Local Acceleration of Neurofilament Transport at Nodes of Ranvier.

Myelinated axons are constricted at nodes of Ranvier. These constrictions are important physiologically because they increase the speed of saltatory nerve conduction, but they also represent potential bottlenecks for the movement of axonally transported cargoes. One type of cargo are neurofilaments, which are abundant space-filling cytoskeletal polymers that function to increase axon caliber. Neurofilaments move bidirectionally along axons, alternating between rapid movements and prolonged pauses. Strikingly, axon constriction at nodes is accompanied by a reduction in neurofilament number that can be as much as 10-fold in the largest axons. To investigate how neurofilaments navigate these constrictions, we developed a transgenic mouse strain that expresses a photoactivatable fluorescent neurofilament protein in neurons. We used the pulse-escape fluorescence photoactivation technique to analyze neurofilament transport in mature myelinated axons of tibial nerves from male and female mice of this strain ex vivo Fluorescent neurofilaments departed the activated region more rapidly in nodes than in flanking internodes, indicating that neurofilament transport is faster in nodes. By computational modeling, we showed that this nodal acceleration can be explained largely by a local increase in the duty cycle of neurofilament transport (i.e., the proportion of the time that the neurofilaments spend moving). We propose that this transient acceleration functions to maintain a constant neurofilament flux across nodal constrictions, much as the current increases where a river narrows its banks. In this way, neurofilaments are prevented from piling up in the flanking internodes, ensuring a stable neurofilament distribution and uniform axonal morphology across these physiologically important axonal domains.SIGNIFICANCE STATEMENT Myelinated axons are constricted at nodes of Ranvier, resulting in a marked local decrease in neurofilament number. These constrictions are important physiologically because they increase the efficiency of saltatory nerve conduction, but they also represent potential bottlenecks for the axonal transport of neurofilaments, which move along axons in a rapid intermittent manner. Imaging of neurofilament transport in mature myelinated axons ex vivo reveals that neurofilament polymers navigate these nodal axonal constrictions by accelerating transiently, much as the current increases where a river narrows its banks. This local acceleration is necessary to ensure a stable axonal morphology across nodal constrictions, which may explain the vulnerability of nodes of Ranvier to neurofilament accumulations in animal models of neurotoxic neuropathies and neurodegenerative diseases.

Modification of Schwann cell gene expression by electroporation in vivo.

Clinical outcomes of nerve grafting are often inferior to those of end-to-end nerve repair. This may be due, in part, to the routine use of cutaneous nerve to support motor axon regeneration. In previous work, we have demonstrated that Schwann cells express distinct sensory and motor phenotypes, and that these promote regeneration in a modality-specific fashion. Intra-operative modification of graft Schwann cell phenotype might therefore improve clinical outcomes. This paper demonstrates the feasibility of electroporating genes into intact nerve to modify Schwann cell gene expression. Initial trials established 70 V, 5 ms as optimum electroporation parameters. Intact, denervated, and reinnervated rat tibial nerves were electroporated with the YFP gene and evaluated serially by counting S-100 positive cells that expressed YFP. In intact nerve, a mean of 28% of Schwann cells expressed the gene at 3 days, falling to 20% at 7 days with little expression at later times. There were no significant differences among the three groups at each time period. Electronmicroscopic evaluation of treated, intact nerve revealed only occasional demyelination and axon degeneration. Intra-operative electroporation of nerve graft is thus a practical means of altering Schwann cell gene expression without the risks inherent in viral transfection.

MiR-463-3p inhibits tibial nerve regeneration via post-transcriptional suppression of SPRR1A.

Background: miRNAs have been involved in neural development, degeneration, and regeneration. MiR-463-3p is expressed in reproductive and nervous systems. In this study, the role of miR-463-3p in tibial nerve injury and regeneration was explored. Materials and methods: A model of tibial nerve injury was established with the crush method, and the levels of miR-463-3p were detected at days 0, 3, 7, 12, 18 and 24 post-injury. Then, primary tibial nerve cells were isolated from newborn mice, and miR-463-3p was respectively overexpressed and knocked down in cultured cells. Behaviors of tibial nerve cells were detected. Furthermore, bioinformatics technology was used to investigate the underlying mechanism. Results: The expression miR-463-3p was robustly increased in the injured tibial nerve in vivo and in tibial nerve cells treated with oxygen-glucose deprivation. The data on gain- and loss-of-function demonstrated that miR-463-3p negatively regulated including neurite length, percentage of cells with neurites, and cell branching in tibial nerve cells. Small proline-rich repeat protein 1 A (SPRR1A), an identified nerve regeneration associated genes, was identified as a target gene of miR-463-3p. Conclusion: Inhibition of miR-463-3p could increase SPRR1A expression in the tibial nerve tissue and improve regeneration of the tibial nerve post-injury in vivo.

