MMP‐9 controls Schwann cell proliferation and phenotypic remodeling via IGF‐1 and ErbB receptor‐mediated activation of MEK/ERK pathway

Phenotypic remodeling of Schwann cells is required to ensure successful regeneration of damaged peripheral axons. After nerve damage, Schwann cells produce an over 100‐fold increase in metalloproteinase‐9 (MMP‐9), and therapy with an MMP inhibitor increases the number of resident (but not infiltrating) cells in injured nerve. Here, we demonstrate that MMP‐9 regulates proliferation and trophic signaling of Schwann cells. Using in vivo BrdU incorporation studies of axotomized sciatic nerves of MMP‐9?/? mice, we found increased Schwann cell mitosis in regenerating (proximal) stump relative to wild‐type mice. Treatment of cultured primary Schwann cells with recombinant MMP‐9 suppressed their growth, mitogenic activity, and produced a dose‐dependent, biphasic, and selective activation of ERK1/2, but not JNK and p38 MAPK. MMP‐9 induced ERK1/2 signaling in both undifferentiated and differentiated (using dbcAMP) Schwann cells. Using inhibitors to MEK and trophic tyrosine kinase receptors, we established that MMP‐9 regulates Ras/Raf/MEK—ERK pathways through IGF‐1, ErbB, and PDGF receptors. We also report on the early changes of MMP‐9 mRNA expression (within 24 h) after axotomy. These studies establish that MMP‐9 controls critical trophic signal transduction pathways and phenotypic remodeling of Schwann cells. © 2009 Wiley‐Liss, Inc.     

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     

Short‐term low‐frequency electrical stimulation enhanced remyelination of injured peripheral nerves by inducing the promyelination effect of brain‐derived neurotrophic factor on Schwann cell polarization

Electrical stimulation (ES) has been found to aid repair of nerve injuries and have been shown to increase and direct neurite outgrowth during stimulation. However, the effect of ES on peripheral remyelination after nerve damage has been investigated less well, and the mechanism underlying its action remains unclear. In the present study, the crush‐injured sciatic nerves in rats were subjected to 1 hr of continuous ES (20 Hz, 100 μsec, 3 V). Electron microscopy and nerve morphometry were performed to investigate the extent of regenerated nerve myelination. The expression profiles of P0, Par‐3, and brain‐derived neurotrophic factor (BDNF) in the injuried sciatic nerves and in the dorsal root ganglion neuron/Schwann cell cocultures were examined by Western blotting. Par‐3 localization in the sciatic nerves was determined by immunohistochemistry to demonstrate Schwann cell polarization during myelination. We reported that 20‐Hz ES increased the number of myelinated fibers and the thickness myelin sheath at 4 and 8 weeks postinjury. P0 level in the ES‐treated groups, both in vitro and in vivo, was enhanced compared with the controls. The earlier peak of Par‐3 in the ES‐treated groups indicated an earlier initiation of Schwann cell myelination. Additionally, ES significantly elevated BDNF expression in nerve tissues and in cocultures. ES on the site of nerve injury potentiates axonal regrowth and myelin maturation during peripheral nerve regeneration. Furthermore, the therapeutic actions of ES on myelination are mediated via enhanced BDNF signals, which drive the promyelination effect on Schwann cells at the onset of myelination. © 2010 Wiley‐Liss, Inc.     

Fibroblast‐derived tenascin‐C promotes Schwann cell migration through β1‐integrin dependent pathway during peripheral nerve regeneration

