Light Microscopy

ULTRASTRUCTURAL STUDIES OF INH-INDUCED NEUROPATHY IN RATS III. REPAIR AND REGENERATION

WILLIM W. SCHLAEPFER, M.D,* AND HERMANN HAGER, M.D., PH.D. From the Department of Electron Microscopy, Deutsche Forschungsanstalt fur Psychiatrie (MaxPkmnchlnstitut), Munich, Germany

The inadequacy of histologic techniques for the simultaneous recording of the interaction of all peripheral nerve components during regeneration and repair has contributed to our present ignorance of several fundamental factors. Extensive investigations by light microscopy have resulted in disagreements over several fundamental aspects of these processes.' 2 The electron microscope offers the opportunity for a more accurate demonstration of the evolution of the reactions. Several observations of regeneration following wallerian degeneration have been made with this instrument.8-'0 The Isoniazid (isonicotinic acid hydrazide, INH) induced peripheral neuropathy of the rat is characterized by a primary axonal alteration and a subsequent breakdown of the myelin sheath." 12 These degenerative processes are accompanied and succeeded by reparative and regenerative phenomena of both parenchymal and mesenchymal elements. The specific activity of fibroblasts, axons, and Schwann cells in the degenerated nerve constitute the basis for this report. MATERIAL

AND

METHODS

Twenty female Sprague-Dawley rats were fed 350 mg. Isoniazid per kg. daily per os for periods varying from 6 to i6 days. Three rats were treated for 14 days and sacrificed on the 44th, I20th and I54th days. The sciatic nerves of experimental and 4 normal control rats were removed and examined by light, phase and electron microscopy. The fixative for electron microscopy was i per cent buffered osmium tetroxide; methacrylate and Epon resin were used as embedding media. Part of the material was stained with lead hydroxide.

RESULTS

Light Microscopy An examination of the 6- to i6-day old nerve lesion demonstrated a variable focal disruption of axons and myelin sheaths. Increased celThis work was partially supported by United States Public Health Service Grant 5482 and Special Fellowship in Neuropathology (BT-794), National Institute of Neurological Diseases and Blindness. Accepted for publication, March 24, I964. * Present address: Department of Pathology, Cornell Medical School, New York, N.Y.

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lularity occurred in these regions, characterized primarily by the presence of cells with large round or oval nuclei surrounded by pale, irregular cytoplasm. A mild perivascular mononuclear cell infiltrate was also evident; no evidence of phagocytosis was seen, however. Examination of conventionally stained sections of the 44-day-old lesion showed a marked proliferation of hyperchromatic and elongated Schwann cell nuclei (Fig. i). The cells had a tendency to arrange themselves in narrow longitudinal bundles. The less hyperchromatic and more oval nuclei of fibroblasts were also apparent, but these were not significantly increased in number. Macrophages were scattered throughout and were also localized in perivascular spaces; these were characterized by eccentric nuclei and foamy cytoplasm. Numerous small and delicate nerve fibers could be seen. The I20- and 154-day-old lesions also showed a mild increase of Schwann cell nuclei, but these were not arranged in longitudinal rows. Numerous macrophages containing myelin granules appeared throughout the tissue and in the perivascular spaces. Myelin-stained sections showed a large number of small-caliber myelin sheaths. Phase Contrast Microscopy The 6- to i6-day-old lesions demonstrated a variable disruption of axons and collapse of the myelln sheaths.11"2 In the I3- and i6-day-old lesions a focal increase of round to oval Schwann cell nuclei was noted. These cells often contained myelin debris. The 44-day-old lesions exhibited an increase and elongation of Schwann cell nuclei and regenerating myelinated nerve fibers. The latter in cross section were characterized by a very thin myelin sheath embedded in a large cytoplasmic mass. The I20- and I54-day-old lesions showed a persistence of phagocytes and a predominance of small-caliber myelin sheaths (Fig. 2). The normal myelin sheath was distinctly larger than that of the regenerated fibers.