Automated characterization of nerve fibers labeled fluorescently: determination of size, class and spatial distribution.

Morphological classification of nerve fibers could help interpret the assessment of neural regeneration and the understanding of selectivity of nerve stimulation. Specific populations of myelinated nerve fibers can be investigated by retrograde tracing from a muscle followed by microscopic measurements of the labeled fibers at different anatomical levels. Gastrocnemius muscles of adult rats were injected with the retrograde tracer Fluoro-Gold. After a survival period of 3 days, cross-sections of spinal cords, ventral roots, sciatic, and tibial nerves were collected and imaged on a fluorescence microscope. Nerve fibers were classified using a variation-based criterion acting on the distribution of their equivalent diameters. The same criterion was used to classify the labeled axons using the size of the fluorescent marker. Measurements of the axons were paired to those of the entire fibers (axons+myelin sheaths) in order to establish the correspondence between so-established axonal and fiber classifications. It was found that nerve fibers in L6 ventral roots could be classified into four populations comprising two classes of Aalpha (denoted Aalpha1 and Aalpha2), Agamma, and an additional class of Agammaalpha fibers. Cut-off borders between Agamma and Agammaalpha fiber classes were estimated to be 5.00+/-0.09 microm (SEM); between Agammaalpha and Aalpha1 fiber classes to be 6.86+/-0.11 microm (SEM); and between Aalpha1 and Aalpha2 fiber classes to be 8.66+/-0.16 microm (SEM). Topographical maps of the nerve fibers that innervate the gastrocnemius muscles were constructed per fiber class for the spinal root L6. The major advantage of the presented approach consists of the combined indirect classification of nerve fiber types and the construction of topographical maps of so-identified fiber classes.

Effects of tripolar TENS on slow and fast motoneurons: a preliminary study using H-reflex recovery curve method.

This study aimed at examining the effect of tripolar TENS of vertebral column on the activity of slow and fast motoneurons on 10 healthy non-athlete women aged 22.7 +/- 2.21 yrs. H-reflex recovery curve of soleus (slow) and gastrocnemius (fast) muscles were recorded before and after applying tripolar TENS. For recording of this curve, rectangular paired stimuli were applied on tibial nerve (with 40-520 ISI, frequency of 0.2 Hz and pulse width of 600 micros). Our findings showed that maximum H-reflex recovery in gastrocnemius muscle appeared in the shorter ISI, while in soleus muscle, it appeared in the longer ISI and its amplitude slightly decreased after applying tripolar TENS. It is suggested that tripolar TENS excites not only the skin but also Ia and Ib afferents in the dorsal column. A Synaptic interaction of these afferents in spinal cord causes the inhibition of type I MNs and facilitation of type II MNs. This effect can be used in muscle tone modulation.

A device for the electrophysiological recording of peripheral nerves in response to stretch.

The functional consequences of nervous tissue subjected to mechanical loads are of vital importance in successful clinical outcomes and in tissue engineered applications. In this paper, we developed a new ex vivo device that permitted the recording of nerve action potentials while the nerve was subjected to elongation. Experimental results showed guinea pig nerves to possess an inherent tolerance to mild stretch. The mean elongation at which the compound action potential (CAP) amplitude began to decrease was found to be 8.3 +/- 0.56%. The CAP response to stretch was immediate beyond this threshold. After 17.5 +/- 0.74% elongation, the CAP levels decreased to approximately 50% of its uninjured values. When allowed to relax, the CAP recovered almost completely within minutes. Based on the temporal scale of the CAP response and the presence of oxygen during testing, we conclude that the initial mechanism to CAP degradation cannot be ischemia. Since it is inherently difficult to study mechanical damage independent of hemodynamic factors in vivo, the developed model could be used to further elucidate the injury mechanisms of stretch-induced trauma. The design of the ex vivo chamber will also permit the administration and assessment of pharmacological agents on electrical conduction in various deformation conditions.

Neural tissue engineering: a self-organizing collagen guidance conduit.