Peripheral nerve regeneration requires precise coordination and dynamic interaction among various types of cells in the tissue. It remains unclear, however, whether the cellular crosstalk between fibroblasts and Schwann cells (SCs) is related to phenotype modulation of SCs, a critical cellular process after peripheral nerve injury. In this study, microarray analysis revealed that a total of 6,046 genes were differentially expressed in the proximal nerve segment after sciatic nerve transection in rats, and bioinformatics analysis further identified tenascin‐C (TNC), an extracellular matrix (ECM) protein, as a key gene regulator. TNC was abundantly produced by nerve fibroblasts accumulating at the lesion site, rather than by SCs as usually expected. TNC significantly promoted SC migration without effects on SC proliferation in primary culture. In co‐culture of fibroblasts and SCs, inhibition of TNC expression either by siRNA transfection or antibody blockade could suppress SC migration, while TNC‐stimulated SC migration was mediated by TNC binding to β1‐integrin receptor in SCs through activation of Rac1 effectors. The in vivo evidence showed that exogenous TNC protein enhanced SC migration and axonal regrowth. Our results highlight that TNC‐mediated cellular interaction between fibroblasts and SCs may regulate SC migration through β1‐integrin‐dependent pathway during peripheral nerve regeneration. GLIA 2016;64:374–385     

Acetyl-11-keto-beta-boswellic acid promotes sciatic nerve repair after injury: molecular mechanism

Previous studies showed that acetyl-11-keto-beta-boswellic acid (AKBA), the active ingredient in the natural Chinese medicine Boswellia, can stimulate sciatic nerve injury repair via promoting Schwann cell proliferation. However, the underlying molecular mechanism remains poorly understood. In this study, we performed genomic sequencing in a rat model of sciatic nerve crush injury after gastric AKBA administration for 30 days. We found that the phagosome pathway was related to AKBA treatment, and brain-derived neurotrophic factor expression in the neurotrophic factor signaling pathway was also highly up-regulated. We further investigated gene and protein expression changes in the phagosome pathway and neurotrophic factor signaling pathway. Myeloperoxidase expression in the phagosome pathway was markedly decreased, and brain-derived neurotrophic factor, nerve growth factor, and nerve growth factor receptor expression levels in the neurotrophic factor signaling pathway were greatly increased. Additionally, expression levels of the inflammatory factors CD68, interleukin-1β, pro-interleukin-1β, and tumor necrosis factor-α were also decreased. Myelin basic protein- and β3-tubulin-positive expression as well as the axon diameter-to-total nerve diameter ratio in the injured sciatic nerve were also increased. These findings suggest that, at the molecular level, AKBA can increase neurotrophic factor expression through inhibiting myeloperoxidase expression and reducing inflammatory reactions, which could promote myelin sheath and axon regeneration in the injured sciatic nerve.     

Granulocyte‐macrophage colony‐stimulating factor improves mouse peripheral nerve regeneration following sciatic nerve crush

Peripheral nerve injuries severely impair patients’ quality of life as full recovery is seldom achieved. Upon axonal disruption, the distal nerve stump undergoes fragmentation, and myelin breaks down; the subsequent regeneration progression is dependent on cell debris removal. In addition to tissue clearance, macrophages release angiogenic and neurotrophic factors that contribute to axon growth. Based on the importance of macrophages for nerve regeneration, especially during the initial response to injury, we treated mice with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) at various intervals after sciatic nerve crushing. Sciatic nerves were histologically analyzed at different time intervals after injury for the presence of macrophages and indicators of regeneration. Functional recovery was followed by an automated walking track test. We found that GM‐CSF potentiated early axon growth, as indicated by the enhanced expression of growth‐associated protein at 7 days postinjury. Inducible nitric oxide synthase expression increased at the beginning and at the end of the regenerative process, suggesting that nitric oxide is involved in axon growth and pruning. As expected, GM‐CSF treatment stimulated macrophage infiltration, which increased at 7 and 14 days; however, it did not improve myelin clearance. Instead, GM‐CSF stimulated early brain‐derived neurotrophic factor (BDNF) production, which peaked at 7 days. Locomotor recovery pattern was not improved by GM‐CSF treatment. The present results suggest that GM‐CSF may have beneficial effects on early axonal regeneration.     

