Peripheral Axon Regeneration Research Articles

Collagen filaments as a scaffold for nerve regeneration

This article describes repair of peripheral nerve defect using collagen filaments instead of tubes. Many tube-shaped nerve guides induce regeneration of severed peripheral nerve axons within a limited distance. Substantial regeneration of nerve axons has not been reported without a tubular conduit. Here we show the regeneration of peripheral nerve axons along filaments of collagen without a tube. Cables of collagen filaments were grafted to repair 20-mm defects of rat sciatic nerves. Nerve autografts and collagen tubes were grafted as controls. The mean number and the mean fiber diameter of regenerated myelinated axons were approximately 4800 and 3.3 μm in the distal end of the nerve autograft at 8 weeks postoperatively while in the distal end of the collagen-filaments nerve guide, they were approximately 5500 and 2.3 μm. Collagen tubes failed to bridge the nerve defect. Histologic studies suggest that nerve axons regenerated substantially along the collagen filaments. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 400–405, 2001

Collagen filaments as a scaffold for nerve regeneration.

This article describes repair of peripheral nerve defect using collagen filaments instead of tubes. Many tube-shaped nerve guides induce regeneration of severed peripheral nerve axons within a limited distance. Substantial regeneration of nerve axons has not been reported without a tubular conduit. Here we show the regeneration of peripheral nerve axons along filaments of collagen without a tube. Cables of collagen filaments were grafted to repair 20-mm defects of rat sciatic nerves. Nerve autografts and collagen tubes were grafted as controls. The mean number and the mean fiber diameter of regenerated myelinated axons were approximately 4800 and 3.3 microm in the distal end of the nerve autograft at 8 weeks postoperatively while in the distal end of the collagen-filaments nerve guide, they were approximately 5500 and 2.3 microm. Collagen tubes failed to bridge the nerve defect. Histologic studies suggest that nerve axons regenerated substantially along the collagen filaments.

Non-neuronal cells are not the limiting factor for the low axonal regeneration in C57BL/6J mice.

Peripheral axonal regeneration was investigated in adult male mice of the C57BL/6J (C), BALB/cJ (B) and A/J (A) strains and in their F1 descendants using a predegenerated nerve transplantation model. Four types of transplants were performed: 1) isotransplants between animals of the C, B and A strains; 2) donors of the C strain and recipients of the C x B and C x A breeding; 3) donors of the B strain and recipients of the C x B breeding, and 4) donors of the A strain and recipients of the C x A breeding. Donors had the left sciatic nerve transected and two weeks later a segment of the distal stump was transplanted into the recipient. Four weeks after transplantation the regenerated nerves were used to determine the total number of regenerated myelinated fibers (TMF), diameter of myelinated fibers (FD) and myelin thickness (MT). The highest TMF values were obtained in the groups where C57BL/6J mice were the donors (C to F1 (C x B) = 4658 +/- 304; C to F1 (C x A) = 3899 +/- 198). Also, A/J grafts led to a significantly higher TMF (A to F1 (C x A) = 3933 +/- 565). Additionally, isotransplant experiments showed that when the nerve is previously degenerated, C57BL/6J mice display the largest number of myelinated fibers (C to C = 3136 +/- 287; B to B = 2759 +/- 170, and A to A = 2835 +/- 239). We also observed that when C57BL/6J was the graft donor, FD was the highest and MT did not differ significantly when compared with the other groups. These morphometric results reinforce the idea that Schwann cells and the nerve environment of C57BL/6J provide enough support to the regenerative process. In this respect, the present results support the hypothesis that the non-neuronal cells, mainly Schwann cells, present in the sciatic nerve of C57BL/6J mice are not the main limiting factor responsible for low axonal regeneration.

Embryonic cord transplants in peripheral nerve restore skeletal muscle function.