Electron Microscopy A granular disintegration of the axoplasm was followed by a collapse of the myelin structure within Schwann cell cytoplasm. There were, in turn, decomposition of the myelin debris and its removal by macrophages."12 Evidence of fibroblastic activation was first noted at 13 and i6 days. This was characterized by an elaboration of rough-surfaced endoplasmic reticulum, an increase of RNP granule content and the appearance of irregular "lipid droplets." Differentiation between fibroblasts and histiocytes was most difficult; long, slender processes characteristic of fibroblasts, however, were often seen within the tissue, and no

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phagocytic particles were noted within these cells. In the 44-, 120- and I54-day-old lesions, a significant increase of collagen fibers was observed within the endoneurium (Fig. 3). The slender fibroblastic processes were not conspicuous here, although phagocytes were numerous and an increase of mast cells was observed. A superficial basement membrane was a distinguishing characteristic at the periphery of the Schwann cells. The presence of myelin breakdown products and reactive changes in the cytoplasm of these cells was an important feature during INH-induced neuropathy.12 In addition, a characteristic early change was the formation of numerous slender Schwann cell processes (Fig. 4). These extensions often contained a delicate cytoplasm poor in RNP granules and organelles and occasionally with a fine filamentous ground substance. Several small processes of this nature were enclosed by a single basement membrane, thus forming bundles. The interspaces in the bundles were usually constant and narrow. A tendency toward central separation of individual processes resulted in widening of intercellular gaps. The extracellular space thus formed often showed a fine granular and filamentous background separable from the extracellular endoneurial space by the basement membrane of the bundle (Figs. 4 and 5). The ultrastructural features of axoplasm and the organelle-poor cytoplasm of proliferating Schwann cells were often identical. A distinguishing feature between unmyelinated and regenerating axons was the mesaxon relationship. The nature of occasional solitary processes in the endoneurium could not be ascertained. In the I 6- and 44-day-old lesions, multiple and single axons were embedded within the cytoplasm of a single Schwann cell (Figs. 6 and 7). The cells containing multiple axons characterized unmyelinated nerve fibers. In altered nerves, individual mesaxons occasionally exhibited widened intercellular gaps. Spiral wrapping of the mesaxon around the axon often appeared in nerve fibers with a single axon embedded within a single Schwann cell process (Fig. 7). In the i6- and 44-day-old lesions, many myelinated fibers were characterized by thin myelin sheaths, looseness of spiral wrappings of myelin lamellas, prominence of outer and inner mesaxons, and the complete enclosure of the myelin layer by Schwann cell cytoplasm (Fig. 6). These features were not observed in the 120- and I54-day-old lesions. Here, the most remarkable feature was a relative and absolute increase in the number of smaller nerve fibers (Fig. 3). Occasional longitudinal sections through the nodes of Ranvier at I6 and 44 days demonstrated an increased distance between apposed myelin sheaths and a disproportionate number of myelin lamellas. An unusual finding in Schwann cells containing several axons was a large intracytoplasmic

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space exhibiting an irregular network of moderately dense granules. This was bordered by a membrane, the outer aspect of which was studded with RNP granules (Fig. 6). DISCUSSION The degenerative process in INH-induced neuropathy of rats is accompanied not only by regenerative but also by reparative phenomena. The fibrosis in the endoneurial space may be considered in this category. During the early stage of the lesion, an increase of fibroblasts and their processes is followed by a moderate increase of collagen fibers in the endoneurium of the regenerated nerve. Similar collagenization was noted following wallerian degeneration.13 This has been considered a potential deterrent to the complete functional restoration of severed peripheral nerves.14 It is well known that in collagen formation the long, slender fibroblastic processes disappear following the production of collagen. Therefore, a discrepancy would eventually be expected between the amount of collagen and the number of detectable fibroblastic processes. This should not lead to the conclusion that Schwann cells necessarily participate in the formation of collagen.7'15"16 A marked proliferation of Schwann cells was observed by light microscopy at 44 days. This is an important phenomenon in the regeneration of nerve. Quantitative studies of wallerian degeneration have shown a 13-fold increase of Schwann cell nuclei during the initial 25 days following transection.17 The stimulus for this proliferative activity has been considered to be the loss of the inhibitory influence of the associated axon, a process believed to be the basis for Schwannoma formation.'8 Support for this hypothesis may be found in the observation that Schwann cells fail to grow in tissue culture of normal nerve explants whereas an abundance of growth occurs with degenerating nerve tissue.'920 In addition, Schwann cell proliferation has been believed to terminate on contact with regenerating axon structures.7 The behavior of Schwann cells as sedentary or proliferative and migratory cells has been discussed by Lubinska.21 Schwann cell proliferation was characterized by an arrangement of nuclei into longitudinal rows similar to the "Biu.ngner bands" of wallerian degeneration.22 The electron microscopic equivalent of this proliferation consisted of a bundle of Schwann cell processes surrounded by a single basement membrane. A separation of individual processes within the basement membrane-ensheathed bundle resulted in an enlarged intercellular gap. This space was usually centrally located and frequently contained a fine granular and filamentous matrix (Fig. 5). The pattern was unique and differed from the rare intracellular space in Schwann cell cytoplasm (Fig. 6) reported following wallerian de-

