Regeneration Of Nerve Fibers Research Articles

Nerve Guidance by a Decellularized Fibroblast Extracellular Matrix

Spinal cord and peripheral nerve injuries require the regeneration of nerve fibers across the lesion site for successful recovery. Providing guidance cues and soluble factors to promote neurite outgrowth and cell survival can enhance repair. The extracellular matrix (ECM) plays a key role in tissue repair by controlling cell adhesion, motility, and growth. In this study, we explored the ability of a mesenchymal ECM to support neurite outgrowth from neurons in the superior cervical ganglia (SCG). Length and morphology of neurites extended on a decellularized fibroblast ECM were compared to those on substrates coated with laminin, a major ECM protein in neural tissue, or fibronectin, the main component of a mesenchymal ECM. Average radial neurite extension was equivalent on laminin and on the decellularized ECM, but contrasted with the shorter, curved neurites observed on the fibronectin substrate. Differences between neurites on fibronectin and on other substrates were confirmed by fast Fourier transform analyses. To control the direction of neurite outgrowth, we developed an ECM with linearly aligned fibril organization by orienting the fibroblasts that deposit the matrix on a polymeric surface micropatterned with a striped chemical interface. Neurites projected from SCGs appeared to reorient in the direction of the pattern. These results highlight the ability of a mesenchymal ECM to enhance neurite extension and to control the directional outgrowth of neurites. This micropatterned decellularized ECM architecture has potential as a regenerative microenvironment for nerve repair.

Olfactory ensheathing glia cell therapy and tubular conduit enhance nerve regeneration after mouse sciatic nerve transection

The regenerative potential of the peripheral nervous system (PNS) is widely known, but functional recovery, particularly in humans, is seldom complete. Therefore, it is necessary to resort to strategies that induce or potentiate the PNS regeneration. Our main objective was to test the effectiveness of Olfactory Ensheathing Cells (OEC) transplantation into a biodegradable conduit as a therapeutic strategy to improve the repair outcome after nerve injury. Sciatic nerve transection was performed in C57BL/6 mice; proximal and distal stumps of the nerve were sutured into the collagen conduit. Two groups were analyzed: DMEM (acellular grafts) and OEC (1×105/2μL). Locomotor function was assessed weekly by Sciatic Function Index (SFI) and Global Mobility Test (GMT). After eight weeks the sciatic nerve was dissected for morphological analysis. Our results showed that the OEC group exhibited many clusters of regenerated nerve fibers, a higher number of myelinated fibers and myelin area compared to DMEM group. The G-ratio analysis of the OEC group showed significantly more fibers on the most suitable sciatic nerve G-ratio index. Motor recovery was accelerated in the OEC group. These data provide evidence that the OEC therapy can improve sciatic nerve functional and morphological recovery and can be potentially translated to the clinical setting.

Decellularized nerve xenograft combined with lycium barbarum polysaccharide for repair of sciatic nerve defect in rats

Objective To explore the effect of lycium barbarum polysaccharide (LBP) and decellularized nerve xenograft combination on nerve regeneration and recovery in repairing rat sciatic nerve defects. Methods Xenogeneic acellular nerve scaffolds were prepared by chemical extraction of tibial nerves from rabbits. A 10 mm left sciatic nerve defect model was created in 90 healthy adult Sprague-Dawley rats who were randomly divided into 5 groups: blank group (n=18), model group (n=18), LBP group (n=18), scaffold group (n=18), LBP and scaffold combination group (n=18). Two weeks after model creation. The sciatic nerve defect in LBP, scaffold and combination groups was repaired either by LBP, by scaffold, or by the LBP xenograft combination. After the second surgery, the rats survived for 4, 8 and 12 weeks, respectively. Gross observation of the scaffold, nerve conduction study to measure sciatic nerve motor nerve conduction velocity (MNCV), and neuromorphometry analysis of the graft were carried out at these three time points. Results Neuroma formation at the proximal nerve end was observed in the model group and LBP group. Adhesion of nerve ends to the surrounding tissue was more severe in the model group than in the LBP group. The sizes of the nerve grafts in the scaffold group and combination group were comparable. Proficient vascularization on the surface of these grafts was seen. There was only slight adhesion to the surrounding tissue. The regenerated nerve fibers in the combination group were denser and better oriented. Motor nerve conduction velocity recovery and the number of myelinated nerve fibers in the grafts were measured at 12 weeks postoperatively for blank, model, LBP, scaffold and combination groups, respectively. The differences among the groups were significant (P<0.05). Conclusion The combination of LBP and xenogeneic acellular nerve scaffold enhances peripheral nerve regeneration and recovery when used to repair nerve defects. Key words: Sciatic nerve; Nerve regeneration; Xenogeneic acellular nerve scaffold; Lycium barbarum polysaccharide

