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Reactions of unmyelinated nerve fibers to injury. An ultrastructural study
BRAIN RESEARCH
297
REACTIONS OF U N M Y E L I N A T E D N E R V E FIBERS TO INJURY. A N ULTRASTRUCTURAL STUDY
GARTH M. BRAY, JEAN-MARIE PEYRONNARD AND ALBERT J. AGUAYO Division of Neurology, Departmentof Medicine, the Montreal GeneralHospital and McGill University, Montreal, Que. (Canada)
(Accepted January 18th, 1972)
INTRODUCTION Although unmyelinated nerve fibers are an important component of sensory and autonomic nerves, their pathologic reactions have been overshadowed by changes in myelinated nerve fibers. Prompted by observations suggesting predominant involvement of unmyelinated nerve fibers in certain neuropathies 1,7 studies of unmyelinated nerve fiber responses to injury were undertaken. The rabbit anterior mesenteric nerve was chosen for these studies because more than 99 ~o of its fibers are unmyelinatedl0, 30. The present report is a survey of the ultrastructural changes which occur along the distal stump of this nerve between 12 h and 2 weeks following transection. MATERIALSAND METHODS Nerve injury. Adult albino rabbits were anesthetized with a mixture of air and
ether inhaled through an endotracheal tube. A median laparotomy was performed under aseptic conditions and with the aid of an operating microscope, the anterior mesenteric nerve identified on the posterior aspect of the anterior mesenteric artery. Although there were occasional branches along its course, the main portion of this nerve extended for approximately 1 cm beyond its ganglion before dividing into several small branches (Fig. 1). The anterior mesenteric nerve was measured in situ and sectioned with sharp scissors at the junction of its anatomical ganglion and the main nerve trunk. To ensure that nerve section was as complete as possible, the ganglion was carefully lifted and inspected both before and after transection. The surgical gap between the proximal and distal nerve stumps was greater than 1 mm in all cases. The anterior mesenteric nerve was sectioned in 14 rabbits; 4 animals served as controls. Tissue processing. At various times between 12 h and 2 weeks following injury, animals were reanesthetized and the anterior mesenteric nerves exposed and fixed in situ for 3-5 min with chilled 3 9/0 glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, Brain Research, 42 (1972) 297-309
298
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L. . . . . . . . . . . . . . . . .
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lOmm. Fig. 1. Schematic representation of an anterior mesenteric nerve and ganglion showing that most, but not all, ganglionic cells are proximal to the plane of section (S).
using either local bathing or systemic perfusion of the fixative. Ganglia and nerves were then dissected, immersed in fixative and kept at 4 °C for up to 48 h. Under a dissecting microscope, the nerve was divided into 1 mm transverse segments. Each segment was further divided longitudinally into small blocks, post-fixed with osmium tetroxide, and embedded in epoxy resin according to standard methods. For phase microscopy, sections 1/~m thick were cut on ultramicrotomes with glass knives, fixed on glass slides, stained with p-phenylenediamine, and mounted in glycerine. For electron microscopy, ultrathin sections showing silver interference colors were cut with diamond knives, mounted on copper grids and stained with lead citrate. Quantitative methods. Series of non-overlapping electron micrographs of transverse nerve sections were printed at a final magnification of × 10,000 and, as previously described 1,s,2a, were used to determine axonal diameters. Size-frequency histograms for these unmyelinated nerve fibers were prepared. The number of axons included in individual axon-Schwann cell complexes was also determined from these electron micrographs; such complexes were defined as one or more axons enclosed by Schwann cell cytoplasm and its external lamina, without regard to the presence or absence of a Schwann nucleus in the same complexes. RESULTS
Immediately after section, both ends of the severed nerve separated approximately 1 ram, presumably due to elastic retraction. This gap began to close by 24 h after injury. By 48 h after injury, the gap was bridged by thin strands of tissue. After 6 days, there was a slight enlargement at the site of transection; this enlargement was more obvious at 2 weeks. Brain Research, 42 (1972) 297-309
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Fig. 2. Individual fascicles in cross-section from anterior mesenteric nerves, a, Control, showing only two small myelinated fibers in the entire fascicle, b, Proximal portion of distal stump two days after transection, showing dense swollen axons, c, Distal stump one week after transection showing increased numbers of nuclei. Phase micrographs. × 400. Phase microscopy
In cross-section, normal anterior mesenteric nerves consisted of 8-10 round oval fascicles enclosed by perineurial connective tissue (Fig. 2a). As previously described10, a0, less than 1% of axons in this nerve were found to be myelinated. Occasional ganglionic cells were observed along the anterior mesenteric nerve beyond its main ganglion. Examination of serial sections of an entire nerve and ganglion embedded in paraffin indicated that approximately 5 % of ganglionic cells were located in the main nerve trunk. Thus, axons arising from these cells would not be transected by a lesion at the level chosen for this study. In the distal stump of injured anterior mesenteric nerves, a fascicular arrangement o f nerve fibers was observed. Dense axonal enlargements were present in the proximal 1 mm of the distal stump 12 h after injury. By 48 h, these swellings were observed 4 mm from the plane of section (Fig. 2b), while more distal portions of the nerve appeared normal. Swollen axons were still present 4 days after transection, but few were observed one week after injury when increased numbers of nuclei were the most obvious change seen by phase microscopy (Fig. 2c). Similar changes were seen at 2 weeks with only occasional swollen axons present. Electron microscopy Normal anterior mesenteric nerves The distribution of axons within Schwann cells was similar to other unmyelinated autonomic nerve fibers (Fig. 3). Individual unmyelinated axons were usually separated by Schwann cell cytoplasm and plasma membranes; in contrast to other sympathetic nerves zT, it was unusual for two or more axons to be situated within the same Schwann cell trough. Single axons surrounded by Schwann cell cytoplasm were occasionally seen, presumably due to interchanging of such axons from one Schwann cell to anotherL Brain Research, 42 (1972) 297-309
Fig. 3. Normal anterior mesenteric nerve in cross-section showing clusters of axons enclosed by Schwann cells. Axonal mitochondria, neurofilaments and microtubules can be identified. Axonal cytoplasm is less electron-dense than Schwann cell cytoplasm. An external lamina surrounds each Schwann cell. Electron micrograph, x 8,500.
Fig. 4. Anterior mesenteric nerve two days after injury. Cross-section approximately 2 mm from plane of section shows marked swelling of many axons with accumulation of mitochondria and electron dense bodies. Several normal appearing axons are present. Ekclrcn micrcgral:h. ,~: 10,6C0.
