Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration.

At present, clinical strategies to repair injured peripheral nerve concentrate on efforts to attain primary suture of the cut nerve ends. If this is not possible, autografts are used to unite the separated nerve segments. Both strategies are based on the recognition that the Schwann cells resident in the peripheral nerve trunk play a crucial role in the regenerative process. Neither strategy may be feasible, however, in extensive or multiple injuries because the amount of autograft material is limited, and allografts are subject to immune rejection. Artificially produced nerve bridges constructed of autologous Schwann cells seeded in guidance channels could be used to overcome these limitations. In the present experiments, the potential of Schwann cells derived from adult nerves and seeded in permselective guidance channels to promote neurite regeneration across an 8 mm nerve gap was evaluated in transected rat sciatic nerves. Immunological sequalae were evaluated by comparing Schwann cells from syngeneic and heterologous rat strains. Schwann cells from either adult outbred (Sprague-Dawley, CD) rats or inbred (Fisher, F) rats were suspended in a Matrigel solution at a density of 80 x 10(6) cells/ml (CD) or 40, 80, or 120 x 10(6) cells/ml (F-40, F-80, and F-120 channels, respectively). Channels containing Schwann cells were compared to sciatic nerve autografts, empty channels, or channels filled with Matrigel alone. One day after seeding permselective synthetic guidance channels with a Schwann cell suspension, a central cable of Schwann cells oriented along the axis of the tube was formed due to syneresis of the hydrogel. By 3 weeks postimplantation, regenerating axons had grown into all channels and autografts. Sciatic nerve autografts supported extensive regeneration, containing 4-5 x 10(4) myelinated axons at the graft midpoint. The ability of channels containing syngeneic Schwann cells to foster regeneration was dependent on the Schwann cell seeding density. At the channel's midpoint, the myelinated axon population in F-120 tubes was intermediate between that in sciatic nerve autografts and F-80 channels, and was significantly higher than in F-40 or control channels. The nerve cable in Schwann cell-containing tubes consisted of larger, more organotypic fascicles than acellular control channels. In contrast, heterologous (CD) Schwann cells elicited a strong immune reaction that impeded nerve regeneration. The present study shows that cultured adult syngeneic Schwann cells seeded in permselective synthetic guidance channels support extensive peripheral nerve regeneration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biodegradable magnesium wire promotes regeneration of compressed sciatic nerves

Magnesium(Mg) wire has been shown to be biodegradable and have anti-inflammatory properties. It can induce Schwann cells to secrete nerve growth factor and promote the regeneration of nerve axons after central nervous system injury. We hypothesized that biodegradable Mg wire may enhance compressed peripheral nerve regeneration. A rat acute sciatic nerve compression model was made, and AZ31 Mg wire(3 mm diameter; 8 mm length) bridged at both ends of the nerve. Our results demonstrate that sciatic functional index, nerve growth factor, p75 neurotrophin receptor, and tyrosine receptor kinase A m RNA expression are increased by Mg wire in Mg model. The numbers of cross section nerve fibers and regenerating axons were also increased. Sciatic nerve function was improved and the myelinated axon number was increased in injured sciatic nerve following Mg treatment. Immunofluorescence histopathology showed that there were increased vigorous axonal regeneration and myelin sheath coverage in injured sciatic nerve after Mg treatment. Our findings confirm that biodegradable Mg wire can promote the regeneration of acute compressed sciatic nerves.     

Isolation of activated adult Schwann cells and a spontaneously immortal Schwann cell clone.

Successful mammalian peripheral nerve regeneration is dependent on activated Schwann cells. Schwann cells facilitate neuronal regrowth through the production of tropic cell membrane molecules, neurotrophins, and extracellular matrix components. To better understand Schwann cell function in the regenerating nerve, we have designed a method of isolating proliferating adult Schwann cells from the injured rat sciatic nerve. Relying on the mitotic signal that is present after a crush injury, we can obtain sufficient numbers of dividing Schwann cells within one week of initial culture. A spontaneously immortal Schwann cell clone (iSC) was observed in and isolated from one of these primary cultures. These cells were transformed at a time of maximal Schwann cell activation in response to injury. Both the primary Schwann cells and the iSC have been characterized as Schwann cells by morphology, immunohistochemistry and gene expression.     

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.     

