Axolemma is a mitogen for human Schwann cells

The mechanisms responsible for the induction of Schwann cell proliferation in peripheral nerves undergoing wallerian degeneration and segmental demyelination are not understood. To determine whether contact with axolemma stimulates mitosis of human Schwann cells, cultured Schwann cells from spinal roots obtained postmortem and from sural nerve biopsy specimens were incubated with axolemmal fractions prepared from human spinal cord or from adult rat central nervous system. Schwann cell proliferation was estimated by autoradiographic assay of tritiated thymidine incorporation. Schwann cell labeling indices after exposure to human or rat axolemmal fractions ranged from 26.7 to 59.9%; labeling indices of Schwann cells cultured without axolemmal fraction were 9.8 to 22.4%. The stimulation index, or ratio of Schwann cell labeling index with axolemmal fraction to that without axolemmal fraction, ranged from 1.97 to 3.40. This study demonstrates that both human and rat axolemma are capable of stimulating human Schwann cell replication in vitro.     

Specific and potent mitogenic effect of axolemmal fraction on schwann cells from rat sciatic nerves in serum-containing and defined media

Using cultures of Schwann cells from neonatal rat sciatic nerves, we examined the mitogenic activity of an axolemmal fraction from adult rat CNS. Axolemmal fraction proved a potent mitogen, stimulating [³H]thymidine incorporation into Schwann cell DNA 13.5 fold over control values when axolemmal fraction equivalent to 16 μg of protein per culture microwell or more was added. Half maximal stimulation was obtained with addition of axolemmal fraction equivalent to 4 μg of protein. The concentration-dependence and magnitude of the mitogenic response of the cultured cells were nearly identical whether they were maintained in vitro for 1 day or for 2 weeks prior to addition of the axolemmal fraction. A study of the time-course of the effect of axolemmal fraction on Schwann cell mitosis showed that maximal [³H]thymidine incorporation took place during the fifth day after addition of axolemmal fraction. Axolemmal fraction also produced stimulation of [³H]thymidine incorporation into Schwann cells, seeded and cultured in a serum-free defined medium. Though the concentration-dependence of the mitogenic effect in the absence of serum was similar to that in a serum-containing medium, maximal stimulation in the defined medium was only 2.8-fold. The mitogenic activity of axolemmal fraction was rapidly and almost totally inactivated by sonication or homogenization, and was partially lost after exposure to heat. The mitogenic activities of plasma membrane fragments from rat skeletal muscle or rat erythrocytes, andof mitochondrial fragments (the major contaminant of the axolemmal fraction) were one-tenth that of axolemmal fraction or less. In contrast to glial growth factor prepared from bovine pituitaries (GGF-BP), which stimulates proliferation of both fibroblasts and Schwann cells, axolemmal fraction induced proliferation of Schwann cells but not of endoneurial fibroblasts; cultures treated with axolemmal fraction demonstrated a 3-fold increase in Schwann cell population in 10 days without detectable increase in number of fibroblasts. Also in contrast to GGF-BP, the mitogenic effect of which is considerably enhanced by simultaneous addition of cholera toxin to the medium, cholera toxin had no effect on the Schwann cell proliferative response to axolemmal fraction.     

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.     

Gangliosides modulate Schwann cell proliferation and morphology

We examined the effect of gangliosides on Schwann cell cultures isolated from neonatal rat sciatic nerves. Addition of gangliosides (GM1, GM3, and ganglioside mixture) at concentrations between 0.25 and 2 mg/ml significantly diminished both the baseline rate of proliferation of the Schwann cells and their response to two types of mitogens, the axolemmal fragments and derivatives of adenosine 3'-5'-monophosphate (cAMP). Gangliosides, the sialic acid residue of which had been removed, were highly toxic to the Schwann cells, which went to indicate that sialic acid is necessary to produce the inhibitory effects. Gangliosides also produced prominent changes in the morphological appearance of the Schwann cells. Most of the Schwann cells treated with gangliosides had an elongated shape with long processes and an alignment of end-to-end or side-by-side cell adhesion. These effects of gangliosides apparently were not mediated by cAMP, since intracellular cyclic adenosine monophosphate (cAMP) of Schwann cells at a basal- and forskolin-stimulated level was not altered by the exogenous gangliosides. These findings indicate that the direct effect of gangliosides on Schwann cells should also be considered as a background mechanism of ganglioside-induced facilitation of neuronal regeneration.     

