Influence of non-neuronal cells on regeneration of the rat sciatic nerve

The ability of the rat sciatic nerve to regenerate into a previously frozen distal nerve segment was studied and compared to regeneration after a crush lesion. The regeneration rate in the frozen segment was 1.9 mm/day, which was approximately half of that observed after a crush lesion (3.3 mm/day). If an unfrozen nerve segment was left intact beyond the frozen section, the rate of regeneration increased to 3.2 mm/day. However, a fresh nerve segment sutured along the frozen segment did not significantly affect the rate of regeneration. Incorporation of [3H]thymidine in the regenerating nerve, analyzed after 1, 3 and 6 days, showed an increased labelling in the frozen segment. This increase spread from the proximal nerve segment into the frozen section. In nerves where a segment was left intact beyond the frozen section, [3H]thymidine incorporation was seen to enter the frozen section from both sides. The spreading of [3H]thymidine incorporation appeared to correlate with the rate of regeneration. However, the same pattern of incorporation could be observed in nerves where regeneration was detained by a transection. The results suggest that Schwann and/or other cells which invade the frozen nerve segment affect the rate of axonal elongation, and that the migration of these cells occurs independently of regenerating fibers.     

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.     

IGF-I expression is decreased in LIF-deficient mice after peripheral nerve injury

We investigated the regulation of insulin-like growth factor 1 (IGF-1) expression after sciatic nerve crush using leukemia inhibitory factor (LIF)-deficient mice. One day post-crush, IGF-1 mRNA levels were lower in the LIF-deficient mouse nerve than in the wild type nerve. IGF-1 protein, analyzed by immunohistochemistry, was also decreased 1 day post-crush in LIF-deficient nerves relative to wild type nerves. By 3 days post-crush, IGF-1 immunoreactivity was induced in Schwann cells to equivalent levels in both types of nerve. After crush, IGF-1 expression was also found in mast cells, and these were initially decreased in the LIF-deficient mice. Thus, LIF appears to regulate IGF-1 expression in the peripheral nerve basally and early in the regeneration response in vivo.     

Adhesion and proliferation are enhanced in vitro in Schwann cells from nerve undergoing Wallerian degeneration

Proliferation of Schwann cells during nerve degeneration or regeneration is well documented in vivo. We investigated whether the proliferative response of Schwann cells to injury is retained in vitro. Using 5-month-old male C57BL mice, Schwann cells were isolated from sciatic nerves under 3 experimental conditions: (1) uninjured, (2) after permanent nerve-transection, or (3) after nerve-crush, which permits axonal regeneration. Schwann cells rarely attached to polylysine-coated coverslips when isolated from uninjured or 1 day posttransection/crush nerves. The number of adherent cells increased when Schwann cells were isolated 3 days after nerve-transection or -crush. When cells were isolated from transected nerves, cell adhesion reached a peak 2 weeks after the injury and then declined. Maximal attachment of Schwann cells occurred when the cells were isolated 2-4 weeks after nerve-crush. The percentage of Schwann cells with spreading processes corresponded closely with the number of thymidine-labeled cells at 1 day in vitro. The in vitro capacity of cells to spread and incorporate thymidine reached maximal levels at 5 days posttransection/crush. Capacity of cells to spread and incorporate thymidine subsequently decreased with time following transection. However, a biphasic elevation in cell spreading and thymidine incorporation was observed in Schwann cells isolated from crushed nerves. Maximal growth of Schwann cells in vitro occurred at 1-2 weeks posttransection and at 1-4 weeks postcrush. Adhesion and spreading of Schwann cells were promoted by coating coverslips with laminin or fibronectin. Preincubation of Schwann cells with soluble laminin or fibronectin prevented the initial cell attachment induced by the corresponding protein. Our results suggest that Schwann cells from injured nerves possess binding sites for laminin and fibronectin, which are, in part, responsible for the enhanced adhesion of Schwann cells in vitro. This study provides a new method for preparation of Schwann cells from peripheral nerves of adult mice.     

