Ferreira I L, Duarte C B, Neves A R, Carvalho A P
Center for Neuroscience of Coimbra, University of Coimbra, Portugal.
Neurochem Int. 1998 Apr;32(4):387-96. doi: 10.1016/s0197-0186(97)00099-5.
The aim of this study was to determine whether culture-conditioned medium (CCM) can prevent neuronal damage caused by excitotoxicity or by "chemical ischemia" in cultured chick retina cells. Excitotoxic conditions were obtained by incubating retina cells with glutamate or kainate and "chemical ischemia" was induced by metabolic inhibition. In this case, cultures were briefly exposed to sodium cyanide, to block oxidative phosphorylation and iodoacetic acid, to block glycolysis. The assessment of neuronal injury was made spectrophotometrically by quantification of cellularly reduced MTT. Stimulation of retina cells with glutamate or kainate in serum deprived culture medium (BME-FCS), lead to a decrease in the MTT metabolism that was dependent on the time of exposure to the toxic agents. CCM prevented cell damage, either when present during the stimulation period or during the recovery period. This protection was more prominent in the case of kainate-induced neuronal death. "Chemical ischemia" also lead to a decrease of the MTT metabolism in a time-dependent manner and CCM protected retina cells from "ischemia"-induced lesions when present during the stimulation period and during the recovery period. The protective effect of CCM was partially decreased by the tyrosine kinase inhibitor, genistein, when the cells were stimulated with kainate, but not with glutamate, or when the cells were subjected to "chemical ischemia". CCM protected retina cells against both the acute and the delayed toxicity induced by either glutamate or kainate, or by "chemical ischemia", when present during both the insult and the recovery period. The presence of survival factors in the media may effectively inhibit the cell death signals generated by glutamate receptor activation or by "chemical ischemia".
本研究的目的是确定培养条件培养基（CCM）是否能够预防培养的鸡视网膜细胞中由兴奋性毒性或“化学性缺血”引起的神经元损伤。通过用谷氨酸或海藻酸盐孵育视网膜细胞来获得兴奋性毒性条件，并用代谢抑制诱导“化学性缺血”。在这种情况下，将培养物短暂暴露于氰化钠以阻断氧化磷酸化，并暴露于碘乙酸以阻断糖酵解。通过对细胞内还原的MTT进行定量，用分光光度法评估神经元损伤。在无血清培养基（BME - FCS）中用谷氨酸或海藻酸盐刺激视网膜细胞，导致MTT代谢降低，这取决于暴露于毒性剂的时间。CCM在刺激期或恢复期存在时均可预防细胞损伤。在海藻酸盐诱导的神经元死亡的情况下，这种保护作用更为显著。“化学性缺血”也以时间依赖性方式导致MTT代谢降低，并且CCM在刺激期和恢复期存在时可保护视网膜细胞免受“缺血”诱导的损伤。当用海藻酸盐而非谷氨酸刺激细胞时，或当细胞遭受“化学性缺血”时，酪氨酸激酶抑制剂染料木黄酮会部分降低CCM的保护作用。当CCM在损伤期和恢复期均存在时，它可保护视网膜细胞免受谷氨酸或海藻酸盐或“化学性缺血”诱导的急性和延迟毒性。培养基中存活因子的存在可能有效抑制由谷氨酸受体激活或“化学性缺血”产生的细胞死亡信号。
