4S RNA is transported axonally in normal and regenerating axons of the sciatic nerves of rats

Experiments were designed to determine if following injection of [³H]uridine into the lumbar spinal cord of the rat, [³H]RNA could be demonstrated within axons of the sciatic nerve, and if 4S RNA is the predominant RNA species present in these axons.

In one experiment the left sciatic nerve of a rat was crushed. Two days later 170 μCi of [³H]uridine was injected into the vicinity of the lumbar ventral horn cells. Ten days after injection, rats were sacrificed and sciatic nerves were prepared for autoradiography. Photomicrographs were taken of labeled areas of intact and regenerating nerves and grains were counted over Schwann cells, myelin, axons and other unspecified areas. In both intact and regenerating sciatic nerves more than 20% of the silver grains were associated with motor axons and approximately 40% were found over cytoplasm of Schwann cells surrounding these axons. These data indicate an intra-axonal localization of RNA in sciatic nerve axons, as well as an active transfer of RNA precursors from axons to their surrounding Schwann cells.

In separate studies, the left sciatic nerve was crushed and 10 days later [³H]-uridine was bilaterally injected intraspinally into 6 rats. Four control rats were sacrificed at 14 or 20 days after injection. In the remaining 2 rats the sciatic nerve was cut 14 days after injection and the distal part of the nerve was allowed to degenerate for 6 days before sacrificing the rat. Thus, the distal portion of the nerve contained Schwann cells labeled by axonal transport but lacked intact axons. RNA was isolated from experimental and control nerve segments by hot phenol extraction and ethanol precipitation. RNA species (28S, 18S and 4S) were separated by polyacrylamide gel electrophoresis and radioactivity was measured in a liquid scintillation counter. Control groups had RNA profiles similar to those already described²⁰, with greater than 30% of the radioactivity present as 4S RNA. The proximal portions of nerve taken from the group in which nerves were cut, had a similar amount of radioactivity present as 4S RNA. However, in the distal segments of these nerves (in which the axons had degenerated thus creating an ‘axon-less’ nerve) the amount of radioactivity in the 4S peak decreased to approximately 15% of the total RNA, suggesting that 4S RNA is the predominant if not the only RNA present in these axons. These results strongly indicate that both intact and regenerating sciatic nerves of rats selectively transport 4S RNA along their motor axons.     

Schwann cell basal lamina and nerve regeneration

Nerve segments approximately 7 mm long were excised from the predegenerated sciatic nerves of mice, and treated 5 times by repetitive freezing and thawing to kill the Schwann cells. Such treated nerve segments were grafted into the original places so as to be in contact with the proximal stumps. The animals were sacrificed 1, 2, 3, 5, 7 and 10 days after the grafting. The grafts were examined by electron microscopy in the middle part of the graft, i.e. 3-4 mm distal to the proximal end and/or near the proximal and distal ends of the graft. In other instances, the predegenerated nerve segments were minced with a razor blade after repetitive freezing and thawing. Such minced nerves were placed in contact with the proximal stumps of the same nerves. The animals were sacrificed 10 days after the grafting. Within 1-2 days after grafting, the dead Schwann cells had disintegrated into fragments. They were then gradually phagocytosed by macrophages. The basal laminae of Schwann cells, which were not attacked by macrophages, remained as empty tubes (basal lamina scaffolds). In the grafts we examined, no Schwann cells survived the freezing and thawing process. The regenerating axons always grew out through such basal lamina scaffolds, being in contact with the inner surface of the basal lamina (i.e. the side originally facing the Schwann cell plasma membrane). No axons were found outside of the scaffolds. One to two days after grafting, the regenerating axons were not associated with Schwann cells, but after 5-7 days they were accompanied by Schwann cells which were presumed to be migrating along axons from the proximal stumps. Ten days after grafting, proliferating Schwann cells observed in the middle part of the grafts had begun to sort out axons. In the grafts of minced nerves, the fragmented basal laminae of the Schwann cells re-arranged themselves into thicker strands or small aggregations of basal laminae. The regenerating axons, without exception, attached to one side of such modified basal laminae. Collagen fibrils were in contact with the other side, indicating that these modified basal laminae had the same polarity in terms of cell attachment as seen in the ordinary basal laminae of the scaffolds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis of myelin proteins and ultrastructural investigations in regenerating rat sciatic nerve