The growth of motoneurones and their neurites in relation to Schwann cells harvested from sciatic nerve

A study has been made of the effects of Schwann cells isolated from neonatal sciatic nerve on motoneurones in culture. Motoneurones were identified in dissociated spinal cord cultures from 6-day avian embryos by prior retrograde labelling with rhodamine-latex microspheres. Schwann cells trebled the expression of long neurites in these motoneurones over that in control media as well as maintaining them viable for 24 h. A transformed Schwann cell line, RN22, produced similar results. These effects were mediated by a soluble factor(s) released from the Schwann cells which was distinct from nerve growth factor. Schwann cells exerted this initiation of neurites on homogeneous cultures of motoneurones, indicating that other spinal cord cells are not necessary to mediate this effect. The observations are discussed in terms of the hypothesis that Schwann cells guide motor axons during regeneration and formation of limb and muscle nerves.     

Laminin molecules in freeze-treated nerve segments are associated with migrating Schwann cells that display the corresponding alpha6beta1 integrin receptor

Isolated acellular nerve segments protected from migration of Schwann cells and the acellular nerve segments joined with the distal nerve stumps were prepared by a repeated freeze-thaw procedure in the rat sciatic nerves. The presence of laminin-1 and -2, as well as alpha6 and beta1 integrin chains, was detected by indirect immunohistochemistry in the sections through acellular nerve segments at 7 and 14 days after cryotreatment. The position of basal laminae and Schwann cells was identified by immunostaining for collagen IV and S-100 protein, respectively. The isolated cryo-treated segment without living Schwann cells (S-100-) did not display immunoreactivity for laminins and integrin chains, while the basal lamina position was verified through the whole segment by immunostaining for collagen IV. The absence of immunostaining for laminin-1 and -2 in cryo-treated nerve segment was verified by Western blot analysis. A crucial diminution of laminin-1 and -2 in the cryo-treated nerve segment of 10-mm length did not abolish the growth and maturation of axons. The greater part of nerve segment connected with the nerve stump displayed no immunohistochemical staining for S-100, corresponding with absence of Schwann cells. The border region of the nerve segment contained Schwann cells (S-100+) migrating from the near-freeze undamaged part of the distal nerve stump. In addition to immunostaining for S-100 protein, the migrating Schwann cells displayed immunostaining for laminins (-1, and -2) and integrin chains (alpha6 and beta1). The results indicate that the presence of laminin molecules in the acellular nerve segments prepared by the repeated freeze-thaw procedure is related with the migrating Schwann cells. The immunostaining for laminins and integrin chains, which constitute one of integrin receptor, suggests an autocrine and/or paracrine utilization of laminin molecules in the promotion of Schwann cell migration.     

Gene transfer to schwann cells after peripheral nerve injury: A delivery system for therapeutic agents

We transferred a reporter gene to Schwann cells to test whether they might serve as an endoneurial delivery system for therapeutic proteins. A replication-defective adenoviral vector carrying the gene for β-galactosidase (lacZ) was injected into the distal segment of intact or crushed sciatic nerves of adult rats, and the expression of lacZ was histochemically assessed. Less than 1% of the Schwann cells became reactive in intact nerves, but up to 18% of the proliferating Schwann cells of injured nerves expressed lacZ. Gene expression decayed with time but might persist for up to 2 months. It was enhanced by immunosuppression: daily cyclosporin A injections reduced both proliferation of Schwann cells and lymphocytic infiltration of the nerve, whereas tolerance induced by a single intrathymic injection of the vector 4 days after birth abolished the inflammatory response but not the proliferation of Schwann cells. The vector itself did not impede axonal regeneration. The results indicate that adenoviral gene transfer to Schwann cells in injured nerves is possible and suggest that induced production of neurotrophic factor may represent a therapeutic supplement to surgical nerve repair.     