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generation.8 A similar structure has been observed by conventional microscopy in regeneration following wallerian degeneration.23 The latter study indicated a persistence of sheath membrane or neurilemma on the external surface of the Schwann cell during degeneration and proliferation of Schwann cells within these tube-like remnants. The space between proliferating Schwann cells was considered a pathway for regenerating axons. However, it is also possible that this tubular arrangement of processes could represent a phase of regeneration in which a longitudinal direction is given to the Schwann cell proliferation. Thus the large extracellular space within the bundle of Schwann cell processes could represent the initial separation of the processes into individual units. These later could become associated with the regenerating axons in the surrounding endoneurial space. A positive identification of regenerating axons frequently presents some difficulty. The differentiation of axons and Schwann cell processes is often based upon the absence of RNP granules, paucity of vesicles and organelles, and the filamentous arrangement of ground substance in the axoplasm. These criteria are not always reliable since a fine filamentous pattern has been noted in the ground substance of Schwann cell cytoplasm 24'25 and the distal portions of doubly transected peripheral nerves have shown the presence of Schwann cell processes with "axoplasmic" features.6 Electron microscopic studies of regenerating axons in the limb buds of salamanders have shown the young axons to have an emptyappearing cytoplasm containing a few small vesicles and fine fibers; occasional bulbous enlargements exhibit numerous small vesicles and mitochondria.26 The growth cones in regenerating mammalian nerve have demonstrated numerous microvesicles, mitochondria and multivesicular bodies in terminal axon sprouts.5'27 These organelle features were not encountered in the present study. Consequently, the presence of the mesaxon or the surrounding myelin sheath was felt to offer the only reliable means of identification of the axon. Because of the difficulty in identifying young axons, the anatomic pathway of the regenerating axon could not be definitely established. Axon-like structures were occasionally suspended within the endoneurial space. It is also possible, however, that the regenerating axons may have grown within the bundles of Schwann cell processes since several of the processes were indistinguishable from axoplasmic substance. Ultrastructural studies of regeneration following wallerian degeneration support this hypothesis.9 Conventional microscopy has suggested that the regenerating axons either penetrate Schwann cell cytoplasm, grow into the extracellular space within the Biingner bands, or cling to the outside surface of Schwann cells within the endoneurium.21'28'29 The proliferative phase of Schwann cells was followed by close inter-

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action between these cells and regenerating axons. The engulfment of the axon by Schwann cell cytoplasm resulted in mesaxon formation. The frequent appearance of multiple axons within a single Schwann cell probably reflected the presence of unmyelinated fibers. The enclosure of a single axon within a single Schwann cell process was followed by spiral wrapping of the mesaxon around the central axon, thereby forming a myelin sheath. Young myelinated nerve fibers were characterized by prominent inner and outer mesaxons, loose wrapping of the myelin about the axon and a wide seam of Schwann cell cytoplasm around the newly formed myelin sheath. The node of Ranvier of these nerve fibers showed an unusually wide separation of adjacent myelin sheaths and a disproportion of their thicknesses. These features were similar to those described in the histogenesis of myelin.30 The pattern has also been noted during regeneration following wallerian degeneration.3'4'6 It is likely, therefore, that the fundamental process of myelin formation is operative during both the histogenesis and regeneration of peripheral nerve. SUMMARY

The reparative and regenerative processes in INH-induced neuropathy of rats were investigated by light, phase contrast and electron microscopy. These were characterized by mild fibrosis of the endoneurium, proliferation of Schwann cells within basement membraneensheathed longitudinal bundles, separation of individual processes within bundles, regeneration of axons and the interaction of Schwann cell processes with axons and a resulting mesaxon and myelin sheath formation. The anatomic pathway of axonal regeneration was not ascertained although several possible sites were considered. A production of collagen fibers by Schwann cells was not seen. I. 2.

