Extracellular fluid conditioned during peripheral nerve regeneration stimulates Schwann cell adhesion, migration and proliferation

Schwann cell movement and proliferation occur during peripheral nerve regeneration and remyelination. We asked whether soluble factors promoting these activities were present in fluid surrounding rat sciatic nerves regenerating across a 10-mm gap bridged by a silicone tube. In this model, regenerated and remyelinated axons extend across the gap by 28 days following nerve transection and tube implantation. Fluid conditioned by cells participating in nerve regeneration (RCF) was assayed for its ability to promote Schwann cell adhesion, migration and proliferation in vitro. RCFs collected at post-transectional days 1-28 were equally effective in promoting Schwann cell-substratum adhesion. In contrast, the motility-promoting activity of RCF was minimal at 1-2 days following nerve-transection, peaked at 7 days and remained elevated through 21 days. The RCF peak response was 87-fold greater than control. Schwann cell proliferative activity of RCF exhibited peaks of activity at 1 and 14 days post-transection. The biological potency of this fluid for each activity assayed in vitro correlated well with the behavior of Schwann cells chronicled during nerve repair in vivo. These findings suggest that soluble factors promoting Schwann cell adhesion, migration, and proliferation accumulate extracellularly during peripheral nerve regeneration and remyelination.     

Miconazole enhances nerve regeneration and functional recovery after sciatic nerve crush injury

Introduction: Improving axonal outgrowth and remyelination is crucial for peripheral nerve regeneration. Miconazole appears to enhance remyelination in the central nervous system. In this study we assess the effect of miconazole on axonal regeneration using a sciatic nerve crush injury model in rats. Methods: Fifty Sprague‐Dawley rats were divided into control and miconazole groups. Nerve regeneration and myelination were determined using histological and electrophysiological assessment. Evaluation of sensory and motor recovery was performed using the pinprick assay and sciatic functional index. The Cell Counting Kit‐8 assay and Western blotting were used to assess the proliferation and neurotrophic expression of RSC 96 Schwann cells. Results: Miconazole promoted axonal regrowth, increased myelinated nerve fibers, improved sensory recovery and walking behavior, enhanced stimulated amplitude and nerve conduction velocity, and elevated proliferation and neurotrophic expression of RSC 96 Schwann cells. Discussion: Miconazole was beneficial for nerve regeneration and functional recovery after peripheral nerve injury. Muscle Nerve 57 : 821–828, 2018     

Long non-coding RNA NONMMUG014387 promotes Schwann cell proliferation after peripheral nerve injury

Schwann cells play a critical role in peripheral nerve regeneration through dedifferentiation and proliferation. In a previous study, we performed microarray analysis of the sciatic nerve after injury. Accordingly, we predicted that long non-coding RNA NONMMUG014387 may promote Schwann cell proliferation after peripheral nerve injury, as bioinformatic analysis revealed that the target gene of NONMMUG014387 was collagen triple helix repeat containing 1(Cthrc1). Cthrc1 may promote cell proliferation in a variety of cells by activating Wnt/PCP signaling. Nonetheless, bioinformatic analysis still needs to be verified by biological experiment. In this study, the candidate long non-coding RNA, NONMMUG014387, was overexpressed in mouse Schwann cells by recombinant adenovirus transfection. Plasmid p HBAd-MCMV-GFP-NONMMUG014387 and p HBAd-MCMV-GFP were transfected into Schwann cells. Schwann cells were divided into three groups: control(Schwann cells without intervention), Ad-GFP(Schwann cells with GFP overexpression), and Ad-NONMMUGO148387(Schwann cells with GFP and NONMMUGO148387 overexpression). Cell Counting Kit-8 assay was used to evaluate proliferative capability of mouse Schwann cells after NONMMUG014387 overexpression. Polymerase chain reaction and western blot assay were performed to investigate target genes and downstream pathways of NONMMUG014387. Cell proliferation was significantly increased in Schwann cells overexpressing lnc RNA NONMMUG014387 compared with the other two groups. Further, compared with the control group, m RNA and protein levels of Cthrc1, Wnt5 a, ROR2, Rho A, Rac1, JNK, and ROCK were visibly up-regulated in the Ad-NONMMUGO148387 group. Our findings confirm that long non-coding RNA NONMMUG014387 can promote proliferation of Schwann cells surrounding the injury site through targeting Cthrc1 and activating the Wnt/PCP pathway.     