Dorsal root ganglion-derived Schwann cells combined with poly（lactic-co-glycolic acid）/chitosan conduits for the repair of sciatic nerve defects in rats

Schwann cells, nerve regeneration promoters in peripheral nerve tissue engineering, can be used to repair both the peripheral and central nervous systems. However, isolation and puriifcation of Schwann cells are complicated by contamination with ifbroblasts. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. In this study, we collected dorsal root ganglia from neonatal rats from which we obtained highly puriifed Schwann cells using serum-free melanocyte culture medium. The purity of Schwann cells （〉95%） using our method was higher than that using standard medium containing fetal bovine serum. The obtained Schwann cells were implanted into poly（lactic-co-glycolic acid）/chi-tosan conduits to repair 10-mm sciatic nerve defects in rats. Results showed that axonal diameter and area were signiifcantly increased and motor functions were obviously improved in the rat sciatic nerve tissue. Experimental ifndings suggest that serum-free melanocyte culture medium is conducive to purify Schwann cells and poly（lactic-co-glycolic acid）/chitosan nerve conduits combined with Schwann cells contribute to restore sciatic nerve defects.     

Induction of parathyroid hormone-related peptide following peripheral nerve injury: role as a modulator of Schwann cell phenotype

Parathyroid hormone-related peptide (PTHrP) is widely distributed in the rat nervous system, including the peripheral nervous system, where its function is unknown. PTHrP mRNA expression has recently been shown to be significantly elevated following axotomy of sympathetic ganglia, although the role of PTHrP was not investigated. The role of PTHrP in peripheral nerve injury was investigated in this study using the sciatic nerve injury model and dorsal root ganglion (DRG) explant model of nerve regeneration. We find that PTHrP is a constitutively secreted peptide of proliferating Schwann cells and that the PTHrP receptor (PTH1R) mRNA is expressed in isolated DRG and in sciatic nerve. Using the sciatic nerve injury model, we show that PTHrP is significantly upregulated in DRG and in sciatic nerve. In addition, in situ hybridization revealed significant localization of PTHrP mRNA to Schwann cells in the injured sciatic nerve. We also find that PTHrP causes a dramatic increase in the number of Schwann cells that align with and bundle regrowing axons in explants, characteristic of immature, dedifferentiated Schwann cells. In addition to stimulating migration of Schwann cells along the axonal membrane, PTHrP also stimulates migration on a type 1 collagen matrix. Furthermore, treatment of purified Schwann cell cultures with PTHrP results in the rapid phosphorylation of the cAMP response element protein, CREB. We propose that PTHrP acts by promoting the dedifferentiation of Schwann cells, a critical requirement for successful nerve regeneration and an effect consistent with known PTHrP functions in other cellular differentiation programs.     

Skin-derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection

The regeneration in the peripheral nervous system is often incomplete and the treatment of severe lesions with nerve tissue loss is primarily aimed at recreating nerve continuity. Guide tubes of various types, filled with Schwann cells, stem cells, or nerve growth factors are attractive as an alternative therapy to nerve grafts. In this study, we evaluated whether skin-derived stem cells (SDSCs) can improve peripheral nerve regeneration after transplantation into nerve guides. We compared peripheral nerve regeneration in adult rats with sciatic nerve gaps of 16 mm after autologous transplantation of GFP-labeled SDSCs into two different types of guides: a synthetic guide, obtained by dip coating with a L-lactide and trimethylene carbonate (PLA-TMC) copolymer and a collagen-based guide. The sciatic function index and the recovery rates of the compound muscle action potential were significantly higher in the animals that received SDSCs transplantation, in particular, into the collagen guide, compared to the control guides filled only with PBS. For these guides the morphological and immunohistochemical analysis demonstrated an increased number of myelinated axons expressing S100 and Neurofilament 70, suggesting the presence of regenerating nerve fibers along the gap. GFP positive cells were found around regenerating nerve fibers and few of them were positive for the expression of glial markers as S-100 and glial fibrillary acidic protein. RT-PCR analysis confirmed the expression of S100 and myelin basic protein in the animals treated with the collagen guide filled with SDSCs. These data support the hypothesis that SDSCs could represent a tool for future cell therapy applications in peripheral nerve regeneration.     

Synthesis of leukemia inhibitory factor in injured peripheral nerves and their cells

The time and site of induction of leukemia inhibitory factor mRNA in injured rat sciatic nerves and its regulation in Schwann cells and fibroblasts from neonatal rat nerves were investigated. Leukemia inhibitory factor mRNA is induced at the lesion site within 6 h of sciatic nerve transection but only after 24 h in the more distal segments. In vitro, interleukin-1β increases the concentration of leukemia inhibitory mRNA in nerve fibroblasts but not in Schwann cells. Changes in leukemia inhibitory factor mRNA concentration in injured nerves and peripheral nerve cells are similar to those for nerve growth factor mRNA.     

