The Changes Of Cortial Neurons NO On Vitro Oxygen-glucose Deprivation Injury Model In Neurons And Its Effect To Caspase-3

Objective: To study the changes of cortial neurons NO on vitro oxygen-glucose deprivation injury modelin rat neurons and its effect to neuronal caspase-3.Methods：The cortical tissue was digested and the cells were planted in the medium containing10%fetalbovine serum, and were given maintenance feeding with neurobasal medium containing B27after four toeight hours. The morphology was observed under phase-contrast microscope. Immunohistochemistry wereapplied to identify the expression of NSE in neurons. The cortial neurons cultured for7d were randomdivided into three groups. There were the control group, the oxygen-glucose deprivation group and theL-NAME (NOS inhibitor) group. NO content in medium was detected by ultraviolet spectrophotometry.Expression of NSE and caspase-3were measured by western blot. Caspase-3mNRA level was detected byreverse transcription polymerase chain reaction（RT-PCR）.Results:1.The neurons were cultured successfully, and the purity of neurons was above90%.2.The NO content on the oxygen-glucose deprivation group (1.77±0.17umoL/L)was significantlyhigher than the control group (0.93±0．15umoL/L) and the L-NAME group (0.99±0.10umol／L)（p<0.05）.There was no significant difference between the control group and theL-NAME group (p>0.05).3. Caspase-3protein and mRNA levels of the control group and the L-NAME group were lowerthan the oxygen-glucose deprivation group (p<0.05). There was no significant differencebetween the control group and the L-NAME group (p>0.05).4. NSE protein level of the oxygen-glucose deprivation group was lower than the control groupand the L-NAME group (p<0.05). There was no significant difference between the controlgroup and the L-NAME group (p>0.05).Conclusions:1. Establishing a serum-free primary culture method for cortical neurons of new-born rats.The neurons were purified by method of differential attachment. This technique is aneasy and practical tool for primary culture of new-born rats cortical neurons with highpurity, and can be used an in vitro model of research..2. Oxygen-glucose deprivation may lead to excessive production of NO in cortial neurons,which had excited toxicity to neurons.3. The L-NAME can decrease the high expression of caspase-3caused by oxygen-glucosedeprivation injury model in neurons. The intervention of NOS synthetic inhibitor hadneuroprotective effects.