Regulation of glucose transport in cultured Schwann cells

Glucose is the major source of metabolic energy in the peripheral nerve. Energy derived from glucose is mostly utilized for axonal repolarization. One route by which glucose may reach the axon is by crossing the Schwann cells that initially surround the axons. Considering the ability of neurons to control many glial cell functions, we postulated that Schwann cell glucose transporters might be transiently regulated by axonal contact. Glucose transport was studied in a cultured, differentiated rat Schwann cell line stably expressing SV40 T antigen regulated by a synthetic mouse metallothionein promoter. 3[H]-2-deoxy-D-glucose uptake was measured in cultured cells in basal and in various experimental conditions. Glucose transporter gene expression was determined after RNA isolation from cultured cells through Northern and RNAse protection assay. In vitro, Schwann cells were found to express high-affinity, insulin-insensitive, facilitative glucose transporters and predominantly GLUT1 mRNA. Schwann cell 2-deoxyglucose uptake was increased by axolemmal membranes or forskolin but unchanged by elevated glucose levels. Regulation of Schwann cell glucose transporters by axolemma and their resistance to glucose-induced down-regulation suggest extrinsic rather than intrinsic regulation that might enhance Schwann cell vulnerability to glucotoxicity.     

Evidence that the axolemmal mitogen for cultured Schwann cells is a positively charged, heparan sulfate proteoglycan-bound, heparin-displaceable molecule

Treatment of axolemma with pH 9 buffer results in a pellet enriched two-fold in the mitogen for cultured Schwann cells. Heparitinase treatment releases 8% of the mitogen into solution, while heparin selectively solubilizes the mitogen, resulting in an extract which has a specific mitogenic activity approximately 2.5 times greater than the mitogenicity of the starting axolemma membrane. These data support a model in which the axolemmal mitogen is a positively charged molecule associated with negatively charged sulfated proteoglycans.     

Experimental lead neuropathy: inorganic lead inhibits proliferation but not differentiation of Schwann cells

Schwann cells were prepared from the sciatic nerves of newborn rats and cultured in a monolayer. Addition of lead acetate at concentrations between 0.4 and 10.0 micrograms/ml, levels comparable to those occurring in neural tissues and physiological fluids of lead-intoxicated rats, diminished both the baseline rate of proliferation of the Schwann cells and their response to the mitogens, axolemmal fragments, glial growth factor, and the adenosine 3':5'-cyclic monophosphate (cAMP) analogues 8-bromo-cAMP and dibutyryl-cAMP. This demonstrates a direct toxic effect of inorganic lead on Schwann cells. Lead acetate in this concentration range did not, however, inhibit the cAMP analogue-induced appearance of the "myelin marker" lipid galactocerebroside on the surfaces of the cultured Schwann cells.     

Evidence for the colocalization of the axonal mitogen for Schwann cells and oligodendrocytes

Axons that normally will encounter either CNS or PNS glia have been shown to contain a powerful mitogen for both Schwann cells and oligodendrocytes. The normally nonmyelinated, nonglial ensheathed cerebellar granule cells have been shown to possess a proliferative signal for Schwann cells, suggesting that a glial mitogen is common to all axons. To determine if a glial mitogen capable of stimulating both Schwann cells and oligodendrocytes is colocalized on all types of axons we have (1) cocultured granule cells with oligodendrocytes, (2) incubated oligodendrocytes with granule cell membranes, and (3) evaluated the ability of heparin extracts of granule cell membranes, splenic nerve microsomes, and axolemma-enriched fractions isolated from rat and bovine CNS to stimulate mitosis of cultured oligodendrocytes. Neither the intact granule cells nor the granule cell membrane fraction stimulated cultured oligodendrocytes to divide. However, heparin extracts of the granule cell membranes were significantly mitogenic to the cultured oligodendrocytes. Heparin extracts of splenic nerve microsomes were more mitogenic than the comparable extract obtained from bovine CNS axolemma-enriched fractions. These results suggest that the neuronal mitogen for oligodendroglia is colocalized with the neuronal mitogen for Schwann cells.     

