left sciatic. One central and distal fine silk thread suture placed; fairly good approximation attained. One layer of Cargile membrane wrapped about the transplant and resected nerve ends; well applied, forming closely fitting tube. Both wounds closed. May 19, 1919, killed. Dog very good condition; uses left hind leg well; no neurotrophic changes left hind foot. On exposing the left sciatic, the region of the nerve transplant is located by reason of the distinct central bulbous enlargement. Transplant has the appearance of a normal nerve; no material increase of connective tissue about it. No trace of Cargile membrane. Calf muscles exposed; these have the appearance of normal muscle. After freeing nerve and transplant from the bed, on slowly cutting nerve central to transplant, vigorous contraction of calf and the plantar foot muscle noted. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.-In longitudinal sections through the central wound, central bulb distinctly evidenced by crisscrossing and tangling of the neuraxes of this region; line of central wound not clearly demarked. Numerous neuraxes pass from central bulb to and through the transplant. In cross sections of the transplanted nerve funicular structure and sheaths clearly are maintained. Numerous new nerve fibers observed with the transplant. Nearly all of the neuraxes observed in cross sections of the transplant found within its sheaths; only a few small scattered nerve funiculi found in the connective tissue surrounding the transplant. Numerous nerve fibers, myelinated and nonmyelinated, can be traced to the distal sciatic in which they are found in all of its several funiculi about equally distributed.
EXPERIMENT No. 234.- Dog No. 30; large; full grown; 44 days. August 15, 1918, right sciatic exposed and internal popliteal bundle freed. Left ulnar exposed and freed. A segment 2.1 cm. length taken from the left ulnar transplanted to the right internal popliteal. One central and one distal waxed, fine silk thread suture placed; good approximation. Two layers of Cargile membrane which had been stored several days in 70 per cent alcohol, then for 24 hours in absolute alcohol and before use spread on a dry, sterile towel until dry, wrapped about the nerve and the resected nerve ends. (Cargile membrane thus treated is in subsequent experiments referred to as alcoholized Cargile membrane.) Cargile membrane well applied; close-fitting tube formed. Both wounds closed. September 28, dog found dead in the morning; seemed fairly well the day before; skin disease; moderate emaciation. Wound well healed. On exposing the right sciatic, external popliteal found free. Region of the
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transplant in the internal popliteal easily located by reason of the presence of the Cargile membrane, which appeared wrapped about the nerve, forming a closely fitting sheath which was only slightly adherent to the surrounding connective tissue. It seemed evident that there had taken place no absorption of the alcoholized Cargile membrane. Nerve and the transplant with the Cargile membrane removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; hematoxylin and eosin; safranine and licht grün.
Microscopic findings.- Alcoholized Cargile membrane evident in all of the sections, cross and longitudinal, taken at different levels in the transplant and through central and distal wounds. In cross sections of the transplant, the two layers of the membrane appear as undulating membranes of fibrous tissue, which show no evidence of undergoing absorption. On both faces of the membranes newly formed, vascularized connective tissue observed. Cargile membranes found to overlap central and distal wounds for a distance of 6 mm. to 8 mm. The transplanted nerve segment found well united to resected nerve ends. In cross sections of the transplant, it may be observed that the funicular arrangement is well maintained, as also the perineural sheath of the funiculi. Between these perineural sheaths
FIG. 233.- Cross section of auto-nerve transplant, wrapped with two layers of alcoholized Cargile membrane, Experiment No. 234, 44 days after operation. Tissue fixed in formalin and stained in iron-hematoxylin. The deeply stained and folded lines in the figure represent sections of the Cargile membrane. Note the absence of marked connective tissue proliferation and the Cargile membrane newly formed connective tissue is found. In this connective tissue numerous small nerve funiculi observed; these are limited in distribution peripherally by the Cargile membrane.
EXPERIMENT No. 235.- Dog No. 56; large; full grown; 172 days. July 24, 1918, left sciatic exposed; internal popliteal bundle freed. The right ulnar exposed and freed. A segment of 4 cm. length taken from the right ulnar transplanted to the left internal popliteal. One central and distal waxed, fine silk thread suture placed. Central approximation good; distal fair, improved by epineural suture. Transplant and the resected nerve ends wrapped in two layers of alcoholized Cargile membrane; well applied, forming closely fitting tube. Adrenalin used to control oozing; dry, clean field. Both wounds closed. January 11, 1919,dog had been very active. Found dead in the morning, having hung himself on a tie rope. No neurotrophic changes left hind foot. On exposing the left sciatic external popliteal found free; only very loosely adherent to operated internal popliteal. Operated internal popliteal appears of relatively large size. Closer inspection reveals that the alcoholized Cargile membrane had not been absorbed, and is surrounded by a thin layer of connective tissue only loosely adherent to the surrounding tissue. No distinct central bulb evident,
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Cargile membrane extending over central suture. Calf muscles exposed; these present the appearance of normal muscles. The whole of the sciatic nerve, with transplant and the Cargile membrane sheath removed in one piece and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining, though pale, attained.
Microscopic findings.- In all of the several series of sections, though especially in cross sections of the transplant, the two layers of the Cargile membrane distinctly observed, surrounded by a thin layer of fairly dense connective tissue. The membranes have the structural appearance of closely felted fibrous tissue, though silver stain used does not differentiate this tissue clearly. The funiculi of the transplanted nerve segment evident. Numerous myelinated and nonmyelinated nerve fibers observed within the transplant. Between the denser connective tissue enveloping the Cargile membrane and the perineural sheaths, a looser connective tissue observed; in this, here and there, groups of fat cells seen. In this looser connective tissue, smaller and larger nerve funiculi seen. Regenerating nerve fibers traced through the distal wound into the distal popliteal nerve, in which there may be ob- served, in all of the funiculi, numerous myelinated and nonmyelinated nerve fibers.
FIG. 234.- Cross section of auto-nerve transplant, wrapped in alcoholized Cargile membrane, Experiment No. 236, terminated 272 days after the operation; pyridine-silver preparation. The Cargile membrane as a wavy, undulating layer embedded in the fibrous tissue surrounding the nerve trunk. Note the neurotization of the transplanted nerve segment as seen in the figure
EXPERIMENT No. 236.- Dog No. 35; small dog; full grown; 272 days. August 20, 1918,right sciatic exposed and the internal popliteal bundle freed. Left ulnar exposed and freed. A segment of 3 cm. length taken from the left ulnar transplanted to the right internal popliteal. One central and distal waxed, fine silk thread suture placed. Only fair central and distal approximation attained. One layer of alcoholized Cargile membrane wrapped about trans-plant and the resected nerve ends; fairly smooth tube formed. Both wounds closed. May19, 1919, killed. Dog in very good condition. No neurotrophic changes right hind foot. On exposing the right sciatic, external popliteal found free. Quite distinct central bulb on internal popliteal noted. Macroscopically, no trace of Cargile membrane. Moderate increase of connective tissue about operated internal popliteal in region of the transplant recorded. Calf muscles exposed; these have the appearance and size of normal muscles. After freeing operated internal popliteal and transplant and cutting the external popliteal
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near head of fibula, on slowly cutting with scissors the nerve central to the transplant, good contraction of calf and foot muscles observed. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good differential silver staining attained.
Microscopic findings.-In longitudinal sections of the central wound, distinct central bulb, evidenced by twisting and intercrossing of neuraxes, noted. Numerous myelinated and nonmyelinated nerve fibers enter the transplant. In cross sections of the transplant 1 cm.distal to the central wound, evidence of the Cargile membrane made out in the form of a wavy, undulating membrane of fibrous tissue enveloped in a fairly dense layer of fibrous tissue. This not so clearly observed in longitudinal sections of the transplant. The funiculi of the transplanted nerve segment clearly demarked; many new nerve fibers observed within these funiculi; both myelinated and nonmyelinated. A looser connective tissue intervenes between the perineural sheaths and the peripheral denser layer of the fibrous tissue enveloping the Cargile membrane. In this a few small nerve funiculi observed. Numerous myelinated and nonmyelinated nerve fibers observed in the distal popliteal in all of its nerve funiculi.
EXPERIMENT No. 237.-Dog No. 32; medium size; full grown; 274 days. August 17,1918, right sciatic exposed and the internal popliteal bundle freed. The left ulnar exposed and freed. A segment of 2.1 cm. length taken from the left ulnar transplanted to the resected right internal popliteal. One central and distal waxed, fine silk thread suture placed. "Fair"'central and distal approximation attained. Two layers of alcoholized Cargile membrane wrapped about the transplant and resected nerve ends; good tube formed distally, not so good centrally. Both wounds closed. May 19, 1919, killed. Dog in very good condition; uses right hind foot well; no neurotrophic changes right hind foot. On exposing the right sciatic, external popliteal bundle found free. Moderate increase of connective tissue about operated internal popliteal in region of the transplant. No macroscopic evidence of Cargile membrane. Calf muscles exposed; these have the size and appearance of normal muscles. After cutting the external popliteal near head of fibula and completely freeing the sciatic and the transplant from the bed, on slowly cutting with scissors the nerve central to the transplant, good contraction of calf and foot muscles observed. Sciatic and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining in only part of the series; the remainder pale.
Microscopic findings.-In cross sections of the transplanted nerve segment made about1 cm. distal to the central wound, there is observed a dense fibrous tissue layer separated from the epineural and perineural sheaths of the transplant by looser connective tissue. This outer, denser layer of connective tissue may contain remnants of the Cargile membrane; if so, these are not clearly differentiated in the silver stain. Numerous myelinated and non-myelinated nerve fibers within the funiculi of the transplanted nerve segment observed; as also in the connective tissue surrounding the transplant. Numerous myelinated and non-myelinated nerve fibers can be traced through the transplant and distal wound to the distal popliteal.
EXPERIMENT No. 238.-Dog No. 31; large; full grown; 275 days. August 16, 1918, right sciatic exposed; internal popliteal bundle freed. The left ulnar exposed and freed. A segment of 3.6 cm. length taken from the left ulnar transplanted to the resected right internal popliteal. Quite a little bleeding; controlled. One central and distal waxed, fine silk thread suture placed; good approximation. Two layers of alcoholized Cargile membrane wrapped about the transplant and resected nerve ends; well applied, forming close-fitting tubular sheath. Both wounds closed. May 19, 1919, killed. Dog in very good condition; used right hind foot well; no neurotrophic changes right foot. On exposing the right sciatic, external popliteal found free. Only moderate increase of connective tissue about operated internal popliteal, especially transplant region, observed; here somewhat adherent to underlying muscle. No macroscopic evidence of Cargile membrane noted. Small spindle-shaped central bulb found. Calf muscles exposed; these have the appearance and size of normal muscles. Nerve and transplant freed from bed. On slowly cutting with scissors nerve central to the transplant, contraction of calf muscles and movement of toes observed. The nerve and the transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Fair differential silver staining attained.
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Microscopic findings.- In longitudinal sect ions of the central wound area, central bulb is evidenced structurally by twisting and crisscrossing of neuraxes. In cross sections of the transplant, it is observed that the funicular structure of the nerve is well maintained; each funiculus with its perineural sheath. Numerous myelinated and nonmyelinated nerve fibers observed within these sheaths. A relatively dense layer of fibrous tissue surrounds the transplant; in this layer no distinct evidence of the Cargile membrane noted, except near distal wound; here, in a series of longitudinal sections, remnants of Cargile membrane seen. A layer of looser connective tissue intervenes between the perineural sheaths and the peripheral layer of denser fibrous tissue; in this looser fibrous layer many small funiculi of nerve fibers observed. Numerous myelinated and nonmyelinated nerve fibers can be traced through the distal wound into the distal popliteal nerve.
EXPERIMENT No. 239.- Dog No. 52; medium size; full grown; 300 days. July 19, 1918, left sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment of the right ulnar of 3.8 cm. length transplanted to the resected left internal popliteal. One central and distal waxed, fine silk thread suture placed; very good central and distal approximation. Four layers of alcoholized Cargile membrane wrapped about the transplant and the resected nerve ends. Wound not quite dry; a small amount of blood with Cargile membrane sheath, especially near distal suture. Both wounds closed. May 15, 1919, killed. Dog in very good condition; uses left hind foot well; no neurotrophic changes. On exposing the left sciatic, it is observed that the external popliteal is moderately adherent to the operated internal popliteal, especially near distal wound. Moderate increase of connective tissue about the transplant. Transplant appears relatively thick in its middle third; it could not be determined on gross inspection whether a portion of the Cargile membrane sheath had not been absorbed. Calf muscles exposed; these have appearance and size of normal muscles. Nerve and transplant completely freed from bed. Observer was unavoidably interrupted and could not test the functional return of muscles until about one-half hour after dog was killed. Cutting the nerve central to transplant causes only very feeble contraction of the calf muscles; no toe movement noted. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining central portions, distal only fair.
Microscopic findings.- In longitudinal sections of the central wound area, evidence of central bulb observed, this evidenced by the twisting and crisscrossing of neuraxes in region of central wound and central stump proximal thereto. Many nerve fibers traced to the central end of the transplant. In cross sections of the transplant taken about 1 cm. distal to the central wound, the appearance presented suggests the possibility that two nerve segments were transplanted ulnar and median or ulnar and musculocutaneous in that two relatively large nerves, distinctly separated and surrounded by a relatively dense layer of connective tissue, are observed. No distinct evidence of the Cargile membrane sheath noted in this denser fibrous layer. Between the perineural sheaths of the nerve transplant and the peripheral denser fibrous sheath a looser connective tissue intervenes; in this are found numerous small nerve funiculi. Numerous myelinated and nonmyelinated nerve fibers can be traced through the transplant and distal wound into the distal popliteal, in which they are found in large numbers.
Our experimental observations relating to the use of Cargile membranes as a wrapping for operated or liberated peripheral nerves confirms the observations of Craig and Ellis79 in so far as concerns the early absorption of both the unchromatized and the chromatized Cargile membrane such as is furnished commercially for use in surgical practice. A number of our experiments are not here listed owing to the death of animals from intercurrent infectious disease. A number were of relatively short duration and permitted observation as to the early disappearance of the Cargile membrane, when placed in tissue. In a number of discarded experiments there was wound infection. Taking into consideration these experiments not listed it is our observation that Cargile membrane when wrapped about a peripheral nerve and inclosed
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within an aseptic wound disintegrates and is absorbed within 10 davs to 15 days. Experiments No. 226 and No. 227 may serve to show that a Cargile membrane when wrapped about a nerve incites relatively little connective-tissue proliferation. Leucocytes are evident in the tissue fluids within and outside of the membrane. By the middle of the second month after the operation (Experiment No. 228) there could be found no trace of the two layers of the Cargile membrane used in this transplant. It is especially during the first two months after the operation of peripheral nerve suture or of nerve transplantation that a membranous structure wrapped about a nerve would be most effective in preventing ingrowth of connective tissue and prevent the spread of down-growing neuraxes. Downgrowth of central neuraxes is not pronounced until toward the end of the second week, although evident prior to this time. Cargile membrane as found in trade, therefore, can not exert much influence one way or the other when used as a wrapping about an operated peripheral nerve. Four of the experiments (No. 230 to No. 233) were carried on respectively for nearly a year. In these experiments there was found quite complete repair of the peripheral nerve through the transplant, and, as the records show, not very much increase of connective tissue in the region of the transplant. The increase in the connective tissue noted is evident in an increase in the density of the epineural sheath as also in the formation of a layer of fibrous tissue which appears to have replaced the Cargile membrane.
