The axonal transport of slowly migrating (3H)leucine labelled proteins and the regeneration rate in regenerating hypoglossal and vagus nerves of the rabbit

The axonal transport of slowly migrating [³H]leucine labelled proteins was studied in regenerating hypoglossal and vagus nerves of the rabbit 1–4 weeks after a nerve crush. In normal nerves, as well as in the nerves contralateral to nerve crush, a transport rate of 4–5 mm/day for the hypoglossal nerve and 20–25 mm/day for the vagus nerve was calculated.The axonal transport of slowly migrating labelled proteins was increased in the regenerating hypoglossal nerve as compared to the contralateral 1 week after a nerve crush; this was mainly due to an increased transport rate.In contrast, the axonal transport of slowly migrating labelled proteins was decreased in the regenerating vagus nerve as compared to the contralateral nerve 1 week after a nerve crush. The results indicate a decreased transport rate and a decreased amount of material transported on the regenerating side. Changes in axonal transport in both hypoglossal and vagus nerves were less pronounced after 4 weeks of regeneration.Rapidly migrating [³H]leucine labelled proteins were transported into regenerating hypoglossal and vagus nerves distal to the crush zone, indicating axonal transport in the growing axons. The regeneration rate was estimated to be about 3 mm/day in the vagus nerve after 2–4 weeks of regeneration and about 4 mm/day in the hypoglossal nerve after 1 week of regeneration.The levels of acetyl-CoA: choline O-acetyltransferase (ChAc, E.C. 2.3.1.6) and acetylcholinesterase (AChE, E.C. 3.1.1.7) activity were decreased in regenerating vagus nerve the first week after a nerve crush.     

The initial period of peripheral nerve regeneration and the importance of the local environment for the conditioning lesion effect

The aim of this study was to investigate the early period of neurite outgrowth in the regenerating rat sciatic nerve and to determine if the non-neuronal cells were important for the conditioning lesion effect. Regeneration distance was evaluated with the pinch-reflex test 6 h to 5 days after a test crush lesion. The regeneration velocity accelerated during approximately 3 days, whereupon outgrowth continued with a constant velocity. In unconditioned nerves the initial delay was 2.8 h and the constant rate of regeneration was 3.2 mm/day. In nerves with a distal conditioning lesion the initial delay was 2.4 h and the rate of regeneration increased by 52%. When the test crush was applied at the same place as the conditioning crush the initial delay was 1.9 h and the rate of regeneration increased by 61%. The conditioning lesion effect was not influenced by the distance between the cell body and the conditioning crush lesion. Furthermore, the conditioning lesion effect could not be expressed if conditioned axons grew into a freeze injured nerve section. Incorporation of [3H]thymidine increased in the regenerating nerve segment. The increase occurred earlier if this segment had been subjected to a conditioning crush lesion. The results of these experiments showed that peripheral neurites start to regenerate within a few hours after an injury, suggesting that growth cone formation is independent of the cell body reaction. A conditioning crush lesion increases the regeneration velocity and its acceleration, and the conditioning lesion effect cannot be expressed in the absence of living Schwann and other non-neuronal cells.     

The axonal transport of (3H)fucose labelled glycoproteins in normal and regenerating peripheral nerves

The axonal transport of [³H]fucose labelled glycoproteins was studied in the hypoglossal and vagus nerves of the rabbit. These glycoproteins were transported with the rapid phase of axonal transport at a rate of about 300 mm/day and 400 mm/day in the hypoglossal and vagus nerves respectively.The amount of labelled rapidly migrating glycoproteins was increased in the regenerating hypoglossal nerve as compared to the contralateral nerve 1–4 weeks after a nerve crush. In contrast, the amount of transported labelled glycoproteins in the regenerating vagus nerve was decreased 1–4 weeks after a nerve crush. There was no indication of any change in the transport rate of the rapid phase of glycoproteins during regeneration in any of the nerves.The subcellular distribution of labelled glycoproteins accumulating proximal to a ligature applied to the regenerating and contralateral hypoglossal nerve showed a similar and preferential labelling of the microsomal fraction both on the regenerating and contralateral side 1 week after a nerve crush.     

