Near-terminus axonal structure and function following rat sciatic nerve regeneration through a collagen-GAG matrix in a ten-millimeter gap

The objectives of this study were to evaluate the regenerated axon structure at near-terminal locations in the peroneal and tibial branches 1 year following implantation of several tubular devices in a 10-mm gap in the adult rat sciatic nerve and to determine the extent of recovery of selected sensory and motor functions. The devices were collagen and silicone tubes implanted alone or filled with a porous collagen-glycosaminoglycan matrix. Intact contralateral nerves and autografts were used as controls. Nerves were retrieved at 30 and 60 weeks postoperatively for histological evaluation of the number and diameter of regenerated axons proximal and distal to the gap and in the tibial and peroneal nerve branches, near the termination point. Several functional evaluation methods were employed: gait analysis, pinch test, muscle circumference, and response to electrical stimulation. A notable finding was that the matrix-filled collagen tube group had a significantly greater number of large-diameter myelinated axons (> or =6 microm in diameter) in the distal nerve branches than any other group, including the autograft group. These results were consistent with previously reported electrophysiological measurements that showed that the action potential amplitude for the A fibers in the matrix-filled collagen tube group was greater than for the autograft control group. Functional testing revealed the existence of both sensory and motor recovery following peripheral nerve regeneration through all devices; however, the tests employed in this study did not show differences among the groups with regeneration. Electrical stimulation in vivo showed that threshold parameters to elicit muscle twitch were the same for reinnervating and control nerves. The investigation is of importance in showing for the first time the superiority of a specific fully resorbable off-the-shelf device over an autograft for bridging gaps in peripheral nerve, with respect to the near-terminus axonal structure.     

Transplantation of mesenchymal stem cells from human umbilical cord versus human umbilical cord blood for peripheral nerve regeneration

BACKGROUND: Mesenchymal stem cells (MSCs) appear to be a good alternative to Schwann cells in the treatment of peripheral nerve injury. Fetal stem cells, like umbilical cord blood (UCB) and umbilical cord (UC) stem cells, have several advantages over adult stem cells.OBJECTIVE: To assess the effects of UC-derived MSCs (UCMSCs) and UCB-derived MSCs (UCBMSCs) in repair of sciatic nerve defects. DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at the laboratory of Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital, from July to December 2009. MATERIALS: UCMSCs were provided by the Research Institute of Biotechnology, Dongguk University. UCBMSCs were provided by the Laboratory of Stem Cells and Tumor Biology, College of Veterinary Medicine, Seoul National University. Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco-BRL, USA. METHODS: Seven-week-old Sprague-Dawley rats were randomly and evenly divided into three groups: DMEM, UCBMSCs, and UCMSCs. A 10-mm defect in the left sciatic nerve was constructed in all rats. DMEM (15 μL) containing 1 × 106 UCBMSCs or UCMSCs was injected into the gap between nerve stumps, with the surrounding epineurium as a natural conduit. For the DMEM group, simple DMEM was injected. MAIN OUTCOME MEASURES: At 7 weeks after sciatic nerve dissection, dorsal root ganglia neurons were labeled by fluorogold retrograde labeling. At 8 weeks, electrophysiology and histomorphometry were performed. At 2, 4, 6, and 8 weeks after surgery, sciatic nerve function was evaluated using gait analysis.RESULTS: The UCBMSCs group and the UCMSCs group exhibited similar sciatic nerve function and electrophysiological indices, which were better than the DMEM group, as measured by gait analysis (P < 0.05). Fluorogold retrograde labeling of sciatic nerve revealed that the UCBMSCs group demonstrated a higher number of labeled neurons; however, the differences were not significant. Histomorphometric indices were similar in the UCBMSCs and UCMSCs groups, and total axon counts, particularly axon density (P < 0.05), were significantly greater in the UCBMSCs and UCMSCs groups than in the DMEM group. CONCLUSION: Transplanting either UCBMSCs or UCMSCs into axotomized sciatic nerves could accelerate and promote sciatic nerve regeneration over 8 weeks. Both treatments had similar effects on nerve regeneration.     