An experiment study of the effect of high frequency electrotome on preventing the formation of painful neuroma

Objective To investigate the feasibility and practicability of high frequency electrotome cutting and (or) coagulation of nerve stumps in preventing painful neuroma formation. Methods Fifty-six 3-month-old Sprague-Dawley rats were randomly divided into four groups with 14 rats each. Depending on group assignment, the sciatic nerve was cut by a blade, by electric cutting with a high frequency electrotome, by electric cutting and coagulation with the electrotome, by a blade and electric coagulation with the electrotome. Postoperative observation and evaluations included the incidence of autotomy, the diameter of the nerve stump, nerve fiber regeneration and orientation at the nerve stump, and immunohistochemical staining of nerve growth factor (NGF) expression at the sciatic nerve stump. Results The blade and electric coagulation group had the lowest autotomy level, the latest autotomy onset, and the lowest incidence of severe autotomy. The nerve stump did not show obvious enlargement, neither was it adhered to the surrounding tissue. On the contrary the electric cutting group had the highest autotomy level, the shortest average onset time of autotomy and the highest incidence of severe autotomy. There was obvious neuroma formation at the nerve stump which was tightly adhered to the surrounding tissue. The differences between these two groups were significant (P<0.05). Immunohistochemical staining demonstrated NGF expression in the nerve stumps of rats in all 4 groups. The expression level was the lowest in the blade and electric coagulation group and highest in the electric cutting group. Conclusion Cutting nerve with a sharp blade and coagulating it with a high frequency electrotome is an effective method to prevent the formation of painful neuroma. This method is not only simple but also cost efficient. Key words: Neuroma; Electrocoagulation; Stump pain; Animal experimentation

Chitosan crosslinked flat scaffolds for peripheral nerve regeneration

Chitosan (CS) has been widely used in a variety of biomedical applications, including peripheral nerve repair, due to its excellent biocompatibility, biodegradability, readily availability and antibacterial activity. In this study, CS flat membranes, crosslinked with dibasic sodium phosphate (DSP) alone (CS/DSP) or in association with the γ-glycidoxypropyltrimethoxysilane (CS/GPTMS_DSP), were fabricated with a solvent casting technique. The constituent ratio of crosslinking agents and CS were previously selected to obtain a composite material having both adequate mechanical properties and high biocompatibility.In vitro cytotoxicity tests showed that both CS membranes allowed cell survival and proliferation. Moreover, CS/GPTMS_DSP membranes promoted cell adhesion, induced Schwann cell-like morphology and supported neurite outgrowth from dorsal root ganglia explants.Preliminary in vivo tests carried out on both types of nerve scaffolds (CS/DSP and CS/GPTMS_DSP membranes) demonstrated their potential for: (i) protecting, as a membrane, the site of nerve crush or repair by end-to-end surgery and avoiding post-operative nerve adhesion; (ii) bridging, as a conduit, the two nerve stumps after a severe peripheral nerve lesion with substance loss.A 1 cm gap on rat median nerve was repaired using CS/DSP and CS/GPTMS_DSP conduits to further investigate their ability to induce nerve regeneration in vivo. CS/GPTMS_DSP tubes resulted to be more fragile during suturing and, along a 12 week post-operative lapse of time, they detached from the distal nerve stump. On the contrary CS/DSP conduits promoted nerve fiber regeneration and functional recovery, leading to an outcome comparable to median nerve repaired by autograft.

