Myelinating Schwann cells and Netrin-1 control intra-nervous vascularization of the developing mouse sciatic nerve.

Peripheral nerves are vascularized by a dense network of blood vessels to guarantee their complex function. Despite the crucial role of vascularization to ensure nerve homeostasis and regeneration, the mechanisms governing nerve invasion by blood vessels remain poorly understood. We found, in mice, that the sciatic nerve invasion by blood vessels begins around embryonic day 16 and continues until birth. Interestingly, intra-nervous blood vessel density significantly decreases during post-natal period, starting from P10. We show that, while the axon guidance molecule Netrin-1 promotes nerve invasion by blood vessels via the endothelial receptor UNC5B during embryogenesis, myelinated Schwann cells negatively control intra-nervous vascularization during post-natal period.

Polydatin protects Schwann cells from methylglyoxal induced cytotoxicity and promotes crushed sciatic nerves regeneration of diabetic rats.

Oxidative stress plays the main role in the pathogenesis of diabetes mellitus and peripheral neuropathy. Polydatin (PD) has been shown to exhibit strong antioxidative and antiinflammatory effects. At present, no research has focused on the possible effects of PD on Schwann cells and impaired peripheral nerves in diabetic models. Here, we used an in vitro Schwann cell damage model induced by methylglyoxal and an in vivo diabetic sciatic nerve crush model to study problems in such an area. In our experiment, we demonstrated that PD potently alleviated the decrease of cellular viability, prevented reactive oxygen species generation, and suppressed mitochondrial depolarization as well as cellular apoptosis in damaged Schwann cells. Moreover, we found that PD could upregulate Nrf2 and Glyoxalase 1 (GLO1) expression and inhibit Keap1 and receptor of AGEs (RAGE) expression of damaged Schwann cells. Finally, our in vivo experiment showed that PD could promote sciatic nerves repair of diabetic rats. Our results revealed that PD exhibited prominent neuroprotective effects on Schwann cells and sciatic nerves in diabetic models. The molecular mechanisms were associated with activating Nfr2 and GLO1 and inhibiting Keap1 and RAGE.

Comprehensive Analysis of Age-related Changes in Lipid Metabolism and Myelin Sheath Formation in Sciatic Nerves.

To investigate the molecular changes related to myelin formation and lipid metabolism in the sciatic nerve in Sprague Dawley (SD) rats during aging. Thirty-six healthy male SD rats were divided into five groups according to age: 1 week, 1 month, 6 months, 12 months, and 24 months. Sciatic nerves were collected from 1-month-old and 24-month-old SD rats (n = 3) to perform next-generation sequencing (NGS) and bioinformatics analysis. Specimens from each group were harvested and analyzed by qPCR, Western blotting, and transmission electron microscopy (TEM). Protein-protein interaction (PPI) networks of differentially expressed mRNAs (DEmRNAs) related to myelin and lipid metabolism were constructed. DEmRNAs in subnetworks were verified using qPCR. A total of 4580 DEmRNAs were found during aging. The top enriched GO biological processes were primarily clustered in cholesterol and lipid metabolism, including the cholesterol biosynthetic process (RF = 3.16), sterol biosynthetic process (RF = 3.03), cholesterol metabolic process (RF = 2.15), sterol metabolic process (RF = 2.11), fatty acid biosynthetic process (RF = 2.09), and lipid biosynthetic process (RF = 1.79). The mRNA levels of MBP, PMP22, and MPZ were downregulated during aging, while the protein expression of MBP showed an increasing trend. The TEM results showed thin myelin sheaths and an increased number of unmyelinated axons in the 1-week-old rats, and the sheaths became thickened with degenerated axons appearing in older animals. Forty PPI subnetworks related to lipid metabolism were constructed, including one primary subnetwork and two smaller subnetworks. The hub genes were mTOR in sub-network 1, Akt1 in sub-network 2, and SIRT1 in sub-network 3. No gene expression was found consistent with the sequencing results, while in the downregulated genes, AKT1, CEBPA, LIPE, LRP5, PHB, and Rara were significantly downregulated in 24-month-old rats. Lipid metabolism might play an important role in maintaining the structure and physiological function in sciatic nerves during aging and could be candidates for nerve aging research.

Mesenchymal Stem Cell Treatment Perspectives in Peripheral Nerve Regeneration: Systematic Review.

