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.     

Axonal signals regulate expression of glia maturation factor-beta in Schwann cells: an immunohistochemical study of injured sciatic nerves and cultured Schwann cells

Glia maturation factor-beta (GMF-beta) is a 17 kDa protein purified and sequenced from bovine brains. Using the monoclonal antibody G2-09 directed against GMF-beta, we previously demonstrated endogenous GMF-beta in astroblasts, Schwann cells, and their tumors in culture. In the present study, we have used indirect immunofluorescence microscopy with G2-09 to examine the effects of transection, crush, and regeneration of sciatic nerve on the expression of GMF-beta in Schwann cells in situ and to study the time course of GMF-beta induction in Schwann cells in vitro. For comparison, a parallel study was carried out with monoclonal antibodies directed against nerve growth factor (NGF) receptor. We found that (1) neither GMF-beta nor NGF receptor was detectable in intact sciatic nerves, (2) all Schwann cells of the distal segment of the transected nerve expressed GMF-beta as early as 3 d after axotomy that persisted up to 3 weeks, (3) axonal regeneration repressed the Schwann cell expression of GMF-beta, (4) isolated Schwann cells derived from rat sciatic and adult human sural nerves developed intracellular GMF-beta in culture following an initial lag period, and (5) the induction of Schwann cell NGF receptor coincided temporally with that of GMF-beta in the transected nerve and in culture. These results show that the expression of GMF-beta in Schwann cells, as is the case with the NGF receptor, is induced by the loss of the normal axon-Schwann cell contact. We propose that the induction of GMF-beta, as well as NGF receptor, in Schwann cells after nerve injury plays a role in axonal regeneration.     

Substrate-bound nerve growth factor promotes neurite growth in peripheral nerve

Nerve growth factor (NGF), in addition to its well-known effects as a soluble neurite growth-promoting factor, also appears to promote the elongation of neurites when it is adsorbed to tissue culture substrates. Peripheral nerve Schwann cells appear to possess a receptor for NGF on their surfaces which is induced substantially after axotomy. We have found that the adsorption of NGF onto cryostat sections of the distal stump of previously severed sciatic nerve enhances neurite growth over this tissue. This finding, coupled with the two previous observations, suggests that Schwann cell surface NGF receptors serve to bind to NGF-like growth factors so as to provide favorable surfaces for regenerating peripheral nerve axons.     

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

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

Expression of CHL1 and L1 by neurons and glia following sciatic nerve and dorsal root injury

Cell adhesion molecules (CAMs), particularly L1, are important for axonal growth on Schwann cells in vitro. We have used in situ hybridization to study the expression of mRNAs for L1 and its close homologue CHL1, by neurons regenerating their axons in vivo, and have compared CAM expression with that of GAP-43. Adult rat sciatic nerves were crushed (allowing functional regeneration), or cut and ligated to maintain axonal sprouting but prevent reconnection with targets. In other animals lumbar dorsal roots were transected to produce slow regeneration of the central axons of sensory neurons. In unoperated animals L1 and CHL1 mRNAs were expressed at moderate levels by small- to medium-sized sensory neurons and L1 mRNA was expressed at moderate levels by motor neurons. Many large sensory neurons expressed neither L1 nor CHL1 mRNAs and motor neurons expressed little or no CHL1 mRNA. Neither motor nor sensory neurons showed any obvious upregulation of L1 mRNA after axotomy. Increased CHL1 mRNA was found in motor neurons and small- to medium-sized sensory neurons 3 days to 2 weeks following sciatic nerve crush, declining toward control levels by 5 weeks when regeneration was complete. Cut and ligation injuries caused a prolonged upregulation of CHL1 mRNA (and GAP-43 mRNA), indicating that reconnection with target tissues may be required to signal the return to control levels. Large sensory neurons did not upregulate CHL1 mRNA after axotomy and thus regenerated within the sciatic nerve without producing CHL1 or L1. Dorsal root injuries caused a modest, slow upregulation of CHL1 mRNA by some sensory neurons. CHL1 mRNA was also upregulated by many presumptive Schwann cells in injured nerves and by some satellite cells around large sensory neurons after sciatic nerve injuries and was transiently upregulated by some astrocytes in the degenerating dorsal columns after dorsal rhizotomy.     

