Estrogen and progesterone stimulate Schwann cell proliferation in a sex- and age-dependent manner.

The effects of estrogen and progesterone on Schwann cell proliferation were studied in cultured segments of the rat sciatic nerve from adult male, female, and newborn rats, by measurement of [3H thymidine incorporation or bromo-deoxy-uridine- (BrdU)-labelling and immunocytochemistry. Estrogen (100 nM-500 nM) enhanced [3H] thymidine incorporation in segments from male and newborn rats, while it had no effect on segments from female rats. Progesterone stimulated thymidine incorporation in segments from female and newborn rats (100 nM-500 nM), but caused only a small proliferative response in Schwann cells from male rats at high concentrations. The proliferative effects of estrogen and progesterone were blocked when the segments were cultured in the presence of inhibitors of their respective receptors, ICI 128 780 and zk 112994. The data suggest that Schwann cells possess distinct receptors for estrogen and progesterone and that these receptors may be involved in the control of Schwann cell proliferation. It also shows that the response of Schwann cells to sex hormones varies with sex and perhaps also with age.     

Insulin and the insulin-like growth factors I and II are mitogenic to cultured rat sciatic nerve segments and stimulate [3H]thymidine incorporation through their respective receptors

The factors that control proliferation of Schwann cells during peripheral nerve regeneration are not yet known. In this study we investigated the effects of insulin, insulin-like growth factor I and II (IGF-I and IGF-II), IGF-I analogues, and factors that interfere with their respective receptors, on [³H]thymidine incorporation into cultured nerve segments from the rat sciatic nerve. Segments cultured in nM (0.1–1.7 nM) concentrations of insulin, truncated IGF-I (tIGF-I), long R³IGF-I, or IGF-II exhibited an increase in [³H]thymidine incorporation compared with control segments. IGF-II was most potent. JB1, an IGF-I antagonist, counteracted the effects of tIGF-I and insulin. The results suggest that non-neuronal cells in the nerve segment, probably Schwann cells, possess distinct receptors for insulin, IGF-I, and IGF-II and that these receptors may be involved in the control of Schwann cell proliferation during peripheral nerve regeneration. © 1996 Wiley-Liss, Inc.     

Sciatic nerve regeneration is accelerated in galectin-3 knockout mice

The success of peripheral nerve regeneration depends on intrinsic properties of neurons and a favorable environment, although the mechanisms underlying the molecular events during degeneration and regeneration are still not elucidated. Schwann cells are considered one of the best candidates to be closely involved in the success of peripheral nerve regeneration. These cells and invading macrophages are responsible for clearing myelin and axon debris, creating an appropriate route for a successful regeneration. After injury, Schwann cells express galectin-3, and this has been correlated with phagocytosis; also, in the presence of galectin-3, there is inhibition of Schwann-cell proliferation in vitro. In the present study we explored, in vivo, the effects of the absence of galectin-3 on Wallerian degeneration and nerve-fiber regeneration. We crushed the sciatic nerves of galectin-3 knockout and wild-type mice, and followed the pattern of degeneration and regeneration from 24 h up to 3 weeks. We analyzed the number of myelinated fibers, axon area, fiber area, myelin area, G-ratio and immunofluorescence for β-catenin, macrophages and Schwann cells in DAPI counterstained sections. Galectin-3 knockout mice showed earlier functional recovery and faster regeneration than the wild-type animals. We concluded that the absence of galectin-3 allowed faster regeneration, which may be associated with increased growth of Schwann cells and expression of β-catenin. This would favor neuron survival, followed by faster myelination, culminating in a better morphological and functional outcome.     