We report a novel implantable device that will deliver a tethered aligned collagen guidance conduit containing Schwann cells into a peripheral nerve injury site. Cells (Schwann cells and fibroblasts) incorporated into tethered rectangular collagen gels contracted and resulted in uniaxial alignment. This tissue-engineered construct was tested in three-dimensional culture and demonstrated the ability to guide neurite extension from dissociated dorsal root ganglia. A silicone tube was adapted to provide tethering sites for an implantable construct such that uniaxial cell-generated tension resulted in the formation of a bridge of aligned collagen fibrils, with a resident Schwann cell population. The potential of this device for surgical nerve regeneration was assessed in a 5-mm defect in a rat sciatic nerve model. Neural regeneration through this device was significantly greater than in controls, demonstrating that this system has potential both as a simple robust clinical implant and as a three-dimensional engineered tissue model.

Nerve cross-bridging to enhance nerve regeneration in a rat model of delayed nerve repair.

There are currently no available options to promote nerve regeneration through chronically denervated distal nerve stumps. Here we used a rat model of delayed nerve repair asking of prior insertion of side-to-side cross-bridges between a donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) nerve stump ameliorates poor nerve regeneration. First, numbers of retrogradely-labelled TIB neurons that grew axons into the nerve stump within three months, increased with the size of the perineurial windows opened in the TIB and CP nerves. Equal numbers of donor TIB axons regenerated into CP stumps either side of the cross-bridges, not being affected by target neurotrophic effects, or by removing the perineurium to insert 5-9 cross-bridges. Second, CP nerve stumps were coapted three months after inserting 0-9 cross-bridges and the number of 1) CP neurons that regenerated their axons within three months or 2) CP motor nerves that reinnervated the extensor digitorum longus (EDL) muscle within five months was determined by counting and motor unit number estimation (MUNE), respectively. We found that three but not more cross-bridges promoted the regeneration of axons and reinnervation of EDL muscle by all the CP motoneurons as compared to only 33% regenerating their axons when no cross-bridges were inserted. The same 3-fold increase in sensory nerve regeneration was found. In conclusion, side-to-side cross-bridges ameliorate poor regeneration after delayed nerve repair possibly by sustaining the growth-permissive state of denervated nerve stumps. Such autografts may be used in human repair surgery to improve outcomes after unavoidable delays.

Tracing axons in the peripheral nerve using lacZ gene recombinant adenovirus and its application to regeneration of the peripheral nerve.

The usefulness of recombinant adenovirus with LacZ to trace axons in the peripheral nervous system was investigated. Recombinant adenovirus with LacZ was applied to the cut end of the tibial nerve in rats. The LacZ gene product (B-galactosidase) filled axons of the tibial nerve, which permitted the continuous long-range tracing of axons. Further, the branching and the direction of the branches could also be examined. Labeled axons in the tibial nerves ran parallel to each other without branching and kept this relative position in the tibial and the sciatic nerve. When the virus was introduced to the regenerating nerve using a silicon tube, the regenerating fibers grew tortuously with short branches in the bulge at the proximal end of the silicon tube. The fibers grew straight in the tube and passed through the bulge at the distal end of the tube without branching. These observations indicate that the LacZ gene recombinant adenovirus is a useful tracer for the study of the peripheral nervous system and of the regeneration processes.

Organization of hindlimb nerve projections to the rat spinal cord: a choleragenoid horseradish peroxidase study.

The aim of the present study has been to investigate the projections of hindlimb muscle afferent fibers to the spinal cord with particular emphasis on the ventral horn and the column of Clarke. Following transections of the appropriate ventral roots, injections of the B-subunit of cholera toxin conjugated to horseradish peroxidase were made into the tibial, peroneal, hamstring, superior gluteal, femoral, and obturator nerves in one group of adult rats. In another group of rats, similar experiments were done with intact ventral roots in order to map the location in the ventral horn of the motoneuron cell columns supplying each investigated nerve. An extensive overlap was found for the different nerve projections to Rexed's laminae V-VII. A somatotopic organization of the nerve projections was seen in the lamina IX cell groups of the ventral horn as well as in the column of Clarke, even though an overlap existed. The densest primary afferent projection from each injected nerve was to its homonymous motoneurons. Only a small to moderate overlap between the projections of the tributary branches of the sciatic nerve was found in the ventral horn, whereas the obturator and femoral nerve projections showed more profound overlap. In the column of Clarke, hindlimb nerves innervating distal muscles projected medially, and nerves innervating proximal muscles projected laterally.

