Expression of HGF and cMet in the peripheral nervous system of adult rats following sciatic nerve injury

Hepatocyte growth factor (HGF) exhibits neurotrophic properties on different types of neuron, including motor, sensory and parasympathetic neurons. We demonstrate that sciatic nerve ligation induces an increase of the HGF receptor, c-met, mRNA in the distal segment of the sciatic nerve to the ligation site and a delayed elevation in the proximal segment. Immunohistochemical analysis revealed co-localization of cMet and GFAP and indicates that Schwann cells express cMet in the sciatic nerve after injury. HGF mRNA was detected in the spinal cord and DRG, and nerve injury did not alter the expression. These data demonstrate that the expression of HGF and cMet in the peripheral nervous system shows the unique pattern of regulation following nerve injury.     

Netrin-1 and peripheral nerve regeneration in the adult rat

Axonal guidance during development of the nervous system is thought to be highly regulated through interactions of axons with attractive, repulsive, and trophic cues. Similar mechanisms regulate axonal regeneration after injury. The netrins have been shown to influence the guidance of several classes of developing axons. Although netrins have been implicated as axonal guidance cues in the developing peripheral nervous system, there has been no direct evidence of netrin-1 expression in either developing or adult peripheral nerve. The present study utilized competitive PCR and immunohistochemistry to demonstrate the localization of netrin-1 within adult rat sciatic nerve. The expression of netrin-1 mRNA and protein was compared for normal or regenerated sciatic nerve 2 weeks following either a crush or a transection and repair injury. The PCR data show that netrin-1 mRNA is normally expressed at low levels in peripheral nerve, and similar low levels are found 2 weeks following a crush injury. However, 2 weeks following nerve transection and repair there is approximately a 40-fold increase in netrin-1 mRNA levels. Immunohistochemistry data show that Schwann cells are the major source of netrin-1 protein in peripheral nerve. Our results suggest that netrin-1 mRNA levels are profoundly affected during peripheral nerve injury and regeneration. The localization of netrin-1 to Schwann cells suggests that this protein is strategically situated to influence axon regeneration in adult peripheral nerve.     

The 4C5 antigen is associated with Schwann cell migration during development and regeneration of the rat peripheral nervous system

The monoclonal antibody 4C5 recognizes a cell surface antigen of the developing central nervous system (CNS) and peripheral nervous system (PNS). In vitro antibody perturbation experiments have shown that the 4C5 antigen is involved in horizontal and vertical migration processes of granule cells during development of the rodent cerebellum. Moreover, results concerning the cellular localization and temporal expression of the 4C5 antigen during development and after injury of the rat sciatic nerve suggested that it may participate in Schwann cell migrations that occur during the above processes. To test this possibility, we examined the effects of our function-blocking antibody on Schwann cell migration in three in vitro bioassays: in tissue cultures from developing sciatic nerve, in dorsal root ganglion cultures on cryostat sections of normal or denervated adult sciatic nerve, and in pure Schwann cell cultures. The results showed that the presence of monoclonal antibody 4C5 in all the above culture systems strongly inhibited Schwann cell migration, indicating that the 4C5 antigen participates in migration processes that take place during development and regeneration of the peripheral nervous system. Moreover, staining of migrating Schwann cells in the presence of monoclonal antibody 4C5 with rhodamine-phalloidin showed that 4C5 antigen activity is associated with actin cytoskeletal organization of these cells, and more specifically with lamellipodia formation.     

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.     

Baculoviral inhibitor of apoptosis protein repeat-containing protein 3 delays early Wallerian degeneration after sciatic nerve injury

Wallerian degeneration is a complex biological process that occurs after nerve injury,and involves nerve degeneration and regeneration.Schwann cells play a crucial role in the cellular and molecular events of Wallerian degeneration of the peripheral nervous system.However,Wallerian degeneration regulating nerve injury and repair remains largely unknown,especially the early response.We have previously reported some key regulators of Wallerian degeneration after sciatic nerve injury.Baculoviral inhibitor of apoptosis protein repeat-containing protein 3(BIRC3)is an important factor that regulates apoptosis-inhibiting protein.In this study,we established rat models of right sciatic nerve injury.In vitro Schwann cell models were also established and subjected to gene transfection to inhibit and overexpress BIRC3.The data indicated that BIRC3 expression was significantly up-regulated after sciatic nerve injury.Both BIRC3 upregulation and downregulation affected the migration,proliferation and apoptosis of Schwan cells and affected the expression of related factors through activating c-fos and ERK signal pathway.Inhibition of BIRC3 delayed early Wallerian degeneration through inhibiting the apoptosis of Schwann cells after sciatic nerve injury.These findings suggest that BIRC3 plays an important role in peripheral nerve injury repair and regeneration.The study was approved by the Institutional Animal Care and Use Committee of Nantong University,China(approval No.2019-nsfc004)on March 1,2019.     

