Active Src expression is induced after rat peripheral nerve injury

The non-receptor-type Src tyrosine kinases are key components of intracellular signal transduction that are expressed at high levels in the nervous system. To improve understanding of the cascades of molecular events underlying peripheral nerve regeneration, we analyzed active Src expression in the crushed or cut rat sciatic nerves using a monoclonal antibody (clone 28) that recognizes the active form of Src tyrosine kinases, including c-Src and c-Fyn. Western blots showed that active Src expressed in the normal sciatic nerve transiently increased up to threefolds after both types of injury. Immunohistochemistry using clone 28 showed that axonal components are the primary sites of active Src expression in the normal sciatic nerve. Soon after both types of injury, active Src was abundantly expressed in Schwann cells of the segments distal to the injury site. The expression of active Src in the cells decreased with restoration of the axon-Schwann cell relationship and eventually became depleted to very low levels after crushing, but was sustained at high levels in the cut model until the end of the experiment. Regenerated axons consistently expressed active Src throughout nerve regeneration and these eventually became the major sites of active Src expression in the crushed nerve. Among the Src tyrosine kinases, active c-Src selectively increased after crushing according to immunoprecipitation and immunoblotting analyses. Due to its potent biological activity, the increased amounts of the active form of Src probably enhance axonal regrowth, the Schwann cell response, and axon-Schwann cell contact for peripheral nerve regeneration.     

Transforming growth factor beta 1, a cytokine with regenerative functions

We review the biology and role of transforming growth factor beta 1(TGF-β1) in peripheral nerve injury and regeneration, as it relates to injuries to large nerve trunks(i.e., sciatic nerve, brachial plexus), which often leads to suboptimal functional recovery. Experimental studies have suggested that the reason for the lack of functional recovery resides in the lack of sufficient mature axons reaching their targets, which is a result of the loss of the growth-supportive environment provided by the Schwann cells in the distal stump of injured nerves. Using an established chronic nerve injury and delayed repair animal model that accurately mimics chronic nerve injuries in humans, we summarize our key findings as well as others to better understand the pathophysiology of poor functional recovery. We demonstrated that 6 month TGF-β1 treatment for chronic nerve injury significantly improved Schwann cell capacity to support axonal regeneration. When combined with forskolin, the effect was additive, as evidenced by a near doubling of regenerated axons proximal to the repair site. We showed that in vivo application of TGF-β1 and forskolin directly onto chronically injured nerves reactivated chronically denervated Schwann cells, induced their proliferation, and upregulated the expression of regeneration-associated proteins. The effect of TGF-β1 and forskolin on old nerve injuries is quite impressive and the treatment regiment appears to mediate a growth-supportive milieu in the injured peripheral nerves. In summary, TGF-β1 and forskolin treatment reactivates chronically denervated Schwann cells and could potentially be used to extend and prolong the regenerative responses to promote 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.     

Biodegradable magnesium wire promotes regeneration of compressed sciatic nerves

Magnesium(Mg) wire has been shown to be biodegradable and have anti-inflammatory properties. It can induce Schwann cells to secrete nerve growth factor and promote the regeneration of nerve axons after central nervous system injury. We hypothesized that biodegradable Mg wire may enhance compressed peripheral nerve regeneration. A rat acute sciatic nerve compression model was made, and AZ31 Mg wire(3 mm diameter; 8 mm length) bridged at both ends of the nerve. Our results demonstrate that sciatic functional index, nerve growth factor, p75 neurotrophin receptor, and tyrosine receptor kinase A m RNA expression are increased by Mg wire in Mg model. The numbers of cross section nerve fibers and regenerating axons were also increased. Sciatic nerve function was improved and the myelinated axon number was increased in injured sciatic nerve following Mg treatment. Immunofluorescence histopathology showed that there were increased vigorous axonal regeneration and myelin sheath coverage in injured sciatic nerve after Mg treatment. Our findings confirm that biodegradable Mg wire can promote the regeneration of acute compressed sciatic nerves.     

