Rat Sciatic Nerve Schwann Cells Research Articles

Nylon as an in vitro scaffold for three-dimensional study of neural cells.

The requirement for improved modeling of cells in culture for study of cell-to-cell interactions has led to an increased focus on three-dimensional (3D) in vitro models. In this study, NG108-15 neural cells and rat sciatic nerve Schwann cells were successfully grown as monocultures and as a coculture on the biocompatible nylon mesh substrates. This has allowed proliferation and interactions between the two cell types to be monitored on the mesh over time. Electrophysiological recordings validated the NG108-15 neural cell differentiation which confirmed the similarity of recorded action potentials in two-dimensional culture environments. A variety of assays have been used, demonstrating the use of nylon as an appropriate substrate for neural cell in vitro studies, and nylon can easily be imaged using inverted microscopy. These studies confirm the suitability of the nylon 3D culture presented for modeling neural cell behavior. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1575-1584, 2018.

Immunohistochemical localization of the sigma 1 receptor in Schwann cells of rat sciatic nerve

The sigma-1 (σ 1) receptors can bind different psychotropic drugs and have been implicated in schizophrenia, depression and dementia. The cloning of the σ 1-receptor has allowed to obtain specific antibodies and, in a recent immunohistochemical study, we demonstrated that, in addition to neurons, the σ 1-receptor is located in oligodendrocytes [Brain Res. 961 (2003) 92.]. In the present study using in vivo and in vitro techniques, we demonstrate the localization of the σ 1-receptor in Schwann cells. Double immunofluorescence studies showed that σ 1-receptor co-localized with S100 protein, a specific marker of Schwann cells, in both rat sciatic nerve Schwann cells and Schwann cells in cultures. The σ 1-receptor immunoreactivity was seen in the cytoplasm and paranodal region formed by these cells, but not in myelin itself. The presence of σ 1-receptor in oligodendrocytes and Schwann cells is discussed on the basis on recent findings involving this receptor in lipid metabolism, compartmentalization and transport to the plasma membrane, thus suggesting a role for σ 1-receptor signaling in myelination.

Developmental increase of aspartoacylase in oligodendrocytes parallels CNS myelination

Canavan disease, an autosomal-recessive neurogenetic disorder, is caused by mutations in aspartoacylase, an enzyme that deacetylates N-acetylaspartate to generate free acetate in the brain. Earlier studies have shown that aspartoacylase is primarily restricted to myelin synthesizing cells (oligodendroglia) in the CNS. These findings have led us to investigate the developmental expression of aspartoacylase gene in the rat brain in an attempt to shed more light on the role of this enzyme in myelination. In situ hybridization using a 35S riboprobe based on murine aspartoacylase cDNA was used in this study. The probe hybridized mostly to the white matter tracts with different densities depending on the age of the animal and region of the brain examined. Little or no hybridization signals were detected in the 1-day-old rats, whereas the signal was clearly detectable in most of the white matter regions of the CNS in the 11-day-old rats. The signal density markedly increased at postnatal day 17, the peak of myelination. Thereafter, the hybridization signals decreased somewhat but still could be observed in the adult animals. Thus, the developmental expression pattern of aspartoacylase gene in the postnatal brain closely parallels myelination in the CNS. In the CNS, the hybridization signal of ASPA appeared to be restricted primarily to oligodendrocytes, the primary myelin synthesizing cell type in the CNS. However, the signal was not detectable in rat sciatic nerve (Schwann cells) of the peripheral nervous system. These findings indicate that the role of N-acetylaspartate in myelin synthesis is restricted to the CNS. Furthermore, they provide additional support for the acetate deficiency hypothesis of Canavan disease and also make a stronger case for acetate supplementation as an immediate and inexpensive therapy for Canavan disease.

Expression of the neurofibromatosis 2 tumor suppressor gene product, merlin, in Schwann cells.

