Abstract OBJECTIVE: To investigate the effects of mild hypothermia on sequential events of neuronal apoptosis following hypoxic-ischemic brain damage (HIBD) in neonatal rats. METHODS: A model of HIBD was prepared by ligating the left common carotid artery in 7-day-old rats, followed by 8% hypoxia exposure. HIBD rats were randomly assigned into a hypothermia group (rectal temperature = 33 ℃) and a normothermia group (rectal temperature = 36 ℃). TUNEL, Haematoxylin and Eosin, and Nissl staining were used to detect neuronal apoptosis. Western blotting, RT-PCR and enzyme activity measurement were used to evaluate the changes of plasma and mitochondrial cytochrome c (Cyt c), caspase-3 mRNA expression and caspase-3 enzyme activity, respectively. RESULTS: The number of apoptotic cells in the ipsilateral hemisphere of the hypothermia group was significantly reduced compared with that of the normothermia group at 72 hrs post-HI (6.4±1.7% vs 25.3 ± 1.5%) (P<0.01).Analysis of Western blotting showed that Cyt c levels increased in the cytosolic fraction, but decreased significantly in the mitochondrial fraction in the ipsilateral hemisphere of the hypothermia group at 24, 48 and 72 hrs of HI insult compared with the normothermia group (P<0.05). Caspase-3 mRNA increased significantly after 24 hrs post-HI in the normothermia group, and this change became more pronounced with time. Mild hypothermia treatment decreased significantly caspase-3 mRNA expression at 24, 48 and 72 hrs post-HI (P<0.05). Caspase-3 activity gradually increased 2 hrs after HI insult and peaked at 24 hrs in the normothermia group. Mild hypothermia treatment resulted in a significant reduction in caspase-3 activity in the ipsilateral hemisphere, with an optimal effect produced at 24 hrs post-HI (2.42 ± 0.5 RFU vs 34.7 ± 3.2 RFU; P＜0.01). CONCLUSIONS: Mild hypothermia treatment attenuates neuronal apoptosis following HIBD, possibly through a reduction in Cyt c release from mitochondria and an inhibition of caspase-3 mRNA expression and its enzyme activity. ［Chin J Contemp Pediatr, 2007, 9 (1) :37-41］
