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.     

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.     

The role of extracellular matrix in peripheral nerve regeneration: a wound chamber study

Summary A wound chamber model was used for the study of the interaction between axon, Schwann cell and extracellular matrix during peripheral nerve regeneration. Impermeable silicone tubes, 8 mm long and 1.4 mm in internal diameter were sutured to transected rat sciatic nerve and the contents of the tubes were removed at intervals for chemical, histological, immunocytochemical and electron microscopic studies. There was an initial phase of fluid accumulation and the formation of a fibrin/fibronectin clot or cable which connected the cut ends of the nerve. The chamber fluid was shown to have a protein profile similar to that of rat serum. Schwann cells, endothelial cells and fibroblasts migrated first into the cable, apparently mediated by cell-fibrin interaction. Axons buried within the Schwann cell cytoplasm were led into the cable but an axon-fibrin interaction was not observed. After 1 week, the fibrin matrix underwent dissolution, with replacement by collagen. This marked the onset of myelination and the organization of nerve fibers into fascicles. The findings from the present study suggest that the interactions between axon and Schwann cell and between Schwann cell and a changing extracellular matrix are the essential driving force in nerve growth and differentiation during peripheral nerve regeneration.Supported by a grant from the National Science Council of R. O. C. (NSC 80-0412-B075-67)     

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     

Histological assessment in peripheral nerve tissue mgineering

The histological analysis of peripheral nerve regeneration is one of the most used methods to demonstrate the success of the regeneration through nerve conduits. Nowadays, it is possible to evaluate different parameters of nerve regeneration by using histological, histochemical, immunohistochemical and ultrastructural techniques. The histochemical methods are very sensible and are useful tools to evaluate the extracellular matrix remodeling and the myelin sheath, but they are poorly specific. In contrast, the immunohistochemical methods are highly specific and are frequently used for the identification of the regenerated axons, Sehwann cells and proteins associated to nerve regeneration or neural linage. The ultrastructural techniques offer the possibility to perform a high resolution morphological and quantitative analysis of the nerve regeneration. However, the use of a single histological method may not be enough to assess the degree of regeneration, and the combination of different histological techniques could be necessary.     

Initial upregulation of growth factors and inflammatory mediators during nerve regeneration in the presence of cell adhesive peptide-incorporated collagen tubes

Neurotrophic factors play an important modulatory role in axonal sprouting during nerve regeneration involving the proliferation of hematogenous and Schwann cells in damaged tissue. We have exposed lesioned sciatic nerves to a collagen prosthesis with covalently bonded small cell adhesive peptides Arg-Gly-Asp-Ser (RGDS), Lys-Arg-Asp-Ser (KRDS), and Gly-His-Lys (GHK) to study local production of growth factors and cytokines in the regenerating tissues. Western/enzyme-linked immunosorbent assay (ELISA) studies were performed after 10 days of regeneration, when the tubular prosthesis is filled with fibrous matrix infiltrated by hematogenous cells and proliferating Schwann cells with growth factors produced locally. Regeneration was also analyzed by morphometrical methods after 30 days. The quantification of growth factors and proteins by ELISA revealed that there was an enhanced expression of the neurotrophic factors nerve growth factor (NGF) and neurotrophins (NT-3 and NT-4) in the regenerating tissues. This was further established by Western blot to qualitatively analyze the presence of the antigens in the regenerating tissues. Schwann cells were localized in the regenerating tissues using antibodies against S-100 protein. Other growth factors including growth-associated protein 43 (GAP-43), apolipoprotein E (Apo E), and pro-inflammatory cytokine like interleukin-1alpha (IL-1alpha) expression in the peptide groups were evaluated by ELISA and confirmed by Western blotting. Cell adhesive integrins in the proliferating cells were localized using integrin-alpha V. The combined results suggest that the early phase of regeneration of peripheral nerves in the presence of peptide-incorporated collagen tubes results in the enhanced production of trophic factors by the recruited hematogenous cells and Schwann cells, which in turn help in the secretion of certain vital trophic and tropic factors essential for early regeneration. Furthermore, hematogenous cells recruited within the first 10 days of regeneration help in the production of inflammatory mediators like interleukins that in turn stimulate Schwann cells to produce NGF for axonal growth.     

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.     

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.     

Impaired nerve regeneration in reeler mice after peripheral nerve injury

Reelin, an extracellular matrix protein, plays an important role in the regulation of neuronal migration and cortical lamination in the developing brain. Little is known, however, about the role of this protein in axonal regeneration. We have previously shown that Reelin is secreted by Schwann cells in the peripheral nerve compartment during postnatal development and that it is up-regulated following nerve injury in adult mice. In this work, we generated mice deficient in Reelin ( reeler ) that express yellow fluorescent protein (YFP) in a subset of neurons and examined the axonal regeneration following nerve crush. We found that axonal regeneration was significantly altered compared with wild-type mice. By contrast, retrograde tracing with Fluorogold dye after sciatic nerve crush was unaffected in these mutants, being comparable with normal axonal transport observed in wild-type. These results indicate that the absence of Reelin impairs axonal regeneration following injury and support a role for this protein in the process of peripheral nerve regeneration.     

Neurite-promoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers

The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cell stained with anti-fibronectin and anti-laminin. Wihtin the degenerated distal nerve segment, cells characteristics of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth. Affinity-purified anti-laminin antibody failed to block chamber fluid NPF activity while completely blocking the neurite-promoting activity of laminin. These two results suggested that chamber fluid NPF activity did not consist of individual molecules of either fibronectin or laminin. The spatial and temporal distribution of insoluble fibronectin and laminin and the temporal correlation between chamber fluid NPF accumulation and neurite outgrowth support the possibility that these agents influence regenerative events including axonal elongation in vivo.     