3-N-Butylphthalide mitigates high glucose-induced injury to Schwann cells:association with nitrosation and apoptosis

A high glucose state readily causes peripheral axon atrophy, demyelination, loss of nerve fiber function, and delayed regeneration. However, few studies have examined whether nitration is also critical for diabetic peripheral neuropathy. Therefore, this study investigated the effects of high glucose on proliferation, apoptosis, and 3-nitrotyrosine levels of Schwann cells treated with butylphthalide. In addition, we explored potential protective mechanisms of butylphthalide on peripheral nerves. Schwann cells were cultured in vitro with high glucose then stimulated with the peroxynitrite anion inhibitors uric acid and 3-n-butylphthalide for 48 hours. Cell Counting Kit-8 and flow cytometry were used to investigate the effects of uric acid and 3-n-butylphthalide on proliferation and apoptosis of Schwann cells exposed to a high glucose environment. Effects of uric acid and 3-n-butylphthalide on levels of 3-nitrotyrosine in Schwann cells were detected by enzyme-linked immunosorbent assay. The results indicated that Schwann cells cultured in high glucose showed decreased proliferation, but increased apoptosis and intracellular 3-nitrotyrosine levels. However, intervention with uric acid or 3-n-butylphthalide could increase proliferation of Schwann cells cultured in high glucose, and inhibited apoptosis and intracellular 3-nitrotyrosine levels. According to our data, 3-n-butylphthalide may inhibit cell nitrification and apoptosis, and promote cell proliferation, thereby reducing damage to Schwann cells caused by high glucose.     

Spatiotemporal microRNA profile in peripheral nerve regeneration:miR-138 targets vimentin and inhibits Schwann cell migration and proliferation

While the peripheral nervous system has regenerative ability,restoration of sufficient function remains a challenge.Vimentin has been shown to be localized in axonal growth fronts and associated with nerve regeneration,including myelination,neuroplasticity,kinase signaling in nerve axoplasm,and cell migration;however,the mechanisms regulating its expression within Schwann cell(SC) remain unexplored.The aim of this study was to profile the spatial and temporal expression profile of micro RNA(mi RNA) in a regenerating rat sciatic nerve after transection,and explore the potential role of mi R-138-5 p targeting vimentin in SC proliferation and migration.A rat sciatic nerve transection model,utilizing a polyethylene nerve guide,was used to investigate mi RNA expression at 7,14,30,60,and 90 days during nerve regeneration.Relative levels of mi RNA expression were determined using microarray analysis and subsequently validated with quantitative real-time polymerase chain reaction.In vitro assays were conducted with cultured Schwann cells transfected with mi RNA mimics and assessed for migratory and proliferative potential.The top seven dysregulated mi RNAs reported in this study have been implicated in cell migration elsewhere,and GO and KEGG analyses predicted activities essential to wound healing.Transfection of one of these,mi RNA-138-5 p,into SCs reduced cell migration and proliferation.mi R-138-5 p has been shown to directly target vimentin in cancer cells,and the luciferase assay performed here in rat Schwann cells confirmed it.These results detail a role of mi R-138-5 p in rat peripheral nerve regeneration and expand on reports of it as an important regulator in the peripheral nervous system.     

Schwann cell migration is integrin‐dependent and inhibited by astrocyte‐produced aggrecan

Schwann cells transplantation has considerable promise in spinal cord trauma to bridge the site of injury and for remyelination in demyelinating conditions. They support axonal regeneration and sprouting by secreting growth factors and providing a permissive surface and matrix molecules while shielding axons from the inhibitory environment of the central nervous system. However, following transplantation Schwann cells show limited migratory ability and they are unable to intermingle with the host astrocytes. This in turn leads to formation of a sharp boundary and an abrupt transition between the Schwann cell graft and the host tissue astrocytes, therefore preventing regenerating axons from exiting the graft. The objective of this study was to identify inhibitory elements on astrocytes involved in restricting Schwann cell migration. Using in vitro assays of cell migration, we show that aggrecan produced by astrocytes is involved in the inhibition of Schwann cell motility on astrocytic monolayers. Knockdown of this proteoglycan in astrocytes using RNAi or digestion of glycosaminglycan chains on aggrecan improves Schwann cell migration. We further show aggrecan mediates its effect by disruption of integrin function in Schwann cells, and that the inhibitory effects of aggrecan can overcome by activation of Schwann cell integrins. © 2010 Wiley‐Liss, Inc.     