Changes in cytoskeletal gene expression affect the composition of regenerating axonal sprouts elaborated by dorsal root ganglion neurons in vivo

The effect of a change in neurofilament (NF) and tubulin gene expression on the elongation of axonal sprouts by adult rat sensory neurons was examined. Distal sciatic nerve crush axotomy was used to initiate changes in cytoskeletal gene expression in lumbar dorsal root ganglion (DRG) neurons. In situ hybridization of DRG neurons with 35S-labeled cDNA probes revealed a significant reduction in the level of mRNAs for the low-molecular weight-NF protein and a significant increase in the level of beta tubulin mRNAs by 2 weeks after axotomy. A novel modification of the axonal transport paradigm was used to examine the biochemical composition of the regenerating axons formed by primed and unprimed DRG neurons. Primed neurons (which had sustained a crush axotomy of the distal sciatic nerve 2 weeks earlier) and unprimed (normal) neurons were labeled by microinjection of 35S-methionine and then stimulated to regenerate axons by a crush located very close to the DRG. In this paradigm, axonal sprouts that formed after the proximal crush axotomy incorporated radiolabeled, slow axonally transported proteins as they elongated. Fluorographs of SDS-PAGE revealed that the regenerating axonal sprouts of primed DRG cells incorporated and conveyed significantly less labeled NF protein than did the regenerating axons of unprimed DRG neurons. Electron microscopy revealed that the regenerating axonal sprouts of primed DRG cells contained numerous microtubules but very few identifiable NFs compared with the regenerating sprouts of unprimed DRG neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Axonal transport and localization of B-50/GAP-43-like immunoreactivity in regenerating sciatic and facial nerves of the rat

Neurons that can regenerate their axons following axotomy increase their synthesis and axonal transport of a growth-associated protein, called GAP-43, which has been shown to be identical to the synaptic phosphoprotein B-50. The function of B-50/GAP-43 to the process of regeneration is unknown. We used a polyclonal, affinity-purified antibody against B-50 to study the axonal transport and localization of B-50/GAP-43-like immunoreactivity (B50LI) in the regenerating sciatic and facial nerves of adult rats. Quantitative data were obtained by densitometry of the B-50 band in immunoblots of nerve segments, which had been run on SDS-polyacrylamide gels. In the regenerating sciatic nerve, anterograde accumulation at a collection ligature was 3.0 times higher than retrograde accumulation. The mobile fraction of B50LI was only 0.28 of total B50LI and traveled with a mean anterograde velocity of 5.3 mm/hr. B50LI distribution in the newly regenerated portion of the nerve revealed maximal B50LI levels midway between the position of the crush and the fastest-growing axons. Immunocytochemistry of this portion of the nerve demonstrated B50LI to be associated with regenerating axons but also to a large extent with extra-axonal structures outlining the Schwann cell bands of Büngner. This zone of B50LI-positive Schwann cell bands was found to extend more distally in nerves in which regeneration had processed longer, e.g., up to 5 mm distal to the crush after 3 d and 8 mm after 4 d. Further distal to this zone, many fine regenerating axonal profiles could be detected with B-50 antibody, but were neurofilament negative. These findings raise the possibility of an extra-axonal function of B-50/GAP-43, as this protein might be secreted from regenerating axons and might play a role in axon-Schwann cell interactions during axonal maturation.     

Schwann cell-autonomous role of neuropilin-2

Neuropilins and group A plexins are components of receptor complexes for class 3 semaphorins, gradients of which help to guide migration of neural progenitor cells and axonal growth cones during development. We demonstrated previously that neuropilins and class 3 semaphorins are induced in sciatic nerve by crush or transection. We now report that in cultured rat Schwann cells, expression of mRNA encoding neuropilin-2 (NRP2) and plexin-A3 (PlexA3), proteins involved in semaphorin-3F (Sema3F) signal transduction, is diminished markedly by forskolin, an adenylate cyclase activator that, like axonal contact, induces Schwann cell synthesis of myelin lipids and proteins. Interestingly, Schwann cell expression of mRNA encoding NRP1, which participates in Sema3A signaling, is not downregulated by forskolin. Antibodies that recognize ectodomains of NRP2 but not control antibodies prevented cultured Schwann cells from aligning in parallel and forming columns. These results are consistent with the view that in nerves undergoing Wallerian degeneration, Schwann cell NRP2 facilitates assembly of Schwann cells into the tubular aggregates (bands of Büngner) that guide regenerating axons.     