3. 4.

5. 6.

REFERENCES GUTH, L. Regeneration in the mammalian peripheral nervous system. Physiol. Rev., I956, 36, 441-478. RAMON Y CAJAL, S. Degeneration and Regeneration of the Nervous System. MAY, R. M. (translator). Hafner Publishing Co., New York, I928, Vol. I. TERRY, R. D., and HARKIN, J. C. Regenerating peripheral nerve sheaths following wallerian degeneration. Exper. Cell Res., 1957, 13, I93-I97. GLIMSTEDT, G., and WOHLFAHRT, G. Electron microscopic studies on peripheral nerve regeneration. Lunds Univ. Arsskr. N.F. Avd. 2, I960, 56, I-22. ESTABLE, C.; ACOSTA-FERREIRA, W., and SOTELO, J. R. An electron microscope study of the regenerating nerve fibers. Ztschr. Zellforsch., I957, 46, 387-399. WECHSLER, W., and HAGER, H. Elektronenmikroskopische Untersuchung der Wallerschen Degeneration des peripheren Saugetiernerven. Beitr. path. Anat., I962, i26, 352-380.

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7. BARTON, A. A. An electron microscopic study of degeneration and regeneration of nerve. Brain, I962, 85, 799-808. 8. OHMI, S. Electron microscopy of peripheral nerve regeneration. Ztschr. Zellforsch., I962, 56, 625-63I. 9. NATHANIEL, E. J. H., and PEASE, D. C. Regenerative changes in rat dorsal roots following wallerian degeneration. J. Ultrastruct. Res., I963, 9, 533-549. IO. BLU1MCKE, S. Zur Morphologie und Genese des Leitgewebes peripherer Nervenfaserregenerate. II. Elektronenoptische Befunde aus der Nervennarbe. Zentrabi. allg. Path., I963, I04, 24I-255. II. SCHLAEPPER, W. W., and HAGER, H. Ultrastructural studies of INH-induced experimental neuropathy in rats. I. Early axonal changes. Am. J. Path., I964, 45, 209-219. I2. SCHLAEPFER, W. W., and HAGER, H. Ultrastructural studies of INH-induced experimental neuropathy in rats. II. Alteration and decomposition of the myelin sheath. Am. J. Path., I964, 45, 423-433. I3. ABERCROmBIE, M., and JOHNSON, M. L. Collagen content of rabbit sciatic nerve during wallerian degeneration. J. Neurol. Neurosurg. & Psychiat., I946, 9, II3-II8. I4. WEISS, P. The technology of nerve regeneration: A review. Sutureless tubulation and related methods of nerve repair. J. Neurosurg., I944, I, 400-450. E. J. H. Fibrillogenesis by Reactive Schwann Cells in RegeneraNATHANIEL, I5. ting Dorsal Roots. In: Electron Microscopy. Fifth International Congress for Electron Microscopy, Aug. 29-Sept. 2, I962. BREESE, S. S., JR. (ed.). Academic Press, Inc., New York, I962, Vol. 2, N-7. I6. NATHANIEL, E. J. H., and PEASE, D. C. Collagen and basement membrane formation by Schwann cells during nerve regeneration. J. Ultrastruct. Res., I963, 9, 55o-56o. 17. ABERCROMBIE, M., and JOHNSON, M. L. Quantitative histology of wallerian degeneration. I. Nuclear population in rabbit sciatic nerve. J. Anat., I946, 8o, 37-50. I8. MASSON, P. Experimental and spontaneous schwannomas (peripheral gliomas). Am. J. Path., 1932, 8, 367-388. I9. INGEBRIGTSEN, R. A contribution to the biology of peripheral nerves in transplantation. II. Life of peripheral nerves of mammals in plasma. J. Exper. Med., I9I6, 23, 25I-264. 20. ABERCROMBIE, M., and JOHNSON, M. L. Outwandering of cells in tissue cultures of nerves undergoing wallerian degeneration. J. Exper. Biol., I942, I9, 266-283. 2I. LUBINSKA, L. Sedentary and migratory states of Schwann cells. Exper. Cell Res., I96I, Suppl. 8, 74-90. 22. BfUNGNER, 0. v. tYber die Degenerations- und Regenerationsvorgange an Nerven nach Verletzungen. Beitr. path. Anat., I89I, IO, 321-393. 23. HOLMES, W., and YOUNG, J. Z. Nerve regeneration after immediate and delayed suture. J. Anat., I942, 77, 63-96. 24. ELFVIN, L. G. Electron-microscopic investigation of filament structures in unmyelinated fibers of cat splenic nerve. J. Ultrastruct. Res., i96i, 5, 5i-64. 25. PICK, J. On the submicroscopic organization of the myelinated sympathetic nerve fibers in the frog (Rana pipiens). Anat. Rec., i962, I44, 295-325. 26. HAY, E. D. The fine structure of nerves in the epidermis of regenerating salamander limbs. Exper. Cell Res., I960, I9, 299-317.