Poly(D,L-Lactide-Co-Glycolide) Tubes With Multifilament Chitosan Yarn or Chitosan Sponge Core in Nerve Regeneration

The influence of different kinds of nerve guidance conduits on regeneration of totally transected rat sciatic nerves through a 7-mm gap was examined. Five different types of conduits made of chitosan and poly(D,L-lactide-co-glycolide) (PLGA) were constructed and tested invivo. We divided 50 animals into equal groups of 10, with a different type of conduit implanted in each group: chitosan sponge core with an average molecular mass of polymer (Mv) of 287 kDa with 7 channels in a PLGA sleeve, chitosan sponge core with an Mv of 423kDa with 7 channels in a PLGA sleeve, chitosan sponge core (Mv, 423 kDa) with 13 channels in a PLGA sleeve, chitosan multifilament yarn in a PLGA sleeve, and a PLGA sleeve only. Seven weeks after the operation, we examined the distance covered by regenerating nerve fibers, growing of nerves into the conduit's core, and intensity and type of inflammatory reaction in the conduit, as well as autotomy behavior (reflecting neuropathic pain intensity) in the animals. Two types of conduits were allowing nerve outgrowth through the gap with minor autotomy and minor inflammatory reactions. These were the conduits with chitosan multifilament yarn in a PLGA sleeve and the conduits with 13-channel microcrystalline chitosan sponge in a PLGA sleeve. The type of chitosan used to build the nerve guidance conduit influences the intensity and character of inflammatory reaction present during nerve regeneration, which in turn affects the distance crossed by regenerating nerve fibers, growing of the nerve fibers into the conduit's core, and the intensity of autotomy in the animals.

Tumor necrosis factor-α antagonist suppresses local inflammatory reaction and facilitates olfactory nerve recovery following injury.

Olfactory dysfunction is a common finding in head trauma due to injury to the olfactory nerve. We previously reported that anti-inflammatory treatment with steroids improves recovery outcome in olfactory nerve injury models. Clinically, however, steroid administration is not recommended in the acute phase of head injury cases because of concerns regarding its side effects. Tumor necrosis factor (TNF-α) is known to play a key role in inflammatory response to injury. The present study examines if the inhibition of TNF-α can facilitate functional recovery in the olfactory system following injury. Olfactory nerve transection (NTx) was performed in olfactory marker protein (OMP-tau-lacZ) mice to establish injury models. We measured TNF-α gene expression in the olfactory bulb using semi-quantitative and real time polymerase chain reaction (PCR) assays and found that they increase within hours after NTx injury. A TNF-α antagonist (etanercept) was intraperitoneally injected immediately after the NTx and histological assessment of recovery within the olfactory bulb was performed at 5-70 days. X-gal staining labeled OMP in the degenerating and regenerating olfactory nerve fibers, and immunohistochemical staining detected the presence of reactive astrocytes and macrophages/microglia. Etanercept-injected mice showed significantly smaller areas of injury-associated tissue, fewer astrocytes and macrophages/microglia, and an increase in regenerating nerve fibers. Olfactory function assessments using both an olfactory avoidance behavioral test and evoked potential recordings showed improved functional recovery in etanercept-injected animals. These findings suggest that inhibition of TNF-α could provide a new therapeutic strategy for the treatment of olfactory dysfunction following head injuries.

Autocrine fibronectin from differentiating mesenchymal stem cells induces the neurite elongation in vitro and promotes nerve fiber regeneration in transected spinal cord injury.

Extracellular matrix (ECM) expression is temporally and spatially regulated during the development of stem cells. We reported previously that fibronectin (FN) secreted by bone marrow mesenchymal stem cells (MSCs) was deposited on the surface of gelatin sponge (GS) soon after culture. In this study, we aimed to assess the function of accumulated FN on neuronal differentiating MSCs as induced by Schwann cells (SCs) in three dimensional transwell co‐culture system. The expression pattern and amount of FN of differentiating MSCs was examined by immunofluorescence, Western blot and immunoelectron microscopy. The results showed that FN accumulated inside GS scaffold, although its mRNA expression in MSCs was progressively decreased during neural induction. MSC‐derived neuron‐like cells showed spindle‐shaped cell body and long extending processes on FN‐decorated scaffold surface. However, after blocking of FN function by application of monoclonal antibodies, neuron‐like cells showed flattened cell body with short and thick neurites, together with decreased expression of integrin β1. In vivo transplantation study revealed that autocrine FN significantly facilitated endogenous nerve fiber regeneration in spinal cord transection model. Taken together, the present results showed that FN secreted by MSCs in the early stage accumulated on the GS scaffold and promoted the neurite elongation of neuronal differentiating MSCs as well as nerve fiber regeneration after spinal cord injury. This suggests that autocrine FN has a dynamic influence on MSCs in a three dimensional culture system and its potential application for treatment of traumatic spinal cord injury. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1902–1911, 2016.