Induction of postnatal schwann cell death by the low-affinity neurotrophin receptor in vitro and after axotomy.

Schwann cells express the low-affinity neurotrophin receptor (p75), but no role for either the neurotrophins or their cognate receptors in Schwann cell development has been established. We have found that Schwann cells isolated from postnatal day 1 (P1) or P2 mice that were p75-deficient exhibited potentiated survival compared to wild-type cells after growth factor and serum withdrawal. There was, however, no disparity in the survival of p75-deficient and wild-type Schwann cells isolated at embryonic day 15, suggesting that the death-inducing effects of p75 are developmentally regulated. A comparable degree of cell death was also observed in the sciatic nerves of both wild-type and p75-deficient mice at P1. However, 24 hr after axotomy, there was a 13-fold increase in the percentage of apoptotic nuclei in the distal nerve stumps of the transected sciatic nerves of neonatal wild-type but not p75-deficient mice. The expression of both the p75 and nerve growth factor (NGF) genes was upregulated after axotomy in neonatal wild-type nerves. Collectively, these results suggest that NGF-mediated activation of p75 is likely to be an important mediator of Schwann cell apoptosis in the context of peripheral nerve injury.     

NGFR-mRNA expression in sciatic nerve: a sensitive indicator of early stages of axonopathy

Expression of the low-affinity nerve growth factor receptor (NGFR) in the sciatic nerve (particularly Schwann cells) is high during development but is downregulated upon establishment of the mature axon-Schwann cell relationship. NGFR is re-expressed by Schwann cells if this relationship is altered by degeneration of axons (axotomy) or myelin (tellurium intoxication). To determine the sensitivity of NGFR expression to axonal injury, we have assayed NGFR-mRNA levels in proximal and distal regions of nerves exposed to the axonopathic agents acrylamide and isoniazid, as well as in proximal and distal stumps of axotomized nerves. NGFR-mRNA was elevated in all three models and correlated regionally with sites of axonal perturbation. In distal regions of acrylamide- and isoniazid-intoxicated nerves, NGFR-mRNA was elevated at least 2 days prior to visible signs of axonal degeneration as assayed by morphological techniques utilizing light microscopy. NGFR-mRNA was also elevated in proximal regions of axotomized and acrylamide-intoxicated nerves prior to signs of axonal degeneration. In these models, increased mRNA expression correlated with alterations in the size distribution of axonal cross sections. The common response in all of these situations indicates that NGFR expression, in addition to being a marker for axonal degeneration, is also a sensitive indicator of less profound perturbations in normal axon-Schwann cell interactions, including early stages of axonopathy. We suggest that assay for NGFR-mRNA may be utilized as a rapid and simple method (relative to more labor-intensive morphological methods) to screen for peripheral neurotoxicity. Additionally, regional analysis (distal versus proximal) may give insight into the sequence of events involved in the neuropathology of such disorders.     

Basic Behavior of Migratory Schwann Cells in Peripheral Nerve Regeneration

In axonal regeneration after a peripheral nerve injury, Schwann cells migrate from the two nerve ends and at last form a continuous tissue cable across the gap which guides the axons toward the bands of Büngner. However, the behavior of migratory Schwann cells and their possible role are obscure. Using a film model in which the proximal stump of a transected nerve in mice was sandwiched between two thin plastic films, we analyzed neural regeneration in the early phase up to the 6th day after axotomy. Regenerating neurites emerged from the nodes of Ranvier adjacent to the axotomized nerve stump within 3 h after axotomy and extended along the parent nerve onto the film. All of the regenerating neurites on the surface of the film consisted of naked axons for at least 2 days after axotomy. Thereafter, Schwann cells from the proximal nerve migrated along a network of the regenerating axons and then closely attached to the axons, ensheathing them. Some of the Schwann cells advanced ahead of the axonal growth cones and were distributed over regions in which axonal extension was not yet present. As calculated from the time course of regenerating neurites, the velocity of axonal regeneration showed two phases: an initial slow phase (77 μm/day) up to the 2nd post-operative day followed by a faster phase (283 μm/day). The first observation of Schwann cells coincided with the onset of the second phase. In addition, the length of regenerating axons on the surface of the film containing many Schwann cells was significantly greater than that on the surface where Schwann cells were not yet present. It meant that migratory Schwann cells stimulated axons to elongate for a longer distance. Furthermore, Schwann cells from a distal stump showed a stronger ability to accelerate the axonal outgrowth than these from a proximal stump.     

