In vitro study of axonal migration and myelination of motor neurons in a three-dimensional tissue-engineered model

Primary motor neurons are difficult to study in conventional culture systems because of their short-term survival without trophic support from glia. In addition, axonal migration on a two-dimensional Petri dish does not reflect the three-dimensional (3D) environment in vivo. A unique in vitro 3D model of motor nerve regeneration was developed to study motor neuron axonal migration and myelination. Mouse spinal cord motor neurons were seeded on a collagen sponge populated with Schwann cells and fibroblasts. This fibroblast-populated sponge was intended to mimic the connective tissue through which motor axons have to elongate in vivo. Addition of conventional neurotrophic supplements was not required for motor neuron survival but was necessary to promote deep neurite outgrowth, as assessed by immunostaining of neurofilament M. A vigorous neurite elongation was detected inside the sponge after only 14 days of neuron culture, reaching more than 850 microm. The model also allowed the maturation of motor fibers as one-third of them were positive for neurofilament H. Neurites growing in the sponge were subject to myelination when Schwann cells were present, as shown by myelin basic protein immunostaining and electron microscopy. We demonstrated in this model the spontaneous formation of numerous thick myelin sheaths surrounding motor fibers after long-term culture (28 days). Thus, this model might be a valuable tool to study the effect of various cells and/or attractive or repulsive molecules on motor neurite outgrowth in vitro and also for the study of myelination and pathogenesis of motor neuron diseases.     

Rho-kinase inhibition enhances axonal regeneration after peripheral nerve injury

In injured adult neurons, the process of axonal regrowth and reestablishment of the neuronal function have to be activated. We assessed in this study whether RhoA, a key regulator of neurite elongation, is activated after injury to the peripheral nervous system. RhoA is activated in motoneurons but not in Schwann cells after mouse sciatic nerve injury. To examine whether the activation of RhoA and its effector, Rho-kinase, retards axon regeneration of injured motoneurons, we employed a Rho-kinase inhibitor, fasudil. Amplitudes of distally evoked compound muscle action potentials are increased significantly faster after axonal injury in mice treated with fasudil compared with controls. Histological analysis shows that fasudil treatment increases the number of regenerating axons with large diameter, suggesting that axon maturation is facilitated by Rho-kinase inhibition. In addition, fasudil does not suppress the myelination of regenerating axons. These findings suggest that RhoA/Rho-kinase may be a practical molecular target to enhance axonal regeneration in human peripheral neuropathies.     

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.     

Role of neuregulin-1 in peripheral nerve injury

Peripheral nerve injury in humans often leads to permanent functional deficits. Schwann cells play an important role in the recovery of peripheral nerve injury by ensheathing axons and providing various neurotrophic factors. Neuregulin-1 (NRG-1) provides axonal signals, which allow dedifferentiation and rapid proliferation of Schwann cells. Subsequently, NRG-1 promotes axonal myelination and influences myelin thickness. Moreover, NRG-1 plays a critical role in synapse formation in the neuromuscular junction. These effects, together, suggest that NRG-1 promotes recovery of peripheral nerve injury.     

Role of inflammatory cytokines in peripheral nerve injury

Inflammatory events occurring in the distal part of an injured peripheral nerve have, nowadays, a great resonance. Investigating the timing of action of the several cytokines in the important stages of Wallerian degeneration helps to understand the regenerative process and design pharmacologic intervention that promotes and expedites recovery. The complex and synergistic action of inflammatory cytokines finally promotes axonal regeneration. Cytokines can be divided into pro-and anti-inflammatory cytokines that upregulate and downregulate, respectively, the production of inflammatory mediators. While pro-inflammatory cytokines are expressed in the first phase of Wallerian degeneration and promote the recruitment of macrophages, anti-inflammatory cytokines are expressed after this recruitment and downregulate the production of all cytokines, thus determining the end of the process. In this review, we describe the major inflammatory cytokines involved in Wallerian degeneration and the early phases of nerve regeneration. In particular, we focus on interleukin-1, interleukin-2, interleukin-6, tumor necrosis factor-β, interleukin-10 and transforming growth factor-β.     