Synthesis of leukemia inhibitory factor in injured peripheral nerves and their cells

The time and site of induction of leukemia inhibitory factor mRNA in injured rat sciatic nerves and its regulation in Schwann cells and fibroblasts from neonatal rat nerves were investigated. Leukemia inhibitory factor mRNA is induced at the lesion site within 6 h of sciatic nerve transection but only after 24 h in the more distal segments. In vitro, interleukin-1β increases the concentration of leukemia inhibitory mRNA in nerve fibroblasts but not in Schwann cells. Changes in leukemia inhibitory factor mRNA concentration in injured nerves and peripheral nerve cells are similar to those for nerve growth factor mRNA.     

Expression and glycanation of the NG2 proteoglycan in developing, adult, and damaged peripheral nerve

We have investigated expression of the axon growth-inhibitory proteoglycan NG2 in peripheral nerve. In the adult, NG2 was present on endoneurial and perineurial fibroblasts, but not on Schwann cells. At birth, peripheral nerve NG2 was heavily glycanated, but was much less so in the adult. In vitro, sciatic nerve fibroblasts also produced heavily glycanated NG2. After peripheral nerve injury in rats and humans, an accumulation of NG2-positive cells was observed at the injury site. In the rat, there was an increase in NG2 glycanation for at least 2 weeks following injury. In mixed cultures of Schwann cells and peripheral nerve fibroblasts, the axons preferred to grow on the Schwann cells and seldom crossed onto the fibroblasts. Three-dimensional cultures of sciatic nerve fibroblasts were inhibitory to the growth of dorsal root ganglion axons. Inhibition of proteoglycan synthesis made the cells more permissive. NG2 may play a part in blocking axon regeneration through scar tissue in injured human peripheral nerve.     

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.     

Endogenous BDNF is required for myelination and regeneration of injured sciatic nerve in rodents

Following a peripheral nerve injury, brain-derived neurotrophic factor (BDNF) and the p75 neurotrophin receptor are upregulated in Schwann cells of the Wallerian degenerating nerves. However, it is not known whether the endogenous BDNF is critical for the functions of Schwann cells and regeneration of injured nerve. Treatment with BDNF antibody was shown to retard the length of the regenerated nerve from injury site by 24%. Histological and ultrastructural examination showed that the number and density of myelinated axons in the distal side of the lesion in the antibody-treated mice was reduced by 83%. In the BDNF antibody-treated animals, there were only distorted and disorganized myelinated fibres in the injured nerve where abnormal Schwann cells and phagocytes were present. As a result of nerve degeneration in BDNF antibody-treated animals, subcellular organelles, such as mitochondria, disappeared or were disorganized and the laminal layers of the myelin sheath were loosened, separated or collapsed. Our in situ hybridization revealed that BDNF mRNA was expressed in Schwann cells in the distal segment of lesioned nerve and in the denervated muscle fibres. These results indicate that Schwann cells and muscle fibres may contribute to the sources of BDNF during regeneration and that the deprivation of endogenous BDNF results in an impairment in regeneration and myelination of regenerating axons. It is concluded that endogenous BDNF is required for peripheral nerve regeneration and remyelination after injury.     

4S RNA is transported axonally in normal and regenerating axons of the sciatic nerves of rats

Experiments were designed to determine if following injection of [³H]uridine into the lumbar spinal cord of the rat, [³H]RNA could be demonstrated within axons of the sciatic nerve, and if 4S RNA is the predominant RNA species present in these axons.