Human amnion tissue injected with human umbilical cord mesenchymal stem cells repairs damaged sciatic nerves in rats

Human umbilical cord mesenchymal stem cells,incorporated into an amnion carrier tubes,were assessed for nerve regeneration potential in a rat nerve defect model.Damaged nerves were exposed to human amnion carriers containing either human umbilical cord mesenchymal stem cell (cell transplantation group)or saline(control group).At 8,12,16 and 20 weeks after cell implantation,the sciatic functional index was higher in the cell transplantation group compared with the control group.Furthermore,electrophysiological examination showed that threshold stimulus and maximum stimulus intensity gradually decreased while compound action potential amplitude gradually increased.Hematoxylin-eosin staining showed that regenerating nerve fibers were arranged in nerve tracts in the cell transplantation group and connective tissue between nerve tracts and amnion tissue reduced over time.Gastrocnemius muscle cell diameter,wet weight and restoration ratio were increased.These data indicate that transplanted human umbilical cord mesenchymal stem cells,using the amnion tube connection method,promote restoration of damaged sciatic nerves in rats.     

A novel chronic nerve compression model in the rat

Current animal models of chronic peripheral nerve compression are mainly silicone tube models. However, the cross section of the rat sciatic nerve is not a perfect circle, and there are differences in the diameter of the sciatic nerve due to individual differences. The use of a silicone tube with a uniform internal diameter may not provide a reliable and consistent model. We have established a chronic sciatic nerve compression model that can induce demyelination of the sciatic nerve and lead to atrophy of skeletal muscle. In 3-week-old pups and adult rats, the sciatic nerve of the right hind limb was exposed, and a piece of surgical latex glove was gently placed under the nerve. N-butyl-cyanoacrylate was then placed over the nerve, and after it had set, another piece of glove latex was placed on top of the target area and allowed to adhere to the first piece to form a sandwich-like complex. Thus, a chronic sciatic nerve compression model was produced. Control pups with latex or N-butyl-cyanoacrylate were also prepared. Functional changes to nerves were assessed using the hot plate test and electromyography. Immunofluorescence and electron microscopy analyses of the nerves were performed to quantify the degree of neuropathological change. Masson staining was conducted to assess the degree of fibrosis in the gastrocnemius and intrinsic paw muscles. The pup group rats subjected to nerve compression displayed thermal hypoesthesia and a gradual decrease in nerve conduction velocity at 2 weeks after surgery. Neuropathological studies demonstrated that the model caused nerve demyelination and axonal irregularities and triggered collagen deposition in the epineurium and perineurium of the affected nerve at 8 weeks after surgery. The degree of fibrosis in the gastrocnemius and intrinsic paw muscles was significantly increased at 20 weeks after surgery. In conclusion, our novel model can reproduce the functional and histological changes of chronic nerve compression injury that occurs in humans and it will be a useful new tool for investigating the mechanisms underlying chronic nerve compression.     

Myelinated fiber regeneration after crush injury is retarded in sciatic nerves of aging mice

To compare nerve regeneration in young adult and aging mice, the right sciatic nerves of 6- and 24-month-old mice were crushed at the sciatic notch. Two weeks later, both groups of mice were perfused with an aldehyde solution, and, after additional fixation, the sciatic nerves were processed so that the transverse sections of each nerve subsequently studied by light and electron microscopy included the entire posterior tibial fascicle 5 mm distal to the crush site. The same level was sectioned in unoperated contralateral nerves; these nerves served as controls. Electron micrographs and the Bioquant Image Analysis System IV were used to measure areas of posterior tibial fascicles and count the number of myelinated axons, the number of unmyelinated axons, and their frequency in Schwann cell units. In aging mice, the total number of regenerating myelinated axons was significantly reduced, but totals of regenerating unmyelinated axons in aging and young adults did not differ significantly. In aging mice, the frequency of Schwann cells that contained a single unmyelinated axon was greater, suggesting that before myelination began, Schwann cell ensheathment of axons also was slowed. After axotomy by a crush injury, the area of the posterior tibial fascicle was less than that in young adults and the distal disintegration of myelin sheath remnants also appeared to be retarded. The results indicate that responses of neurons, axons, and Schwann cells could be important in slowing the regeneration of myelinated fibers found in sciatic nerves from aging mice.     