施万细胞在修复外周神经损伤中作用机制的研究进展

外周神经损伤后若不能及时准确的修复,则会导致外周神经功能的永久丧失。目前研究显示施万细胞(SC)参与外周神经损伤后碎片清除、轴突和髓鞘再生以及靶器官再支配过程中,外周神经损伤后SC被迅速激活进入修复过程,经历一系列动态的细胞重塑变化,转化为修复表型,促进神经再生、引导对靶器官再支配,从而恢复神经功能,其中有许多信号通路,转录调节因子等调控这些过程。基于此,该文系统总结了SC在外周神经再生过程中的研究进展,为深入研究外周神经修复提供新的方法和策略。     

Abnormalities in the axonal cytoskeleton induced by a connexin32 mutation in nerve xenografts

The X-linked form of Charcot-Marie-Tooth neuropathy is associated with mutations in the connexin32 (Cx32) gene. The functional role of Cx32 in Schwann cells and the relationship of these mutations to the progressive axonal loss and distal limb weakness seen in this disease have not been elucidated. To investigate the local influence of Schwann cells bearing the Cx32 gene defect on axonal cytoskeleton and the myelination process, the nerve xenograft model was used to transfer a Cx32 missense mutation (Glu102Gly) from human to an in vivo myelination system in nude mice. Twelve nerve grafts from two family members with Cx32 mutations and 17 grafts from three healthy individuals were generated by end-to-end anastomosis of approximately 6-mm sural nerve fascicles into the cut ends of the sciatic nerve in nude mice. Specimens were examined at 2, 4, 8, 12, and 16 weeks. Ultrastructural morphometric analysis showed Schwann cells with Cx32 mutation have a profound effect on the nude mice axons, resulting in an increase in neurofilament density, a depletion of microtubules associated with fragmentation of smooth axonal reticulum, and increased vesicles and mitochondria. At 16 weeks, axonal enlargement was evident within the proximal part of the graft; axonal atrophy, degeneration, and fiber loss were seen in distal-graft and host segments. The myelination process was not affected. We conclude that Cx32 mutation impairs a modulatory function of Schwann cells on axons, resulting in profound cytoskeletal alterations leading to distal axonal degeneration. These observations emphasize the role of impaired Schwann cell-axon interactions in the pathogenesis of hereditary neuropathies. J. Neurosci. Res. 51:174–184, 1998. © 1998 Wiley-Liss, Inc.     

The myelin protein MAL affects peripheral nerve myelination: a new player influencing p75 neurotrophin receptor expression

The myelin and lymphocyte protein (MAL) is a raft-associated membrane protein predominantly expressed by oligodendrocytes and Schwann cells. Here we show that MAL regulates myelination in the peripheral nervous system. In mice overexpressing MAL, myelination was retarded and fibers were hypomyelinated, whereas myelination in MAL knockout mice was accelerated. This was not due to impaired Schwann cell proliferation, differentiation or axonal sorting. We found that the expression level of p75 neurotrophin receptor mRNA and protein was strongly reduced in developing sciatic nerves in MAL-overexpressing mice. This reduction is well correlated with the observed alterations in myelination initiation, speed of myelination and alterations in Remak bundle development. Our results suggest a functional role for MAL in peripheral myelination by influencing the expression of membrane components that mediate axon-glia interaction during ensheathment and myelin wrapping.     

Role of the extracellular matrix in myelination of peripheral nerve.

Assembly of the extracellular matrix (ECM) has been tightly linked to compact myelin formation in the peripheral nervous system. We recently demonstrated that myelination of dorsal root ganglion (DRG) axons by Schwann cells may occur in the absence of basal lamina. We have now determined whether laminin deposition occurs around myelinating SC, even though basal lamina has not been assembled. DRG/SC co-cultures were prepared from E15 rat embryos and incubated in fully defined medium (B27) with and without ascorbic acid for 21-24 days. Cultures were stained with a rabbit anti-laminin antibody and examined by laser confocal fluorescence microscopy. Myelination occurred in both groups. In the presence of ascorbic acid, there was dense even laminin staining around myelinating SC. In the absence of ascorbic acid, laminin staining was also present but was irregular and less dense. DRG and SC were co-cultured without ascorbic acid in the presence or absence of a function blocking anti-beta(1) integrin receptor antibody. The antibody completely inhibited myelination. Finally, DRG/SC co-cultures were prepared both with and without ascorbic acid and incubated under control conditions or in the presence of continual, gentle motion. Movement in the absence of ECM significantly inhibited myelination. This demonstrates that laminin deposition on the surface of SC but not ECM assembly is required for formation of compact myelin. ECM is required to provide mechanical stability during the process of myelination.     

Transplantation of olfactory ensheathing cells promotes axonal regeneration and functional recovery of peripheral nerve lesion in rats

Introduction: Olfactory ensheathing cells (OECs) hold promise for cell therapy because they may promote regeneration of the central nervous system. However, OECs have been less studied after peripheral nerve injury (PNI). The purpose of this investigation was to determine the effect of OEC transplantation on a severe sciatic nerve (SN) lesion. Methods: OECs were injected in rats after section and 2‐cm resection of the SN. Results: Three months after therapy, muscle strength and morphometric studies showed complete restoration of the contractile properties of the gastrocnemius and complete repair of the SN. Immunohistochemistry and RT‐PCR studies indicated an increase in the presence of neurotrophic factors. Interestingly, tracking of green fluorescent protein (GFP)‐positive OECs showed that no OECs were present in the SN. Discussion: Our results demonstrate that, after severe PNI, OECs have remarkable potential for nerve regeneration by creating a favorable microenvironment. Muscle Nerve, 2011     