In one experiment the left sciatic nerve of a rat was crushed. Two days later 170 μCi of [³H]uridine was injected into the vicinity of the lumbar ventral horn cells. Ten days after injection, rats were sacrificed and sciatic nerves were prepared for autoradiography. Photomicrographs were taken of labeled areas of intact and regenerating nerves and grains were counted over Schwann cells, myelin, axons and other unspecified areas. In both intact and regenerating sciatic nerves more than 20% of the silver grains were associated with motor axons and approximately 40% were found over cytoplasm of Schwann cells surrounding these axons. These data indicate an intra-axonal localization of RNA in sciatic nerve axons, as well as an active transfer of RNA precursors from axons to their surrounding Schwann cells.

In separate studies, the left sciatic nerve was crushed and 10 days later [³H]-uridine was bilaterally injected intraspinally into 6 rats. Four control rats were sacrificed at 14 or 20 days after injection. In the remaining 2 rats the sciatic nerve was cut 14 days after injection and the distal part of the nerve was allowed to degenerate for 6 days before sacrificing the rat. Thus, the distal portion of the nerve contained Schwann cells labeled by axonal transport but lacked intact axons. RNA was isolated from experimental and control nerve segments by hot phenol extraction and ethanol precipitation. RNA species (28S, 18S and 4S) were separated by polyacrylamide gel electrophoresis and radioactivity was measured in a liquid scintillation counter. Control groups had RNA profiles similar to those already described²⁰, with greater than 30% of the radioactivity present as 4S RNA. The proximal portions of nerve taken from the group in which nerves were cut, had a similar amount of radioactivity present as 4S RNA. However, in the distal segments of these nerves (in which the axons had degenerated thus creating an ‘axon-less’ nerve) the amount of radioactivity in the 4S peak decreased to approximately 15% of the total RNA, suggesting that 4S RNA is the predominant if not the only RNA present in these axons. These results strongly indicate that both intact and regenerating sciatic nerves of rats selectively transport 4S RNA along their motor axons.     

Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration.

At present, clinical strategies to repair injured peripheral nerve concentrate on efforts to attain primary suture of the cut nerve ends. If this is not possible, autografts are used to unite the separated nerve segments. Both strategies are based on the recognition that the Schwann cells resident in the peripheral nerve trunk play a crucial role in the regenerative process. Neither strategy may be feasible, however, in extensive or multiple injuries because the amount of autograft material is limited, and allografts are subject to immune rejection. Artificially produced nerve bridges constructed of autologous Schwann cells seeded in guidance channels could be used to overcome these limitations. In the present experiments, the potential of Schwann cells derived from adult nerves and seeded in permselective guidance channels to promote neurite regeneration across an 8 mm nerve gap was evaluated in transected rat sciatic nerves. Immunological sequalae were evaluated by comparing Schwann cells from syngeneic and heterologous rat strains. Schwann cells from either adult outbred (Sprague-Dawley, CD) rats or inbred (Fisher, F) rats were suspended in a Matrigel solution at a density of 80 x 10(6) cells/ml (CD) or 40, 80, or 120 x 10(6) cells/ml (F-40, F-80, and F-120 channels, respectively). Channels containing Schwann cells were compared to sciatic nerve autografts, empty channels, or channels filled with Matrigel alone. One day after seeding permselective synthetic guidance channels with a Schwann cell suspension, a central cable of Schwann cells oriented along the axis of the tube was formed due to syneresis of the hydrogel. By 3 weeks postimplantation, regenerating axons had grown into all channels and autografts. Sciatic nerve autografts supported extensive regeneration, containing 4-5 x 10(4) myelinated axons at the graft midpoint. The ability of channels containing syngeneic Schwann cells to foster regeneration was dependent on the Schwann cell seeding density. At the channel's midpoint, the myelinated axon population in F-120 tubes was intermediate between that in sciatic nerve autografts and F-80 channels, and was significantly higher than in F-40 or control channels. The nerve cable in Schwann cell-containing tubes consisted of larger, more organotypic fascicles than acellular control channels. In contrast, heterologous (CD) Schwann cells elicited a strong immune reaction that impeded nerve regeneration. The present study shows that cultured adult syngeneic Schwann cells seeded in permselective synthetic guidance channels support extensive peripheral nerve regeneration.(ABSTRACT TRUNCATED AT 400 WORDS)     

A decline in glial cell-line-derived neurotrophic factor expression is associated with impaired regeneration after long-term Schwann cell denervation.

