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.     

p38 MAPK activation promotes denervated Schwann cell phenotype and functions as a negative regulator of Schwann cell differentiation and myelination

Physical damage to the peripheral nerves triggers Schwann cell injury response in the distal nerves in an event termed Wallerian degeneration: the Schwann cells degrade their myelin sheaths and dedifferentiate, reverting to a phenotype that supports axon regeneration and nerve repair. The molecular mechanisms regulating Schwann cell plasticity in the PNS remain to be elucidated. Using both in vivo and in vitro models for peripheral nerve injury, here we show that inhibition of p38 mitogen-activated protein kinase (MAPK) activity in mice blocks Schwann cell demyelination and dedifferentiation following nerve injury, suggesting that the kinase mediates the injury signal that triggers distal Schwann cell injury response. In myelinating cocultures, p38 MAPK also mediates myelin breakdown induced by Schwann cell growth factors, such as neuregulin and FGF-2. Furthermore, ectopic activation of p38 MAPK is sufficient to induce myelin breakdown and drives differentiated Schwann cells to acquire phenotypic features of immature Schwann cells. We also show that p38 MAPK concomitantly functions as a negative regulator of Schwann cell differentiation: enforced p38 MAPK activation blocks cAMP-induced expression of Krox 20 and myelin proteins, but induces expression of c-Jun. As expected of its role as a negative signal for myelination, inhibition of p38 MAPK in cocultures promotes myelin formation by increasing the number as well as the length of individual myelin segments. Altogether, our data identify p38 MAPK as an important regulator of Schwann cell plasticity and differentiation.     

Phenotypic changes of Schwann cells on the proximal stump of injured peripheral nerve during repair using small gap conduit tube

Dedifferentiation of Schwann cells is an important feature of the response to peripheral nerve injury and specific negative myelination reg-ulators are considered to have a major role in this process. However, most experiments have focused on the distal nerve stump, where the Notch signaling pathway is strongly associated with Schwann cell dedifferentiation and repair of the nerve. We observed the phenotypic changes of Schwann cells and changes of active Notch signaling on the proximal stump during peripheral nerve repair using small gap conduit tubulization. Eighty rats, with right sciatic nerve section of 4 mm, were randomly assigned to conduit bridging group and control group (epineurium suture). Glial fibrillary acidic protein expression, in myelinating Schwann cells on the proximal stump, began to up-reg-ulate at 1 day after injury and was still evident at 5 days. Compared with the control group, Notch1 mRNA was expressed at a higher level in the conduit bridging group during the first week on the proximal stump. Hes1 mRNA levels in the conduit bridging group significantly increased compared with the control group at 3, 5, 7 and 14 days post-surgery. The change of the Notch intracellular domain shared a simi-lar trend as Hes1 mRNA expression. Our results confirmed that phenotypic changes of Schwann cells occurred in the proximal stump. The differences in these changes between the conduit tubulization and epineurium suture groups correlate with changes in Notch signaling.This suggests that active Notch signaling might be a key mechanism during the early stage of neural regeneration in the proximal nerve stump.     

Endogenous bioelectric fields: a putative regulator of wound repair and regeneration in the central nervous system

Studies on a variety of highly regenerative tissues, including the central nervous system(CNS) in non-mammalian vertebrates, have consistently demonstrated that tissue damage induces the formation of an ionic current at the site of injury. These injury currents generate electric fields(EF) that are 100-fold increased in intensity over that measured for uninjured tissue. In vitro and in vivo experiments have convincingly demonstrated that these electric fields(by their orientation, intensity and duration) can drive the migration, proliferation and differentiation of a host of cell types. These cellular behaviors are all necessary to facilitate regeneration as blocking these EFs at the site of injury inhibits tissue repair while enhancing their intensity promotes repair. Consequently, injury-induced currents, and the EFs they produce, represent a potent and crucial signal to drive tissue regeneration and repair. In this review, we will discuss how injury currents are generated, how cells detect these currents and what cellular responses they can induce. Additionally, we will describe the growing evidence suggesting that EFs play a key role in regulating the cellular response to injury and may be a therapeutic target for inducing regeneration in the mammalian CNS.     