The neurofibromatosis 2 (NF2) tumor suppressor gene encodes a protein with sequence similarity to a family of molecules linking cell membrane proteins to the actin cytoskeleton. Individuals affected with NF2 develop schwannomas at an increased frequency. In this paper, we report that merlin is expressed in Schwann cells, where it is localized in vitro to the cell membrane by immunohistochemistry and subcellular fractionation. Exogenous expression of merlin fragments confirms this subcellular distribution and suggests that both the N-terminal and C-terminal portions of the molecule are required for this localization. In addition, merlin is expressed in rat sciatic nerve Schwann cells at paranodal membranes, where it colocalizes with RhoA. Lastly, expression of the NF2 gene increases during postnatal rat sciatic nerve development, consistent with its role as a negative growth regulator for Schwann cells. These results collectively suggest that merlin may function at the cell surface to modulate cell growth in Schwann cells and to link cell membrane proteins to the cytoskeleton.

Melanocortin analogue Org2766 binds to rat Schwann cells, upregulates NGF low-affinity receptor p75, and releases neurotrophic activity

Binding of the stable melanocortin(4-9) analogue, Org2766 [Met(O2)-Glu-His-Phe-D-Lys-Phe] to cultured rat sciatic nerve Schwann cells was demonstrated using a biotinylated derivative in semiquantitative histochemical and CELISA assays. Org2766 bound to Schwann cells, but not to fibroblasts, and was displaced maximally by unlabeled Org2766, alpha-MSH and ACTH(1-24). Displacement of Org2766 from the binding sites was considerably reduced by N- and C-truncation of the peptide. Specific binding of Org2766 was also demonstrated in the immortal rat Schwann cell line SCL4.1/F7 and was more pronounced in cells displaying a differentiated morphology. Org2766 and alpha-MSH increased cyclic AMP content of Schwann cells but neither stimulated DNA synthesis when applied alone. However, in the presence of a priming (subthreshold) concentration of the mitogen, cholera toxin, Org2766 and alpha-MSH caused a delayed increase in DNA synthesis. Org2766 did not modulate the expression of several differentiation-related Schwann cell markers. However, Org2766 increased immunoreactivity for p75 low-affinity NGF receptor on Schwann cells and evoked the release of neurotrophic factor(s) that synergized with NGF in stimulating neurite outgrowth in rat DRG neurons. The results indicate that Schwann cells are a primary target for the action of Org2766 and provide evidence for an indirect mechanism by which melanocortins might stimulate neurite sprouting in regenerating peripheral nerve axons.

The beta 1 subunit mRNA of the rat brain Na+ channel is expressed in glial cells.

Although the molecular characteristics of glial Na+ channels are not well understood, recent studies have shown the presence of mRNA for rat brain Na+ channel alpha subunits in astrocytes and Schwann cells. In this study, we asked whether the mRNA for the rat brain Na+ channel beta 1 subunit is expressed in glial cells. We performed in situ hybridization using a complementary RNA probe for the coding regions of the rat brain Na+ channel beta 1 subunit mRNA and detected beta 1 subunit mRNA in cultured rat optic nerve astrocytes and sciatic nerve Schwann cells. The beta 1 subunit was amplified by reverse transcription-polymerase chain reaction in rat optic and sciatic nerves, which lack neuronal somata but contain astrocytes and Schwann cells, respectively. Doublet bands of the beta 1 subunit mRNA were amplified from both optic and sciatic nerves. Through the cloning and sequencing of these bands, we confirmed the amplification of a mRNA highly homologous to the previously cloned rat brain Na+ channel beta 1 subunit (beta 1.1) and a novel form of the beta 1 subunit mRNA (beta 1.2), which is closely homologous to beta 1.1 but contains an additional 86-nucleotide insert in 3' noncoding regions. Two beta 1 subunit mRNAs were also amplified from rat brain and skeletal muscle, but not from rat liver or kidney. These results indicate that rat brain Na+ channel beta 1 subunit mRNAs are expressed in glial cells as well as in neurons.

