Effects Of Lithium On Proliferation, Survival And Function Of Adult Rat Schwann Cell In Vitro

Schwann cells are the myelinating glial cells in the PNS and play a key role in Wallerian degeneration and subsequent regeneration due to their ability to dedifferentiate, migrate, proliferate, express growth promoting factors, and myelinate regenerating axons. In animal models of SCI, grafting Schwann cells or peripheral nerve into the lesion site has been shown to promote axonal regeneration and myelination. Combinatorial strategies with schwann cells and other emerging strategies for spinal cord repair to maximize axonal regeneration and functional recovery.Activated Schwann cell is one of the optimal cell candidates for the repair after SCI. Numerous NTFs secreted by AASCs can improve the microenvironment in the lesion site and promote functional recovery of spinal cord injured rats. Importantly, the development of in vitro systems to harvest human Schwann cells presents a unique opportunity for autologous transplantation in the clinic. To employ Schwann cells for transplantation into the spinal cord, large numbers of cells will be necessary.Recent studies revealed a mechanism that ties together these disparate effects of lithium. Lithium acts through multiple pathways to inhibit glycogen synthetase kinase-3 beta (GSK3b) and turn on cell growth and protection programs. Lithium also stimulates proliferation of stem cells, including bone marrow and neural stem cells in the subventricular zone, striatum, and forebrain. More Importantly, Lithium has low mutagenic and carcinogenic risk. Moreover we also noticed that 2-Mercaptoethanol may induce cell proliferation and enhance DNA synthesis and leukemia inhibitory factor can regulate cell differentiation, proliferation and phenotype. Therefore, we investigate the Effect of lithium and embryonic stem cell culture medium, respectively, on activated Schwann cells proliferation and survival in vitro and got the following results:1. Culture, purification and biological characters of adult Schwann cells from rat predegenerated sciatic nerve sciatic nerves from adult male rat were transected at the midthigh。Seven days later,the distal segment of the transected nerves were removed and used for adult SCs cultures。A simple, inexpensive method for purifying SCs, in which various harvested cell types showed different rates of attachment to the coated laminin culture ware in the initial stage of primary culture, was developed.2. The purified Schwann cells still exhibited a spindle-like shape with bipolar or tripolar morphology at 3 div, 5div, and align themselves parallel to each other in confluent cultures at 7div and 10div. Immunostaining for S100 antibodies showed that the staining was evident in the cell soma and the processes. The proliferation of purified SCs was monitored by visualizing Brdu incorporation using immunocytochemistry. The flow cytometric results which provided quantitative evidence of SCs purity was about 93% at 7 days in vitro. And within 10 day, SCs purity could reach above 91%.3. lower concentrations of lithium 0.1mM did not increase the total number of cells compared to the basal control level. Whereas the total number of cells cultured with both 1mM and 5mM lithium chloride was significantly higher than the control culture. Moreover,Treatment with lithium 10mM sharp decreased the number of cells compared with control,Suggested that high concentrations of lithium may be toxic to SCs. The total number of cells cultured with embryonic stem cell culture medium treatment significantly higher than the control culture.4. Five millimole of lithium maximally stimulated SCs proliferation in culture. Treatment with 5mM Lithium significantly increased the number of BrdU-positive Cells compared with control at both 5 day and 9 day. Likewise, the number of BrdU-positive cells cultured induced by 5mMLiCl cultures or condition medium was significantly higher than the control culture at different time points .5. The ability of these cells to be amplified still retain normal functionality is crucial. We demonstrated that when SCs were allowed to directly contact neurons, SCs could direct the orientation of regrowing neurits, and that axons grew parallel to the long axis of underlying fusiform SCs. In addition, both neuron bodies and axons preferred SCs over artificial substrates in the direct contact coculture.6. To test the ability of the amplified SCs to relate to neuronal surfaces, cells were periodically seeded onto DRG neurite beds and maintained for 7-14 days. To better visualize myelin formed in the cultures, selected cultures were immunostained for the myelin basic protein-MBP. When cells were planted onto neurites, they were seen to line up along the neurites and proliferate. Within the first week, the SCs flattened out on the neurites, extending processes along the axons. Myelin was first observed at 14d. And then, the number of myelin segments increased with time. At the ultrastructural level, some of the differentiated schwann cells formed a mesaxon that spiraled up to 3 turns around an axon at this stage of 14 DIV. Several very thin membranous with no compacted myelin were revealed around an axons. Compact myelin was revealed in co-cultures at 35 DIV. Many cases of segregated large axons were associated 1:1 with SCs and were wrapped with 10-30 layers of compast myelin sheath.From above results we can draw the conclusions that Lithium (1mM, 5mM) and embryonic stem cell culture medium, respectively, could improve significantly activated Schwann cells proliferation in vitro. However, higher concentrations of lithium at 10mM did not increase proliferation. Activated Schwann cells stimulated by lithium could support neurite outgrowth and myelinate axons in vitro.