Synthesis of progesterone in Schwann cells: regulation by sensory neurons

In peripheral nerves, progesterone synthesized by Schwann cells has been implicated in myelination. In spite of such an important function, little is known of the regulation of progesterone biosynthesis in the nervous system. We show here that in rat Schwann cells, expression of the 3 beta-hydroxysteroid dehydrogenase and formation of progesterone are dependent on neuronal signal. Levels of 3 beta-hydroxysteroid dehydrogenase mRNA and synthesis of [3H]progesterone from [3H]pregnenolone were low in purified Schwann cells prepared from neonatal rat sciatic nerves. However, when Schwann cells were cultured in contact with sensory neurons, both expression and activity of the 3 beta-hydroxysteroid dehydrogenase were induced. Regulation of 3 beta-hydroxysteroid dehydrogenase expression by neurons was also demonstrated in vivo in the rat sciatic nerve. 3 beta-hydroxysteroid dehydrogenase mRNA was present in the intact nerve, but could no longer be detected 3 or 6 days after cryolesion, when axons had degenerated. After 15 days, when Schwann cells made new contact with the regenerating axons, the enzyme was re-expressed. After nerve transection, which does not allow axonal regeneration, 3 beta-hydroxysteroid dehydrogenase mRNA remained undetectable. The regulation of 3 beta-hydroxysteroid dehydrogenase mRNA after lesion was similar to the regulation of myelin protein zero (P0) and peripheral myelin protein 22 (PMP22) mRNAs, supporting an important role of locally formed progesterone in myelination.     

Osteopontin: a novel axon-regulated Schwann cell gene.

Osteopontin (OPN) is a RGD-containing glycoprotein with cytokine-like, chemotactic, and pro-adhesive properties. During wound healing, OPN is abundantly expressed by infiltrating macrophages and has been implicated in posttraumatic tissue repair. To delineate a role in the regenerative response to axotomy we examined the expression of OPN in Wallerian degeneration of the sciatic nerve in rats. Unexpectedly, we found high constitutive expression of OPN by myelinating Schwann cells (SCs) in uninjured control nerves. OPN mRNA expression was confirmed in primary cultures of rat SCs. Upon axotomy, SC-expressed OPN in the degenerating distal nerve stump transiently increased during the first days after injury, but was continuously downregulated thereafter, reaching its minimum at Day 14. Macrophages invading axotomized nerves were OPN-negative. During late stages after axotomy, SC-OPN was reexpressed in regenerating but not permanently transected nerves. We also found OPN expression by myelinating SCs in human sural nerves with a dramatic reduction in severe axonal polyneuropathies. Taken together, our study identifies OPN as a novel Schwann cell gene regulated by axon-derived signals. The lack of OPN induction in infiltrating macrophages indicates fundamental differences in tissue repair between axonal injury in the peripheral nervous system and structural lesions in other organ systems.     

Dissociated neurons regenerate into sciatic but not optic nerve explants in culture irrespective of neurotrophic factors

Explants of adult or 10-day-old rat sciatic and optic nerves were implanted as "bridges" through a silicon grease seal in a three-compartment chamber culture system, leading from a narrow center chamber to two adjacent side chambers. Dissociated newborn rat sympathetic or sensory neurons were plated into the center chamber and grown in the presence of optimal concentrations of nerve growth factor (NGF). By light microscopy, nerve fibers were seen to grow out of the sciatic nerve explants in the side chambers after 2 to 3 weeks. Electron microscopy showed large numbers of axons present inside the sciatic nerves, irrespective of the presence and number of living Schwann cells. Besides their tendency to fasciculate, axons grew with high preference on Schwann cell membranes and the Schwann cell side of the basal lamina, a situation identical to in vivo regeneration. In contrast to the sciatic nerves, no axons could be found under any condition in the optic nerves. This result points to the existence of extremely poor, non-permissive substrate conditions in the differentiated optic nerves which cannot be overcome by the strong fiber outgrowth-promoting effects of NGF.     