Regulation of protease nexin-1 and angiotensin II receptor subtype 1 expression: Inverse relationship in experimental models of nerve injury

The up-regulation of PN-1 following nerve lesion has been investigated in vitro in cultures of dorsal root ganglion (DRG) explants, sciatic nerve segments, and isolated Schwann cells. In the first culture model, Schwann cells associated with neuronal processes synthesized small amounts of PN-1. Injury of the neurites emerging from the DRGs led to enhanced levels of PN-1 in Schwann cells located distal to the lesion site where degeneration of neuronal processes took place. In cultured sciatic nerve segments, PN-1 synthesis increased with a time-course comparable to that in ganglion explants following lesion. In the third model, PN-1 levels gradually rose in isolated Schwann cells during the first 3-8 days in culture. Dissociation of Schwann cells from the sciatic nerve therefore causes an effect similar to nerve damage. Impairment of Schwann cell-neuron interactions was followed by a reduction in the expression levels of the angiotensin II (Ang II) receptor subtype AT₁ in all three systems studied. Since the neuropeptide Ang II is able to repress PN-1 synthesis in cultured Schwann cells, loss of neuronal contact might decrease their responsiveness to Ang II, thus resulting in PN-1 upregulation by default. © 1995 Wiley-Liss, Inc.     

Time-dependent effects of insulin on Schwann cell proliferation in the in vitro regenerating adult frog sciatic nerve

The present study showed that insulin (0.01 μg/ml, ≈? 2 nM) inhibited [³H]-thymidine incorporation in support cells, most likely Schwann cells, of the cultured frog sciatic nerve. A 25–35% inhibition took place in regenerating nerve preparations as well as in preparations devoid of neuronal protein synthesis, i.e., in isolated 5 mm nerve segments and in gangliectomized nerves, suggesting that the effect was direct and not mediated via the neuronal cells. The inhibition by insulin was time-dependent in that an effect was seen after 4 days but not at shorter or at longer periods of culturing. In separate experiments biotinylated insulin was shown to be taken up by Schwann cells in the regenerating nerve. Addition of serum increased the [³H]-thymidine incorporation severalfold and abolished the inhibitory action of insulin. Our results suggest that insulin, at a certain stage of the regeneration programme, exerts a direct, inhibitory effect on the proliferation of the Schwann cells in the cultured frog sciatic nerve. © 1993 Wiley-Liss, Inc.     

Pre-degenerated peripheral nerves co-cultured with bone marrow-derived cells：a new technique for harvesting high-purity Schwann cells

Schwann cells play an important role in the peripheral nervous system, especially in nerve repair following injury, so artificial nerve regeneration requires an effective technique for obtaining purified Schwann cells. In vivo and in vitro pre-degeneration of peripheral nerves have been shown to obtain high-purity Schwann cells. We believed that in vitro pre-degeneration was simple and controllable, and available for the clinic. Thus, we co-cultured the crushed sciatic nerves with bone marrow-derived cells in vitro. Results demonstrated that, 3 hours after injury, a large number of mononuclear cells moved to the crushed nerves and a large number of bone marrow-derived cells infiltrated the nerve segments. These changes promoted the degradation of the nerve segments, and the dedifferentiation and proliferation of Schwann cells. Neural cell adhesion molecule and glial fibrillary acidic protein expression were detected in the crushed nerves. Schwann cell yield was 9.08 ± 2.01 × 104/mg. The purity of primary cultured Schwann cells was 88.4 ± 5.79%. These indicate a successful new method for obtaining Schwann cells of high purity and yield from adult crushed sciatic nerve using bone marrow-derived cells.     

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.     

人参皂甙Rb1对体外培养雪旺细胞作用的实验研究

目的研究人参皂甙Rb1对体外培养雪旺细胞增殖分化的影响,探讨其促进神经再生的作用和机制。方法取8个月月龄的新西兰兔的坐骨神经雪旺细胞体外培养第2代,加入不同浓度的人参皂甙Rb1,继续培养10d,显微镜观察计数,绘制各自的增殖曲线。在36h和72h对各组细胞进行流式细胞分析。结果活细胞计数显示:含20μg/ml人参皂甙Rb1组的倍增时间为5.3d,明显优于对照组(P<0.01)。用含20μg/ml人参皂甙Rb1培养36h、72h后雪旺细胞增殖的流式细胞分析,处于S期的雪旺细胞较对照组明显增高(P<0.01)。结论人参皂甙Rb1具有促进体外培养雪旺细胞快速增殖分化的作用,有助于损伤神经的再生。     

Reduction of proliferating non-neuronal cells in dried nerve segments partly impairs nerve regeneration