Skin‐derived precursor schwann cell myelination capacity in focal tibial demyelination

Introduction: Skin‐derived precursor cells (SKPs) are neural crest progenitor cells that can attain a Schwann cell–like phenotype through in vitro techniques (SKP‐SCs). We hypothesized that SKP‐SCs could produce mature myelin and, in doing so, facilitate the recovery of a focal demyelination injury. Methods: We unilaterally injected DiI‐labeled, green fluorescent protein (GFP)‐producing SKP‐SCs into the tibial nerves of 10 adult Lewis rats (with contralateral media control), 9 days after bilateral doxorubicin injury (0.38 μg). Tibial compound motor action potentials (CMAPs) were followed for 57 days. A separate morphometric cohort also included a Schwann cell injection group. Results: SKP‐injected nerves recovered fastest in terms of electrophysiology and morphometry. SKP‐SCs formed morphologically mature myelin, accounting for 15.3 ± 5.3% of the total myelin in SKP‐SC–injected nerves. Conclusions: SKP‐SCs are robustly capable of myelination. They improve the recovery of a focal tibial nerve demyelination model by myelinating a measured percentage of axons. Muscle Nerve 50:262–272, 2014     

GABA and its B‐receptor are present at the node of Ranvier in a small population of sensory fibers,implicating a role in myelination

The γ‐aminobutyric acid (GABA) type B receptor has been implicated in glial cell development in the peripheral nervous system (PNS), although the exact function of GABA signaling is not known. To investigate GABA and its B receptor in PNS development and degeneration, we studied the expression of the GABA_B receptor, GABA, and glutamic acid decarboxylase GAD65/67 in both development and injury in fetal dissociated dorsal root ganglia (DRG) cell cultures and in the rat sciatic nerve. We found that GABA, GAD65/67, and the GABA_B receptor were expressed in premyelinating and nonmyelinating Schwann cells throughout development and after injury. A small population of myelinated sensory fibers displayed all of these molecules at the node of Ranvier, indicating a role in axon–glia communication. Functional studies using GABA_B receptor agonists and antagonists were performed in fetal DRG primary cultures to study the function of this receptor during development. The results show that GABA, via its B receptor, is involved in the myelination process but not in Schwann cell proliferation. The data from adult nerves suggest additional roles in axon–glia communication after injury. © 2014 Wiley Periodicals, Inc.     

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.     

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.     

Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk-1 receptor

Vascular endothelial growth factor (VEGF) is an angiogenic factor that stimulates axonal outgrowth. Here we used in situ hybridization and immunocytochemistry to study the VEGF receptor flk-1 in cultured superior cervical ganglia (SCG) and dorsal root ganglia (DRG) from adult mice, and also the effects of VEGF on regeneration in vitro. Neurons in both ganglia contained the flk-1 receptor and showed an increased mRNA expression and immunoreactivity for flk-1 after 48 h in culture. In SCG, but not in DRG, double immunostaining for flk-1 and VEGF revealed coexpression in many neurons, implying that VEGF may exert both autocrine and paracrine actions. One proportion of the flk-1-positive neurons in DRG stained positive for the large neuron marker RT97 and another proportion expressed calcitonin gene-related peptide (CGRP). Small IB4-positive neurons were devoid of flk-1 immunoreactivity. Most flk-1-positive neurons in the DRG, but not in the SCG, were also immunoreactive to neuropilin-1. VEGF was found to stimulate axonal outgrowth from DRG, both by an action on the growing axons and the nerve cell bodies. The latter effect could be mediated by retrograde axonal transport as revealed by the use of a two compartment system to assay axonal outgrowth. We also found that the VEGF-induced axonal outgrowth was blocked by the flk-1 inhibitor SU5416. The results strongly suggest that VEGF acts as a neurotrophic factor and plays an important role during the regeneration of peripheral nerves.     

Local control of axonal properties by Schwann cells: neurofilaments and axonal transport in homologous and heterologous nerve grafts.