Myelin protein synthesis, as well as ultrastructural and morphometric changes in regenerating peripheral nerve, was studied. Sciatic nerves of rats were crushed unilaterally; sham-operated nerves of the contralateral side served as controls. For the in vivo experiments, rats were killed at selected periods after the nerves were crushed (30, 60, 90, and 120 days); seven days prior to killing, the animals were injected intravenously with L-[4,5-3H]leucine. For the in vitro experiments, proximal and distal segments of sciatic nerve and equivalent sham-operated nerves were labeled with 3H-amino acid mixture 90 days after axotomy. Purified myelin was isolated from nerve segments; specific radioactivity and gel electrophoretic patterns of proteins were analyzed. Cross-sectional electron microscope (EM) preparations of proximal, distal, and contralateral segments of nerves also were examined. Results showed that the incorporation of labeled amino acids into total myelin proteins was enhanced significantly in the distal segment of sciatic nerves at all of the periods of regeneration studied. The yield of myelin protein per mm distal nerve segment increased as regeneration proceeded. The remyelination of fibers early after nerve crush was weak, whereas it gradually attained the normal range 90-120 days after axotomy. Morphometric analysis of myelin sheath thickness of regenerating axons was consistent with the data obtained for myelin protein synthesis.     

Multipotentiality of Schwann cells in cross-anastomosed and grafted myelinated and unmyelinated nerves: quantitative microscopy and radioautography.

Cross-anastomoses and autogenous grafts of unmyelinated and myelinated nerves were examined by electron microscopy and radioautography to determine if Schwann cells are multipotential with regard to their capacity to produce myelin or to assume the configuration seen in unmyelinated fibres. Two groups of adult white mice were studied. (A) In one group, the myelinated phrenic nerve and the unmyelinated cervical sympathetic trunk (CST) were cross-anastomosed in the neck. From 2 to 6 months after anastomosis, previously unmyelinated distal stumps contained many myelinated fibres while phrenic nerves joined to proximal CSTs became largely unmyelinated. Radioautography of distal stumps indicated that proliferation of Schwann cells occurred mainly in the first few days after anastomosis but was also present to a similar extent in isolated stumps. (B) In other mice, CSTs were grafted to the myelinated sural nerves in the leg. One month later, the unmyelinated CSTs became myelinated and there was no radioautographic indication of Schwann cell migration from the sural nerve stump to the CST grafts. Thus, Schwann cell proliferation in distal stumps is an early local response independent of axonal influence. At later stages, axons from the proximal stumps cause indigenous Schwann cells in distal stumps from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously myelinated nerves become associated with unmyelinated fibres. Consequently, the regenerated distal nerve resembled the proximal stump. It is suggested that this change is possible because Schwann cells which divide after nerve injury reacquire the developmental multipotentiality which permits them to respond to aoxonal influences.     

Schwann cell migration through freeze-killed peripheral nerve grafts without accompanying axons

Summary Freeze-dried tibial nerve grafts were anastomosed to either the proximal stump or the distal stump of severed tibial nerves in adult inbred Fischer rats. In the case of grafts attached to the proximal stump the tibial nerve was ligated three times, the most distal ligature from the spinal cord being 1 cm from the site of anastomosis. In both types of experiment Schwann cells were, therefore, free to enter the initially acellular grafts without accompanying axons. The grafts were examined 17 days to 12 weeks after operation. Immunofluorescence for S-100 protein was used to evaluate the distance migrated by the Schwann cells and electron microscopy was used to examine the morphology of the cells which invaded the grafts. Schwann cell migration was similar from the proximal and distal stumps. The migrating Schwann cells formed columns which resembled bands of Bungner. They were found mainly, but not exclusively, inside the pre-existing basal lamina tubes left behind by the killed nerve fibres. Some Schwann cells secreted a thin, patchy basal lamina even though they lacked axonal contact. Schwann cell columns became partially compartmentalized by fibroblast processes. Myelin and other debris were removed most rapidly in those parts of the grafts penetrated by large numbers of Schwann cells. The maximum distance the Schwann cells penetrated into the grafts was 8.5 mm and this was achieved by 6 to 8 weeks after operation. This is about half the maximum distance migrated by Schwann cells accompanying regenerating axons through similar grafts. The reasons why Schwann cells migrate shorter distances without axons and the significance of these results for the interpretation of axonal regeneration experiments using acellular grafts are discussed.Supported by a grant from the Medical Research Council     

The role of Schwann cells in the regeneration of peripheral nerve axons through muscle basal lamina grafts