Nerve growth factor signaling of p75 induces differentiation and ceramide-mediated apoptosis in Schwann cells cultured from degenerating nerves.

In peripheral nerve regeneration or remyelination, immature Schwann cells expressing p75(NTR) play cardinal roles in the support and regeneration of axons (Griffin JW, Hoffman PN. Peripheral Neuropathy 361-376, 1993). Only one of four to six Schwann cells participate in remyelination of damaged or regenerating axons. The rest of the cells, or supernumerary Schwann cells, show severe atrophy and gradually decrease in number, reestablishing a 1:1 axon-Schwann cell relationship (Said G, Duckett S. Acta Neuropathol (Berl) 53:173-179, 1981). Recent reports demonstrated that severely atrophied supernumerary Schwann cells are eliminated by apoptosis during axonal regeneration or remyelination (Hirata H, Hibasami H. Apoptosis 3:353-360, 1998; Berciano MT, Calle E. Acta Neuropathol (Berl) 95:269-279, 1998). The mechanism to induce selective death of supernumerary Schwann cells without causing any damage to axon-associated Schwann cells or axons remains to be determined. In this article, we report that p75(NTR), the low-affinity receptor for all members of neurotrophins, signals both cell differentiation and apoptosis through intracellular ceramide elevation. The final response is dependent on the intracellular ceramide level and Schwann cells modulate their response by changing expression level of p75(NTR). This effect was selective for nerve growth factor (NGF). Taken together, the present study suggests that NGF contributes both to phenotypic regulation and to elimination of the dedifferentiated Schwann cells, while supporting survival or regeneration of certain types of axons during peripheral nerve repair or regeneration.     

Rapamycin promotes Schwann cell migration and nerve growth factor secretion

Rapamycin, similar to FK506, can promote neural regeneration in vitro. We assumed that the mechanisms of action of rapamycin and FK506 in promoting peripheral nerve regeneration were similar. This study compared the effects of different concentrations of rapamycin and FK506 on Sc hwann cells and investigated effects and mechanisms of rapamycin on improving peripheral nerve regeneration. Results demonstrated that the lowest rapamycin concentration(1.53 nmol/L) more significantly promoted Schwann cell migration than the highest FK506 concentration(100 μmol/L). Rapamycin promoted the secretion of nerve growth factors and upregulated growth-associated protein 43 expression in Schwann cells, but did not significantly affect Schwann cell proliferation. Therefore, rapamycin has potential application in peripheral nerve regeneration therapy.     

Proliferating immature Schwann cells contribute to nerve regeneration after ischemic peripheral nerve injury

Schwann cells exhibit a high degree of plasticity in adult peripheral nerves after mechanical injury; they have, therefore, been implicated in promoting nerve regeneration. However, Schwann cell behavior after ischemic injury has not yet been elucidated. To determine how Schwann cell plasticity may contribute to recovery from ischemic neuropathy, we used a rat model in which ischemia was induced in the tibial nerve by a 5-hour occlusion of the supplying arteries. Proliferation of immature Schwann cells that emerged in the injured nerve was evaluated by double immunostaining for the p75 neurotrophin receptor and proliferating cell nuclear antigen. The number of proliferating cell nuclear antigen/p75 neurotrophin receptor double-positive cells increased significantly in 1 to 2 weeks after ischemia and subsequently decreased by 4 weeks. During this time, the postmitotic Schwann cells differentiated into mature cells, as demonstrated with bromodeoxyuridine incorporation, which facilitated axon guidance and subsequent axon remyelination. These results suggest the emergence and proliferation of immature Schwann cells that contribute to nerve regeneration after ischemic injury. The manipulation of this population of proliferating immature Schwann cells may be a useful strategy for treating ischemic peripheral neuropathy.     