Characterization of a Schwann cell neurite‐promoting activity that directs motoneuron axon outgrowth

Schwann cells support and facilitate axonal growth during development and successful regeneration in the peripheral nerve. In the regenerating rat sciatic nerve, Schwann cells provide a trophic milieu for primary sensory, sympathetic, and motoneurons. We have characterized a neurotrophic activity produced by adult rat sciatic nerve Schwann cells and a spontaneously immortal Schwann cell clone (iSC). This activity elicits neurite outgrowth from chick embryo explants of both CNS and PNS. The iSC activity has been concentrated by cation-exchange chromatography and compared to known neurotrophins in bioassay. Pooled bound fractions elicit neurite outgrowth from sympathetic, ciliary and motoneurons. In collagen matrix cocultures of iSC and E4 ventral horn (before motor axon extension to muscle targets), the iSC activity can direct the initial axonal extension from motoneurons. The data presented suggest that Schwann cell-produced activity may mediate motoneuron axonal extension before contact with their peripheral source of neurotrophin.

Effects of acrylamide on subcellular distribution of elements in rat sciatic nerve myelinated axons and Schwann cells

Electron probe X-ray microanalysis was used to determine whether experimental acrylamide (ACR) neuropathy involves deregulation of subcellular elements (Na, P, S, Cl, K, Ca and Mg) and water in Schwann cells and small, medium and large diameter myelinated axons of rat sciatic nerve. Results show that in proximal but not distal sciatic nerve, ACR treatment (2.8 mM in drinking water) was associated with an early (15 days of exposure), moderate increase in mean axoplasmic K concentrations (mmol/kg) of medium and small diameter fibers. However, all axons in proximal and distal nerve regions displayed small increases in dry and wet weight contents of axoplasmic Na and P. As ACR treatment progressed (up to 60 days of exposure), Na and P changes persisted whereas proximal axonal K levels returned to control values or below. Alterations in mitochondrial elemental content paralleled those occurring in axoplasm. Schwann cells in distal sciatic nerve exhibited a progressive loss of K, Mg and P and an increase in Na, Cl and Ca. Proximal glia displayed less extensive elemental modifications. Elemental changes observed in axons are not typical of those associated with cell injury and might reflect compensatory or secondary responses. In contrast, distal Schwann cell alterations are consistent with injury, but whether these changes represent primary or secondary mechanisms remains to be determined.

Mitogenic response of rat Schwann cells to fibroblast growth factors is potentiated by increased intracellular cyclic AMP levels.

Rat sciatic nerve Schwann cells either do not proliferate, or proliferate very slowly, in medium containing 10% fetal bovine serum (FBS). They were previously shown to respond only to a limited number of mitogens associated with cells of central and peripheral nervous systems, which appeared to be distinct from FGFs and PDGF, and to agents that raise intracellular cAMP levels. In a basal medium consisting of 75% DMEM, 25% Ham's F-12, 5 nM sodium selenite, 50 microM 2-amino ethanol, and 2 mM histidine, supplemented with 5% FBS, we showed that aFGF, bFGF, and PDGF were all capable of stimulating Schwann cell growth and the stimulation was greatly potentiated by forskolin and dibutyryl-cAMP. In addition, pretreating culture surface with purified matrix proteins such as laminin, fibronectin, or type 1 collagen, was necessary for obtaining a better cellular response to the mitogenesis of these growth factors even in 10% FBS. Our results clearly indicated that providing a suitable medium and substratum, aFGF, bFGF and PDGF are mitogens for rat sciatic nerve Schwann cells in medium with and without forskolin or dibutyryl-cAMP.