Evacuated muscle is a possible substitute for nerve autografts in the repair of damaged peripheral nerves. Previous experiments have shown that killed or evacuated muscle grafts are as effective as nerve autografts for bridging gaps of up to 4 cm between proximal and distal nerve stumps. Evacuated muscle grafts are made of extracellular matrix components, which are good substrates for axon growth in vitro. However, experiments in vivo have generally demonstrated that live Schwann cells are essential for successful axon regeneration. In the present experiments we have used immunohistochemical techniques with anti-S100 and anti-neurofilament antibodies to visualize axon growth and Schwann cell migration into muscle grafts over the first 10 days following grafting. We only saw axons growing into grafts accompanied by Schwann cells, and most though not all Schwann cells were associated with axons. Schwann cell migration from the proximal stump in association with axons was much faster and more extensive than from the distal stump. We examined muscle grafts over the first 20 days after grafting by electron microscopy. Regenerating axons were always associated with Schwann cells, which were mostly in the basal lamina-lined tubes left by the evacuated myofibrils. A comparison between evacuated muscle grafts and grafts in which the muscle had been killed but not evacuated revealed that 7 days after grafting there were more than twice as many regenerated axons in and distal to the evacuated grafts, but that by 20 days the numbers of axons were similar in the two groups.     

Neurofibromatosis xenografts. Contribution to pathogenesis

We transplanted Schwann cells of 3 patients with neurofibromatosis from neurofibromas, sural nerve, and from a malignant schwannoma into sciatic nerves of immunoincompetent mice. Three and six months later, the grafts and distal nerve segments contained normal myelinated fibers. After rendering host animals immune competent again, neurofibroma and malignant schwannoma Schwann cells were rejected, but grafts retained normally myelinated fibers indicating that these were of mouse origin. Sural nerve Schwann cells from a neurofibromatosis patient were rejected also leaving naked axons in the grafted segments showing that human Schwann cells from the sural nerve of one patient had invested and myelinated the regenerating mouse axons. The nature of putative signals passing between axons and Schwann cells might be elucidated by the combination of human and animal cells in immunoincompetent host nerves. Hypothetical signals for myelination of mouse axons were normally received by sural nerve Schwann cells of a patient with neurofibromatosis, but not by Schwann cells from neurofibromas or malignant schwannomas.     

AN ULTRASTRUCTURAL STUDY OF THE EARLY STAGES OF AXONAL REGENERATION THROUGH RAT NERVE GRAFTS

Segments of rat sciatic nerve 5 mm long were removed and either maintained alive in tissue culture medium or killed by freeze-drying. Twenty-four h later the nerve segments were replaced as autografts. Animals were killed 3-14 days after grafting. Grafts of cultured nerves (C-grafts) always contained many living cells. Grafts of freeze-dried nerves (FD-grafts) contained few living cells at 3 days, but were repopulated by 7 days. A few regenerating axons were identified in the most proximal parts of 3 day C-grafts and by 14 days many myelinated axons extended to the distal ends. Axons were absent from 3 and 7 day FD-grafts, but by 14 days some non-myelinated axons extended to the distal end of such grafts. Regenerating axons were always associated with Schwann cells. Small perineurial compartments were formed at the junctional zones of all grafts and throughout the FD-grafts. Revascularization of the FD-grafts was delayed when compared to that in C-grafts. Fenestrated capillaries were observed in both types of graft. These experiments demonstrate that axons regenerate through FD-grafts that have been repopulated by cells and the grafts probably lack the normal perineurial and blood/nerve diffusion barriers. The significance of these results is discussed in relation to the requirements for successful axonal regeneration.     

Presence and regulation of transforming growth factor beta mRNA and protein in the normal and lesioned rat sciatic nerve

