Pathophysiology of mitochondrial control for neuronal death and brain protection

缺血性神经元死亡是由线粒体介导的。我们已经证明，线粒体膜电位表现出特定的行为，取决于缺血影响的级别。培养的大鼠海马神经元在短期缺氧缺糖(OGD)后观察到线粒体超极化。由于超极化可能与随后的死亡模式有关，线粒体膜电位的作用可能与胞内钙缓冲能力和调节细胞凋亡级联反应的PKB信号通路有关。1.培养的海马神经元在DISH中与葡萄糖树(30OGD，120OGD)厌氧孵育30或120min后，线粒体膜电位的作用可能与胞内钙缓冲能力有关。OGD后，神经元负载Fluo-3或Rhod-2，监测胞浆和线粒体钙浓度([Ca^&lt；2&gt；]c，[Ca^&lt；2&gt；]m)。为评价线粒体膜电位，用三甲基罗丹明(TmRe)(四甲基罗丹明，乙酯，高氯酸盐)Lo…更有说服力。用0.5 mM谷氨酸诱导神经元去极化，谷氨酸负荷使[Ca^&lt；2&gt；]c急剧升高，随后对照组和30OGD组[Ca^&lt；2&gt；]m升高。达到峰值后，30OGD组较对照组[Ca；2；gt；c]c下降更快，30OGD组[Ca2；m]m进行性升高，而对照组则无明显变化。30OGD组归一化荧光曲线下面积(AUCf)显著低于对照组，而Rhod-2归一化荧光曲线下面积(AUcr)显著大于对照组。在120OGD时，线粒体[Ca^&lt；2&gt；]c和[Ca^&lt；2&gt；]m的增加是钝化的，30OGD后线粒体的钙缓冲能力增强。这种缓冲能力可能与短期缺血发作后的预适应有关。2 OGD和Akt我们采用ELISA法检测磷酸化Akt。短期OGD(30min)增加Akt的磷酸化，而长时间的OGD(120min)则抑制Akt的磷酸化。我们现在通过使用Western blotting来确认这一明显的变化。较少

Ischemic neuronal death is mediated by mitochondria. We have demonstrated that mitochondrial membrane potential showed a specific behavior depending on the grade of ischemic impact. Mitochondrial hyperpolarization has been observed after short-term oxygen-glucose deprivation (OGD) in cultured rat hippocampal neurons. Since hyperpolarization putatively links to subsequent death mode, the role of mitochondrial membrane potential may relate to cytosolic Ca^<++> [Ca^<2+>]c buffering capacity, and signaling pathway of PKB, which regulate apoptotic cascade.1 Cytosolic Ca^<++> [Ca^<2+>]c buffering capacity after OGDHippocampal neurons cultured in dish were anaerobically incubated for 30 or 120 min with glucose-tree medium (30OGD, 120OGD). After OGD, neurons were loaded fluo-3 or rhod-2 for monitoring cytosolic and mitochondrial Ca^<++> concentration ([Ca^<2+>]c, [Ca^<2+>]m). To evaluate mitochondrial membrane potential (MMP), TMRE (tetramethylrhodamie, ethyl ester, perchloratye (TMRE)) was lo … More aded. Neurons were exposed to 0.5mM glutamate to induce depolarization.The [Ca^<2+>]c, was steeply increased by glutamate load and increase in [Ca^<2+>]m followed in control and 30OGD. The [Ca^<2+>]c in 30OGD more rapidly decreased than that in control after it reached peak, and [Ca^<2+>]m progressively increased in 30OGD, while it remained constant in control. The area under the curve of normalized fluorescence of fluo-3 (AUCf) in 30OGD was significantly lower than that oin control, while the area under the curve of normalized fluorescence of rhod-2 (AUCr) in 30OGD was significantly larger than control. In 120OGD, increase in [Ca^<2+>]c and [Ca^<2+>]m was blunted.Mitochondrial Ca buffering capacity after 30OGD was enhanced. This buffering capacity may link to preconditioning after short term ischemic episode.2 OGD and AktWe employed ELISA for detection of phosphorylated Akt. Short-term OGD(30min) increased phosphorylation of Akt, while longer OGD (120min) suppressed phosphorylation. We are now confirming this distinct change by using Western blotting. Less