Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron
Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron
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DOI：
10.1016/s0197-0186(02)00228-0
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发表时间：
2003-08-01
影响因子：
4.2
通讯作者：
Iwao, Y
中科院分区：
文献类型：
--
作者：
Iijima, T;Mishima, T;Iwao, Y
缺血限制了氧和葡萄糖向细胞的输送，并干扰了线粒体膜电位（MMP）的维持。MMP调节高能磷酸盐的产生和凋亡级联。因此，MMP是决定神经元命运的重要参数。根据缺血性影响的分级，MMP的时程差异尚未阐明。在短期氧糖剥夺前和后，监测MMP和细胞内ATP含量。将接种在用于荧光显微镜检查的35 mm有孔培养皿中的原代海马培养物置于密封室中进行厌氧孵育。100%氮气连续流入腔室，并更换无葡萄糖培养基，使氧-葡萄糖剥夺（OGD）的条件，从而推断缺血。MMP通过电压依赖性染料JC-1的荧光在荧光显微镜下进行评估。在指定的OGD时间段后，在接种于96孔板中的海马培养物中通过荧光素酶反应评价细胞内ATP含量。在OGD期间，MMP降低至0.72 +/- 0.03（标准化JC-1荧光），然后在30 min OGD后60 min复氧期间增加至超极化水平1.99 +/- 0.12。OGD 60 min后MMP下降，复氧时偶恢复。OGD和复氧90分钟后，MMP降低，再也没有恢复。在30 min OGD和30 min OGD后30 min复氧后，细胞内ATP含量分别为8.1 +/- 6.6和3.2 +/- 1.9%; 60 min OGD未显著改变这些水平（7.1 +/- 5.8，2.6 +/- 0.5%）。OGD后的超极化并不伴随ATP的产生。这一观察结果表明，在复氧和FoF 1-ATP合酶的干扰电子再进入内膜的抑制。OGD后能量产生系统的病理发现可能为解释缺血后能量衰竭提供线索。(C)2003爱思唯尔科技有限公司版权所有。
Ischemia limits the delivery of oxygen and glucose to cells and disturbs the maintenance of mitochondrial membrane potential (MMP). MMP regulates the production of high-energy phosphate and apoptotic cascading. Thus, MMP is an important parameter determining the fate of neurons. Differences in the time course of MMP according to the grading of the ischemic impact have not been clarified. MMP and intracellular ATP contents were monitored before and after short-term oxygen-glucose deprivation. A primary hippocampal culture seeded in a 35 mm fenestrated dish for fluorescence microscopy was mounted in a sealed chamber for an anaerobic incubation. A continuous flow of 100% nitrogen into the chamber and a replacement of glucose-free medium allowed the condition of oxygen-glucose deprivation (OGD), thereby extrapolating ischemia. MMP was evaluated by the fluorescence of a voltage-dependant dye, JC-1, under fluorescence microscopy. The intracellular ATP content was evaluated in a hippocampal culture seeded in a 96-well plate by the luciferin-luciferase reaction after a designated period of OGD. During OGD, MMP decreased to 0.72 +/- 0.03 (normalized JC-1 fluorescence), then increased to the hyperpolarized level 1.99 +/- 0.12 during 60 min reoxygenation after 30 min OGD. MMP after 60 min OGD decreased and recovered occasionally during reoxygenation. After 90 min OGD and reoxygenation, MMP was reduced and never recovered. The intracellular ATP content was 8.1 +/- 6.6 and 3.2 +/- 1.9% after 30 min OGD and 30 min reoxygenation following 30 min OGD, respectively; 60 min OGD did not significantly change these levels (7.1 +/- 5.8, 2.6 +/- 0.5%). Hyperpolarization after OGD did not accompany ATP production. This observation suggests the inhibition of electron reentry into an inner membrane during reoxygenation and the disturbance of FoF1-ATP synthase. This pathological finding of an energy-producing system after OGD may provide a clue to explain post-ischemic energy failure. (C) 2003 Elsevier Science Ltd. All rights reserved.