The results attained in the experiments in which "alcoholized Cargile membrane" was used were wholly unexpected. The method of storing unusedportions of Cargile membrane in alcohol was resorted to as a ready means for keeping sterile portions of the membrane not used at any one experiment. Much surprise was experienced on exposing the operated nerve in Experiment No.234, 44 days after the operation, to find the region of the operation completely surrounded by a layer of Cargile membrane, which, like the nerve itself, above and below the seat of the operation was only very loosely attached to the surrounding tissue. It had been anticipated in this experiment that no trace of the Cargile membrane would be found. On consulting the records it was found that the Cargile membrane used in this case had been stored in alcohol prior to use in the operation. In Experiment No. 234, which terminated approximately one and one-half months after operation, the two layers of Cargile membrane wrapped about the nerve at the time of operation appear not to have been affected by the tissue fluids nor by phagocytic action. The two layers of Cargile membrane and the nerve funiculi of the transplanted nerve segment are occupied by cellular, loose connective tissue. In the connective tissue are found small bundles of neuraxes, confined within the Cargile membrane. To what extent the presence of the alcoholized Cargile membrane would operate to prevent the ingrowth of connective tissue can only be conjectured. It should be borne in mind that the connective tissue found in the regions of the central and distal wounds in the early stages of nerve repair after resection and bridging by a nerve transplant, or in the one wound after simple suture, is histogenetically to a large extent derived from the connective tissue of the severed nerve trunk, its endo-, peri-, and epineurium. Even after the most favorable primary nerve suture, in aseptic tissue, with desired
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approximation of severed nerve ends, embryonic connective tissue which progresses toward the stage of connective tissue fibril formation is formed between the severed nerve ends. Slight hemorrhage with formation of blood clot increases the connective tissue formation in proportion to clot formation. Wrapping with Cargile or other membranous structures does not prevent this. The connective tissue found between the central stump of a resected nerve and the central end of a nerve transplant, at the time when the central neuraxes begin to grow toward the periphery during the first 10 days to 14 days after the operation, is not materially influenced by the presence or absence of an alcoholized Cargile membrane. In an operation for nerve transplantation in which a nerve segment of perhaps 6 cm. to 8 cm. in length is used as a nerve bridge the central neuraxes would not reach the region of the distal wound until after the expiration of from 8 weeks to 12 weeks after the operation, at which time the connective tissue at the distal wound would be quite fully organized. There seems warrant for the statement that in the distal wound region the presence of the alcoholized Cargile membrane would influence the ingrowth and differentiation of the connective tissue. In Experiment No. 235, terminated somewhat over five months after the operation, the two layers of alcoholized Cargile membrane used at the operation were still evident macroscopically at the time the nerve was exposed and removed for study. The Cargile membrane was found surrounded by a thin layer of connective tissue only loosely adherent to the surrounding tissue. Between the Cargile membrane and the perineural sheaths of the funiculi of the nerve transplant, there is found a loose areolar tissue containing here and there fat cells arranged in small groups or singly. Denser areolar connective tissue surrounds the external surface of the Cargile membrane. In this experiment the small bundles of neuraxes found outside of the perineural sheaths of the nerve transplant are noted in the loose areolar tissue observed within the Cargile membrane. In experiments terminated approximately nine months after the operation (Experiments No. 236 to No. 239) in which alcoholized Cargile membrane was used to wrap the nerve in the field of operation, either as single layer or as several layers, the Cargile membrane was not recognized macroscopically at the time the nerves were removed for study nor with any degree of certainty microscopically in sections. A layer of relatively dense fibrous tissue, in the form of wavy, undulating, fibrous bands, more readily recognized in cross sections of the transplant region, appear to have replaced the Cargile membrane. Within this layer there is an area of looser areolar tissue with groups of fat cells, separating the denser layer from the transplanted nerve trunk. In each of the four experiments of relatively long duration it is worthy of note that the funicular structure of the transplanted nerve segments was well maintained; neurotization of the distal segment of the operated nerve had been obtained through down growth of central neuraxes, which pass in the main through the funiculi of the transplanted nerve segments.
The experiments in which alcoholized Cargile membrane was used as a sheath for the operated nerve in the field of nerve repair seem to us to warrant the deduction that Cargile membrane stored in alcohol after the manner here
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detailed remains unabsorbed when inclosed in aseptic wounds for a period extending at least four months. Alcoholized Cargile membrane was not found to incite unduly connective tissue proliferation; it remains closely adherent to the nerve transplant and the resected nerve ends and does retain within the limits of the membrane the down-growing neuraxes. The presence of the alcoholized Cargile membrane does not appear to influence the down-growth of the central neuraxes through the funiculi of the nerve transplant. Our experiments seem to us to warrant the conclusion that the use of alcoholized Cargile membrane, prepared as above stated, and used in double or triple layers as a sheath for wrapping nerve trunks at the suture line after the nerve suture or as a wrapping for a nerve transplant when such sheathing is deemed necessary, deserves consideration in operations for peripheral nerve repair.
SERIES NO. 17
AUTO-NERVE TRANSPLANTS, WRAPPED IN AUTO-FASCIAL SHEATHS OF FASCIA LATA
In a series of auto-nerve transplants, the transplant and the central and distal wound regions were insheathed with a layer of fascia lata, taken at the time of operation from the same animal. In these experimental operations the sciatic nerve of a dog was resected and bridged by a nerve transplant taken from the ulnar of the opposite side of the same dog. After thus bridging a nerve defect, the fascia lata on the side on which the sciatic nerve was operated upon was exposed through a long incision, denuded of its subcutaneous covering and a piece 5 cm. to 6 cm. long and 2 cm. wide excised, rinsed in warm sterile salt solution and applied as a sheath inclosing the nerve transplant and the central and distal wounds. A close-fitting tube was formed by placing central and distal stay sutures and intervening half mattress sutures. The inner surface of the fascia lata was placed adjacent the nerve. The operation was somewhat tedious and necessitated the making of three rather extensive surface wounds. The three wounds were closed and, in the majority of the experiments, healed by primary union. Auto-fascial sheaths have been quite extensively used in surgery otherwise than in connection with peripheral nerve repair. Denk 82 used fascia lata tubulization in connection with cases of neurolysis in the Balkan Wars. The cases are not discussed and the value of the fascial sheaths is not clearly brought out. Döpfner 83 and Hirschel 84 advocate the use of fascial sheaths in nerve repair. Kredel 83 had opportunity to reoperate a case 24 days after a fascial sheath was placed about the tibialis which bad been liberated and longitudinally incised. It was found that the fascial sheath which had been loosely applied had contracted so as to fit the nerve closely. He expresses the fear that a fascial sheath may contract so as to strangulate the nerve. Kirk and Lewis 88 present a series of experimental observations in which fascial sheaths were used to form a tubular suture. It was found that neuraxes of central origin would pass through the lumen of the fascial tube to reach the distal segment. Their experiments conceived for a different purpose are comparable in so far as concerns the behavior of a fascial sheath in relation with a wounded peripheral nerve.
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The protocols of experiments on auto-fascial sheaths for auto-nerve transplants are as follows:
PROTOCOLS
EXPERIMENT No. 240.- Dog No. 18; large; full grown; 14 days. May 15, 1918, left sciatic exposed and freed. Right ulnar exposed and freed. Two segments of the right ulnar, each having 3 cm. length, transplanted to the resected left sciatic. For each segment, one central and distal suture of fine Chinese silk placed; good central, "fair" distal approximation-
FIG. 235.- Cross section of an auto-nerve transplant, wrapped in an auto-fascial sheath, Experiment No. 240, terminated 14 days after operation; formalin fixation iron-hematoxylin staining. Note the funicular structure of the two ulnar nerves used as transplants. The tube of fascia is clearly evident in the figure. Epineural fibrous tissue is materially increased
tion attained. The fascia lata of the left leg exposed and a piece 4.5 cm. long and 2 cni.wide exsected and wrapped about the nerve transplants and resected. nerve ends, with theinner surface of the fascia lata adjacent to the nerve. Central and distal. stay sutures of Chinese silk and two intervening half mattress sutures placed, forming a fascial tube. The three wounds closed. May 29, dog found dead in the morning. The fascial and ulnar wounds open. Sciatic wound, deep wound healed, superficial wound several stitches have given away. On exposing the left sciatic, fascial sheath found well in place; surrounded by newly
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formed connective tissue; adherent to the surrounding tissue and the underlying muscle. Fascial sheath presents a glistening white appearance. Deep wound not congested; no distinct evidence of infection noted. Sciatic and transplant and fascial sheath removed and fixed in neutral formalin. Serial sections stained in iron-hematoxylin and picro-fuchsin.
Microscopic findings.-In longitudinal sections of the central and distal wound areas, it is noted that the distal end of the central stump and the central end of the distal stump present early degenerative changes of the nerve fibers. In cross sections of the transplant and the fascial sheath, the outer surface of the fascial sheath is seen to be covered with coagulum, extravasated red blood cells, numerous leucocytes and newly formed connective tissue. The cross cut transplanted nerves are clearly demarked; their perineural sheaths appear as thickened. Between the inner surface of the fascial sheath and the nerve transplants, newly formed connective tissue, containing many leucocytes and phagocytic cells,
FIG. 236.- Cross section of auto-nerve transplant wrapped in auto-fascial sheath, Experiment No. 241, terminated 15 days after operation; formalin fixation, iron-hematoxylin staining. The very complete auto-fascial tube formed is evident in the section. Connective tissue proliferation is clearly recognized but is not excessive observed. The nerve fibers of the nerve transplant do not present the same type of degeneration as do the nerve fibers of the distal sciatic; fragmentation of the myelin not so far advanced.
EXPERIMENT No. 241.-Dog No. 17; medium size; full grown; 15 days. August 13.1918, right sciatic exposed and freed. Left ulnar exposed and freed. A segment of 3.6 cm. length taken from the left ulnar transplanted to the resected sciatic. One central and distal waxed, fine silk thread suture placed; good approximation. A portion of the fascia lata taken from the right leg of the same dog wrapped about the transplant and resected nerve ends. Three half mattress sutures placed. Good fascial tube formed. The three wounds closed. August 28, killed. Dog not well since operation. All three wounds in part open; superficial skin wounds, deep wounds healed. On exposing the right sciatic, deep wound found healed; appears not to be infected. Fascial sheath and transplant found well in place. Fascial sheath surrounded by newly formed connective tissue; adherent to surrounding tissue; evidence of sanguineous fluid within the fascial sheath. Sciatic and transplant
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plant and fascial sheath removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchin; safranine and licht grün.
Microscopic findings.-In cross sections of the transplant and the fascial sheath, trans- planted nerve found clearly demarked, its perineural sheaths appear thickened. On both surfaces of the fascial sheath newly formed connective tissue, containing leucocytes, extravasated red blood cells, and coagulum. The tendon cells of the transplanted fascial sheath present normal shape and staining reaction.
EXPERIMENT No. 242.- Dog No. 40; medium size; not quite full grown; 22 days. August 19, 1918, right sciatic exposed and internal popliteal freed. Left ulnar exposed and freed. A segment of 3 cm. length taken from the left ulnar transplanted to the right internal popliteal. One central and distal waxed, fine silk thread suture placed; approximation not good. Central and distal epineural suture improves approximation. An auto-fascial sheath taken from the right fascia lata of the same dog, wrapped about the transplant and resected nerve ends. One central and distal stay sutures and three intervening half mattress silk sutures placed. Good fascial tube formed. The three wounds closed. September 10, killed. Much emaciated; severe skin disease; trophic ulcer over right hip. Sciatic wound not completely healed, several stitches had given away; deep wound seemed healed. On exposing the right sciatic, slight evidence of infection of deep wound noted; parts congested. Transplant and fascial sheath found well in place, surrounded by connective tissue and adherent to underlying muscle. External popliteal found adherent to side of fascial sheath. Sciatic, transplant, and fascial sheath removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-In cross sections of the transplant and fascial sheath, fascial sheath found surrounded by newly formed connective tissues containing numerous leucocytes. Between transplanted nerve and fascial sheath newly formed connective tissue, numerous leucocytes, and extravasated blood cells observed. In longitudinal sections of the central and distal wound areas, leucocytes and phagocytic cells especially numerous in the region of central and distal wounds. Distal nerve in process of degeneration. Fragmentation of myelin and breaking down of the nerve fibers of the transplanted nerves not of the same nature as in distal popliteal; proliferation of sheath cells not noted.
EXPERIMENT No. 243.- Dog No. 57; small dog; full grown; 46 days. July 25, 1918, left sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment 3.2 cm. length taken from the right ulnar transplanted to the resected left internal popliteal. Centrally one through-and-through suture and one epineural suture, distally one suture of waxed, fine silk thread placed; approximation good. Adrenalin used; dry field. Fascial sheath taken from the fascia lata of the left leg of the same dog wrapped about transplant and resected nerve ends. Central and distal stay sutures and three intervening half mattress sutures placed. Good, even fascial tube formed. The three wounds closed. Toward the end of July developed skin disease; had not been well some weeks. September 9, the dog found dead in the morning. On exposing the left sciatic, external popliteal found free. Moderate increase of connective tissue about sheath. Distinct central bulb showing through central end of the fascial tube. Sciatic, transplant, and fascial sheath removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.- In cross sections through the transplant and the fascial sheath there is noted a fairly dense layer of fibrous tissue surrounding the fascial sheath. Sheath in close relation to perineural sheath of transplanted nerve segment, very little connective tissue intervening. Neurolemma sheaths of transplanted nerve segment evident; appear thickened. Certain ones contain ovoids of myelin and phagocytes filled with lipoid granules; others contain syncytial protoplasmic bands; these also observed outside of neurolemma sheaths. Distinct central bulb evidenced structurally. Fascial sheath extends over the central bulb.
EXPERIMENT No. 244.-Dog No. 55; large, full grown; 47 days. July 22, 1918, left sciatic exposed and the internal popliteal freed. The right ulnar exposed and freed. A segment of 3 cm. length taken from the right ulnar transplanted to the left internal popliteal. One central and distal waxed, fine silk thread suture placed; fairly good approximation
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attained. A fascial sheath taken from the facsia lata of the left leg of the same dog wrapped about the transplant and resected nerve ends. One central and distal stay sutures and two intervening half mattress sutures placed. Good tube formed. Dry field. The three wounds closed. September 8, dog found dead in the morning; much emaciated; skin disease. On exposing the left sciatic external popliteal found moderately adherent to the operated internal popliteal; easily dissected. Fascial sheath found well in place, distinct increase of connective tissue about it; adherent to underlying muscle. Distal internal popliteal present a distinct, light-yellow color. Nerve and transplant and the facial sheath removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin ; safranine and licht grün.
Microscopic findings.- In cross sections of transplanted nerve and sheath, it is observed that a relatively thick laver of fibrous tissue surrounds fascial sheath. Between the fascial sheath and the transplanted nerve, and blending with the perineural sheath of the same, distinct layer of fairly dense fibrous tissue. In longitudinal sections embracing central and distal wounds, numerous leucocytes observed within the fascial sheath. In cross sections of the nerve transplant, increase in the amount of endoneural connective tissue noted. Within the old neurolemma sheaths, in many instances, one, two, three, or four small medullated fibers observed; other neurolemma sheaths distended with detritus derived from breaking down nerve fibers. Numerous nucleated, syncytial protoplasmic strands noted within the transplant in longitudinal sections of the same.
EXPERIMENT No. 245.- Dog No. 38; medium size; full grown; 61 days. June 21, 1918, left sciatic exposed; internal popliteal freed. The right ulnar exposed and freed. A segment of 2.5 cm. length taken from the right ulnar transplanted to the left internal popliteal. One central and distal waxed, fine silk thread suture placed; good approximation. Fascial sheath taken from the fascia lata of the left leg of the same dog wrapped about the transplant and resected nerve ends. One central and distal stay suture and one half mattress suture placed. Adrenalin used to control oozing. The three wounds closed. August 22, dog used in the morning for another operation; found dead 1.30 p. in.; did not recover from second operation. Dog in good condition; slight toe-drop left hind foot; neurotrophic ulcer dorsum of left foot. Sciatic and other wounds well healed. On exposing the left sciatic, the external popliteal found firmly adherent to the fascial sheath about operated internal popliteal. Fascial sheath clearly demarked; marked increase of connective tissue about it. Distinct central bulb evident through proximal end of the fascial sheath. Calf muscles found very atrophic. Nerve and the fascial sheath removed and fixed in ammoniated alcohol for piridine-silver staining. Quite good differential silver staining attained.
Microscopic findings.-In cross sections of the transplant and the fascial sheath, sheath found in close relation to the transplanted nerve segment; a thin layer of fibrous tissue surrounds the sheath. Endoneural connective tissue of the transplant distinctly increased. New neuraxes, which in longitudinal sections of the central wound can be traced from the distal end of the bulbous enlargment into the transplant, in cross sections are found arranged in the form of numerous very small nerve funiculi, often containing only a few nerve fibers, and without special fibrous sheath, but separated by endoneural connective tissue. New neuraxes traced through the transplant and through distal wound into the distal stump. In cross sections of the distal popliteal, scattered neuraxes found in all of its funiculi, in many instances more than one in old neurolemma sheath. Beginning regeneration of distal popliteal through transplant attained.
EXPERIMENT No. 246.-Dog No. 41; small dog; full grown; 31 days. July 28, 1919, left sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment of the right ulnar of 3.5 cm. length transplanted to the resected left internal popliteal. One central and distal waxed, fine silk thread suture placed. Good central approximation; distal partly pulled out as the sheath was being applied, a fascial sheath taken from the left fascia lata of the same dog wrapped about the transplant and resected nerve ends. One central and distal stay suture and one half mattress suture applied. Good fascial tube formed. Wounds clean and dry. All three wounds closed. August 28, dog found dead in the morning; had not been well for some time; much emaciated; small neutrophic
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ulcer on dorsum of left foot. On exposing the left sciatic, external potpliteal found quite adherent to fascial sheath. Fascial sheath well in place;- moderate increase of connective tissue about it noted. Found loosely adherent to underlying muscle. Sciatic and transplant with fascial sheath removed and fixed in ammoniated alcohol for pyridine-silver staining. Only fair differential neuraxis staining attained. Tissues not well embedded; sections much torn.