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Fig. 5. Two days after injury. Reactive Schwann cell changes and details of a swollen axon are shown. Electron micrograph, x 14,500.
Fig. 6. Three days after injury. A swollen axon is enclosed only by a Schwann cell external lamina. The axon contains clumps of neurofilaments (F), microtubules (T), dense bodies (M), and lamellar structures (arrow). Electron micrograph, x 12,800.
302
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Fig. 7. Four days after injury. Axonal plasma membranes, Schwann cell cytoplasm and external lamina enclose swollen axons containing irregular tubular structures and occasional larger vesicles. Electron micrograph. :< 14,000.
Injured anterior mesenteric nerves (1) Axonal changes. The dense, swollen axons identified on phase microscopy contained aggregations of mitochondria, multivesicular bodies, membranous lamellar structures, and amorphous electron-dense material (Fig. 4). Twenty-four hours after injury, most mitochondria in swollen axons were normal in appearance but were haphazardly arranged rather than in their usual orientation parallel to the long axis of the axon. By 48 h mitochondria showed diverse degenerative changes - - increased density, bizarre shapes and loss of cristae (Fig. 5). Neurofilaments and microtubules could not be identified in many swollen axons; in others, however, they were observed in clumps (Fig. 6). Axonal plasma membranes usually appeared to be intact. Large pale axons, containing irregularly arranged microtubutes were a frequent finding 4 days after nerve section, particularly in the midportion of the anterior mesenteric nerve (Fig. 7). These pale axons were frequently enclosed within the same Schwann cell as axons with dense inclusions, and occasionally both types of axonal change could be seen in the same axon. In more distal portions of the anteriormesenteric nerve trunk (5-10 m m from the level of transection) many axons appeared Brain Research, 42 (1972) 297-309
Fig. 8. Six days after injury. Most axons appear normal but there are increased numbers of axons per Schwann cell. Redundant external lamina are present (arrows). Electron micrograph, x 10,600.
Fig. 9. Six days after injury. Many axons appear normal but one is markedly enlarged, containing mitochondria and numerous dense-core granules, as well as large, electron-dense amorphous material enclosed by single membranes. Electron micrograph, x 21,300.
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normal 4 days after transection. However, occasional denervated Schwann cell processes and redundant external lamina were observed. One and two weeks after nerve section most axons were arranged in clusters within Schwann cells (Fig. 8). Each axon-Schwann cell complex appeared to contain an increased number of axons and, in contrast to control anterior mesenteric nerves, more than one axon was often found within the same Schwann cell trough (Figs. 8, 10). Frequently one axon within each cluster was larger than all others. The contents of individual axons appeared normal. Axons were usually distinguishable from Schwann celt processes by the relative pallor of their cytoplasm, the prominence of neurofilaments and neurotubules and by their mesaxon 7. One week after nerve section, some large axons contained numerous dense-core vesicles measuring between 50 and 75 nm in diameter (Fig. 9). In addition there were large electron-dense single membrane-bound profiles resembling autophagic vacuoles (Fig. 9). (2) Schwann cell changes. Schwann cell cytoplasm surrounding swollen axons was thinned and stretched; large portions of the axonal surface were often covered only by Schwann cell external lamina (Fig. 6). Reactive changes in Schwann cell cytoplasm were observed as early as two days after nerve section; enlarged mitochondria, free ribosomes, rough endoplasmic reticulum and Golgi networks became prominent (Fig. 5). By one week after transection, when there were increased numbers of small axons the configuration of individual axon-Schwann ceil complexes was round
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Fig. 10. Size-frequency histograms prepared for 400 unmyelinated nerve fibers from proximal and distal control anterior mesenteric nerves (cross-hatched bars) and proximal anterior mesenteric nerves 2 weeks after transection (solid bars).
Brain Research, 42 (1972) 297-309
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ULTRASTRUCTURAL REACTIONS OF UNMYELINATED NERVES
or oval. Proliferation of Schwann cells within residual external lamina (bands of B~ingner) was infrequently observed. Denervated Schwann cell processes were infrequently seen and pockets of collagen were not observed within Schwann cells. (3) Other reacting cells. There was no evidence of infiltration by leukocytes or histiocytes, although occasional cells containing necrotic debris were observed. Unlike Schwann cells, these macrophages lacked external lamina and did not enclose axons.
Quantitative studies Control anterior mesenteric nerve fibers ranged from 0.1 to 1.8 #m in axonal diameter. Size-frequency histograms and medians were similar for proximal and distal ends of the anterior mesenteric nerve trunk (Fig. 10). Two weeks after transection, the size-frequency histogram was distorted by increased numbers of small axons approximately 0.4 # m in diameter and the median fiber diameter was reduced from 0.84 to 0.65/zm. Ratios of axons to axon-Schwann cell complexes were determined for proximal and distal control anterior mesenteric nerves as well as proximal anterior mesenteric nerve one and two weeks after transection (Fig. ll). Using the M a n n Whitney U test ~, it was shown that there was no significant difference in these ratios for proximal and distal portions of control nerve while there was a significant increase in the ratio for anterior mesenteric nerves two weeks after transection.
CONTROL
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NUMBER OF AXONS
Fig. I 1. Numbers of unmyelinated axons per axon-Schwann cell complex for proximal and distal control anterior mesenteric nerves as well as transected anterior mesenteric nerves one and two weeks after transection. Histograms were prepared from 200 non-selected axon-Schwann cell complexes. Using the Mann-Whitney U test, there was no significant difference between histograms for proximal and distal nerve segments or between control and one-week transected nerves (P > 0.05); differences between histograms for control and two-week transected nerves were significant (P < 0.001). The large number of complexes with only one axon probably represent axons transferring from one Schwann cell to anotbera; thus, the number of axon-Schwann cell complexes is much larger than the actual number of Schwann cells.