The rapid atrophy of skeletal muscle after denervation severely compromises efforts to restore muscle function. We have transplanted embryonic day 14-15 (E14-E15) ventral spinal cord cells into adult Fischer rat tibial nerve stump to provide neurons for reinnervation. Our aim was to evaluate medial gastrocnemius reinnervation physiologically because this transplant strategy will only be effective if the reinnervated muscle contracts, generates sufficient force to induce joint movement, and is fatigue resistant enough to shorten repeatedly. Twelve weeks posttransplantation, brief duration electrical stimuli applied to the transplants induced medial gastrocnemius contractions that were strong enough to produce ankle movement in 4 of 12 rats (33%). The force of these four "low-threshold" reinnervated muscles and control muscles declined only gradually during five hours of intermittent, supramaximal stimulation and without depression of EMG potential area, which is strong evidence of functional neuromuscular junctions and fatigue resistant muscles. Sectioning of the medial gastrocnemius nerves confirmed that these contractions were innervation dependent. Weakness in low-threshold reinnervated muscles (8% control force) related to incomplete reinnervation, reductions in muscle fiber size, specific tension, and/or the presence of nonfunctional neuromuscular junctions. Muscle reinnervation achieved using this novel transplantation strategy may salvage completely denervated muscle and may provide the potential to evoke limb movement when injury or disease precludes or delays peripheral axon regeneration.

Results of nerve sutures in the wrist in children

Wounds involving the peripheral nerves cause serious damage in the upper limb. The prognosis for recovery remains uncertain despite microsurgery. It is commonly accepted that the results are far better in children than in adults. We sought to confirm this idea on the basis of objective recovery criteria, discriminating between peripheral axonal regeneration and the child's own capacity to adapt. In other words, do nerves regenerate better in children? The clinical results of 25 nerve sutures of the wrist, in children under 15, were analysed with a minimum follow-up of 12 months. The subjects were reviewed clinically and had an electromyogram. The resulting data was compared to that for adults found in the literature. Clinically, the overall function of the hand was always satisfactory. The sensory results were often excellent (S4 or S3+ in 23 cases/25). The mean value muscular testing was between M2 and M3. With respect to the EMG results, the values we recorded were rarely representative of the functional results. The motor recordings were mostly poor, demonstrating that the nerve supply was compensated by a phenomenon involving collateral innervation. The poor results of peripheral nerve surgery are only due to the quality of the restored innervation. Dellon and Mackinnon demonstrated that they were also due to the incapacity of the nerve centres to integrate a change in the profile of sensory information. It would therefore seem that not only the quantity of nerve tissue repair is insufficient, but also the quality. Children have a superior cerebral capacity to adapt than adults and probably benefit from better cortical acquisition processes and are thus capable of putting the changes in the nerve messages to better use. The analysis of the clinical and EMG results also reveals a trend demonstrated partial nerve repair in children. We think that the functional results suggest that children have a better central capacity to adapt.

Tanycytes Transplanted into the Adult Rat Spinal Cord Support the Regeneration of Lesioned Axons

During past years a number of therapeutic strategies have been developed in order to stimulate axonal regeneration after traumatic injuries of the spinal cord. Recently, encouraging data have been obtained by grafting specific glial cells such as Schwann cells or olfactory ensheathing glial cells, known to support the regeneration of peripheral or central axons, respectively. In a recent series of studies, we have shown that tanycytes, a particular glial cell type present in the mediobasal hypothalamus, were able to support the regeneration of a variety of axons innervating this region. The aim of the present study was to determine whether tanycytes could also support the regeneration of lesioned spinal axons. Cultured hypothalamic tanycytes and cortical astrocytes were prelabeled with Fast blue (FB) and grafted into the thoracic spinal cord of adult rats. Three weeks after the transplantation, the animals were fixed and spinal cord sections treated for multiple fluorescence detection of the FB-labeled transplanted cells on the one hand and of various glial and neuronal markers on the other hand. We show here that in all the spinal cords examined, transplanted tanycytes or astrocytes formed large spherical clusters of about 0.5 mm in diameter, located in the mediolateral spinal cord layer. The immunodetection of glial markers showed that transplanted astrocytes exhibited intense immunostaining for both glial fibrillary acidic protein (GFAP) and vimentin (VIM), whereas transplanted tanycytes were intensely immunostained for VIM, but GFAP negative. The immunodetection of axonal markers showed that contrasting with astrocyte transplants, tanycyte transplants were invaded by numerous axonal fibers. These data indicate that tanycyte transplants may represent a useful therapeutic tool for the reparation of the lesioned spinal axons.

Nerve growth factor-hypersecreting Schwann cell grafts augment and guide spinal cord axonal growth and remyelinate central nervous system axons in a phenotypically appropriate manner that correlates with expression of L1.