Traumatic peripheral nerve lesions affect hundreds of thousands of patients every year; their consequences are life-altering and often devastating and cause alterations in movement and sensitivity. Spontaneous peripheral nerve recovery is often inadequate. In this context, nowadays, cell therapy represents one of the most innovative approaches in the field of nerve repair therapies. The purpose of this systematic review is to discuss the features of different types of mesenchymal stem cells (MSCs) relevant for peripheral nerve regeneration after nerve injury. The published literature was reviewed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A combination of the keywords "nerve regeneration", "stem cells", "peripheral nerve injury", "rat", and "human" were used. Additionally, a "MeSH" research was performed in PubMed using the terms "stem cells" and "nerve regeneration". The characteristics of the most widely used MSCs, their paracrine potential, targeted stimulation, and differentiation potentials into Schwann-like and neuronal-like cells are described in this paper. Considering their ability to support and stimulate axonal growth, their remarkable paracrine activity, their presumed differentiation potential, their extremely low immunogenicity, and their high survival rate after transplantation, ADSCs appear to be the most suitable and promising MSCs for the recovery of peripheral nerve lesion. Clinical considerations are finally reported.

FGF21 impedes peripheral myelin development by stimulating p38 MAPK/c-Jun axis.

Fibroblast growth factor 21 (FGF21) as a metabolic stress hormone, is mainly secreted by the liver. In addition to its well-defined roles in energy homeostasis, FGF21 has been shown to promote remyelination after injury in the central nervous system. In the current study, we sought to examine the potential roles of FGF21 in the peripheral nervous system (PNS) myelination. In the PNS myelin development, Fgf21 expression was reversely correlated with myelin gene expression. In cultured primary Schwann cells (SCs), the application of recombinant FGF21 greatly attenuates myelination-associated gene expression, including Oct6, Krox20, Mbp, Mpz, and Pmp22. Accordingly, the injection of FGF21 into neonatal rats markedly mitigates the myelination in sciatic nerves. On the contrary, the infusion of the anti-FGF21 antibody accelerates the myelination. Mechanistically, both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) were stimulated by FGF21 in SCs and sciatic nerves. Following experiments including pharmaceutical intervention and gene manipulation revealed that the p38 MAPK/c-Jun axis, rather than ERK, is targeted by FGF21 for mediating its repression on myelination in SCs. Taken together, our data provide a new aspect of FGF21 by acting as a negative regulator for the myelin development process in the PNS via activation of p38 MAPK/c-Jun.

Effectiveness of electrical stimulation on nerve regeneration after crush injury: Comparison between invasive and non-invasive stimulation.

Several studies have investigated the use of invasive and non-invasive stimulation methods to enhance nerve regeneration, and varying degrees of effectiveness have been reported. However, due to the use of different parameters in these studies, a fair comparison between the effectiveness of invasive and non-invasive stimulation methods is not possible. The present study compared the effectiveness of invasive and non-invasive stimulation using similar parameters. Eighteen Sprague Dawley rats were classified into three groups: the iES group stimulated with fully implantable device, the tES group stimulated with transcutaneous electrical nerve stimulation (TENS), and the injury group (no stimulation). The iES and tES groups received stimulation for 6 weeks starting immediately after the injury. Motor function was evaluated using the sciatic functional index (SFI) every week. The SFI values increased over time in all groups; faster and superior functional recovery was observed in the iES group than in the tES group. Histological evaluation of the nerve sections and gastrocnemius muscle sections were performed every other week. The axon diameter and muscle fiber area in the iES group were larger, and the g-ratio in the iES group was closer to 0.6 than those in the tES group. To assess the cause of the difference in efficiency, a 3D rat anatomical model was used to simulate the induced electric fields in each group. A significantly higher concentration and intensity around the sciatic nerve was observed in the iES group than in the tES group. Vector field distribution showed that the field was orthogonal to the sciatic nerve spread in the tES group, whereas it was parallel in the iES group; this suggested that the tES group was less effective in nerve stimulation. The results indicated that even though rats in the TENS group showed better recovery than those in the injury group, it cannot replace direct stimulation yet because rats stimulated with the invasive method showed faster recovery and superior outcomes. This was likely attributable to the greater concentration and parallel distribution of electric field with respect to target nerve.

Nanofibrous Nerve Conduits with Nerve Growth Factors and Bone Marrow Stromal Cells Pre-Cultured in Bioreactors for Peripheral Nerve Regeneration.