In the peripheral nervous system, regeneration of motor and sensory axons into chronically denervated distal nerve segments is impaired compared to regeneration into acutely denervated nerves. In order to find possible causes for this phenomenon we examined the changes in the expression pattern of the glial cell-line-derived neurotrophic factor (GDNF) family of growth factors and their receptors in chronically denervated rat sciatic nerves as a function of time with or without regeneration. Among the GDNF family of growth factors, only GDNF mRNA expression was rapidly upregulated in Schwann cells as early as 48 h after denervation. This upregulation peaked at 1 week and then declined to minimal levels by 6 months of denervation. The changes in the protein expression paralleled the changes in the expression of the GDNF mRNA. The mRNAs for receptors GFRalpha-1 and GFRalpha-2 were upregulated only after maximal GDNF upregulation and remained elevated as late as 6 months. There were no significant changes in the expression of GFRalpha-3 or the tyrosine kinase coreceptor, RET. When we examined the expression of GDNF in a delayed regeneration paradigm, there was no upregulation in the distal chronically denervated tibial nerve even when the freshly axotomized peroneal branch of the sciatic nerve was sutured to the distal tibial nerve. This study suggests that one of the reasons for impaired regeneration into chronically denervated peripheral nerves may be the inability of Schwann cells to maintain important trophic support for both motor and sensory neurons.     

Identification of key genes involved in axon regeneration and Wallerian degeneration by weighted gene co-expression network analysis

Peripheral nerve injury repair requires a certain degree of cooperation between axon regeneration and Wallerian degeneration.Therefore,investigating how axon regeneration and degeneration work together to repair peripheral nerve injury may uncover the molecular mechanisms and signal cascades underlying peripheral nerve repair and provide potential strategies for improving the low axon regeneration capacity of the central nervous system.In this study,we applied weighted gene co-expression network analysis to identify differentially expressed genes in proximal and distal sciatic nerve segments from rats with sciatic nerve injury.We identified 31 and 15 co-expression modules from the proximal and distal sciatic nerve segments,respectively.Functional enrichment analysis revealed that the differentially expressed genes in proximal modules promoted regeneration,while the differentially expressed genes in distal modules promoted neurodegeneration.Next,we constructed hub gene networks for selected modules and identified a key hub gene,Kif22,which was up-regulated in both nerve segments.In vitro experiments confirmed that Kif22 knockdown inhibited proliferation and migration of Schwann cells by modulating the activity of the extracellular signal-regulated kinase signaling pathway.Collectively,our findings provide a comparative framework of gene modules that are co-expressed in injured proximal and distal sciatic nerve segments,and identify Kif22 as a potential therapeutic target for promoting peripheral nerve injury repair via Schwann cell proliferation and migration.All animal experiments were approved by the Institutional Animal Ethics Committee of Nantong University,China(approval No.S20210322-008)on March 22,2021.     

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.     

The mRNA of transferrin is expressed in Schwann cells during their maturation and after nerve injury

Transferrin, the iron carrier protein, has been shown to be involved in oligodendroglial cell differentiation in the central nervous system but little is known about its role in the peripheral nervous system. In the present work, we have studied the presence of transferrin and of its mRNA in rat sciatic nerves and in Schwann cells isolated at embryonic and adult ages as well as during the regeneration process that follows nerve crush. We have also studied the correlation between the expression of the mRNAs of transferrin and the expression of mature myelin markers in the PNS. We show that transferrin is present in whole sciatic nerves at late stages of embryonic life as well as at postnatal day 4 and in adult rats. We demonstrate for the first time, that in normal conditions, the transferrin mRNA is expressed in Schwann cells isolated from sciatic nerves between embryonic days 14 and 18, being absent at later stages of development and in adult animals. In adult rats, 3 days after sciatic nerve crushing, the mRNA of transferrin is expressed in the injured nerve, but 7 days after injury its expression disappears. Transferrin protein in the sciatic nerve closely follows the expression of its mRNA indicating that under these circumstances, it appears to be locally synthesized. Transferrin in the PNS could have a dual role. During late embryonic ages it could be locally synthesized by differentiating Schwann cells, acting as a pro-differentiating factor. A similar situation would occur during the regeneration that follows Wallerian degeneration. In the adult animals on the other hand, Schwann cells could pick up transferrin from the circulation or/and from the axons, sub serving possible trophic actions closely related to myelin maintenance.     