Changes in the Number of Regenerating Myelin Fibers in Damaged Nerves in Rats after Allotransplantation of Dissociated Embryonic Central Nervous System Rudiments

Experiments were performed on six female and 36 male adult Wistar rats to compare the influences of dissociated cells obtained from different embryonic CNS rudiments on the growth of regenerating nerve fibers in damaged recipient nerves. Sciatic nerves were damaged by application of a ligature, after which some animals received injections of suspensions of cells into the proximal nerve segment, using cells obtained by dissociation of spinal cord or anterior cerebral vesicle fragments from rats at 15 days of development. These experiments showed that injection of dissociated cells from embryonic spinal cord rudiments but not neocortex rudiments into the damaged nerve led to increases in number of myelinated nerve cells in recipients at 60 days.

An Experimental Study on the Treatment of Spinal Cord Injury in Rats by Tissue Engineering Method that Uses ADSCs Modified by BDNF and NT-3 Double Genes Combing the use of SFCS

Introduction Adipose-derived stem cells (ADSCs) possess the potential of multi-differentiation which could differentiate into neuronal cells and Schwann cells under certain conditions. If we inoculate the ADSCs modified by Brain derived neurophic factor(BDNF) and Neurotrophin-3(NT-3)double genes on the silk fibroin/chitosan (SFCS), and transplant it into 2 mm pre-made spinal cord transection animal model, we can take advantage of the characteristic of ADSCs which could express BDNF and NT-3 stably after being double gene modified and the advantage of tissue engineering, we look forward to providing a new treatment method for spinal cord injury (SCI), which thereby could reduce SCI morbidity and mortality rate, so as to build up a solid theoretical foundation for the clinical treatment of SCI. Material and Methods According to the preliminary design, animal experiment is divided into seven groups: Group A: SFCS/ADSCs/Lenti-BDNF-GFP-NT-3-RFP, namely, BDNF and NT-3 double genes co-transfect ADSCs combining SFCS group; Group B: SFCS/ADSCs/Lenti-BDNF-GFP, namely, BDNF single gene transfect ADSCs combining SFCS group; Group C: SFCS/ADSCs/ Lenti-NT-3-RFP, namely, NT-3 single gene transfect ADSCs combining SFCS group; Group D: SFCS/ADSCs/Lenti-eGFP, namely, empty vector transfect ADSCs combining SFCS group; Group E:SFCS, namely, single SFCS group; Group F:only cut off spinal cord and without treatment; Group G: normal control group, just open the vertebral plate without any treatment. Group A, B, C, D and E transplant the scaffold into 2 mm spinal cord transection defect rat model. Group F cuts off spinal cord and makes 2 mm defect, Group G is normal group. Do a BBB scoring at 1 w, 4 w, 8 w and 12 w after surgery to analyze functional recovery, execute the rats 12 w later, take out the scaffolds, test them by HE staining, immunehistochemical staining (NSE, GAP-43, Caspase-3, GFAP), LFB staining, RT-PCR and Western blot, the purpose of which is to observe the effect of tissue engineering on the recovery of SCI. Results Group A (BDNF and NT-3 double genes modified ADSCs combining the use of SFCS group) has a better repair effect on rats spinal cord transection injury treatment, which reveals higher BBB scores, increased GAP-43 expression, more positive NSE cells, decreased expression of GFAP and Caspase-3. At the same time denser regenerating nerve fiber could move through the scaffold, and the most important thing is the repair effect of Group A is better than single gene modified Group B and Group C, which is better than Group D (empty vector transfect ADSCs combining SFCS group), which is better than Group E (single use SFCS), which is better than Group F (just cut off spinal cord and without treatment). Conclusion Under the guidance of tissue engineering method, ADSCs modified by BDNF and NT-3 double genes combining the use of SFCS can significantly reduce the inflammatory response after injury, inhibit scar hyperplasia and cell apoptosis and promote nerve fibers regeneration and the differentiation of ADSCs into neurons in vivo, so as to improve the repair effect of rat spinal cord transection injuries, which provides a new method for the treatment of SCI.

