Antioxidants are necessary for myelination of dorsal root ganglion neurons, in vitro

We have demonstrated that myelination of dorsal root ganglion (DRG) axons occurs in a fully defined, serum-free medium (B27). This implies that there may be components in B27 medium that support myelination. To determine which of the components in B27 were essential for myelination, we systematically removed components from B27 until myelination was lost. We added these components to a fully defined minimal medium (N2) that supports neuron survival but not myelination. When antioxidants were removed from B27, myelination was lost. However, the individual antioxidants did not induce myelination when added to N2 medium. Addition of ascorbic acid along with the B27 antioxidants was sufficient to induce myelination in N2 medium, which was enhanced by retinyl acetate. Removal of vitamin E from B27 caused a partial loss of myelination, and addition of vitamin E to N2 medium containing ascorbic acid induced partial myelination. Addition of serum to the B27 myelinating medium inhibited myelination completely. These results indicate that antioxidants are important for myelination, in vitro. Vitamin E may play an important role. Use of a serum-free medium may be beneficial for in vitro myelination studies because serum has unknown inhibitory effects.     

Expression of dystroglycan and the laminin-alpha 2 chain in the rat peripheral nerve during development

In Schwann cells, the transmembrane glycoprotein beta-dystroglycan comprises the dystroglycan complex, together with the extracellular glycoprotein alpha-dystroglycan, which binds laminin-2 (alpha 2/beta 1/gamma 1), a major component of the Schwann cell basal lamina. To provide clues to the biological functions of the interaction of the dystroglycan complex with laminin-2 in peripheral nerves, we investigated the expression of beta-dystroglycan and the laminin-alpha 2 chain in rat sciatic nerve during development by immunoblot, immunofluorescence, and immunoelectron microscopic studies. The expression of beta-dystroglycan and the laminin-alpha 2 chain in the rat sciatic nerve was low and not confined to the Schwann cell outer membrane from embryonic day 18 to birth, when there was only an immature basal lamina assembly and no compact myelin formation by Schwann cells. However, the expression of these proteins increased markedly and became clearly localized to the Schwann cell outer membrane between birth and postnatal day 7, when both basal lamina assembly and compact myelin formation by Schwann cells progressed rapidly. From postnatal day 7 to adult, there was no remarkable change in the expression of these proteins. Our results support the hypothesis that the dystroglycan complex functions as an adhesion apparatus, binding the Schwann cell outer membrane with the basal lamina, and suggest that the dystroglycan complex plays a role in Schwann cell myelination through its interaction with laminin-2.     

Rapid growth of regenerating axons across the segments of sciatic nerve devoid of Schwann cells

The characteristic response of Schwann cells (SC) accompanies peripheral nerve injury and regeneration. To elucidate their role, the question of whether or not regenerating axons can elongate across the segments of a peripheral nerve devoid of SC was investigated. Rat sciatic nerve was crushed so that the continuity of SC basal laminae was not interrupted. A segment about 15 mm long distal to the crush was either repeatedly frozen/thawed to eliminate SC or scalded by moist heat which, in addition, denatured the proteins in the SC basal laminae, too. Both sensory and motor axons grew rapidly across the frozen/thawed segment of the nerve. Their rate of elongation was reduced by only 30% in comparison to control crushed nerves. SC were not present along the path of growing axons adhering tightly to the bare SC basal laminae. The rate of elongation of regenerating sensory and motor axons in scalded nerve segments was eight times lower than in control crushed nerves. SC were present in that part of the scalded region that had been invaded by the regenerating axons but no further distally. These results suggest that acellular basal laminae of SC provide very good, although not optimal, conditions for elongation of regenerating sensory and motor axons. If biochemical integrity of the basal lamina is destroyed, the regenerating axons must be accompanied or preceded by viable SC. and axon elongation rate is significantly reduced.     