Schwann cells contribute to efficient axonal regeneration after peripheral nerve injury and, when grafted to the central nervous system (CNS), also support a modest degree of central axonal regeneration. This study examined (1) whether Schwann cells grafted to the CNS exhibit normal patterns of differentiation and association with spinal axons and what signals putatively modulate these interactions, and (2) whether Schwann cells overexpressing neurotrophic factors enhance axonal regeneration. Thus, primary Schwann cells were transduced to hypersecrete human nerve growth factor (NGF) and were grafted to spinal cord injury sites in adult rats. Comparisons were made to nontransfected Schwann cells. From 3 days to 6 months later, grafted Schwann cells exhibited a phenotypic and temporal course of differentiation that matched patterns normally observed after peripheral nerve injury. Schwann cells spontaneously aligned into regular spatial arrays within the cord, appropriately remyelinated coerulospinal axons that regenerated into grafts, and appropriately ensheathed but did not myelinate sensory axons extending into grafts. Coordinate expression of the cell adhesion molecule L1 on Schwann cells and axons correlated with establishment of appropriate patterns of axon-Schwann cell ensheathment. Transduction of Schwann cells to overexpress NGF robustly increased axonal growth but did not otherwise alter the nature of interactions with growing axons. These findings suggest that signals expressed on Schwann cells that modulate peripheral axonal regeneration and myelination are also recognized in the CNS and that the modification of Schwann cells to overexpress growth factors significantly augments their capacity to support extensive axonal growth in models of CNS injury.

Induction of microtubule-associated protein 1B expression in Schwann cells during nerve regeneration

Microtubule-associated protein 1B (MAP1B) is expressed at high levels during development of the nervous system and is localized primarily in neurons while specific phosphorylated isoforms of MAP1B are localized exclusively in growing axons. The levels of MAP1B are down regulated in most regions of the adult CNS, but remain high in neurons and axons of the PNS. This study demonstrates that the expression of MAP1B is induced in adult Schwann cells following sciatic nerve lesion and regeneration. High levels of both mRNA and the MAP1B protein were detected in Schwann cells associated with the axotomized distal stump. Expression of MAP1B was also observed in cultured primary Schwann cells from neonatal rats. The properties of the MAP1B protein in cultured Schwann cells were further characterized by Western blot analysis using specific antibodies that recognize the N-terminal, middle and C-terminal domains of MAP1B. All of these antibodies detected a protein of 320–340 kDa demonstrating that MAP1B expressed by Schwann cells is very similar, or identical, to MAP1B expressed by neurons. The phosphorylation of MAP1B in Schwann cells was also studied using monoclonal antibodies (mAb) that recognize specific phosphorylation epitopes. The results indicated that the expression of MAP1B in Schwann cells exhibited a differential phosphorylation state that was recognized by mAb 1B6 but not by other mAbs, including 1B-P, 150 and RT97, that recognize phosphorylated MAP1B in growing axons. We therefore conclude that MAP1B is expressed in Schwann cells during both development and axonal regeneration, suggesting that the developmental pattern of MAP1B in these cells is recapitulated in adult Schwann cells during the early stages of regeneration and remyelination of injured peripheral axons. The presence of MAP1B in Schwann cells may support morphological changes of these cells, particularly the formation of processes prior to their differentiation into myelin forming Schwann cells.

Control of Schwann Cell Survival and Proliferation: Autocrine Factors and Neuregulins

Postnatal rat Schwann cells secrete factors that prevent the programmed cell death (PCD) of low-density Schwann cells in serum-free culture. These autocrine survival signal(s) do not promote Schwann cell proliferation. Moreover, while NRG and bFGF, which promote proliferation, both rescue a subpopulation of neonatal Schwann cells from PCD, they do not rescue freshly isolated Schwann cells from older animals; other known protein factors tested also do not mimic the autocrine signal. These results suggest that Schwann cells switch their survival dependency around the time of birth from axonal signals such as NRG to autocrine signals. Such an arrangement would be advantageous for the regeneration of peripheral axons following injury. We also compared NRG-induced Schwann cell proliferation using autocrine signals or serum to promote survival. The autocrine signals increase the rate of NRG-stimulated proliferation of low-density Schwann cells in serum-free medium, whereas serum inhibits proliferation by inhibiting both the production of survival signals and the expression of erbB2 and erbB3 receptors; these inhibitions are all reversed by forskolin. In contrast, forskolin has no effect on proliferation when the cells are exposed to high levels of autocrine factors.