To determine whether drying of nerve grafts can affect axonal outgrowth and proliferation of non-neuronal cells, nerve segments dried for 0-60 min were used as nerve grafts to bridge a gap in transected rat sciatic nerves. Axonal outgrowth was measured by pinch reflex test and confirmed by immunocytochemical staining of neurofilaments. The proliferation of non-neuronal cells was measured by incorporation of BrdU in the dried nerve segments. Drying of the nerve segment for 60 min reduced the length of axonal outgrowth to 66 and 76% 3 and 6 days, respectively, after the grafting procedure. At that time point the number of proliferating cells was reduced by 51%. It is concluded that the number of proliferating non-neuronal cells is reduced in dried nerve segments which only partly impairs axonal outgrowth. Factors other than Schwann cells are probably important for an optimal mileue for regeneration in nerve grafts.     

Ca2+ involvement in activation of extracellular-signal-regulated-kinase 1/2 and m-calpain after axotomy of the sciatic nerve

Detailed mechanisms behind regeneration after nerve injury, in particular signal transduction and the fate of Schwann cells(SCs), are poorly understood. Here, we investigated axotomy-induced activation of extracellular-signal-regulated kinase-1/2(ERK1/2; important for proliferation) and m-calpain in vitro, and the relation to Ca2+ deletion and Schwann cell proliferation and death after rat sciatic nerve axotomy. Nerve segments were cultured for up to 72 hours with and without ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid(EGTA). In some experiments, 5-bromo-2′-deoxyuridine(Brd U) was added during the last 24 hours to detect proliferating cells and propidium iodide(PI) was added at the last hour to detect dead and/or dying cells. Immunohistochemistry of sections of the cultured nerve segments was performed to label m-calpain and the phosphorylated and activated form of ERK1/2. The experiments revealed that immunoreactivity for p-ERK1/2 increased with time in organotypically cultured SCs. p-ERK1/2 and m-calpain were also observed in axons. A significant increase in the number of dead or dying SCs was observed in nerve segments cultured for 24 hours. When deprived of Ca2+, activation of axonal m-calpain was reduced, whereas p-ERK1/2 was increased in SCs. Ca2+ deprivation also significantly reduced the number of proliferating SCs, and instead increased the number of dead or dying SCs. Ca2+ seems to play an important role in activation of ERK1/2 in SCs and in SC survival and proliferation. In addition, extracellular Ca2+ levels are also required for m-calpain activation and up-regulation in axons. Thus, regulation of Ca2+ levels is likely to be a useful method to promote SC proliferation.     

Activation of extracellular-signal-regulated kinase-1/2 precedes and is required for injury-induced Schwann cell proliferation

Activation of extracellular-signal-regulated kinase-1/2 (Erk1/2) by phosphorylation to p-Erk1/2, and proliferation of Schwann cells were investigated in the rat sciatic nerve by immunohistochemistry. Axotomy in vivo and culturing of nerve segments in vitro resulted in a rapid (30 min) increase of p-Erk1/2 in Schwann cells with peaks at 2 and 24 h. Proliferation measured by bromodeoxy uridine incorporation and immunostaining in vivo and in vitro 48 h after axotomy showed an increase in Schwann cell proliferation at the sites of Erk1/2 activation. The Erk1/2 inhibitor U0126 inhibited both the increase in p-Erk1/2 and the bromodeoxy uridine incorporation. We suggest that an increase in p-Erk1/2 is required for nerve injury-induced proliferation of Schwann cells.     

Nanoparticles carrying neurotrophin-3-modified Schwann cells promote repair of sciatic nerve defects

Schwann cells and neurotrophin-3 play an important role in neural regeneration,but the secretion of neurotrophin-3 from Schwann cells is limited,and exogenous neurotrophin-3 is inactived easily in vivo.In this study,we have transfected neurotrophin-3 into Schwann cells cultured in vitro using nanoparticle liposomes.Results showed that neurotrophin-3 was successfully transfected into Schwann cells,where it was expressed effectively and steadily.A composite of Schwann cells transfected with neurotrophin-3 and poly(lactic-co-glycolic acid) biodegradable conduits was transplanted into rats to repair 10-mm sciatic nerve defects.Transplantation of the composite scaffold could restore the myoelectricity and wave amplitude of the sciatic nerve by electrophysiological examination,promote nerve axonal and myelin regeneration,and delay apoptosis of spinal motor neurons.Experimental findings indicate that neurotrophin-3 transfected Schwann cells combined with bridge grafting can promote neural regeneration and functional recovery after nerve injury.     