Brain Research, 42 (1972) 297-309
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DISCUSSION
Following transection of anterior mesenteric nerves, the two most striking changes were the development of axonal swellings in the proximal portion of the distal stump during the first 4 days and the appearance of multiple small axons with an increased axon-Schwann cell ratio thereafter. Axons react to transection and other forms of trauma by enlargement of the segments both proximal and distal to the point of injury 5. Electron microscopic features of such axonal swellings have been described in peripheral nerve fibers 3,'-'2, and spinal cord 16. Specifc aspects of these swollen axons such as lysosomal activity 11, mitochondrial accumulations z4, and quantitative correlations of axonal swelling and organelle content 19, have been studied. Axonal swellings have also been described in proximal and distal segments of unmyelinated sympathetic nerves studied up to 24 h after constriction 14,15. Axonal swellings in the distal segments of transected or compressed myelinated nerves are confined to the proximal few mm of the distal stulnp3: similar findings were observed in the present study. Ultrastructural features of the axonal swellings in unmyetinated nerves of this study were similar to axonal swellings previously described in predominantly myelinated nerves following injury. Accumulations of mitochondria3,15,33, 34 and various electron dense structures thought to be lysosomes 11 have been reported. Similar disintegration of neurofilaments, microtubules and endoplasmic reticulum as well as preservation of axolemmal plasma membranes have been observed in peripheral myelinated axons 3. Mire et al. zo have demonstrated that unmyelinated axons in tissue culture show focal dilatations in response to transection or nutritional deprivation. They also presented evidence that such swellings, which unlike in vivo axonal enlargements did not contain organelles, were related to alterations in axonal Na~-K + transport mechanisms. It remains to be determined whether such mechanisms are involved in the development in vivo of axonal swellings such as those observed in the present study. Degenerative changes are less dramatic in distal portions of transected nerves beyond the segments in which axonal swelling occurs. It has been suggested that, in myelinated fibers, axons disappear before their myelin sheaths have disintegrated 17,32. Roth and Richardson z8 studied the degeneration of unmyelinated sympathetic nerve fibers in the rat iris and observed that such axons had disappeared by 48 h after cervical sympathectomy. In the present study a similar loss of unmyelinated nerve fibers was suggested by the presence of occasional denervated Schwann cell processes and isolated external lamina beyond the first 4 mm of the distal stump. This finding was confirmed by the quantitative studies. The precise fate of these fibers cannot be definitely established from our material because only the main trunk of the anterior mesenteric nerve, approximately 1 cm in length, was available for study. Furthermore, the predominance of regenerating axons by 6 days after transection made assessment of such changes difficult. However, Iwayama 13 has presented evidence that such axons are absorbed within Schwann cells with only amorphous dense bodies remaining. By 6 days after nerve transection, multiple small axons with increased axonBrain Research, 42 (1972) 297-309
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Schwann cell ratios were prominent. Although such small axons have been interpreted as degenerative 31, it is more likely that they represent regenerating axons growing from the proximal stump because there was bridging of the gap between the two ends of severed nerves at this time. It has also been shown that similar small axons grow from the proximal stump of a transected nerve which has been isolated from its distal stump by siliconized rubber tubes2, 4. It is possible that some of the small axons may have developed from nerve fibers whose ganglion cells were located distal to the plane of section but this would not likely account for a significant number of fibers. The propensity of autonomic nerve fibers for regeneration within one week of injury has been previously demonstrated 21. In the present study, these regenerative changes were even more prominent two weeks after injury, confirming physiologic evidence for the capacity of unmyelinated nerves for rapid longitudinal growth12, 21. The calculations of Hopkins 12 that such regeneration occurred in unmyelinated nerves at a rate of 1-2 mm a day are consistent with our observations. This rapid regeneration of unmyelinated fibers may also explain earlier conclusions that such nerve fibers did not undergo degeneration when transected or crushed 17,26. Small axons of size and arrangement within Schwann cells similar to those observed as early as 6 days after injury in the present study have been observed in certain human and experimental neuropathies24. ~9. Because they are also observed in the human fetus at 9-16 weeks ~5, they are presumed to represent an embryonal tbrm of nerve regeneration and are generally called axonal sprouts. Their occurrence in the present study suggests that a regression to this form of neural development follows axonal transection. In submammalian species which are capable of regrowing severed limbs, nerve fiber regeneration occurs by extension of enlarged proximal nerve fiber stumps, called end-bulbs or growth cones 18. Although the ultrastructural characteristics of such a form of nerve growth has been delineated in regenerating newt limb¢ 8 and in tissue culture preparations of ganglion cells 35, distinction between degenerating axons and growth cones is difficult in injured mammalian nerves because of the overlap of these two types of axonal change 3. Thus, it is not possible to state with certainty from our material whether or not growth cones were actually present. Longitudinal tissue sections might have been helpful in assessing this aspect of nerve regeneration; however, we found it practically impossible to obtain true longitudinal sections of any useful length in tissue sections of such small nerve fibers. Thus, this study has surveyed the responses to injury in the distal stump of an unmyelinated nerve during the first 2 weeks following transection. The early occurrence of axonal enlargements near the point of injury, similar to those observed in injured myelinated nerves, and regeneration by clusters of small axons have been demonstrated. It has not been possible to determine the mechanism whereby distal portions of the transected axons disappear or the precise degenerative, or regenerative nature of the large, pale swollen axons observed 4 days after transection. Further elucidation of these changes must await studies in which degenerating and regenerating axons can be isolated from each other.
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SUMMARY
Reactions of unmyelinated nerves to injury were studied in the distal stumps of rabbit anterior mesenteric nerves following transection. These nerves, chosen because they are almost exclusively unmyelinated, were examined by phase contrast and electron microscopy at intervals from 12 h to 2 weeks after transection. Swollen axons containing mitochondria and other organelles were prominent in the proximal few mm of the distal stump of anterior mesenteric nerve trunks during the first 4 days after transection. As early as 6 days after injury, regenerative changes consisting of numerous small axons with an increased axon-Schwann cell ratio were observed; there was little trace of degenerating axons, or their debris. Thus the capacity of unmyelinated nerve fibers for rapid regeneration has been demonstrated. It is anticipated that this delineation of reactions in unmyelinated nerves will contribute to a greater understanding of functional and morphologic abnormalities in disorders of peripheral nerves. ACKNOWLEDGEMENTS
The technical assistance of Miss Wendy Downey, Miss Sandra Harrington, Mrs. Adrienne Liberman and Miss Jane Trecarten is acknowledged with gratitude. This project was supported by grants from the Medical Research Council and Muscular Dystrophy Association of Canada.