Local administration of vasoactive intestinal peptide after nerve transection accelerates early myelination and growth of regenerating axons

Our goal was to determine whether local injections of vasoactive intestinal peptide (VIP) promote early stages of regeneration after nerve transection. Sciatic nerves were transected bilaterally in 2 groups of 10 adult mice. In the first group, 15 microg (20 microL) of VIP were injected twice daily into the gap between transected ends of the right sciatic nerve for 7 days (4 mice) or 14 days (6 mice). The same number of mice in the second group received placebo injections (20 microL of 0.9% sterile saline) in the same site, twice daily, for the same periods. After 7 days, axon sizes, relationships with Schwann cells and degree of myelination were compared in electron micrographs of transversely sectioned distal ends of proximal stumps. Fourteen days after transection, light and electron microscopy were used to compare and measure axons and myelin sheaths in the transection gap, 2-mm distal to the ends of proximal stumps. Distal ends of VIP-treated proximal stumps contained larger axons 7 days after transection. More axons were in 1:1 relationships with Schwann cells and some of them were surrounded by thin myelin sheaths. In placebo-treated proximal stumps, axons were smaller, few were in 1:1 relationships with Schwann cells and no myelin sheaths were observed. In VIP-treated transection gaps, measurements 14 days after transection showed that larger axons were more numerous and their myelin sheaths were thicker. Our results suggest that in this nerve transection model, local administration of VIP promotes and accelerates early myelination and growth of regenerating axons.     

Evidence for an early role for myelin-associated glycoprotein in the process of myelination

The expression of myelin-associated glycoprotein (MAG) in purified rat Schwann cells following coculture with dorsal root ganglion neurons was compared with the expression of galactocerebroside (GalC) and Po using immunocytochemistry. In defined serum-free medium, lacking ascorbic acid, in which Schwann cells proliferate but neither ensheathe nor myelinate axons, axonal interaction up-regulated the cell surface expression of MAG and GalC but not of Po. Excision of neuronal cell bodies resulted in a down-regulation of both MAG and GalC from the Schwann cell surface. When cocultures were switched to complete medium (serum plus ascorbic acid) to promote myelination, Schwann cells committed to form myelin continued to express high levels of MAG and GalC on their surface, but nonmyelinating Schwann cells down-regulated MAG and GalC. There was significant MAG immunoreactivity associated with the external aspect of the apparent nodal region of developing myelin sheaths. Permeabilization prior to immunostaining revealed that all of the Schwann cell cytoplasmic processes of nascent internodes were significantly stained with anti-MAG antibodies before the appearance of Po immunoreactivity. The amount of MAG on the surface of mature myelin segments was reduced compared with developing myelin segments, but there was a considerable amount of anti-MAG staining in the paranodes and Schmidt-Lanterman incisures. The time of expression and localization of MAG indicates that it may be a critical molecule in the process by which the Schwann cell engulfs an axon destined to be myelinated and establishes the extent of the future internode.     

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.     

Role of axons in the regulation of P0 biosynthesis by Schwann cells.

The role of axons in the expression of the major myelin glycoprotein, P0, has been investigated using neuron/Schwann cell cultures. These cultures were either nonmyelinating or myelinating due to growth in defined medium or in medium containing serum and chick embryo extract, respectively. The neurons and Schwann cells used in the studies were derived from embryonic day 15 rat dorsal root ganglia (DRG), and the Schwann cells from these ganglia are shown not to synthesize appreciable levels of P0 prior to growth in culture. Myelinating cultures of Schwann cells and neurons grown together for 18-21 days synthesize P0 that is readily identified by immunoblotting. The nonmyelinating cultures, which do not assemble basal lamina, also synthesize P0 that is detectable by either [3H]mannose precursor incorporation or by immunoblotting. The steady-state level of P0 in the nonmyelinating cultures is less than that of the myelinating cultures, and the P0 that is synthesized by the former appears to be catabolized shortly after its biosynthesis. Since nonmyelinating Schwann cells synthesize P0 when in contact with neurites in vitro, we have examined the ability of such nonmyelinating cells to express the glycoprotein in vivo. Very little steady-state P0 is detected in immunoblots of the adult rat cervical sympathetic trunk (CST), a nerve in which approximately 99% of the axons are nonmyelinated. Similarly, the amounts of [3H]mannose and [3H]amino acids that are incorporated into newly synthesized P0 are much lower in the CST than in the adult sciatic nerve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of ADAM10 in axonal outgrowth and myelination of the peripheral nerve