Axonal signals regulate the differentiation of non-myelin-forming Schwann cells: an immunohistochemical study of galactocerebroside in transected and regenerating nerves

Little is known about the factors involved in directing and maintaining the divergent differentiation of the 2 major Schwann cell variants, myelin and non-myelin-forming cells, in peripheral nerves. There is strong evidence that the differentiation of myelin-forming cells depends critically on cell-cell signaling through contact with appropriate axons. In this paper we ask whether this remarkable dependence of the Schwann cell on axonal contact for full differentiation is unique to those cells that form myelin or whether axonal signaling is also an important factor in the differentiation of non-myelin-forming Schwann cells. Sciatic nerves or cervical sympathetic trunks of adult rats were either transected or crushed and the axons allowed to degenerate and, in the case of crushed nerves, to regenerate into the distal stump for periods of time varying from 2 d to 9 weeks. The distal stump of the nerve was excised at specific times, the Schwann cells dissociated and immunolabeled with antibodies to galactocerebroside. In the sciatic nerve, which contains a mixture of non-myelin-forming and myelin-forming Schwann cells, transection resulted in a loss of galactocerebroside expression from the surface of all the Schwann cells in the distal stump over a 9 week period, irrespective of their original phenotype. In crushed sciatic nerves, where axons were allowed to regrow into the distal stumps, the number of Schwann cells expressing immunohistochemically detectable quantities of galactocerebroside in the stump declined over the first 3 weeks, but by 9 weeks after crush the total percentage of galactocerebroside-positive cells in the nerve had risen to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Axons modulate the expression of transforming growth factor-betas in Schwann cells

We have investigated the expression of transforming growth factor (TGF)-β1,-β2, and -β3 in developing, degenerating, and regenerating rat peripheral nerve by immunohistochemistry and Northern blot analysis. In normal adult sciatic nerve, TGF-β1, -β2, and -β3 are detected in the cytoplasm of Schwann cells, and the levels of TGF-β1 and -β3 mRNAs are constant during post-natal development. When sciatic nerves are transected to cause axonal degeneration and prevent axonal regeneration, the level of TGF-β1 mRNA in the distal nerve-stump increases markedly and remains elevated, whereas the level of TGF-β3 mRNA falls modestly and remains depressed. When sciatic nerves are crushed to cause axonal degeneration and allow axonal regeneration, the level of TGF-β1 mRNA initially increases as axons degenerate, and then falls as axons regenerate. TGF-β2 mRNA was not detected in developing or lesioned sciatic nerves at any time. Cultured Schwann cells have high levels of TGF-β1 mRNA, the amount of which is reduced by forskolin, which mimicks the effect of axonal contact. These data demonstrate that Schwann cells express TGF-β1, -β2, and -β3, and that TGF-β1 and -β3 mRNA predominate over TGF-β2 mRNA in peripheral nerve. Axonal contact and forskolin decrease the expression of TGF-β1 in Schwann cells. © 1993 Wiley-Liss, Inc.     

Morphological evidence for a transport of ribosomes from Schwann cells to regenerating axons

Recently, we showed that Schwann cells transfer ribosomes to injured axons. Here, we demonstrate that Schwann cells transfer ribosomes to regenerating axons in vivo. For this, we used lentiviral vector-mediated expression of ribosomal protein L4 and eGFP to label ribosomes in Schwann cells. Two approaches were followed. First, we transduced Schwann cells in vivo in the distal trunk of the sciatic nerve after a nerve crush. Seven days after the crush, 12% of regenerating axons contained fluorescent ribosomes. Second, we transduced Schwann cells in vitro that were subsequently injected into an acellular nerve graft that was inserted into the sciatic nerve. Fluorescent ribosomes were detected in regenerating axons up to 8 weeks after graft insertion. Together, these data indicate that regenerating axons receive ribosomes from Schwann cells and, furthermore, that Schwann cells may support local axonal protein synthesis by transferring protein synthetic machinery and mRNAs to these axons.     

