Wallerian degeneration of peripheral nerve. Age-dependent loss of nerve lipids

The age-dependent loss of the major peripheral nerve lipids (cholesterol, phospholipid, and total galactolipid) was quantitated over a period of 9 weeks of Wallerian degeneration induced by surgical transection of rabbit sciatic nerves in animals of several ages. Proportionate losses of these lipids were determined by calculating the content of each lipid on a per nerve and on a per gram fresh weight basis remaining after a given period of Wallerian degeneration as a percent of original normal values at several time following surgery. The proportionate loss of each lipid from the distal stump was the most prompt and the most complete in nerves transected at 2 weeks of age, and the least in nerves transected at 20 weeks of age. The prompter clearance of these lipids from younger than older degenerating nerve gives convincing evidence that the suggestion from light-microscopic studies of faster clearance of neural debris in younger than in older animals is correct. A possible relationship between these biochemical findings and the phenomenon of greater functional recovery from peripheral nerve injury in younger than in older subjects is discussed.     

Antibody to transforming growth factor beta reduces collagen production in injured peripheral nerve

Epineurial scarring in peripheral nerve after injury inhibits normal axonal regeneration primarily due to fibroblast deposition of type I collagen. The transforming growth factor beta (TGF-beta) family is an important class of signaling molecules that has been shown to stimulate fibroblasts to produce collagen. The aim of this study was to design a prototypic therapeutic system in which the neutralization of TGF-beta in crushed rat sciatic nerve would decrease collagen formation. A total of 45 experimental Lewis rats were used. Group 1 animals (20 rats) sustained a unilateral crush injury to the sciatic nerve with injection of phosphate buffer solution. Group 2 animals (20 rats) sustained a unilateral crush injury to the sciatic nerve with injection of phosphate-buffered saline and goat, anti-rat, panspecific TGF-beta antibody. Group 3 control animals (five rats) underwent only exposure of sciatic nerve with injection of antibody. All animals were killed at 14 days and sciatic nerve specimens were harvested at that time. Slides of experimental tissue were processed using a 35S-labeled oligomer for procollagen alpha-1 mRNA, then dipped in photographic emulsion and examined by darkfield autoradiography. Morphometric analysis of pixel counts was then performed. A significant reduction in total pixel count per high-power field and in total number of fibroblasts per high-power field was found in crushed rat sciatic nerve treated with anti-TGF-beta antibody when compared with those treated only with phosphate-buffered saline. These findings are consistent with successful reduction in procollagen induction after a crush injury by topical administration of blocking antibody against transforming growth factor beta. The concept of growth factor blockade for therapeutic collagen reduction is attractive in the context of nerve injury, and the current article provides a model for future clinical application.     

Muscle reinnervation following neonatal nerve crush. Interactive effects of glycosaminoglycans and insulin-like growth factor-I

This study shows that glycosaminoglycans promote muscle reinnervation following neonatal sciatic nerve injury. Such an effect appears to be mediated by insulin-like growth factor-1. The glycosaminoglycan moiety of proteoglycans is a constituent of the basal lamina active on nerve regeneration by means of the interaction with laminin and with several growth factors. We have previously shown that supplementation of glycosaminoglycans affects neuronal degeneration and regeneration. In this study we report that following neonatal lesion of the rat sciatic nerve glycosaminoglycan treatment promoted extensor digitorum longus muscle reinnervation with consequent improvement of muscle morphology. In saline-treated rats, reinnervation was only partial and there was a marked muscle fibre atrophy. In addition glycosaminoglycan treatment of lesioned rats increased insulin-like growth factor-I messenger RNA and protein in the reinnervated muscle, and insulin-like growth factor-I and insulin-like growth factor binding protein-3 plasma levels. Similarly, treatment of nerve lesioned rats with insulin-like growth factor-I promoted muscle reinnervation and prevention of muscle fibre atrophy, higher levels of insulin-like growth factor-I in the reinnervated muscle and of insulin-like growth factor-I and insulin-like growth factor binding proteins in plasma. These data suggest that glycosaminoglycans are potent stimulants of muscle reinnervation and that their effects may be mediated by increased levels of insulin-like growth factor-I.     