Microscopic findings.-In cross sections of the transplant and the fascial sheath, it is found that the sheath is surrounded by dense layers of fibrous tissue. Sheath in close relation to nerve transplant, very little connective tissue intervening. The transplanted nerve presents areas in which the old neurolemma sheaths are either broken down or distended with detritus and phagocytic cells with lipoid globules. Only a few neuraxes can be traced from the distal end of the central stump, through the transplant, to the distal popliteal. This is no doubt in part accounted for by the imperfect differentiation of neuraxes attained in the pyridine-silver staining.
EXPERIMENT No. 247.- Dog No. 4; medium size; full grown; 66 days. June 13, 1918, right sciatic exposed; internal popliteal freed. Left ulnar exposed and freed. A segment of2.5 cm. length taken from the left ulnar transplanted to the resected right internal popliteal. One central and distal waxed, fine silk thread suture placed; good approximation. A fascial sheath taken from the right fascia lata of the same dog wrapped about the transplant and the resected nerve ends. One central and distal stay suture and three intervening sutures placed. Wound not quite dry, slight oozing, which was not fully controlled. The three wounds closed. August 18, killed. Much emaciated; had not been active for several days; no neurotrophic changes right hind foot. On exposing the right sciatic, the external popliteal is found adherent to the fascial sheath. Fascial sheath found well in place; forms closely fitting tube, surrounded by fibrous tissue; only moderately adherent to underlying muscle. Quite distinct central bulb, evident through the transplant. Sciatic and transplant with fascial sheath removed and fixed in ammoniated alcohol for pyridine-silver staining. Distal posterior tibial fixed in neutral formalin. Good differential neuraxis staining attained. Tissues not well embedded; sections torn.
Microscopic findings.-In cross sections of the nerve transplant and the fascial sheath, it may be observed that the sheath is surrounded by a relatively thick layer of fairly dense fibrous tissue. In alternate cross and longitudinal sections of operated nerve, it may be seen that neuraxes pass from the distal end of the central bulb, through the transplant into the distal popliteal.
EXPERIMENT No. 248.- Dog No. 40; medium size; dog not quite full grown; 65 days. July 7, 1918, ]eft sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment of 3.0 cm. length taken from the right ulnar transplanted to the resected left internal popliteal. One central and distal suture of No. 00 catgut placed. Approximation not satisfactory; not as good as when waxed, fine silk thread is used for suture. A fascial sheath taken from the left fascia lata of the same dog wrapped about the transplant and resected nerve ends. Central and distal stay sutures and one intervening suture all Of fine silk thread placed. Good fascial tube formed. Dry wound. The three wounds closed. September 10, killed. Dog much emaciated, severe skin disease. On exposing the left sciatic, external popliteal found adherent along side of the fascial sheath. Fascial sheath found well in place; its proximal and distal ends not clearly demarked; moderate increase of connective tissue about it; adherent to underlying muscle. No distinct central bulb made out. Calf muscles exposed; atrophic; do not respond when nerve is cut. Sciatic and the transplant. with the fascial sheath removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good differential neuraxes staining attained.
Microscopic findings.- Only indistinct central bulb evidenced structurally. In cross sections of the transplant and sheath, sheath is found surrounded by a relatively loose layer of fibrous tissue. The transplanted nerve clearly demarked, its perineural sheath not materiallv thickened, between these perineural sheaths and the inner surface of the fascial sheath a layer of loose fibrous tissue. In the transplanted nerve many new neuraxes, singly or in small bundles, observed; endoneural connective tissue only moderately increased. These
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new neuraxes can be traced through the transplant and distal wound into the distal popliteal to the level of the calf muscles. Partial regeneration of the distal popliteal attained.
EXPERIMENT No. 249.-Dog No. 17; medium size, full grown; 106 days. May 14, 1918, left sciatic exposed. Right ulnar exposed. Two segments, each measuring 1.6 cm. taken from the right ulnar transplanted to the resected right sciatic. One central and distal suture of fine Chinese silk for each segment placed; only fair approximation attained. A fascial sheath taken from the fascia lata of the left side of the same dog wrapped about the transplants and the resected nerve ends. One central and distal stay suture and continuous over and over suture between, placed. The three wounds closed. All wounds healed well. August 28, killed. Dog had not been well for some time; nearly moribund when killed. On exposing the left sciatic, moderate increase of connective tissue about tile transplant is found; adherent to underlying muscle. Fascial sheath distinctly evident; its central and distal limits not distinctly made out. Nerve distal to transplant presents the appearance of normal nerve. On exposure, the calf muscles seem still somewhat atrophic and are of pale red color. On slowly cutting the sciatic central to sheath and transplant, no contraction of calf muscles; this may in part be accounted for by condition of dog when killed. Sciatic with the transplant and sheath removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential neuraxis staining attained.
Microscopic findings.-Distinct central bulb evidenced structurally. In cross sections of the transplants and sheath, the fascial sheath appears not to have been reduced in thickness and is found surrounded by a layer of relatively dense fibrous tissue. The two transplanted ulnar segments clearly demarked within the fascial tube. Their perineural sheath relatively thick, but the funicular structure is not lost. Very little connective tissue found between the perineural sheaths and the fascial sheath. Each of the transplant nerve segments, as seen in cross sections, contains numerous new neuraxes; in about equal number in two nerve segments. These neuraxes can, in sections, be traced through the distal wound to the distal sciatic, found approximately equally distributed through its several funiculi. Regeneration of the central end of distal sciatic attained.
EXPERIMENT No. 250.- Dog No. 42; large dog; full grown; 268 days. August 21, 1918, right sciatic exposed; internal popliteal freed. Left ulnar exposed and freed. A segment of3 cm. length taken from the left ulnar transplanted to the right internal popliteal. One central and distal waxed, fine silk thread suture placed. Centrally good approximation; distally a second, an epineural, suture placed; approximation fair. Fascial sheath taken from the fascia lata of the right side of the same dog, wrapped about transplant and the resected nerve ends. Central and distal stay sutures and five intervening half mattress sutures placed. Good tube formed. The three wounds closed. May 16, 1919, killed. Dog in good condition; walks well, but does not climb stairs as easily as normal dog; no neurotrophic changes right hind foot. On exposing the right sciatic external popliteal bundle found free. Quite marked increase of connective tissue about the transplant noted, so that fascial sheath is not clearly made out. Calf muscles exposed; these have the appearance and size and color of normal muscle. Cutting of nerve central to transplant causes good contraction of calf muscles; movement of toes feeble and indistinct. Nerve and transplant with sheath fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.- In cross sections of the transplant and sheath, fascial sheath is clearly demarked; does not appear to have been reduced in thickness by absorption; surrounded by a relatively dense layer of fibrous tissue, containing here and there small lobules of adipose tissue. The transplanted ulnar segment presents a funicular structure with perineural sheaths thickened, and endoneural connective tissue materially increased. A loose connective tissue intervenes between perineural sheaths and fascial sheath; in this there may be observed numerous small funiculi of nerve fibers delimited peripherally by the fascial sheath. The funiculi of the transplanted nerve contain numerous new neuraxes scattered or in small bundles. In series of alternate cross and longitudinal sections, new neuraxes can be traced to the distal popliteal in which, in each of the several funiculi, there may be observed numerous both myelinated and nonmyelinated nerve fibers. Very complete regeneration of the distal popliteal through the transplant attained.
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EXPERIMENT No. 251.-Dog No. .39; medium size; full grown; 276 lays. August 14, 1918, right sciatic exposed; internal popliteal freed. Left ulnar exposed and freed. A segment of 3.5 cml. length taken from the left ulnar transplanted to the resected right internal popliteal. One central and distal waxed, fine silk thread suture placed; good approximation, Fascial sheath taken from the right fascia lata of the same dog wrapped about the transplant and the resected nerve ends. The piece of fascia cut a little too narrow at one end so that complete fascial tube could not be made. Central and distal stay sutures and two intervening half mattress sutures placed. Three wounds closed. May 16, 1919, killed. Dog in very good condition; used right hind leg and foot well; no neurotrophic changes. On exposing the right sciatic, the external popliteal found free. Operated internal popliteal in
FIG. 237.-Cross section of auto-nerve transplant, wrapped in auto-fascial sheath, Experiment No. 2,50, terminated 268 days after the operation. Nerve funiculi of transplant are maintained and fully neurotized. Auto-fascial sheath still distinctly evident somewhat over six months after operation
the region of the transplant and sheath surrounded by quite dense connective tissue and adherent to the underlying muscle. Fascial sheath not clearly made out. Calf muscles exposed; these have appearance, both as to size and color, of normal muscle. Sciatic freed from bed and external popliteal resected. On slowly cutting with scissors nerve central to the transplant, good contraction of calf and foot muscles noted. Sciatic, transplant and sheath removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential neuraxis staining attained.
Microscopic findings.-In cross sections of the transplant and sheath, the fascial sheath is found well in place; appears not to have been reduced in thickness by absorption; presents essentially the same structure as in short-time experiments of this series; surrounded by a
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distinct layer of fibrous tissue in which many fat cells are seen. The transplanted nerve segment is clearly demarked, having retained its funicular structure. The perineural sheaths of transplant found thickened and for nearly the whole circumference in relation with inner surface of the fascial sheath; very little connective tissue intervening. New neuraxes traced from central stump through the transplant into distal popliteal in which are found numerous myelinated and nonmyelinated nerve fibers arranged singly or in small bundles and separated by endoneural connective tissue which is distinctly increased in amount. Almost complete regeneration of the distal popliteal through the transplant attained.
EXPERIMENT No. 252.-Dog No. 42; large dog; full grown; 318 days. July 2, 1918, left sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment of 3.6 cm. length of right ulnar transplanted to the resected left internal popliteal. One central and distal waxed, fine silk thread suture placed; good approximation. Fascial sheath taken from the left fascia lata of the same dog wrapped about transplant and the resected nerve ends. One central and distal stay suture, no intervening sutures placed. Good tube formed. Field not quite dry; oozing controlled by use of adrenalin. The three wounds closed. May16, 1919, killed. Dog in very good condition, walks well but does not climb stairs as easily as normal dog; no neurotrophic changes left hind foot. Left sciatic exposed; skill adherent along wound line. External popliteal found free. Operated internal popliteal, transplant and sheath surrounded by quite dense layer of connective tissue and adherent to underlying muscle. Fascial sheath not clearly made out. Calf muscles exposed; these have the appearance and size of normal muscle. Cutting of sciatic, after removing external popliteal and removing nerve from bed, central to the transplant, calls forth good contraction of calf and foot muscles. Sciatic, transplant and sheath removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential neuraxis staining attained.
Microscopic findings.-Structural evidence of well-developed central bulb. Down-growing central neuraxes cross and recross distal part of the bulbous enlargement; evidence of resistance spiral noted. Many neuraxes observed as passing to central end of transplant. In cross sections of the transplant and sheath, fascial sheath is clearly made out for the greater part of the circumference, to one side, it would appear that the lip of the fascial sheath separated, admitting an ingrowth of connective tissue. The transplanted nerve segment clearly demarked, with funicular structure retained. Fairly dense connective tissue intervenes between the several funiculi and the inner surface of the fascial sheath. Numerous myelinated and nonmyelinated neuraxes are transmitted by the transplant to the distal popliteal, cross and longitudinal sections of which present an appearance which resembles that of a nearly completely regenerated nerve.
EXPERIMENT No. 253.-Dog. No. 39; medium size; full grown; 326 days. June 24,1918, left sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment of 3.2 cm. length of the right ulnar transplanted to the left internal popliteal. On central and distal waxed, fine silk thread suture placed; good approximation. Fascial sheath taken from the left fascia lata of the same dog wrapped about transplant and the resected nerve ends. One central and distal and two intervening half mattress sutures placed. Good tube formed. Wound not quite dry; oozing controlled by use of adrenalin. The three wounds closed. May 16, 1919, killed. Dog in very good condition; rises left leg well; no neurotrophic changes. On exposing left sciatic, external popliteal found free. Operated internal popliteal surrounded by relatively dense layer of fibrous tissue, adherent to underlying muscle. Distal nerve has the appearance of normal nerve. Calf muscles exposed; these have the appearance of normal muscle. After freeing sciatic from bed and cutting the external popliteal, slowly cutting with scissors the sciatic central to trans-plant, calls forth good contraction of calf and foot muscles. Nerve, transplant and sheath removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential neuraxes staining attained.
Microscopic findings.-Central neuraxes pass without special line of demarcation to trans-plant. In cross sections of transplant and sheath, fascial sheath found well in place and not materially reduced in thickness through absorption; covered on its outer surface by distinct layer of fibrous tissue. The transplanted nerve segment clearly demarked; perineural sheath thickened, these in close relation to inner surface of the fascial sheath. Numerous small
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funiculi of nerve fibers in the connective tissue within the fascial sheath, but outside of perineural sheath, observed; especially to one side. Numerous neuraxes, both myelinated and nonmyelinated, transmitted by the transplant to the distal popliteal, which in cross and longitudinal sections presents the appearance of a nearly completely regenerated nerve.
The experiments of Series No. 17, 14 in number, are sufficiently varied as to time under observation and sufficiently numerous to admit of formulating certain general deductions relative to auto-fascial sheaths. Experiments No. 250 to No. 253 were under observation for periods varying, respectively, from somewhat over 8 months to nearly 11 months, and in each of these experi-ments it is noted that the fascial sheath could be clearly made out in microscopic sections of the transplant region. These four experiments were studied chiefly with reference to the behavior of the down-growing neuraxes, which were thus fixed for pyridine-silver staining. In preparations stained after this method the fascicular structure of the transplanted fascial sheaths could be clearly made out, the cellular elements were not clearly differentiated. Therefore it can not be stated whether the fixed connective tissue cells-tendon cells-of the fascial sheaths were maintained after transplantation. Lewis and Davis 87 have shown that a fascial tube remains patent for an extended period. In a case in which a fascial tube was used in tendon repair, the sheath was evident 255 days after the operation. In these experiments of long duration there is no distinct evidence of a secondary contraction of the fascial tube. There is found a layer of areolar tissue between the inner surface of the fascial sheath and the perineural sheaths of the funiculi of the transplanted nerve segments. This varies in thickness in the several experiments and also indifferent regions of the same experiment. The newly formed nerve fibers found within the funiculi of the nerve transplant are of normal appearance and were especially numerous in Experiment No. 253, terminated 326 days after the operation at which the fascial sheath was placed. In this experiment the fascial sheath was well maintained and there was found relatively little loose areolar tissue within the fascial sheath. The contraction noted by Kredel 85 in the case of neurolysis of the tibialis in which the fascial sheath was found contracted 24 days later is not conclusive. It may be asked whether the correct operative procedure was undertaken in the first place. It may be repeated that these experiments seem to indicate that there is relatively little secondary contraction of a fascial sheath. From the experiments here reported it is evident that an auto-fascial sheath of fascia lata does incite connective tissue proliferation even in an aseptic wound in healthy tissue, a wound passing in the main through intermuscular planes. It seems quite impossible to remove a piece of fascia lata without having extravasated blood cells and tissue fragments adhere especially to its outer surface, the more so if a layer of subcutaneous fat is removed with the fascia, as has been recommended. The inner surface is quite smooth if the portion removed is correctly oriented. In all of our experiments a distinct development of fibrous tissue is noted surrounding the fascial sheath. This layer is quite cellular in the experiments of short duration and in it there are found many leucocytes and extravasated red blood cells. Incertain of the experiments the fascial sheath was found adherent to the muscle bed, to the extent necessitating dissection; this shows connective tissue proliferation.
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This newly formed connective tissue surrounding the fascial sheaths, even in aseptic wounds in healthy tissue, on subsequent contraction and cicatrization may be regarded as having deleterious effect on the structural and functional regeneration of an injured nerve, sheathed by fascial sheath, rather than the secondary contraction of the fascial sheath itself. While thus inciting connective tissue growth, the fascial sheath does appear to retard the growth of connective tissue into the central and distal wound and the immediate environs of a nerve transplant. Experiment No. 252 bears on this indirectly. In this experiment, studied 318 days after the primary operation, the fascial tube appears to have opened along the side. There was an ingrowth of connective tissue through this cleft, to the extent that a fairly dense layer of con-nective tissue intervenes between the nerve funiculi and the inner surface of the fascial sheath, much more so than when the fascial sheath remained closed in tube form as in the majority of the experiments. However, even in this experiment there was found good neurotization of the distal segment through the nerve transplant.