Axonal transport of actin and regeneration rate in non-myelinated sensory nerve fibres

The relationship was examined between the rate of regeneration and rate of axonal transport of actin in the sensory fibres of the rabbit vagus nerve. Regeneration rate, determined as the distance moved by [35S]methionine-radiolabelled, fast-transported proteins beyond a crush, was about 3 mm/day. The rate of transport of actin, identified by two-dimensional polyacrylamide gel electrophoresis with fluorography, and DNase affinity chromatography, was 25-30 mm/day. No slower rate of actin transport comparable with regeneration rate, could be found in either control or regenerating nerves. While the provision of actin, by slow axonal transport, to the axonal growth cone may be essential for nerve regeneration, the regeneration rate is not directly controlled by the rate of actin transport.     

Nerve repair and axonal transport: Outgrowth delay and regeneration rate after transection and repair of rabbit hypoglossal nerve

The axonal transport and distribution of the fast phase of [³H]leucine-labeled proteins were used to monitor the outgrowth delay and regeneration rate in rabbit hypoglossal nerves 5–21 days after crush or transection. The transected nerves were repaired with mesothelial chambers or epineurial sutures. Radiolabeled proteins were transported into regenerating axons in the distal nerve segment after an initial delay of 2.5 days for crushed nerves and after a delay (initial and scar delays) of 4.8 and 5.7 days for sutured and mesothelial chamber-reconnected nerves, respectively. Regeneration rate was 3.5 mm/day after a crush and 2 mm/day after a transection with either type of repair. Total radioactivity was greateer in both crushed and repaired nerves than in their contralateral controls. Transported radioactivity accumulated at the site of the lesions. This accumulation was greater and persisted longer in repaired nerves than in crushed ones. The difference in regenerative response after different types of trauma with respect to changes in axonal transport is emphasized.     

Comparison of posttranslational protein modification by amino acid addition after crush injury to sciatic and optic nerves of rats

Posttranslational protein modifications by the addition of amino acids are reactions which occur in intact sciatic and optic nerves of rats. The nerves differ, however, in that 2 h after crush injury these reactions are activated in sciatic but not in optic nerves. As sciatic nerves will eventually regenerate, whereas optic nerves will not, we have proposed that the activation of these reactions is correlated with the ability of a nerve to regenerate. The current experiments examined the posttranslational addition of amino acids to proteins at times greater than 2 h after nerve crush, during sciatic nerve regeneration and optic nerve degeneration. We also examined the optic nerve for morphologic correlates to changes in protein modification and partially characterized the proteins modified by [3H]Lys in the regenerating sciatic nerve using two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In a segment of sciatic nerve taken from a region just proximal to the site of crush, protein modification by covalent addition of [3H]Arg, [3H]Lys and [3H]Leu increased during both posttraumatic (2 h postcrush) and regenerative (6 days and 14 days postcrush) stages. Two-dimensional PAGE of [3H]Lys modified sciatic nerve proteins 6 days after crush injury showed labeling of proteins having molecular masses in the 18,000- to 20,000-, 30,000- to 40,000-, and 80,000- to 100,000-Da ranges, with neutral or basic isoelectric points (pI 7.1 to 8.0). In the retinal portion of the crushed optic nerve, incorporation of the same amino acids was unchanged or depressed to 21 days postcrush, except at 6 days postcrush when the incorporation of all three amino acids into proteins was increased threefold. These increases correlated with the appearance of terminal end bulbs in the portion of nerve analyzed. Histological examination of each nerve 2 h postcrush showed marked edema in the optic but not the sciatic nerve, a condition which may be related to the ability of sciatic and inability of optic nerves to activate protein modification reactions.     

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

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

Regeneration of motor axons in the rat sciatic nerve studied by labeling with axonally transported radioactive proteins.

Labeling regenerating axons with axonally transported radioactive proteins provides information about the location of the entire range of axons from the fastest growing ones to those which are trapped in the scar. We have used this technique to study the regeneration of motor axons in the rat sciatic nerve after a crush lesion. From 2 to 14 days after the crush the lumbar spinal cord was exposed by laminectomy and multiple injections of [3H]proline were made stereotactically in the ventral horn. Twenty-four hours later the nerves were removed and the distribution of radioactivity along the nerve was measured by liquid scintillation counting. There was a peak of radioactivity in the regenerating axons distal to the crush due to an accumulation of label in the tips of these axons. After a delay of 3.2 +/- 0.2 (S.E.) days, this peak advanced down the nerve at a rate of 3.0 +/- 0.1 (S.E.) mm/day. The leading edge of this peak, which marks the location of the endings of the most rapidly growing labeled fibers, moved down the nerve at a rate of 4.4 +/- 0.2 mm/day after a delay of 2.1 +/- 0.2 days; this is the same time course as that of the most rapidly regenerating sensory axons in the rat sciatic nerve, measured by the pinch test. Another peak of radioactivity at the crush site, presumed to represent the ends of unregenerated axons or misdirected sprouts, declined rapidly during the first week, and more slowly thereafter.     