Delayed Immediate Early Gene Responses Precede Delayed Initiation Of Regeneration In Diabetic Nerve

Nerve fiber regeneration is impaired in diabetic nerve and contributes to the relentless nerve fiber loss characterizing this disorder. Immediate early gene responses constitute the initial response to nerve injury and include upregulation of NGF and IGF-1 primarily by Schwann cells. These responses are believed to initiate macrophage recruitment necessary for initiation of axonal regeneration. We examined NGF, IGF-1 and CNTF mRNA in sciatic nerve at 10 timepoints (0.5hr to 24d) following sciatic nerve crush in diabetic BB/W-rats. The peak of the immediate upregulation of IGF-1 and NGF occurred at 0.5 and 6 hrs respectively in control nerves and was delayed to 24 hrs and 2d for IGF-1 and NGF respectively in diabetic nerve. Also the expression of NGF p75 receptor was significantly attenuated in diabetic nerve. CNTF mRNA showed an immediate downregulation following nerve crush with no significant differences between control and diabetic rats. These findings suggest that attenuations of the immediate gene responses of para- and autocrine IGF-1 and NGF in diabetic nerve may be responsible for the earlier reported defect in macrophage recruitment and delayed initiation of nerve fiber 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.     

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.     

Growth of axons into developing muscles of the chick forelimb is preceded by cells that stain with Schwann cell antibodies

A study has been made of the development of limb and muscle nerves in relation to the first appearance of Schwann cells in the flexor digitorum profundus (fdp) and flexor carpi ulnaris (fcu) muscles of the avian forelimb. Schwann cells were identified by immunofluorescent techniques with antibodies to the glycoprotein HNK-1. Myotubes and nerves were identified by using antibodies to myosin and to neurofilament, respectively. At stage 24/25 the brachialis longus inferior (Bli n) and superior (Bls n) nerve trunks within proximal regions of the forelimb were surrounded by Schwann cells. These cells extended in a column for a distance of approximately 100 microns beyond the growing ends of nerves. At stage 26 both interosseus nerve (in n) and the medial-ulnar nerve (m-u n) had formed from the Bli n; each of these branches was surrounded by Schwann cells, which again extended approximately 100 microns beyond the growing ends of the nerves. By stage 26/27 the fdp and fcu muscles were clearly delineated by groups of myotubes. No nerves were detected within these groups; however, Schwann cells were observed between the myotubes. At stage 27 axons had left the in n and m-u n and grown into the fdp and fcu muscles, respectively. These axons were surrounded by Schwann cells. The present observations show that Schwann cells are located ahead of the main limb and muscle nerves as they grow into the fdp and fcu muscles of the limb. It is possible that these Schwann cells play a role in guiding nerves to their correct muscles in the developing chick forelimb.     

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.     

Influence of laminin-2 on Schwann cell-axon interactions

The dy/dy mouse suffers from a form of muscular dystrophy caused by a substantial reduction in laminin alpha2-chain protein, a major component of both muscle and Schwann cell basal laminae. This article examines the effect of laminin alpha2 deficiency on Schwann cell-axon interactions both in vivo at varying intervals after nerve crush, and in vitro, in cocultures of neurons and Schwann cells. The morphological spectrum of aberrant Schwann cell-axon associations seen in uncrushed dy/dy sciatic nerves was recapitulated during regeneration: myelination of regenerating axons was delayed compared with the process in unaffected mice and the relatively few myelin sheaths which were formed in dy/dy distal nerve stumps were often uncompacted. In vitro, Schwann cells dissociated from adult dy/dy sciatic nerves predictably failed to express detectable laminin alpha2-chain and displayed an unusual multipolar morphology. Branching of neurites, in terms both of numbers of terminal branches and of complexity of branching, from dorsal root ganglia neurons grown on dy/dy Schwann cells, was significantly less extensive than that seen when neurons were cocultured with Schwann cells from unaffected littermates, but this effect was reversed by exogenous laminin-2. Our results lend strong support to the view that laminin-2 is essential for establishing and/or maintaining Schwann cell-axon interactions, in normal and in regenerating nerves.     