Determination of the membrane potential of cultured mammalian schwann cells and its sensitivity to potassium using a thiocarbocyanine fluorescent dye

The membrane potential of cultured rat sciatic nerve Schwann cells was determined with conventional microelectrode and voltage-sensitive fluorescent dye, Di-S-C3(5), optical techniques. The value for membrane potential obtained with microelectrodes was -42.1 +/- 4.7 mV (n = 8). Using optically determined fluorescent intensity changes caused by changes in external potassium ion concentration, in the presence or absence of valinomycin (null point method), the membrane potential was estimated at -45.7 +/- 6.2 mV (n = 7); with a gramicidin and valinomycin double ionophore method it was -52.2 +/- 9.1 (n = 4). The membrane potential of Schwann cells was found to be potassium sensitive at and above the physiological range of [K+] at 27.5 mV/10x delta[K+], which is approximately half the Nernstian value. This result suggests that other ion permeabilities strongly influence the resting membrane potential of cultured Schwann cells. Since Na+ had little effect on the membrane potential, it is concluded that Cl- is a likely candidate for the other permeant ionic species. The optical method has been shown to be a useful tool for the systematic study of the membrane potential of Schwann cells in culture and for the characterization of its ionic basis and regulation.

Schwann cell proliferation in vitro is under negative autocrine control.

In healthy adult peripheral nerve, Schwann cells are believed to be generally quiescent. Similarly, cultures of isolated rat sciatic nerve Schwann cells hardly proliferate in serum-supplemented medium. The possibility that Schwann cells negatively regulate their own proliferation was supported by the demonstration that conditioned media from Schwann cell cultures inhibited the proliferation of mitogen-stimulated test cultures. The inhibition could be complete, was dose dependent, and was exhibited when the test Schwann cells were under the influence of different types of mitogens such as cholera toxin, laminin, and living neurons. The inhibition of proliferation was completely reversible and a rapid doubling of cell number resulted when treatment with conditioned medium was withdrawn from mitogen-stimulated Schwann cells. Conditioned medium from cholera toxin-stimulated and immortalized Schwann cell cultures contained less antiproliferative activity than that found in medium from quiescent Schwann cell cultures. However, media conditioned by two actively proliferating rat Schwannoma cell lines were rich sources of antiproliferative activity for Schwann cells. Unlike the mitogen-stimulated Schwann cells, whose proliferation could be inhibited completely, the immortalized and transformed Schwann cell types were nearly unresponsive to the antiproliferative activity. The antiproliferative activity in Schwann and Schwannoma cell conditioned media was submitted to gel filtration and SDS-PAGE. The activity exists in at least two distinct forms: (a) a high molecular weight complex with an apparent molecular mass greater than 1,000 kD, and (b) a lower molecular weight form having a molecular mass of 55 kD. The active 55-kD form could be derived from the high molecular weight form by gel filtration performed under dissociating conditions. The 55-kD form was further purified to electrophoretic homogeneity. These results suggest that Schwann cells produce an autocrine factor, which we designate as a "neural antiproliferative protein," which completely inhibits the in vitro proliferation of Schwann cells but not that of immortalized Schwann cells or Schwannoma lines.

Identification of a lipid-anchored heparan sulfate proteoglycan in Schwann cells.