A new method for studies of the effects of locally applied drugs on peripheral nerve regeneration in vivo

An in vivo technique which allows local application of drugs to the regenerating rat sciatic nerve for several days is presented. The sciatic nerve was transected at the knee level and a crush lesion was made proximal to the transection. The crush lesion and the nerve segment distal to it was enclosed in a chamber made of silicone tubing (STC). The STC was perfused via a catheter connected to a miniosmotic pump. Regeneration was evaluated with the 'pinch-test' at 3, 4 and 6 days after the enclosure of the nerve. The rate of regeneration in the STC-surrounded nerve segment was 3.5 mm/day after an initial delay of 1.6 days which are values similar to those in uncovered nerves with crush lesions. Perfusion of the STC with either vinblastine, cycloheximide, actinomycin D or mitomycin C inhibited regeneration. The effects were confined to the STC-covered region. Leakage of drugs was too small to affect the nerve outside the chamber. The results suggest that regeneration requires proliferation and protein synthesis in the cells surrounding the growing axons. This technique could be useful for studies of the local effects of various drugs, specific antibodies, potential growth factors etc. on regeneration of peripheral nerves.     

Rapid growth of regenerating axons across the segments of sciatic nerve devoid of Schwann cells

The characteristic response of Schwann cells (SC) accompanies peripheral nerve injury and regeneration. To elucidate their role, the question of whether or not regenerating axons can elongate across the segments of a peripheral nerve devoid of SC was investigated. Rat sciatic nerve was crushed so that the continuity of SC basal laminae was not interrupted. A segment about 15 mm long distal to the crush was either repeatedly frozen/thawed to eliminate SC or scalded by moist heat which, in addition, denatured the proteins in the SC basal laminae, too. Both sensory and motor axons grew rapidly across the frozen/thawed segment of the nerve. Their rate of elongation was reduced by only 30% in comparison to control crushed nerves. SC were not present along the path of growing axons adhering tightly to the bare SC basal laminae. The rate of elongation of regenerating sensory and motor axons in scalded nerve segments was eight times lower than in control crushed nerves. SC were present in that part of the scalded region that had been invaded by the regenerating axons but no further distally. These results suggest that acellular basal laminae of SC provide very good, although not optimal, conditions for elongation of regenerating sensory and motor axons. If biochemical integrity of the basal lamina is destroyed, the regenerating axons must be accompanied or preceded by viable SC. and axon elongation rate is significantly reduced.     

Acute treatment with pulsed electromagnetic fields and its effect on fast axonal transport in normal and regenerating nerve

The mechanism whereby low-frequency electromagnetic fields accelerate axonal regrowth and regeneration of peripheral nerve after crush lesion is not known. One candidate is an alteration in axonal transport. In this study we exposed unoperated rats for 15 min/day, and rats that had undergone a crush lesion of the sciatic nerve, for 1 hr/day for 2 days, to 2-Hz pulsed electromagnetic fields. To label fast transported proteins, [³H]-proline was microinjected into the spinal cord, and the sciatic nerves were removed 2, 3.5, and 5 hr later. The rates of fast axonal transport were obtained for animals in all groups by counting sequential 2-mm segments of nerves. The following transport rates were found: in unoperated normal sciatic nerve not exposed to PEMF, 373 ± 14 mm/day; in unoperated normal nerve exposed to PEMF, 383 ± 14 mm/day; in sham crush nerves not exposed to PEMF, 379 ± 19 mm/day; in sham crush nerve exposed to PEMF, 385 ± 17 mm/day; in crushed nerves not exposed to PEMF, 393 ± 16 mm/ day; and in crushed nerves exposed to PEMF, 392 ± 15 mm/day. The results of these experiments indicate that (1) a crush injury to the sciatic nerve does not alter the rate of fast axonal transport, and (2) low-frequency pulsed electromagnetic fields do not alter fast axonal transport rates in operated (crush) or unoperated sciatic nerves. © 1995 Wiley-Liss, Inc.     