REFERENCES 1 AGUAYO,A., NAIR, C. P. V., AND BRAY, G. M., Peripheral nerve abnormalities in the Riley-Day syndrome, Arch. Neurol. (Chic.), 24 (1971) 106-116. 2 AOUAYO,A., PEYRONNARD,J. M., MARTIN,J. B., AND BRAY, G. M., Responses of unmyelinated nerves to axonal transection and neuronal loss. An uttrastructural study. In J. D~M~DT (Ed.), New Developments in EMG, Proc. IVth Int. Congr. Electromyography, Brussels, 12-15 September, Karger, Basel, 1972, in press. 3 BLUMCKE,S., NIEDORF, H. R., AND RODE, J., Axoplasmic alterations in the proximal and distal stumps of transected nerves, Acta neuropath. (Berl.), 7 (1966) 44-61. 4 BRAY,G. M., AGUAYO,A., AND PEYRONNARD,J. M., Regeneration of unmyelinated nerve fibers, Clin. Res., 19 (1971) 798, 5 CAJAL, S. RAM6N Y, Degeneration and Regeneration of the Nervous System (Transl. by R. May), Oxford Univ. Press, London, 1928. 6 CAMPBELL, R. C., Statistics for Biologists, Cambridge Univ. Press, Cambridge, 1967, p. 44. 7 DYCK, P. J., AND LAMaERT, E. H., Dissociated sensation in amyloidosis, Arch. Neurol. (Chic.), 20 (1969) 490-507. 8 DYCK, P. J., ELLEFSON,R. D., LAIS,A. C., SMITH, R. C., TAYLOR, W. F., AND VAN DYKE, R. A., Histologic and lipid studies of sural nerves in inherited hypertrophic neuropathy. Preliminary report of a lipid abnormality in nerve and liver in Dejerine-Sottas disease, Mayo Ciin. Proc., 45 (1970) 286-327. 9 LAMES,R. A., ANDGAMBLE,H. J., Schwann cell relationships in normal cutaneous nerves, J. Anat. (Lond.), 106 (1970) 417-435. 10 HESS,A., The fine structure and morphologic organization of non-myelinated nerve fibers, Proc. roy. Soc. B, 144 (1956) 496-506. 11 HOLTZMAN,E., AND NOVIKOFF,A. B., Lysosomes in the rat sciatic nerve following crush, J. Cell Biol., 27 (1965) 651-669.
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12 HOPKINS,A. P., Conduction in regenerating unmyelinated nerve fibers, The Physiologist, 13 (1970) 224. 13 IwAYAMA,T.,Ultrastructuralchangesinthenervesinnervatingthecerebralarteryafter sympathectomy, Z. Zellforsch., 109 (1970) 465-480. 14 KAPELLER,K., AND MAYOR,D., An electron microscopic study of the early changes proximal to a constriction in sympathetic nerve, Proc. roy. Soc. B, 172 (1969) 39-51. 15 KAPELLER,K., AND MAYOR, D., An electron microscopic study of the early changes distal to a constriction in sympathetic nerve, Proc. roy. Soc. B, 172 (1969) 53-63. 16 LAMPERT,P. W., A comparative electron microscopic study of reactive, degenerating, regenerating and dystrophic axons, J. Neuropath. exp. Neurol., 26 (1967) 345-368. 17 LEE,J. C.-Y., Electron microscopy of WaUerian degeneration, J. comp. Neurol., 120 (1963) 65-79. 18 LENTZ, T. L., Fine structure of nerves in the regenerating limb of the newt Triturus, Amer. J. Anat., 121 (1967) 647-670. 19 MARTINEZ,A. J., AND FRIEDE, R. L., Accumulation of axoplasmic organelles in swollen nerve fibers, Brain Research, 19 (1970) 183-198. 20 MIRE, J. J., HENDELMAN,W. J., AND BUNGE, R. P., Observations on a transient phase of focal swelling in degenerating unmyelinated nerve fibers, J. Cell Biol., 45 (1970) 9-22. 21 MURRAY,J. G., AND THOMPSON,J. W., The occurrence and function of collateral sprouting in the sympathetic nervous system of the cat, J. Physiol. (Lond.), 135 (1957) 133-162. 22 NATHANIEL,E. J. H., ANDPEASE,D. C., Degenerative changes in the rat dorsal roots during Wallerian degeneration, J. Ultrastruct. Res., 9 (1963) 511-532. 23 OCHOA,J., AND MAIR,W. G. P., The normal sural nerve in man. I. Ultrastructure and numbers of fibers and cells, Acta neuropath. (Berl.), 13 (1969) 197-216. 24 OCHOA, J., Isoniazid neuropathy in man; quantitative electron microscope study, Brain, 93 (1970) 831-850. 25 OCHOA,J., The sural nerve of the human foetus; electron microscope observations and counts of axons, J. Anat. (Lond.), 108 (1971) 231-245. 26 OI-IMI,S., Electron microscopy of peripheral nerve regeneration, Z. Zellforsch., 56 (1962) 625-631. 27 PETERS, A., PALAY, S. L., AND WEBSTER, H. DEF., The Fine Structure of the Nervous System, Harper and Row, New York, 1970, p. 78. 28 ROTH, C. D., AND RICHARDSON,K. C., Electron microscopical studies on axonal degeneration in the rat iris following ganglionectomy, Amer. J. Anat., 124 (1969) 341-359. 29 SCHR6DER,J. M., Die Hyperneurotisation Biangnerscher B~inder bei der experimentellen Isoniazid Neuropathie: Phasenkontrast und elektronenmikroskopische Untersuchungen, Virchows Arch. Abt. Zellpath., I (1968) 131-156. 30 SIMPSON,S. A., AND YOUNG, J. Z., Regeneration of fiber diameter after cross unions of visceral and somatic nerves, J. Anat. (Lond.), 79 (1945) 48-65. 31 TAXI,J., l~tude au microscope 61ectronique de la d6g6n6rescence Wali6rienne des fibres nerveuses amy61iniques, C. R. Acad. Sci. (Paris), 248 (1959) 2796-2798. 32 THOMAS,P. K., Changes in the endoneurial sheaths of peripheral myelinated nerve fibres during Wallerian degeneration, J. Anat. (Lond.), 98 (1964) 175-182. 33 VIAL, J. D., The early changes in the axoplasm during Watlerian degeneration, J. Biophys. Biochem. Cytol., 4 (1958) 551-555. 34 WEBSTER,H. DEF., Transient focal accumulation ofaxonal mitochondria during the early stages of Wallerian degeneration, J. Cell Biol., 12 (1962) 361-377. 35 YAMADA,K. M., SPOONER, B. S., AND WESSELLS,N. K., Ultrastructure and function of growth cones and axons of cultured nerve cells, J. Cell Biol., 49 (1971) 614--635.