Nerve transplantation shows that motor end-plate disease is not a primary Schwann cell defect

Motor end-plate diseased (MED) mice have altered nerve impulse conduction velocities and refractory periods. To test whether these pathological properties are caused by a primary Schwann cell defect, nerves were transplanted from MED and wildtype (WT) animals onto WT recipients. The donor origin of cells in the regenerated nerve was assessed by prelabeling with [3H]thymidine and by electrophoretic analysis of glucose phosphate isomerase allotypes. Nerve fiber regeneration through MED and WT implants was equally efficient. No difference was found in nerve conductivities of MED and WT grafts. Therefore a primary defect in the Schwann cells of the MED mouse is unlikely.     

Insulin and the insulin-like growth factors I and II are mitogenic to cultured rat sciatic nerve segments and stimulate [3H]thymidine incorporation through their respective receptors

The factors that control proliferation of Schwann cells during peripheral nerve regeneration are not yet known. In this study we investigated the effects of insulin, insulin-like growth factor I and II (IGF-I and IGF-II), IGF-I analogues, and factors that interfere with their respective receptors, on [³H]thymidine incorporation into cultured nerve segments from the rat sciatic nerve. Segments cultured in nM (0.1–1.7 nM) concentrations of insulin, truncated IGF-I (tIGF-I), long R³IGF-I, or IGF-II exhibited an increase in [³H]thymidine incorporation compared with control segments. IGF-II was most potent. JB1, an IGF-I antagonist, counteracted the effects of tIGF-I and insulin. The results suggest that non-neuronal cells in the nerve segment, probably Schwann cells, possess distinct receptors for insulin, IGF-I, and IGF-II and that these receptors may be involved in the control of Schwann cell proliferation during peripheral nerve regeneration. © 1996 Wiley-Liss, Inc.     

P0 gene expression in Schwann cells is modulated by an increase of cAMP which is dependent on the presence of axons.

The role of cAMP in the regulation of P0 gene expression was investigated in Schwann cells of normal, regenerated, and permanently transected rat sciatic nerve. Forskolin treatment of endoneurial segments of rat sciatic nerve resulted in increased cAMP and P0 mRNA levels in normal and regenerated nerves but not in permanently transected nerves, where axonal regeneration is prevented. This increase of cAMP and P0 mRNA occurred within 30 and 90 min, respectively. P0 mRNA levels in the endoneurial segment of the permanently transected nerve were not increased with dibutyryl cAMP. The Schwann cells of the permanently transected nerve, however, retained the ability to myelinate 15 embryonic day (E15) dorsal root ganglia (DRG) neuron and neurite networks cultured in vitro. P0 mRNA levels increased within 4 days in transected endoneurium segments cocultured with E15 DRG neurons and neurites and further increased in 21 day myelinating cocultures. Although cAMP was not detectable in 4 day cocultures, it increased to detectable levels in 21 day cultures, suggesting that cAMP is involved in the myelinating process. These results indicate that the presence of the axon is required for the observed increase of cAMP and P0 mRNA levels and suggest that the increase of cAMP occurs within the axon which then presumably activates a different Schwann cell second messenger pathway to induce P0 gene expression.     

Membrane-bound CSPG mediates growth cone outgrowth and substrate specificity by Schwann cell contact with the DRG neuron cell body and not via growth cone contact