Gelsolin is required for macrophage recruitment during remyelination of the peripheral nervous system

Reorganization of the actin cytoskeleton is necessary for Schwann cell proliferation, migration and for the morphological changes associated with sorting, ensheathing and myelination of axons. Such reorganization requires regulated severing and depolymerization of actin filaments. Gelsolin is an actin filament severing protein expressed in many cell types including Schwann cells. Using Gelsolin knockout mice, we investigated the role of this protein in the myelination and remyelination of the peripheral nervous system. Our results show that although gelsolin is not necessary for developmental myelination, it is required for timely remyelination of the sciatic nerve following crush injury. Gelsolin is necessary for macrophage motility in culture, and its absence is likely to impair the recruitment of macrophages to the injury site. © 2009 Wiley‐Liss, Inc.     

Schwann cell remyelination is restricted to astrocyte-deficient areas after transplantation into demyelinated adult rat brain

The ability to generate large numbers of Schwann cells from a peripheral nerve biopsy makes them potential candidates for the clinical application of cell transplantation to enhance remyelination in human demyelinating disease. Transplant-derived Schwann cell remyelination has previously been demonstrated in the spinal cord but not for demyelinated axons in the brain, a more likely site for initial clinical intervention. We have transplanted Schwann cells from male neonatal rat sciatic nerves into ethidium bromide-induced areas of demyelination in the deep cerebellar white matter of adult female rats. The extent of Schwann cell remyelination 28 days after transplantation was significantly increased in lesions that received direct injections of Schwann cells compared with non-transplanted lesions. Using in situ hybridisation to identify the rat Y chromosome, transplanted male cells were found to co-localise with the P0 immunoreactive area of Schwann cell remyelination. Combined immunohistochemistry and in situ hybridisation confirmed that many remyelinating Schwann cells were transplant-derived. P0 immunoreactivity and transplanted male cells were found in GFAP-negative, astrocyte-free areas. Transplanted Schwann cells were not identified outside of transplanted lesions, nor did they did not contribute to remyelination of a lesion at a distance from the site of transplantation. Our findings indicate that demyelinated axons in the adult brain can be remyelinated by transplanted Schwann cells but that migration and remyelination are restricted to areas from which astrocytes are absent.     

Repulsive migration of schwann cells induced by slit‐2 through Ca2+‐dependent RhoA‐Myosin signaling

Schwann cells migrate along axons before initiating myelination during development and their migration facilitates peripheral nerve regeneration after injury. Axon guidance molecule Slit‐2 is highly expressed during peripheral development and nerve regeneration; however, whether Slit‐2 regulates the migration of Schwann cells remains a mystery. Here we show that Slit‐2 receptor Robo‐1 and Robo‐2 were highly expressed in Schwann cells in vitro and in vivo. Using three distinct migration assays, we found that Slit‐2 repelled the migration of cultured Schwann cells. Furthermore, frontal application of a Slit‐2 gradient to migrating Schwann cells first caused the collapse of leading front, and then reversed soma translocation of Schwann cells. The repulsive effects of Slit‐2 on Schwann cell migration depended on a Ca²⁺ signaling release from internal stores. Interestingly, in response to Slit‐2 stimulation, the collapse of leading front required the loss of F‐actin and focal adhesion, whereas the subsequent reversal of soma translocation depended on RhoA‐Rock‐Myosin signaling pathways. Taken together, we demonstrate that Slit‐2 repels the migration of cultured Schwann cells through RhoA‐Myosin signaling pathways in a Ca²⁺‐dependent manner.     

Purification of Schwann cells by selection of p75 low affinity nerve growth factor receptor expressing cells from adult peripheral nerve

The intrinsic capacity of Schwann cells to promote regeneration after limited peripheral nerve lesions has been successfully transferred to extensive peripheral nerve injuries and central nervous system lesions by autologous transplantation strategies. However, both the intrinsic ability of axotomized neurons to regenerate and the permissiveness of the parenchyma surrounding the acute injury site diminish over time. Therefore, the autologous transplantation mode requires a fast and effective method to isolate Schwann cells from peripheral nerve biopsies. Here, we report a method to purify p75 low affinity nerve growth factor receptor (p75LNGFr) expressing Schwann cells from peripheral nerve biopsies in adult rats using magnetic-activated cell separation (MACS). After 1 week of nerve degeneration in culture, nerve fragments were dissociated resulting in mixed cultures containing Schwann cells and fibroblasts. After incubation with specific anti-p75LNGFr antibodies and secondary magnetic bead conjugated antibodies followed by one cycle of MACS, 95% pure Schwann cell cultures were generated as confirmed by flow-cytometry and immunocytochemistry. In contrast to established methods, MACS separation of p75LNGFr expressing cells allows the reliable purification of Schwann cells within 9 days after biopsy employing direct selection of Schwann cells rather than fibroblast depletion assays. Therefore, this method represents an effective and fast means to generate autologous Schwann cells for clinical transplantation strategies aiming for axon repair and remyelination.     