The general conclusion seems warranted that an auto-fascial sheath is very slowly absorbed, evidence of its persistence having been noted nearly a year after it was placed at operation in the wound in healthy tissue. However, even in aseptic wounds in healthy tissue there is noted a distinct proliferation of the surrounding connective tissue, which would prejudice against the use of fascial sheaths in connection with operations of nerve repair, especially where such operations pass through cicatricial tissue. It would appear that auto-fascial sheaths incite more connective tissue proliferation than does alcoholized Cargile membrane, the application of which is technically much simpler and serves every purpose that could be gained on use of a fascial sheath.
SERIES NO. 18
AUTO-NERVE TRANSPLANTS WRAPPED IN FORMALIZED ARTERIAL SHEATHS
This short series of experiments was suggested through records of clinical and experimental observations in which arteries or veins, used either fresh or after fixation and taken as auto-, homo-, or heterogenous vessels were placed asa wrapping or as a tube to ensheath the suture lines in operations for repair of peripheral nerves. To review in extenso the literature involved is not here justifiable. The brief experimental observations of Foramitti, 88 have influenced later operators and experimenters and may thus be given brief consideration here, the more so since we have followed his method of preparing arterial tubes. though Büngner 27 some years earlier had used a segment of a sterilized human brachial artery to bridge a nerve section. We shall consider "tubular sutures"under Series No. 20, in connection with which series the bridging of nerve defects by means of tubular structures will be considered. Ensheathing a nerve suture after severance, or a nerve transplant with a tubular structure is not a " tubular suture" in the correct use of this term. Three animals were operated by Foramitti. Both fresh and hardened arteries were used, either applied to a liberated nerve, by cutting the vessel wall longitudinally and slipping the vessel wall over one end of a resected nerve, making a central nerve flap and moving the arterial
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segment so as to cover the field of operation: Or. as a tubular suture. The longest observation, that of tubular suture, extended only six weeks. Foramitti found that on use of both fresh an(l hardened arteries, the inclosed peripheral nerve tissues was only lightly adherent to the wall of the artery. The experiments of Foramitti are not conclusive and leave further doubt when he states "die physiologische Function des Nerven war hergestellt," in speaking of a section and sutured sciatic of the dog studied three weeks after operation. We have used in this series only prepared arterial walls, taken from the carotid of large dogs, and used for the purpose of making a tubular sheath after nerve transplantation. The arteries used were prepared as follows: The carotids of large dogs were stretched over glass tubes of suitable size, were then fixed in 5 per cent to 10 per cent formalin solution for 48 hours, washed in water for 24 hours, boiled in distilled water for 20 minutes, and then stored in 70 percent to 95 percent alcohol. They were stored on glass rods in the alcohol in wide mouthed, glass-stoppered bottles, for several days to several weeks depending on the time when they were used at operation. Before use at operation an arterial segment of required length was slipped from the glass rod, cut longitudinally along a line and placed in sterile salt solution for about 30 minutes. The arterial sheath thus prepared was then wrapped about the nerve transplant, sutured in place and allowed to extend 5 mm. to 8 mm. over the central and divided suture lines, and was fixed in place by central and distal stay sutures and intervening half mattress sutures, using for suture fine, waxed silk thread. Experiments with fresh arteries or veins used as sheaths were not made: neither have we tested autogenous nor heterogenous vessels. Foramitti recommended the use of formalized arteries of the calf. With the treatment given the tissue, namely, formalin fixation, boiling for 20 minutes and storage in 95 percent alcohol, it is to be questioned whether homogenous vessels obtained from amputations or even at autopsy (in selected cases) could not be used for ensheathing operated nerves in human surgery. The experiments of Eden 89 of inserting the central and distal ends of the resected anterior crural through fine slits into the femoral artery or vein, with the circulation maintained, did not seem to us worthy of repetition.
The protocols of experiments on the use of formalized arterial walls for the purpose of ensheathing nerve transplants are as follows:
PROTOCOLS
EXPERIMENT No. 254.- Dog No. 50; large dog; full grown; 6 days. July 17, 1918, left sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment of 3 cm. length transplanted to the resected left internal popliteal. One central and distal waxed, fine silk thread suture placed; centrally fair, distally good approximation attained. A segment of formalized artery of a dog, split longitudinally along one side, placed so as to surround the transplant and the resected nerve ends. A central and distal stay suture and three intervening half mattress sutures placed. Adrenalin used to obtain dry field. Wounds closed. July 23, dog found dead in the morning. The superficial wound seemed healed. On reopening wound, evidence of infection noted in deep wound; parts congested. Transplant and tube surrounded by sanguineous exudate. Arterial sheath found well in place. The nerve transplant and the arterial sheath removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
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Microscopic findings.- As seen in cross sections of the transplant and sheath, arterial sheath forms a well-closed tube with edges overlapping. About the sheath blood coagulum, tissue detritus, numerous wandering leucocytes and phagocytes. Within the sheath relatively few leucocytes. In longitudinal sections of the region of the central and distal wounds leucocvtes have penetrated for a distance into the central and distal stump, and central and distal ends of the transplant, where they are found between the nerve fibers as also within the neurolemma sheaths. The nerve fibers of the transplanted nerve segment present little evidence of beginning degeneration. Their myelin sheaths stain pale, but as yet no distinct evidence of myelin fragmentation. Proliferation of sheath cell nuclei not observed.
EXPERIMENT No. 255.-Dog No. 48; medium size; full grown; 57 days. July 15, 1918, left sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment of 2.5 cm. length of right ulnar transplanted to the resected left internal popliteal. One central and distal waxed, fine silk thread suture placed; good approximation. An arterial sheath, formed by cutting longitudinally a segment of formalized carotid artery of a dog and wrapped about transplant and resected nerve ends placed. One central and distal stay suture and two intervening half mattress sutures used. Good tube formed. Dry field after using adrenalin. Wounds closed. September 10, dog found dead in the morning; much emaciated. On exposing the left sciatic, arterial sheath is found well in place. Proximal and distal ends of sheath covered with connective tissue and adherent to epineural sheath of the resected nerve. No material increase of connective tissue about sheath. Nerve, transplant and sheath fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-In cross sections of sheath and transplant, arterial sheath clearly recognized in sections; centrally the lips of the arterial sheath have rolled in so that to one side of the transplanted nerve a segment is exposed; distally arterial sheath forms a tube completely inclosing transplant. In section it may be observed that the elastic tissue of the intima and media, and the fibrous tissue of the adventitia are best preserved; the in- voluntary muscle of the media still recognizable, though many muscle cells appear fragmented. Only a thin layer of fibrous tissue surrounds the arterial sheath. The transplanted nerve segment has retained its funicular structure, with perineural sheaths materially thickened. Dense fibrous tissue intervenes between nerve and arterial sheath, especially centrally, where sheath is incomplete. Many small medullated fibers found within the transplant.
EXPERIMENT No. 256.-Dog No. 45; small dog; full grown; 63 days. July 12, 1918, left sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment of the right ulnar of 3 cm. length transplanted to the resected left internal popliteal. One central and distal waxed, fine silk thread suture placed. Centrally fair, distally good approximation attained. An arterial tubular sheath formed by splitting longitudinally a segment of formalized carotid artery of a dog, and wrapping the same about the transplant and the resected nerve ends. Stay sutures and two intervening half mattress sutures placed. Good tube formed. Wounds closed. September 13, dog nearly moribund; killed. Much emaciated; severe skin disease. On exposing left sciatic, arterial sheath is found well in place. The two half mattress sutures appear to have given away, since a slit can be recognized along one side of "tube," for nearly its whole length. No material increase of connective tissue about the sheath. Nerve, transplant, and arterial sheath removed and fixed in ammoniated alcohol for pyridine-silver staining. Only faint silver staining attained; especially in the region of the transplant.
Microscopic findings.-In cross sections, it may be observed that the arterial sheath forms a closely fitting tube surrounding the transplant. A very thin fibrous tissue sheath surrounds the arterial sheath. Arterial wall not so clearly defined in silver-stained preparations as when other fixatives and certain other staining methods are used. However, in the silver preparation it is possible to differentiate the arterial wall and especially its elastic tissue. The perineural sheath of the transplanted nerve segment in close relation to the inner surface of the arterial sheath, very little connective tissue intervening. In series of longitudinal and cross sections, it is possible to trace numerous neuraxes from the central
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stump through the transplant and distal wound to the distal popliteal in which a few scattered neuraxes can be traced distally for a distance of about 2 cm.
EXPERIMENT No. 257.- Dog No. 49; medium size; full grown; 117 days. July 16, 1918, left sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment of the right ulnar of 3.5 cm. length transplanted to the resected left internal popliteal. One central and distal waxed, fine silk thread suture placed; good approximation. An arterial sheath made by cutting longitudinally along one side, a segment of a formalized carotid artery of a clog and wrapping same about transplant and resected nerve ends. Stay sutures and three half mattress sutures placed. Good tube formed. Dry field obtained after using adrenalin. Wounds closed. November 10, dog found dead in the morning, much emaciated; severe skin disease. Oni exposing the left sciatic, arterial sheath found well in place; no material increase of connective tissue about sheath. No distinct central bulb noted. Condition of calf muscles not recorded. Nerve, transplant, and sheath removed and fixed in ammoniated alcohol for pyridine-silver staining. Faint but fairly good differential neuraxis staining attained.
Microscopic findings.-In cross sections of the transplant and sheath, it is observed that arterial sheath forms complete tube about transplanted nerve segment and is surrounded by only a thin layer of fibrous tissue. Sheath in close relation with the perineural sheaths of the transplanted nerve segment, the funicular structure of which is retained; the endoneural connective tissue of the funiculi materially increased. In alternate cross and longitudinal sections, numerous new nueraxes may be traced from the central stump through the transplant to the distal popliteal.
EXPERIMENT No. 258.- Dog No. 43; small dog; full grown; 130 days. July 11, 1918, left sciatic freed. Right ulnar freed. A segment of the right ulnar of approximately 3 cm. length transplanted to the resected left sciatic. One central and distal waxed, fine silk thread suture placed. Approximation fair; a small amount of blood exudate in central nerve wound. An arterial sheath made by splitting longitudinally a segment of a formalize carotid artery of a dog and wrapping same about the transplant and the resected nerve ends. Stay sutures and several mattress sutures placed. Good tube formed. Wounds closed. November 18, dog seemed fairly well in the morning; found dead 4 p. m.; seemed in good condition; well nourished; no neurotrophic changes on left foot. On exposing the left sciatic, arterial sheath found well in place, moderate increase of connective tissue about it. No distinct central bulb, only slight enlargement noted. Calf muscles exposed; these slightly atrophic, but of good color. Nerve, transplant, and sheath removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.- In cross sections of the transplant and sheath, arterial sheath is found to have been well maintained; forming closely fitting tube about the transplant; with scarcely any connective tissue between transplanted nerve segment and inner surface of sheath and only a thin layer of fibrous tissue surrounding it. In cross and longitudinal sections, it is to be observed that new neuraxes in large numbers may be traced from the central stump through the transplant to the distal popliteal in which numerous neuraxes are found in its peripheral distribution, namely, to the posterior tibial several centimeters above the heel and into the small interfascicular nerve branches in the calf muscles. Regeneration of distal popliteal through the transplant attained.
EXPERIMENT No. 259.- Dog No. 44; small dog; full grown; 243 days. July 12, 1918, left sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment of the right ulnar of 3 cm. length transplanted to the resected left internal popliteal. One central and distal waxed, fine silk thread suture placed. Central suture gave way; resutured, one through-and-through suture and one epineural suture; central approximation fair, distal good. An arterial sheath made by splitting longitudinally a segment of the formalized carotid artery of a dog and wrapping the same about the transplant and the resected nerve ends. Two stay sutures and three half mattress sutures placed. Good tube formed. Dry field after use of adrenalin. Wounds closed. March 12, 1919, killed. Dog in good condition; no neurotrophic changes left foot; uses left hind foot and leg well. On exposing the left sciatic no macroscopic evidence of arterial sheath found. No material increase of connective tissue about operated internal popliteal; nerve only moderately adherent to underlying
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muscle. Only small central bulb noted. Calf muscles exposed; these present the appearance of normal muscle. After freeing the operated nerve from bed and slowly cutting with scissors central to the transplant good contraction of calf muscles observed, but only feeble and indistinct movement of the toes noted. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential neuraxis staining attained.
Microscopic findings.-Scarcely any structural evidence of a central bulb noted. In longitudinal sections of the central and distal wound region and in cross and longitudinal sections of the transplant no distinct structural evidence of the arterial sheath noted. In the silver stained preparations the presence or absence of the sheath is not readily determined. The transplant and regions of the nerve wounds surrounded by a relatively thin layer of fibrous tissue in which no clear determination of the elastic tissue of the arterial sheath could be gained. In cross sections of the transplant it is to be observed that the endoneural connective tissue is very materially increased. The numerous neuraxes, which can be traced from the central stump through the transplant, within the transplant are found arranged in small funiculi, separated by endoneural connective tissue. These neuraxes can be traced to the distal popliteal, in which they are found in large numbers. Very complete regeneration of the distal popliteal through the transplant attained.
This series of experiments is unsatisfactory to the extent that such a large per cent of the experimental animals died from intercurrent disease not related to the experiments, that a function test could not be made in certain of the experiments in which such test would have been desirable. The whole series could be and was used to determine morphology. It was found, confirming Foramitti, that a formalized arterial tube incites relatively little connective tissue proliferation when placed in normal tissue in aseptic wounds. In the experiments of long standing, the arterial tube was found surrounded by only a relatively thin layer of fibrous tissue which was only loosely adherent to adjacent connective tissue. Between the perineural sheath of the funiculi of the transplanted nerve segment and the inner surface of the arterial sheath there was found only a relatively small amount of areolar tissue. The arterial sheath was clearly made out macroscopically in Experiment No. 258 somewhat over four months after it was placed about the operated nerve. It is more particularly the elastic tissue of the vessel wall that resists absorption, forming a compact tubular structure easily recognized in cross sections of the transplant. There is no evidence of a secondary contraction of the vessel wall after it is placed in the tissue. It is recognized, of course, that a tubular structure which persists for a period of approximately 4 months has fulfilled any useful purpose which may be hypothecated to it, when considered in connection with the wound region in a peripheral nerve. In Experiment No. 259, of nearly 8 months' duration, the arterial sheath used at the operation could not be made out macroscopically nor was it recognized in sections, stained after the pyridine-silver method. The neurotization of the distal segment was very satisfactory in all of the experiments in which an auto-nerve transplant was ensheathed in a formalized arterial tube, especially so in experiments observed for a time sufficient to admit of downgrowth of central neuraxes. Attention is particularly called to the protocols of Experiments Nos. 258 and 259 in this regard. Since formalized arterial sheaths are so easily prepared and may be kept on hand in 70 percent to 95 percent alcohol, ready for use on need, the method deserves consideration in surgical practice when sheathing of the suture line in nerve suture or of a nerve transplant after bridging a nerve defect is deemed
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necessary. However, this type of sheathing presents no advantage over a sheath or tube formed with a double or triple layer of an alcoholized Cargile membrane, which is more easily prepared and applield. A formalized arterial wall used as sheath or tube appears to incite less connective tissue proliferation than does an auto-fascial sheath or tube as judged by the experimental evidence presented.