LncRNA BC088259 promotes Schwann cell migration through Vimentin following peripheral nerve injury

Schwann cell, the major glial cell in the peripheral nervous system, plays an essential role in peripheral nerve regeneration. However, the regulation of Schwann cell behavior following nerve injury is insufficiently explored. According to the development of high-throughput techniques, long noncoding RNAs (lncRNAs) have been recognized. Accumulating evidence shows that lncRNAs take part in diverse biological processes and diseases. Here, by microarray analysis, we identified an upregulated lncRNA profile following sciatic nerve injury and focused on BC088259 for further investigation. Silencing or overexpression of BC088259 could affect Schwann cell migration. Mechanistically, BC088259 might exert this regulatory role by directly binding with Vimentin. Collectively, our study not only revealed a set of upregulated lncRNAs following nerve injury but also identified a new functional lncRNA, BC088259, which was important for Schwann cell migration, providing a therapeutic avenue toward peripheral nerve injury.     

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.     

Dorsal root ganglion neurons up-regulate the expression of laminin-associated integrins after peripheral but not central axotomy

The favorable prognosis of regeneration in the peripheral nervous system after axonal lesions is generally regarded as dependent on the Schwann cell basal lamina. Laminins, a heterotrimeric group of basal lamina molecules, have been suggested to be among the factors playing this supportive role. For neurons to utilize laminin as a substrate for growth, an expression of laminin binding receptors, integrins, is necessary. In this study, we have examined the expression of laminin binding integrin subunits in dorsal root ganglion (DRG) neurons after transection to either their peripherally projecting axons, as in the sciatic nerve, followed by regeneration, or the centrally projecting axons in dorsal roots, followed by no or weak regenerative activity. In uninjured DRG, immunohistochemical staining revealed a few neurons expressing integrin subunit alpha6, whereas integrin subunits alpha7 and foremost beta1 were expressed in a majority of neurons. After an injury to the sciatic nerve, mRNAs encoding all three integrins were up-regulated in DRG neurons. By anterograde tracing, immunoreactivity for all studied integrins was also found in association with growing axons after a sciatic nerve crush lesion in vivo. In contrast, mRNA levels remained constant in DRG neurons after a dorsal root injury. Together with previous findings, this suggests that integrin subunits alpha6, alpha7, and beta1 have an important role in the regenerative response following nerve injury and that the lack of regenerative capacity following dorsal root injury could in part be explained by the absence of response in integrin regulation.     

Upregulation of Ryk expression in rat dorsal root ganglia after peripheral nerve injury

To study changes of Ryk expression in dorsal root ganglia (DRG) after peripheral nerve injury, we set up an animal model of unilateral sciatic nerve lesioned rats. Changes of Ryk protein expression in DRG neurons after unilateral sciatic nerve injury were investigated by immunostaining. Changes of Ryk mRNA were also tested by semi-quantitative PCR concurrently. We found, both at the level of protein and mRNA, that Ryk could be induced in cells of ipsilateral DRG after unilateral sciatic nerve lesion. Further investigation by co-immunostaining confirmed that the Ryk-immunoreactive (Ryk-IR) cells were NeuN-immunoreactive (NeuN-IR) neurons of DRG. We also showed the pattern of Ryk induction in DRG neurons after sciatic nerve injury: the number of Ryk IR neurons peaked at 2 weeks post-lesion and decreased gradually by 3 weeks post-lesion. The proportions of different sized Ryk IR neurons were also observed and counted at various stages after nerve lesion. Analysis of Ryk mRNA by RT-PCR showed the same induction pattern as by immunostaining. Ryk mRNA was not expressed in normal or contralateral DRG, but was expressed 1, 2 and 3 weeks post-lesion in the ipsilateral DRG. Ryk mRNA levels increased slightly from 1 to 2 weeks, decreased then by 3 weeks post-lesion. These results indicate that Ryk might be involved in peripheral nerve plasticity after injury. This is a novel function apart from its well-known fundamental activity as a receptor mediating axon guidance and outgrowth.     