Skin-derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection

The regeneration in the peripheral nervous system is often incomplete and the treatment of severe lesions with nerve tissue loss is primarily aimed at recreating nerve continuity. Guide tubes of various types, filled with Schwann cells, stem cells, or nerve growth factors are attractive as an alternative therapy to nerve grafts. In this study, we evaluated whether skin-derived stem cells (SDSCs) can improve peripheral nerve regeneration after transplantation into nerve guides. We compared peripheral nerve regeneration in adult rats with sciatic nerve gaps of 16 mm after autologous transplantation of GFP-labeled SDSCs into two different types of guides: a synthetic guide, obtained by dip coating with a L-lactide and trimethylene carbonate (PLA-TMC) copolymer and a collagen-based guide. The sciatic function index and the recovery rates of the compound muscle action potential were significantly higher in the animals that received SDSCs transplantation, in particular, into the collagen guide, compared to the control guides filled only with PBS. For these guides the morphological and immunohistochemical analysis demonstrated an increased number of myelinated axons expressing S100 and Neurofilament 70, suggesting the presence of regenerating nerve fibers along the gap. GFP positive cells were found around regenerating nerve fibers and few of them were positive for the expression of glial markers as S-100 and glial fibrillary acidic protein. RT-PCR analysis confirmed the expression of S100 and myelin basic protein in the animals treated with the collagen guide filled with SDSCs. These data support the hypothesis that SDSCs could represent a tool for future cell therapy applications in peripheral nerve regeneration.     

Peripheral nerve regeneration is delayed in neuropilin 2-deficient mice

Peripheral nerve transection or crush induces expression of class 3 semaphorins by epineurial and perineurial cells at the injury site and of the neuropilins neuropilin-1 and neuropilin-2 by Schwann and perineurial cells in the nerve segment distal to the injury. Neuropilin-dependent class 3 semaphorin signaling guides axons during neural development, but the significance of this signaling system for regeneration of adult peripheral nerves is not known. To test the hypothesis that neuropilin-2 facilitates peripheral-nerve axonal regeneration, we crushed sciatic nerves of adult neuropilin-2-deficient and littermate control mice. Axonal regeneration through the crush site and into the distal nerve segment, repression by the regenerating axons of Schwann cell p75 neurotrophin receptor expression, remyelination of the regenerating axons, and recovery of normal gait were all significantly slower in the neuropilin-2-deficient mice than in the control mice. Thus, neuropilin-2 facilitates peripheral-nerve axonal regeneration.     

Diabetic neuropathy: Electrophysiological and morphological study of peripheral nerve degeneration and regeneration in transgenic mice that express IFNβ in β cells

Diabetic neuropathy is one of the most frequent complications in diabetes but there are no treatments beyond glucose control, due in part to the lack of an appropriate animal model to assess an effective therapy. This study was undertaken to characterize the degenerative and regenerative responses of peripheral nerves after induced sciatic nerve damage in transgenic rat insulin I promoter / human interferon beta (RIP/IFNβ) mice made diabetic with a low dose of streptozotocin (STZ) as an animal model of diabetic complications. In vivo, histological and immunohistological studies of cutaneous and sciatic nerves were performed after left sciatic crush. Functional tests, cutaneous innervation, and sciatic nerve evaluation showed pronounced neurological reduction in all groups 2 weeks after crush. All animals showed a gradual recovery but this was markedly slower in diabetic animals in comparison with normoglycemic animals. The delay in regeneration in diabetic RIP/IFNβ mice resulted in an increase in active Schwann cells and regenerating neurites 8 weeks after surgery. These findings indicate that diabetic‐RIP/IFNβ animals mimic human diabetic neuropathy. Moreover, when these animals are submitted to nerve crush they have substantial deficits in nerve regrowth, similar to that observed in diabetic patients. When wildtype animals were treated with the same dose of STZ, no differences were observed with respect to nontreated animals, indicating that low doses of STZ and the transgene are not implicated in development of the degenerative and regenerative events observed in our study. All these findings indicate that RIP/IFNβ transgenic mice are a good model for diabetic neuropathy. Muscle Nerve, 2010     

17β-Estradiol stimulates regeneration of sciatic nerve in female mice

In ovariectomized mice with and without estrogen replacement, regeneration of the sciatic nerve after crush injury was studied. Functional recovery, quantified with sciatic functional index was significantly accelerated in estrogen-treated mice throughout the regeneration. On semi-thin sections of sciatic nerves in estrogen-treated mice we registered a greater total number of regenerating nerve fibers at the first week, and a higher mean axonal area at the third week of regeneration. Our results demonstrated that estrogen treatment enhances regeneration of the sciatic nerve.     