The central nervous system and peripheral nervous system (CNS/PNS) contain factors that inhibit axon regeneration, including myelin-associated glycoprotein (MAG), the Nogo protein, and chondroitin sulfate proteoglycan (CSPG). They also contain factors that promote axon regeneration, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Axon regeneration into and within the CNS fails because the balance of factor favors inhibiting regeneration, while in the PNS, the balance of factor favors promoting regeneration. The balance of influences in the CNS can be shifted toward promoting axon regeneration by eliminating the regeneration-inhibiting factors, overwhelming them with regeneration-promoting factors, or making axon growth cones non-receptive to regeneration-inhibiting factors. The present in vitro experiments, using adult rat dorsal root ganglion (DRG) neurons, were designed to determine whether the regeneration-inhibiting influences of Schwann cell CSPG are mediated via Schwann cell membrane contact with the DRG neuron cell body or their growth cones. The average longest neurite of neurons in cell body contact with Schwann cells was 7.4-fold shorter than those of neurons without Schwann cell-neuron cell body contact (naked neurons), and the neurites showed substrate specificity, growing only on the Schwann cell membranes and not extending onto the laminin substrate. The neurites of naked neurons showed no substrate specificity and extended over the laminin substrate, as well as onto and off the Schwann cells. After digesting the Schwann cell CSPG with the enzyme C-ABC, neurons in cell body contact with Schwann cells extended neurites the same length as those of naked neurons, and their neurites showed no substrate selectivity. Further, the neurites of naked neurons were not longer than those of naked neurons not exposed to C-ABC. These data indicate that the extent of neurite outgrowth from adult rat DRG neurons and substrate specificity of their growth cone is mediated via contact between the Schwann cell membrane-bound CSPG and the DRG neuron cell body and not with their growth cones. Further, there was no apparent influence of diffusible or substrate-bound CSPG on neurite outgrowth. These results show that eliminating the CSPG of Schwann cells in contact with the cell body of DRG neurons eliminates the sensitivity of their growth cones to the CSPG-induced outgrowth inhibition. This may in turn allow the axons of these neurons to regenerate through the dorsal roots and into the spinal cord.     

PC12 neurite regeneration and long-term maintenance in the absence of exogenous nerve growth factor in response to contact with Schwann cells

We have investigated mouse and rat ganglionic Schwann cells as possible sources of neurite outgrowth-promoting factors by co-culturing Schwann cells with nerve growth factor (NGF)-responsive PC12 pheochromocytoma cells primed by pretreatment with NGF. NGF-primed PC12 cells are capable of neurite regeneration when provided with an appropriate neurite promoting factor such as NGF. When primed PC12 cells were co-cultured with Schwann cells in the absence of exogenous NGF, PC12 cells that directly contacted Schwann cells became enlarged and flattened, attaining a neuron-like morphology within one day. When contact with Schwann cells was established, PC12 cells regenerated neurites by the first day of co-culture and these were maintained throughout the experiments (7 weeks). Most PC12 cells cultured in the same collagen-coated dishes with Schwann cells, but not directly in contact with them, failed to regenerate neurites. Instead, they began to proliferate, forming cell clusters. Neurite regeneration by PC12 cells in contact with Schwann cells was not blocked by antibody to NGF. These results demonstrate the presence of a neurite-promoting activity localized to the vicinity of the Schwann cell surface which is capable of eliciting regeneration and long-term maintenance of PC12 neurites in the absence of exogenous NGF. This activity does not appear to be due to NGF.     

The fate of Schwann cell basement membranes in permanently transected nerves

After peripheral nerve fiber degeneration, Schwann cell basement membranes (SCBM) persist, maintain the columnar orientation of multiplying Schwann cells, and provide pathways for regenerating axons to original target tissue. Despite the putative importance of these functions, there is little quantitative information on SCBM in chronic denervation. The number, integrity, shape, and size of SCBM were evaluated in transverse electron micrographs of peroneal nerves of groups of mice at various times after permanent sciatic nerve transection. With increasing time after transection, the SCBM fragment, the fragments become shorter, dispersed throughout the endoneurium and partially disappear. The discontinuity, dispersion, and partial disappearance of SCBM, which worsens with time after nerve transection, alters the scaffold by which neurites grow back to formerly innervated or appropriate target tissue. Without reaching appropriate target tissue, regenerating fibers may fail to develop or undergo retrograde atrophy and degeneration. The progressive changes of the SCBM may contribute to the demonstrated poor regeneration when nerve reconnection is delayed, or when proximal nerves are reconnected, so that much time may elapse before neurites grow back to distal nerve.