Myelinated sensory and alpha motor axon regeneration in peripheral nerve neuromas

Histochemical staining for carbonic anhydrase and cholinesterase (CE) activities was used to analyze sensory and motor axon regeneration, respectively, during neuroma formation in transected and tube-encapsulated peripheral nerves. Median-ulnar and sciatic nerves in the rodent model permitted testing whether a 4 cm greater distance of the motor neuron soma from axotomy site or intrinsic differences between motor and sensory neurons influenced regeneration and neuroma formation 10, 30, and 90 days later. Ventral root radiculotomy confirmed that CE-stained axons were 97% alpha motor axons. Distance significantly delayed axon regeneration. When distance was negligible, sensory axons grew out sooner than motor axons, but motor axons regenerated to a greater quantity. These results indicate regeneration differences between axon subtypes and suggest more extensive branching of motor axons within the neuroma. Thus, both distance from injury site to soma and inherent motor and sensory differences should be considered in peripheral nerve repair strategies.     

Blood flow in mobilized nerves: results in a rabbit sciatic nerve model

The purpose of this study was to evaluate the immediate effect on capillary blood flow of surgical mobilization of 15 cm of the rabbit sciatic and tibial nerve. Capillary nerve blood flow was determined with 16-micron radioactive microspheres. Thirty-seven rabbits were divided into six groups. In the control group A (n = 7), the in situ nerve blood flow was determined. In group B (n = 7), the nerve was mobilized, leaving only the proximal and distal endoneurial vascular supplies; blood flow increased compared with in situ values in most segments. In group C (n = 5), the nerve was mobilized as in group B, but also transected distally; blood flow was markedly decreased in the distal segments but was maintained up to a diameter-to-length ratio of 1:63. In group D (n = 6), the nerve was not mobilized, but was transected proximally and distally; blood flow increased in all segments. In group E (n = 5), the nerve was mobilized and transected proximally and distally, leaving only nerve branches intact; blood flow was significantly higher in segments of the nerve from which nerve branches originated with the nerve receiving blood flow through its branches. In group F (n = 6), the nerve was mobilized, all extrinsic vessels except one were transected, and the proximal and distal nerve was transected; flow was maintained to a diameter:length ratio of 1:41 from the source of blood flow. Therefore, it does appear that long lengths of nerve may be mobilized and transposed while maintaining sufficient blood flow.     

Fracture healing and callus innervation after peripheral nerve resection in rats

The effects of femoral and sciatic nerve resection on fracture healing and innervation of the fracture callus were studied using a stable fracture model. In 34 rats the right tibia was subjected to a standardized closed fracture and stabilized with a modular intramedullary nail. In half of the animals, resection of 1 cm of the femoral and sciatic nerves was performed (nerve resection group), whereas the other animals had sham operations (sham group). To avoid unequal load-bearing between the two groups, all fractured hindlimbs were immobilized in a plaster of Paris cast. The trial was terminated after 5 weeks of fracture healing. Callus size was scored radiographically, and bone mineralization was measured by 85-strontium incorporation. Seven rats from each group had immunohistochemical examination for neural regeneration and ingrowth. Antisera for protein gene product 9.5, neurofilaments, neural growth associated protein 43/B-50, calcitonin gene related peptide, and substance P were used. The mechanical properties of the healing fractures were recorded in a three-point cantilever bending test. After 5 weeks, the normally innervated, fractured tibias had regained approximately 50% strength compared with the unfractured side, in comparison with only 20% in the animals that had nerve resection. Although the fracture calluses were mechanically weaker, they were significantly larger in the nerve resection group, indicating defects in tissue composition or organization rendered by the nerve injury. The mineralization rate, as measured by 85-strontium incorporation, was the same in the two groups. However, the nerve resection did not provide complete denervation but changed the innervation pattern of the healing fracture, as the density of sensory nerve fibers immunostaining for substance P and neurofilaments was less in the group with femoral and sciatic nerve resection. The results suggest that intact innervation is essential for normal fracture healing because nerve injury induced a large, but mechanically insufficient, fracture callus.     

Induction of axon-like processes from axotomized retinal ganglion cells of adult hamsters after intravitreal injection of sciatic nerve exudate

We have shown previously that intravitreous transplantation of a peripheral nerve (PN) graft stimulates a subpopulation of retinal ganglion cells (RGCs) to sprout axon-like processes. The present study examined whether the effect of PN graft was due to diffusible factors released from the graft. Injection of sciatic nerve exudate into vitreous of the eye induced the formation of axon-like processes. Significantly more RGCs with axon-like processes were stained after injection of exudate from the distal stump than the proximal stump of the transected sciatic nerve. Thus, our results showed that trophic factors released from the intravitreous PN can induce the formation of axon-like processes and that more trophic factors are secreted from the distal than the proximal stump of the transected sciatic nerve.     