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297
REACTIONS OF U N M Y E L I N A T E D N E R V E FIBERS TO INJURY. A N ULTRASTRUCTURAL STUDY
GARTH M. BRAY, JEAN-MARIE PEYRONNARD AND ALBERT J. AGUAYO Division of Neurology, Departmentof Medicine, the Montreal GeneralHospital and McGill University, Montreal, Que. (Canada)
(Accepted January 18th, 1972)
INTRODUCTION Although unmyelinated nerve fibers are an important component of sensory and autonomic nerves, their pathologic reactions have been overshadowed by changes in myelinated nerve fibers. Prompted by observations suggesting predominant involvement of unmyelinated nerve fibers in certain neuropathies 1,7 studies of unmyelinated nerve fiber responses to injury were undertaken. The rabbit anterior mesenteric nerve was chosen for these studies because more than 99 ~o of its fibers are unmyelinatedl0, 30. The present report is a survey of the ultrastructural changes which occur along the distal stump of this nerve between 12 h and 2 weeks following transection. MATERIALSAND METHODS Nerve injury. Adult albino rabbits were anesthetized with a mixture of air and
ether inhaled through an endotracheal tube. A median laparotomy was performed under aseptic conditions and with the aid of an operating microscope, the anterior mesenteric nerve identified on the posterior aspect of the anterior mesenteric artery. Although there were occasional branches along its course, the main portion of this nerve extended for approximately 1 cm beyond its ganglion before dividing into several small branches (Fig. 1). The anterior mesenteric nerve was measured in situ and sectioned with sharp scissors at the junction of its anatomical ganglion and the main nerve trunk. To ensure that nerve section was as complete as possible, the ganglion was carefully lifted and inspected both before and after transection. The surgical gap between the proximal and distal nerve stumps was greater than 1 mm in all cases. The anterior mesenteric nerve was sectioned in 14 rabbits; 4 animals served as controls. Tissue processing. At various times between 12 h and 2 weeks following injury, animals were reanesthetized and the anterior mesenteric nerves exposed and fixed in situ for 3-5 min with chilled 3 9/0 glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, Brain Research, 42 (1972) 297-309
298
~
G.M. BRAYet aL S"tSROXIMAL '( DISTAL
L. . . . . . . . . . . . . . . . .
J
lOmm. Fig. 1. Schematic representation of an anterior mesenteric nerve and ganglion showing that most, but not all, ganglionic cells are proximal to the plane of section (S).
using either local bathing or systemic perfusion of the fixative. Ganglia and nerves were then dissected, immersed in fixative and kept at 4 °C for up to 48 h. Under a dissecting microscope, the nerve was divided into 1 mm transverse segments. Each segment was further divided longitudinally into small blocks, post-fixed with osmium tetroxide, and embedded in epoxy resin according to standard methods. For phase microscopy, sections 1/~m thick were cut on ultramicrotomes with glass knives, fixed on glass slides, stained with p-phenylenediamine, and mounted in glycerine. For electron microscopy, ultrathin sections showing silver interference colors were cut with diamond knives, mounted on copper grids and stained with lead citrate. Quantitative methods. Series of non-overlapping electron micrographs of transverse nerve sections were printed at a final magnification of × 10,000 and, as previously described 1,s,2a, were used to determine axonal diameters. Size-frequency histograms for these unmyelinated nerve fibers were prepared. The number of axons included in individual axon-Schwann cell complexes was also determined from these electron micrographs; such complexes were defined as one or more axons enclosed by Schwann cell cytoplasm and its external lamina, without regard to the presence or absence of a Schwann nucleus in the same complexes. RESULTS
Immediately after section, both ends of the severed nerve separated approximately 1 ram, presumably due to elastic retraction. This gap began to close by 24 h after injury. By 48 h after injury, the gap was bridged by thin strands of tissue. After 6 days, there was a slight enlargement at the site of transection; this enlargement was more obvious at 2 weeks. Brain Research, 42 (1972) 297-309
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Fig. 2. Individual fascicles in cross-section from anterior mesenteric nerves, a, Control, showing only two small myelinated fibers in the entire fascicle, b, Proximal portion of distal stump two days after transection, showing dense swollen axons, c, Distal stump one week after transection showing increased numbers of nuclei. Phase micrographs. × 400. Phase microscopy
In cross-section, normal anterior mesenteric nerves consisted of 8-10 round oval fascicles enclosed by perineurial connective tissue (Fig. 2a). As previously described10, a0, less than 1% of axons in this nerve were found to be myelinated. Occasional ganglionic cells were observed along the anterior mesenteric nerve beyond its main ganglion. Examination of serial sections of an entire nerve and ganglion embedded in paraffin indicated that approximately 5 % of ganglionic cells were located in the main nerve trunk. Thus, axons arising from these cells would not be transected by a lesion at the level chosen for this study. In the distal stump of injured anterior mesenteric nerves, a fascicular arrangement o f nerve fibers was observed. Dense axonal enlargements were present in the proximal 1 mm of the distal stump 12 h after injury. By 48 h, these swellings were observed 4 mm from the plane of section (Fig. 2b), while more distal portions of the nerve appeared normal. Swollen axons were still present 4 days after transection, but few were observed one week after injury when increased numbers of nuclei were the most obvious change seen by phase microscopy (Fig. 2c). Similar changes were seen at 2 weeks with only occasional swollen axons present. Electron microscopy Normal anterior mesenteric nerves The distribution of axons within Schwann cells was similar to other unmyelinated autonomic nerve fibers (Fig. 3). Individual unmyelinated axons were usually separated by Schwann cell cytoplasm and plasma membranes; in contrast to other sympathetic nerves zT, it was unusual for two or more axons to be situated within the same Schwann cell trough. Single axons surrounded by Schwann cell cytoplasm were occasionally seen, presumably due to interchanging of such axons from one Schwann cell to anotherL Brain Research, 42 (1972) 297-309
Fig. 3. Normal anterior mesenteric nerve in cross-section showing clusters of axons enclosed by Schwann cells. Axonal mitochondria, neurofilaments and microtubules can be identified. Axonal cytoplasm is less electron-dense than Schwann cell cytoplasm. An external lamina surrounds each Schwann cell. Electron micrograph, x 8,500.
Fig. 4. Anterior mesenteric nerve two days after injury. Cross-section approximately 2 mm from plane of section shows marked swelling of many axons with accumulation of mitochondria and electron dense bodies. Several normal appearing axons are present. Ekclrcn micrcgral:h. ,~: 10,6C0.
ULTRASTRUCTURAL REACTIONS OF UNMYELINATED NERVES
301
Fig. 5. Two days after injury. Reactive Schwann cell changes and details of a swollen axon are shown. Electron micrograph, x 14,500.