miR-30c promotes Schwann cell remyelination following peripheral nerve injury

Differential expression of miRNAs occurs in injured proximal nerve stumps and includes miRNAs that are firstly down-regulated and then gradually up-regulated following nerve injury. These miRNAs might be related to a Schwann cell phenotypic switch. miR-30c, as a member of this group, was further investigated in the current study. Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1, 4, 7, 14, 21, and 28 days post injury for analysis. Following sciatic nerve injury, miR-30c was down-regulated, reaching a minimum on day 4, and was then upregulated to normal levels. Schwann cells were isolated from neonatal rat sciatic nerve stumps, then transfected with miR-30c agomir and co-cultured in vitro with dorsal root ganglia. The enhanced expression of miR-30c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells. We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of miR-30c agomir on myelin sheath regeneration. Fourteen days after surgery, sciatic nerve stumps were harvested and subjected to immunohistochemistry, western blot analysis, and transmission electron microscopy. The direct injection of miR-30c stimulated the formation of myelin sheath, thus contributing to peripheral nerve regeneration. Overall, our findings indicate that miR-30c can promote Schwann cell myelination fol-lowing peripheral nerve injury. The functional study of miR-30c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.     

The effects of claudin 14 during early Wallerian degeneration after sciatic nerve injury

Claudin 14 has been shown to promote nerve repair and regeneration in the early stages of Wallerian degeneration(0–4 days) in rats with sciatic nerve injury, but the mechanism underlying this process remains poorly understood. This study reported the effects of claudin 14 on nerve degeneration and regeneration during early Wallerian degeneration. Claudin 14 expression was up-regulated in sciatic nerve 4 days after Wallerian degeneration. The altered expression of claudin 14 in Schwann cells resulted in expression changes of cytokines in vitro. Expression of claudin 14 affected c-Jun, but not Akt and ERK1/2 pathways. Further studies revealed that enhanced expression of claudin 14 could promote Schwann cell proliferation and migration. Silencing of claudin 14 expression resulted in Schwann cell apoptosis and reduction in Schwann cell proliferation. Our data revealed the role of claudin 14 in early Wallerian degeneration, which may provide new insights into the molecular mechanisms of Wallerian degeneration.     

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.     

Tissue kallikrein protects rat hippocampal CA1 neurons against cerebral ischemia/reperfusion‐induced injury through the B2R‐Raf‐MEK1/2‐ERK1/2 pathway

We have documented that tissue kallikrein (TK) prevents neurons from hypoxia/reoxygenation injury through the B2R‐ERK1/2 pathway and the antihypoxic function of TK through Homer1b/c‐ERK1/2 signaling pathways. The present study investigates the molecular mechanisms of exogenous TK activation of the B2R‐ERK1/2 pathway through the β‐arrestin‐2 assembled B2R‐Raf‐MEK1/2 signaling module in vivo. The cresyl violet staining results indicated that exogenous TK protected the rat hippocampal CA1 neurons against cerebral ischemia/reperfusion (I/R) injury. The immunoprecipitation (IP) and immunoblotting (IB) results revealed that exogenous TK upregulated the β‐arrestin‐2 assembled B2R‐Raf‐MEK1/2 signaling module and upregulated the phosphorylation of Raf (p‐Raf), MEK1/2 (p‐MEK1/2), and ERK1/2 (p‐ERK1/2). Meanwhile, exogenous TK upregulated the expression of nuclear factor‐κB (NF‐κB), depressed the release of cytochrome c (Cyt c) and bax from mitochondria to the cytosol, and depressed the activation of caspase‐3. Take together, our results suggest that exogenous TK attenuated the cerebral I/R induced rat hippocampal CA1 neurons injury through activating the β‐arrestin‐2 assembled B2R‐Raf‐MEK1/2 signaling module and that the activated B2R‐Raf‐MEK1/2 signaling module could upregulate the expression of NF‐κB, decrease the release of cytochrome c and bax from mitochondria to the cytosol, and depress the activation of caspase‐3. © 2014 Wiley Periodicals, Inc.     