The α-chemokine CXCL14 is up-regulated in the sciatic nerve of a mouse model of Charcot–Marie–Tooth disease type 1A and alters myelin gene expression in cultured Schwann cells

At present the pathogenesis of CMT1A neuropathy, caused by the overexpression of PMP22, has not yet been entirely understood. The PMP22-overexpressing C61 mutant mouse is a suitable animal model, which mimics the human CMT1A disorder. We observed that myelin gene expression in the sciatic nerve of the C61 mouse was up-regulated at postnatal day 4 to 7 (P4–P7). When investigating the morphology of peripheral nerves in C61 and wildtype mice at early stages of postnatal development, hypermyelination could be detected in the femoral quadriceps and sciatic nerve of transgenic animals at postnatal day 7 (P7). In order to identify genes, other than Pmp22, that are modulated in sciatic nerve of P7 transgenic mice, we applied microarray technology. Amongst the regulated genes, the gene encoding the α-chemokine CXCL14 was most prominently up-regulated. We report that Cxcl14 was expressed exclusively by Schwann cells of the sciatic nerve, as well as by cultured Schwann cells triggered to differentiate. Furthermore, in cultured Schwann cells CXCL14 modulated the expression of myelin genes and altered cell proliferation. Our findings demonstrate that early overexpression of PMP22, in a mouse model of CMT1A, results in a strong up-regulation of CXCL14, which seems to play a novel regulatory role in Schwann cell differentiation.     

Autocrine action of BMP2 regulates expression of GDNF-mRNA in sciatic Schwann cells

Schwann cell is a cell type that forms myelin sheath and provides trophic supports for neuronal cells by producing neurotrophic factors in both normal and traumatic situations. It was recently reported that after lesion of sciatic nerve, mRNA for glial cell line-derived neurotrophic factor (GDNF) is induced in nonneuronal cells in the nerve. However, the mechanism regulating GDNF-mRNA has remained largely unknown. In the present study, we searched for factors regulating the GDNF-mRNA expression in Schwann cells. First, we found that after transfer into explant culture as an in vitro lesion model, sciatic nerve segments began to express mRNA for bone morphogenetic protein-2 (BMP2) concomitantly with the induction of GDNF-mRNA. Treatment of the Schwann cells isolated from the sciatic nerve with combination of BMP2 and retinoic acid (RA) dramatically induced GDNF-mRNA, while BMP2 or RA alone had no effect. Furthermore, ionomycin, a calcium ionophore, which had even stronger activity on the induction of GDNF-mRNA also induced also BMP2-mRNA in cultured Schwann cells. Effects of inhibitors of intracellular signaling pathways such as protein kinase C inhibitor and MAPKK inhibitor suggested that the molecular mechanism of the induction of GDNF-mRNA is distinct from that of BMP2-mRNA. These results suggest that the Schwann cell-produced BMP2 plays an important role in the induction of GDNF after nerve injury in an autocrine fashion.     

Synthesis of leukemia inhibitory factor in injured peripheral nerves and their cells

The time and site of induction of leukemia inhibitory factor mRNA in injured rat sciatic nerves and its regulation in Schwann cells and fibroblasts from neonatal rat nerves were investigated. Leukemia inhibitory factor mRNA is induced at the lesion site within 6 h of sciatic nerve transection but only after 24 h in the more distal segments. In vitro, interleukin-1β increases the concentration of leukemia inhibitory mRNA in nerve fibroblasts but not in Schwann cells. Changes in leukemia inhibitory factor mRNA concentration in injured nerves and peripheral nerve cells are similar to those for nerve growth factor mRNA.     

