【摘要】 目的　探讨全身低温对缺氧缺血性脑损伤的保护作用及其机理。方法　结扎 7日龄Wistar大鼠左侧颈总动脉后吸入 7 7%氧气 6 0min制成脑缺氧缺血 (HI)模型 ,随机分成低温组(30℃ ,n =18)和常温组 (36℃ ,n =18)给予维持恒定目标温度 10h。在HI后 2 4h ,每组 8只取脑进行匀浆用于半胱天冬酶 2 ,3(caspase 2 ,3)活性测定和Western蛋白印迹检查 ,其余每组 10只在HI后 72h处死 ,取脑进行石蜡包埋 ,冠状切片 10 μm用于活性caspase 3,凋亡诱导因子 (AIF) ,发夹寡核苷酸探针原位杂交 (HPP)检测凋亡及相关因子 ,微管相关蛋白 2 (MAP 2 )免疫组化染色用于计算脑梗塞体积和海马CA1神经元丢失 ,HE染色计算脑损伤积分。结果　常温组在HI后 2 4h损伤侧大脑半球caspase 2 ,3的活性 [(2 7 7± 14 7)、(94 9± 5 3 1) pmol/(min·mg蛋白 ) ]均明显高于正常对照组 [(7 6± 0 7)、(12 9± 0 5 ) pmol/(min·mg蛋白 ) ]和低温干预组 [(7 9± 3 4 )、(2 1 1± 18 7)pmol/(min·mg蛋白 ) ],P <0 0 1;而低温组与正常对照组相比无明显差异。Western蛋白印迹结果显示低温组caspase 3的激活受到明显抑制。免疫组化显示常温组活性caspase 3及AIF的阳性细胞数(中位数 14 8 5 ;2 2 /视野 )明显高于低温组 (中位数 4 8 5 ;9/视?更多还原

【Abstract】 Objective Recent studies suggest that hypothermia may be a potential treatment for perinatal hypoxic-ischemic （HI） brain damage. But the mechanisms of this effect are not well known. In the present study, the protective effect of systemic hypothermia as well as effect on apoptosis and associated biochemical events were investigated on neonatal rats with HI brain damage. Methods Seven-day-old Wistar rats were subjected to left carotid artery ligation and hypoxia was persisted for 60 min. Immediately at the end of hypoxia, the animals were maintained either at 36℃ or 30℃ for 10 h at random. Caspase-2, 3 activity in brain homogenate was detected with Western blotting at 24 h post-HI （ n =8 for each group）. Immunoactivity of microtubule-associated protein-2 （MAP-2）, active caspase-3, apoptosis inducing factor （AIF） and oligonucleotide hairpin probe staining were detected at 72 h post-HI. The infarct volume, neuronal loss in CA₁ sector of hippocampus as well as brain injury scoring were calculated according to MAP-2 staining and hematoxylin and eosin staining. Results Caspase-2, 3 activities were much higher in the normothermia group [（27.7±14.7）, （94.9±53.1） pmol/（min·mg protein）] at 24 h post-HI than those of hypothermia [（7.9±3.4）, （21.1±18.7） pmol/（min·mg protein）] and normal control groups [（7.6±0.7）, （12.9±0.5） pmol/（min·mg protein）] （ P< 0.01）. The activities were not significantly different between hypothermia group and normal control group. Western blotting showed that caspase-3activation process was blocked by hypothermia. The number of active caspase-3 and AIF positive cells in the cortex of ipsilateral hemisphere was much higher in the normothermia group （median:148.5; 22 /field） than that of hypothermia group （median: 48.5; 9/field） （ P< 0.05）. The number of apoptotic cells as judged by oligonucleotide hairpin probe labeling was much higher in normothermia group （median: 144/field） than that of hypothermia group （median: 133/field） （ P< 0.05）. The brain injury scoring, infarct volume and neuronal loss in CA₁ area of hippocampus were much less in the hypothermia group [10.4±2.9; 40.5±34.8)mm 3; 25.7±11.5]than that of normothermia group [14.2±3.5; （73.9±22.4） mm 3; 37.4±10.6, P< 0.05]. Conclusions Systemic hypothermia for 10 h after hypoxia-ischemia seemed to be effective in reducing brain damage and the mechanism is associated with alteration of apoptotic pathway.更多还原