Peripheral nerve injury (PNI) was the leading cause of permanent dysfunction in movement and sensation. Synthesized nerve guide conduits (NGCs) with Schwann Cells (SCs) can help peripheral nerve regeneration. However, poor accessibility of SCs and lack of full coverage of seeded cells on NGCs can lead to failure of nerve regeneration across long gaps and full functional recovery. To overcome these limitations, bone marrow stromal cells (BMSCs) and a novel culture method were proposed in the current study. BMSCs were harvested and seeded on a never growth factor (NGF)-loaded PCL nanofibrous NGCs and cultured with a rotary cell culture system (RCCS) before implantation. The NGCs were tested in vitro with PC-12 cells to validate the bioactivity of released NGF and to access its ability to promote neurite extension. Also, the NGCs were tested in vivo with rat sciatic nerve model to exam its potential in bridging the long gap (15 mm segmental defect). The efficacy of the NGCs was investigated based on the results of the functional test, electrophysiology test, muscle atrophy, and histological analysis. The results of in vitro PC-12 cell study confirmed the bioactivity of released NGF and showed a significant increase in the neurite extension with the help of PEG-diamine and BSA. These results showed that the novel loading method could preserve the bioactivity of growth factors and achieve a sustained release in vitro. Besides, the results of the in vivo study exhibited a significant increase with the combination of all additives. These results showed that with the help of NGF and RCCS, the NGCs with the seeded BMSCs could enhance peripheral nerve regeneration across long nerve injury gaps.

Enhancement of sciatic nerve regeneration with dual delivery of vascular endothelial growth factor and nerve growth factor genes.

BACKGROUND: Peripheral nerve injury is one common clinical disease worldwide, in which sciatic nerve is anatomically the most challenging to regenerate given its length and large cross-sectional area. For the present, autologous nerve grafting remains to be the most ideal strategy when treating with sciatic nerve injury. However, this method sacrifices healthy nerves and requires highly intensive surgery, still calling for other advanced alternatives for nerve grafting. RESULTS: In this study, we utilized previously well-established gene delivery system to dually deliver plasmid DNA (pDNA) encoding vascular endothelial growth factor (VEGF) and nerve growth factor (NGF), exploring therapeutics for sciatic nerve injury. Low-molecular-weight branched polyethylenimine (bPEI) was constructed as the backbone structure of gene vectors, and it was further crosslinked to synthesize degradable polycations via the conjugation of dialdehydes. Potential synergistic effect between VEGF and NGF proteins were observed on rat sciatic nerve crush injury model in this study. CONCLUSIONS: We concluded that dual delivery of plasmid VEGF and NGF as gene therapy could enhance sciatic nerve regeneration.

The Ormdl genes regulate the sphingolipid synthesis pathway to ensure proper myelination and neurologic function in mice.

Sphingolipids are membrane and bioactive lipids that are required for many aspects of normal mammalian development and physiology. However, the importance of the regulatory mechanisms that control sphingolipid levels in these processes is not well understood. The mammalian ORMDL proteins (ORMDL1, 2 and 3) mediate feedback inhibition of the de novo synthesis pathway of sphingolipids by inhibiting serine palmitoyl transferase in response to elevated ceramide levels. To understand the function of ORMDL proteins in vivo, we studied mouse knockouts (KOs) of the Ormdl genes. We found that Ormdl1 and Ormdl3 function redundantly to suppress the levels of bioactive sphingolipid metabolites during myelination of the sciatic nerve. Without proper ORMDL-mediated regulation of sphingolipid synthesis, severe dysmyelination results. Our data indicate that the Ormdls function to restrain sphingolipid metabolism in order to limit levels of dangerous metabolic intermediates that can interfere with essential physiological processes such as myelination.

GDNF-ADSCs-APG embedding enhances sciatic nerve regeneration after electrical injury in a rat model.