A number of axonal properties, including slow axonal transport and neurofilament phosphorylation, are altered in a mutant mouse strain with a Schwann cell deficiency, the Trembler. The Trembler phenotype is associated with poor myelination and reduced axonal caliber in the peripheral nervous system, but the genetic lesion has not yet been identified. To determine whether changes in axonal properties resulted from a direct action of Schwann cells on the axon, a segment of sciatic nerve from myelin-deficient Trembler mouse was grafted into the sciatic nerve of a normal mouse and normal axons were allowed to regenerate. Normal axons surrounded by Trembler Schwann cells are reduced in diameter, but resume their original diameter distal to the graft. Neurofilament transport was also affected locally in sciatic nerves with Trembler grafts into normal nerve. The velocity of neurofilament transport was not significantly different from controls in portions of the nerve proximal to the Trembler graft, but there was a reduction in neurofilament transport rates upon entering the Trembler graft. This was accompanied by an increase in the ratio of neurofilament over tubulin in the case of the Trembler graft, suggesting both a slowing of the neurofilament and an increase in the rate of tubulin transport. Using heterologous grafts of Trembler nerve segments into wildtype nerves, Schwann cells were shown to locally influence axonal caliber, neurofilament organization, and slow axonal transport. These observations emphasize the importance of glial cells in modulating neuronal structure and functions, as well as focusing attention on the role of glia in the etiology of neuropathologies that alter the neuronal environment.     

Peripheral but not central axotomy promotes axonal outgrowth and induces alterations in neuropeptide synthesis in the nodose ganglion of the rat

We investigated the effects of central and peripheral axotomy of the sensory neurons in the nodose ganglion on neurite outgrowth and neuropeptide expression. Axonal outgrowth was studied in ganglia subjected to a conditioning lesion of the vagus nerve 6 days prior to in vitro explantation. In such cultures, a conditioning effect, i. e. a shorter initial delay and faster axonal outgrowth, was observed after peripheral axotomy, while central axotomy had no effect. Neuropeptide expression was measured by immunocytochemistry 3 days after axotomy. Peripheral axotomy induced an increase in the number of neurons expressing the C-terminal flanking peptide of neuropeptide Y (C-PON), galanin (GAL) and vasoactive intestinal peptide (VIP). In contrast, central axotomy did not affect neuropeptide expression. These results suggest that both axonal outgrowth and expression of neuropeptides in the sensory neurons of the nodose ganglion could be regulated by the contact of the cells with their peripheral, but not their central targets.     

Monocarboxylate transporter 1 in Schwann cells contributes to maintenance of sensory nerve myelination during aging

Schwann cell (SC)-specific monocarboxylate transporter 1 (MCT1) knockout mice were generated by mating MCT1 ^f/f mice with myelin protein zero (P0)-Cre mice. P0-Cre^+/−, MCT1 ^f/f mice have no detectable early developmental defects, but develop hypomyelination and reduced conduction velocity in sensory, but not motor, peripheral nerves during maturation and aging. Furthermore, reduced mechanical sensitivity is evident in aged P0-Cre^+/−, MCT1 ^f/f mice. MCT1 deletion in SCs impairs both their glycolytic and mitochondrial functions, leading to altered lipid metabolism of triacylglycerides, diacylglycerides, and sphingomyelin, decreased expression of myelin-associated glycoprotein, and increased expression of c-Jun and p75-neurotrophin receptor, suggesting a regression of SCs to a less mature developmental state. Taken together, our results define the contribution of SC MCT1 to both SC metabolism and peripheral nerve maturation and aging.     

Increased expression of pp60c-src protein-tyrosine kinase during peripheral nerve regeneration.

Since little is known about the intracellular changes that take place in response to Schwann cell-neuron interactions that occur during neurite outgrowth and myelination, we investigated the expression of a protein-tyrosine kinase, pp60c-src, during peripheral nerve regeneration through a silicone tube. Segments of regenerated nerve, extracted at various times following nerve-transection, showed an induction of in vitro c-src kinase activity as measured by autophosphorylation of immunoprecipitated pp60c-src. This activity occurred at 7 days following nerve transection coincident with the onset of neurite outgrowth in vivo. This kinase activity, which peaked out between 21 and 35 days and decreased thereafter, appeared to be associated with axonal growth and myelination, but not mitogenesis in the tube. Analysis of c-src proteins levels by Western blot showed a similar expression profile as that of the kinase activity. Qualitatively, the expression of an immunoreactive c-src band, migrating slightly slower than pp60, was detected in extracts of regenerating nerve segments as well as in the corresponding L4 and L5 dorsal root ganglia. This protein may be the CNS neuronal-specific form (pp60+) of the c-src protein. In situ hybridization revealed that Schwann cells and sensory and motor neurons associated with the regenerated sciatic nerve were positive for c-src mRNA during regeneration possibly accounting for the increased src protein expression during regeneration. Since the increased expression of pp60c-src in regenerated nerve segments coincides with both axonal sprouting and myelination, our findings suggest that the c-src protein may play a role in Schwann cell-neuron interactions which facilitate the occurrence of these events during regeneration. In addition, although pp60+ is generally not detectable in the mature PNS, our findings show that this protein may be induced during conditions of PNS differentiation which promote neurite outgrowth.