Effects found in a one-year oral toxicity study in Sprague-Dawley rats of a novel 5-HT1A receptor partial agonist for the treatment of anxiety in humans

Regeneration of motor axons is enhanced if they have sprouted prior to nerve injury. We examined whether sensory axon regeneration and recovery of pain response was affected by previous collateral sprouting. In the experimental group of rats, the right saphenous, tibial, and sural nerves were transected and ligated. The peroneal nerve was left to sprout into the adjacent denervated skin. Two months later, the axons of the peroneal nerve were crushed in the sciatic nerve. In the control group, the right sciatic nerve was crushed at the same time that the saphenous, tibial, and sural nerves were transected. Recovery of pain response in the foot was determined by the skin pinch test. Sensory axon elongation rate was measured by the nerve pinch test. The number of myelinated axons was determined in nerve cross sections stained by Azur blue. Recovery of pain sensitivity in the animals of the experimental group was delayed for 2-3 weeks in comparison to the control group. Moreover, the spatial pattern of pain response in the experimental group was irregular, displaying residual regions of insensitive skin which were not present in controls. The elongation rate of regenerating sensory axons in the experimental group was not decreased, and the number of myelinated axons in the peroneal nerves was even about 10% higher than in the control group. Therefore, we assume that the terminal arborization of the neurilemmal tubes pertaining to the former axon sprouts delayed regrowth of sensory axon terminals in the skin.     

Application of sciatic functional index in nerve functional assessment

In order to confirm the reliability of the sciatic functional index (SFI) in the rat, SFI, muscle strength, electrophysiological, and morphometric assessments were carried out from the 10th day to the sixth month after nerve injury or repair. The results showed that the SFI has a positive correlation with all tested indices of muscle strength, electrophysiology, and morphology (r = 0.925-0.996, P < 0.01 or P < 0.001). These results indicate that the SFI is a reliable index for evaluating rat sciatic nerve regeneration and can be widely used.     

Neurotoxic effects of sodium nitroprusside in rat hippocampal slices

The effect of a permanent transection on myelin gene expression in a regenerating sciatic nerve and in an adult sciatic nerve was compared to establish the degree of axonal control exerted upon Schwann cells in each population. First, the adult sciatic nerve was crushed, and the distal segment allowed to regenerate. At 12 days post-crush, the sciatic nerve was transected distal to the site of crush to disrupt the Schwann cell-axonal contacts that had reformed. Messenger RNA (mRNA) levels coding for five myelin proteins were assayed in the distal segment of the crush-transected nerve after 9 days and were compared to corresponding levels in the distal segments of sciatic nerves at 21 days post-crush and 21 days post-transection using Northern blot and slot-blot analysis. Levels of mRNAs found in the distal segment of the transected and crush-transected nerve suggested that Schwann cells in the regenerating nerve and in the mature adult nerve are equally responsive to axonal influences. The crush-transected model allowed the genes that were studied to be classified according to their response to Schwann cell-axonal contact. The levels of mRNAs were 1) down-regulated to basal levels (P0 and MBP mRNAs), 2) down-regulated to undetectable levels (myelin-associated glycoprotein mRNAs), 3) upregulated (mRNAs encoding 2'3'-cyclic nucleotide phosphodiesterase and beta-actin), or 4) not stringently controlled by the removal of Schwann cell-axonal contact (proteolipid protein mRNAs). This novel experimental model has thus provided evidence that the expression of some of the important myelin genes during peripheral nerve regeneration is dependent on continuous signals from the ingrowing axons.     

Prolonged intercostal nerve blockade in sheep using controlled-release of bupivacaine and dexamethasone from polymer microspheres