Enrichment of Schwann cell cultures from neonatal rat sciatic nerve by differential adhesion

A novel method of Schwann cell purification from neonatal rat sciatic nerve has been developed using differential adhesion. After enzymatic and mechanical dissociation, the cell digest is allowed to settle on polylysine-coated glass coverslips for 30 min with intermittent shaking. After an 18-h incubation, bipolar cells comprise 95% of the non-adherent population. Indirect immunofluorescence with the cell-specific markers rabbit anti-galactocerebroside and rabbit anti-bovinr-P-2 basic protein antiserum confirmed light microscopic identification of these bipolar cells as Schwann cells. Rabbit anti-human fibronectin specifically labeled fibroblasts which comprised< 5% of the cell population, but did not bind to Schwann cells. Schwann cells isolated by differential adhesion were injected into a rabbit. When absorbed with cultured rat skin fibroblasts, serum from this rabbit specifically surface labeled 99% of the bipolar and round cells after 18 h and 5 days in vitro and also labeled Schwann cells in fetal rat dorsal root ganglia cultures, but not fibroblasts or neurons.     

Platelet-derived growth factor and regulation of Schwann cell proliferation in vivo.

To examine the role of platelet-derived growth factor (PDGF) in the in vivo regulation of Schwann cell proliferation, steady-state levels of mRNAs encoding PDGF A and B chains, and PDGF alpha and beta receptors were measured in immature and adult rat sciatic nerves and in cultured rat Schwann cells. PDGF B chain and PDGF beta receptor mRNAs are present in immature rat sciatic nerves and to a lesser extent in adult rat nerves. Short-term cultures of neonatal rat Schwann cells express PDGF beta receptor mRNA, but not PDGF B chain mRNA, and are stimulated to synthesize DNA by addition of PDGF BB to the medium. These data indicate that PDGF BB is a developmentally regulated paracrine growth factor for rat Schwann cells. Very long-term cultures of rat Schwann cells, which have lost normal dependence on exogenous growth factors, express PDGF B chain mRNA as well as mRNAs encoding the PDGF alpha and beta receptors, suggesting that, under these circumstances, PDGF BB also act as an autocrine growth factor. PDGF A chain mRNA is present in both immature and adult rat sciatic nerves and is expressed by primary and secondary cultures of rat Schwann cells as well. However, because the abundance of PDGF alpha receptor mRNA is very low in rat Schwann cells, PDGF AA is not likely to be a significant autocrine growth factor for rat Schwann cells.     

Tissue culture studies of neurofibromatosis: effects of axolemmal fragments and cyclic adenosine 3',5'-monophosphate analogues on proliferation of Schwann-like and fibroblast-like neurofibroma cells

Six dermal neurofibromas obtained from 5 patients with neurofibromatosis were dissociated and the cells were plated on polylysine-coated glass. Two principal cell types were observed in the cultures: elongated and bipolar Schwann-like cells (SLCs), and polymorphic flattened fibroblast-like cells (FLCs). Indirect immunofluorescence demonstrated that SLCs expressed surface laminin but not surface fibronectin; FLCs expressed surface fibronectin but were only weakly positive for surface laminin. Tritiated thymidine autoradiography demonstrated that cultured SLCs proliferated slowly (labeling index, 0.7 to 4.0%), whereas FLCs divided more rapidly (labeling index, 7.5 to 26.4%). Axolemmal fragments prepared from human or rat central nervous system specimens adhered to SLCs derived from each of the 6 neurofibromas, but not to FLCs. Axolemmal fragments induced a marked proliferative response of SLCs from 2 of the 6 neurofibromas but had no effect on proliferation of SLCs from the other 4 neurofibromas or FLCs from any of the 6 neurofibromas. In one patient from whom 2 neurofibromas were obtained, SLCs from one neurofibroma responded to axolemmal fragments, while SLCs from the other did not. Treatment of the cultures with 0.1 mM cyclic adenosine 3'5'-monophosphate (cAMP) analogue, 8-bromo cAMP, caused marked inhibition of proliferation of both SLCs and FLCs derived from all 6 neurofibromas. The same concentration of another cAMP analogue, dibutyryl cAMP, inhibited proliferation of SLCs but not of FLCs.     