Autocrine action of BMP2 regulates expression of GDNF-mRNA in sciatic Schwann cells

Schwann cell is a cell type that forms myelin sheath and provides trophic supports for neuronal cells by producing neurotrophic factors in both normal and traumatic situations. It was recently reported that after lesion of sciatic nerve, mRNA for glial cell line-derived neurotrophic factor (GDNF) is induced in nonneuronal cells in the nerve. However, the mechanism regulating GDNF-mRNA has remained largely unknown. In the present study, we searched for factors regulating the GDNF-mRNA expression in Schwann cells. First, we found that after transfer into explant culture as an in vitro lesion model, sciatic nerve segments began to express mRNA for bone morphogenetic protein-2 (BMP2) concomitantly with the induction of GDNF-mRNA. Treatment of the Schwann cells isolated from the sciatic nerve with combination of BMP2 and retinoic acid (RA) dramatically induced GDNF-mRNA, while BMP2 or RA alone had no effect. Furthermore, ionomycin, a calcium ionophore, which had even stronger activity on the induction of GDNF-mRNA also induced also BMP2-mRNA in cultured Schwann cells. Effects of inhibitors of intracellular signaling pathways such as protein kinase C inhibitor and MAPKK inhibitor suggested that the molecular mechanism of the induction of GDNF-mRNA is distinct from that of BMP2-mRNA. These results suggest that the Schwann cell-produced BMP2 plays an important role in the induction of GDNF after nerve injury in an autocrine fashion.     

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.     

ERK磷酸化对糖尿病周围神经病雪旺氏细胞Netrin-1表达水平及凋亡的影响

目的 探讨ERK通路对糖尿病周围神经病雪旺氏细胞凋亡以及Netrin-1表达水平的影响。方法 建立动物和细胞模型分析ERK通路对雪旺氏细胞以及Netrin-1表达水平的影响。结果 观察糖尿病模型小鼠坐骨神经组织中p-ERK的表达增加,ERK抑制剂PD98059抑制高糖诱导雪旺氏细胞中ERK的磷酸化,即ERK通路促进糖尿病周围神经疾病的发生; 糖尿病模型小鼠坐骨神经组织中Netrin-1表达减少,ERK抑制剂PD98059增加高糖刺激中雪旺氏细胞中Netrin-1的表达,即ERK抑制剂通过增加Netrin-1的表达来发挥神经保护作用; PD98059可逆转高糖诱导雪旺氏细胞中Caspase-3的表达和细胞凋亡,即ERK抑制剂降低雪旺氏细胞凋亡,从而防止糖尿病周围神经疾病的发展。结论 ERK抑制剂可抑制糖尿病周围神经疾病雪旺氏细胞的凋亡以及增加Nertin-1的表达,从而发挥保护作用     

Schwann cells transduced with a lentiviral vector encoding Fgf‐2 promote motor neuron regeneration following sciatic nerve injury

Fibroblast growth factor 2 (FGF‐2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF‐2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF‐2 specifically increases motor neuron axonal growth. To further explore the potential capability of FGF‐2 to promote axon regeneration, we produced a lentiviral vector (LV) to overexpress FGF‐2 (LV‐FGF2) in the injured rat peripheral nerve. Cultured Schwann cells transduced with FGF‐2 and added to collagen matrix embedding spinal cord or DRG explants significantly increased motor but not sensory neurite outgrowth. LV‐FGF2 was as effective as direct addition of the trophic factor to promote motor axon growth in vitro. Direct injection of LV‐FGF2 into the rat sciatic nerve resulted in increased expression of FGF‐2, which was localized in the basal lamina of Schwann cells. To investigate the in vivo effect of FGF‐2 overexpression on axonal regeneration after nerve injury, Schwann cells transduced with LV‐FGF2 were grafted in a silicone tube used to repair the resected rat sciatic nerve. Electrophysiological tests conducted for up to 2 months after injury revealed accelerated and more marked reinnervation of hindlimb muscles in the animals treated with LV‐FGF2, with an increase in the number of motor and sensory neurons that reached the distal tibial nerve at the end of follow‐up. GLIA 2014;62:1736–1746     

Autophagic myelin destruction by schwann cells during wallerian degeneration and segmental demyelination