BACKGROUND: Previous work from the authors' group characterized a prolonged percutaneous blockade of the sciatic nerve in rats using bupivacaine-dexamethasone microspheres. The goals of the current study are to examine the (1) efficacy of bupivacaine microspheres with and without dexamethasone for intercostal blockade in sheep; (2) scaling of dose and duration with a 100-fold increase in body size from rats to sheep; (3) local toxicity and adverse systemic reactions to bupivacaine microspheres with and without dexamethasone. METHODS: Intercostal blocks were performed percutaneously in sedated sheep. Sensory blockade was measured at repeated time points by absent flinch response to skin pinch. Plasma bupivacaine concentrations were measured using high performance liquid chromatography. Chest wall specimens were examined by light microscopy. RESULTS: The duration of intercostal blockade increased with bupivacaine dose for animals receiving from 8 to 80 mg/kg of microspheres with and without dexamethasone. At each dose, microspheres containing dexamethasone had a longer duration of block than microspheres without dexamethasone. From 8 to 80 mg/kg, the mean duration of block with bupivacaine-dexamethasone microspheres increased from 4 to 13 days. Plasma concentrations of bupivacaine remained 10-fold below the convulsive EC50 concentration for sheep. Chest wall histology showed a significant granulomatous reaction around bupivacaine microspheres but not around bupivacaine-dexamethasone microspheres. CONCLUSIONS: A single administration of bupivacaine-dexamethasone microspheres produces an effective chest wall analgesia of several days' duration. This may prove useful clinically for thoracic surgery or trauma.     

Comparative dose-dependence study of FK506 and cyclosporin A on the rate of axonal regeneration in the rat sciatic nerve

The new immunosuppressant drug FK506 (Tacrolimus) increases the rate of nerve regeneration in vivo (Gold et al., 1994; Gold et al., 1995). In the present study, we have examined the dose-dependence of FK506's ability to enhance nerve regeneration. In the first set of experiments, rats received daily s.c. injections of FK506 (2 mg/kg, 5 mg/kg or 10 mg/kg) for 18 days after a sciatic nerve crush injury. Signs of functional recovery in the hind feet appeared earlier than in saline-treated control rats at all three FK506 dosage; recovery was maximally accelerated in the 5-mg/kg group. Light microscopy at 18 days after nerve crush revealed more regenerating myelinated fibers in FK506-treated rats than in controls; this was most apparent in the 5-mg/kg group. Morphometric analysis of axonal areas in the soleus nerve confirmed that axonal calibers were maximally increased in the 5-mg/kg group. In the second set of experiments, the rate of axonal regeneration was determined by radiolabeling the L5 dorsal root ganglion. Regeneration rate for sensory axons was maximally increased (by 34%) in the 5-mg/kg group. In contrast, cyclosporin A (10 or 50 mg/kg; dosages were selected on the basis of the 1/10 lower potency of cyclosporin A) did not significantly alter the rate of axonal regeneration. Cyclosporin A (50 mg/kg) also failed to increase functional recovery or axonal calibers in the soleus nerve. Because the two drugs share a common mechanism for producing immunosuppression (i.e., calcineurin inhibition), these results indicate that FK506's nerve regenerative property involves a distinct, calcineurin-independent mechanism.     

Fish oil supplementation prevents diabetes-induced nerve conduction velocity and neuroanatomical changes in rats

Diabetic neuropathy has been associated with a decrease in nerve conduction velocity, Na,K-ATPase activity and characteristic histological damage of the sciatic nerve. The aim of this study was to evaluate the potential effect of a dietary supplementation with fish oil [(n-3) fatty acids] on the sciatic nerve of diabetic rats. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (n = 20) were fed a nonpurified diet supplemented with either olive oil (DO) or fish oil (DM), and control animals (n = 10) were fed a nonpurified diet supplemented with olive oil at a daily dose of 0.5 g/kg by gavage for 8 wk. Nerves were characterized by their conduction velocity, morphometric analysis and membrane Na, K-ATPase activity. Nerve conduction velocity, as well as Na,K-ATPase activity, was improved by fish oil treatment. A correlation was found between these two variables (R = 0.999, P < 0.05). Moreover, a preventive effect of fish oil was observed on nerve histological damage [endoneurial edema, axonal degeneration (by 10-15%) with demyelination]. Moreover, the normal bimodal distribution of the internal diameter of myelinated fibers was absent in the DO group and was restored in the DM group. These data suggest that fish oil therapy may be effective in the prevention of diabetic neuropathy.     

Long-term repopulating ability of xenogeneic transplanted human fetal liver hematopoietic stem cells in sheep

