Delayed peripheral nerve repair: methods,including surgical ‘cross-bridging' to promote nerve regeneration

Despite the capacity of Schwann cells to support peripheral nerve regeneration, functional recovery after nerve injuries is frequently poor, especially for proximal injuries that require regenerating axons to grow over long distances to reinnervate distal targets. Nerve transfers, where small fascicles from an adjacent intact nerve are coapted to the nerve stump of a nearby denervated muscle, allow for functional return but at the expense of reduced numbers of innervating nerves. A 1-hour period of 20 Hz electrical nerve stimulation via electrodes proximal to an injury site accelerates axon outgrowth to hasten target reinnervation in rats and humans, even after delayed surgery. A novel strategy of enticing donor axons from an otherwise intact nerve to grow through small nerve grafts(cross-bridges) into a denervated nerve stump, promotes improved axon regeneration after delayed nerve repair. The efficacy of this technique has been demonstrated in a rat model and is now in clinical use in patients undergoing cross-face nerve grafting for facial paralysis. In conclusion, brief electrical stimulation, combined with the surgical technique of promoting the regeneration of some donor axons to ‘protect' chronically denervated Schwa nn cells, improves nerve regeneration and, in turn, functional outcomes in the management of peripheral nerve injuries.     

The role of exosomes in peripheral nerve regeneration

Peripheral nerve injuries remain problematic to treat, with poor functional recovery commonly observed. Injuries resulting in a nerve gap create specific difficulties for axonal regeneration. Approaches to address these difficulties include autologous nerve grafts (which are currently the gold standard treatment) and synthetic conduits, with the latter option being able to be impregnated with Schwann cells or stem cells which provide an appropriate micro-environment for neuronal regeneration to occur. Transplanting stem cells, however, infers additional risk of malignant transformation as well as manufacturing difficulties and ethical concerns, and the use of autologous nerve grafts and Schwann cells requires the sacrifice of a functioning nerve. A new approach utilizing exosomes, secreted extracellular vesicles, could avoid these complications. In this review, we summarize the current literature on exosomes, and suggest how they could help to improve axonal regeneration following peripheral nerve injury.     

Biological conduit small gap sleeve bridging method for peripheral nerve injury: regeneration law of nerve fibers in the conduit

The clinical effects of 2-mm small gap sleeve bridging of the biological conduit to repair peripheral nerve injury are better than in the traditional epineurium suture, so it is possible to replace the epineurium suture in the treatment of peripheral nerve injury. This study sought to identify the regeneration law of nerve fibers in the biological conduit. A nerve regeneration chamber was constructed in models of sciatic nerve injury using 2-mm small gap sleeve bridging of a biodegradable biological conduit. The results showed that the biological conduit had good histocompatibility. Tissue and cell apoptosis in the conduit apparently lessened, and regenerating nerve fibers were common. The degeneration regeneration law of Schwann cells and axons in the conduit was quite different from that in traditional epineurium suture. During the prime period for nerve fiber regeneration(2–8 weeks), the number of Schwann cells and nerve fibers was higher in both proximal and distal ends, and the effects of the small gap sleeve bridging method were better than those of the traditional epineurium suture. The above results provide an objective and reliable theoretical basis for the clinical application of the biological conduit small gap sleeve bridging method to repair peripheral nerve injury.     

Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

Human umbilical cord-derived mesenchymal stem cells(h UCMSCs) represent a promising young-state stem cell source for cell-based therapy. h UCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the paracrine effects of h UCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that h UCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with h UCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These findings suggest that paracrine action may be a key mechanism underlying the effects of h UCMSCs in peripheral nerve repair.     

Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury

The transient receptor potential cation channel subfamily V member 1(TRPV1) provides the sensation of pain(nociception). However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517(300 mg/kg), a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immunofluorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clusters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.     

Exogenous nerve growth factor protects the hypoglossal nerve against crush injury

Studies have shown that sensory nerve damage can activate the p38 mitogen-activated protein kinase(MAPK)pathway,but whether the same type of nerve injury after exercise activates the p38MAPK pathway remains unclear.Several studies have demonstrated that nerve growth factor may play a role in the repair process after peripheral nerve injury,but there has been little research focusing on the hypoglossal nerve injury and repair.In this study,we designed and established rat models of hypoglossal nerve crush injury and gave intraperitoneal injections of exogenous nerve growth factor to rats for 14 days.p38MAPK activity in the damaged neurons was increased following hypoglossal nerve crush injury;exogenous nerve growth factor inhibited this increase in acitivity and increased the survival rate of motor neurons within the hypoglossal nucleus.Under transmission electron microscopy,we found that the injection of nerve growth factor contributed to the restoration of the morphology of hypoglossal nerve after crush injury.Our experimental findings indicate that exogenous nerve growth factor can protect damaged neurons and promote hypoglossal nerve regeneration following hypoglossal nerve crush injury.     