The pluripotency of adipose-derived stem cells (ADSCs) makes them appropriate for tissue repair and wound healing. Owing to the repair properties of autologous platelet-rich gel (APG), which is based on easily accessible blood platelets, its clinical use has been increasingly recognized by physicians. The aim of this study was to investigate the effect of combined treatment with ADSCs and APG on sciatic nerve regeneration after electrical injury. To facilitate the differentiation of ADSCs, glial cell line-derived neurotrophic factor (GDNF) was overexpressed in ADSCs by lentivirus transfection. GDNF-ADSCs were mingled with APG gradient concentrations, and in vitro, cell proliferation and differentiation were examined with 5-ethynyl-2'-deoxyuridine staining and immunofluorescence. A rat model was established by exposing the sciatic nerve to an electrical current of 220 V for 3 seconds. Rat hind-limb motor function and sciatic nerve regeneration were subsequently evaluated. Rat ADSCs were characterized by high expression of CD90 and CD105, with scant expression of CD34 and CD45. We found that GDNF protein expression in ADSCs was elevated after Lenti-GDNF transfection. In GDNF-ADSCs-APG cultures, GDNF was increasingly produced while tissue growth factor-ß was reduced as incubation time was increased. ADSC proliferation was augmented and neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) expression were upregulated in GDNF-ADSCs-APG. In addition, limb motor function and nerve axon growth were improved after GDNF-ADSCs-APG treatment. In conclusion, our study demonstrates the combined effect of ADSCs and APG in peripheral nerve regeneration and may lead to treatments that benefit patients with electrical injuries.

Exosomes from human adipose-derived stem cells promote sciatic nerve regeneration via optimizing Schwann cell function.

Human adipose-derived stem cells (ASCs) have a potential for the treatment of peripheral nerve injury. Recent studies demonstrated that stem cells can mediate therapeutic effect by secreting exosomes. We aimed to investigate the effect of human ASCs derived exosomes (ASC-Exos) on peripheral nerve regeneration in vitro and in vivo. Our results showed after being internalized by Schwann cells (SCs), ASC-Exos significantly promoted SC proliferation, migration, myelination, and secretion of neurotrophic factors by upregulating corresponding genes in vitro. We next evaluated the efficacy of ASC-Exo therapy in a rat sciatic nerve transection model with a 10-mm gap. Axon regeneration, myelination, and restoration of denervation muscle atrophy in ASC-Exos treated group was significantly improved compared to vehicle control. This study demonstrates that ASC-Exos effectively promote peripheral nerve regeneration via optimizing SC function and thereby represent a novel therapeutic strategy for regenerative medicine and nerve tissue engineering.

Comparison of the regeneration induced by acellular nerve allografts processed with or without chondroitinase in a rat model.

There have been various studies about the acellular nerve allograft (ANA) as the alternative of autologous nerve graft in the treatment of peripheral nerve defects. As well as the decellularization process methods of ANA, the various enhancement methods of regeneration of the grafted ANA were investigated. The chondroitin sulfate proteoglycans (CSPGs) inhibit the action of laminin which is important for nerve regeneration in the extracellular matrix of nerve. Chondroitinase ABC (ChABC) has been reported that it enhances the nerve regeneration by degradation of CSPGs. The present study compared the regeneration of ANA between the processed without ChABC group and the processed with ChABC group in a rat sciatic nerve 15 mm gap model. At 12 weeks postoperatively, there was not a significant difference in the histomorphometric analysis. In the functional analysis, there were no significant differences in maximum isometric tetanic force, wet muscle weight of tibialis anterior. The processed without ChABC group had better result in ankle contracture angle significantly. In conclusion, there were no significant differences in the regeneration of ANA between the processed without ChABC group and the processed with ChABC group.

Investigation of axonal regeneration of Triturus ivanbureschi by using physiological and proteomic strategies.

Peripheral nerve injuries are frequently observed and successful treatment depends mainly on the injury type, location of the damage, and the elapsed time prior to treatment. The regenerative capacity is limited only to the embryonic period in many mammalian tissues, but urodele amphibians do not lose this feature during adulthood. The main purpose of this study is to define the recovery period after serious sciatic nerve damage of a urodele amphibian, Triturus ivanbureschi. Experimental transection damage was performed on the sciatic nerves of T. ivanbureschi specimens. The recovery period of sciatic nerves were investigated by walking track analysis, electrophysiological recordings, and bottom-up proteomic strategies at different time points during a 35-day period. A total of 34 proteins were identified related to the nerve regeneration process. This study showed that the expression levels of certain proteins differ between distal and proximal nerve endings during the regeneration period. In distal nerve stumps, transport proteins, growth factors, signal, and regulatory molecules are highly expressed, whereas in proximal nerve stumps, neurite elongation proteins, and cytoskeletal proteins are highly expressed.

Tanshinone IIA attenuates nerve structural and functional damage induced by nerve crush injury in rats.