SERIES NO. 19
AUTO-NERVE TRANSPLANTS WRAPPED IN AUTOGENOUS-FAT FLAP
Relatively frequent reference is made in literature to the use of pedicled or nonpedicled fat sheaths or flaps in connection with operation for the repair of peripheral nerves or of neurolysis. This method of sheathing an operated nerve was for a time especially recommended in case the nerve repair or liberation was undertaken in the presence of scar tissue. The behavior of a fat flap or sheath, either pedicled or nonpedicled, has not been considered experimentally so far as we are able to ascertain. It has been the intention to extend this series of experiments but for various reasons further experiments were not undertaken. In one of the two experiments, the fat membrane was taken in the region of fascia lata through a skin wound made for this purpose; in the other the fat membrane was taken through the wound exposing the sciatic. The fat membrane was thus taken from subcutaneous tissue. There is of necessity free oozing of blood on liberating a fat layer, with consequent adherence of blood coagulum to the tissue removed. The fat membrane, which had an average thickness of 5 mm., was rinsed in sterile salt solution before it was wrapped about the nerve transplant. It was held in place by several stay sutures made with fine, waxed silk thread. The fat sheath thus made was without pedicle and was autogenous tissue, slightly cooled during manipulation and rinsing in the saline solution. The protocols of the two experiments under this series are as follows:
PROTOCOLS
EXPERIMENT No. 260.-Dog No. 54; large dog; not full grown; 4 days. July 22, 1918, left sciatic exposed and the internal popliteal freed. The right ulnar exposed and freed. A segment of the right ulnar of 3.8 cm. length transplanted to the resected left internal popliteal. One central and distal waxed, fine silk thread suture placed; good approximation. A layer of subcutaneous fat removed from the region of the fascia lata of the left leg of the same dog, trimmed to proper size and rinsed in warm, sterile saline solution to remove the adherent blood, was placed under the transplant and the resected nerve ends and folded over so as to form a fat sheath. Two proximal and two distal stay sutures and three intervening sutures placed; fairly even fat sheath formed. Wounds closed. July 26, dog found dead in the morning. Sciatic wound found open in part; field congested: a small amount of pus noted. Fat sheath of yellow-brown color; soft and pliable. Nerve transplant and fat sheath removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-In cross sections of the transplant and fat sheath, it may be observed that the nerve transplant is necrotic and as seen in longitudinal sections of the central wound, had torn free from the central stump. The fat sheath is found to have been penetrated from all sides and through its entire thickness by numerous leucocytes and phagocytes. Many of the fat cells appear collapsed and empty of fat globule; others partially so. The interlobular connective tissue of the fat sheath presents evidence of beginning
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necrosis, evidenced by fragmentation and curling of fiber bundles, especially of elastic tissue.
EXPERIMENT No. 261.- Dog No. 47; medium size; full grown; 240 days. July 15, 1918, left sciatic exposed; internal popliteal freed. Right ulnar exposed and freed. A segment 2.5 cm. length from the right ulnar transplanted to the resected left internal popliteal. One central and distal suture of waxed, fine silk thread placed; good approximation. A layer of subcutaneous fat removed from the region of the wound, trimmed and washed in warmed, sterile saline solution, placed under nerve and transplant and folded over to form a tube; central and distal stay sutures and several intervening sutures placed. Wounds closed. Sciatic wound healed slowly; stitches did not give away, but small droplets of liquid fat appeared to escape from wound; no infection. March 12, 1919, killed. Dog in good condition; no neulotrophic changes left hind foot. On exposing left sciatic dense, fibrous tissue was found in the line of the wound, and dense fibrous tissue surrounded the operated nternal popliteal. No evidence of fat sheath noted; this replaced by dense fibrous tissue. Large central bulb observed. Nerve in region of transplant of smaller diameter than resected nerve; distal popliteal has the appearance of a normal nerve. Calf muscles exposed; these have nearly recovered full size, are of pale yellow color streaked with light yellow lines. On cutting nerve central to the transplant, distinct though not vigorous contraction of calf muscles observed; feeble toe movements noted. Nerve and transplant removed and fixed in ammoniated alcohol for pyridine-silver staining. Very good silver differentiation attained.
Microscopic findings.- Neither in cross nor longitudinal sections is there observed any evidence of the fat sheath. In the region of central and distal wounds and the intervening transplant, distinct increase of dense fibrous tissue about the nerve. In cross sections of the nerve transplant, distinct increase of the endoneural connective tissue observed. New neuraxes traced from the central stump through the transplant are within the transplant found arranged in small bundles, spaced by intervening connective tissue. New neuraxes found in abundance in the distal internal popliteal in all of its funiculi, traced in peripheral distribution in alternate cross and longitudinal sections. Regeneration of distal popliteal through the transplant with great increase of connective tissue in the field of operation attained.
Experiment No. 260 loses in value by reason of the slight infection of the wound region and the early termination of the experiment by reason of the death of the animal. Attention is called to the relatively early disappearance of the fat from certain of the transplanted fat cells, so that many of these cells appear collapsed, shriveled, and shrunken. There was noted marked leucocyte invasion into the fat flap, perhaps in part to be ascribed to the infection present. It was noted in this experiment and in Experiment No. 261 for a few days after the operation that there was taking place a slight oozing of a lipoid fluid from the wound. It was observed in connection with Experiment No. 261 that the wound healed relatively slowly, though infection was not evident. In this experiment the dog was in good condition throughout the time he was under observation, was active, and was kept nearly eight months after the operation. On exposing the sciatic nerve at the termination of the experiment there was found very marked increase of fibrous tissue in the region of the wound and operated sciatic. This fibrous mass was more dense and more extensive than found after any of the other operations in which the nerve transplant was sheathed. The nerve and the nerve transplant, through which neurotization of the distal segment had taken place, were found embedded in the deeper portion of this fibrous tissue, which was adherent superficially to the skin.
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A general deduction seems hardly justifiable on the basis of a single experiment. However, the results here presented argue against the use of a completely detached, autogenous-fat sheath or flap, in connection with operated or liberated peripheral nerves, since the fat membrane appears to be replaced by dense fibrous tissue, persisting only a relatively short time as a fat layer. It is to be questioned whether a pedicled fat layer used as a sheath for wrapping an operated nerve would be maintained for a longer time as a fat layer and thus serve the purpose hypothecated to it.
NERVE SUTURE
SERIES NO. 20
TUBULAR SUTURE WITH USE OF FORMALIZED ARTERY
Tubular suture in the repair of peripheral nerves with loss of substance has been given extensive consideration and came into use relatively early in the development of operative technique as regards peripheral nerve repair. The term " tubular suture" as here employed recognizes the union of severed nerve ends by means of a tubular structure, which may be empty or filled at the time of use by inserting and maintaining in place the central and distal stumps of the severed nerve within the lumen of the tubular structure, centrally and distally. As previously stated, wrapping a nerve after neurolysis or after suture, or after nerve transplantation with a membranous structure is, correctly speaking, not a tubular suture but should be regarded as a procedure for ensheathing an operated nerve. Tubular structures were suggested as a means of union of severed nerves and are today used, with a view of maintaining a channel along which central neuraxes (monogenetic view) or central and distal neuraxes (polygenetic view) may be enabled to grow, on the theory that such tubular structures along a certain direct path would prevent connective tissue proliferation and organization between the severed and resected nerve ends and at the same time prevent dispersion of the newly formed neuraxes.
Among the various methods tried by Glick 90 for the purpose of bridging nerve defects was that of tubular suture, made by inserting the severed nerve ends into a " Neuber's bone drain. " In no instance did regeneration of the distal stump take place. Vanlair 26 instituted a series of experiments on tubular nerve suture also using "bone drains." In a young dog with the sciatic resected 3 cm. and the ends united by means of a bone drain tubular suture, four months after the operation the tissue intervening between the nerve ends was found to contain many nerve fibers. Nerve fibers were also found in the distal stump. Büngner 27 united the resected and separated nerve ends by means of tubular sutures made from sterilized human brachial artery. At the end of about a month and a half nerve fibers were found in the space between the nerve ends. Huber 30 reported eight experiments in which decalcified bone tubes made from the ulna of chicken were used for purpose of tubular suture of the resected ulnar nerves of dogs. Three of these experiments were observed for a period varying, respectively, from about four months to about five months, and in these the neuraxes regarded as of central origin were found in the central
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end of the distal stump. A relatively early absorption of the bone tube was noted in the experiments of shorter duration. The tubes were found replaced by a relatively loose connective tissue, thus leaving a path of less densely organized connective tissue between the severed nerve ends. These several lines of investigation give experimental foundation for two types of tubular structure perhaps more frequently used in practical surgery than others, namely, bone tubes and arterial tubes. Numerous other tubular structures have been used for this purpose. More than mention of the majority of these is not justified here; nearly all have been or should be discarded, since their use is not. justified experimentally. Mention is here made of tubes formed of iodoform gauze, W olfer; 91 of magnesium tubes, Payr; 92 hardened gelatin tubes, Lotheisen; 93 galalith tubes (case in treated with formalin), Auerbach; 94 rubber tubes and rubber tubes filled with serum, Steinthal; 95 rubber bandages, Meuriot and Platon. 81 These and other tubular structures of like nature are more of academic than practical interest and their consideration need not occupy space here. Brief recognition should be given, if only to condemn it, to the method of tubular suture suggested by Edinger,96 who, largely on theoretic grounds and without sufficient experimental warrant, recommended the use of arteries filled with agar, for the purpose of bridging nerve defects. Edinger appears to have reasoned that if neuraxes would grow into agar or blood serum in tissue culture, a tubular suture filled with agar should prove more serviceable than an empty tube. These "Edinger tubes" were prepared commercially and for a period were used extensively in the German service, even in cases in which a direct suture could have been made. A series of contributions appeared so soon as the observations on the results obtained on the use of the Edinger tube could be ascertained; Stracker 97 and a number appearing in 1917 may be listed here, Hohmann and Spielmeyer, 98 Enderlen and Lobenhoffer, 99 Spitzy, 100 Wollenberg, 101, Blenke, 102 and Eden 103 all of whom discredited use of the Edinger tube even in case the prepared artery was filled with autoserum as was later suggested. This method was not tested experimentally in our laboratory but was condemned on a priori considerations. The experimental work reported on briefly by Hohmann and Spielmeyer, 98 in which arterial tubes filled with agar were tested, led to the conclusion that agar in the arterial tubes appeared to block the downgrowth of neuraxes rather than facilitate their growth. The use of fascial tubes in the repair of peripheral nerves with loss of substance was tested experimentally by Kirk and Lewis. 86 They conclude that a defect in a nerve may be successfully bridged by means of a fascial tube, regeneration taking place entirely through nerve fibers growing from the central stump. They give directions and precautions for use of a fascial tube in the repair of peripheral nerves. Their experiments were controlled by study of histologic sections stained after differential neuraxes staining methods and had been completed so recently that they could be considered a part of these experimental observations.
Tubular nerve suture with the use of vessels is mentioned so frequently in literature on the repair of peripheral nerves and to our knowledge had not been given more than passing consideration experimentally since the few experiments made by Foramitti 88 were recorded, that it seemed to us desirable to make
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use for this purpose of certain of the ulnar nerves resected to obtain auto-nerve transplants for Series No. 16, No. 17 and No. IS as previously reported. In all 13 experiments of tubular suture were made. The arteries used were the carotids of dogs. The arteries were obtained from large dogs after bleeding while uniler anesthesia, were slipped over glass rods of suitable size and fixed in 5 per cent to 10 per cent solution of formalin, for 48 hours: were then washed in water for 24 hours; boiled in distilled water for 20 minutes and placed in70 per cent to 95 per cent alcohol until needed. Before use, pieces of desired length of the artery selected, were slipped from the glass rod and placed in sterile saline solution for about 30 minutes. On applying the formalized arterial tube for purpose of tubular suture the following procedure became routine: A fine, waxed silk suture armed with a fine needle at each end was passed through the central and distal stumps of the resected ulnar from 2 mm. to 3 mm. from the cut ends. The needles of each suture were then passed through the opposing sides of the formalized arterial tube from 7 mm. to 8 mm. from the respective ends of the tube and the ends of the resected nerve, central and distal, drawn into the lumen of the arterial tube and held in place by knotting the silk sutures over one side of the arterial tube. The wound was then closed by using the necessary fascial and skin sutures. In this series of experiments no nerve trans-plant was inserted between the resected nerve ends: the ends of the resected ulnar, from 3 cm. to nearly 5 cm. apart in the several experiments, were merely inserted into the ends of the arterial tubes and kept in place by means of stay sutures.
The protocols for the experiments under Series No. 20, tubular sutures with formalized arterial tubes, are as follows:
PROTOCOLS
EXPERIMENT No. 262.- Dog No. 50; large; full grown; 6 days. July 17, 1918, right ulnar exposed; resected 3.5 cm. The resected ends of the ulnar inserted into the ends of a segment of a formalized carotid artery of a dog, 4.5 cm. length, and held in place by central and distal stay sutures. Dry field obtained after use of adrenalin; fascia stitched over nerve and arterial tube; wound closed. July 23, dog found dead in the morning. Ulnar wound wide open; arterial tube exposed. Distal end of ulnar pulled out from arterial tube. Ulnar and arterial tube removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.- Distal end of central ulnar stump found within the lumen of the arterial tube. In longitudinal sections of this region, the usual degeneration of central stump in region of wound observed. Arterial tube found collapsed to flat ribbonlike structure. Within its lumen, as seen in cross section, is found a small amount of stainable granular precipitate and fine shreds of fibrin; scarcely any cellular elements. Walls of arterial tube well preserved. The looser, outer connective tissue coat of arterial tube invaded by wandering leucocytes, extravasated red blood cells and phagocytes present in large numbers in the tissue surrounding the arterial tube.
EXPERIMENT No. 263.- Dog No. 46; small dog; full grown; 17 days. July 13, 1918, right ulnar exposed and resected 3.5 cm. The resected ends of the ulnar inserted into the ends of a segment of formalized carotid artery of a dog, 4.5 cm. length, and held in place by central and distal stay sutures. Adrenalin used to obtain drv field. Fascia stitched over arterial tube; wound closed. July 31, dog found dead in the morning. Superficial wound healed; a dead place found under skill in operative field; fascia healed over arterial tube. Arterial tube found well in place; distinct bulbous enlargement on central ulnar stump. Arterial tube collapsed; appears as flat band. Arterial tube with ulnar nerve removed
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moved and fixed in neutral formalin. Sections stained in iron-henatoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.-In longitudinal sections, including the distal end of the central stump and the central end of the arterial tube, bulbous end of the central stump found well within arterial tube. Bulbous end of central stump presents early stages of regeneration down-growing neuraxes and rich capillary net. Beyond the end of the nerve, within the arterial tube, felt-work of newly formed fibrous tissue, syncytial protoplasmic bands and capillaries. In cross sections of the arterial tube, about its middle, vessel wall clearly made out; media and greater part of the adventitia intact. Within the lumen of the collapsed tube, newly formed fibrous tissue fibroblasts and here and there capillary sprouts notes; relatively few leucocytes and a stained granular precipitate. Only a thin layer of loose connective tissue surrounds the tube. Distal nerve is found in process of degeneration.
EXPERIMENT No. 264.- Dog No. 51; medium size; full grown; 18 days. July 18, 1918; right ulnar exposed; resected 4 cm. Resected ends of ulnar inserted into the ends of a segment of formalized carotid artery of dog, of 4 cm. length. One central and distal stay suture placed. The diameter of the lumen of the tube greater than that of the nerve. Each end of tube split longitudinally for a distance of 0.5 cm., edges overlapped and held in place by half mattress sutures; ends of tube made to fit nerve closely. Wounds closed. August 5, killed. Dog not in good condition. Ulnar wound well healed. On exposing right ulnar, arterial tube is found well in place, united to the resected nerve ends. Arterial tube surrounded by newly formed connective tissue; tube collapsed. Light pressure on it causes a small amount of sanguineous fluid to escape at the distal end. Ulnar and arterial tube removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good differential neuraxis staining attained. Only central bulb and central end of arterial tube cut; tissue did not embed well, sections torn.
Microscopic findings.-In longitudinal sections of the central bulb and the central end of the arterial tube, the normal ulnar can be traced into the bulbous end, clearly evidenced structurally, this bulbous end surrounded by the arterial tube, which is well preserved. Beyond the distal end of this central bulbous enlargement, small bundles of neuraxes can be traced into newly formed connective tissue continuous with the same, this mainly along the inner wall of the arterial tube, for a distance of about 1 cm.
EXPERIMENT No. 265.- Dog No. 53; small dog; full grown; 41 days. July 20, 1918, light ulnar exposed and resected 4 cm. As tubular suture, used a segment of a formalized carotid artery of a dog, 4 cm. long. One central and distal stay suture placed. Distally a second suture used to narrow the lumen of the tube. Dry field obtained by use of adrenalin. Wound closed. Five to eight days after operation, slight evidence of infection; stitch abscess. Wound treated with tincture of iodine; apparent infection stopped. August 30,dog found dead in the morning; some emaciation; wound well heated. On exposing the right ulnar, arterial tube found well in place: united to resectedc nerve ends. Distinct central bulb seen through tube wall. A small amount of sanguineous fluid noted within the tube. Tube surrounded by newly formed connective tissue; adherent to surrounding tissue. Ulnar and arterial tube removed and fixed in neutral formalin. Sections stained in iron-hema-toxylin and picro-fuchsin; safranine and licht grün.
. Microscopic findings.- In longitudinal sections of area of junction of the central and distal resected nerve ends and arterial tube, it is to be observed that the arterial wall is united to the resected nerve ends by fibrous tissue. The structure of the arterial wall well preserved a concerns elastic and fibrous tissue, with a layer of fairly dense fibrous tissue on its outer surface. In longitudinal sections of the central end of arterial tube, distinct central bulb is evidenced structurally, beyond which very few structures are traced distally within the lumen of the tube. Cross sections of the arterial tube approximately 1.5 cm. from its central bed, shows the tube collapsed and practically empty; scarcely any cellular elements found within the lumen of the tube in this region and distal thereto.