Point Sources of Schwann Cells Result in Growth into a Nerve Entubulation Repair Site in the Absence of Axons: Effects of Freeze-Thawing

Axonal regeneration in the peripheral nervous system requires the comigration of Schwann cells along with or ahead of the growing neurites. The present studies were undertaken to elucidate some of the parameters that influence Schwann cell migration into a nerve wound site. We used a modification of an entubulation repair model with two isolated segments of denervated nerve to show that Schwann cells in vivo can bridge a 10-mm distance in the absence of axons. Two pieces of isolated nerve served as point sources of Schwann cells. A crucial finding was that in order for Schwann cells to bridge this distance both isolated/denervated nerve stumps had to be living. In cases where one nerve stump was frozen Schwann cells were not able to bridge the gap distance, although other cell types made up a tissue bridge between both stumps. We have further shown that as Schwann cells grow into this nerve wound site they stain intensely for the low-affinity NGF receptor p75^NGFR. Related ^{in vitro} experiments showed that Schwann cells actively migrate on cryostat sections of either intact or previously lesioned sciatic nerve, suggesting that they can grow on either axon-containing or axonless areas of nerve tissue. Contrary to migration on sciatic nerve, although Schwann cells attached and survived on cryostat sections of optic nerve, they did not migrate on this substrate. The results of the present experiments suggest that Schwann cells can respond to specific diffusible and substrate bound signals which influence their migration into a nerve repair site.     

Lithium promotes proliferation and suppresses migration of Schwann cells

Schwann cell proliferation,migration and remyelination of regenerating axons contribute to regeneration after peripheral nervous system injury.Lithium promotes remyelination by Schwann cells and improves peripheral nerve regeneration.However,whether lithium modulates other phenotypes of Schwann cells,especially their proliferation and migration remains elusive.In the current study,primary Schwann cells from rat sciatic nerve stumps were cultured and exposed to 0,5,10,15,or 30 mM lithium chloride(LiCl)for 24 hours.The effects of LiCl on Schwann cell proliferation and migration were examined using the Cell Counting Kit-8,5-ethynyl-2′-deoxyuridine,Transwell and wound healing assays.Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine assays showed that 5,10,15,and 30 mM LiCl significantly increased the viability and proliferation rate of Schwann cells.Transwell-based migration assays and wound healing assays showed that 10,15,and 30 mM LiCl suppressed the migratory ability of Schwann cells.Furthermore,the effects of LiCl on the proliferation and migration phenotypes of Schwann cells were mostly dose-dependent.These data indicate that lithium treatment significantly promotes the proliferation and inhibits the migratory ability of Schwann cells.This conclusion will inform strategies to promote the repair and regeneration of peripheral nerves.All of the animal experiments in this study were ethically approved by the Administration Committee of Experimental Animal Center of Nantong University,China(approval No.20170320-017)on March 2,2017.     

Spatiotemporal distribution and function of N‐cadherin in postnatal Schwann cells: A matter of adhesion?

During embryonic development of the peripheral nervous system (PNS), the adhesion molecule neuronal cadherin (N‐cadherin) is expressed by Schwann cell precursors and associated with axonal growth cones. N‐cadherin expression levels decrease as precursors differentiate into Schwann cells. In this study, we investigated the distribution of N‐cadherin in the developing postnatal and adult rat peripheral nervous system. N‐cadherin was found primarily in ensheathing glia throughout development, concentrated at neuron–glial or glial–glial contacts of the sciatic nerve, dorsal root ganglia (DRG), and myenteric plexi. In the sciatic nerve, N‐cadherin decreases with age and progress of myelination. In adult animals, N‐cadherin was found exclusively in nonmyelinating Schwann cells. The distribution of N‐cadherin in developing E17 DRG primary cultures is similar to what was observed in vivo. Functional studies of N‐cadherin in these cultures, using the antagonist peptide INPISGQ, show a disruption of the attachment between Schwann cells, but no interference in the initial or long‐term contact between Schwann cells and axons. We suggest that N‐cadherin acts primarily in the adhesion between glial cells during postnatal development. It may form adherents/junctions between nonmyelinating glia, which contribute to the stable tubular structure encapsulating thin caliber axons and thus stabilize the nerve structure as a whole. © 2010 Wiley‐Liss, Inc.     

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.     