After peripheral nerve crush injury, the fibers of distal nerve segments gradually disintegrate, and axons regrow from the proximal nerve segment, eventually reaching the target organ. However, the axon regeneration is generally not sufficient for the recovery of neurological function, so drug therapy is necessary. In the current study, we explored the effect of Tanshinone IIA in nerve regeneration in a sciatic nerve crush injury model using Sprague Dawley rats. The rats were administered 45 mg/kg of Tanshinone IIA once daily. Motor behavior and tibialis anterior muscle mass were assessed, and histological analysis of the sciatic nerve and lumbar spinal cord were conducted. The results showed that the administration of Tanshinone IIA improved nerve growth and motor function, and resulted in a marked decrease of neuronal death. The findings of this exploratory study suggest that Tanshinone IIA alleviates injury and boosts regeneration after nerve crush injury in a rat model of sciatic nerve injury.

FGF9 modulates Schwann cell myelination in developing nerves and induces a pro-inflammatory environment during injury.

Myelin sheath is critical for the proper functioning of the peripheral nervous system (PNS), which allows the effective conduction of nerve impulses. Fibroblast growth factor 9 (FGF9) is an autocrine and paracrine protein in the fibroblast growth factor family that regulates cell differentiation and proliferation. Fgf9 Schwann cell (SC) conditional knockout mice were developed to detect the role of FGF9 in the PNS. In our study, the absence of Fgf9 led to delayed myelination in early development. The expression of mature SC-related genes decreased, and the expression of genes associated with immature SCs increased in the Fgf9 knockout mice. These data were consistent with the morphology and praxeology we observed during the development of the peripheral nerves. Extracellular-regulated kinases 1/2 (ERK1/2) are key signals for myelination, and our results showed that Fgf9 ablation led to the inactivation of ERK1/2. Further research was performed to detect the role of FGF9 in peripheral nerve injury. In superoxide dismutase 1-G93A mice with Fgf9 SC knockout, we found that Fgf9 ablation inhibited the expressions of Cd68, Il-1ß, and Cd86, which contributed to the degeneration of the axon and myelin sheath.

The TSC1-mTOR-PLK axis regulates the homeostatic switch from Schwann cell proliferation to myelination in a stage-specific manner.

Proper peripheral myelination depends upon the balance between Schwann cell proliferation and differentiation programs. The serine/threonine kinase mTOR integrates various environmental cues to serve as a central regulator of cell growth, metabolism, and function. We report here that tuberous sclerosis complex 1 (TSC1), a negative regulator of mTOR activity, establishes a stage-dependent program for Schwann cell lineage progression and myelination by controlling cell proliferation and myelin homeostasis. Tsc1 ablation in Schwann cell progenitors in mice resulted in activation of mTOR signaling, and caused over-proliferation of Schwann cells and blocked their differentiation, leading to hypomyelination. Transcriptome profiling analysis revealed that mTOR activation in Tsc1 mutants resulted in upregulation of a polo-like kinase (PLK)-dependent pathway and cell cycle regulators. Attenuation of mTOR or pharmacological inhibition of polo-like kinases partially rescued hypomyelination caused by Tsc1 loss in the developing peripheral nerves. In contrast, deletion of Tsc1 in mature Schwann cells led to redundant and overgrown myelin sheaths in adult mice. Together, our findings indicate stage-specific functions for the TSC1-mTOR-PLK signaling axis in controlling the transition from proliferation to differentiation and myelin homeostasis during Schwann cell development.

Nerve Growth and Interaction in Gelfoam® Histoculture: A Nervous System Organoid.

Nestin-expressing hair follicle-associated pluripotent (HAP) stem cells reside mainly in the bulge area (BA) of the hair follicle but also in the dermal papilla (DP). The BA appears to be origin of HAP stem cells. Long-term Gelfoam® histoculture was established of whiskers isolated from transgenic mice, in which there is nestin-driven green fluorescent protein (ND-GFP). HAP stem cells trafficked from the BA toward the DP area and extensively grew out onto Gelfoam® forming nerve-like structures. These fibers express the neuron marker ß-III tubulin-positive fibers and consisted of ND-GFP-expressing cells and extended up to 500 mm from the whisker nerve stump in Gelfoam® histoculture. The growing fibers had growth cones on their tips expressing F-actin indicating that the fibers were growing axons. HAP stem cell proliferation resulted in elongation of the follicle nerve and interaction with other nerves in 3D Gelfoam® histoculture, including the sciatic nerve, trigeminal nerve, and trigeminal nerve ganglion.