The transforming growth factors beta (TGF-β), a family of regulatory polypeptides, are involved in numerous vital processes including inflammation and wound healing. Since repair of a peripheral nerve lesion includes a series of well-defined steps of cellular actions possibly controlled by TGF-βs, and since TGF-β mRNA and immunoreactivity have been found in the normal peripheral nerve, we have examined TGF-β mRNA regulation and protein expression in the lesioned peripheral nerve. Sciatic nerves of adult rats were either crushed (allowing axonal regenration) or transected (to prevent axonal regeneration and to induce Wallerian degeneration in the distal stump). After intervals of 6 hours, 2 and 6 days post-lesion, the rats were sacrificed and each nerve was cut into four segments, two proximal and two distal to the lesion site. TGF-β 1-3 mRNA were determined for each segment. We demonstrate that TGF-ß1 mRNA levels are higher than those of TGF-ß3; the amplitude of mRNA regulation depends on time, type of lesion and localization relative to the lesion site. TGF-ß2 mRNA could not be detected. For TGF-ß1-3 immunocytochemistry, animals were sacrificed 12, 24, 48, 72 hours and 7 and 14 days after surgery. TGF-β immunoreactivity (IR) was observed for all isoforms in lesioned and unlesioned nerves. In the segment directly adjacent to the lesion at its proximal side, an increase of TGF-β-IR became apparent as soon as 12 hours after surgery; it remained elevated during the whole period observed in both models. In the segment adjoining the distal side of the lesion, an increase of TGF-β-IR was observed after 48 hours, which was still present after 14 days. At day 7 after crush or transection, an increase of TGF-β-IR was detected in the most distal segments, which reached its highest levels at the end of our observation period. Our results suggest that the presence of axonal contact might induce an enhancement of TGF-β expression by Schwann cells in the distal stump of a lesioned and regenerating peripheral nerve. Since we demonstrate an increase of TGF-β mRNA and protein expression also in the distal stump of transected nerves where Schwann cells are not able to contact sprouting axons from the proximal part, other regulatory pathways must exist. The acquisition of a “reactive” Schwann cell phenotype after peripheral nerve lesion might involve an upregulation of TGF-β expression. © 1994 Wiley-Liss, Inc.     

The role of Schwann cells and basal lamina tubes in the regeneration of axons through long lengths of freeze-killed nerve grafts

The ability of long acellular nerve grafts to support axonal regeneration was examined using inbred rats. Grafts (40 mm long) of tibial/plantar nerves were used either as live grafts or after freeze-drying to render the grafts acellular. The grafts were sutured to the proximal stump of severed tibial nerves in host animals which were then killed 1-12 weeks later. Axons rapidly regenerated through the living grafts but only extended 10-20 mm into the acellular grafts. This distance was achieved by 6 weeks and thereafter no significant further axonal extension occurred in the acellular grafts. A few naked axons lacking Schwann cell contact were identified in all acellular grafts, but became more numerous near the distal extent of axonal penetration into 6-12 week grafts. These axons contained large numbers of neurofilaments. When the distal 20 mm of 6 week acellular grafts (segments into which axons had not penetrated) were sutured to freshly severed tibial nerves, axons grew readily into the grafted tissue to a maximum distance of 9 mm. It is therefore likely that the limits to axonal regeneration through initially acellular grafts were set by factors intrinsic to the severed nerve. It is suggested that the limited migratory powers of Schwann cells may be one such factor. The concept that basal lamina tubes are not essential for axonal regeneration but may act as low resistance pathways for both axonal elongation and Schwann cell migration is discussed.     

Neurofilament mRNAs are present and translated in the normal and severed sciatic nerve

Local protein synthesis within axons has been studied on a limited scale. In the present study, several techniques were used to investigate this synthesis in sciatic nerve, and to show that it increases after damage to the axon. Neurofilament (NF) mRNAs were probed by RT-PCR, Northern blot and in situ hybridization in axons of intact rat sciatic nerve, and in proximal or distal stumps after sciatic nerve transection. RT-PCR demonstrated the presence of NF-L, NF-M and NF-H mRNAs in intact sciatic nerve, as well as in proximal and distal stumps of severed nerves. Northern blot analysis of severed nerve detected NF-L and NF-M, but not NF-H. This technique did not detect the three NFs mRNAs in intact nerve. Detection of NF-L and NF-M mRNA in injured nerve, however, indicated that there was an up-regulation in response to nerve injury. In situ hybridization showed that NF-L mRNA was localized in the Schwann cell perinuclear area, in the myelin sheath, and at the boundary between myelin sheath and cortical axoplasm. RNA and protein synthesizing activities were always greater in proximal as compared to distal stumps. NF triplet proteins were also shown to be synthesized de novo in the proximal stump. The detection of neurofilament mRNAs in nerves, their possible upregulation during injury and the synthesis of neurofilament protein triplet in the proximal stumps, suggest that these mRNAs may be involved in nerve regeneration, providing a novel point of view of this phenomenon.     

Three dimensional analysis of unmyelinated fibers in normal and pathologic autonomic nerves.

