The Modeliing Of Hypoxic NSCs And The Repair Effect Of Lithium For Hypoxic NSCs

ObjectiveTo establish model of hypoxic NSCs of neonatal rat in vitro and investigate theprothetic effect of lithium for hypoxic NSCs and provide experimental evidence for clinical treatment of HIE with lithium.Methods1.Isolation, identification and cultivation of NSCs of rat: sacrificing SD rats after born24hours, disinfecting with ethanol with a concentration of75%.Hippocampus wasisolated from neonatal SD rats and put it into D-hank’s liquid.Get rid of meninx andvascellum carefully and cut clean brain into pieces with eye scissors,0.25%trypsindigest cells15min in water bath and blow cells，200mesh stainless filter and entrifuged,resuspended and NSCs were cultured in serum free medium. Nestin expression in NSCswas detected by mimunocytochemistry. The proliferation of NSCs was assessed by bothof5-bromodeoxyuridine (5-BrdU) incorporation assay and double-markedimmunofluorescence. The differentiation of NSCs was induced. To identify thedifferentiated NSCs, antibodies of neuron specific neuronal nuclei (NSE) and antibodyof glial fibrillary acidic protein (GFAP) were employed respectively withmimunocytochemistry.2. Modelling of hypoxic NSCs in vitro: in sugar-free medium and hypoxic environment. Applying DMEM sugar-free medium which is similar to the basal medium for thissugar-free medium and applying a mix of CO2with a volume fraction of5%and N2with a volume fraction of95%for the hypoxic environment to do the experiment.Define the observation of cell morphology and trypan blue staining and cck-8as thestandard of whether the modeling is successful or not.3. The repair effect of lithium for hypoxic NSCs: adding lithium chloride withdifferent concentrations into hypoxic NSCs and investigating the repair effect of lithiumchloride for hypoxic NSCs by the observation of cell morphology and cck-8.Results1.Isolation, identification and cultivation of NSCs of rat: On the first day of culturing,we could see the separated cells as round ones and have a good light shielding. On thethird day of culturing, we could see cell clusters as almost round and with loosestructure and various shapes. On the fifth to seventh day, we could see the cellsconnected closely and swell as round balls. The neurospheres grow rapidly after passingon from generation to generation and the balls are uniform and clear. Detected byimmunocytochemistry, positive nestin expressed as NSCs and positive BrdU expressedas NSCs are in the condition of proliferation. By inducing NSCs with serum-freemedium3days, we could see the neurons of NSE positive staining and the cell body islike a triangle with1-2projections. Astrocytes of GFAP staining positive have a shapelike a star and many stubby branches.2. The modelling of hypoxic NSCs：After culturing in sugar-free DMEM medium and amix of CO2with a volume fraction of5%and N2with a volume fraction of95%for anhour, compared to the normal group, the cell morphology is changing for the hypoxiagroup. In the hypoxia group, the amount of NSCs is decreasing and the appearance isirregular or even exploded into floc or debris. By Trypan blue staining, the amount ofdead cells in the hypoxia group is increasing clearly（P<0.05）. By cck-8, the activeness of NSCs for the hypoxia group is decreasing clearly and is positively correlated with thetime of hypoxia（P<0.05）.3. The repair effect of lithium for hypoxic NSCs：After adding lithium chloride withdifferent concentrations in hypoxic NSCs, compared to the hypoxic group, theobservation of cell morphology for controlled group is as below: the amount of NSCs ismore for the controlled group and has a good refraction. By cck-8, the activeness ofNSCs for the controlled group is much more than the hypoxic group(P<0.05).Theactiveness for3mM is stronger than1mM（P<0.05）. Detected by immunocytochemistrywe could see that the proliferation of lithium-intervention group is significantly higherthan the hypoxia group.Conclusions1. The hippocampus of normal SD rats born after1day could culture NSCs in vitro.2. After culturing in sugar-free medium and a mix of CO2with a volume fraction of5%and N2with a volume fraction of95%for an hour, we could establish the modelof hypoxia NSCs.3. Lithium chloride could be used to increase the viability of cell for hypoxia NSCs.4. Lithium chloride could be used to improve the proliferative of cell for hypoxia NSCs.5. The repair effect of lithium chloride for hypoxia NSCs is correlated with the amountof the lithium chloride in a certain amount range.6. Lithium chloride has a good chance of becoming a new medicine for the clinicaltreatment of HIE.