As lysosomal hydrolysis has long been suggested to be responsible for myelin clearance after peripheral nerve injury, in this study, we investigated the possible role of autophagolysosome formation in myelin phagocytosis by Schwann cells and its final contribution to nerve regeneration. We found that the canonical formation of autophagolysosomes was induced in demyelinating Schwann cells after injury, and the inhibition of autophagy via Schwann cell‐specific knockout of the atg7 gene or pharmacological intervention of lysosomal function caused a significant delay in myelin clearance. However, Schwann cell dedifferentiation, as demonstrated by extracellular signal‐regulated kinase activation and c‐Jun induction, and redifferentiation were not significantly affected, and thus the entire repair program progressed normally in atg7 knockout mice. Finally, autophagic Schwann cells were also found during segmental demyelination in a mouse model of inflammatory peripheral neuropathy. Together, our findings suggest that autophagy is the self‐myelin destruction mechanism of Schwann cells, but mechanistically, it is a process distinct from Schwann cell plasticity for nerve repair. GLIA 2016;64:730–742     

Temporal and Spatial Expression of Ciliary Neurotrophic Factor after Peripheral Nerve Injury

Schwann cells in the intact sciatic nerve express high amounts of ciliary neurotrophic factor (CNTF), but 7 days after injury to the nerve expression dramatically decreases. To determine whether this change occurs only in the region of the injury or throughout the whole nerve we examined the spatial and temporal expression of CNTF after a crush injury. One day after injury the amount of CNTF mRNA and protein decreased within the first 4 mm distal to the crush site. This decrease progressed in a centrifugal manner distally until mRNA and protein were scarcely detectable by 7 days. In nerve proximal to the crush site CNTF expression decreased slightly and was still detectable at all sample times. During regeneration CNTF expression remained very low up to 14 days after injury. By 30 days mRNA and protein were detectable and by 60 days CNTF protein was present at normal amounts. Immunohistochemical analysis of normal nerve revealed CNTF localized in outer portion of the cytoplasm of myelin-forming Schwann cells. Three days after injury CNTF coalesced with pockets of cytoplasm in the Schwann cell and by 5 days was barely detectable. Positive staining remained in proximal segments where little or no degeneration occurred. These results demonstrate that CNTF expression in Schwann cells is synchronized with their functional state. CNTF expression decreases with demyelination during Wallerian degeneration and returns to normal following remyelination during regeneration. These findings also suggest that CNTF expression requires intact axon-Schwann cell interactions.     

Enhancement of glutamatergic transmission in the cingulate cortex in response to mild noxious stimuli under a neuropathic pain‐like state

Pain is evoked by noxious body stimulation or through negative emotional events and memories. There are several caveats to the simple proposition that pain and emotion are linked in the cingulate cortex (CG). In this study, we investigated whether mild noxious heat stimuli could affect the neuronal activity in the CG of rats with sciatic nerve ligation. We produced a partial sciatic nerve injury by tying a tight ligature in rats. Seven days after sciatic nerve ligation, rats received mild noxious heat stimuli. Mild noxious heat stimuli produced flinching behaviors in sciatic nerve‐ligated rats, but not sham‐operated rats. In addition, the mild noxious heat stimuli caused a significant increase in the release of glutamate in the CG of nerve‐ligated rats compared with that of sham‐operated rats. Furthermore, phosphorylated‐NR1‐positive cells in this area significantly increased after mild noxious heat stimuli under a neuropathic pain. Under this condition, there were no significant changes in the levels of immediate‐early genes such as c‐fos, c‐jun, JunB, and Fra1 in the CG between nerve‐ligated and sham‐operated rats. However, mild noxious heat stimuli under a neuropathic pain‐like state produced a marked increase in the phosphorylated‐c‐jun (p‐c‐jun) immunoreactivity, which is commonly used to map neurons in the brain that can be activated after N‐methyl‐D ‐aspartate receptor activation. These findings raise the possibility that mild noxious heat stimuli under a peripheral nerve injury may increase the release of glutamate and promote its related postneuronal activity in the CG. Synapse, 2010. © 2010 Wiley‐Liss, Inc.