Effects of repetitive conditioning crush lesions on regeneration of the rat sciatic nerve

The effect of repetitive conditioning lesions was tested on regeneration of the rat sciatic nerve. The nerve was conditioned by crush lesions one, two or three times with an interval of 2 or 4 days between each successive lesion. Axonal elongation was measured 3 days after a final test crush lesion. Two conditioning lesions stimulated axonal elongation more than one, while a third conditioning lesion had no further effect on axonal outgrowth. However, if the number of conditioning lesions were varied within a constant conditioning interval, outgrowth after the test lesion was the same. This suggests that the conditioning interval and not the number of conditioning lesions determined the outgrowth after a test lesion. When the conditioning lesion(s) and the test lesion were made at the same place, outgrowth was longer than if the lesions were spatially separated. Incorporation of [3H]thymidine in the regenerated nerve segment showed that proliferation of non-neuronal cells was initiated by each lesion. By counting the number of cell nuclei this proliferation was shown to correspond to an increase of cells in the regenerating nerve. It is therefore possible that the greater number of non-neuronal cells in the distal nerve segment accounts for the enhanced conditioning lesion effect in nerves where the conditioning and test lesions are made at the same place.     

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.     

Prior collateral sprouting enhances elongation rate of sensory axons regenerating through acellular distal segment of a crushed peripheral nerve

Regenerating axons in crushed peripheral nerves grow through their distal nerve segments even in the absence of Schwann cell support, but their elongation rate is reduced by 30%. We examined whether prior exposure of sensory neurons to trophic factors achieved either by collateral sprouting or regeneration after conditioning lesion could enhance subsequent regeneration of their axons after crush, and compensate for loss of cell support. Collateral sprouting of the peroneal cutaneous sensory axons in the rat was evoked by transection of adjacent peripheral nerves in the hind leg. The segment of the peroneal nerve distal to the crush was made acellular by repeated freezing. Sensory axon elongation rate during regeneration was measured by the nerve pinch test. Prior axonal sprouting for two weeks increased the elongation rate of sensory axons through the acellular distal nerve segment back to normal value observed in control crushed nerves. The number of axons in the acellular distal segment at a fixed distance from the crush site was about 50% greater in sprouting than in control non-sprouting nerves. However, prior sprouting caused no further increase of axon elongation rate in control crushed nerves. Prior collateral sprouting, therefore, could in some respect compensate for loss of cell support in the distal nerve segment after crush lesion. This suggests that loss of cell-produced trophic factors is probably responsible for slower elongation rate through the acellular distal nerve segment. Surprisingly, prior conditioning lesion caused no enhancement of elongation rate of the sensory axons regenerating in the absence of cell support.     

Incorporation of [32P]phosphate into nucleotides of the dorsal root ganglia of regenerating rat sciatic nerve

[32P]Phosphate incorporation into nucleotides of the dorsal root ganglia (DRG) was studied after a crush lesion of the rat sciatic nerve. DRG were labelled during a 2-h, in vitro incubation in a balanced salt solution containing [32P]orthophosphate, 1, 2, 4 and 8 days after the crush lesion. Nucleotides were analyzed by HPLC on an ion-exchange column. An increased incorporation of 32P was found in DRG of the injured nerve for all the studied time periods. This increase was unevenly distributed among the nucleotides. UTP, CTP and ADP showed the largest and most persistent increases in labelling. The specific activity of 4 analyzed nucleotides (ATP, ADP, UTP and CDP) remained constant in DRG from crushed nerves. Thus, the observed increase in 32P-labelling could not solely be due to an increased uptake of label but must also reflect an enhanced metabolism of nucleotides in regenerating DRG. The finding that alterations of nucleotide metabolism could be observed within one day after the crush lesion suggests that this response can be used as a valuable tool for studies of the initial events of regeneration.     

Conduction studies in peripheral cat nerve using implanted electrodes: II. The effects of prolonged constriction on regeneration of crushed nerve fibers

Arrays of chronically implanted electrodes were used to examine the time course of elongation and maturation of peripheral nerve fibers in the cat after crush of the tibial nerve in the proximal calf. Regeneration after crush alone was compared with crush 5 mm proximal to a tight constriction of the nerve. Regeneration was monitored by the progression of excitability along the electrode arrays on the tibial and plantar nerves. The sensitivity was sufficient to record the averaged activity in single nerve fibers allowing detection of the earliest regeneration. The diameters of the fastest regenerating fibers were estimated from the conduction velocity proximal to the site of crush. Both after crush alone, and after crush constriction, small myelinated fibers regenerated in front of large fibers. The rate of elongation after crush alone was 3.2 mm/day, whereas it was slower (P less than 0.02) distal to crush + constriction (2.2 mm/day). In both lesions, the extrapolated delay to onset of regeneration was 8 days. In observations up to 300 days after crush, maturation was delayed or impaired by the constriction, and the compound nerve action potential had a smaller amplitude and a dispersed shape. Transverse sections of nerves after crush + constriction showed a diminished number of large and an increased number of small fibers compared with crush alone, possibly due to persistent branching of regenerated fibers. After both crush alone and crush + constriction, regenerated fibers had similar g ratios, suggesting that myelination developed fully in fibers of diminished diameters.     