17Beta-estradiol stimulates regeneration of sciatic nerve in female mice

In ovariectomized mice with and without estrogen replacement, regeneration of the sciatic nerve after crush injury was studied. Functional recovery, quantified with sciatic functional index was significantly accelerated in estrogen-treated mice throughout the regeneration. On semi-thin sections of sciatic nerves in estrogen-treated mice we registered a greater total number of regenerating nerve fibers at the first week, and a higher mean axonal area at the third week of regeneration. Our results demonstrated that estrogen treatment enhances regeneration of the sciatic nerve.     

Initial upregulation of growth factors and inflammatory mediators during nerve regeneration in the presence of cell adhesive peptide-incorporated collagen tubes

Neurotrophic factors play an important modulatory role in axonal sprouting during nerve regeneration involving the proliferation of hematogenous and Schwann cells in damaged tissue. We have exposed lesioned sciatic nerves to a collagen prosthesis with covalently bonded small cell adhesive peptides Arg-Gly-Asp-Ser (RGDS), Lys-Arg-Asp-Ser (KRDS), and Gly-His-Lys (GHK) to study local production of growth factors and cytokines in the regenerating tissues. Western/enzyme-linked immunosorbent assay (ELISA) studies were performed after 10 days of regeneration, when the tubular prosthesis is filled with fibrous matrix infiltrated by hematogenous cells and proliferating Schwann cells with growth factors produced locally. Regeneration was also analyzed by morphometrical methods after 30 days. The quantification of growth factors and proteins by ELISA revealed that there was an enhanced expression of the neurotrophic factors nerve growth factor (NGF) and neurotrophins (NT-3 and NT-4) in the regenerating tissues. This was further established by Western blot to qualitatively analyze the presence of the antigens in the regenerating tissues. Schwann cells were localized in the regenerating tissues using antibodies against S-100 protein. Other growth factors including growth-associated protein 43 (GAP-43), apolipoprotein E (Apo E), and pro-inflammatory cytokine like interleukin-1alpha (IL-1alpha) expression in the peptide groups were evaluated by ELISA and confirmed by Western blotting. Cell adhesive integrins in the proliferating cells were localized using integrin-alpha V. The combined results suggest that the early phase of regeneration of peripheral nerves in the presence of peptide-incorporated collagen tubes results in the enhanced production of trophic factors by the recruited hematogenous cells and Schwann cells, which in turn help in the secretion of certain vital trophic and tropic factors essential for early regeneration. Furthermore, hematogenous cells recruited within the first 10 days of regeneration help in the production of inflammatory mediators like interleukins that in turn stimulate Schwann cells to produce NGF for axonal growth.     

FK506 enhances regeneration of axons across long peripheral nerve gaps repaired with collagen guides seeded with allogeneic Schwann cells

We assessed the effects of FK506 administration on regeneration after a 6-mm gap repair with a collagen guide seeded with allogeneic Schwann cells (SCs) in the mouse sciatic nerve. SCs were isolated from predegenerated adult sciatic nerves and expanded in culture using a defined medium, before being seeded in the collagen guide embedded in Matrigel. Functional reinnervation was evaluated by noninvasive methods to determine recovery of motor, sensory, and autonomic functions in the hindpaw over 4 months postoperation. Histological analysis of the regenerated nerves was performed at the end of the study. Using simple collagen guides for tubulization repair, treatment with an immunosuppressant dose of FK506 (5 mg/kg/day) resulted in significant improvement of the onset and the degree of reinnervation. While the introduction of allogeneic SCs did not improve regeneration versus a collagen guide filled only with Matrigel, treatment with FK506 allowed for successful regeneration in all the mice and for significant improvement in the levels of functional recovery. Compared with the untreated group, there was greater survival of transplanted pre-labeled SCs in the FK506-treated animals. Morphologically, the best nerve regeneration (in terms of nerve caliber and numbers of myelinated axons) was obtained with SC-seeded guides from FK506-treated animals. Thus, FK506 should be considered as adjunct therapy for various types of tubulization repair.     

Glia maturation factor beta stimulates axon regeneration in transected rat sciatic nerve.

Rat sciatic nerves were bilaterally transected and repaired with an entubulation technique. The nerve interstump gap was filled with either collagen gel or collagen gel mixed with a putative neurotrophic factor (leupeptin, 4-aminopyridine, lipid angiogenic factor or glia maturation factor beta (GMF-beta]. Six weeks after nerve transection, the myelinated distal stump axons were quantified for each nerve. Only the nerves treated with GMF-beta had significantly more axons than the control side.     