Identification of a functional interaction of HMGB1 with Receptor for Advanced Glycation End-products in a model of neuropathic pain

Recent studies indicate that the release of high mobility group box 1 (HMGB1) following nerve injury may play a central role in the pathogenesis of neuropathic pain. HMGB1 is known to influence cellular responses within the nervous system via two distinct receptor families; the Receptor for Advanced Glycation End-products (RAGE) and Toll-like receptors (TLRs). The degree to which HMGB1 activates a receptor is thought to be dependent upon the oxidative state of the ligand, resulting in the functional isoforms of all-thiol HMGB1 (at-HMGB1) acting through RAGE, and disufide HMGB1 (ds-HMGB1) interacting with TLR4. Though it is known that dorsal root ganglia (DRG) sensory neurons exposed to HMGB1 and TLR4 agonists can influence excitation, the degree to which at-HMGB1 signaling through neuronal RAGE contributes to neuropathic pain is unknown. Here we demonstrate that at-HMGB1 activation of nociceptive neurons is dependent on RAGE and not TLR4. To distinguish the possible role of RAGE on neuropathic pain, we characterized the changes in RAGE mRNA expression up to one month after tibial nerve injury (TNI). RAGE mRNA expression in lumbar dorsal root ganglion (DRG) is substantially increased by post-injury day (PID) 28 when compared with sham injured rodents. Protein expression at PID28 confirms this injury-induced event in the DRG. Moreover, a single exposure to monoclonal antibody to RAGE (RAGE Ab) failed to abrogate pain behavior at PID 7, 14 and 21. However, RAGE Ab administration produced reversal of mechanical hyperalgesia on PID28. Thus, at-HMGB1 activation through RAGE may be responsible for sensory neuron sensitization and mechanical hyperalgesia associated with chronic neuropathic pain states.     

Edaravone combined with Schwann cell transplantation may repair spinal cord injury in rats

Edaravone has been shown to delay neuronal apoptosis, thereby improving nerve function and the microenvironment after spinal cord injury. Edaravone can provide a favorable environment for the treatment of spinal cord injury using Schwann cell transplantation. This study used rat models of complete spinal cord transection at T9. Six hours later, Schwann cells were transplanted in the head and tail ends of the injury site. Simultaneously, edaravone was injected through the caudal vein. Eight weeks later, the PKH-26-labeled Schwann cells had survived and migrated to the center of the spinal cord injury region in rats after combined treatment with edaravone and Schwann cells. Moreover, the number of PKH-26-labeled Schwann cells in the rat spinal cord was more than that in rats undergoing Schwann cell transplantation alone or rats without any treatment. Horseradish peroxidase retrograde tracing revealed that the number of horseradish peroxidase-positive nerve fibers was greater in rats treated with edaravone combined with Schwann cells than in rats with Schwann cell transplantation alone. The results demonstrated that lower extremity motor function and neurophysiological function were better in rats treated with edaravone and Schwann cells than in rats with Schwann cell transplantation only. These data confirmed that Schwann cell transplantation combined with edaravone injection promoted the regeneration of nerve fibers of rats with spinal cord injury and improved neurological function.     

Repair of peripheral nerve defects with chemically extracted acellular nerve allografts loaded with neurotrophic factors-transfected bone marrow mesenchymal stem cells

Chemically extracted acellular nerve allografts loaded with brain-derived neurotrophic factor-transfected or ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells have been shown to repair sciatic nerve injury better than chemically extracted acellular nerve allografts alone, or chemically extracted acellular nerve allografts loaded with bone marrow mesenchymal stem cells. We hypothesized that these allografts compounded with both brain-derived neurotrophic factor- and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells may demonstrate even better effects in the repair of peripheral nerve injury. We cultured bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor and/or ciliary neurotrophic factor and used them to treat sciatic nerve injury in rats. We observed an increase in sciatic functional index, triceps wet weight recovery rate, myelin thickness, number of myelinated nerve fibers, amplitude of motor-evoked potentials and nerve conduction velocity, and a shortened latency of motor-evoked potentials when allografts loaded with both neurotrophic factors were used, compared with allografts loaded with just one factor. Thus, the combination of both brain-derived neurotrophic factor and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells can greatly improve nerve injury.     