The disintegrin and metalloproteinase 10 (ADAM10) is a membrane‐anchored metalloproteinase with both proteolytic and disintegrin characteristics. Here, we investigate the expression, regulation, and functional role of ADAM10 in axonal outgrowth and myelination of the peripheral nerve. Expression pattern analysis of 11 ADAM family members in co‐cultures of rat dorsal root ganglia (DRG) neurons and Schwann cells (SCs) demonstrated the most pronounced mRNA expression for ADAM10. In further studies, ADAM10 was found to be consistently upregulated in DRG‐SC co‐cultures before the induction of myelination. Neurons as well as SCs widely expressed ADAM10 at the protein level. In neurons, the expression of ADAM10 was exclusively limited to the axons before the induction of myelination. Inhibition of ADAM10 activity by the hydroxamate‐based inhibitors GI254023X and GW280264X resulted in a significant decrease in the mean axonal length. These data suggest that ADAM10 represents a prerequisite for myelination, although its activity is not required during the process of myelination itself as demonstrated by expression analysis of myelin protein zero (P0) and Sudan black staining. Hence, during the process of myelin formation, ADAM10 is highly upregulated and appears to be critically involved in axonal outgrowth that is a requirement for myelination in the peripheral nerve. © 2009 Wiley‐Liss, Inc.     

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.     

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)     

Abnormalities expressed in long-term cultures of dorsal root ganglia from the dystrophic mouse

We have compared the development in long-term tissue culture of dorsal root ganglia taken from normal and dystrophic mice. Cultures were prepared from late fetal (15–20 days) or neonatal mice of either the C57BL/6 dy^2j/dy^2j dystrophic(dy) or C57BL/6J +/+ (control) strain and maintained until fully myelinated (5 weeks or more). Analysis by light and electron microscopy indicated tha the substantial ensheathment failure present in certain dy nerve roots in vivo is not expressed in cultures; myelination and Schwann cell numbers are comparable to control cultures. On the other hand, many of the subtle abnormalities more recently described in distal parts of peripheral nerves of dystrophic mice are expressed in the dy cultures. These include: (a) discontinuity in the basal lamina surrounding both myelin-forming and non-myelinating Schwann cells; (b) elongated nodes of Ranvier occurring along otherwise well myelinated nerve fibres; (c) relatively short myelin internodes that are increased in thickness as well as irregularities of internodes length along a nerve fiber; (d) Schwann cell nuclei substantially displaced from the central point of myelin internodes; and (e) occasional regions of incomplete ensheathment of unmyelinated nerve fibers. In discussing these observations, we present arguments that the dy nerve lesion may be explained by the presence of an abnormality in the extracellular matrix of the peripheral nerve tissues of the dy mouse.     

Schwann cells and peripheral nervous system myelin in the rat retina

Summary Within the retinal nerve fiber layer of a 6-week-old Sprague-Dawley rat, scattered aggregates of PNS myelinated axons have been found and described. We believe this is likely to represent a normal but rare phenomenon in the rat.     