From 'Nerve Fiber Regeneration' to 'Functional Changes' in the Human Brain-On the Paradigm-Shifting Work of the Experimental Physiologist Albrecht Bethe (1872-1954) in Frankfurt am Main.

Until the beginning 1930’s the traditional dogma that the human central nervous system (CNS) did not possess any abilities to adapt functionally to degenerative processes and external injuries loomed large in the field of the brain sciences (Hirnforschung). Cutting-edge neuroanatomists, such as the luminary Wilhelm Waldeyer (1836–1921) in Germany or the Nobel Prize laureate Santiago Ramón y Cajal (1852–1934) in Spain, debated any regenerative and thus “plastic” properties in the human brain. A renewed interest arose in the scientific community to investigate the pathologies and the healing processes in the human CNS after the return of the high number of brain injured war veterans from the fronts during and after the First World War (1914–1918). A leading research center in this area was the “Institute for the Scientific Study of the Effects of Brain Injuries,” which the neurologist Ludwig Edinger (1855–1918) had founded shortly before the war. This article specifically deals with the physiological research on nerve fiber plasticity by Albrecht Bethe (1872–1954) at the respective institute of the University of Frankfurt am Main. Bethe conducted here his paradigmatic experimental studies on the pathophysiological and clinical phenomena of peripheral and CNS regeneration.

Small fiber pathology--a culprit for many painful disorders?

Small fiber neuropathies (SFNs) are a subgroup of sensory neuropathies that almost exclusively affect thinly myelinated A-delta or unmyelinated C-nerve fibers. Patients with SFN typically report acral burning pain, paresthesias, and dysesthesias, and sometimes itch manifesting particularly at toes and feet. Although neurological examination and standard neurophysiological assessment are normal to only marginally abnormal, special small nerve fiber tests may show functional, electrical, and morphological small fiber pathology. Interestingly, the application of small nerve fiber assessment tools has revealed similar findings also in other painful and nonpainful disorders, such as the complex regional pain syndrome, postherpetic neuralgia, or fibromyalgia syndrome. The diversity in clinical presentation, however, already implies that different pathophysiological mechanisms underlie small nerve fiber degeneration and regeneration in these disorders. This review aims at presenting current knowledge on small nerve fiber research and at intensifying the awareness for SFN vs small fiber pathology as a chance to learn about small nerve fiber pathophysiology.

Transforming growth factor-β3 promotes facial nerve injury repair in rabbits.

The present study investigated the effects of transforming growth factor (TGF)-β3 on the regeneration of facial nerves in rabbits. A total of 20 adult rabbits were randomly divided into three equal groups: Normal control (n=10), surgical control (n=10) and TGF-β3 treatment (n=10). The total number and diameter of the regenerated nerve fibers was significantly increased in the TGF-β3 treatment group, as compared with in the surgical control group (P<0.01). Furthermore, in the TGF-β3 treatment group, the epineurial repair of the facial nerves was intact and the nerve fibers, which were arranged in neat rows, were morphologically intact with visible myelin swelling. However, in the surgical control group, the epineurial repair was incomplete, as demonstrated by: Atrophic nerve fibers, partially disappeared axons and myelin of uneven thickness with fuzzy borders. Electron microscopy demonstrated that the regenerated fibers in the TGF-β3 treatment group were predominantly myelinated, with clear-layered myelin sheath structures and axoplasms rich in organelles. Although typical layered myelin sheath structures were observed in the surgical control group, the myelin sheaths of the myelinated nerve fibers were poorly developed and few organelles were detected in the axoplasms. Neuro-electrophysiological examination demonstrated that, as compared with the surgical control group, the latency period of the action potentials in the TGF-β3 treatment group were shorter, whereas the stimulus amplitudes of the action potentials were significantly increased (P<0.01). The results of the present study suggest that TGF-β3 may improve the regeneration of facial nerves following trauma or injury.