Schwann cell differentiation in vitro: extracellular matrix deposition and interaction

Neonatal rat Schwann cells isolated in culture proliferate slowly and do not form a basement membrane although they express laminin continuously. We demonstrate here that isolated Schwann cells express other basement membrane components, including entactin and heparan sulfate proteoglycan. Treatment with ascorbate, and to a lesser extent with cyclic adenosine 3',5'-monophosphate, modulates the synthesis of extracellular matrix components by cultured Schwann cells. After this treatment, fibronectin and collagen type IV are detected on the Schwann cell surface, which form, with the other components, a membrane-bound extracellular matrix. Electron microscopy shows that these elements are organized in a filamentous matrix which resembles a basement membrane and may be a precursor form of a basement membrane. We also show the effect of complete basement membrane matrices on Schwann cell behavior in culture. These matrices support Schwann cell proliferation in both serum-containing and serum-free media. The extracellular matrix from endothelial cells mimics fibronectin and induces a flat phenotype whereas the reconstituted basement membrane gel from the EHS tumor mimics laminin and allows an elongated phenotype. Thus, the basement membrane matrices interact with Schwann cells in vitro and may play an important role in Schwann cell proliferation in vivo.     

Characteristics of Na+ current in Schwann cells cultured from frog sciatic nerve

Characteristics of voltage-dependent currents in cultured frog Schwann cells were investigated by the whole-cell clamp technique. An inward current was detectable at a membrane potential level more positive than-50 mV and reached a maximum value at about-10 mV, while no rectifying channel was present. The inward current was carried by Na⁺ ions, because the extrapolated reversal potential of the current agreed with the calculated E_Na, and the current was sensitive to tetrodotoxin. The membrane potential for half-maximal inactivation was-82 mV. The inactivation curve indicated that more than 90% of the Na⁺ channels were inactivated at the resting membrane potential, suggesting that the cultured frog Schwann cells could not generate an action potential under physiological conditions. The time constant for the inactivation at a maximum current was 5.3 ms (-10 mV, 13°C). The electrophysiological characteristics of the Na⁺ current in the cultured frog Schwann cells were compared with those in other tissues. This Na⁺ current was quantitatively different from that observed in the amphibian node of Ranvier but was similar to that in the mammalian Schwann or glial cells, especially in the more hyperpolarized half-maximal inactivation potential and in the slower inactivation time course. © 1994 Wiley-Liss, Inc.     

A quantitative morphometric analysis of rat spinal cord remyelination following transplantation of allogenic Schwann cells

Quantitative morphometric techniques were used to assess the extent and pattern of remyelination produced by transplanting allogenic Schwann cells into demyelinated lesions in adult rat spinal cords. The effects of donor age, prior culturing of donor cells, prior lesioning of donor nerves, and host immunosuppression were evaluated by transplanting suspensions of 30,000 acutely dissociated or cultured Schwann cells from neonatal, young adult, or aged adult rat sciatic nerves into X-irradiation and ethidium bromide-induced demyelinated dorsal column lesions, with or without co-transplantation of neonatal optic nerve astrocytes. Three weeks after transplantation, spinal cords were processed for histological analysis. Under all Schwann cell transplant protocols, large areas containing many Schwann cell-like myelinated axon profiles could be readily observed throughout most of the lesion length. Within these "myelin-rich" regions, the vast majority of detectable axons showed a peripheral-like pattern of myelination. However, interaxonal spacing also increased, resulting in densities of myelinated axons that were more similar to peripheral nerve than intact dorsal columns. Freshly isolated Schwann cells remyelinated more axonal length than cultured Schwann cells, and cells from younger donors remyelinated slightly more axon length than cells from older donors, but all Schwann cell transplant protocols remyelinated tens of thousands of millimeters of axon length and remyelinated axons at similar densities. These results indicate that Schwann cells prepared under a variety of conditions are capable of eliciting remyelination, but that the density of remyelinated axons is much lower than the myelinated axon density in intact spinal cords.     