Cooperative roles of BDNF expression in neurons and Schwann cells are modulated by exercise to facilitate nerve regeneration

After peripheral nerve injury, neurotrophins play a key role in the regeneration of damaged axons that can be augmented by exercise, although the distinct roles played by neurons and Schwann cells are unclear. In this study, we evaluated the requirement for the neurotrophin, brain-derived neurotrophic factor (BDNF), in neurons and Schwann cells for the regeneration of peripheral axons after injury. Common fibular or tibial nerves in thy-1-YFP-H mice were cut bilaterally and repaired using a graft of the same nerve from transgenic mice lacking BDNF in Schwann cells (BDNF(-/-)) or wild-type mice (WT). Two weeks postrepair, axonal regeneration into BDNF(-/-) grafts was markedly less than WT grafts, emphasizing the importance of Schwann cell BDNF. Nerve regeneration was enhanced by treadmill training posttransection, regardless of the BDNF content of the nerve graft. We further tested the hypothesis that training-induced increases in BDNF in neurons allow regenerating axons to overcome a lack of BDNF expression in cells in the pathway through which they regenerate. Nerves in mice lacking BDNF in YFP(+) neurons (SLICK) were cut and repaired with BDNF(-/-) and WT nerves. SLICK axons lacking BDNF did not regenerate into grafts lacking Schwann cell BDNF. Treadmill training could not rescue the regeneration into BDNF(-/-) grafts if the neurons also lacked BDNF. Both Schwann cell- and neuron-derived BDNF are thus important for axon regeneration in cut peripheral nerves.     

Ascorbic acid accelerates Wallerian degeneration after peripheral nerve injury

Wallerian degeneration occurs after peripheral nerve injury and provides a beneficial microenvironment for nerve regeneration. Our previous study demonstrated that ascorbic acid promotes peripheral nerve regeneration, possibly through promoting Schwann cell proliferation and phagocytosis and enhancing macrophage proliferation, migration, and phagocytosis. Because Schwann cells and macrophages are the main cells involved in Wallerian degeneration, we speculated that ascorbic acid may accelerate this degenerative process. To test this hypothesis, 400 mg/kg ascorbic acid was administered intragastrically immediately after sciatic nerve transection, and 200 mg/kg ascorbic acid was then administered intragastrically every day. In addition, rat sciatic nerve explants were treated with 200 μM ascorbic acid. Ascorbic acid significantly accelerated the degradation of myelin basic protein-positive myelin and neurofilament 200-positive axons in both the transected nerves and nerve explants. Furthermore, ascorbic acid inhibited myelin-associated glycoprotein expression, increased c-Jun expression in Schwann cells, and increased both the number of macrophages and the amount of myelin fragments in the macrophages. These findings suggest that ascorbic acid accelerates Wallerian degeneration by accelerating the degeneration of axons and myelin in the injured nerve, promoting the dedifferentiation of Schwann cells, and enhancing macrophage recruitment and phagocytosis. The study was approved by the Southern Medical University Animal Care and Use Committee(approval No. SMU-L2015081) on October 15, 2015.     