Adrenergic innervation of the tibial and vagus nerves in rats of different ages.

Adrenergic nerve fibers innervating blood vessels in the epi-perineurium and endoneurium of the tibial and vagus nerves of male Fischer-344 rats of different ages were examined using formaldehyde-induced fluorescence technique and fluorescence microscopy. Between 6 and 24 months of age, no significant difference in the mean density of perivascular innervation in the epi-perineurium of the nerves was found, whereas between 24 months and 30 months the density decreased in both nerve types. The intensity of noradrenaline fluorescence of nerve fibers also was decreased in the oldest age group. In the endoneurium, the density of adrenergic nerve fibers was reduced by 57% at 24 months, and by more than 98% at 30 months as compared with 18 months. Age-related differences were similar in the tibial and vagus nerves. The results suggest that adrenergic neuronal control of the microcirculation in the rat tibial and vagus nerves is lost during aging as it is in other organs of the cardiovascular system.

Chronic effects of topical application of capsaicin to the sciatic nerve on responses of primate spinothalamic neurons.

The responses of 144 spinothalamic tract (STT) cells were recorded in 15 anesthetized macaque monkeys (Macaca fascicularis). Three to 4 weeks prior to the acute experiment, the sciatic nerve was surgically exposed on one or both sides so that capsaicin or vehicle could be applied. Responses of STT cells recorded in 3 experimental groups were compared: untreated (21 cells), vehicle-treated (40 cells), and capsaicin-treated (83 cells). The background activity of cells in the vehicle- and capsaicin-treated groups was the same as in the untreated group (that is, cells on the side contralateral to surgery). Responses to innocuous (BRUSH) and noxious (PINCH) mechanical stimuli were unchanged by vehicle or by capsaicin treatment. However, responses to other noxious (PRESSURE and SQUEEZE) mechanical stimuli were significantly increased in the vehicle-treated group. Compared with a large reference population, all experimental groups showed a significant increase in overall responsiveness to mechanical stimuli (as determined by cluster analysis), greatest in the vehicle-treated group. Responses to noxious heat stimuli were significantly reduced in the capsaicin-treated group for 45 degrees C and 47 degrees C stimuli. Volleys in A fibers, probably A delta fibers, evoked prolonged responses in many STT cells of all treatment groups. Electron microscopic counts of axons in the sciatic nerves of animals treated with capsaicin showed a reduced number of C fibers but no appreciable loss of myelinated axons. This loss of unmyelinated sensory fibers was presumably responsible for the reduction in the responses of the STT cells to noxious heat stimuli. Increased responses to some noxious mechanical stimuli and to A fiber volleys may have been the consequence of several factors, including surgical manipulation, a chemical action of vehicle and a contralateral action of capsaicin treatment.

Time-dependent inhibition of hindlimb somatic afferent transmission within nucleus tractus solitarius: an in vivo intracellular recording study.

In a previous study we demonstrated that hindlimb somatic afferent stimulation evokes excitatory responses from neurons in nucleus tractus solitarius. When paired electrical stimuli were delivered to hindlimb somatic afferents, the unit response to the second stimulus was significantly reduced compared with responses to the first. This temporal response pattern has been termed time-dependent inhibition since responses to the second stimulus recover as the interval separating the first and second stimuli is increased. To examine possible synaptic mechanisms for somatic afferent-evoked time-dependent inhibition, intracellular recordings were made from nucleus tractus solitarius neurons in anesthetized, paralysed rats. Skeletal muscle afferent fibers were activated by electrically stimulating the right tibial nerve in the hindlimb and neuronal responses recorded in the contralateral nucleus of the solitary tract. Time-dependent inhibition of tibial nerve-evoked unit discharge was studied using a conditioning-test stimulation procedure, with the first (conditioning) and second (test) stimuli separated by intervals of 50, 150 and 250 ms. In 49 units that responded to tibial nerve stimulation, 46 were excited and three were inhibited. Among units excited, 25 displayed a unimodal response that had an onset latency of 21.3 +/- 5.9 ms. The remaining 21 units responded with a bimodal discharge pattern characterized by both a short-latency and a long-latency response. The onset latency of the early response was 23.7 +/- 5.3 ms and was not statistically different from the unimodal response onset latency. The onset latency of the late response was 143 +/- 23.9 ms.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved functional recovery of denervated skeletal muscle after temporary sensory nerve innervation.