Transforming growth factor β as a neuronoglial signal during peripheral nervous system response to injury

In contrast to the central nervous system (CNS), the peripheral nervous system (PNS) displays an important regenerative ability which is dependent, at least in part, on Schwann cell properties. The mechanisms which stimulate Schwann cells to adapt their behavior after a lesion to generate adequate conditions for PNS regeneration remain unknown. In this work, we report that adult rat dorsal root ganglion (DRG) neurons are able, after a lesion performed in vivo or when they are dissociated and cultured in vitro, to synthesize transforming growth factor β (TGFβ), a pleiotropic growth factor implicated in wound healing processes and in carcinogenesis. This TGFβ is tentatively identified as the β-1 isoform. Adult rat DRG neurons release a biologically active form of TGFβ which is able to elicit multiple Schwann cell responses including a stimulation to proliferate. Moreover, purified TGFβ-1 produces a Schwann cell morphology alteration and decreases the secretion of tissue-type plasminogen activator (tPA) and enhances the secretion of plasminogen activator inhibitor (PAI) by Schwann cells. This generates conditions which are thought to favor a successful neuritic regrowth. Furthermore, purified TGFβ-1 stimulates type IV collagen mRNA expression in Schwann cells. This subtype of collagen is associated with the process of myelinization. Finally, TGFβ-1 decreases nerve growth factor (NGF) mRNA expression by Schwann cells, an effect which could participate in the maintenance of a distoproximal NGF gradient during nerve regeneration. We propose that neuronal TGFβ plays an essential role as a neuronoglial signal that modulates the response of Schwann cells to injury and participates in the successful regeneration processes observed in the PNS. © 1993 Wiley-Liss, Inc.     

Lack of effects of transforming growth factor-alpha gene knockout on peripheral nerve regeneration may result from compensatory mechanisms.

Transforming growth factor-alpha (TGF-alpha), previously identified as a major member of the epidermal growth factor (EGF) family of growth factors, plays a role in proliferation, differentiation, and survival of neuronal and glial precursors and is implicated in development of the nervous system. However, its roles in nerve injury-induced responses remain obscure. The current study examined roles of endogenous TGF-alpha in peripheral nerve regeneration using sciatic nerve injury models with TGF-alpha knockout mice. Three weeks after a sciatic nerve crush, no significant differences were found between TGF-alpha wild-type and mutant mice in the number of retrogradely labeled L5 dorsal root ganglion (DRG) sensory neurons and L5 spinal cord motor neurons and in the morphology of myelinated regenerating nerve fibers, indicating that TGF-alpha is not essential for sensory and motor nerve regeneration. To assess a possible functional redundancy among TGF-alpha-related ligands in response to a nerve injury, mRNA expression of the EGF family was analyzed by RT-PCR in L4/L5 DRG pools and distal degenerating sciatic nerve segments after sciatic nerve ligation. Prior to and 1 day after ligation, there was a higher level of EGF-R mRNA in DRGs and in nerve in TGF-alpha null mice compared to wild types, and there was an induction of ligand amphiregulin mRNA in DRGs in mutant mice in place of the TGF-alpha upregulation present in wild types. These results indicate that TGF-alpha gene knockout does not affect peripheral nerve regeneration, probably due to a functional redundancy within the EGF family through a compensatory expression mechanism at both the receptor and ligand levels in TGF-alpha knockout mice.     

Dynamic change of Numbl expression after sciatic nerve crush and its role in Schwann cell differentiation

Numbl, as a conserved homolog of Drosophila Numb, has been implicated in early development of the nervous system, but its expression and roles in nervous system lesion and repair remained unknown. Here, we performed an acute sciatic nerve injury model in adult rats and studied the dynamic changes of Numbl expression in the sciatic nerve. Temporally, Numbl expression was sharply decreased after sciatic nerve crush and reached a valley at day 7. Spatially, Numbl was widely expressed in the normal sciatic nerve, including axons and Schwann cells, whereas, after injury, Numbl expression was decreased predominantly in Schwann cells. In vitro, we induced Schwann cell differentiation with cAMP and found that Numbl expression was decreased in the differentiated process. Depletion of Numbl could promote Schwann cell differentiation. In addition, we demonstrated that in vitro myelination was suppressed by overexpression of Numbl in Schwann cells. Collectively, we hypothesized peripheral nerve injury induced a downregulation of Numbl in the sciatic nerve, which was associated with Schwann cell differentiation.