Peripheral nerve regeneration along collagen filaments

This paper describes the regeneration of severed peripheral nerve axons along collagen filaments without a tube. Two thousand collagen filaments were grafted to bridge 20 mm defects of rat sciatic nerve. The number of myelinated axons was approximately 4800 in the distal end of the nerve autograft at 8 weeks postoperatively; while in the collagen-filaments nerve guide it was 5500. The results suggested the collagen filaments guided regenerating axons effectively.     

Nerve regeneration across a 25-mm gap bridged by a polyglycolic acid-collagen tube: a histological and electrophysiological evaluation of regenerated nerves

In the study reported here we have examined the nerve regeneration that occurs over a 25-mm gap using a novel biodegradable nerve guide tube. The tube was a composite of polyglycolic acid (PGA) mesh coated with collagen which was filled with neurotrophic factors. The left sciatic nerve of ten adult cats was dissected. The stumps were connected by the tube, and fixed gap. Histological examinations carried out 4–16 months after implantation of the tube revealed regeneration of well vascularized nerve tissue. Regeneration of both myelinated, unmyelinated axons and Schwann cells was confirmed by electron microscopy 5 months after surgery. Following injection of horseradish peroxidase (HRP) into a site peripheral to the regenerated segment of the sciatic nerves, motoneurons in the ventral horn of the spinal cord, afferent terminals in the medial portion of the dorsal column of the medulla oblongata, and sensory afferent nerve terminals in the dorsal horn of the spinal cord were labelled. Electrophysiological examinations revealed restoration of evoked electromyograms and sensory evoked potentials (SEPs) recorded from the cerebral cortex as well as the spinal cord. We also found that some of the regenerated motor axons exhibited branching in the regenerated segments. In two cases, a single motoneuronal axon from the regenerated side projected to both flexors and extensors, simultaneously. Our results indicate that the PGA-collagen composite tube is a promising tool for use as a nerve guide tube in peripheral nerve regeneration.     

Comparison of fibrin glue, bioresorbable tubing and sutures in peripheral nerve repair

Regeneration of severed rat tibial nerves was functionally and morphologically compared with repair following the use of 3 anastomosis techniques: collagen guide tubes, fibrin glue and conventional microsurgical sutures. In addition, one tibial nerve was crushed in some rats. At ten weekly intervals, functional recovery, assessed by sciatic nerve stimulated evoked contraction of the flexor digitorum muscle, was quicker and more complete following nerve crush than following the anastomosis techniques which were not different from each other. Ten weeks following the surgery, the retrograde transport morphological technique indicated that the anastomosis techniques were not different from each other. The number of labeled tibial motoneurons (tube and suture groups) was significantly less than the crush group, but the glue group was intermediate. Thus, although having less extensive recovery following crush, the quicker and easier techniques of nerve repair, i.e., collagen tubes or fibrin glue, produced comparable anatomical and functional recovery as the more time-consuming, technically demanding microsurgical repair with fine sutures.     

Skeletal muscle-derived cells repair peripheral nerve defects in mice

Skeletal muscle-derived cells have strong secretory function,while skeletal muscle-derived stem cells,which are included in muscle-derived cells,can differentiate into Schwann cell-like cells and other cell types.However,the effect of muscle-derived cells on peripheral nerve defects has not been reported.In this study,5-mm-long nerve defects were created in the right sciatic nerves of mice to construct a peripheral nerve defect model.Adult female C57BL/6 mice were randomly divided into four groups.For the muscle-derived cell group,muscle-derived cells were injected into the catheter after the cut nerve ends were bridged with a polyurethane catheter.For external oblique muscle-fabricated nerve conduit and polyurethane groups,an external oblique muscle-fabricated nerve conduit or polyurethane catheter was used to bridge the cut nerve ends,respectively.For the sham group,the sciatic nerves on the right side were separated but not excised.At 8 and 12 weeks post-surgery,distributions of axons and myelin sheaths were observed,and the nerve diameter was calculated using immunofluorescence staining.The number,diameter,and thickness of myelinated nerve fibers were detected by toluidine blue staining and transmission electron microscopy.Muscle fiber area ratios were calculated by Masson’s trichrome staining of gastrocnemius muscle sections.Sciatic functional index was recorded using walking footprint analysis at 4,8,and 12 weeks after operation.The results showed that,at 8 and 12 weeks after surgery,myelin sheaths and axons of regenerating nerves were evenly distributed in the muscle-derived cell group.The number,diameter,and myelin sheath thickness of myelinated nerve fibers,as well as gastrocnemius muscle wet weight and muscle area ratio,were significantly higher in the muscle-derived cell group compared with the polyurethane group.At 4,8,and 12 weeks post-surgery,sciatic functional index was notably increased in the muscle-derived cell group compared with the polyurethane group.These criteria of the muscle-derived cell group were not significantly different from the external oblique muscle-fabricated nerve conduit group.Collectively,these data suggest that muscle-derived cells effectively accelerated peripheral nerve regeneration.This study was approved by the Animal Ethics Committee of Plastic Surgery Hospital,Chinese Academy of Medical Sciences(approval No.040)on September 28,2016.     

