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

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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.