Schwann cells synthesize both hydrophobic and peripheral cell surface heparan sulfate proteoglycans (HSPGs). Previous analysis of the kinetics of radiolabeling suggested the peripheral HSPGs are derived from the membrane-anchored forms (Carey, D., and D. Evans. 1989. J. Cell Biol. 108:1891-1897). Peripheral cell surface HSPGs were purified from phytic acid extracts of cultured neonatal rat sciatic nerve Schwann cells by anion exchange, gel filtration, and laminin-affinity chromatography. Approximately 250 micrograms of HSPG protein was obtained from 2 X 10(9) cells with an estimated recovery of 23% and an overall purification of approximately 2000-fold. SDS-PAGE analysis indicated the absence of non-HSPG proteins in the purified material. Analysis of heparinase digestion products revealed the presence of at least six core protein species ranging in molecular weight from 57,000 to 185,000. The purified HSPGs were used to produce polyclonal antisera in rabbits. The antisera immunoprecipitated a subpopulation of 35SO4-labeled HSPGs that were released from Schwann cells by incubation in medium containing phosphatidylinositol-specific phospholipase C (PI-PLC); smaller amounts of immunoprecipated HSPGs were also present in phytic acid extracts. In the presence of excess unlabeled PI-PLC-released proteins, immunoprecipitation of phytic acid-solubilized HSPGs was inhibited. SDS-PAGE analysis of proteins immunoprecipitated from extracts of [35S]methionine labeled Schwann cells demonstrated that the antisera precipitated an HSPG species that was present in the pool of proteins released by PI-PLC, with smaller amounts present in phytic acid extracts. Nitrous acid degradation of the immunoprecipitated proteins produced a single 67,000-Mr core protein. When used for indirect immunofluorescence labeling, the antisera stained the external surface of cultured Schwann cells. Preincubation of the cultures in medium containing PI-PLC but not phytic acid significantly reduced the cell surface staining. The antisera stained the outer ring of Schwann cell membrane in sections of adult rat sciatic nerve but did not stain myelin or axonal membranes. This localization suggests the HSPG may play a role in binding the Schwann cell plasma membrane to the adjacent basement membrane surrounding the individual axon-Schwann cell units.

Platelet-derived growth factors and fibroblast growth factors are mitogens for rat Schwann cells.

Rat sciatic nerve Schwann cells in culture respond to a limited range of mitogens, including glial growth factor, transforming growth factors beta-1 and beta-2 (TGF-beta 1, TGF-beta 2), some cell membrane-associated factors, and to agents such as cholera toxin and forskolin which raise intracellular levels of cAMP. These responses require the presence of FCS, which exhibits little or no mitogenic activity in the absence of other factors. However, we recently found that forskolin greatly potentiates the mitogenic signal from TGFs-beta 1 and beta 2, raising the possibility that cAMP might couple other factors to mitogenesis. We have therefore screened a range of candidate mitogens using DNA synthesis assays. Other than TGFs-beta and glial growth factor, none of the factors tested were mitogenic in the presence of 10% serum alone. With the addition of forskolin, however, porcine PDGF, human PDGF, acidic and basic FGF were potent mitogens for rat Schwann cells, stimulating DNA synthesis and increasing cell number. Cholera toxin and dibutyrylcyclicAMP, but not 1,9-dideoxyforskolin, can substitute for forskolin indicating that the mitogenic effect is mediated via adenylyl cyclase activation. Porcine PDGF gave half-maximal stimulation at 15 pM, and human PGDF an equivalent response at 1 nM. Basic FGF was half maximal at 5 pM, acidic FGF at 1 nM. The recognition of PDGFs and FGFs as mitogens for Schwann cells has many implications for the study of Schwann cell proliferation in the development and regeneration of nerves, and in Schwann cell tumorigenesis.

Rat sciatic nerve Schwann cell microcultures: responses to mitogens and production of trophic and neurite-promoting factors.

During embryonic development and in response to injury, the growing axons of peripheral neurons may influence the migration and proliferation of Schwann cells which, in return, may present neurons with a critical supply of factors required for neuronal survival, growth and differentiation. The identification and characterization of agents influencing the proliferation of Schwann cells as well as Schwann cell production of factors affecting neurons is greatly facilitated by the use of in vitro techniques. We describe here a simplified method of obtaining large numbers of purified neonatal rat sciatic nerve Schwann cells for use in generating large numbers of replicate microcultures. We then illustrate the use of these microcultures to examine Schwann cell: i) morphology and survival; ii) proliferation; and iii) production of neuronotrophic and neurite-promoting activities. We report that rat Schwann cells in microculture proliferate in response to serum, laminin and fibronectin, cholera toxin, and chick embryo parasympathetic ciliary neurons. Also, extracts of Schwann cell microcultures contain independently regulated activities which support the survival and neurite outgrowth of peripheral ganglionic neurons.

Regulation of Myelin P0 Glycoprotein Synthesis in Cultured Rat Schwann Cells and Continuous Rat PNS Cell Lines

We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2'-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyryl cyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.