Fig. 6. Three days after injury. A swollen axon is enclosed only by a Schwann cell external lamina. The axon contains clumps of neurofilaments (F), microtubules (T), dense bodies (M), and lamellar structures (arrow). Electron micrograph, x 12,800.
302
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Fig. 7. Four days after injury. Axonal plasma membranes, Schwann cell cytoplasm and external lamina enclose swollen axons containing irregular tubular structures and occasional larger vesicles. Electron micrograph. :< 14,000.
Injured anterior mesenteric nerves (1) Axonal changes. The dense, swollen axons identified on phase microscopy contained aggregations of mitochondria, multivesicular bodies, membranous lamellar structures, and amorphous electron-dense material (Fig. 4). Twenty-four hours after injury, most mitochondria in swollen axons were normal in appearance but were haphazardly arranged rather than in their usual orientation parallel to the long axis of the axon. By 48 h mitochondria showed diverse degenerative changes - - increased density, bizarre shapes and loss of cristae (Fig. 5). Neurofilaments and microtubules could not be identified in many swollen axons; in others, however, they were observed in clumps (Fig. 6). Axonal plasma membranes usually appeared to be intact. Large pale axons, containing irregularly arranged microtubutes were a frequent finding 4 days after nerve section, particularly in the midportion of the anterior mesenteric nerve (Fig. 7). These pale axons were frequently enclosed within the same Schwann cell as axons with dense inclusions, and occasionally both types of axonal change could be seen in the same axon. In more distal portions of the anteriormesenteric nerve trunk (5-10 m m from the level of transection) many axons appeared Brain Research, 42 (1972) 297-309
Fig. 8. Six days after injury. Most axons appear normal but there are increased numbers of axons per Schwann cell. Redundant external lamina are present (arrows). Electron micrograph, x 10,600.
Fig. 9. Six days after injury. Many axons appear normal but one is markedly enlarged, containing mitochondria and numerous dense-core granules, as well as large, electron-dense amorphous material enclosed by single membranes. Electron micrograph, x 21,300.
304
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normal 4 days after transection. However, occasional denervated Schwann cell processes and redundant external lamina were observed. One and two weeks after nerve section most axons were arranged in clusters within Schwann cells (Fig. 8). Each axon-Schwann cell complex appeared to contain an increased number of axons and, in contrast to control anterior mesenteric nerves, more than one axon was often found within the same Schwann cell trough (Figs. 8, 10). Frequently one axon within each cluster was larger than all others. The contents of individual axons appeared normal. Axons were usually distinguishable from Schwann celt processes by the relative pallor of their cytoplasm, the prominence of neurofilaments and neurotubules and by their mesaxon 7. One week after nerve section, some large axons contained numerous dense-core vesicles measuring between 50 and 75 nm in diameter (Fig. 9). In addition there were large electron-dense single membrane-bound profiles resembling autophagic vacuoles (Fig. 9). (2) Schwann cell changes. Schwann cell cytoplasm surrounding swollen axons was thinned and stretched; large portions of the axonal surface were often covered only by Schwann cell external lamina (Fig. 6). Reactive changes in Schwann cell cytoplasm were observed as early as two days after nerve section; enlarged mitochondria, free ribosomes, rough endoplasmic reticulum and Golgi networks became prominent (Fig. 5). By one week after transection, when there were increased numbers of small axons the configuration of individual axon-Schwann ceil complexes was round
MEDI A'8N4Ml AL PROXI
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I
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o_
i ,
i
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Fig. 10. Size-frequency histograms prepared for 400 unmyelinated nerve fibers from proximal and distal control anterior mesenteric nerves (cross-hatched bars) and proximal anterior mesenteric nerves 2 weeks after transection (solid bars).
Brain Research, 42 (1972) 297-309
305
ULTRASTRUCTURAL REACTIONS OF UNMYELINATED NERVES
or oval. Proliferation of Schwann cells within residual external lamina (bands of B~ingner) was infrequently observed. Denervated Schwann cell processes were infrequently seen and pockets of collagen were not observed within Schwann cells. (3) Other reacting cells. There was no evidence of infiltration by leukocytes or histiocytes, although occasional cells containing necrotic debris were observed. Unlike Schwann cells, these macrophages lacked external lamina and did not enclose axons.
Quantitative studies Control anterior mesenteric nerve fibers ranged from 0.1 to 1.8 #m in axonal diameter. Size-frequency histograms and medians were similar for proximal and distal ends of the anterior mesenteric nerve trunk (Fig. 10). Two weeks after transection, the size-frequency histogram was distorted by increased numbers of small axons approximately 0.4 # m in diameter and the median fiber diameter was reduced from 0.84 to 0.65/zm. Ratios of axons to axon-Schwann cell complexes were determined for proximal and distal control anterior mesenteric nerves as well as proximal anterior mesenteric nerve one and two weeks after transection (Fig. ll). Using the M a n n Whitney U test ~, it was shown that there was no significant difference in these ratios for proximal and distal portions of control nerve while there was a significant increase in the ratio for anterior mesenteric nerves two weeks after transection.
CONTROL
TRANSECT ION
MEDIAN 3'8
~
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1
5
10
15
20
25
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10
15
20
25
NUMBER OF AXONS
Fig. I 1. Numbers of unmyelinated axons per axon-Schwann cell complex for proximal and distal control anterior mesenteric nerves as well as transected anterior mesenteric nerves one and two weeks after transection. Histograms were prepared from 200 non-selected axon-Schwann cell complexes. Using the Mann-Whitney U test, there was no significant difference between histograms for proximal and distal nerve segments or between control and one-week transected nerves (P > 0.05); differences between histograms for control and two-week transected nerves were significant (P < 0.001). The large number of complexes with only one axon probably represent axons transferring from one Schwann cell to anotbera; thus, the number of axon-Schwann cell complexes is much larger than the actual number of Schwann cells.