NEUROTROPHIC EFFECTS OF ETIFOXINE

Higher anxiety results in the decreased levels of various neurotrophic factors and enkephalins and in impaired production of proinflammatory cytokines. The anxiolytic etifoxine is used to treat anxiety states and adjustment disorders. Etifoxine modulates the GABAergic transmission and metabolism of neurosteroids. The latter determines the unique neurotrophic and neuroprotective properties of the drug, such as increased expression of neurotrophic factors, regeneration of nerve fibers, and preservation and regeneration of myelin sheaths. Other important pharmacological effects of an etifoxine molecule have been also discovered; these are to relieve allodynia related to 3α-steroids and GABA receptors and to effectively treat cerebral edema, experimental autoimmune encephalitis, and excessive nervous excitability in the presence of alcohol withdrawal. In addition, the chemoreactome simulation of the molecule of etifoxine has established that its attenuated side effects are due to its lower interaction with serotonin, acetylcholine, adrenergic and other neurotransmitter receptors than is shown by benzodiazepines. Etifoxine has been also found to have anti-inflammatory (due to antihistamine and antileukotriene effects) and antitumor activities and an ability to affect hemodynamics and vessel walls. The paper presents a systematic analysis of the results of trials of the neurotrophic properties of etifoxine. It considers how the drug stimulates the expression of neurotrophic factors, accelerates the maturation and regeneration of nerve fibers, and regenerates myelin sheaths. The neurotrophic effects of etifoxine along with its anxiolytic activity will accelerate the recovery of patients with different neurological diseases and enhance the quality of their neurorehabilitation.

Synovial Neuronal Changes in Knee Joint Osteoarthritis

Purpose: The purpose is to study whether pain and inflammation in knee joint osteoarthritis (OA) are associated with local synovial neuronal changes. Methods: Synovial biopsies were harvested from the medial and lateral knee compartments from OA patients undergoing total joint replacement surgery. All patients had predominant pain at the medial joint compartment. Pain and knee joint function were evaluated by knee society score (KSS). Synovial inflammation was analyzed by histopathological analysis and expression of growth associated protein-43 (GAP-43), sensory (SP, CGRP) and autonomic (NPY, VIP, TH) neuropeptides was studied by single and double immunohistochemistry techniques. Results: We observed reduced KSS and increased inflammatory score in synovial membrane of medial knee compartment. A significant increase in GAP-43 [P = 0.001], SP [P = 0.05], CGRP [P = 0.05] and TH [P = 0.05] expression was observed and SP, CGRP and NPY were found to be co-existed predominantly with GAP-43 in synovial membrane collected from medial compared to the lateral knee compartment. Conclusions: Regenerating nerve fibers containing sensory and autonomic neuropeptides are associated with pain and inflammation in knee joint OA.

Application of bone marrow mesenchymal stem cell-derived extracellular matrix in peripheral nerve tissue engineering