Prolonged muscle denervation results in poor functional recovery after nerve repair. The possible protective effect of temporary sensory innervation of denervated muscle, prior to motor nerve repair, has been examined in the rat. Soleus and gastrocnemius muscles were denervated by cutting the tibial nerve, and the peroneal nerve was then sutured to the transected distal tibial nerve stump either immediately or after two, four or six months. In half of the animals with delayed repair, the saphenous (sensory) nerve was temporarily attached to the distal nerve stump. Muscles were evaluated three months after the peroneal-to-tibial union, and were compared with each other, with unoperated control muscles and with untreated denervated muscles. After four to six months of sensory "protection", gastrocnemius muscles weighed significantly more than unprotected muscles, and both gastrocnemius and soleus muscles exhibited better preservation of their structure, with less fiber atrophy and connective tissue hyperplasia. The maximum compound action potentials were significantly larger in gastrocnemius and soleus muscles following sensory protection, irrespective of the delay in motor nerve union. Isometric force, although less than in control animals and in those with immediate nerve repair, remained reasonably constant after sensory protection, while in unprotected muscles there was a progressive and significant decline as the period of denervation lengthened. We interpret these results as showing that, although incapable of forming excitable neuromuscular junctions, sensory nerves can nevertheless exert powerful trophic effects on denervated muscle fibers. We propose that these findings indicate a useful strategy for improving the outcome of peripheral nerve surgery.

A freeze-fracture study of the perineurium in normal and protein-deprived rats.

Observations have been made using the freeze-fracture replication technique on the perineurium of normal and protein-deprived rats in which its permeability barrier function is known to be deficient. The perineurial cells of young normal rats possessed belt-like tight junctions (zonulae occludentes) at the borders and maculae occludentes at sites remote from their borders. In older rats, the zonulae occludentes were more prominent and the maculae occludentes relatively less frequent. No abnormalities were detected in the tight junctions of young rats with early induction of protein deficiency but this may have been related to sampling problems. In older severely protein-deficient animals, although many of the tight junctions were normal, some were abnormal and contained focal regions of dispersed strands. The density of caveolae in the surface membrane of the perineurial cells of older rats with severe protein deficiency was significantly greater than in the control animals. This provides support for the view that the pinocytotic-like vesicles of perineurial cells are involved in transport of substances across the cells. The increased numbers of caveolae in the protein deficient rats may reflect increased transcellular traffic. There were considerable differences in the density of P-face IMPs between the different perineurial lamellae, but the results did not allow a decision to be made as to whether there was a polarization of the cells between their endoneurial and epineurial aspects. No differences were detected in the density of P-face IMPs between the young control and protein-deprived rats. In the perineurium of the older rats with protein deficiency, IMP density was significantly greater in the E face than in the controls but not different in the P face. The delay in the development of enzymatic activity in the perineurium of protein-deficient rats that has been demonstrated histochemically is therefore not paralleled by a reduction in IMPs.

Distribution of adrenergic innervation of blood vessels in peripheral nerve.

The distribution of adrenergic innervation of microvessels in the extrafascicular and endoneurial compartments of rat tibial nerve was examined with glyoxylic acid-induced and formaldehyde-induced histofluorescence methods. Periarterial and arteriolar adrenergic nerves were present in the epineurium-perineurium suggesting that blood flow in the extrafascicular connective tissue is under neurogenic influence. In contrast, blood vessels in the nerve endoneurium were not associated with histofluorescent nerve fibers. The absence of perivascular adrenergic innervation in the endoneurium suggests that regulation of vascular function within the endoneurium is not under neurogenic control.

Axonal elongation through long acellular nerve segments depends on recruitment of phagocytic cells from the near-nerve environment. Electrophysiological and morphological studies in the cat.