Long-term endoneurial changes after nerve transection

Summary Long-term endoneurial changes in the distal stump of transected rat sciatic nerve were examined from 8 to 50 weeks after nerve transection. The morphological alterations were followed both in nerves which were allowed to regenerate and in nerves in which regeneration was prevented by suturing. The nerves prevented from regenerating showed markedly atrophied Schwann cell columns after 20 weeks and a disappearance of some Schwann cell columns after 30 weeks. The surrounding endoneurial fibroblast-like cells gradually lost their delicate cytoplasmic extensions and formed rough fascicles around numerous shrunken Schwann cell columns or around areas from which Schwann cells had apparently disappeared. Inside the fascicles, the Schwann cell loss was replaced by collagen fibrils or occasionally, by a dense accumulation of microfibrils. The loss of endoneurial cytoplasmic processes continued up to 50 weeks, leaving behind patches of thin fibrils around numerous shrunken Schwann cell columns or around collagenous areas where Schwann cells were lost. The endoneurial matrix showed presence of thin 25- to 30-nm collagen fibrils close to shrunken Schwann cell columns up to 50 weeks but in areas with advanced degeneration a shift towards regular 50- to 60-nm collagen fibrils occurred. The degenerated areas resembled those described in Renaut bodies and neurofibromas. Despite suturing of transected nerves to prevent sprouting, occasional regenerating sprouts were noted in the Schwann cell columns. These axons were surrounded in a sheath-like fashion by pre-existing endoneurial cell fascicles covered by a basal lamina. In the reinnervating nerves the endoneurial space gradually lost its compartmentized structures consisting of collagen fibrils and endoneurial fibroblast-like cells. After 20 weeks the endoneurial cells were inconspicuous and the extracelluar matrix consisted mainly of 50- to 60-nm collagen fibrils. During axonal growth and maturation, Schwann cells containing unmyelinated axons surrounded large, myelinated axons in a collar-like fashion. Close to these collars of Schwann cells, thin 25- to 30-nm collagen fibrils were noted in focal areas, even after 50 weeks. Occasionally, numerous clusters of regenerating axonal sprouts were noted in the perineurium. These were surrounded by multiple layers of cells possessing basal lamina. The present results show that after nerve transection the distal stump of the severed nerve shows dynamic changes in the endoneurial space, especially in nerves where reinnervation is prevented. The endoneurial fascicles around occasional axonal sprouts in sutured nerves, representing possibly a delayed type of regeneration, show that axons have a strong ability to grow but on the other hand endoneurial structures are unable to respond normally to axonal growth after advanced degeneration.     

β4 integrin and other Schwann cell markers in axonal neuropathy

Schwann cell gene expression is dynamically regulated after peripheral nerve injury and during regeneration. We hypothesized that the changes in protein expression described after rat peripheral nerve injury could be used to identify single Schwann cell-axon units in human axonal neuropathy. Therefore, we performed immunofluorescence staining on sections of injured rat sciatic nerves compared with sections of neuropathic human sural nerves. We chose the markers β4 integrin, P0 glycoprotein, and glial fibrillary acidic protein (GFAP) to characterize Schwann cells, and neurofilament-heavy (NF-H) to recognize axons. Normal rat or human myelin-forming units demonstrated a sharp ring of β4 staining at their outer surface, P0 staining in the myelin sheath, and NF-H staining in the axon. Acutely denervated rat units transited from broken rings of β4 and P0 staining, to diffuse β4 and absent P0 and NF-H staining. Chronically denervated rat Schwann cells re-expressed β4 more highly, but in a diffuse, non-polarized pattern. In contrast, regenerating units re-expressed β4, P0, and NF-H; β4 staining was polarized to the outer surface of Schwann cells. Finally, GFAP staining increased progressively after injury and decreased during regeneration in the distal nerve stump. In neuropathic human sural nerves, we identified units exhibiting each of these β4, P0, and NF-H staining patterns; the proportion of each pattern correlated best with the extent and chronicity of axonal injury. Thus, synchronous injury of rat sciatic nerve predicts patterns of Schwann cell marker expression in human axonal neuropathy. In addition, the unique changes in the polarity of β4 integrin expression, in combination with changes in P0 and NF-H expression, may distinguish normal from denervated or reinnervated myelin-forming Schwann cells in human sural nerve biopsies. © 1996 Wiley-Liss, Inc.