Skin-derived precursor cells enhance peripheral nerve regeneration following chronic denervation

While peripheral nerves demonstrate the capacity for axonal regeneration, outcome following injury remains relatively poor, especially following prolonged denervation. Since axon-deprived Schwann cells (SCs) in the distal nerve progressively lose their ability to support axonal growth, we took the approach of using skin-derived precursor cells (SKPs) as an accessible source of replacement SCs that could be transplanted into chronically denervated peripheral nerve. In this study, we employed a delayed cross-reinnervation paradigm to assess regeneration of common peroneal nerve axons into the chronically denervated rodent tibial nerve following delivery of SKP-derived SC (SKP-SCs). SKP-SC treated animals exhibited superior axonal regeneration to media controls, with significantly higher counts of regenerated motorneurons and histological recovery similar to that of immediately repaired nerve. Improved axonal regeneration correlated with superior muscle reinnervation, as measured by compound muscle action potentials and wet muscle weights. We therefore conclude that SKPs represent an easily accessible, autologous source of stem cell-derived Schwann cells that show promise in improving regeneration through chronically injured nerves.     

Neurofilament mRNAs are present and translated in the normal and severed sciatic nerve

Local protein synthesis within axons has been studied on a limited scale. In the present study, several techniques were used to investigate this synthesis in sciatic nerve, and to show that it increases after damage to the axon. Neurofilament (NF) mRNAs were probed by RT-PCR, Northern blot and in situ hybridization in axons of intact rat sciatic nerve, and in proximal or distal stumps after sciatic nerve transection. RT-PCR demonstrated the presence of NF-L, NF-M and NF-H mRNAs in intact sciatic nerve, as well as in proximal and distal stumps of severed nerves. Northern blot analysis of severed nerve detected NF-L and NF-M, but not NF-H. This technique did not detect the three NFs mRNAs in intact nerve. Detection of NF-L and NF-M mRNA in injured nerve, however, indicated that there was an up-regulation in response to nerve injury. In situ hybridization showed that NF-L mRNA was localized in the Schwann cell perinuclear area, in the myelin sheath, and at the boundary between myelin sheath and cortical axoplasm. RNA and protein synthesizing activities were always greater in proximal as compared to distal stumps. NF triplet proteins were also shown to be synthesized de novo in the proximal stump. The detection of neurofilament mRNAs in nerves, their possible upregulation during injury and the synthesis of neurofilament protein triplet in the proximal stumps, suggest that these mRNAs may be involved in nerve regeneration, providing a novel point of view of this phenomenon.     

Neurite-promoting influences of proliferating Schwann cells and target-tissues are not prerequisite for rapid axonal elongation after nerve crush

An important role in peripheral nerve regeneration has been ascribed to humoral trophic and tropic agents arising from the nonneuronal cells in the distal nerve stump and the denervated targets. In order to estimate their contribution to axonal elongation after crush injury to the rat sciatic nerve, an in vivo model was designed in which local cellular and target-derived influences were eliminated by 1) freeze-thawing of a long nerve segment distal to the crush site and 2) cutting the nerve far distally to the crush site, but within the frozen-thawed segment, and deflecting the frozen-thawed nerve stump in the opposite direction from its natural course. The sensory and motor axon elongation rate was estimated from the results of the nerve pinch test and choline acetyltransferase distribution along the nerve segment distal to the crush. The elongation rate of regenerating axons in deflected nerve segments, either non-treated or frozen-thawed, was close in magnitude to that obtained when target-derived influences were not eliminated. Neurotropism of axonal targets is therefore of little importance for axon elongation after nerve crush. In the absence of Schwann cells along the axonal path in frozen-thawed nerve segments, the elongation rate of both sensory and motor axons declined by about 40%. This implies that interactions between viable Schwann cells and growth cones of regenerating axons are not prerequisite for rapid axon elongation when Schwann cell basal lamina constitutes the growth substratum. Nevertheless, Schwann cells in Bungner bands possibly enhance the axon elongation rate by humoral or cell surface-mediated mechanisms.     

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.