The Study About Protective Effects Of Aspirin On Injury Induced By Oxygen-Glucose Deprivation/Reperfusion In Rat Cultured Cortical Neurons

Objective:Many previous studies have shown that aspirin have a direct neuroprotective effect, it could against brain ischemia injury, resulting in reducing the infarct size, decreasing the content of inflammatory cells and adhesion molecules and inhibiting cell apoptosis in ischemia area in vivo. In our present study, the injury model was established by oxygen-glucose deprivation in vitro, to investigate the protective effect against oxygen-glucose deprivation of aspirin, the potential neuroprotective mechanism and the optimal dosage of aspirin.Methods:1. Primary cortical neurons were prepared from brains of the newborn Wistar rats within 24 hours; the neurons at 7 days in vitro were used.2. Cortical neurons cultured for 7 days were randomly divided into three groups:①control group, neurons was treated with DMEM/F12 and incubated with 5% CO2 and 95% O2 gas;②injury model group, model of oxygen-glucose deprivation and reperfusion (OGD/R) injury group: neurons were washed with Earle's balanced salt solution without glucose for two times, and put into oxygen deficient box with 2 ml Earle's without glucose in each dish for two hours with 90% N2 gas. After that, the neurons were incubated in DMEM/F12 at 37℃for 24 hours respectively;③Aspirin treatment groups, the model of oxygen-glucose deprivation and reperfusion injury was established by changing non- glucose Earle's, then the aspirin was joined ,which included the three concentration of aspirin (10μmol/L,ASA-L;50μmol/L,ASA-M;100μmol/L,ASA-H);3. Each group was observed by inverted phase contrast microscope; to evaluate the effectiveness of OGD/R model by measurement LDH in the medium; neuron mitochondrion viability was measured by the reduction of 3-(4,5-dimethylthiahiazo-2-yl)-2,5-diphenytetrazoli- um bromide(MTT) and Calcein staining; mitochondrion membrane potential (MMP) and apoptosis were measured by flow cytometry.Results:1. We established a experimented cultured rat's cortical neurons by using the common culture method; the cortical neurons was detected by neuronal specific enolase (NSE) immunocytochemical staining; and the oxygen-glucose deprivation and reperfusion (OGD/R) model of rat's cortical neurons in vitro had been established successfully.2. Primary cortical neurons induced by oxygen-glucose deprivation and reperfusion injury, the LDH activity significantly increased showing great negative correlation to neurons vigor; OGD cultures for 2 hour significantly declined the mitochondrion activity and MMP(P<0.05), increased the percentage of neurons apoptosis and necrosis(P<0.01);compared with OGD/R group, Aspirin treatment after oxygen-glucose deprivation and reperfusion obviously increased cell survival rate and Aspirin could significantly increase the neurons mitochondrion activity and MMP, decrease the percentage of neurons apoptosis and necrosis, with increasing doses of Aspirin, the effects increased linearly with increased dosage.Conclusion:The Aspirin could significantly inhibit the decline in mitochondrion activity and MMP, and had effects of keeping stable of MMP and anti-neurons apoptosis during injury induced by OGD/R, which suggests that aspirin is an effective neuroprotection...