The distal nerve stump plays a central role in the regeneration of peripheral nerve but the relative importance of cellular and humoral factors is not clear. We have studied this question by freezing the tibial nerve distal to a crush lesion in cat. The importance of constituents from the near-nerve environment was assessed by modification of the contact between the tibial nerve and the environment. Silicone cuffs, containing electrodes for electrophysiological assessment of nerve regeneration, were placed around the tibial nerve distal to the crush site. The interaction between long acellular frozen nerve segments (ANS) and the near-nerve environment was ascertained by breaching the silicone cuff to allow access of cellular or humoral components. Tibial nerves were crushed and frozen for 40 mm and enclosed in nerve cuffs with 0.45-microm holes or 2.0-mm holes to allow access of humoral factors or tissue ingrowth, respectively. In a second set of experiments, tibial nerves were crushed and either frozen for 20+20 mm, leaving a 10 mm segment with viable cells in the center (stepping-stone segment) or frozen for 50 mm. These nerves were enclosed in cuffs with 2.0 mm holes corresponding to the viable nerve segment. The regeneration was monitored electrophysiologically by implanted electrodes and after 2 months the nerves were investigated by light and electron microscopy. The results indicate that soluble substances in the near-nerve environment, such as nutrients, oxygen or tropic substances did not exert any independent beneficial effect on the outgrowing axons. However, phagocytic cells entering the acellular segment from the near-nerve environment were crucial for axonal outgrowth in long ANS.

Anin vivo method to prepare normal Schwann cells free of axons and myelin.

A viable population of undifferentiated Schwann cells may be prepared from chronically denervated peripheral nerves. Nerve transection stimulates a sequence of cellular events in distal stumps leading to removal of axons and myelin, and proliferation of Schwann cells. Sealing the ends of nerve stumps prevents reinnervation and leaves daughter Schwann cells residing in longitudinal columns. Schwann cells may be harvested from the endoneurial tissue of the nerve stumps 5-12 weeks after nerve transection. Unlike myelinating cells prepared from intact tissue, where function has been specified by associated axons, Schwann cells obtained from denervated stumps are functionally naive. Their usefulness in analyzing axonal regulation of myelinogenesis and mitosis is therefore suggested.

Axonal elongation through acellular nerve segments of the cat tibial nerve: importance of the near-nerve environment.

Peripheral nerve regeneration is considered to be influenced by structural, cellular and humoral factors in the distal nerve stump. Axonal elongation was, however, not affected by the presence of a 20 mm acellular nerve segment (ANS) distal to a crush lesion in a cat tibial nerve which was shielded from the environment by a silicone cuff [K. Fugleholm, H. Schmalbruch, C. Krarup, Early peripheral nerve regeneration after crushing, sectioning, and freeze studied by implanted electrodes in the cat, J. Neurosci., 14 (1994) 2659-2673]. In the present study axons were challenged to regenerate through crush lesions combined with 30-, 40-, 50-, 60- and 70-mm ANSs. For 30- and 40-mm ANSs, the nerves were shielded by impermeable silicone cuffs containing electrodes for electrophysiological evaluation of axonal elongation. All nerves were examined histologically by light microscopy 9 weeks after the lesion. The elongation through the shielded 30-mm ANS was slower than through a shielded nerve segment with viable cells. In the isolated 40-mm ANS, incomplete Wallerian degeneration and lack of blood vessels were observed, and axonal elongation was severely impaired. Regeneration across 40-70 mm non-shielded ANSs was intact and there was no relation between the number of regenerated fibers and the length of the ANS. There was no reduction in the number of blood vessels in the non-isolated ANSs. The results suggest that regeneration through an isolated acellular nerve segment exceeding 30 mm depends on cellular and humoral support from the near-nerve environment. Thus, the near-nerve environment is crucial for regeneration through long ANSs, and the importance of humoral, cellular and vascular support is discussed.

Schwann cell mitosis in response to regenerating peripheral axons in vivo.

Schwann cell mitosis has been demonstrated in chronically denervated cat tibial nerves re-innervated by axons regenerating from the proximal stump of a coapted peroneal nerve. Thymidine incorporation rose above baseline levels at the axon front, with no detectable increase in more distal regions occupied by denervated Schwann cells. Schwann cells therefore enter S phase upon the arrival of a regenerating axon in vivo as previously described in tissue culture. Intraneural treatment of the denervated distal stump with Mitomycin C prior to re-innervation delayed the subsequent appearance of myelin formation. This supports the notion that axonally stimulated division of Schwann cells is a prerequisite for myelination during nerve regeneration. Axonal advancement was also retarded by drug treatment, possibly because of a reduced level of trophic support provided by the compromised Schwann cells. A comparable absence of myelin and poor re-innervation was found in chemically untreated distal stumps that had been maintained in the denervated state for prolonged periods when Schwann cell columns are known to undergo progressive atrophy. These observations suggest that nerve repair should be delayed for limited periods if efficacious regeneration is desired.