Peripheral Axotomy Induces Long-Term c-Jun Amino-Terminal Kinase-1 Activation and Activator Protein-1 Binding Activity by c-Jun and junD in Adult Rat Dorsal Root GangliaIn Vivo

One of the earliest documented molecular events after sciatic nerve injury in adult rats is the rapid, long-term upregulation of the immediate early gene transcription factor c-Jun mRNA and protein in lumbar dorsal root ganglion (DRG) neurons, suggesting that c-Jun may regulate genes that are important both in the early post-injury period and during later peripheral axonal regeneration. However, neither the mechanism through which c-Jun protein is increased nor the level of its post-injury transcriptional activity in axotomized DRGs has been characterized. To determine whether transcriptional activation of c-Jun occurs in response to nerve injury in vivo and is associated with axonal regeneration, we have assayed axotomized adult rat DRGs for evidence of jun kinase activation, c-Jun phosphorylation, and activator protein-1 (AP-1) binding. We report that sciatic nerve transection resulted in chronic activation of c-Jun amino-terminal kinase-1 (JNK) in L4/L5 DRGs concomitant with c-Jun amino-terminal phosphorylation in neurons, and lasting AP-1 binding activity, with both c-Jun and JunD participating in DNA binding complexes. The timing of JNK activation was dependent on the distance of the axotomy site from the DRGs, suggesting the requirement for a retrograde transport-mediated signal. AP-1 binding and c-Jun protein returned to basal levels in DRGs as peripheral regeneration was completed but remained elevated in the case of chronic sprouting, indicating that c-Jun may regulate target genes that are involved in axonal outgrowth.

Peripheral nerve regeneration through nerve guides seeded with adult Schwann cells

This study tested the usefulness of Schwann cells in the repair of a severed nerve with a biosynthetic bridge or guide. Reinforced collagen nerve guides were used to bridge an 18 mm gap in the sciatic nerve of 21 young adult rats. The animals were divided into three groups and the guides were filled with: (i) more than 0.5 x 10(6) cultured syngeneic adult Schwann cells (group L, n = 12); (ii) less than 0.5 x 10(6) Schwann cells (Group S, n = 6); and (iii) phosphate buffered saline (control, n = 3). Schwann cells were pre-labelled with Hoechst dye. Regeneration was assessed functionally and histologically at 1, 2, 3 and 6 + months after surgery. Group L animals showed numerous regenerated axons surrounded by implanted Schwann cells within the first month. The total number of myelinated fibers (12.5 x 10(3)) remained above normal unoperated values (7 x 10(3)) in long-term animals. Regenerated axons were found in Group S in the third month, but no Hoechst labelled cells were found. The number of myelinated fibers (3.9 x 10(3)) remained below normal values in long-term animals. Control guides failed to support axonal regeneration. Functional recovery was evident at week 20 (Group L) and week 30 (Group S) after surgery, with no difference in function between the two groups by the end of the study. Supplementing guides with Schwann cells enhances regeneration of peripheral axons over a distance normally prohibitive. This effect is greatest in the early stages of regeneration (1-3 months) and is dependent on the number of cells implanted.

Basic fibroblast growth factor promotes extension of regenerating axons of peripheral nerve. In vivo experiments using a Schwann cell basal lamina tube model.

Schwann cell basal lamina tubes serve as attractive conduits for regeneration of peripheral nerve axons. In the present study, by using basal lamina tubes prepared by in situ freeze-treatment of rat saphenous nerve, the effects of exogenously applied basic fibroblast growth factor (bFGF) on peripheral nerve regeneration was examined 2 and 5 days after bFGF administration. Regenerating axons were observed by light and electron microscopy using PGP9.5-immunohistochemistry for specific staining of axons. In addition, the localizations of bFGF and its receptor (FGF receptor-1) were examined by immunohistochemistry using anti-bFGF antibody and anti-FGF receptor-1 antibody, respectively. Regenerating axons extended further in the bFGF-administered segment than in the bFGF-untreated control segment. Electron microscopy showed that regenerating axons grew out unaccompanied by Schwann cells. Findings concerning angiogenesis and Schwann cell migration were very similar between the bFGF treated and control nerve segment. bFGF-immunoreactivity was not detected in the control nerve segment. In contrast, bFGF-immunoreactivity was detected on the basal lamina tubes as well as on the plasmalemma of regenerating axons facing the basal lamina in the bFGF treated nerve segment up to 5 days after administration, suggesting that exogenous bFGF can be retained in the basal lamina for several days after administration. FGF receptor was detected on the plasma membrane of regenerating axons where they abutted the basal lamina. These results indicate that bFGF could promote the extension of early regenerating axons by directly influencing the axons, but not via Schwann cells or angiogenesis.