Electrospun and woven silk fibroin/poly(lactic-coglycolic acid) nerve guidance conduits for repairing peripheral nerve injury

We have designed a novel nerve guidance conduit(NGC) made from silk fibroin and poly(lactic-co-glycolic acid) through electrospinning and weaving(ESP-NGCs). Several physical and biological properties of the ESP-NGCs were assessed in order to evaluate their biocompatibility. The physical properties, including thickness, tensile stiffness, infrared spectroscopy, porosity, and water absorption were determined in vitro. To assess the biological properties, Schwann cells were cultured in ESP-NGC extracts and were assessed by morphological observation, the MTT assay, and immunohistochemistry. In addition, ESP-NGCs were subcutaneously implanted in the backs of rabbits to evaluate their biocompatibility in vivo. The results showed that ESP-NGCs have high porosity, strong hydrophilicity, and strong tensile stiffness. Schwann cells cultured in the ESP-NGC extract fluids showed no significant differences compared to control cells in their morphology or viability. Histological evaluation of the ESP-NGCs implanted in vivo indicated a mild inflammatory reaction and high biocompatibility. Together, these data suggest that these novel ESP-NGCs are biocompatible, and may thus provide a reliable scaffold for peripheral nerve repair in clinical application.     

Femoral nerve regeneration and its accuracy under different injury mechanisms

Surgical accuracy has greatly improved with the advent of microsurgical techniques. However, complete functional recovery after peripheral nerve injury has not been achieved to date. The mechanisms hindering accurate regeneration of damaged axons after peripheral nerve injury are in urgent need of exploration. The present study was designed to explore the mechanisms of peripheral nerve regeneration after different types of injury. Femoral nerves of rats were injured by crushing or freezing. At 2, 3, 6, and 12 weeks after injury, axons were retrogradely labeled using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate(Dil) and True Blue, and motor and sensory axons that had regenerated at the site of injury were counted. The number and percentage of Dil-labeled neurons in the anterior horn of the spinal cord increased over time. No significant differences were found in the number of labeled neurons between the freeze and crush injury groups at any time point. Our results confirmed that the accuracy of peripheral nerve regeneration increased with time, after both crush and freeze injury, and indicated that axonal regeneration accuracy was still satisfactory after freezing, despite the prolonged damage.     

A novel therapeutic target for peripheral nerve injury-related diseases: aminoacyl-tRNA synthetases

Aminoacyl-t RNA synthetases(Amino ARSs) are essential enzymes that perform the first step of protein synthesis. Beyond their original roles, Amino ARSs possess non-canonical functions, such as cell cycle regulation and signal transduction. Therefore, Amino ARSs represent a powerful pharmaceutical target if their non-canonical functions can be controlled. Using Amino ARSs-specific primers, we screened m RNA expression in the spinal cord dorsal horn of rats with peripheral nerve injury created by sciatic nerve axotomy. Of 20 Amino ARSs, we found that phenylalanyl-t RNA synthetase beta chain(FARSB), isoleucyl-t RNA synthetase(IARS) and methionyl-t RNA synthetase(MARS) m RNA expression was increased in spinal dorsal horn neurons on the injured side, but not in glial cells. These findings suggest the possibility that FARSB, IARS and MARS, as a neurotransmitter, may transfer abnormal sensory signals after peripheral nerve damage and become a new target for drug treatment.     

Dynamic expression of Slit1–3 and Robo1–2 in the mouse peripheral nervous system after injury

The Slit family of axon guidance cues act as repulsive molecules for precise axon pathfinding and neuronal migration during nervous system development through interactions with specific Robo receptors.Although we previously reported that Slit1–3 and their receptors Robo1 and Robo2 are highly expressed in the adult mouse peripheral nervous system,how this expression changes after injury has not been well studied.Herein,we constructed a peripheral nerve injury mouse model by transecting the right sciatic nerve.At 14 days after injury,quantitative real-time polymerase chain reaction was used to detect mRNA expression of Slit1–3 and Robo1–2 in L4–5 spinal cord and dorsal root ganglia,as well as the sciatic nerve.Immunohistochemical analysis was performed to examine Slit1–3,Robo1–2,neurofilament heavy chain,F4/80,and vimentin in L4–5 spinal cord,L4 dorsal root ganglia,and the sciatic nerve.Co-expression of Slit1–3 and Robo1–2 in L4 dorsal root ganglia was detected by in situ hybridization.In addition,Slit1–3 and Robo1–2 protein expression in L4–5 spinal cord,L4 dorsal root ganglia,and sciatic nerve were detected by western blot assay.The results showed no significant changes of Slit1–3 or Robo1–2 mRNA expression in the spinal cord within 14 days after injury.In the dorsal root ganglion,Slit1–3 and Robo1–2 mRNA expression were initially downregulated within 4 days after injury;however,Robo1–2 mRNA expression returned to the control level,while Slit1–3 mRNA expression remained upregulated during regeneration from 4–14 days after injury.In the sciatic nerve,Slit1–3 and their receptors Robo1–2 were all expressed in the proximal nerve stump;however,Slit1,Slit2,and Robo2 were barely detectable in the nerve bridge and distal nerve stump within 14 days after injury.Slit3 was highly ex-pressed in macrophages surrounding the nerve bridge and slightly downregulated in the distal nerve stump within 14 days after injury.Robo1 was upregulated in vimentin-positive cells and migrating Schwann cells inside the nerve bridge.Robo1 was also upregulated in Schwann cells of the distal nerve stump within 14 days after injury.Our findings indicate that Slit3 is the major ligand expressed in the nerve bridge and distal nerve stump during peripheral nerve regeneration,and Slit3/Robo signaling could play a key role in peripheral nerve repair after injury.This study was approved by Plymouth University Animal Welfare Ethical Review Board (approval No.30/3203) on April 12,2014.     

