Development of analyzing softwere of neuronal death model in primary cultured cells

在本研究中，我们开发了在计算机中拍摄培养细胞显微照片的软件，通过计数培养细胞来自动分析细胞活力。细胞活力的自动分析加快了细胞计数实验的速度。利用该软件，我们进行了实验，以阐明谷氨酸在培养的大鼠皮层神经元和培养的大鼠视网膜神经元的神经毒性的机制。此外，我们寻找神经保护因子对谷氨酸神经毒性。1.维生素B ^类似物甲钴胺对谷氨酸神经毒性的影响用皮层培养物来检测。<12>结果表明，甲钴胺促进细胞内甲基化与S-腺苷甲硫氨酸，这是形成在代谢途径的甲钴胺。也有人建议，甲钴胺保护皮质文化对谷氨酸神经毒性减少一氧化氮（NO）的神经毒性作用。2.用视网膜培养物检测Zn ^&lt;2 +&gt;对谷氨酸神经毒性的影响。Zn ^（2+）可保护培养物免受N-甲基-D-天冬氨酸（NMDA）受体介导的谷氨酸神经毒性。3.使用视网膜培养物分析NO在谷氨酸神经毒性中的作用。一个低浓度的NO诱导的保护作用，通过减少NMDA受体介导的电流和浓度升高的NO，与氧自由基相互作用，谷氨酸神经毒性，成为有毒的和增强的谷氨酸神经毒性的文化。这些结果表明，视网膜缺血时释放的高浓度谷氨酸导致大量NO的形成，当其过量存在时，其对神经元是有毒的。本研究结果将为中枢神经系统多种神经退行性疾病的神经保护药物的开发研究提供基础参考资料。

In this study, we developed the software for taking the microscopic pictures of cultured cells in a computer to automatically analyze cell viability by counting cultured cells. The automated analysis of cell viability accelerated the speed of cell-counting experiments. Using this softwere, we performed experiments to elucidate the mechanism underlying glutamate neurotoxicity in cultured rat cortical neurons and in cultured rat retinal neurons. Moreover, we searched neuroprotective factors against glutamate neurotoxicity. 1.The effect of methylcobalamin, a vitamin B^<12> analog, on glutamate neurotoxicity was examined using cortical cultures. The results suggests that methylcobalamin promotes intracellular methylation with S-adenosylmethionine, which is formed in the metabolic pathway of methylcobalamin. It is also suggested that methylcobalamin protects cortical cultures against glutamate neurotoxicity by reducing neurotoxic action of nitric oxide (NO). 2.The effect of Zn^<2+> on glutamate neurotoxicity was examined using retinal cultures. Zn^<2+> protected the cultures against glutamae neurotoxicity mediated by N-methyl-D-aspartate (NMDA) receptor. 3.The role of NO in glutamate neurotoxicity was analyzed using retinal cultures. A low concentration of NO induced a protective action against glutamate neurotoxicity by reducing the NMDA receptor-mediated currents and that elevated concentrations of NO,interacting with oxygen radicals, became toxic and enhanced glutamate neurotoxicity in the cultures. These results suggest that high concentration of glutamate released in retinal ischemia causes formation of large amount of NO,which is toxic to neurons when it is present in excess. The results in this study will offer basic reference materials to drive forward developmental research for neuroprotective drugs against many neurodegeneration diseases in central nervous system.