The effect of aging on efferent nerve fibers regeneration in mice

This study evaluates the influence of aging on nerve regeneration and reinnervation of target organs in mice aged 2, 6, 9, 12, 18 and 24 months. In animals of each age group the sciatic nerve was subjected to crush, section or section and suture. Reinnervation of plantar muscles and sweat glands (SG) was evaluated over three months after operation by functional methods. Reappearance of SG secretion and motor responses occurred slightly earlier in young than older mice. The degree of motor and sudomotor reinnervation, with respect to preoperative control values, was also significantly higher in young than old animals. The differences were more pronounced after 12 months of age. The degree of recovery progressively decreased with the severity of the lesion, differences being more marked in older mice. Neurorraphy improved recovery, comparatively more in older than in young mice. These results indicate that, after injuries of peripheral nerves, axonal regeneration and reinnervation are maintained throughout life, but tend to be more delayed and slightly less effective with aging.

Dispersion of regenerating axons across enclosed neural gaps

Tubular prostheses support peripheral axon regeneration across gaps of up to 3 cm in the primate. However, the precision with which axons cross a gap and reinnervate the periphery remains controversial. These experiments use continuous tracing of regenerated rat sciatic nerve axons with HRP-WGA to examine the dispersion of axons as they cross a gap, and the effects on this dispersion of gap distance and fascicular orientation. Proximal and distal tibial and peroneal fascicles were precisely oriented about the longitudinal midplane of a silicon tube, with correct or reversed fascicular alignment and gaps of 2 mm and 5 mm. After 6 weeks of regeneration, HRP-WGA was applied to the distal peroneal fascicle to continuously label its reinnervating axons. These axons tended to grow straight across the tube, with dispersion increasing as a factor of distance when correct fascicular alignment was maintained. However, when fascicular alignment was reversed, axonal dispersion was determined by fascicular size rather than fascicular identity. These experiments provide no evidence for neurotropic interactions promoting "correct" fascicular reinnervation. Progressive axonal dispersion and the absence of factors to promote fascicular specificity should result in an increase of random reinnervation and functional disruption with larger gaps. An enclosed gap is not an acceptable substitute for nerve graft when reconstructing a nerve that serves multiple functions.

Role of macrophages in peripheral nerve degeneration and repair.

A cut or crush injury to a peripheral nerve results in the degeneration of that portion of the axon isolated from the cell body. The rapid degeneration of this distal segment was for many years believed to be a process intrinsic to the nerve. It was believed that Schwann cells both phagocytosed degenerating axons and myelin sheaths and also provided growth factors to promote regeneration of the damaged axons. In recent years, it has become apparent that the degenerating distal segment is invaded by monocytes from the blood. We will review the evidence that these recruited macrophages play a role in both degeneration and regeneration of peripheral nerve axons after injury and consider whether the slow degeneration and poor monocyte recruitment in the central nervous system may contribute to the poor regeneration there.

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

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

Fate of GAP-43 in ascending spinal axons of DRG neurons after peripheral nerve injury: delayed accumulation and correlation with regenerative potential

Proteins characteristic of growing axons often fail to be induced or transported along axons that have been interrupted far from their cell bodies in the adult mammalian CNS. Here, we inquire whether long axons in the mammalian CNS can support efficient axonal transport and deposition of one such protein, GAP-43, when the protein is induced in neuron cell bodies. We have used immunocytochemistry to follow the fate of GAP-43 in dorsal column axons ascending the rat spinal cord from dorsal column axons ascending the rat spinal cord from dorsal root ganglion (DRG) neurons, after synthesis of the protein is induced in these cells by peripheral nerve injury. Sciatic nerve lesions do lead to an accumulation of GAP-43 in dorsal column axons derived from the lumbar DRG. However, in distal segments of these CNS axons, accumulation of GAP-43 is apparent only after a delay of 1-2 weeks, in contrast to its rapid accumulation in axon segments within the PNS environment, suggesting that deposition and stabilization of GAP-43 can be limited by local, posttranslational regulation. GAP-43 immunoreactivity subsides to control levels within 8 weeks after crush lesions that permit peripheral axon regeneration, but remains robust 8 weeks after resection lesions that prevent peripheral regeneration. Accumulation of GAP-43 in cervical dorsal column axons after peripheral nerve injury is closely correlated with the ability of these axons to respond to local cues capable of eliciting axon growth (Richardson and Verge, 1986).

