Pathophysiology of mitochondrial control for neuronal death and brain protection

缺血性神经元死亡是线粒体介导的。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性影响的等级。在培养的大鼠海马神经元短时氧糖剥夺（OGD）后观察到线粒体超极化。由于超极化被认为与随后的死亡模式有关，线粒体膜电位的作用可能与细胞质Ca^<++> [Ca^<2+>]c缓冲能力和调控凋亡级联的PKB信号通路有关用葡萄糖树培养基（30OGD, 120OGD）厌氧培养ogd海马神经元后的胞浆Ca^<++> [Ca^<2+>]c缓冲能力30或120分钟。OGD后，神经元加载fluo-3或rhod-2，监测细胞质和线粒体Ca^<++>浓度（[Ca^<2+>]c, [Ca^<2+>]m）。为了评估线粒体膜电位（MMP），采用了四甲基罗丹米，乙酯，高氯酸盐（TMRE）。用0.5mM谷氨酸刺激神经元去极化。谷氨酸负荷使[Ca^<2+>]c急剧升高，对照组和30OGD组[Ca^<2+>]m的升高紧随其后。30OGD [Ca^<2+>]c在达到峰值后比对照组下降得更快，[Ca^<2+>]m在30OGD逐渐增加，而对照组保持不变。30OGD中fluo-3 （AUCf）归一化荧光曲线下面积显著低于对照组，而30OGD中rhod-2 （AUCr）归一化荧光曲线下面积显著大于对照组。在120OGD时，[Ca^<2+>]c和[Ca^<2+>]m的增加减弱。30OGD后线粒体Ca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OGD和Akt我们采用ELISA法检测磷酸化Akt。短期OGD（30min）增加Akt磷酸化，而较长OGD（120min）抑制磷酸化。我们现在正在用免疫印迹法确认这种明显的变化。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