Regulation of Schwann cell proliferation in cultured segments of the adult rat sciatic nerve

Schwann cell proliferation was studied in cultured segments of the rat sciatic nerve by measurement of [³H] thymidine incorporation or through bromodeoxyuridine-(BrdU)-labelling and immunocytochemistry. The aim was to delineate mechanisms involved in the injury-induced proliferative response of Schwann cells. Removal of extracellular Ca²⁺ by addition of EGTA to the culture medium suppressed [³H] thymidine incorporation as did the calmodulin inhibitor 48/80. The Ca²⁺ ionophore A23187 increased incorporation. Staurosporin, an inhibitor of protein kinase C (PKC), suppressed [³H] thymidine incorporation while phorbol-12-myristate-13-acetate (PMA) enhanced incorporation. Manipulation of the cAMP system showed that increased cAMP levels inhibited proliferation. Inhibition of protein kinase A by HA 1004 increased the incorporation of [³H] thymidine. Immunostaining for BrdU and glial specific markers together with morphological evaluation of myelin association showed that proliferation occurred in Schwann cells. The results are consistent with a model in which Schwann cell proliferation is enhanced by Ca²⁺ through activation of calmodulin-dependent and/or PKCdependent mechanisms. Inhibition is achieved through the cAMP system. Together, these results show that Schwann cells regulate proliferation differently in an integrated environment, e.g. the nerve structure, than in isolation as primary monocultures. J. Neurosci. Res. 52:530–537, 1998. © 1998 Wiley-Liss, Inc.     

微囊化成年兔许旺细胞的体外培养*

背景：研究发现将体外培养扩增的大量许旺细胞种植到神经导管上来引导周围神经再生效果明显,而目前备受关注的微囊化免疫隔离技术,在克服免疫排斥问题上具有明显的优势与实用性。 目的：微囊包裹兔许旺细胞并进行体外培养,观察许旺细胞形态及增殖变化。 设计、时间及地点：体外细胞水平观察实验,于2005-09/2006-05在南昌大学第二附属医院分子实验室完成。 材料：健康成年家兔由南昌大学医学院动物中心提供,微囊发生器由南昌大学医学院神经生物学研究室研制。 方法：使双侧坐骨神经发生Wallerian变性,经改良后反复差速贴附法进行培养,快速分离纯化许旺细胞。海藻酸钠-氯化钡一步成囊法包裹生长良好的第3代许旺细胞。 主要观察指标：倒置显微镜下观察培养过程中微囊化许旺细胞形态变化,MTT检测不同时期微囊化许旺细胞及游离许旺细胞的增殖改变。 结果：在培养过程中可见微囊及其囊内许旺细胞形态均保持完整,微囊未见破损。微囊化后的许旺细胞总体呈现悬浮生长的趋势。培养0,1,3,5,7 d时 MTT检测微囊许旺细胞组与游离许旺细胞组吸光度值差异有显著性意义(P < 0.05)。 结论：微囊包裹的许旺细胞体外可存活较长时间,微囊化许旺细胞相比游离许旺细胞增殖速度有所减慢。     

Lithium promotes proliferation and suppresses migration of Schwann cells

Schwann cell proliferation,migration and remyelination of regenerating axons contribute to regeneration after peripheral nervous system injury.Lithium promotes remyelination by Schwann cells and improves peripheral nerve regeneration.However,whether lithium modulates other phenotypes of Schwann cells,especially their proliferation and migration remains elusive.In the current study,primary Schwann cells from rat sciatic nerve stumps were cultured and exposed to 0,5,10,15,or 30 mM lithium chloride(LiCl)for 24 hours.The effects of LiCl on Schwann cell proliferation and migration were examined using the Cell Counting Kit-8,5-ethynyl-2′-deoxyuridine,Transwell and wound healing assays.Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine assays showed that 5,10,15,and 30 mM LiCl significantly increased the viability and proliferation rate of Schwann cells.Transwell-based migration assays and wound healing assays showed that 10,15,and 30 mM LiCl suppressed the migratory ability of Schwann cells.Furthermore,the effects of LiCl on the proliferation and migration phenotypes of Schwann cells were mostly dose-dependent.These data indicate that lithium treatment significantly promotes the proliferation and inhibits the migratory ability of Schwann cells.This conclusion will inform strategies to promote the repair and regeneration of peripheral nerves.All of the animal experiments in this study were ethically approved by the Administration Committee of Experimental Animal Center of Nantong University,China(approval No.20170320-017)on March 2,2017.