The role of Schwann cells and basal lamina tubes in the regeneration of axons through long lengths of freeze-killed nerve grafts

The ability of long acellular nerve grafts to support axonal regeneration was examined using inbred rats. Grafts (40 mm long) of tibial/plantar nerves were used either as live grafts or after freeze-drying to render the grafts acellular. The grafts were sutured to the proximal stump of severed tibial nerves in host animals which were then killed 1-12 weeks later. Axons rapidly regenerated through the living grafts but only extended 10-20 mm into the acellular grafts. This distance was achieved by 6 weeks and thereafter no significant further axonal extension occurred in the acellular grafts. A few naked axons lacking Schwann cell contact were identified in all acellular grafts, but became more numerous near the distal extent of axonal penetration into 6-12 week grafts. These axons contained large numbers of neurofilaments. When the distal 20 mm of 6 week acellular grafts (segments into which axons had not penetrated) were sutured to freshly severed tibial nerves, axons grew readily into the grafted tissue to a maximum distance of 9 mm. It is therefore likely that the limits to axonal regeneration through initially acellular grafts were set by factors intrinsic to the severed nerve. It is suggested that the limited migratory powers of Schwann cells may be one such factor. The concept that basal lamina tubes are not essential for axonal regeneration but may act as low resistance pathways for both axonal elongation and Schwann cell migration is discussed.     

Studies on the effects of altered PMP22 expression during myelination in vitro

Severe inherited dysmyelinating diseases of the peripheral nervous system, the Charcot-Marie-Tooth type1A disease (CMT1A) and the hereditary neuropathy with liability to pressure palsies (HNPP) are associated with a large DNA duplication or deletion of a chromosomal region containing the peripheral myelin protein 22 (PMP22) gene. It has been suggested that a gene dosage effect involving PMP22 is responsible for the pathological phenotype. We investigated if altered PMP22 expression affects the onset of myelin formation and the ultrastructure of myelin. Rat Schwann cell cultures were stably infected with recombinant retrovirus vectors harboring the rat PMP22 cDNA in sense or antisense orientation. Schwann cells over- or underexpressing PMP22 were cocultured with purified DRG neurons under conditions that promote myelination. We examined PMP22 expression and localization in the myelin forming cultures by RT-PCR, immunohistochemistry and confocal microscopy, and we analyzed myelin ultrastructure by electron microscopy. Our results demonstrate that abnormal levels of PMP22 expression do not impair the early stages of myelination and membrane compaction and do not interfere with the expression of other myelin genes. Our observations further indicate that PMP22 is involved more in controlling myelin thickness and stability than in the events determining the initial steps of myelin formation. J. Neurosci. Res. 48:31–42, 1997. © 1977 Wiley-Liss, Inc.     

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.     

Disappearance of the post-lesional laminin immunopositivity of brain vessels is parallel with the formation of gliovascular junctions and common basal lamina. A double-labelling immunohistochemical study

Previous studies revealed that during development the laminin immunopositivity gradually disappeared from the brain vessels, but temporarily re-appeared in them around lesions. The question of the present study was the correlation between the post-lesional vascular immunopositivity to laminin and the glial reaction. Following stab wounds, double fluorescent immunohistochemical labelling was performed against laminin (using a polyclonal antiserum against laminin 1) and glial fibrillary acidic protein. A number of vessels exhibited intense immunopositivity to laminin within the lesioned tissue. Where these laminin immunopositive vessels entered the perilesional brain substance, the astroglia formed contacts on them, and the separate vascular and glial basal laminae fused. The disappearance of the post-lesional laminin immunopositivity seemed to coincide with these phenomena. When monoclonal antibodies were applied against the beta1 and gamma1 laminin chains, vessels proved to be immunopositive at the lesion, but none in the intact brain tissue. No immunoreactivity was detected in the cases of alpha2 and beta2 chains. The results suggest that the disappearance of laminin immunopositivity may be attributed to that the epitopes become inaccessible for antibodies owing to the formation of gliovascular junctions and common basal lamina between astroglia and vessel. The possible role of an alteration in the laminin composition and the effect of fixation are discussed.     

