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

在本实验中，我们开发了一种能够在体内观察线粒体电位的新系统。使用电位测定染料JC-1测量线粒体电位，该染料在线粒体中以电位依赖的方式积累，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/绿比值表示线粒体电位。将2 μl染料（JC-1, 6 μM；环糊精，20%；DMSO, 0.5%；生理盐水）通过直流电极注射到顶叶颞叶皮层，持续2分钟。在染料加载过程中，直流电位稳定，无扩散下降现象。在一项初步研究中，证实在注射JC-1染料后的24小时内没有引起组织学变化（苏木精-伊红染色）。激发光（每次照射2秒）由150瓦氙灯产生，并通过直径5毫米的光纤传导到皮质表面。在氙灯和光纤之间放置了一个虹膜，以防止染料光氧化。利用安装在带有绿色和红色带通滤波器的微距瞄准镜上的电冷却CCD相机，每隔20秒测量直流记录点附近470 X 470 μm区域内的红色和绿色荧光强度。将指示线粒体电位的红/绿荧光比值归一化为红/绿比值的控制值。我们发现线粒体在缺血期间通过逆转ATP合成酶活性来消耗ATP，这通过消耗ATP损害细胞膜电位，并且使用二氮氧化合物会导致化学预处理，从而降低线粒体电位。

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.