Transforming growth factor beta 1, a cytokine with regenerative functions

We review the biology and role of transforming growth factor beta 1(TGF-β1) in peripheral nerve injury and regeneration, as it relates to injuries to large nerve trunks(i.e., sciatic nerve, brachial plexus), which often leads to suboptimal functional recovery. Experimental studies have suggested that the reason for the lack of functional recovery resides in the lack of sufficient mature axons reaching their targets, which is a result of the loss of the growth-supportive environment provided by the Schwann cells in the distal stump of injured nerves. Using an established chronic nerve injury and delayed repair animal model that accurately mimics chronic nerve injuries in humans, we summarize our key findings as well as others to better understand the pathophysiology of poor functional recovery. We demonstrated that 6 month TGF-β1 treatment for chronic nerve injury significantly improved Schwann cell capacity to support axonal regeneration. When combined with forskolin, the effect was additive, as evidenced by a near doubling of regenerated axons proximal to the repair site. We showed that in vivo application of TGF-β1 and forskolin directly onto chronically injured nerves reactivated chronically denervated Schwann cells, induced their proliferation, and upregulated the expression of regeneration-associated proteins. The effect of TGF-β1 and forskolin on old nerve injuries is quite impressive and the treatment regiment appears to mediate a growth-supportive milieu in the injured peripheral nerves. In summary, TGF-β1 and forskolin treatment reactivates chronically denervated Schwann cells and could potentially be used to extend and prolong the regenerative responses to promote axonal regeneration.     

同种异体神经复合体修复兔周围神经缺损

背景：应用种植许旺细胞的去细胞同种异体神经复合体修复周围神经缺损,探索其对神经再生及功能恢复有更好的促进作用,并且免疫原性非常小。 目的：用种植胎兔许旺细胞的去细胞同种异体神经复合体修复兔缺损的坐骨神经,观察移植神经周围免疫细胞的变化及功能恢复。 　 方法：48只新西兰白兔随机分成实验组和对照组。两组动物均切除一段坐骨神经,造成2.0 cm长的缺损,实验组用种植胎兔许旺细胞的同种异体神经复合体修复坐骨神经;对照组仅用去细胞同种异体神经修复。移植后1,4,8周光镜观察移植段坐骨神经周围肌肉组织中免疫细胞的浸润情况,计数每个高倍视野免疫细胞的数量。移植后4,8,16周大体观察兔的足部溃疡形成及愈合情况,大体观察神经愈合情况;肌电图检查桥接段坐骨神经的传导速度。 结果与结论：手术区局部均未出现明显的排斥反应,实验组足部溃疡愈合情况优于对照组。移植后1周移植段坐骨神经周围肌肉组织中有大量淋巴细胞及巨噬细胞浸润,实验组明显多于对照组(P < 0.05);移植后4周,浸润的免疫细胞两组均较1周后明显减少,实验组减少更明显。移植后8周,浸润的免疫细胞更加减少,但两组间比较差异无显著性意义(P > 0.05)。移植后4周时,两组均未见明显的神经传导,8,16周神经传导速度实验组均优于对照组(P < 0.05)。提示,种植许旺细胞的去细胞同种异体神经复合体免疫原性非常小,对神经再生及功能恢复有更好的促进作用。     

Regeneration of axons from adult rat retinal ganglion cells on cultured Schwann cells is not dependent on basal lamina.