Role for the oligodendrocyte cytoskeleton in myelination

Enriched cultures of rat brain oligodendrocytes were extracted with a buffer that separated the cells into a Triton X-100-soluble fraction and an insoluble cytoskeleton (CSK) residue. The buffer was optimised so that intact microtubules were preserved in the CSK residue. The partition of four myelin proteins between the soluble and the CSK fractions was determined by immunoblotting and immunofluorescence. Immunoblotting showed that two integral membrane proteins of myelin, the proteolipid protein (PLP) and the DM-20 protein, were completely extracted under these conditions. By contrast, a substantial amount of myelin basic protein (MBP) and to a lesser extent 2,3-cyclic nucleotide-3-phosphohydrolase (CNP) remained associated with the CSK residue. The association of these proteins with the CSK was confirmed by immunofluorescence. A remarkable difference in the distribution of microfilaments and microtubules was observed in oligodendrocytes. Immature cells possessed many fine processes that were rich in microfilaments. The cell body of these oligodendrocytes was devoid of microfilaments but did contain microtubules. Furthermore, a close association between CNP and microfilaments and between MBP and microtubules was revealed after detergent lysis. The strong interaction between CNP and filamentous actin was underlined by their concomitant disappearance from the extremities of the cell at a later stage of development when extensive membrane sheets had formed. Mature cells had fewer, thicker processes than younger cells and their processes contained microtubules, not microfilaments. MBP was present throughout the thick processes and the membrane sheets. These observations suggest roles for CNP and MBP at distinct stages of myelin process formation and support a directive role for the oligodendrocyte's CSK in the formation of myelin.     

Endogenous BDNF is required for myelination and regeneration of injured sciatic nerve in rodents

Following a peripheral nerve injury, brain-derived neurotrophic factor (BDNF) and the p75 neurotrophin receptor are upregulated in Schwann cells of the Wallerian degenerating nerves. However, it is not known whether the endogenous BDNF is critical for the functions of Schwann cells and regeneration of injured nerve. Treatment with BDNF antibody was shown to retard the length of the regenerated nerve from injury site by 24%. Histological and ultrastructural examination showed that the number and density of myelinated axons in the distal side of the lesion in the antibody-treated mice was reduced by 83%. In the BDNF antibody-treated animals, there were only distorted and disorganized myelinated fibres in the injured nerve where abnormal Schwann cells and phagocytes were present. As a result of nerve degeneration in BDNF antibody-treated animals, subcellular organelles, such as mitochondria, disappeared or were disorganized and the laminal layers of the myelin sheath were loosened, separated or collapsed. Our in situ hybridization revealed that BDNF mRNA was expressed in Schwann cells in the distal segment of lesioned nerve and in the denervated muscle fibres. These results indicate that Schwann cells and muscle fibres may contribute to the sources of BDNF during regeneration and that the deprivation of endogenous BDNF results in an impairment in regeneration and myelination of regenerating axons. It is concluded that endogenous BDNF is required for peripheral nerve regeneration and remyelination after injury.     

Specific inhibition of secreted NRG1 types I–II by heparin enhances Schwann Cell myelination

Primary cultures of mixed neuron and Schwann cells prepared from dorsal root ganglia (DRG) are extensively used as a model to study myelination. These dissociated DRG cultures have the particular advantage of bypassing the difficulty in purifying mouse Schwann cells, which is often required when using mutant mice. However, the drawback of this experimental system is that it yields low amounts of myelin. Here we report a simple and efficient method to enhance myelination in vitro. We show that the addition of heparin or low molecular weight heparin to mixed DRG cultures markedly increases Schwann cells myelination. The myelin promoting activity of heparin results from specific inhibition of the soluble immunoglobulin (Ig)‐containing isoforms of neuregulin 1 (i.e., NRG1 types I and II) that negatively regulates myelination. Heparin supplement provides a robust and reproducible method to increase myelination in a simple and commonly used culture system. GLIA 2016;64:1227–1234