We previously reported on the successful engraftment and long-term multilineage expression (erythroid, myeloid, lymphoid) of human fetal liver hematopoietic stem cells in sheep after transplantation in utero. That the engraftment of long-term repopulating pluripotent stem cells occurred in these animals was shown here by the fact that transplantation of human CD45+ cells isolated from bone marrow of these chimeric animals into preimmune fetal sheep resulted in engraftment and expression of human cells. Marrow cells were obtained from three chimeric sheep at 3.2-3.6 yr after transplant. The relative percentage of human CD45+ cells present in these marrows was 3.3 +/- 0.32%. A total of 29 x 10(6) CD45+ cells were isolated by panning, pooled, and transplanted into six preimmune sheep fetuses (4.8 x 10(6) cells/fetus). All six recipients were born alive. Hematopoietic progenitors exhibiting human karyotype were detected in marrows of two lambs soon after birth. Cells expressing human CD45 antigen were also detected in blood and marrow of both lambs. Human cell expression has been multilineage and has persisted for > 1 yr. These results demonstrate that the expression of human cells in this large animal model resulted from engraftment of long-term repopulating pluripotent human stem cells.     

p75 neurotrophin receptor induction and macrophage infiltration in peripheral nerve during experimental diabetic neuropathy: possible relevance on regeneration

In this study we examined the expression of the neurotrophin receptor p75 (p75NTR) and the activation of macrophages in the sciatic nerve of rats at different time points after the induction of diabetes with streptozotocin (STZ). Northern blot and immunocytochemical analysis showed that p75NTR was not detectable in the sciatic nerve by Week 2 after STZ treatment. At this time, single nerve fiber immunostaining using ED1 monoclonal antibody revealed that active macrophages were infiltrating the endoneurium, which had a normal morphological aspect. By Weeks 5 and 15 p75NTR mRNA and protein were induced in the endoneurium of diabetic animals. Immunocytochemical analysis of teased single nerve fibers showed that p75NTR protein was distributed uniformly along isolated fibers with no pathological evidence of axonal degeneration or myelin disruption. At this time, cells of the phagocyte lineage had already disappeared from the nerve. These data show that during experimental diabetic neuropathy, the endoneurial induction of p75NTR is localized along isolated nerve fibers showing no morphological alterations, and in time, follows the recruitment of active macrophages in the nerve, suggesting that these cells, directly or through their products, can influence p75NTR induction. This process might play an important role in STZ diabetic neuropathy, as a response to decreased levels of neurotrophins such as NGF and promoting nerve regeneration in the early phases of the disease.     

In vivo performance of a new biodegradable polyester urethane system used as a nerve guidance channel

Biodegradable nerve guidance channels (NGCs) represent a promising alternative to current clinical nerve repair procedures. To be suitable as a NGC material, the polymer system should possess elastomeric properties and degrade at a defined rate without interfering with the regenerating environment. Polymers made of non-crystallizable blocks of poly[glycolide-co-(epsilon-caprolactone)]-diol and crystallizable blocks of poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyvaleric acid]-diol (PHB) can be modulated so as to respond to those criteria. Tubular structures were fabricated from three different types of materials containing either 41, 17 or 8 wt% PHB. Nerve regeneration through a 10 mm long NGC using a transected sciatic nerve model with an 8 mm gap was studied in rats at 4, 12 and 24 weeks. Out of 26 implanted NGCs, 23 contained regenerated tissue cables centrally located within the channel lumen and composed of numerous myelinated axons and Schwann cells. No significant difference in the degree of regeneration was observed between the various channel types. The inflammatory reaction associated with the polymer degradation had not interfered with the nerve regeneration process. Macrophages and giant cells surrounded polymer material remnants. A weight loss of 33, 74 and 88% for polymers containing 41, 17 and 8 wt% PHB was observed after 24 weeks by nuclear magnetic resonance (NMR) anaylsis, respectively. In all cases, the polymer fragments had a porous appearance with multiple surface cracks as evidenced by scanning electron microscopical analysis. Guidance channels made of 8 wt% PHB containing polymer displayed the highest degree of degradation at 24 weeks with only small polymer fragments remaining. The present study suggests that this new biodegradable elastomeric polymeric material holds promises for its utilization as nerve guidance channels.     

Two modes of microtubule-associated protein 1B phosphorylation are differentially regulated during peripheral nerve regeneration

