The Oxidant-antioxidant Change Of Neurons And Neuroprotective Effects Of Citicoline Against Oxygen-Glucose Deprivation And Reperfusion Injury

Part one Study on the method of primary neurons culture and differential vulnerability between hippocampal and cortical neurons to oxygen-glucose deprivation/ ReperfusionObjiective To establish primary culture methods for newborn rat hippocampal and cortical cells, and investigate the differential vulnerability between hippocampal and cortical neurons to oxygenglucose deprive-ation/Reperfusion. Method The hippocampal and cortical neurons were isolated from new born rats, and cultured with serum-free medium combination, and its activity was verified by morphology. Primary hippocampal and cortical neurons were cultured for 8-10d and then were used to establish the oxygen-glucose deprivation/reperfusion(OGD/R) model . The content of dehydrogenase (LDH),glutathione(GSH) and nitric oxide(NO), the activity of nitricoxide synthase (NOS)and glutathion peroxidase(GPx) were measured by assay kits at 3h,6h and 24h after OGD/R. The morphology of survival and apoptotic cells was observed by trypan blue and Hochest33258 staining at 24h after OGD/R. Results Hippocampal neurons had markedly higher activity of NOS than cortical neurons after OGD/R, which coincided with an increase in NO production(P<0.05). The GPx activity and GSH level were declined significantly in hippocampal but not in cortical neurons after OGD/R (P<0.05). After OGD/R, the cell death and apoptotic rates of hippocampal neurons were obviously more than cortical neurons (P<0.05). Conclusion Hippocampal neurons are more vulnerable than cortical neurons after OGD/R. An imbalance between excess prooxidant production (increased NOS activity and NO level) and insufficient antioxidant defenses created by reduced GPx activity and GSH levels may, in part, explain the higher susceptibility to OGD of hippocampalneurons.Part two Neuroprotective effects of citicoline against Oxygen-Glucose Deprivation / Reperfusion injury in cultured hippocampal cellsObjective To demonstrate the protective effects of citicoline against neuronal Oxygen-Glucose Deprivation /Reperfusion injury and to investigate the underlying mechanisms. Methods The rat neurons cultivated as the object of study and then build the model of oxygen-glucose deprivation and reperflision. They were randomly divided into 3 groups: control group, oxygen-glucose deprivation group, citicoline treatment groups(1,10,100umol/L), Metabolic rate of MTT at reperflision 24h,the content of reduced glutathione and the active of glutathion peroxidase were measured by assay kits at reperflision 6h and 24h. Cells were dyed Annexin V-PI and the rate of apoptosis was detected by flow cytometry. Results Compared with OGD/reperfusion group, citicoline treatment after OGD obviously increased GSH level and activity GPx at reperflision 6h (P<0.05); citicoline increased cell survival rate, activity GPx and decreased cell apoptotic rate. Conclusion: citicoline has protective effect on hippocampal neurons against oxygen-glucose deprivation and reperflision injury by increasing the metabolic rate of MTT, raising antioxidation, and decreasing apoptosis.