Figure 3.
A, Western blot analysis of BimL in the MCA territory of rat brains after tFCI. The band of BimL was evident at 24 kDa and peaked 6 hr after reperfusion. C, Control. B, Quantitative analysis (n = 4) showed a significant increase in BimL induction 6 hr after tFCI, compared with the healthy controls (C) (#p < 0.05). O.D., Optical density. C, Okadaic acid (OA) enhanced phosphorylation of BimL proteins. Intracerebroventricular injection of OA (100 ng/kg) was associated with a decrease in electrophoretic mobility of BimL expression (n = 4; #p < 0.05, compared with the vehicle-treated animals). BW, Body weight. D, Representative photomicrographs show immunohistochemistry for BimL in the caudate-putamen of the MCA territory after tFCI. After 6 hr of reperfusion, BimL expression was intensely increased compared with the same region in the nonischemic rat brains (C). After 24 hr of reperfusion, BimL immunoreactivity became weak but was still clearly observed in a considerable number of cells. Scale bar, 50 μm. E, Representative photomicrographs show double immunofluorescent staining for BimL and NeuN. Six hours after reperfusion, BimL-positive cells were observed in the ischemic caudate-putamen. NeuN immunoreactivity showed the distribution of neurons in the same view. Overlapped image of BimL and NeuN immunoreactivity demonstrated BimL-positive cells colocalized primarily with neurons. Scale bar, 8 μm. F, Western blot analyses of BimL in the cytosolic fraction and the mitochondrial fraction after tFCI. BimL was present in the cytosolic fraction from the nonischemic control brains (C) and was significantly induced 6 hr after reperfusion without alternations in its subcellular localization. Cytochrome oxidase (COX) was used as an internal control for the mitochondrial fraction. G, Western blot analysis of BimL in the ischemic MCA territory after intracerebroventricular injection of the vehicle (V) or SP600125, a selective JNK inhibitor. Six hours after tFCI, BimL was significantly increased in the vehicle-treated animals compared with the nonischemic control brains (C), but was inhibited by treatment with SP600125 in a dose-dependent manner. H, Statistical analysis showed a significant decrease in BimL immunoreactivity in the SP600125-treated group (1.0 mg/kg) compared with the vehicle-treated group (V) in the entire MCA territory on the ischemic side (n = 4; *p < 0.05). The statistically significant difference in BimL expression disappeared in the SP600125-treated group at a dose of 0.5 mg/kg, compared with the nonischemic control brains (C) (n = 4; #p < 0.05). Error bars represent mean ± SD.