Erythropoietin promotes Schwann cell migration and assembly of the provisional extracellular matrix by recruiting β1 integrin to the cell surface

In peripheral nerve injury, Schwann cells undergo profound phenotypic modulation, adopting a migratory phenotype and remodeling the extracellular matrix so that it is permissive for axonal regrowth. Erythropoietin (Epo) and its receptor (EpoR) are expressed by Schwann cells after nerve injury, regulating inflammatory cytokine expression and minimizing the duration of neuropathic pain. The mechanism of Epo activity in the injured peripheral nerve remains incompletely understood. Herein, we demonstrate that Epo promotes Schwann cell migration in vitro on fibronectin (FN)‐coated surfaces. Epo also rapidly recruits β1 integrin subunit to the Schwann cell surface by a JAK‐2‐dependent pathway. Although β1 integrin subunit‐containing integrins were not principally responsible for Schwann cell adhesion or migration on FN under basal conditions, β1 gene‐silencing blocked the ability of Epo to promote cell migration. Epo also induced Schwann cell FN expression in vitro and in vivo. The FN was organized into insoluble fibrils by Epo‐treated Schwann cells in vitro and into an extensive matrix surrounding Schwann cells in vivo. Our results support a model in which Epo promotes Schwann cell migration and assembly of the provisional extracellular matrix in the injured peripheral nerve by its effects on integrin recruitment to the cell surface and local FN production. © 2009 Wiley‐Liss, Inc.     

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 challenges and beauty of peripheral nerve regrowth

This review provides an overview of selected aspects of peripheral nerve regeneration and potential avenues to explore therapeutically. The overall coordinated and orchestrated pattern of recovery from peripheral nerve injury has a beauty of execution and progress that rivals all other forms of neurobiology. It involves changes at the level of the perikaryon, coordination with important peripheral glial partners, the Schwann cells, a controlled inflammatory response, and growth that overcomes surprising intrinsic roadblocks. Both regenerative axon growth and collateral sprouting encompass fascinating aspects of this story. Better understanding of peripheral nerve regeneration may also lead to enhanced central nervous system recovery.     

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.     

Early cyclosporin A treatment retards axonal degeneration in an experimental peripheral nerve injection injury model

Injury to peripheral nerves during injections of therapeutic agents such as penicillin G potassium is common in developing countries. It has been shown that cyclosporin A, a powerful immunosuppressive agent, can retard Wallerian degeneration after peripheral nerve crush injury. However, few studies are reported on the effects of cyclosporin A on peripheral nerve drug injection injury. This study aimed to assess the time-dependent efficacy of cyclosporine-A as an immunosuppressant therapy in an experimental rat nerve injection injury model established by penicillin G potassium injection. The rats were randomly divided into three groups based on the length of time after nerve injury induced by cyclosporine-A administration(30 minutes, 8 or 24 hours). The compound muscle action potentials were recorded pre-injury, early post-injury(within 1 hour) and 4 weeks after injury and compared statistically. Tissue samples were taken from each animal for histological analysis. Compared to the control group, a significant improvement of the compound muscle action potential amplitude value was observed only when cyclosporine-A was administered within 30 minutes of the injection injury(P < 0.05); at 8 or 24 hours after cyclosporine-A administration, compound muscle action potential amplitude was not changed compared with the control group. Thus, early immunosuppressant drug therapy may be a good alternative neuroprotective therapy option in experimental nerve injection injury induced by penicillin G potassium injection.