[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.

CONCLUSIONS

Cytochrome C release is one of the early biochemical changes of neuronal apoptosis after hypoxia-ischemia in neonatal rat brain.