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.     

Implantation of cultured sensory neurons and Schwann cells into lesioned neonatal rat spinal cord. I. Methods for preparing implants from dissociated cells

Our goal was to devise methods of implanting defined populations of the cellular constituents of peripheral nerve into regions of spinal cord injury. This objective derived from the knowledge that the cellular environment of peripheral nerve is known to be supportive of axon regeneration from both central and peripheral neurons. Two of the constituents of the peripheral nerve environment known to influence axonal growth are the Schwann cell and extracellular matrix (particularly basal lamina), both of which can be obtained in culture. We describe here large-scale methods of establishing purified populations of rat sensory neurons to which purified populations of Schwann cells were added. These essentially monolayer preparations were then scrolled and cut into lengths of proper shape and size to provide implants for sites of spinal cord injury in newborn rats. We also describe methods enabling the addition of leptomeningeal components to the implants; this addition contributes a proliferating population of vascular endothelial cells (identified by immunostaining) to the otherwise vasculature-free neuron/Schwann cell implant. Light and electron microscopic observations were made to characterize the implants. When the implant was ready for use, it contained Schwann cells that were differentiated, i.e., had begun to ensheathe axons and form basal lamina. The use of a medium containing human plasma to foster endothelial cell growth led to increased neurite fasciculation and Schwann cell migratory activity in the outgrowth, particularly when the neurons and Schwann cells were cultured on leptomeninges. The second paper in this series reports the deportment of these implants and their influence on corticospinal tract growth after placement into regions of dorsal column injury in neonatal rats (Kuhlengel et al., J. Comp. Neurol 293:74-91, 1990).     

Rapamycin promotes Schwann cell migration and nerve growth factor secretion

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

Spatial-Temporal progress of peripheral nerve regeneration within a silicone chamber: Parameters for a bioassay

The spatial-temporal progress of peripheral nerve regeneration across a 10-mmgap within a silicone chamber was examined with the light and electron microscope at 2-mm intervals. A coaxial, fibrin matrix was observed at 1 week with a proximal-distal narrowing that extended beyond the midpoint of the chamber. At 2 weeks, Schwann cells, fibroblasts, and endothelial cells had migrated into the matrix from both nerve stumps. There was a delay of 7–14 days after nerve transection and chamber implantation before regenerating axons appeared in the chamber. At 2 weeks, nonmyelinated axons were seen only in the proximal 1–5 mm of the chamber in association with Schwann cells. Axons reached the distal stump by 3 weeks and a proximal-distal gradient of myelination was observed. These observations define the parameters of a morphologic assay for regeneration in this chamber model which can be used to investigate cellular and molecular mechanisms underlying the success of peripheral nerve regeneration.     

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.     

Classic axon guidance molecules control correct nerve bridge tissue formation and precise axon regeneration

The peripheral nervous system has an astonishing ability to regenerate following a compression or crush injury;however,the potential for full repair following a transection injury is much less.Currently,the major clinical challenge for peripheral nerve repair come from long gaps between the proximal and distal nerve stumps,which prevent regenerating axons reaching the distal nerve.Precise axon targeting during nervous system development is controlled by families of axon guidance molecules including Netrins,Slits,Ephrins and Semaphorins.Several recent studies have indicated key roles of Netrin1,Slit3 and EphrinB2 signalling in controlling the formation of new nerve bridge tissue and precise axon regeneration after peripheral nerve transection injury.Inside the nerve bridge,nerve fibroblasts express EphrinB2 while migrating Schwann cells express the receptor EphB2.EphrinB2/EphB2 signalling between nerve fibroblasts and migrating Schwann cells is required for Sox2 upregulation in Schwann cells and the formation of Schwann cell cords within the nerve bridge to allow directional axon growth to the distal nerve stump.Macrophages in the outermost layer of the nerve bridge express Slit3 while migrating Schwann cells and regenerating axons express the receptor Robo1;within Schwann cells,Robo1 expression is also Sox2-dependent.Slit3/Robo1 signalling is required to keep migrating Schwann cells and regenerating axons inside the nerve bridge.In addition to the Slit3/Robo1 signalling system,migrating Schwann cells also express Netrin1 and regenerating axons express the DCC receptor.It appears that migrating Schwann cells could also use Netrin1 as a guidance cue to direct regenerating axons across the peripheral nerve gap.Engineered neural tissues have been suggested as promising alternatives for the repair of large peripheral nerve gaps.Therefore,understanding the function of classic axon guidance molecules in nerve bridge formation and their roles in axon regeneration could be highly beneficial in developing engineered neural tissue for more effective peripheral nerve repair.     

Thrombospondin expression in nerve regeneration I. comparison of sciatic nerve crush, transection, and long-term denervation

Patterns of expression of the extracellular matrix molecule thrombospondin (TSP) were examined during peripheral nerve regeneration following sciatic nerve crush or transection. In noninjured nerve, was present in the axoplasm, Schwann cells, endoneurium, and perineurium of the adult mouse sciatic nerve. Following nerve crush or nerve transection, levels of TSP rapidly increased distal to the trauma site. Elevated levels of TSP were present distal to regenerating axons, while expression gradually returned to normal proximal to the regenerating axons. When reinnervation was blocked, TSP levels remained high in the endoneurium in excess of 30 days, but TSP was absent by 60 days. Following reanastomosis of the proximal and distal segments after 60 days of denervation, TSP was re-expressed in the distal nerve stump. These results indicate that TSP, which is involved in neuronal migrations in the embryo and neurite outgrowth in vitro, appears to play a role in axonal regeneration in the adult peripheral nervous system.     

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.