The influence of cultured Schwann cells on regeneration through acellular basal lamina grafts

Acellular basal lamina grafts have been shown to be less immunogenic in comparison to cellular grafts, but possess a limited potential for supporting axonal regeneration through them. The present study describes the effect of cultured Schwann cells on enhancing regeneration through acellular grafts. 2 cm long acellular grafts, and in vitro Schwann cell populated acellular grafts were used to repair a surgically created gap in the host peroneal nerve. The transplants were analyzed at 1, 2, 4 and 8 weeks to determine their ability to support axonal regeneration. Host axonal regeneration through Schwann cell cocultured acellular grafts occurred rapidly and was significantly better as compared to non-cultured acellular grafts. The results demonstrate a beneficial effect of Schwann cell culture pretreatment on regeneration through acellular grafts and an improved recovery of the target muscle. The procedure of first preparing acellular grafts with subsequent coculture with Schwann cells offers a novel approach for the repair of injured nervous tissue.     

Peripheral nerve regeneration is delayed in neuropilin 2-deficient mice

Peripheral nerve transection or crush induces expression of class 3 semaphorins by epineurial and perineurial cells at the injury site and of the neuropilins neuropilin-1 and neuropilin-2 by Schwann and perineurial cells in the nerve segment distal to the injury. Neuropilin-dependent class 3 semaphorin signaling guides axons during neural development, but the significance of this signaling system for regeneration of adult peripheral nerves is not known. To test the hypothesis that neuropilin-2 facilitates peripheral-nerve axonal regeneration, we crushed sciatic nerves of adult neuropilin-2-deficient and littermate control mice. Axonal regeneration through the crush site and into the distal nerve segment, repression by the regenerating axons of Schwann cell p75 neurotrophin receptor expression, remyelination of the regenerating axons, and recovery of normal gait were all significantly slower in the neuropilin-2-deficient mice than in the control mice. Thus, neuropilin-2 facilitates peripheral-nerve axonal regeneration.     

Repair of extended peripheral nerve lesions in rhesus monkeys using acellular allogenic nerve grafts implanted with autologous mesenchymal stem cells

Despite intensive efforts in the field of peripheral nerve injury and regeneration, it remains difficult in humans to achieve full functional recovery following extended peripheral nerve lesions. Optimizing repair of peripheral nerve injuries has been hindered by the lack of viable and reliable biologic or artificial nerve conduits for bridging extended gaps. In this study, we utilized chemically extracted acellular allogenic nerve segments implanted with autologous non-hematopoietic mesenchymal stem cells (MSCs) to repair a 40 mm defect in the rhesus monkey ulnar nerve. We found that severely damaged ulnar nerves were structurally and functionally repaired within 6 months following placement of the MSC seeded allografts in all animals studied (6 of 6, 100%). Furthermore, recovery with the MSC seeded allografts was similar to that observed with Schwann cell seeded allografts and autologous nerve grafts. The findings presented here are the first demonstration of the successful use of autologous MSCs, expanded in culture and implanted in a biological conduit, to repair a peripheral nerve gap in primates. Given the difficulty in isolating and purifying sufficient quantities of Schwann cells for peripheral nerve regeneration, the use of MSCs to seed acellular allogenic nerve grafts may prove to be a novel and promising therapeutic approach for repairing severe peripheral nerve injuries in humans.     

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.     

Macrophage polarization in nerve injury: do Schwann cells play a role?

In response to peripheral nerve injury, the inflammatory response is almost entirely comprised of infiltrating macrophages. Macrophages are a highly plastic, heterogenic immune cell, playing an indispensable role in peripheral nerve injury, clearing debris and regulating the microenvironment to allow for efficient regeneration. There are several cells within the microenvironment that likely interact with macrophages to support their function – most notably the Schwann cell, the glial cell of the peripheral nervous system. Schwann cells express several ligands that are known to interact with receptors expressed by macrophages, yet the effects of Schwann cells in regulating macrophage phenotype remains largely unexplored. This review discusses macrophages in peripheral nerve injury and how Schwann cells may regulate their behavior.     

Schwann cell proliferation during Wallerian degeneration is not necessary for regeneration and remyelination of the peripheral nerves: axon-dependent removal of newly generated Schwann cells by apoptosis

Peripheral nerve injury is followed by a wave of Schwann cell proliferation in the distal nerve stumps. To resolve the role of Schwann cell proliferation during functional recovery of the injured nerves, we used a mouse model in which injury-induced Schwann cell mitotic response is ablated via targeted disruption of cyclin D1. In the absence of distal Schwann cell proliferation, axonal regeneration and myelination occur normally in the mutant mice and functional recovery of injured nerves is achieved. This is enabled by pre-existing Schwann cells in the distal stump that persist but do not divide. On the other hand, in the wild type littermates, newly generated Schwann cells of injured nerves are culled by apoptosis. As a result, distal Schwann cell numbers in wild type and cyclin D1 null mice converge to equivalence in regenerated nerves. Therefore, distal Schwann cell proliferation is not required for functional recovery of injured nerves.     