EXPERIMENT No. 266.-Dog No. 57; small dog; full grown; 46 days. July 25, 1918, right ulnar exposed; resected 3.6 cm. A tubular suture made with a 4 cm. long segment of a formalized carotid artery of a dog, held in place by means of one central and distal stay suture. Dry field obtained after use of adrenalin. Wound closed. About 10 days after the operation
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after the wound had healed, the skin of this dog became infected; log not in good condi-tion. September 8, dog found dead in the in morning; emaciated. No neurotropic changes right fore foot. Superficial wound well healed. On exposing nerve, a small pus pocket found over arterial tube; this found to contain a short segment of thread. Arterial tube collapsed; centrally attached to nerve; distally nerve seems to have pulled out of tube. Ulnar and arterial tube removed and fixed in neutral formalin. Sections stained in iron-hematoxylin picro-fuchsin; safranine and licht grün.
Microscopic findings.-In longitudinal sections of central and distal ends of the arterial tube and resected nerve ends, ends of nerve are found to be within the lumen of the arterial tube and united thereto by means of fibrous tissue. Large central bulb evidenced structurally; this ends free within the lumen of the tube. Surrounding end of bulb, numerous leucocytes and phagocytes; very little structural evidence of organized tissue. In cross sections of the arterial tube, approximately its middle wall, of artery presents evidence of disintegration; elastic layers of media present very wavy course and are widely spaced. Within the lumen of the tube, numerous leucocytes and phagocytes; no organized tissue. It is concluded that there was present slight infection in this experiment.
EXPERIMENT No. 267.- Dog No. 55; large; full grown; 47 days. July 22, 1918, right ulnar exposed; resected 3.5 cm. A tubular suture made, using a segment of 4 cm. length of a formalized carotid artery of a dog. One central and distal stay suture placed. Arterial tube cut a little too short so that resected ulnar, after suture, was slightly under tension. Dry field obtained after use of adrenalin. Wound closed. September 8, dog found dead in the morning; much emaciated; severe skin disease. Ulnar wound well healed. On exposing the ulnar, a small dead space is found over the arterial tube with a small amount of sanguineous pus. Arterial tube well in place, united to resected nerve ends. Nerve and arterial tube removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.- In longitudinal sections of the central and distal ends of arterial tube and the resected nerve ends, the arterial tube is found to be well in place united to resected nerve ends by fibrous tissue. Tube wall very well preserved and found of compact structure. Distinct central bulb evidenced structurally, from the distal end of which there is found to extend into the lumen of the tube a newly formed connective tissue consisting of fine interlacing fibers and fibroblasts practically filling the collapsed lumen of the tube and extending within the lumen to the distal nerve segment; arterioles, venules, and capillaries found in this tissue. Within this tissue, traced in successive series from the central bulb, are also found small funiculi of nerve bundles, especially along the inner surface of the arterial tube. These nerve bundles do not extend to the distal nerve, which is degenerated. Sections present no evidence of infection within the lumen of the arterial tube.
EXPERIMENT No. 268.- Dog No. 48; medium size; full grown; 57 days. July 15, 1918, right ulnar exposed and resected approximately 3 cm. A tubular suture made by using a 4 cm. segment of the formalized carotid artery of a dog. One central and distal stay suture placed. Wound closed. September 10, dog found dead in the morning; much emaciated, ulnar wound well healed. On exposing the right ulnar, increase of connective tissue in region of wound noted; possible evidence of deep wound infection not recognized. Arterial tube found well in place, ends adherent to resected nerve ends. Large central bulb within the arterial tube noted. Tube collapsed; surrounded by connective tissue, adherent to surrounding tissues. Ulnar nerve and arterial tube removed and fixed in neutral formalin. Sections stained in iron-hematoxylin and picro-fuchsin; safranine and licht grün.
Microscopic findings.- In central and distal longitudinal sections, arterial tube is found firmly united to resected nerve ends. The large central bulb is found to be partly within and partly without the lumen of the arterial tube; from the distal end of the bulb there extends into the lumen of the tube a vascularized fine fibrillar connective tissue which extends to the distal ulnar. In this tissue small funiculi of nerve fibers may be observed in cross sections of the tube about its middle region. The distal ulnar stump completely degenerated.
EXPERIMENT No. 269.- Dog No. 45; medium size; full grown; 63 days. July 12, 1918, right ulnar exposed; resected 3.5 cm. A tubular suture made by using a segment of 4.5 cm. length of a formalized carotid artery of a dog. One central and distal stay suture placed.
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Arterial tube was cut a little too long; when sutured in place was found bent a little in its middle portion. Wound closed. September 13, killed. Dog in poor condition; emaciated; skin disease. Superficial wound well healed. On exposing nerve, slight evidence of deep wound infection noted. Arterial tube found pulled free from central ulnar stump; distally united to distal ulnar segment. The central stump found to end in large central bulb, from the free end of which no nerve fibers can be traced distalward. Only central bulb removed and fixed in ammoniated alcohol for pyridine-silver staining. Pale but differential silver staining attained.
Microscopic findings.-In longitudinal sections of the central bulb, distinct structural evidence of the central end of the arterial tube found in the peripheral connective tissue sheath of the bulb; arterial tube appears to have been torn rather than to have pulled free from the central stump. A few neuraxes can be traced beyond the distal end of the bulb extending for a short distance into the surrounding connective tissue.
EXPERIMENT No. 270.- Dog No. 49; small dog; full grown; 117 days. July 16, 1918, right ulnar exposed; resected 4 cm. A tubular suture made by using a segment of 4.5 cm. length of formalized carotid artery of a dog; one central and distal stay suture. Quite a little venous bleeding finally controlled by use of adrenalin; dry field; wound closed. November 10, dog found dead in the morning; emaciated; no neurotrophic changes right fore foot. On exposing the right ulnar, arterial tube is found well in place, firmly united to resected nerve ends. Only moderate amount of connective tissue surrounds nerve ends and arterial tube; only loosely adherent to the surrounding tissue; tube collapsed. Distinct bulbous enlargement of the distal end of the central ulnar stump. A segment of the central ulnar. arterial tube and 10 cm. of distal ulnar removed, fixed in ammoniated alcohol for pyridine- silver staining. Good differential silver staining attained; tissue not well embedded; difficult to cut after silver impregnation.
Microscopic findings.- In a series of longitudinal sections including the central bulb and central end of the arterial tube, distinct central bulb evidenced structurally, from the distal end of which many neuraxes, grouped in small funiculi, separated by connective tissue pass distally in the lumen of the arterial tube. As seen in cross sections at successive levels, neuraxes more abundant in the central than in the distal part of the arterial tube, though numerous neuraxes reach the distal end of the arterial tube and pass into the central end of the distal ulnar, in which they may be traced at least 5 cm. distal. In the distal ulnar the neuraxes are fairly evenly distributed through the several funiculi. Partial regeneration of the distal ulnar through arterial tubular suture.
EXPERIMENT No. 271.- Dog No. 43; small dog; full grown; 130 days. July 11, 1918, right ulnar exposed and resected 3 cm. A tubular suture made by using a segment of 4 cm. length of the formalized carotid artery of a dog; one central and distal stay suture placed; distally an additional half mattress suture used, to narrow the lumen of the tube. Dry field obtained by using adrenalin. Wound closed. November 18, dog seemed fairly well in the morning; found dead in the afternoon. Seemed in good condition; no neurotrophic changes right fore foot. On exposing the right ulnar, arterial tube is found well in place; it seems of smaller diameter than when used; firmly united to resected nerve ends; only partly collapsed. Only moderate amount of connective tissue found about the tube; loosely adherent. Large central bulb noted; central end of distal ulnar only slightly enlarged. Distal ulnar of small diameter, but has not the appearance of a degenerated nerve. Fore leg muscles supplied by ulnar exposed; they are found only slightly atrophic and of pale red color. Ulnar nerve and arterial tube removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation attained.
Microscopic findings.-In series of cross and longitudinal sections, the wall of the arterial tube found well preserved. Wall of tube found contracted and presenting a number of folds. In cross section it is observed that the lumen of the tube contains fairly dense fibrous tissue which in the sections is retracted slightly from the inner wall of the tube. Within this connective tissue core, and occupying the central field proximally, more to one side distally, are found numerous small funiculi of nerve fibers. These may be traced, becoming less numerous distally, to and into the distal ulnar in which a relatively small number of neuraxes, about equally distributed through the several funiculi, may be traced distally for several
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centimeters; to the limit of the sections. Partial regeneration of distal ulnar through arterial tube attained.
EXPERIMENT No. 272.- Dog No. 47; medium size; full grown; 240 days. July 15, 1918, right ulnar exposed; resected a little over 3 cm. A tubular suture made by using a segment of about 4 cm. length of a formalized carotid artery of a dog; one central and distal stay suture placed. Dry field. Wound closed. March 12, 1919, killed. Dog in good condition; no neurotrophic changes. Ulnar nerve exposed, first as it passes over elbow. Pressing and touching nerve in this region with forceps, calls forth good contraction of forearm muscles supplied by ulnar. After exposing forearm muscles, cutting the nerve below the elbow causes good contraction; individual muscles seen to contract. The ulnar was then exposed centralward. Arterial tube found well in place and firmly united to resected ends of ulnar. Quite large central bulb encased in central end of arterial tube; central end of distal ulnar not materially enlarged. Ulnar with arterial tube removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.- In cross sections of the arterial tube, this is found collapsed to nearly form of ribbon; vessel wall found well preserved; not materially reduced in size by absorption. In a series of longitudinal sections including the central bulb and central end of the arterial tube, it may be observed that from the distal end of the bulb there extend downward numerous small, intercrossing funiculi of nerve fibers, certain of which are myelinated. Within the lumen of the tube, best seen in cross sections, only small amount of fibrous tissue separated these larger and smaller nerve funiculi, which can be traced in decreasing number to the distal ulnar in which they extend distalward in relatively large number. Fairly complete regeneration of distal ulnar, through formalized arterial tube attained.
EXPERIMENT No. 273.- Dog No. 44; small dog; full grown; 243 days. July 12, 1918, right ulnar exposed; resected 3.5 cm. A tubular suture made by using a segment of 4 cm. length of a formalized carotid artery of a dog; one central and distal stay suture placed. Dry field after use of adrenalin. Wound closed. March 12, 1919, killed. Dog in very good condition. No neurotrophic changes. Right ulnar first exposed near elbow, after freeing from connective tissue and then slowly cutting with scissors, feeble contraction of forearm muscles observed. On exposing the ulnar centralward, it was observed that the arterial tube was attached to the central ulnar stump. The arterial tube could be easily traced for distance of a little more than 2 cm., but appeared to have pulled free from the distal ulnar stump. Several fine nerve bundles could be traced in the connective tissue beyond the distal end of the arterial tube, but could not be followed to the distal stump; some intervening connective tissue having unfortunately been removed before the condition was recognized. It would seem that even though the distal tubular suture had given way, certain of the down-growing neuraxes led to the neighborhood of the distal ulnar stump, had reached and penetrated it, with a result of partial regeneration of the nerve. As the experiment, beyond the function test, owing to accident, was incomplete, tissue was not removed for microscopic study.
EXPERIMENT No. 274.- Dog No. 52; medium size; full grown; 300 days. July 19,1918, right ulnar exposed and resected 4 cm. A tubular suture made, using a segment of 4.5 cm. length of a formalized carotid artery of a dog; one central and distal stay suture placed. Dry field; wound closed. May 15, 1919, killed. Dog in good condition. No neurotrophic changes; appears to use right fore leg as well as left. On exposing the ulnar, it is found that the arterial tube remained connected with the distal ulnar stump, was much collapsed and folded to one side, the central end of the arterial tube having pulled free from the central ulnar stump; presumably soon after the operation. Large bulbous enlargment on the distal end of the central ulnar, surrounded by quite dense fibrous tissue. No nerve bundles could be traced beyond the bulb. The distal ulnar segment completely degenerated, and gives no response on cutting or crushing at the level of the elbow. Central ulnar bulb removed for study of neuroma. Experiment not successful as far as showing tubular suture is concerned. The fact that a formalized artery may remain embedded in normal tissuefor more than seven months, is of interest.
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In this series, in which the use of formalized, arterial, tubular nerve sutures is considered, it is observed that the formalized arteries placed in aseptic wounds in normal tissue at the time of operation remain unabsorbed for a relatively long time, for a period of approximately 8 months, thus fulfilling very well certain of the requirements of a tubular nerve suture, namely, that of remaining unabsorbed until the time when central neuraxes may have opportunity to reach the distal stump. Experiment No. 272 is the most satisfactory of those of this series observed for a long time after operation, in this case 240 days. A distance of 3 cm. was successfully bridged by means of tubular suture as attested by function tests and by histologic examination. The arterial tube was found well in place, though much collapsed, with lumen nearly obliterated. Within the lumen there was found a small amount of connective tissue and in this numerous small funiculi of nerve fibers, which in the main present a longitudinal course. In Experiments No. 270 and No. 271,of approximately 4 months' duration, neuraxes of central origin can be traced through the lumen of the arterial tube to the distal segment in which they may be followed for a short distance. In a number of the experiments of shorter duration, the observations were terminated by death of the animals from disease not related to the experiment. These observations are listed serially as to time, and the tissues obtained were studied so far as circumstances permitted. It will be noted that distinct neuromata found on the distal end of the central stump and within the lumen of the arterial tubes are described for nearly all of the experiments. There is found within the lumen of the tubes a scanty development of connective tissues which appears to have arisen from fibroblasts derived from the central and distal nerve stumps, rather than from the tissue surrounding the arterial tubes. Whether sheath cells participate in the formation of the syncytial net found within the tube in early stages has not been determined. This fibrous tissue forms a scaffolding on which the neuraxes proceed distalward. It is of special interest to note, and it confirms in a very satisfactory manner the monophyletic view of the regeneration of the distal stump, that the neuraxes within the lumen of the vessel appear first near the central bulb, later progressively more distant the longer the time intervening between the operation and the time of observation. The distal segment remains degenerated until the central neuraxes have reached in their downgrowth the distal portion of the arterial tube and thus the central end of the distal stump.
These experiments may serve to show that tubular suture may, under favorable conditions, serve to convey neuraxes from the central to the distal stump of a severed and resected nerve. This has been shown for tubular suture made with decalcified bone tubes, Huber, 30 for fascial tubes by Kirk and Lewis, 86 and for formalized arterial tubes in the experiments here recorded. However, the method of tubular suture can not be recommended for adoption in surgical practice since other methods for bridging nerve defects offer greater assurance for success. " Tubulization offers a single large path for the down-growth of neuraxes and, for this reason, is mechanically inferior to nerve trans-plantation for nerve regeneration, since the latter method offers numerous small paths which serve as individual conducting tubules for the neuraxes"(Stookey) ."
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SERIES NO. 21
TENSION SUTURES; RESECTED NERVES SUTURED UNDER EXTREME TENSION, WITH OR WITHOUT SECONDARY WRAPPING IN ALCOHOLIZED CARGILE MEMBRANE OR FORMALIZED ARTERIAL SHEATHS
In this series of 13 experiments the ulnar nerve of dogs, which had been resected for the purpose of obtaining auto-nerve transplants to be used in Series No. 16, No. 17, and No. 18, was used for the purpose of testing nerve suture under tension, the ends of the resected nerve being brought together as closely as possible by direct suture. It is clearly recognized in surgical practice that a distance of separation of severed and resected nerve ends to the extent of 2 cm. to3 or 4 cm. of one of the major extremity nerves can be overcome, as a rule, by liberation of the nerve and the application of traction, so that a suture may be placed. The extent of approximation which can be attained in this way varies with the nerve and, to some extent, with the location of the injury. We have reached the conclusion, based on experimental observations, that nerves can be freely mobilized without material injury or causing degeneration. In many of our experiments on the sciatic of dogs, after exposing the nerve for its entire length from the sciatic notch to the popliteal space, and then liberating the nerve from its bed and separating the two popliteal branches for their entire length, the internal popliteal branch alone was operated upon, resected, and other-wise treated while the other branch remained intact. In such procedure, the external popliteal branch, though freely manipulated and perhaps under tension, showed no sign of degeneration and functioned without interruption. We feel warranted in saying that quite extensive liberation of a human nerve is permissible if necessity demands and that this is without material injury to the nerve nor does it lessen the progress of regeneration. In every nerve injury with severance of continuity and loss of nerve substance an attempt should be made to approximate the severed nerve ends and perform a simple suture. The extent of tension permitted in connection with a nerve suture is a question of judgment, and experience will enable decision for each case; no general rule can be given not applicable to general surgical procedure. Owing to the unsatisfactory reports found in the literature relative to various methods of procedure for bridging nerve defects surgeons have shown a timidity and hesitancy with regard to adopting methods of nerve transplantation and have attempted by direct suture to maintain in apposition nerve ends after loss of substance when, to accomplish such, the united nerve ends and sutures were under tension to a degree not justified. Such a condition we have endeavored to simulate in this series of experiments. A resection in the ulnar of a dog above the elbow to the extent of from 2.5 cm. to nearly 5 cm. is a relatively much greater loss of nerve substance than a segment of equal length removed from the human ulnar. In our experiments, after the resection of the ulnar nerve for purpose of obtaining an auto-nerve transplant which was used to bridge a defect in the sciatic of the same dog, followed by wrapping the region of the transplant after one of the methods described under Series No. 16, No.17, and No. 18, the operation on the sciatic nerve was completed to closure of the skin wound; in the meantime protection was given to the ulnar wound.