Two major modes of MAP1B phosphorylation (I and II), respectively recognized by monoclonal antibodies 150 and 125, have been related to remodeling and formation of processes in the mature nervous system. To gain insight into the cytoskeletal modifications underlying peripheral nerve regeneration, the pattern of expression of both MAP1B phosphorylated modes was studied during this process. Sciatic nerves from adult Wistar rats were crushed and animals allowed to survive for 5, 7, 10 or 14 days. After those survival periods, damaged and undamaged sciatic nerves, dorsal root ganglia (DRG), and spinal cords, were subjected to immunohistochemistry and Western blot, using antibodies 150 and 125. At all survival periods analysed, MAP1B phosphorylated at mode I was concentrated at the distal region of regenerating nerves whereas mode II phosphorylation underwent an overall decrease in regenerating axons that was less evident in more proximal nerve regions. Very high levels of MAP1B phosphorylated at mode II were detected in the bodies of DRG neurons and in bodies and dendrites of spinal motor neurons. This phosphorylation mode was also encountered in some Schwann cells and oligodendroglia associated with more proximal regions of regenerating axons. In this study we conclude that MAP1B was differentially phosphorylated depending on the cell type, subcellular compartment and stage of the regenerative process and discuss the possible functional implications that differential expression of each MAP1B phosphorylation mode might have during nerve regeneration.     

In utero gene therapy: transfer and long-term expression of the bacterial neo(r) gene in sheep after direct injection of retroviral vectors into preimmune fetuses

We investigated whether directly injecting retroviral vectors into preimmune fetuses could result in the transfer and long-term expression of exogenous genes. Twenty-nine preimmune sheep fetuses were injected with helper-free retroviral vector preparations. Twenty-two fetuses survived to term, 4 of which were sacrificed at birth. Of the remaining 18 animals, 3 were controls and 15 had received vector preparations. Twelve of these 15 animals demonstrated transduction of hematopoietic cells when blood and marrow were analyzed by neo(r)-specific PCR. Eight experimental sheep have been followed for 5 years, during which time we have consistently observed proviral DNA and G418-resistant hematopoetic progenitors. The G418-resistant colonies were positive when analyzed by neo(r)-specific PCR. neo(r) gene expression was also demonstrated using several immunological and biochemical methods. The transduction of hematopoietic stem cells was confirmed when lambs transplanted with bone marrow from in utero-transduced sheep exhibited neo(r) activity in marrow and blood. Vector distribution was widespread in primary animals without pathology. PCR analysis indicates that the germ line was not altered. These studies demonstrate that direct injection of an engineered retrovirus is a feasible means of safely delivering a foreign gene to a developing fetus and achieving long-term expression without modifying the germ line of the recipient.     

Identification of peptides inhibiting enzyme I of the bacterial phosphotransferase system using combinatorial cellulose-bound peptide libraries

The effect of hyperbaric oxygen (HBO) treatment on regeneration of the rat sciatic nerve was studied. The sciatic nerve was crushed with a pair of pliers and the animals were either left untreated or subjected to a series of 45-min exposures to 100% O2 at 3.3 atm absolute pressure at 0, 4, and 8 h postoperatively and then every 8 h. Regeneration was evaluated using the pinch-reflex test at 3, 4, or 5 days following surgery and with neurofilament staining at 4 days. The regeneration distances at all time points were significantly longer in animals exposed to hyperbaric oxygen treatment independent of the evaluation procedure. A short initial period of the same HBO treatment schedule, with no more treatments after 25 h, appeared as effective as when treatments were maintained being given every 8 h until evaluation. We conclude that HBO treatment stimulates axonal outgrowth following a nerve crush lesion.     

Quantitative immunohistochemical changes in the endocrine pancreas of nonobese diabetic (NOD) mice

Two-centimeter nerve allografts were transplanted across a major histocompatibility barrier from donor ACI rats into a 0.5-cm gap in the sciatic nerve of recipient Lewis rats and immunosuppressed with FK506, 2 mg/kg per day for 3 months. One group of animals continued to receive intermittent immunosuppression with FK506, 2 mg/kg twice a week for another 2 months, whereas the second group of animals received no further immunosuppression in order to determine whether rejection of nerve allografts can still occur after immunosuppression is withdrawn, even after the axons have regenerated through the nerve graft. The sciatic function index improved from -76.3 at 3 months to -46.6 at 5 months in those animals continuing to receive intermittent immunosuppression, but only improved to -66.8 at 5 months when immunosuppression was discontinued. Similarly, somatosensory evoked potentials demonstrated an improvement in relative latency from 2.3 msec at 3 months to 0.34 msec at 5 months in animals continuing to receive intermittent immunosuppression, but only improved to 1.29 msec at 5 months when immunosuppression was discontinued. Nerve allografts continuing to receive intermittent immunosuppression showed no signs of rejection by light or electron microscopy and no significant difference compared with isografts, whereas nerve allografts whose immunosuppression had been stopped at 3 months showed mild signs of rejection, less regeneration, and a smaller number of nerve fibers.(ABSTRACT TRUNCATED AT 250 WORDS)