The technique of serial-section electron microscopy and diagrammatic three dimensional reconstructions has been used to assess normal and pathological unmyelinated nerve fibers from a peripheral autonomic nerve: the rat cervical sympathetic trunk. Within this predominantly unmyelinated nerve, there is a complex arrangement of axons into longitudinally oriented bundles brought together by chains of Schwann cells. Each bundle is subdivided into smaller components by the cytoplasmic processes of Schwann cells; such subdivisions, which are basal lamina-enclosed masses of Schwann cell cytoplasm, when viewed on cross-sectional electron micrographs, are termed Schwann cell units. The size and shape of each Schwann cell unit varies along the length of fibres, but the diameter of individual axons shows little variation over the segments studied. Axonal branching was not observed in normal unmyelinated nerves. Crush injury and x-irradiation produces different patterns of alteration in the axon-Schwann cell relationships of unmyelinated nerves. Following crush injury, Schwann cell processes increase in diameter and contain numerous small diameter axonal sprouts. Many of the regenerating axons remain thin while others reacquire a normal diameter. X-irradiation affects Schwann cells leading to retraction of their processes and the appearance of naked axonal segments.     

Local administration of vasoactive intestinal peptide after nerve transection accelerates early myelination and growth of regenerating axons

Our goal was to determine whether local injections of vasoactive intestinal peptide (VIP) promote early stages of regeneration after nerve transection. Sciatic nerves were transected bilaterally in 2 groups of 10 adult mice. In the first group, 15 microg (20 microL) of VIP were injected twice daily into the gap between transected ends of the right sciatic nerve for 7 days (4 mice) or 14 days (6 mice). The same number of mice in the second group received placebo injections (20 microL of 0.9% sterile saline) in the same site, twice daily, for the same periods. After 7 days, axon sizes, relationships with Schwann cells and degree of myelination were compared in electron micrographs of transversely sectioned distal ends of proximal stumps. Fourteen days after transection, light and electron microscopy were used to compare and measure axons and myelin sheaths in the transection gap, 2-mm distal to the ends of proximal stumps. Distal ends of VIP-treated proximal stumps contained larger axons 7 days after transection. More axons were in 1:1 relationships with Schwann cells and some of them were surrounded by thin myelin sheaths. In placebo-treated proximal stumps, axons were smaller, few were in 1:1 relationships with Schwann cells and no myelin sheaths were observed. In VIP-treated transection gaps, measurements 14 days after transection showed that larger axons were more numerous and their myelin sheaths were thicker. Our results suggest that in this nerve transection model, local administration of VIP promotes and accelerates early myelination and growth of regenerating axons.     

Laminin molecules in freeze-treated nerve segments are associated with migrating Schwann cells that display the corresponding alpha6beta1 integrin receptor

Isolated acellular nerve segments protected from migration of Schwann cells and the acellular nerve segments joined with the distal nerve stumps were prepared by a repeated freeze-thaw procedure in the rat sciatic nerves. The presence of laminin-1 and -2, as well as alpha6 and beta1 integrin chains, was detected by indirect immunohistochemistry in the sections through acellular nerve segments at 7 and 14 days after cryotreatment. The position of basal laminae and Schwann cells was identified by immunostaining for collagen IV and S-100 protein, respectively. The isolated cryo-treated segment without living Schwann cells (S-100-) did not display immunoreactivity for laminins and integrin chains, while the basal lamina position was verified through the whole segment by immunostaining for collagen IV. The absence of immunostaining for laminin-1 and -2 in cryo-treated nerve segment was verified by Western blot analysis. A crucial diminution of laminin-1 and -2 in the cryo-treated nerve segment of 10-mm length did not abolish the growth and maturation of axons. The greater part of nerve segment connected with the nerve stump displayed no immunohistochemical staining for S-100, corresponding with absence of Schwann cells. The border region of the nerve segment contained Schwann cells (S-100+) migrating from the near-freeze undamaged part of the distal nerve stump. In addition to immunostaining for S-100 protein, the migrating Schwann cells displayed immunostaining for laminins (-1, and -2) and integrin chains (alpha6 and beta1). The results indicate that the presence of laminin molecules in the acellular nerve segments prepared by the repeated freeze-thaw procedure is related with the migrating Schwann cells. The immunostaining for laminins and integrin chains, which constitute one of integrin receptor, suggests an autocrine and/or paracrine utilization of laminin molecules in the promotion of Schwann cell migration.     