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DISCUSSION
Following transection of anterior mesenteric nerves, the two most striking changes were the development of axonal swellings in the proximal portion of the distal stump during the first 4 days and the appearance of multiple small axons with an increased axon-Schwann cell ratio thereafter. Axons react to transection and other forms of trauma by enlargement of the segments both proximal and distal to the point of injury 5. Electron microscopic features of such axonal swellings have been described in peripheral nerve fibers 3,'-'2, and spinal cord 16. Specifc aspects of these swollen axons such as lysosomal activity 11, mitochondrial accumulations z4, and quantitative correlations of axonal swelling and organelle content 19, have been studied. Axonal swellings have also been described in proximal and distal segments of unmyelinated sympathetic nerves studied up to 24 h after constriction 14,15. Axonal swellings in the distal segments of transected or compressed myelinated nerves are confined to the proximal few mm of the distal stulnp3: similar findings were observed in the present study. Ultrastructural features of the axonal swellings in unmyetinated nerves of this study were similar to axonal swellings previously described in predominantly myelinated nerves following injury. Accumulations of mitochondria3,15,33, 34 and various electron dense structures thought to be lysosomes 11 have been reported. Similar disintegration of neurofilaments, microtubules and endoplasmic reticulum as well as preservation of axolemmal plasma membranes have been observed in peripheral myelinated axons 3. Mire et al. zo have demonstrated that unmyelinated axons in tissue culture show focal dilatations in response to transection or nutritional deprivation. They also presented evidence that such swellings, which unlike in vivo axonal enlargements did not contain organelles, were related to alterations in axonal Na~-K + transport mechanisms. It remains to be determined whether such mechanisms are involved in the development in vivo of axonal swellings such as those observed in the present study. Degenerative changes are less dramatic in distal portions of transected nerves beyond the segments in which axonal swelling occurs. It has been suggested that, in myelinated fibers, axons disappear before their myelin sheaths have disintegrated 17,32. Roth and Richardson z8 studied the degeneration of unmyelinated sympathetic nerve fibers in the rat iris and observed that such axons had disappeared by 48 h after cervical sympathectomy. In the present study a similar loss of unmyelinated nerve fibers was suggested by the presence of occasional denervated Schwann cell processes and isolated external lamina beyond the first 4 mm of the distal stump. This finding was confirmed by the quantitative studies. The precise fate of these fibers cannot be definitely established from our material because only the main trunk of the anterior mesenteric nerve, approximately 1 cm in length, was available for study. Furthermore, the predominance of regenerating axons by 6 days after transection made assessment of such changes difficult. However, Iwayama 13 has presented evidence that such axons are absorbed within Schwann cells with only amorphous dense bodies remaining. By 6 days after nerve transection, multiple small axons with increased axonBrain Research, 42 (1972) 297-309
ULTRASTRUCTURAL REACTIONS OF UNMYELINATED NERVES
307
Schwann cell ratios were prominent. Although such small axons have been interpreted as degenerative 31, it is more likely that they represent regenerating axons growing from the proximal stump because there was bridging of the gap between the two ends of severed nerves at this time. It has also been shown that similar small axons grow from the proximal stump of a transected nerve which has been isolated from its distal stump by siliconized rubber tubes2, 4. It is possible that some of the small axons may have developed from nerve fibers whose ganglion cells were located distal to the plane of section but this would not likely account for a significant number of fibers. The propensity of autonomic nerve fibers for regeneration within one week of injury has been previously demonstrated 21. In the present study, these regenerative changes were even more prominent two weeks after injury, confirming physiologic evidence for the capacity of unmyelinated nerves for rapid longitudinal growth12, 21. The calculations of Hopkins 12 that such regeneration occurred in unmyelinated nerves at a rate of 1-2 mm a day are consistent with our observations. This rapid regeneration of unmyelinated fibers may also explain earlier conclusions that such nerve fibers did not undergo degeneration when transected or crushed 17,26. Small axons of size and arrangement within Schwann cells similar to those observed as early as 6 days after injury in the present study have been observed in certain human and experimental neuropathies24. ~9. Because they are also observed in the human fetus at 9-16 weeks ~5, they are presumed to represent an embryonal tbrm of nerve regeneration and are generally called axonal sprouts. Their occurrence in the present study suggests that a regression to this form of neural development follows axonal transection. In submammalian species which are capable of regrowing severed limbs, nerve fiber regeneration occurs by extension of enlarged proximal nerve fiber stumps, called end-bulbs or growth cones 18. Although the ultrastructural characteristics of such a form of nerve growth has been delineated in regenerating newt limb¢ 8 and in tissue culture preparations of ganglion cells 35, distinction between degenerating axons and growth cones is difficult in injured mammalian nerves because of the overlap of these two types of axonal change 3. Thus, it is not possible to state with certainty from our material whether or not growth cones were actually present. Longitudinal tissue sections might have been helpful in assessing this aspect of nerve regeneration; however, we found it practically impossible to obtain true longitudinal sections of any useful length in tissue sections of such small nerve fibers. Thus, this study has surveyed the responses to injury in the distal stump of an unmyelinated nerve during the first 2 weeks following transection. The early occurrence of axonal enlargements near the point of injury, similar to those observed in injured myelinated nerves, and regeneration by clusters of small axons have been demonstrated. It has not been possible to determine the mechanism whereby distal portions of the transected axons disappear or the precise degenerative, or regenerative nature of the large, pale swollen axons observed 4 days after transection. Further elucidation of these changes must await studies in which degenerating and regenerating axons can be isolated from each other.
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SUMMARY
Reactions of unmyelinated nerves to injury were studied in the distal stumps of rabbit anterior mesenteric nerves following transection. These nerves, chosen because they are almost exclusively unmyelinated, were examined by phase contrast and electron microscopy at intervals from 12 h to 2 weeks after transection. Swollen axons containing mitochondria and other organelles were prominent in the proximal few mm of the distal stump of anterior mesenteric nerve trunks during the first 4 days after transection. As early as 6 days after injury, regenerative changes consisting of numerous small axons with an increased axon-Schwann cell ratio were observed; there was little trace of degenerating axons, or their debris. Thus the capacity of unmyelinated nerve fibers for rapid regeneration has been demonstrated. It is anticipated that this delineation of reactions in unmyelinated nerves will contribute to a greater understanding of functional and morphologic abnormalities in disorders of peripheral nerves. ACKNOWLEDGEMENTS
The technical assistance of Miss Wendy Downey, Miss Sandra Harrington, Mrs. Adrienne Liberman and Miss Jane Trecarten is acknowledged with gratitude. This project was supported by grants from the Medical Research Council and Muscular Dystrophy Association of Canada.