The ability of sciatic nerve grafts to support in vivo regeneration of retinal ganglion cell axons in the adult rat raises the question of which peripheral nerve constituents may be required to promote this unexpected central regenerative response. Prime candidates for this role include the surface of the Schwann cell and components of extracellular matrix present in peripheral nerve trunks. To determine the relative importance of Schwann cells and their basal lamina in promoting retinal ganglion cell axon regeneration in the mammalian visual system, we have used an in vitro model. This approach allowed analysis of the abilities of defined peripheral nerve constituents to promote in vitro outgrowth of neurites from explants of adult rat retina harvested 7 to 10 days after in vivo optic nerve crush. Neurite outgrowth was assessed by neurofilament immunofluorescence after 3 to 20 days in vitro. Culture substrata, consisting of isolated Schwann cells (SC), Schwann cells with their assembled extracellular matrix (SC + ECM), or isolated extracellular matrix from which the Schwann cells had been removed (ECM), were prepared by first co-culturing rat Schwann cells with embryonic dorsal root ganglion neurites on a layer of type I collagen, and then manipulating the cultures to produce the desired substrata. Type I collagen alone did not support neurite growth from adult rat retina. SC and SC + ECM supported regeneration of axons from retinal explants at average growth rates of 18 and 30 microns/h, respectively. Isolated ECM was a poor substrate for retinal neurite growth; the few neurites that gained access to this material grew at rates averaging less than 3 microns/h. These observations suggest that regeneration of adult mammalian retinal ganglion cell axons through peripheral nerve grafts (in vivo) is primarily dependent on neurite-promoting factors present on the surface of Schwann cells and does not require organized extracellular matrix.     

Netrin-1 and peripheral nerve regeneration in the adult rat

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

Peripheral nerve regeneration is impeded by interleukin-1 receptor antagonist released from a polymeric guidance channel.

Interleukin-1 receptor antagonist (IL-1ra), a true antagonist of the interleukin-1 (IL-1) receptors, is released by activated macrophages and binds specifically to the IL-1 receptors without triggering IL-1 effects. Following peripheral nerve axotomy, activated macrophages release IL-1, which induces the expression of nerve growth factor (NGF) mRNA in Schwann cells. IL-1ra may therefore impede peripheral nerve regeneration by blocking the NGF-mediated effect of IL-1. Peripheral nerve regeneration occurring through polymeric guidance channels releasing IL-1ra was investigated in a 4-mm gap transected mouse sciatic nerve model. Cohorts of five animals were implanted with tubes releasing either bovine serum albumin (BSA), BSA with IL-1ra, or BSA with deactivated IL-1ra (dIL-1ra) for 4 weeks. In vitro release kinetics indicated that after an initial burst, IL-1ra release was linear for the next 3 1/2 weeks. Following implantation of a polymeric guidance channel, a regenerated cable bridged the nerve gap in all animals. The cables were similar in size and were composed of nerve microfascicles containing both unmyelinated and myelinated axons in association with their Schwann cells. Tissue regenerated in tubes releasing BSA-IL-1ra contained, however, significantly fewer myelinated and unmyelinated axons and blood vessels than did tubes releasing BSA alone or BSA-dIL-1ra. We conclude that a naturally occurring antagonist of IL-1 receptors impedes peripheral nerve regeneration, suggesting that macrophages play an essential role in controlling peripheral nerve regeneration through the release of stimulatory and/or inhibitory molecules.     

Impulse magnetic stimulation facilitates synaptic regeneration in rats following sciatic nerve injury

The current studies describing magnetic stimulation for treatment of nervous system diseases mainly focus on transcranial magnetic stimulation and rarely focus on spinal cord magnetic stimula-tion.Spinal cord magnetic stimulation has been confirmed to promote neural plasticity after injuries of spinal cord,brain and peripheral nerve.To evaluate the effects of impulse magnetic stimulation of the spinal cord on peripheral nerve regneration,we compressed a 3 mm segment located in the middle third of the hip using a sterilized artery forceps to induce ischemia.Then,all animals un-derwent impulse magnetic stimulation of the lumbar portion of spinal crod and spinal nerve roots daily for 1 month.Electron microscopy results showed that in and below the injuryed segment,the inflammation and demyelination of neural tissue were alleviated,apoptotic cells were reduced,and injured Schwann cells and myelin fibers were repaired.These findings suggest that high-frequency impulse magnetic stimulation of spinal cord and corresponding spinal nerve roots promotes synaptic regeneration following sciatic nerve injury.     

Expression of Endopeptidase-24.11 (Common Acute Lymphoblastic Leukaemia Antigen CDI0) in the Sciatic Nerve of the Adult Rat After Lesion and During Regeneration