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.     

Synthetic nerve graft containing collagen and synthetic Schwann cells inproves functional, electrophysiological, and histological parameters of peripheral nerve regeneration

Current methods of peripheral nerve repair are to directly suture cut nerve stumps, or to bridge large gaps with an autograft repair. Autograft-associated problems include donor site morbidity and limited supply. Many of the present limitations of nerve repair might be overcome by expanding the patients own Schwann cells in vitro, then combining the cells with other neuro-tropic and -trophic materials into an Artificial Nerve Graft (ANG) for bridging a nerve gap. In this 4.5 month experiment, a rat peroneal nerve model with a 10 mm gap was used to evaluate the effect of live Schwann cells on peripheral nerve regeneration. Nerve gaps were repaired with cellular ANGs containing live Schwann cell, dead Schwann cell, or mixed fibroblast/Schwann cell populations suspended in a collagen I matrix, and with sutured autografts or ANGs containing just collagen or medium. Regenerated nerves were evaluated by walking track analysis, qualitative and quantitative histology, and electrophysiology. Overall, the autograft was the best repair method, while the ANG containing live Schwann cells was statistically superior to other ANG repair methods. This study demonstrates that an ANG containing cultured syngeneic Schwann cells improves functional, histological, and electrophysiological parameters of peripheral nerve regeneration.     

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.     

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

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

The role of neuregulin-1 in the response to nerve injury

Axons and Schwann cells exist in a highly interdependent relationship: damage to one cell type invariably leads to pathophysiological changes in the other. Greater understanding of communication between these cell types will not only give insight into peripheral nerve development, but also the reaction to and recovery from peripheral nerve injury. The type III isoform of neuregulin-1 (NRG1) has emerged as a key signaling factor that is expressed on axons and, through binding to erbB2/3 receptors on Schwann cells, regulates multiple phases of their development. In adulthood, NRG1 is dispensable for the maintenance of the myelin sheath; however, this factor is required for both axon regeneration and remyelination following nerve injury. The outcome of NRG1 signaling depends on interactions with other pathways within Schwann cells such as Notch, integrin and cAMP signaling. In certain circumstances, this signaling pathway may be maladaptive; for instance, direct binding of Mycobacterium leprae onto erbB2 receptors produces excessive activation and can actually promote demyelination. Attempts to modulate this pathway in order to promote nerve repair will therefore need to give consideration to the exact isoform used, as well as how it is processed and the context in which it is presented to the Schwann cell.     

Role of endogenous Schwann cells in tissue repair after spinal cord injury

Schwann cells are glial cells of peripheral nervous system, responsible for axonal myelination and ensheathing, as well as tissue repair following a peripheral nervous system injury. They are one of several cell types that are widely studied and most commonly used for cell transplantation to treat spinal cord injury, due to their intrinsic characteristics including the ability to secrete a variety of neurotrophic factors. This mini review summarizes the recent findings of endogenous Schwann cells after spinal cord injury and discusses their role in tissue repair and axonal regeneration. After spinal cord injury, numerous endogenous Schwann cells migrate into the lesion site from the nerve roots, involving in the construction of newly formed repaired tissue and axonal myelination. These invading Schwann cells also can move a long distance away from the injury site both rostrally and caudally. In addition, Schwann cells can be induced to migrate by minimal insults (such as scar ablation) within the spinal cord and integrate with astrocytes under certain circumstances. More importantly, the host Schwann cells can be induced to migrate into spinal cord by transplantation of different cell types, such as exogenous Schwann cells, olfactory ensheathing cells, and bone marrow-derived stromal stem cells. Migration of endogenous Schwann cells following spinal cord injury is a common natural phenomenon found both in animal and human, and the myelination by Schwann cells has been examined effective in signal conduction electrophysiologically. Therefore, if the inherent properties of endogenous Schwann cells could be developed and utilized, it would offer a new avenue for the restoration of injured spinal cord.