REFERENCES 1 AGUAYO,A., NAIR, C. P. V., AND BRAY, G. M., Peripheral nerve abnormalities in the Riley-Day syndrome, Arch. Neurol. (Chic.), 24 (1971) 106-116. 2 AOUAYO,A., PEYRONNARD,J. M., MARTIN,J. B., AND BRAY, G. M., Responses of unmyelinated nerves to axonal transection and neuronal loss. An uttrastructural study. In J. D~M~DT (Ed.), New Developments in EMG, Proc. IVth Int. Congr. Electromyography, Brussels, 12-15 September, Karger, Basel, 1972, in press. 3 BLUMCKE,S., NIEDORF, H. R., AND RODE, J., Axoplasmic alterations in the proximal and distal stumps of transected nerves, Acta neuropath. (Berl.), 7 (1966) 44-61. 4 BRAY,G. M., AGUAYO,A., AND PEYRONNARD,J. M., Regeneration of unmyelinated nerve fibers, Clin. Res., 19 (1971) 798, 5 CAJAL, S. RAM6N Y, Degeneration and Regeneration of the Nervous System (Transl. by R. May), Oxford Univ. Press, London, 1928. 6 CAMPBELL, R. C., Statistics for Biologists, Cambridge Univ. Press, Cambridge, 1967, p. 44. 7 DYCK, P. J., AND LAMaERT, E. H., Dissociated sensation in amyloidosis, Arch. Neurol. (Chic.), 20 (1969) 490-507. 8 DYCK, P. J., ELLEFSON,R. D., LAIS,A. C., SMITH, R. C., TAYLOR, W. F., AND VAN DYKE, R. A., Histologic and lipid studies of sural nerves in inherited hypertrophic neuropathy. Preliminary report of a lipid abnormality in nerve and liver in Dejerine-Sottas disease, Mayo Ciin. Proc., 45 (1970) 286-327. 9 LAMES,R. A., ANDGAMBLE,H. J., Schwann cell relationships in normal cutaneous nerves, J. Anat. (Lond.), 106 (1970) 417-435. 10 HESS,A., The fine structure and morphologic organization of non-myelinated nerve fibers, Proc. roy. Soc. B, 144 (1956) 496-506. 11 HOLTZMAN,E., AND NOVIKOFF,A. B., Lysosomes in the rat sciatic nerve following crush, J. Cell Biol., 27 (1965) 651-669.
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12 HOPKINS,A. P., Conduction in regenerating unmyelinated nerve fibers, The Physiologist, 13 (1970) 224. 13 IwAYAMA,T.,Ultrastructuralchangesinthenervesinnervatingthecerebralarteryafter sympathectomy, Z. Zellforsch., 109 (1970) 465-480. 14 KAPELLER,K., AND MAYOR,D., An electron microscopic study of the early changes proximal to a constriction in sympathetic nerve, Proc. roy. Soc. B, 172 (1969) 39-51. 15 KAPELLER,K., AND MAYOR, D., An electron microscopic study of the early changes distal to a constriction in sympathetic nerve, Proc. roy. Soc. B, 172 (1969) 53-63. 16 LAMPERT,P. W., A comparative electron microscopic study of reactive, degenerating, regenerating and dystrophic axons, J. Neuropath. exp. Neurol., 26 (1967) 345-368. 17 LEE,J. C.-Y., Electron microscopy of WaUerian degeneration, J. comp. Neurol., 120 (1963) 65-79. 18 LENTZ, T. L., Fine structure of nerves in the regenerating limb of the newt Triturus, Amer. J. Anat., 121 (1967) 647-670. 19 MARTINEZ,A. J., AND FRIEDE, R. L., Accumulation of axoplasmic organelles in swollen nerve fibers, Brain Research, 19 (1970) 183-198. 20 MIRE, J. J., HENDELMAN,W. J., AND BUNGE, R. P., Observations on a transient phase of focal swelling in degenerating unmyelinated nerve fibers, J. Cell Biol., 45 (1970) 9-22. 21 MURRAY,J. G., AND THOMPSON,J. W., The occurrence and function of collateral sprouting in the sympathetic nervous system of the cat, J. Physiol. (Lond.), 135 (1957) 133-162. 22 NATHANIEL,E. J. H., ANDPEASE,D. C., Degenerative changes in the rat dorsal roots during Wallerian degeneration, J. Ultrastruct. Res., 9 (1963) 511-532. 23 OCHOA,J., AND MAIR,W. G. P., The normal sural nerve in man. I. Ultrastructure and numbers of fibers and cells, Acta neuropath. (Berl.), 13 (1969) 197-216. 24 OCHOA, J., Isoniazid neuropathy in man; quantitative electron microscope study, Brain, 93 (1970) 831-850. 25 OCHOA,J., The sural nerve of the human foetus; electron microscope observations and counts of axons, J. Anat. (Lond.), 108 (1971) 231-245. 26 OI-IMI,S., Electron microscopy of peripheral nerve regeneration, Z. Zellforsch., 56 (1962) 625-631. 27 PETERS, A., PALAY, S. L., AND WEBSTER, H. DEF., The Fine Structure of the Nervous System, Harper and Row, New York, 1970, p. 78. 28 ROTH, C. D., AND RICHARDSON,K. C., Electron microscopical studies on axonal degeneration in the rat iris following ganglionectomy, Amer. J. Anat., 124 (1969) 341-359. 29 SCHR6DER,J. M., Die Hyperneurotisation Biangnerscher B~inder bei der experimentellen Isoniazid Neuropathie: Phasenkontrast und elektronenmikroskopische Untersuchungen, Virchows Arch. Abt. Zellpath., I (1968) 131-156. 30 SIMPSON,S. A., AND YOUNG, J. Z., Regeneration of fiber diameter after cross unions of visceral and somatic nerves, J. Anat. (Lond.), 79 (1945) 48-65. 31 TAXI,J., l~tude au microscope 61ectronique de la d6g6n6rescence Wali6rienne des fibres nerveuses amy61iniques, C. R. Acad. Sci. (Paris), 248 (1959) 2796-2798. 32 THOMAS,P. K., Changes in the endoneurial sheaths of peripheral myelinated nerve fibres during Wallerian degeneration, J. Anat. (Lond.), 98 (1964) 175-182. 33 VIAL, J. D., The early changes in the axoplasm during Watlerian degeneration, J. Biophys. Biochem. Cytol., 4 (1958) 551-555. 34 WEBSTER,H. DEF., Transient focal accumulation ofaxonal mitochondria during the early stages of Wallerian degeneration, J. Cell Biol., 12 (1962) 361-377. 35 YAMADA,K. M., SPOONER, B. S., AND WESSELLS,N. K., Ultrastructure and function of growth cones and axons of cultured nerve cells, J. Cell Biol., 49 (1971) 614--635.
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