Fig. 3.

Evaluation of mitochondrial injury after ischemia. A, Representative microphotographs showing mitochondrial accumulation of the fluorescent probe, Rh 123, and the effects of mitochondrial membrane ionophores DNP and valinomycin.Top, middle, and bottomrows are photographs singly exposed to Rh 123, both doubly exposed to Rh 123 and counterstained by Hoechst 33258, and with high-powered magnification, respectively. Small red particles represent the fluorescent signals of Rh 123 accumulated by the transmembrane potential of mitochondria, suggesting the existence of viable mitochondria. This accumulation of Rh 123 was diminished by pretreatment with ionophores DNP and valinomycin. The vesicular fluorescent signals were observed to be weakened and decreased by the preincubation of the ionophores. Scale bars: 25 μm, top and middle; 10 μm,bottom. B, Fluorescent photomicrographs in both nonischemic (top) and ischemic hemispheres (bottom) of wild-type (Wt;left) and Sod2 knock-out mutant mice (right). Note that, in both wild-type and knock-out mutant mice, some cells were Rh 123-negative in the nonischemic area; Rh 123-positive cells also were observed in the ischemic brain tissue. Thus, not all of the cells without the small Rh 123 fluorescent particles necessarily indicate a mitochondrial injury. Scale bar, 10 μm. C, Ratio of the number of Rh 123-positive cells to that of total cells in the lateral caudoputamen of the ischemic hemisphere, a possible ischemic core, in the present ischemia model at 4 and 24 hr ischemia. Values are mean ± SE; *p < 0.05, **p < 0.01, and ***p < 0.001 versus control, ANOVA.^†p < 0.05 versus wild-type, ANOVA. At 4 hr ischemia, the ratio of Rh 123-positive cells was inclined to decrease as compared with the control value, but no significant difference was observed between wild-type and knock-out mutant mice. However, these cells decreased in both groups at 24 hr ischemia and were significantly less in knock-out mutants than in wild-type mice.