Success of regeneration of peripheral nerve axons in rats after injury at different postnatal ages

Electrophysiological experiment have been carried out on rats to see if the age at which a peripheral nerve injury occurs influences the success of regeneration. The assessment was made on the basis of two measures of peripheral nerve regeneration; the extent to which axons manage to grow across the injury site and into the distal stump, and their ability to resupply cutaneous structures with functional endings. Regeneration after nerve transection of both myelinated and unmyelinated axons was studied. The results showed that, apart from rats injured when 2 weeks old, the age at which injury occurred, over the range 4-40 weeks, had little bearing on the overall success of skin reinnervation. The 2-week-old rats showed significantly poorer recovery.

Aberrant reinnervation

Although great emphasis is placed on providing a satisfactory conduit for regeneration of peripheral axons after nerve repair, the quality of functional restoration is influenced as much by the quality as the quantity of axonal regeneration. Misdirected regeneration is so commonly encountered that motor axons appear to enter and regenerate to muscles in an almost random manner. Thus, when there are several choices, as usually is the case with more proximal nerve or plexus repairs, misdirected reinnervation accounts in many incidences for a poor quality of functional restoration. The regenerative capacities of type I and type II motor axons appear to differ. Proprioceptors and other sensory axons have been shown to reinnervate inappropriate end organs. Consequently, deranged central reflex modulation and disturbed orderly recruitment of motor units according to the size principle also contributes to this problem. Central re-education or adaptation to misdirected regeneration does not occur to any appreciable extent.

Expression of growth-associated protein B-50 (GAP43) in dorsal root ganglia and sciatic nerve during regenerative sprouting

Recently it has been shown that B-50 is identical to the neuron-specific, growth-associated protein GAP43. The present study reports on the fate of B-50/GAP43 mRNA and B-50/GAP43 protein, determined by radioimmunoassay, in a rat model of peripheral nerve regeneration (sciatic nerve crush) over a period of 37 and 312 d, respectively. Moreover, the effects of repeated subcutaneous injection of the neurotrophic peptide Org.2766 (an ACTH4-9 analog) and of a conditioning lesion on B-50/GAP43 protein levels in the regenerating nerve and dorsal root ganglia (DRG) were investigated. Both treatments enhanced the functional recovery as evidenced by a foot-flick withdrawal test. Immunocytochemical analysis using antineurofilament antibodies revealed a peptide-induced increase in the number of outgrowing sprouts in the sciatic nerve. Both the peptide and the conditioning lesion amplified the crush lesion-induced increase in B-50 protein content in the nerve as determined by radioimmunoassay. B-50 protein levels seem to correlate proportionally with the number of sprouts. In the DRG of the crushed sciatic nerve, the time course of B-50 expression was studied. B-50 mRNA was quantified from Northern blots. A linear increase up to 10 times the basal level of B-50 mRNA was observed 2 d postsurgery, followed by a gradual decline to normal levels at day 37. The first significant rise in B-50 mRNA level became apparent between 8 and 16 hr after placement of the crush lesion. The first significant rise in B-50 protein level occurred 40 hr after the crush lesion, reaching a plateau of 3 times the basal level between day 6 and 20. B-50 protein levels in DRG cell bodies remained elevated up to 60 d after crush, a period much longer than that observed for B-50 mRNA. Thus, during a later phase of peripheral axonal regeneration, the presence of B-50 appears to be prolonged, probably by an increase in half-life and not so much by enhanced transcription. Treatment with Org.2766 did not affect the B-50/GAP43 levels in DRG cell bodies during the first 6 d following crush. Conditioning lesion resulted in a DRG B-50/GAP43 protein amount at the same level as in rats 14 d after the test lesion. B-50/GAP43 levels in DRG are probably influenced by the rapid axonal transport of the protein, as has been reported by others.