Event Abstract Back to Event Application of bone marrow mesenchymal stem cell-derived extracellular matrix in peripheral nerve tissue engineering Yun Gu1, Zhenmeiyu Li1, Chenbin Xue1, Shen Yi1 and Xiaosong Gu1 1 Nantong University, Jiangsu Key Laboratory of Neuroregeneration, China Objective: To advance molecular and cellular therapy into the clinic for peripheral nerve injury, modification of neural scaffolds with the extracellular matrix (ECM) of peripheral nerves has been established as a promising alternative to direct inclusion of support cells and/or growth factors within a neural scaffold, while the cell-derived ECM proves to be superior to the tissue-derived ECM in modification of neural scaffolds. Based on the fact that bone marrow mesenchymal stem cells (BMSCs), just like Schwann cells, are adopted as support cells within a neural scaffold, in this study, we used BMSCs as parent cells to generate ECM for application in peripheral nerve tissue engineering. Methods: A chitosan nerve guidance conduit (NGC) and silk fibroin filamentous fillers were respectively prepared for co-culture with purified BMSCs respectively, followed by decellularization to stimulate ECM deposition. The ECM modified NGC and lumen fillers were then assembled into a chitosan/silk fibroin-based, BMSC-derived ECM modified neural scaffold, which was implanted into rats to bridge a 10-mm long sciatic nerve gap. After nerve grafting 12 weeks, a series of histological and functional assessments were conducted to compare the regenerative outcomes between MSC-ECM and Scaffold groups. Results: (1) Light and electron micrography were used to observe the topological structure of the BMSC-derived ECM, and immunostaining was performed to identify the protein composition of the BMSC-derived ECM. These results showed that the ECM ultrastructure consistied of a dense meshwork of filamentous and granular materials. (2)A animal model of peripheral nerve injury was adopted in this study to investigate the performances of the BMSC-derived ECM modified neural scaffolds. Anti-NF immunostaining demonstrated that the length or the number of regenerating nerve fibers was significantly greater in MSC-ECM group than in Scaffold group. Electrophysiological assessments were used to evaluate the effects of BMSC-derived ECM modified neural scaffolds on functional recovery. In this study, we noted that the regenerative outcomes were significantly larger in MSC-ECM group than in Scaffold group, suggesting that BMSC-derived ECM had promoting effects on motor function restoration. Finally, morphological observation of regenerated nerve fibers under TEM and the related morphometric analysis revealed that myelin sheath formation was promoted by BMSC-derived ECM modified scaffolds as compared to that by plain scaffolds. Collectively, the comprehensive data from histological and functional examinations suggest that regenerative outcomes achieved by our engineered neural scaffold were better than those by a plain chitosan/silk fibroin scaffold, and suggested the benefits of BMSC-derived ECM for peripheral nerve repair. Conclusion: We have prepared a novel neural scaffold for peripheral nerve repair. A chitosan NGC and silk fibroin filamentous fillers were respectively prepared, and then co-cultured with purified BMSCs respectively, followed by decellularization to stimulate ECM deposition. The ECM modified NGC and lumen fillers were assembled into a chitosan/SF-based, BMSC-derived ECM modified neural scaffold, which was used to bridge a 10 mm long sciatic nerve gap in rats. Histological and functional assessments after scaffold grafting showed that regenerative outcomes achieved by our developed scaffold were better than those by a plain chitosan/silk fibroin scaffold. National Natural Science Foundation of China (Grant Nos. 81200932 and 81471255); Basic Research Program of Jiangsu Education Department (Grant No. 14KJA310004)

Schwannomas provide insight into the role of p75(NTR) and merlin in Schwann cells following nerve injury and during regeneration.

Peripheral nerve injury leads to Wallerian degeneration of severed axons, leaving the Schwann cell (SC) sheath behind. Denervated SCs may then either survive and remyelinate a regenerating axon, or they may undergo cell death. Because SCs provide trophic support and guidance cues to regenerating nerve fibers, SC loss severely hampers nerve regeneration (Hall, 1986). Thus, significant work has sought to characterize the molecular mechanisms underlying SC fate following peripheral nerve injury. A deeper understanding of these mechanisms has recently been derived from a somewhat unexpected source; cell survival signaling in benign schwannoma tumors has yielded insight into survival signaling in denervated SCs. This recent evidence implicates a pathway involving the single transmembrane neurotrophin receptor p75NTR and Moesin-ezrin-radixin-like protein (merlin, also known as schwannomin or neurofibromin 2), a membrane-cytoskeleton scaffolding protein linking the cellular membrane to the actin cytoskeleton.

Low-density Lipoprotein Receptor-related Proteins in a Novel Mechanism of Axon Guidance and Peripheral Nerve Regeneration