Entubulation repair with protein additives increases the maximum nerve gap distance successfully bridged with tubular prostheses

The major objective of the experiments reported in this paper was to test the hypothesis that the maximum distance that peripheral nervous system (PNS) axons can regenerate through a tubular prosthesis may be increased by specific modifications to the internal environment of the prosthesis. The sciatic nerve of adult male rats was transected and proximal and distal nerve stumps were sutured into a silicone tube 20-25 mm in length. The silicone tubes were implanted empty, or the lumen was filled with collagen or a laminin-containing gel. Following 4-16 weeks survival time animals were sacrificed and the contents of the silicone tubes were processed for histological identification of myelinated and unmyelinated axons. All of the tubes with additives, but one of the initially empty tubes, displayed a regenerated nerve cable within the tube. Retrograde labeling studies were carried out to prove that some of the axons present in the regenerated nerve cables arose from primary motor and sensory neurons. These results show that specific modifications to the microenvironment of regenerating PNS axons can affect the success or failure of tubular prostheses for nerve repair.     

Magnetically Aligned Collagen Gel Filling a Collagen Nerve Guide Improves Peripheral Nerve Regeneration

Bioresorbable collagen nerve guides filled with either magnetically aligned type I collagen gel or control collagen gel were implanted into 4- or 6-mm surgical gaps created in the sciatic nerve of mice and explanted 30 and 60 days postoperation (dpo) for histological and immunohistochemical evaluation. The hypothesis was that contact guidance of regenerating axons and/or invading nonneuronal cells to the longitudinally aligned collagen fibrils would improve nerve regeneration. The criterion for regeneration was observation of regenerating myelinated fibers distal to the nerve guide. Consistent with previous studies showing poor regeneration in 6-mm gaps at 60 dpo with entubulation repair, only one of six mice exhibited regeneration with control collagen gel. In contrast, four of four mice exhibited regeneration with magnetically aligned collagen gel, including the appearance of nerve fascicle formation. The numbers of myelinated fibers were less than the uninjured nerve in all groups, however, which may have been due to rapid resorption of the nerve guides. An attempt to increase the stability of the collagen gel, and thereby the directional information presented by the aligned collagen fibrils, by crosslinking the collagen with ribose before implantation proved detrimental for regeneration.     

Abnormal nerve regeneration in galactose neuropathy

Nerve regeneration across a 10 mm gap through an implanted silicone tube was delayed in galactose-fed rats two and four weeks after transecting the nerve. This experimental metabolic neuropathy resembles diabetic neuropathy in which nerve regeneration is also delayed. Experiments were performed by introducing opposite ends of divided sciatic nerves into close-fitting silicone tubes, leaving a 10 mm gap. Growth of neurites across this gap was monitored by electron microscopy performed in sections at regular intervals of 2 mm from proximal to distal stumps. After two weeks some difference was apparent; axons advanced 1.4 +/- 0.4 mm in galactose-fed rats versus 3.5 +/- 1.5 mm in controls. Myelination did not progress beyond 1 mm in galactose-fed rats. Differences were greater between the two groups at four weeks. The growth of axons in galactose rats was 3.5 +/- 0.2 mm versus 9.4 +/- 0.1 mm in control nerves. In addition the size of the regenerating stump was much greater in control rats. Qualitative differences were also noticed during electron microscopic comparison of control and galactose-treated rats. The dystrophic axons seen in treated rats had abnormal electron-dense organelles, lamellated bodies, vesicles and tubular structures, as well as numerous glycogen granules. Abnormalities of spatial orientation were also noted. Unlike control axons which grew parallel with the long axis of the tube, regenerating axons in experimental animals were seen deviating from the axis at 90 degrees angles. Both immature sprouts and myelinating axons showed abnormal plasticity. Ultrastructural differences were also noted in Schwann cells, macrophages and vessels.