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27. WECHSLR, W., and HAGER, H. Elektronenmikroskopische Befunde zur Feinstruktur von Axonveranderungen regenerierended Nervenfasem im Nervus ischiadicus der weissen Ratte. Acta Neuropath., I962, 7, 489-506. 28. BIELSCHOWSKY, M. Ailgemeine Histologie und Histopathologie des Nervensystems. In: Handbuch der Neurologie. BuMKE, 0., and FOERSTER, 0. (eds.). J. Springer, Berlin, I935, Vol. I, p. I64. 29. WEDDELL, G. Axonal regeneration in cutaneous nerve plexuses. J. Anat., 1942, 77, 49-62. 30. GEREN, B. B. The formation from the Schwann cell surface of myelin in the peripheral nerves of chick embryos. Exper. Cell. Res., 1954, 7, 558-562. We would like to acknowledge the capable technical assistance of Misses Susanne Luh,

Brunhilde Friedrich, Christa Stark and Ingrid Reichel.

LEGENDS FOR FIGURES FIG. I. Rat sciatic nerve, INH, 44 days. Proliferated and elongated Schwann cell nuclei tend to be arranged in longitudinal rows. Small round nuclei of macrophages are also seen. Hematoxylin and eosin stain. X I20. FIG. 2. Cross section of sciatic nerve, INH, I54 days. There is predominance of small-caliber myelin sheaths and persistence of macrophages (m) containing granular debris. Phase contrast microscopy. X I,500. FIG. 3. Cross section of sciatic nerve, INH, 154 days. An increase of collagen (C) content in the endoneurium and persistent debris-laden macrophages (M) are manifest. Small-caliber nerve fibers (n) predominate. N, normal nerve fibers. X 4,000.

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FIG. 4. Cross section of a bundle of proliferating Schwann cell processes, INH, i6 days. These are enclosed by a single basement membrane (BM). Some of the processes (PI) contain numerous RNP granules while others (P2) exhibit few granules or formed organelles. Separation of the processes results in a widened intercellular space (arrow). C, collagen; F, fibroblast. X I5,000. FIG 5. Incomplete cross section of a bundle of proliferating Schwann cell processes (P), INH, i6 days. Separation of processes has produced a large central extracellular space (ECS) which has a fine granular and filamentous background pattern and is enclosed by the basement membrane (BM) of the bundle. F, fibroblast; MS, myelin sheath. X i6,ooo.

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FIG. 6. INH, 44 days. Several individual Schwann cells (S) contain multiple axons (A); each axon is characterized by a mesaxon (m). A recently myelinated nerve fiber (N) shows a looseness of the myelin lamellas. An unusual intracytoplasmic space (SP) within a Schwann cell contains an irregular network of dense granules and is bordered by a membrane whose outer border is studded with RNP granules. X I5,000. FIG. 7. INH, 44 days. Cross section of a bundle of proliferating Schwann cell processes (P). Two axons (A) are embedded within individual Schwann cell processes. A spiral wrapping of the mesaxon (m) around the axons can be seen. BM. basement membrane; C, collagen; MS, myelin sheath. X 28.000.