The low-density lipoprotein receptor-related protein receptors 1 and 2 (LRP1 and LRP2) are emerging as important cell signaling mediators in modulating neuronal growth and repair. We examined whether LRP1 and LRP2 are able to mediate a specific aspect of neuronal growth: axon guidance. We sought to identify LRP1 and LRP2 ligands that could induce axonal chemoattraction, which might have therapeutic potential. Using embryonic sensory neurons (rat dorsal root ganglia) in a growth cone turning assay, we tested a range of LRP1 and LRP2 ligands for the ability to guide growth cone navigation. Three ligands were chemorepulsive: α-2-macroglobulin, tissue plasminogen activator, and metallothionein III. Conversely, only one LRP ligand, metallothionein II, was found to be chemoattractive. Chemoattraction toward a gradient of metallothionein II was calcium-dependent, required the expression of both LRP1 and LRP2, and likely involves further co-receptors such as the tropomyosin-related kinase A (TrkA) receptor. The potential for LRP-mediated chemoattraction to mediate axonal regeneration was examined in vivo in a model of chemical denervation in adult rats. In these in vivo studies, metallothionein II was shown to enhance epidermal nerve fiber regeneration so that it was complete within 7 days compared with 14 days in saline-treated animals. Our data demonstrate that both LRP1 and LRP2 are necessary for metallothionein II-mediated chemotactic signal transduction and that they may form part of a signaling complex. Furthermore, the data suggest that LRP-mediated chemoattraction represents a novel, non-classical signaling system that has therapeutic potential as a disease-modifying agent for the injured peripheral nervous system.

Morphological study of Schwann cells remyelination in contused spinal cord of rats

ObjectiveTo study the role and effect of Schwann cells (SCs) remyelination in contused spinal cord. MethodsGreen fluorescence protein expressing-SCs were transplanted into the epicenter, rostral and caudal tissues of the injury site at 1 week after the spinal cords were contused. At 6 weeks, the spinal cords were removed for cryosections, semithin sections and ultrathin sections, and then immunocytochemical staining of myelin basic protein (MBP), P0 protein (P0) and S100 protein (S100) was carried out on the cryosections. Qualitative and semiquantitative analyses were performed on the cryosections and semithin sections. Ultrastructure of myelinated fibers was observed on the ultrathin sections under electron microscope. ResultsTransplanted SCs and myelinated fibers immunocytochemically labeled by MBP, P0 as well as S100 distributed in whole injured area. The quantity of myelinated fibers labeled by the three myelin proteins showed no statistical difference, however, which was significantly larger than that of controls. On the semithin sections, the experimental group demonstrated more myelinated fibers in the injured area than the controls, but the fibers had smaller diameter and thinner myelin sheath under electron microscope. ConclusionSCs can promote regeneration of injured nerve fibers and enhance remyelination, which may be histological basis of SCs-mediated functional repair of injured spinal cords.

Sciatic endometriosis induces mechanical hypersensitivity, segmental nerve damage, and robust local inflammation in rats.

Endometriosis is a common cause of pain including radicular pain. Ectopic endometrial tissue may directly affect peripheral nerves including the sciatic, which has not been modelled in animals. We developed a rat model for sciatic endometriosis by grafting a piece of autologous uterine tissue around the sciatic nerve. Control animals underwent a similar surgery but received a graft of pelvic fat tissue. The uterine grafts survived and developed fluid-filled cysts; the adjacent nerve showed signs of swelling and damage. Mechanical and cold hypersensitivity and allodynia of the ipsilateral hindpaw developed gradually over the first 2weeks after the surgery, peaked at 2-5weeks, and was almost resolved by 7weeks. Control animals showed only minor changes in these pain behaviours. Histological signs of inflammation in the uterine graft and in the adjacent nerve were observed at 3weeks but were resolving by 7weeks. In vivo fibre recording showed increased spontaneous activity, especially of C-fibres, in sciatic nerve proximal to the uterine graft. Several pro-inflammatory cytokines including interluekin-18, VEGF, fractalkine, and MIP-1α, were elevated in the uterine graft plus sciatic nerve samples, compared to samples from normal nerve or nerve plus fat graft. Growth associated protein 43 (GAP43), a marker of regenerating nerve fibres, was observed in the adjacent sciatic nerve as well as in the uterine graft. This model shared many features with other rat models of endometriosis, but also had some unique features more closely related to neuropathic pain models. Some especially painful forms of endometriosis are essentially neuropathic, because peripheral nerves are directly affected by nearby ectopic endometrial tissue. We modelled endometriosis by implanting autologous uterine tissue around rat sciatic nerve. We observed mechanical and cold pain behaviours along with signs of inflammation and nerve damage and increased pro-inflammatory cytokines at the implant site. Pain behaviours correlated with signs of nerve inflammation and damage rather than with cyst survival.