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The details of the operation on ulnar nerve varied somewhat in the several experiments in this series. In the majority of the operations a silk suture was passed through the central and distal stumps of the resected nerve and then, after liberating the nerve centrally and distally, however, without freeing the distal ulnar from its fibrous bed at the elbow, tension was applied mainly by means of the suture and the nerve ends approximated as nearly as could be without breaking the suture or tearing it our. In some of the experiments it was possible to bring the nerve ends in relatively close approximation, in others a distance of 1 cm. Or even more intervened between the nerve ends. In many of the experiments the field of suture was wrapped with several layers of alcoholized Cargile membrane with about 1 cm. Of the adjacent nerve ends included. This was to closely wrapped about the nerve to form a close-fitting tube. In carotid artery of a dog, was slipped over the central nerve stump prior to suture of the nerve ends, and after the suture was completed was placed over the region of the tension suture and the adjacent nerve ends. In other experiments no protection was given to the suture region. Purposely no attempt was made to immobilize the operated foreleg in these experiments. The tension used in bringing together the resected nerve ends in these experiments. it is thought, was no greater than that used by certain operators in the repairof injured nerves with loss of substance in humans; and in this respect our experiments are comparable to their operations.
PROTOCOLS
EXPERIMENT No. 275.- Dog No. 40; medium size; not full grown; 22 days. August19, 1918, left ulnar exposed; resected nearly 4 cm.; resected ends retracted so that they were 4.6 cm. apart. One waxed, silk thread suture was passed through each end of the resected ulnar and then brought together as closely as possible; the sutures tied. The suture line was wrapped with two layers of alcoholized Cargile membrane, forming a tube about 2 cm. long. Fascia stitched over nerve; wound closed. The left forearm was not immobilized. September 10, dog killed; much emaciated. Ulnar wound healed. On removing skin and fascia over the ulnar field, it was found that the tension suture had given way; the resected nerve ends were found to be about 3 cm. apart. The central stump terminated in a well-developed bulbous enlargement. The alcoholized Cargile membrane was found partly spread out, embedded in connective tissue. The central bulbous end removed and fixed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.-Longitudinal sections show typical structure of amputation neuroma.
EXPERIMENT No. 276.- Dog No. 30; large; full grown; 44 days. August 15, 1918, left ulnar exposed; resected 3 cm. One through-and-through silk suture passed and resected nerve ends brought together and suture tied. As knot was being tied, nerve ends slipped apart to the extent of 5 mm. The suture line wrapped with double layer of alcoholized Cargile membrane. Fascia stitched over nerve; wound closed. September 28, dog found dead in the morning. Ulnar wound well healed. After removing skin and fascia over ulnar field, it was observed that the suture had given way; resected ulnar ends nearly 3 cm. a part. Large bulbous enlargement found on central ulnar stump. Cargile membrane found adherent to distal stump; folded over and embedded in fibrous tissue. Portions of central and distal ulnar stump removed and fixed in neutral formalin.
Microscopic findings.- Well stained neuroma showing typical neuroma structure; small funiculi of nerve fibers found in connective tissue distal to neuroma.
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EXPERIMENT No. 277.- Dog No. 3; medium size; full grown; 56 days. June 11, 1918, left ulnar exposed and resected approximately- 2.5 cm. One through-and-through silk suture passed and resected nerve ends brought together and tied; resected nerve ends 2 minn. apart Two layers of Cargile membrane wrapped about suture line and about 1 cm. of resected nerve ends. Fascia stitched over nerve and wound closed. Left foreleg not immobilized. August 6, killed. Dog in good condition. On exposing the ulnar, it is found that the resected nerve ends are still in close approximation; about as at the close of operation. No trace of Cargile membrane found. Firm connective tissue surrounds and unites the resected nerve ends. Distinct large central bulb noted. Large segments of central and distal nerve ends with intervening connective tissue removed and fixed in ammoniated alcohol for pyridine-silver staining. Unsuccessful differentiation attained; tissues not well embedded; sections torn.
Microscopic findings.- No trace of Cargile membrane in sections. In longitudinal sec-tions through the wound region, a few new neuraxes distinguished in the central end of the distal ulnar stump. In cross sections of distal ulnar 2 cm. distal to wound, nerve presents the appearance of a degenerated nerve.
EXPERIMENT No. 278.- Dog. No. 41; small dog; full grown; 61 days. June 28, 1918, right ulnar resected 4 cm. One through-and-through waxed, silk suture passed, and under tension resected nerve ends brought together and suture tied. After suture was tied the resected nerve ends were found to be 1 cm. apart. Fascia stitched over the nerve and wound closed. August 28, dog found dead in the morning. Wound well healed. On exposing the right ulnar, it was observed that the suture had given way; the resected nerve ends were found to be about 3 cm. apart. Large bulbous end on central ulnar stump observed; no nerve bundles can be traced beyond it distally. The central ulnar bulb was removed and fixed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.- Typical neuroma evidenced structurally.
EXPERIMENT No. 279.- Dog No. 38; medium size; full grown; 61 days. June 21, 1918, right ulnar exposed; resected approximately 3 cm. One through and through silk thread suture passed and resected nerve ends brought together under tension. After tying knot, approximation of nerve ends good. Fascia stitched over nerve and wound closed. Foreleg not immobilized. August 22, dog reoperated in the morning; found dead early afternoon; seemed in very good condition. On exposing the right ulnar, it is evident the suture had given away; resected nerve ends found nearly 3 cm. apart. Very large bulbous enlargement noted on end of central ulnar stump. No nerve fibers can be traced from the distal end of this bulb. Central ulnar and bulb removed and fixed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.- Very large neuroma, with typical neuroma structure evidenced structurally.
EXPERIMENT No. 280.- Dog No. 40; medium sized dog; full grown; 47 days. July 25,1918, right ulnar exposed and resected 4 cm. One through-and-through suture of No. 00 cat-gut passed and tension made; catgut suture broke. A double silk thread suture passed and under tension the resected nerve ends brought together; suture tied. Fascia stitched over the nerve; wound closed. Right foreleg not immobilized. September 10, killed. Dog much emaciated; skin disease. On exposing the right ulnar, it is evident that the suture had given away; resected nerve ends about 3 cm. apart. Large central ulnar bulb; within it, suture is recognized. Central ulnar and central end of distal ulnar stump removed and fixed in ammoniated alcohol for pyridine-silver staining.
Microscopic findings.- Large central neuroma evidenced structurally; suture included in the sections. Distal ulnar degenerated.
EXPERIMENT No. 281.-Dog No. 56; large dog; full grown; 172 days. July 24, 1918, right ulnar exposed and resected 4 cm. A segment of a formalized carotid artery of a dog slipped over the central ulnar stump, which was freed from bed for sufficient length to admit of this. One through-and-through waxed, silk thread suture passed, and under tension resected nerve ends brought together and suture tied. After tying knot, resected nerve ends found to be 1.5 cm. apart. The formalized arterial tube was then slipped down over the suture and the resected nerve ends; fascia stitched and the wound closed. January 11, 1919, during
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the night, dog hung himself on tic rope; was in good condition. On exposing the right ulnar, arterial tube is found to be well in place; with central and distal ulnar stumps firmly fixed within the arterial tube. Black, tension suture clearly seen through wall of arterial tube. No material increase of connective tissue about arterial tube and nerve observed. Large central ulnar bulb noted; distal ulnar has the appearance of a normal nerve. Fore-leg muscles supplied by ulnar not atrophic and good color. Dog had been dead some hours so could not test muscles as to functional return. Central and distal ulnar and arterial tube removed and fixed in ammoniated alcohol for pyridine-silver staining. Good differential silver staining attained.
Microscopic findings.-In longitudinal sections of the central ulnar stump and the arterial tube, vessel wall was found to be well preserved and firmly united to the central ulnar stump. Neuraxes can be traced from the distal end of the ulnar bulb through the arterial tube to the distal ulnar stump. In cross sections of the arterial tube in the interval between the two ulnar stumps, the suture can be clearly made out, as also numerous small nerve bundles separated by connective tissue; much as in a tubular suture without tension suture. In the distal ulnar, numerous new neuraxes are observed in all of the funiculi.
EXPERIMENT No. 282.- Dog No. 35; medium size; full grown; 269 days. August 20, 1918, left ulnar exposed; resected 4 cm. One through-and-through waxed, silk thread suture passed, and under tension resected nerve ends brought together to within 8 mm. Suture line wrapped with two layers of alcoholized Cargile membrane. Fascia stitched over the nerve; wound closed. Right foreleg not immobilized. May 16, 1919, killed. Dog in very good condition. On exposing the left ulnar, it is evident that the suture gave way; perhaps soon after the operation; resected nerve ends separated. Central ulnar stump found to end in large bulb, from which some fine nerve bundles could be traced through the connective tissue to the distal ulnar stump; which shows slight bulbous enlargement. No trace of the suture or Cargile membrane could be detected. Foreleg muscles supplied by the ulnar present normal color, though are slightly atrophic. A segment of the central and distal ulnar with intervening connective tissue and fine nerve bundles removed and fixed in ammoniated alcohol for pyridine-silver staining. Differential though faint silver staining attained.
Microscopic findings.- In longitudinal sections of the distal end of the central stump and the fibrous tissue 1 cm. distal thereto, it is observed that the alcoholized Cargile membrane wrapped about the suture line at the time of operation, came to lie one side of the distal end of the central stump when the suture gave way, where it is found embedded in fibrous tissue. In cross sections of the connective tissue field, found between the resected nerve ends, about 1.5 cm. distal to the central stump, the Cargile membrane is again observed, embedded in dense fibrous tissue and to one side there may be seen numerous small intercrossing bundles of nerve fibers which may be traced to the central end of the distal ulnar stump, in which scattered neuraxes are to be found.
EXPERIMENT No. 283.- Dog No. 39; medium size; full grown; 275 days. August 14, 1918, left ulnar exposed; resected 4 cm. One through-and-through waxed, silk thread suture passed, and under tension nerve ends approached to within about 1 cm.; suture tied. The suture line wrapped with double layer of alcoholized Cargile membrane. Fascia stitched over nerve; wound closed. Left foreleg not immobilized. May 16, 1919, killed. Dog in good condition. On exposing the left ulnar, it is evident that the suture had given way. The central ulnar stump is found to end in large bulb, from the distal end of which a few small nerve bundles can be traced into the connective tissue for a short distance beyond the bulb, but can not be traced to the distal ulnar stump. No trace of suture or Cargile membrane seen. Distal ulnar presents the appearance of a degenerated nerve. The central bulb and the central end of the distal ulnar stump removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation attained.
Microscopic findings.- In longitudinal sections of the central ulnar bulb and the con- nective tissue distal thereto, it is to be observed that numerous small nerve funiculi, having very tortuous course, may be observed in the connective tissue and the surrounding adipose tissue. In cross and longitudinal sections of the distal ulnar stump, a few scattered new neuraxes are observed, not evenly distributed through the several funiculi. These become less numerous distalward.
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EXPERIMENT No. 284.- Dog No. 31; medium size; full grown; 276 days. August 16, 1918, right ulnar exposed and resected 4.7 cm. One through-and-through waxed, silk thread suture passed, and under tension resected nerve ends brought together to within a little less than 2 cm. The resected nerve ends and the suture region wrapped in three layers of alcoholized Cargile membrane. Fascia stitched over nerve; wound closed. Right foreleg not immobilized. May 19, 1919, killed. Dog in very good condition. On exposing the right ulnar, it is evident that the suture had given way. Central ulnar is found to end in a large bulb, from the distal end of which several fine nerve bundles pass distalward in the connective tissue, spreading out fan-shaped over the adjacent muscle. None of these nerve bundles could be traced to the distal ulnar stump, which appears to end free and is only very slightly enlarged. The distal ulnar presents the appearance of a degenerated nerve. Central ulnar bulb and the central end of the distal ulnar stump removed and fixed in ammoniated alcohol for pyridine-silver staining. Differential, but faint silver staining attained.
Microscopic findings.- From the distal end of a large central ulnar bulb, evidenced structurally, numerous small nerve funiculi can be traced into the connective tissue; these having a very tortuous course. Remnants of the alcoholized Cargile membrane, embedded in fibrous tissue, noted. In the distal ulnar stump only a few scattered neuraxes, found mainly in one large funiculus, noted; very few in the several other funiculi observed.
EXPERIMENT No. 285.- Dog No. 32; medium size; full grown; 273 days. August 16, 1918, left ulnar exposed; resected 3.4 cm. One through-and-through waxed, silk thread suture passed, and under tension nerve ends approached until they nearly touched; suture tied. Suture line wrapped with two layers of alcoholized Cargile membrane. Oozing in field not fully controlled. Fascia stitched over nerve; wound closed. Foreleg not immobilized. May 16,1919, killed. Dog in very good condition. On exposing the left ulnar, it is found that the central ulnar stump ends in a long spindle-shaped bulb which leads directly to the distal ulnar stump. It is evident that the suture maintained. Traces of the Cargile membrane made out, though not clearly, since area is surrounded by connective tissue. Distal ulnar presents the appearance of a normal nerve. Foreleg muscles supplied by ulnar present the appearance of normal muscle. Central and distal ulnar segment removed and fixed in ammoniated alcohol for pyridine-silver staining. Good silver differentiation attained.
Microscopic findings.- In longitudinal sections, including the central ulnar bulb, suture line and central end of distal ulnar stump, it can be clearly seen that in this case the tension suture did not give way, since in the sections the sections of the suture thread can clearly be made out and are found in proper position. Distinct evidence is had of the Cargile membrane, wrapped about the suture line at the time of operation. This is found embedded in fibrous issue. Neuraxes coming from the central ulnar stump can be traced directly into the distal ulnar stump, in which they may be traced to the region of muscle innervation. In cross sections of the distal ulnar at the level of the elbow, numerous neuraxes distributed evenly through all of the funiculi, are to be observed. Very complete regeneration of the distal ulnar obtained.
EXPERIMENT No. 286.- Dog No. 39; medium size; full grown; 326 days. June 24, 1918, right ulnar exposed; resected 3.2 cm. One through-and-through silk thread suture passed. Under tension the resected nerve ends brought together to within 1 cm.; suture tied. The suture lines wrapped with two layers of Cargile membrane. Fascia stitched over nerve; wound closed. Foreleg not immobilized. May 16, 1919, killed. Dog in very good condition. On exposing the ulnar nerve, it is evident suture had given way; ends of resected ulna; separated. Central ulnar stump found to end in large bulb, from the distal end of which fine nerve bundles may be traced about 2 cm. distal over the adjacent muscle, but do not appear to reach the distal ulnar stump. Distal ulnar presents the appearance of a degenerated nerve. Central ulnar bulb and distal ulnar stump fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation attained.
Microscopic findings.- From the distal end of the large central ulnar bulb, numerous small nerve funiculi can be traced distalward in the connective tissue, having very tortuous course. In cross and longitudinal sections of the distal ulnar stump, at successive levels to the elbow, only scattered neuraxes are found and these are not found evenly distributed through the several funiculi. Partial but very incomplete regeneration of the distal ulnar attained.
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EXPERIMENT No. 287.- Dog No. 2; medium size; full grown; 342 days. June 12, 1918, left ulnar exposed; resected 2.5 cm. One through-and-through silk thread suture passed. Under tension the resected nerve ends brought together until they meet; suture tied. Suture line wrapped with two layers of Cargile membrane. Fascia stitched over the nerve; wound closed. May 20, 1919, killed. Dog in very good condition. On exposing the left ulnar, evident suture had given way; resected ulnar ends found separated. Central ulnar ends in large bulb, from the distal end of which there can be traced several fine nerve bundles which are found to spread out fan-shaped over the adjacent muscle. These fine nerve bundles could not with certainty be traced to the distal ulnar. The distal ulnar presents the appearance of a partially regenerated nerve. On slowly cutting the distal ulnar at the level of the elbow, after exposing the forearm muscles supplied by ulnar, feeble contraction of these muscles is noted. Certain of the central fibers appear to have reached the distal ulnar segment. Central and distal ulnar and intervening connective tissue removed and fixed in ammoniated alcohol for pyridine-silver staining. Fairly good silver differentiation attained.
