Dux E, Oschlies U, Uto A, Kusumoto M, Siklos L, Joo F, Hossmann K A
Max-Planck-Institute for Neurological Research, Department of Experimental Neurology, Cologne, Germany.
Acta Neuropathol. 1996 Sep;92(3):264-72. doi: 10.1007/s004010050517.
The effect of serum proteins on glutamate-induced mitochondrial calcium accumulation was studied in primary cortical and hippocampal cultures using oxalate-pyroantimonate staining with electron microscopy. Cultures were prepared from rat embryos on gestational day 17-19 and cultivated for 8 days in minimal essential medium (MEM) containing 5% native horse serum. At this time cultures were exposed for 5 min to 100 micro M or 1.0 mM glutamate, followed by recovery in either serum-free or serum-containing culture medium. Mitochondrial calcium accumulation was assessed before glutamate treatment, at the end of glutamate exposure, and after 5 min, 30 min, 6 h and 24 h of recovery. Under control conditions and at the end of glutamate exposure, mitochondria contained only a few calcium deposits. If cultures were placed in serum-free medium after glutamate treatment, mitochondria were progressively loaded with calcium. At 5 min after glutamate exposure mitochondrial calcium deposits were prominent in both cortical and hippocampal cultures, followed by a further steady increase and neuronal death within 24 h. When cultures were allowed to recover after glutamate treatment in serum-containing MEM, calcium sequestration and ultrastructural changes of mitochondria were essentially absent, and neurons survived. No differences between cortical and hippocampal cultures were observed. The data demonstrate that prevention of glutamate neurotoxicity by serum proteins is associated with prevention of post-glutamate mitochondrial calcium accumulation.
利用草酸盐 - 焦锑酸盐染色结合电子显微镜技术，在原代皮质和海马培养物中研究了血清蛋白对谷氨酸诱导的线粒体钙积累的影响。培养物取自妊娠第17 - 19天的大鼠胚胎，在含有5%天然马血清的最低限度基本培养基（MEM）中培养8天。此时，将培养物暴露于100微摩尔或1.0毫摩尔谷氨酸中5分钟，然后在无血清或含血清的培养基中恢复。在谷氨酸处理前、谷氨酸暴露结束时以及恢复5分钟、30分钟、6小时和24小时后评估线粒体钙积累情况。在对照条件下以及谷氨酸暴露结束时，线粒体仅含有少量钙沉积物。如果在谷氨酸处理后将培养物置于无血清培养基中，线粒体钙含量会逐渐增加。谷氨酸暴露后5分钟，皮质和海马培养物中的线粒体钙沉积物都很明显，随后进一步稳定增加，24小时内神经元死亡。当在含血清的MEM中谷氨酸处理后让培养物恢复时，线粒体的钙螯合和超微结构变化基本不存在，神经元存活。未观察到皮质和海马培养物之间的差异。数据表明，血清蛋白对谷氨酸神经毒性的预防与谷氨酸后线粒体钙积累的预防有关。
