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Therapeutic measures of brain ischemia from the point of view of mitochondrial potential.

从线粒体电位的角度治疗脑缺血的措施。

基本信息

批准号：
16390452
负责人：
MORITA Kiyoshi
金额：
$ 7.74万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390452/
关键词：
Mitochondria Brain ischemia Respiratory chain エネルギー動態

项目摘要

In the present experiment, we developed a new system which enables to observe mitochondrial potential in vivo. Mitochondrial potential was measured using a potentiometric dye, JC-1, which accumulates in a potential-dependent manner in mitochondria and subsequently forms J-aggregates from monomers. Since J-aggregates and monomers fluoresce red (590 nm) and green (530 nm), respectively, with excitation light (485 nm), the ratio of red / green indicates mitochondrial potential. Dye loading was performed by injecting 2 μl of dye (JC-1, 6 μM ; cyclodextrine, 20% ; DMSO, 0.5% ; in physiological saline) into the parieto-temporal cortex through the DC electrode over a period of 2 minutes. During the dye loading, DC potential was stable and no spreading depression was observed. In a pilot study, it was confirmed that no histological change (hematoxylin-eosin staining) was induced during a period of 24 hours after the injection of JC-1 dye. The excitation light (2 seconds exposure in each emission) was produced by a 150-watt xenon lamp and was conducted to the cortical surface through a optical fiber (diameter, 5 mm). An iris was placed between the xenon lamp and the optical fiber to prevent photooxidation of the dye. The intensities of red fluorescence and green fluorescence in an area (470 X 470 μm) adjacent to the DC-recording site were measured every 20 seconds using an electrically cooled CCD camera mounted on a macro scope with green and red bandpass filters. The ratio of red / green fluorescence, indicative of mitochondrial potential, was normalized to that of the control value of the red / green ratio. We found that mitochondria consume ATP during ischemia by reversing ATP synthetase activity, which compromises cellular membrane potential by consuming ATP and that the mitochondrial potential is decreased with use of Diazoxide, which induces chemical preconditioning.

在本实验中，我们开发了一种新的系统，可以在体内观察线粒体电位。使用电位染料JC-1测量线粒体电位，JC-1以电位依赖性方式在线粒体中积累，随后从单体形成J-聚集体。由于J-聚集体和单体在激发光（485 nm）下分别发出红色（590 nm）和绿色（530 nm）荧光，因此红色/绿色的比率指示线粒体电位。通过DC电极在2分钟内将2 μl染料（JC-1，6 μM ;环糊精，20% ; DMSO，0.5% ;在生理盐水中）注射到顶叶-颞叶皮质中进行染料加载。在染料负载期间，DC电位稳定并且没有观察到扩散抑制。在初步研究中，证实在注射JC-1染料后24小时内未诱导组织学变化（苏木精-伊红染色）。激发光（每次发射暴露2秒）由150瓦氙灯产生，并通过光纤（直径5 mm）传导至皮质表面。在氙灯和光纤之间放置光圈以防止染料的光氧化。使用安装在具有绿色和红色带通滤光片的宏观显微镜上的电冷却CCD相机，每20秒测量一次DC记录位点附近区域（470 × 470 μm）中的红色荧光和绿色荧光强度。将指示线粒体电位的红色/绿色荧光的比率标准化为红色/绿色比率的对照值。我们发现，线粒体消耗ATP在缺血过程中通过逆转ATP合成酶的活性，这损害细胞膜电位消耗ATP和线粒体电位降低与使用二氮嗪，诱导化学预处理。