新生大鼠脑缺氧缺血后细胞色素C释放与凋亡的关系

目的　探讨未成熟脑在缺氧缺血 (HI)后细胞色素C释放的时相变化及细胞色素C释放与半胱天冬酶的激活和DNA损伤的关系。方法　 7日龄Wistar新生大鼠在缺氧缺血后不同时间点处死取脑 ,部分脑组织进行手工匀浆分离胞浆和线粒体成分进行Western蛋白印迹检查 ,部分进行组织切片用于细胞色素C免疫组化染色和免疫荧光双染色。结果　Western蛋白印迹显示线粒体中细胞色素C在HI后 1h未见明显变化 ,HI后 14hHI侧较对侧 (H)减少 4 6 % ;而HI侧脑组织胞浆中细胞色素C的含量在HI后 1h已明显增加 ,在HI后 14h增加更明显。免疫组化结果显示大脑皮层细胞色素C阳性细胞数在HI后 1h为 8 4± 1 8个 /高倍视野 ,明显高于正常对照的 1 5± 0 8个 /高倍视野(P <0 0 1) ,并随HI后恢复时间的延长阳性细胞逐渐增加 ,在HI后 14h达到峰值 (2 9 0± 5 2个 /高倍视野 ) ;而TUNEL阳性细胞数在HI后 1h为 14± 3个 /高倍视野 ,明显高于正常对照的 1 5± 0 8个 /高倍视野 (P <0 0 1) ,在HI后 2 4h达到峰值 (2 86± 86个 /高倍视野 )。荧光染色发现细胞色素C释放与活性半胱天冬酶 3仅在HI后早期同时染色 ,并且阳性细胞表现为核浓缩 ,随HI后恢复时间的延长 ,细胞色素C的阳性细胞明显少于活性半胱天冬酶 3的阳性细胞 ;细胞色素C与TUNEL

Cytochrome C release and apoptosis in neonatal rat cerebral hypoxia-ischemia

OBJECTIVE: To study the relation of cytochrome C release from mitochondria to cytosol and neuronal apoptosis after cerebral hypoxia-ischemia (HI) in neonatal rats. METHODS: Hypoxia-ischemia was induced in 7-day-old rat pups by ligation of left carotid artery and 7.7% oxygen was inhaled for 55 min. The pups were sacrificed and the brains were taken out at different recovery time. Some of the brains were homogenized and cellular fraction of mitochondria and cytosol was isolated with different speed centrifugation. The cellular fraction was used for Western blotting. Some of the brains were sectioned and stained with antibody against cytochrome C and TUNEL as well as double labeling with different combinations. RESULTS: Western blots showed that cytochrome C in mitochondria was not reduced significantly at 1 h, but reduced markedly at 14 h in ipsilateral hemisphere post-HI. However, the immunoreactivity of cytochrome C in cytosol was increased markedly at 1 h post-HI and reached peak at 14 h post-HI. The number of cytochrome C positive cells in the cortex was increased significantly at 1 h (8.4 +/- 1.8/visual field) compared to normal control (1.5 +/- 0.8/visual field) (P < 0.01) and reached peak at 14 h (29.0 +/- 5.2/visual field) post-HI. The number of TUNEL positive cells increased significantly at 1 h post-HI (14 +/- 3/visual field) compared to normal control (1.5 +/- 0.8/visual field) (P < 0.01) and reached peak at 24 h (286 +/- 86/visual field). The double labeling of cytochrome C and active caspase-3 showed that they colocalized well at 3 h after HI. Furthermore, the positive cells showed nuclei condensation. There were more active caspase-3 positive cells at late recovery (24 h and on) after HI. The double labeling of cytochrome C and TUNEL showed only part of Positive cells colocalized. The cells with cytochrome C strong staining showed TUNEL negative or weakly positive. The cells with TUNEL strong staining showed weakly cytochrome C staining. CONCLUSION: Cytochrome C release is one of the early biochemical changes of neuronal apoptosis after hypoxia-ischemia in neonatal rat brain.