Astroglial proliferation and phenotype are modulated by neuronal plasma membrane

The rate of proliferation of rat astroglia cultured in a serum-free medium, estimated by tritiated thymidine radioautography, was diminished by more than 50% by addition of rat central nervous system axolemmal fragments to the culture medium. Addition of the axolemmal fragments also induced a phenotypic alteration of the cultured astroglia, from cells of irregular shape containing a fine meshwork of intracytoplasmic glial fibrils to star-shaped cells with thicker, cable-like glial fibrils.     

Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects

Tissue-engineering as laboratory based alternative to human autografts and allografts provides "custom made organs" cultured from patient's material. To overcome the limited donor nerve availability different biologic nerve grafts were engineered in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into acellular autologous matrices: veins, muscles, nerves, and epineurium tubes. Autologous nerve grafts, and the respective biogenic material without Schwann cells served as control. After 6 weeks regeneration was assessed clinically, histologically and morphometrically. The PCR analysis showed that the implanted Schwann cells remain within all the grafts. A good regeneration was noted in the muscle-Schwann cell-group, while regeneration quality in the other groups (with or without Schwann cells) was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. All venous and epineurium grafts had a more disorganized regeneration. Seemingly, the lack of endoneural tube like structures in vein grafts lead to impaired regeneration. And, apparently, the beneficial effects of implanted Schwann cells into a large luminal structure can only be demonstrated to a limited extent if endoneural like structures are lacking. A tube offers less area for Schwann cell adhesion and it is more likely to collapse. This underlines the role of the basal lamina, or at least an inner structure acting as scaffold in axonal regeneration. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biogenic graft with basal lamina tubes as pathway for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

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.     

Pathways of migration of transplanted Schwann cells in the demyelinated mouse spinal cord

We have studied the behavior of Schwann cells transplanted at a distance from an induced myelin lesion of the adult mouse spinal cord. These transplanted cells were mouse Schwann cells arising from an immortalized cell line (MSC80) which expresses several Schwann cell phenotypes including the ability to produce myelin. The behavior of MSC80 cells was compared to that of purified rat Schwann cells transplanted in the same conditions. Schwann cells were labeled in vitro with the nuclear fluorochrome Hoechst 33342 and were transplanted at distances of 2-8 mm from a lysolecithin-induced myelin lesion in the spinal cord of shiverer and normal mice. Our results show that transplanted MSC80 cells migrated toward the lesion, in both shiverer and normal mouse spinal cord, preferentially along the ependyma, meninges, and blood vessels. They also migrated along white matter tracts but traveled a longer distance in shiverer (8 mm) than in normal (2-3 mm) white matter. Using these different pathways, MSC80 cells arrived within the lesion of shiverer and normal mouse spinal cord at the average speed of 166 p, m/hr (8 mm/48 hr). Migration was most efficient along the ependyma and the meninges where it attained up to 250 μm/hr. Migration was much slower in white matter tracts (95 μm/hr ± 54 in the shiverer and only 38 pm/hr ± 3 in the normal mouse). We also provide evidence for the specific attraction of MSC80 cells by the lysolecithin-induced lesion since (1) their number increased progressively with time in the lesion, and (2) MSC80 cells left their preferential pathways of migration specifically at the level of the lesion. Finally, combining the Hoechst Schwann cell labeling method with the immunohistochemical detection of the peripheral myelin protein, PO, we show that some of the MSC80 cells which have reached the lesion participate in myelin repair in both shiverer and normal lesioned mouse spinal cord. A series of control experiments performed with rat Schwann cells indicate that the migrating behavior of transplanted MSC80 cells was identical to that of purified but non-immortalized rat Schwann cells. © 1993 Wiley-Liss, Inc.     

Ultrastructural and cytochemical characteristics of cultured rat Schwann cells

Summary Schwann cell cultures were established from sciatic nerve of 3 day-old rats. Described are the ultrastructural, histochemical and ultracytochemical properties of amyelic cultured rat Schwann cells. Ultrastructural characteristics of the cultured Schwann cells are compared to the Schwann cells of 3 day-old and adult rat sciatic nerve. These findings serve as a basis for comparison when studying experimentally induced alterations in the cultured Schwann cells as well as changes due to myelination in vitro.