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.     

In vitro biocompatibility of three chitosan/polycation composite materials for nerve regeneration

BACKGROUND:It has been reported that chitosan nerve conduits could support axon elongation and improve relevant function during in vivo nerve regeneration. OBJECTIVE: To investigate in vitro biocompatibility of three novel, chitosan/polycation composite materials for nerve regeneration in cultured mouse Schwann cells and PC12 cells. DESIGN, TIME AND SETTING: The observational, control experiments for nerve tissue engineering were performed at the Department of Biological Sciences and Biotechnology of Tsingh...     

Schwann cells genetically engineered to express PSA show enhanced migratory potential without impairment of their myelinating ability in vitro

Schwann cells, the myelin-forming cells of the PNS, are attractive candidates for remyelination therapy as they can remyelinate CNS axons. Yet their integration in CNS tissue appears hampered, at least in part, by their limited motility in the CNS environment. As the polysialylated (PSA) form of NCAM regulates migration of neural precursors in the CNS and is not expressed by developing Schwann cells, we investigated whether conferring sustained expression of PSA to Schwann cells derived from postnatal rats enhances their motility. Cells were transduced with a retrovirus encoding polysialyl-transferase STX, an enzyme that synthesizes PSA on NCAM. Migration of wild type and transduced cells expressing STX or the marker gene alkaline phosphatase was examined using a gap bridging assay in dissociated cells and by grafting cells in slice cultures of postnatal brain. Migration of PSA expressing cells was significantly increased in both models, as compared to control cells, and this effect was abolished by endoneuraminidase-N stripping of PSA. PSA-positive Schwann cells retained the ability to differentiate in vitro and expressed the Krox20 and P zero myelination markers. When grafted in neonatal cerebellar slices, STX-transduced cells started to myelinate Purkinje cell axons like control cells and make myelin internodes after 2 to 3 weeks. PSA was redistributed on the cell membrane and downregulated during differentiation in pure Schwann cell cultures and slice co-cultures. Thus, migratory properties of PNS myelin-forming cells within the CNS can be enhanced without altering their differentiation program. This finding may be beneficial for the development of remyelination therapies.     

GDNF‐treated acellular nerve graft promotes motoneuron axon regeneration after implantation into cervical root avulsed spinal cord

T‐H. Chu, L. Wang, A. Guo, V. W‐K. Chan, C. W‐M. Wong and W. Wu (2012) Neuropathology and Applied Neurobiology 38, 681–695 GDNF‐treated acellular nerve graft promotes motoneuron axon regeneration after implantation into cervical root avulsed spinal cord It is well known that glial cell line‐derived neurotrophic factor (GDNF) is a potent neurotrophic factor for motoneurons. We have previously shown that it greatly enhanced motoneuron survival and axon regeneration after implantation of peripheral nerve graft following spinal root avulsion. Aims: In the current study, we explore whether injection of GDNF promotes axon regeneration in decellularized nerve induced by repeated freeze‐thaw cycles. Methods: We injected saline or GDNF into the decellularized nerve after root avulsion in adult Sprague–Dawley rats and assessed motoneuron axon regeneration and Schwann cell migration by retrograde labelling and immunohistochemistry. Results: We found that no axons were present in saline‐treated acellular nerve whereas Schwann cells migrated into GDNF‐treated acellular nerve grafts. We also found that Schwann cells migrated into the nerve grafts as early as 4 days after implantation, coinciding with the first appearance of regenerating axons in the grafts. Application of GDNF outside the graft did not induce Schwann cell infiltration nor axon regeneration into the graft. Application of pleiotrophin, a trophic factor which promotes axon regeneration but not Schwann cell migration, did not promote axon infiltration into acellular nerve graft. Conclusions: We conclude that GDNF induced Schwann cell migration and axon regeneration into the acellular nerve graft. Our findings can be of potential clinical value to develop acellular nerve grafting for use in spinal root avulsion injuries.