The Effects of Glutamate Receptor Antagonists on Cultured Cerebral Cortical Neurons of Neonatal Mouse Damaged by Oxidative Stress

ABSTRACT

PURPOSE: To evaluate neurotoxic effects induced by oxygen-radicals, which were generated by adding xanthine oxidase(XO) and hypoxanthine(HX), and protective effects of glutamate receptor antagonist such as MK-801 and 6-cyano-7-nitroquinoxaline(CNQX). METHODS: Dissociated cell cultures were prepared from cerebrum of neonatal mouse. Tissues were dissected and diced into small pieces in phosphate buffered saline and were incubated at 37degrees C. Isolated cells were resuspended in Eagle's minimum essential medium and plated poly-L-lysine coated plastic coverslips in 96 well multichambers at a cell density of 3x105 cells/well. Cells were grown in a 5% CO2/95% air atmosphere at 37degrees C. Cytotoxic effects were examined in cerebral cortical neurons cultured for 3 hours in media containing various concentration of XO and HX. The protective effects of glutamate receptor antagonist were also examined by MTT assay and neurofilament enzymeimmunoassay(EIA). Microscopic examinations were also done. RESULTS: Oxygen radicals markedly induced decrement of the cell viability of cultured mouse cerebral cortical neurons in a dose-dependent manner. Midpoint cytotoxicity value was 30mU/ml XO/0.1mM HX, when mouse cerebral cortical neurons were incubated for 3 hours with various concentrations of XO and HX. The number of cells and neurites was decreased when cerebral cortical neurons were cultured for 3 hours in a medium containing 30mU/ml XO/0.1mM HX. MK- 801 was very effective in blocking oxidant-induced neurotoxicity, while CNQX falied to show any protective effect in these cultures. CONCLUSION: It is suggested that oxygen radicals are neurotoxic, and selective N-methyl-D-aspartate antagonists such as MK-801 are very effective in protecting neurotoxicity induced by oxygen radicals in cultured cerebral cortical neurons of neonatal mouse.