Myelin‐specific proteolipid protein is expressed in myelinating schwann cells but is not incorporated into myelin sheaths

Contrary to widely held beliefs, the gene encoding proteolipid protein (PLP), the major structural protein of central nervous system myelin, is expressed in Schwann cells and their tumors. Proteolipid mRNA was identified in human acoustic neuromas and in rat and rabbit sciatic nerves using a human PLP cDNA as a probe. Proteolipid protein itself was shown to be present in human and rat sciatic nerve Schwann cells by immunofluorescence microscopy and by Western blot analysis using antisera raised to a synthetic PLP polypeptide. Although easily detected in the Schwann cell body, PLP was not detected in the peripheral myelin itself, suggesting that the PLP is preferentially excluded from this portion of the Schwann cell membrane.

Schwann cells stimulated to proliferate in the absence of neurons retain full functional capability

Schwann cells from neonatal rat sciatic nerve can be maintained and grown in culture in the absence of neurons. We are interested in substantially expanding such cultures for use in the study of Schwann cells, their growth responses, and their interactions with neurons. However, it was important to determine if expanded cell populations retained their distinguishing biological properties and their ability to differentiate when recombined with neurons. Therefore, we have compared the functional properties of extensively expanded populations of sciatic nerve Schwann cells to those of embryonic dorsal root ganglion (DRG) Schwann cells that had been briefly expanded in vitro in the continuous presence of ganglion neurons. Sciatic nerve Schwann cells were cultured and purified according to the methods of Brockes et al. (1979). A combination of crude glial growth factor and forskolin was found to act synergistically in providing maximal stimulation of Schwann cell proliferation. Sciatic nerve Schwann cells that were continuously expanded for at least 2 months were compared to Schwann cells derived from fetal dorsal root ganglia. The results indicate that the complement of secreted proteins from both cell populations, either in isolation or recombined with neurons, was essentially identical; both cell populations expressed the cell-surface antigens laminin and Ran 1 (217C antibody); after seeding onto DRG neurons, both cell populations associated with neuronal processes with the same time course; and under identical nutrient conditions, both cell populations were observed to exhibit a comparable capacity for myelination of DRG axons in vitro. Thus, methods used to establish primary cultures of rat sciatic nerve Schwann cells and to expand secondary cultures in vitro in the absence of neurons preserve basic Schwann cell functions.

Tissue Culture Studies of Schwann Cell Proliferation and Differentiation

Neonatal rat sciatic nerve Schwann cells in monolayer culture are stimulated to proliferate and to express a lipid and a protein characteristic of myelin by agents which raise intracellular cyclic adenosine 3',5'-monophosphate. Both glial growth factor and axolemmal fragments increase the rate of mitosis of cultured rat and human Schwann cells. Rat Schwann cell mitosis is enhanced by a soluble factor produced by concanavalin A-stimulated blood mononuclear cells and inhibited by lead salts. Schwann-like cells cultured from human dermal and plexiform neurofibromas resemble normal human Schwann cells in phenotype and response to mitogens.

Ultrastructure and immunocytochemistry of rat Schwann cells and fibroblasts in vitro

The ultrastructure of dissociated rat sciatic nerve Schwann cells and fibroblasts has been examined and correlated at the morphologic and immunocytochemical levels. In agreement with previous studies, the two cell types were readily distinguishable morphologically on the basis of cell shape and size but no unique structural feature could be found. Schwann cells in this system essentially lacked basal laminae but did show some membrane specializations reminiscent of the in vivo situation. Fibroblasts were exceedingly rich in actin webs beneath the plasmalemma. Immunocytochemically, only Schwann cells stained with anti-Ran-1 antisera and fibroblasts only with anti-Thy-1. Both staining patterns were exclusively associated with the surface membrane. The findings provide a basis for future studies with this system to which neuronal elements may be added in an attempt to initiate myelinative events.