Heparanase attenuates axon degeneration following sciatic nerve transection.

Axon degeneration underlies many nervous system diseases; therefore understanding the regulatory signalling pathways is fundamental to identifying potential therapeutics. Previously, we demonstrated heparan sulphates (HS) as a potentially new target for promoting CNS repair. HS modulate cell signalling by both acting as cofactors in the formation of ligand-receptor complexes and in sequestering ligands in the extracellular matrix. The enzyme heparanase (Hpse) negatively regulates these processes by cleaving HS and releasing the attached proteins, thereby attenuating their ligand-receptor interaction. To explore a comparative role for HS in PNS axon injury/repair we data mined published microarrays from distal sciatic nerve injury. We identified Hpse as a previously unexplored candidate, being up-regulated following injury. We confirmed these results and demonstrated inhibition of Hpse led to an acceleration of axonal degeneration, accompanied by an increase in ß-catenin. Inhibition of ß-catenin and the addition of Heparinase I both attenuated axonal degeneration. Furthermore the inhibition of Hpse positively regulates transcription of genes associated with peripheral neuropathies and Schwann cell de-differentiation. Thus, we propose Hpse participates in the regulation of the Schwann cell injury response and axo-glia support, in part via the regulation of Schwann cell de-differentiation and is a potential therapeutic that warrants further investigation.

A histone deacetylase 3-dependent pathway delimits peripheral myelin growth and functional regeneration.

Deficits in Schwann cell-mediated remyelination impair functional restoration after nerve damage, contributing to peripheral neuropathies. The mechanisms mediating block of remyelination remain elusive. Here, through small-molecule screening focusing on epigenetic modulators, we identified histone deacetylase 3 (HDAC3; a histone-modifying enzyme) as a potent inhibitor of peripheral myelinogenesis. Inhibition of HDAC3 enhanced myelin growth and regeneration and improved functional recovery after peripheral nerve injury in mice. HDAC3 antagonizes the myelinogenic neuregulin-PI3K-AKT signaling axis. Moreover, genome-wide profiling analyses revealed that HDAC3 represses promyelinating programs through epigenetic silencing while coordinating with p300 histone acetyltransferase to activate myelination-inhibitory programs that include the HIPPO signaling effector TEAD4 to inhibit myelin growth. Schwann cell-specific deletion of either Hdac3 or Tead4 in mice resulted in an elevation of myelin thickness in sciatic nerves. Thus, our findings identify the HDAC3-TEAD4 network as a dual-function switch of cell-intrinsic inhibitory machinery that counters myelinogenic signals and maintains peripheral myelin homeostasis, highlighting the therapeutic potential of transient HDAC3 inhibition for improving peripheral myelin repair.

Glial M6B stabilizes the axonal membrane at peripheral nodes of Ranvier.

Glycoprotein M6B and the closely related proteolipid protein regulate oligodendrocyte myelination in the central nervous system, but their role in the peripheral nervous system is less clear. Here we report that M6B is located at nodes of Ranvier in peripheral nerves where it stabilizes the nodal axolemma. We show that M6B is co-localized and associates with gliomedin at Schwann cell microvilli that are attached to the nodes. Developmental analysis of sciatic nerves, as well as of myelinating Schwann cells/dorsal root ganglion neurons cultures, revealed that M6B is already present at heminodes, which are considered the precursors of mature nodes of Ranvier. However, in contrast to gliomedin, which accumulates at heminodes with or prior to Na+ channels, we often detected Na+ channel clusters at heminodes without any associated M6B, indicating that it is not required for initial channel clustering. Consistently, nodal cell adhesion molecules (NF186, NrCAM), ion channels (Nav1.2 and Kv7.2), cytoskeletal proteins (AnkG and ßIV spectrin), and microvilli components (pERM, syndecan3, gliomedin), are all present at both heminodes and mature nodes of Ranvier in Gpm6b null mice. Using transmission electron microscopy, we show that the absence of M6B results in progressive appearance of nodal protrusions of the nodal axolemma, that are often accompanied by the presence of enlarged mitochondria. Our results reveal that M6B is a Schwann cell microvilli component that preserves the structural integrity of peripheral nodes of Ranvier.