Axonal transport of glycerophospholipids in regenerating sciatic nerve of the rat during aging

The effect of age upon the axoplasmic transport of glycerophospholipids has been studied using as a model the regenerating sciatic nerve of young (2-month-old), young adult (6-month-old), middle-aged (16-month-old), and aged (20-month-old) male rats. The right sciatic nerve was crushed 0.5 mm down the incisura ischiadica. Four and nine days after the lesion, a mixture of [2-3H] glycerol and [methyl-14C] choline was bilaterally injected into the spinal cord, at a level of the L4-L5 vertebrae. The animals were killed 18 hr after the isotope injection. Proximal and distal portions of crushed nerve and of contralateral sham-operated ones were dissected and consecutive 5-mm segments were subjected to lipid extraction and analysis. The findings of the present study are summarized as follows: (1) The accumulation of labeled lipid material axonally transported four days after nerve injury was mainly located at the crush site in young, young adult, middle-aged, and aged rats. The accumulation of both 3H-glycerolipids and 14C-choline phospholipids in postcrush segments was markedly higher for young and young adult than for aged rats, four and nine days after crush; (2) the average rate of axonal regeneration, determined between days 4 and 9 following crush injury was 3.6 and 4.2 mm/day for 2-month-old and 6-month-old rats, respectively; it decreased to the value of 2.5 mm/day for 16-20-month-old rats.     

CYTOFLUOROMETRIC QUANTIFICATION OF SOMATOPETAL AXONAL TRANSPORT: EFFECTS OF A CONDITIONING LESION AND 2,5-HEXANEDIONE

Cytofluorometric quantification of axonally transported fluorescein isothiocyanate (FITC)-labelled wheat germ agglutinin (WGA) from the injection site in the snout area was performed in the facial nucleus at various times during regeneration of one of the facial nerves. Measurements were made on single neurons on both operated and non-operated sides in three different groups of mice 8, 12 and 16 days after a nerve crush, Group 1: (control group) animals with a nerve crush, Group 2: animals with a conditioning lesion (nerve injury) made 3 days before the nerve crush, and Group 3: animals exposed daily to 2,5-hexanedione from 2 weeks before nerve crush until killing. A conditioning lesion caused a more rapid return of transport in regenerating nerves but there was no evidence for an increase in the total amount of transported FITC-WGA. For mice exposed to 2,5-hexanedione a transient increase of tracer transport in regenerating nerves could be demonstrated on day 12 after nerve crush. On day 16, however, a reduction of transport was seen in both operated and non-operated nerves. This study shows that it is possible experimentally to manipulate the influx of macromolecules to the nerve cell body from the periphery during nerve regeneration, and the present method offers the opportunity to study quantitatively the effects of various treatments on reinnervation of a muscle.     

Insulin-like growth factor (IGF-1) as a stimulator of regeneration in the freeze-injured rat sciatic nerve

The effect of insulin-like growth factor (IGF-1) on the ability of the rat sciatic nerve to regenerate into a freeze-injured nerve segment was investigated. The freeze-injured segment was perfused for 6 days with Ringer solution and different concentrations of IGF-1, dispensed by a subcutaneously implanted osmotic minipump. At a pump concentration of 50, 100 and 200 micrograms IGF-1/ml the regeneration length increased with 14, 25 and 26%, respectively, as measured by the pinch test and by immunocytochemical staining for neurofilaments (NF) in the growing neurites. Schwann cells invading the freeze-injured segment were visualized by immunostaining for S-100 protein. In nerves perfused with Ringer solution alone the Schwann cells were present as far as the neurites had regenerated, while neurites seemed to grow slightly ahead of the Schwann cells in the nerves perfused with IGF-1. Incorporation of [3H]thymidine increased in IGF-1-treated nerves. However, IGF-1 perfusion did not increase thymidine incorporation when outgrowth of neurites was detained by a transection proximal to the freeze-injured area. The results suggest that IGF-1 affects regeneration by local stimulation of the growing neurites and that IGF-1 stimulates the proliferation of non-neuronal cells indirectly.     

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.