Endopeptidase-24.11, which is identical with the common acute lymphoblastic leukaemia antigen CD1O (CALLA), is a cell surface Zn²⁺ metalloprotease that regulates peptide-induced responses in different tissues, including the nervous and immune systems. In the peripheral nervous system, high levels of the enzyme are present in all neonatal and early postnatal Schwann cells, while as myelination proceeds it is gradually suppressed in the majority of cells that form myelin but retained in non-myelin-forming cells in the adult animal. In the present study we have investigated the effects of transection, crush and regeneration of the adult rat sciatic nerve on the expression of the endopeptidase by Schwann cells in situ. Endopeptidase-24.11 was monitored by immunocytochemistry using the monoclonal anti-endopeptidase antibody 23811. For comparison, a parallel study was carried out with a monoclonal antibody directed against the rat nerve growth factor receptor. We found that (i) all Schwann cells of the distal segment re-expressed endopeptidase-24.11 as early as 4 days after axotomy, the level of immunostaining reaching a maximum after 2 weeks, (ii) axonal regeneration repressed Schwann cell expression of endopeptidase-24.11, and (iii) the induction of the nerve growth factor receptor followed a similar pattern to that of endopeptidase-24.11 in the transected and crushed nerve. Enzymatic amplification of endopeptidase-24.11 cDNA from normal and axotomized adult rat sciatic nerve confirmed the expression of endopeptidase-24.11 in these tissues. Our results show that the expression of endopeptidase-24.11 in Schwann cells, as is the case with the nerve growth factor receptor, is induced by the loss of the normal axon-Schwann cell contact. The significant increase in the expression of endopeptidase-24.11 by Schwann cells after axonal damage suggests that the enzyme could play a role in axonal regeneration.     

Nanofibrous nerve conduits for repair of 30-mm-long sciatic nerve defects*

It has been confirmed that nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit can promote peripheral nerve regeneration in rats. However, its efficiency in repair of over 30-mm-long sciatic nerve defects needs to be assessed. In this study, we used a nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit to bridge a 30-mm-long gap in the rat sciatic nerve. At 4 months after nerve conduit implantation, regenerated nerves were macroscopically observed and histologically assessed. In the nanofibrous graft, the rat sciatic nerve trunk had been reconstructed by restoration of nerve continuity and formation of myelinated nerve fiber. There were Schwann cells and glial cells in the regenerated nerves. Masson’s trichrome staining showed that there were no pathological changes in the size and structure of gastrocnemius muscle cells on the operated side of rats. These findings suggest that nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit is suitable for repair of long-segment sciatic nerve defects.     

Skin-derived precursor cells enhance peripheral nerve regeneration following chronic denervation

While peripheral nerves demonstrate the capacity for axonal regeneration, outcome following injury remains relatively poor, especially following prolonged denervation. Since axon-deprived Schwann cells (SCs) in the distal nerve progressively lose their ability to support axonal growth, we took the approach of using skin-derived precursor cells (SKPs) as an accessible source of replacement SCs that could be transplanted into chronically denervated peripheral nerve. In this study, we employed a delayed cross-reinnervation paradigm to assess regeneration of common peroneal nerve axons into the chronically denervated rodent tibial nerve following delivery of SKP-derived SC (SKP-SCs). SKP-SC treated animals exhibited superior axonal regeneration to media controls, with significantly higher counts of regenerated motorneurons and histological recovery similar to that of immediately repaired nerve. Improved axonal regeneration correlated with superior muscle reinnervation, as measured by compound muscle action potentials and wet muscle weights. We therefore conclude that SKPs represent an easily accessible, autologous source of stem cell-derived Schwann cells that show promise in improving regeneration through chronically injured nerves.     

Exenatide promotes regeneration of injured rat sciatic nerve

Damage to peripheral nerves results in partial or complete dysfunction. After peripheral nerve injuries, a full functional recovery usually cannot be achieved despite the standard surgical repairs. Neurotrophic factors and growth factors stimulate axonal growth and support the viability of nerve cells. The objective of this study is to investigate the neurotrophic effect of exenatide(glucagon like peptide-1 analog) in a rat sciatic nerve neurotmesis model. We injected 10 μg/d exenatide for 12 weeks in the experimental group(n = 12) and 0.1 m L/d saline for 12 weeks in the control group(n = 12). We evaluated nerve regeneration by conducting electrophysiological and motor functional tests. Histological changes were evaluated at weeks 1, 3, 6, and 9. Nerve regeneration was monitored using stereomicroscopy. The electrophysiological and motor functions in rats treated with exenatide were improved at 12 weeks after surgery. Histological examination revealed a significant increase in the number of axons in injured sciatic nerve following exenatide treatment confirmed by stereomicroscopy. In an experimentally induced neurotmesis model in rats, exenatide had a positive effect on nerve regeneration evidenced by electromyography, functional motor tests, histological and stereomicroscopic findings.