Microscopic findings.- Very large central ulnar bulb evidenced structurally, from the distal end of which numerous small nerve funiculi, having very tortuous course, can be traced into the connective tissue. No trace of Cargile membrane observed. In cross and longitudinal sections of the distal ulnar there are observed a good number of new neuraxes, fairly evenly distributed through the several funiculi; these are clearly made out in cross sections of the distal ulnar at the level of the elbow. Fairly complete regeneration of the distal ulnar attained.
In 10 of the 13 experiments of tension sutures, it is quite evident that the tension sutures gave way, permitting the nerve ends to separate and to withdraw centrally or distally or both from the tubular sheath applied. This separation of nerve ends apparently occurred within a few days after the operation. The pressure of the suture on the epineural and perineural con- nective tissue in the line of tension can not help but weaken these fibrous tissue layers and cause them to give way in the direction of the suture pull. Immobilization of the part may lessen the tendency of the tension suture to tear and obviate certain gross strains but can not prevent the direct action of the suture on the fibrous sheaths of the nerve. In the experiments in which the tension suture gave way, the approximation of the nerve ends, made for the purpose of facilitating the down-growing central neuraxes to reach the distal stump was probably lost before the regenerative process was well under way. Of interest are the records of the experiments in which it seemed apparent that the tension sutures had given way, which had been kept for periods varying in the several experiments (No. 282, No. 283, No. 286 and No. 287) from about 9 months to nearly 11 months. In all of these experiments the nerve ends were found separated to nearly the distance recorded at the time the tension suture was applied. In each case there was found a large central bulb and from this there could be traced a variable number of larger and smaller nerve bundles, spread out fan-shaped on the muscle bed or winding their way in tortuous course through the connective tissue. It was not possible to trace these small nerve funiculi to the distal stump, but in sections of the central end of the distal stump neuraxes were found scattered through the funiculi, clearly differentiated in pyridine-silver preparations. These observations show the extent to which down-growing neuraxes will grow in connective tissue. The manner of the downgrowth of these neuraxes confirms the view that the tension sutures gave way within a few days after operation. The downgrowth of neuraxes in connective beds, in intermuscular septa, in the connective tissue between muscle fasciculi and over fascial layers, centimeters
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distal to the central nerve bulb, in several of these experiments reaching the distal stump, explains the manner of spontaneous regeneration after nerve severance, a phenomena of not unusual occurrence. The downgrowth of central neuraxes in the manner above indicated needs to be considered in judging by the mere "return of function" in experimental and in clinical work the worth of operative procedures used for bridging nerve defects; the down-growing neuraxes in the experiment or in the clinical case in question may have reached the distal stump in spite of the operative procedure. A careful control, based on a study of serial sections stained by methods giving differential neuraxis staining is necessary before the results of a particular method of peripheral nerve repair can be evaluated.
In three of the experiments the tension sutures maintained. In Experiment No. 277, the left ulnar nerve was resected only 2.5 cm. With a single suture the nerve ends were brought together to within 2 mm. On exposing the ulnar 54 days after the operation the nerve ends were found in approximately the same relative position. A relatively small number of new neuraxes were found in the central end of the distal stump having passed the connective tissue layer intervening between the nerve ends. In Experiment No. 281, examined somewhat over five months after the operation, the right ulnar had been resected 4 cm. By tension suture the ends were brought together, but on tying had again separated to the extent of 15 mm. In this case a formalized arterial tube had been slipped over the suture region, the experiment was thus to some extent a tubular suture. At the time the ulnar was exposed the suture in place could be seen through the wall of the tubular sutures also found well in place. This dog had accidentally hung himself on the tie rope; the nerve could thus not be tested functionally. In pyridine-silver preparation numerous new neuraxes were found in the distal ulnar which could be traced to the central nerve bulb, through the arterial tube. It is clear that in this experiment the tension suture maintained and that the tubular suture prevented dispersion of the down-growing neuraxes. The neurotization of the distal ulnar was quite complete. In Experiment No. 285, examined approximately nine months after the operation, the left ulnar was resected to the extent of 3.4 cm. and under tension suture the nerve ends were approached until they nearly touched; two layers of alcoholized Cargile membrane were wrapped about the suture line. On exposing the ulnar a long spindle-shaped central nerve-bulb was found to lead directly to the distal ulnar segment. In sections, the suture was found in place. Numerous neuraxes derived from the central stump and found distributed quite evenly through the several funiculi of the distal ulnar segment could be traced to the level of the elbow. These three experiments indicate that should a tension suture maintain, regeneration from the central stump is in no way inhibited by the tension to which the nerve ends are subjected. It is evident from the study of microscopic sections that the tubular sheaths employed facilitate materially the down growth of the central neuraxes. Tension sutures, wrapped with alcoholized Cargile membrane or formalized arterial tube, while in a measure successful in a limited number of experiments of this series, are not to be recommended in preference to other methods of bridging nerve defects, notably nerve transplantation, which offers greater assurance of success.
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GENERAL CONCLUSIONS
1. The experimental observations here presented, controlled for the great majority of the experiments through microscopic study of either the entire operated nerve or so much thereof as was deemed advantageous, treated en bloc by the pyridine-silver method, cut and mounted serially and stained with special reference to neuraxis differentiation, when considered collectively, present convincing evidence in support of the monogenetic view of the regener-ation of peripheral nerves degenerated as a consequence of mechanical or chemical injury. According to this view, regeneration of the distal, degenerated segment of a peripheral nerve is through downgrowth of neuraxes sprouts or buds derived from the undegenerated neuraxes of the central stump. The series of experiments on nerve transplantation, and more particularly such in which homogenous nerve segments were used to bridge nerve defects, appear to us to warrant the deduction that the sine qua non of peripheral nerve regeneration is the downgrowth of central neuraxes, since the sheath cells of the transplanted nerve fibers presented no evidence of latent vitality as manifested by proliferation and appeared to be without biological significance so far as growth of neuraxes through the transplanted nerve fiber is concerned. That down-growing neuraxes may grow relatively long distances in the absence of sheath cells derived from either the central or distal stump of a divided nerve seems to be established by these experimental observations, the experiments confirming deductions drawn from observations on tissue culture and experimental embryological studies (Harrison) to the effect that sheath cells are not essential to the growth of the neuraxes. The ultimate relation of sheath cells to the budding and growing neuraxes and the ultimate relation of the down-growing neuraxes to the syncytial protoplasmic bands (Büngner's " bandfasern "), the end product of Wallerian degeneration of peripheral nerve fibers, has not been conclusively determined. The pyridine-silver methods in common with other silver precipitation methods of neuraxis staining, while permitting excellent neuraxis differentiation, are not methods which would be selected for the study of finer cytologic details. Other methods of histologic study were not given consideration. The reason for this series of experimental studies on peripheral nerve repair did not warrant extended duplication of experiments and of necessity influenced the special fields of inquiry. Our observations answer only in a very general way and not decisively the question of the intraprotoplasmic or the extraprotoplasmic position of the new neuraxes. The end-discs found in our preparations, so far as can be determined, present the same form and structure as those found on the end of nerve fibers growing in tissue culture (Harrison) and are of a diameter which is several times greater than the diameter of the protoplasmic syncytial bands. A decisive answer to the question of the relation of the down-growing neuraxes and the syncytial protoplasmic bands of degenerated peripheral nerves is perhaps not to be
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found as a result of study of sections, but rather to be sought in experimental observations on the regeneration of nerve fibers devoid of sheath cells.
2. These experimental observations warrant the conclusion that bridging a nerve defect by means of a nerve transplant is at legitimate operation and that on the whole the most satisfactory results are to be obtained by use of auto-genous-nerve transplants. For purpose of clinical surgery a multiple or cable auto-nerve transplant is recommended using several segments of one or of more than one cutaneous nerve, which may be sacrificed without material loss of function. The question of the particular cutaneous sensory nerve to be selected for purpose of nerve bridge is not material and depends largely on the convenience of the operators; the question of the funicular arrangement of the nerve selected is of quite secondary importance; whether the central or distal end of the nerve segment to be transplanted is placed centralward it is not necessary to consider. The sum total of the nerve segments transplanted should approximate in cross diameter that of the nerve bridged; experimental evidences at hand showing clearly the value of accurate end-to-end approximation of the cut surfaces in placing a nerve bridge. The suture material found experimentally to be the most serviceable is a fine, waxed silk thread, threaded on fine round and half-curved needles, which are passed through the nerve and transplant and adjusted so as to obtain accurate apposition of the cut surfaces of the nerve ends. In many of the experiments in which sutures were placed, the suture line and the adjacent nerve segments were cut longitudinally into serial sections. It is surprising to note the slight evidence of connective tissue reaction, even in experiments examined a long time after the operation. The down-growing neuraxes derived from the central stump, penetrate the transplant through the central wound and course distally within the funiculi of the transplanted nerve segments and to a large extent within the neurolemma sheaths of the transplanted nerve fibers. Such down-growing neuraxes as reach the distal wound pass through this and penetrate the distal segment of the nerve. A histologic study of material obtained from numerous experiments on bridging nerve defects with nerve transplants, even when made from autogenous tissue, convinces that the function of the nerve transplant is to large extent merely mechanical, in that it offers numerous microscopic tubular structures, which maintain for a relatively long period and serve for the down-growth of central neuraxes. Even in autogenous peripheral nerve tissue when used as a nerve bridge the sheath cells of the transplanted nerve fibers are with-out biological significance. The value of a nerve transplant is proportionate to the extent in which the neurolemma sheaths are patent relatively early after transplantation, and it is a question whether the superiority of autogenous and homogenous transplants as against heterogenous-nerve transplants is not to be ascribed to the relative potency of the neurolemma sheaths relatively early after transplantation rather than to other ascribed causes. The funicular pattern of the central segment of a bridged nerve is to a large extent lost as the down-growing neuraxes pass through the central wound. The neuraxes reaching the distal end of the transplant meet with further rearrangement on passing through the distal wound. Thus there is no possibility of maintaining the funicular pattern of a given nerve regenerated through a nerve transplant. In so far as
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has been determined experimentally, there is no reason to assume that the down-growing neuraxes manifest any selectivity on reaching the peripheral segment after bridging (or after a primary or secondary nerve suture). The neuraxes derived from efferent neurones no doubt enter the neurolemma sheaths found in the distal segment of both efferent and afferent peripheral nerve fibers and vice versa. It is assumed that chance brings at least as many branches to homologous nerve fibers as to heterogenous nerve fibers. A very great increase in the number of new neuraxes found in the peripheral end of the central stump permits many new nerve-fiber branches to go astray in the scar tissue, the transplant, and in the peripheral stump and still leave a sufficient number to admit of structural and functional regeneration.
3. Experimental observation warrants the conclusion that the placing of a homogenous-nerve transplant is an operation which is justified. This operative procedure is made more available to clinical surgery through the convincing experimental results obtained with stored homogenous nerve transplants, and especially with nerve transplants stored in sterile liquid petrolatum. The downgrowth of central neuraxes through the funiculi and the neurolemma sheaths of nerve fibers of homogenous-nerve transplants stored in liquid petrolatum is quite as rapid and very nearly as good as when fresh auto-nerve transplants were used. The possibility of thus storing peripheral nerve tissue when opportunity presents itself makes the operation of homo-genous-nerve transplantation to bridge nerve defects more available as a surgical procedure. This operation obviates the necessity of making a second wound, as is generally necessary when it is desired to obtain autogenous nerve tissue. The method of making a multiple or cable-homogenous-nerve transplant, of using a number of smaller nerve bundles rather than one large nerve, is here suggested; this has not been tested experimentally but commends itself when considered in the light of observations on cable-auto-nerve transplants. The use of homogenous nerve tissue, stored in 50 per cent alcohol for the purpose of nerve bridge, deserves serious consideration. The experimental results obtained are encouraging. The simplicity of this method of storage commends itself.
4. The experimental evidence presented with reference to heterogenous-nerve transplants confirms the earlier observations of Huber that regeneration of a severed nerve with loss of substance may take place through a heterogenous-nerve transplant. So far as can be judged on gross inspection and on study of histologic sections, the source of a nerve transplant does not materially influence the fibrous union of the respective end of the nerve transplant and the resected nerve. If a satisfactory suture is made with desired end-to-end approximation of the cut surfaces the fibroblastic reaction appears essentially the same whether auto-, homo-, or heterogenous nerve segments are used to bridge the nerve defects, suggesting the conclusion that histogenetically the fibrous tissue of the wound regions is derived primarily from the parent nerve and not from the connective tissue elements of the nerve transplant. The source of the nerve transplant does not per se appear to influence the extent of connective tissue proliferation in the field surrounding the nerve transplant. On gross inspection the heterogenous-nerve transplant appears to serve the
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purpose of a nerve bridge quite as satisfactorily as an autogenous- or homogenous-nerve transplant. However, on study of an extended series of microscopic sections of tissue, removed in experiments in which heterogeronous-nerve trans-plants were made, differentially stained for determination of neuraxes, the conviction grows that while a certain relatively small per cent of down-growing central neuraxes may pass through the neurolemma sheaths of the nerve fibers found within the funiculi of the transplanted nerve segment, a much larger per cent of down-growing neuraxes are found in the connective tissue sheaths of the nerve transplant and in the immediately surrounding connective tissue. Many of these extrafunicular down-growing neuraxes, found in the connective tissue either as single fibers or as small bundles of fibers, reach the region of the distal wound and through it bring about neurotization of the distal segment of the nerve. So far as may be determined from a study of pyridine-silver stained preparations of the extensive series of experimental observations on nerve transplantation, the fragmentation of the neuraxes and the myelin, their dissolution, and removal in the transplanted nerves no matter whether auto-,homo-, or heterogenous nerve tissue is considered, is not the same as in the process of Wallerian degeneration as found concurrent in the distal segment. The participation of the sheath cells of the transplanted nerve fibers in the fragmentation and removal of the neuraxes and myelin has not been established and seems quite secondary. The conclusion seems warranted that neither in auto-, homo-, nor in heterogenous nerve transplants are the sheath cells of the transplant of biological significance in the regeneration of the nerve. The much more favorable results obtained experimentally on use of autogenous and homogenous transplants, even when the latter have been stored for extended periods before use, than when heterogenous-nerve tissue is used, can not be attributed to the behavior of the sheath cells in the respective experiment, since this difference extends to heterogenous-nerve transplants stored in alcohol. The experimental evidence presented argues for the elimination of the use of heterogenous-nerve transplants as a surgical procedure.
5. When sheathing of a nerve is deemed necessary after neurolysis, nerve suture or nerve transplantation or other operative procedure on peripheral nerves, the use of alcoholized Cargile membrane in double or triple layers deserves consideration as a surgical procedure. Experimental observations indicate that it remains in the tissue 5 to 6 months without absorption and in healthy tissue and aseptic wounds without causing fibrous tissue proliferation. Alcoholized Cargile membrane is readily prepared, is easily applied, and is pliable when bathed in the tissue juices. There was no opportunity for testing experimentally the behavior of alcoholized Cargile membrane in the presence of scar tissue. The several series of experimental observations in which various substances were used for ensheathing operated nerves permit the conclusion that alcoholized Cargile membrane used for the purpose of a tubular sheath in peripheral nerve repair is to be given preference over tubular sheaths prepared from formalized arterial tubes, auto-fascial sheaths prepared from fascia lata, and a fat sheath or fat membrane; it is more readily prepared and applied and incites less connective tissue proliferation. Cargile membrane as generally available is useless for this purpose by reason of its early absorption.
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6. Experimental evidence is at hand to substantiate the view that regeneration of a severed nerve with loss of substance can take place through a tubular suture. The experiments in which downgrowth of central neuraxes through the lumen of a tubular suture was obtained offer convincing evidence in support of the monogenetic view of peripheral nerve regeneration. The experiments of tubular suture in the repair of severed nerves with loss of substance are of academic interest. While regeneration through a tubular suture is possible, this method of operative procedure does not commend itself in surgical practice, since it is less certain of favorable results than when auto- or homo- nerve transplants are used.
7. Experimental observations contraindicate the application of tension sutures in the repair of severed peripheral nerves.
8. Amputation neuromata form at the distal end of the central stump in aseptic wounds relatively early after nerve severance, and are regarded as a thwarted attempt at nerve regeneration. The injection of absolute alcohol into the nerve about 2.5 cm. above the place of section in experimental observations prevents neuroma formation.
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