The numbers of unmyelinated and myelinated axons in normal and regenerated rat saphenous nerves

Counts have been made of the numbers of unmyelinated and myelinated axons in the proximal and distal stumps of regenerated rat saphenous nerves and from equivalent sites in normal nerves. In the proximal part of normal nerves there were averages of 1 045 myelinated axons and 4 160 unmyelinated ones. Regenerated nerves contained the same number of myelinated axons in their proximal stumps but there was a 40% reduction in the unmyelinated axon count. In the distal stumps of these nerves the myelinated axon count had increased by an average of 620; this comes about because some regenerated myelinated axons support more than one process in the distal stump. In contrast, the number of unmyelinated axons was reduced further, from a mean of 2 476 in the proximal stump to one of 2 219.

The sizes of Schwann cell units in the normal and regenerated nerves were also noted. Schwann cell units in the proximal and distal stumps of the regenerated nerves were smaller than those in the normal ones.

These changes associated with unmyelinated axons in regenerated nerves are likely to contribute to the sensory, vasomotor and sudomotor abnormalities that sometimes occur after peripheral nerve injury and regeneration.     

Axonal branching following crush lesions of peripheral nerves of rat

Branching of myelinated and unmyelinated nerve fibers in normal and regenerating personal and soleus nerves was studied by light and electron microscopy. There were at most 2% more myelinated and 13% more unmyelinated axons in the distal as compared with the proximal nerve segments. Two to four weeks after a crush lesion the distal axons became 2-3 times more numerous; thereafter their number decreased. The number of axons in the proximal nerve segment did not change. The number of myelinated sprouts in most regenerated nerves equalled the number of myelinated fibers in the proximal nerve, while the number of unmyelinated axons after 12-19 weeks was 18-60% higher than normal. Branching was not restricted to the crush region. The results indicate that following a crush lesion all axons branch but only branches of unmyelinated fibers persist for a prolonged period of time. It is tentatively suggested that regenerating axons branch when searching for a target and that when contact is made with the target this prevents additional branching and eliminates redundant branches. Myelinated axons are guided by existing Schwann cells, whereas unmyelinated axons do not follow predetermined pathways; this may explain their greater tendency to form permanent branches.     

Ganglioside synthesis and transport in regenerating sensory neurons of the rat sciatic nerve

The sciatic nerves of rats were crushed with fine forceps and allowed to survive for 3 or 7 days, at which time the 5th lumbar dorsal root ganglion was injected with [3H]glucosamine. Animals were killed 18 h later and the nerves proximal and distal to the crush site were cut into 3 mm segments. Gangliosides were purified from these segments, and radioactivity was separately measured in gangliosides, neutral glycolipids and glycoproteins. For all 3 fractions, radioactivity was distributed similarly between the crush site and point of maximum axonal elongation. A second smaller peak of ganglioside radioactivity was seen to span a few segments immediately distal to the point of maximum axonal elongation. We propose two possible explanations for this: (1) it represents ganglioside synthesis by Schwann cells (from blood-borne [3H]glucosamine) as part of the mitogenic response of these cells to the reappearance of axons; or (2) recently synthesized, transported gangliosides are released from the growth cone and taken up by adjacent mitogenic Schwann cells.     

Axonal degeneration and regeneration in sensory roots in a genital herpes model

Summary In a mouse model of genital herpes simplex virus type 2 (HSV-2) infection, roots of the lower spinal cord were examined 5 days to 6 months after inoculation. Using immunoperoxidase methods on paraffin sections, viral antigen was found in sensory ganglia, their proximal roots and distal nerves on days 5 and 6 after infection. In Epon sections, most mice had focal sensory root abnormalities in lower thoracic, lumbar or sacral levels. At days 7 and 10, lesions showed chiefly nerve fiber degeneration, particularly of large myelinated fibers. At 2 weeks, lesions contained relatively large bundles of small unmyelinated fibers with immature axon-Schwann cell relationships. From 3 to 6 weeks, lesions again contained many more small unmyelinated fibers than normal but, in increasing proportions, axons in bundles were isolated from their neighbors by Schwann cell cytoplasm, and Schwann cells having 11 relationships with axons showed mesaxon or thin myelin sheath formation. At later times, the proportion of small unmyelinated axons decreased in parallel with increased numbers of small myelinated axons. By 6 months, affected roots showed a relative reduction in large myelinated fibers, increased proportions of small myelinated fibers and Schwann cell nuclei. Numbers of unmyelinated fibers were reduced relative to 3- to 6-week lesions. Axonal degeneration and regeneration appears to be the chief pathological change in sensory roots in this model. If regenerated fibers arise from latently infected neurons, then establishment of latency is not a relatively silent event, but is associated with major long-lasting, morphologically detectable effects.