【摘要】 目的　探讨亚低温治疗对新生大鼠缺氧缺血脑损伤 (HIBD)线粒体功能的保护作用及对细胞凋亡的影响。方法　模型动物随机分为常温缺氧缺血组 (肛温 37℃ )和亚低温缺氧缺血组 (肛温 33℃ ) ;对照组为假手术动物 ,于缺氧缺血后 0、2、6、2 4、4 8、72h检测线粒体ATP合成量 ,观察线粒体的超微结构变化 ,并用原位缺口末端标记法 (TUNEL)结合HE染色和Nissl染色检测脑细胞死亡。结果　 37℃缺氧缺血组结扎侧大脑组织线粒体ATP合成量 2h开始下降 ,2 4h下降最明显达正常对照组 36 % (4 5 .0± 6 .1vs 12 2 .5± 6 .7,P <0 .0 1) ,亚低温治疗能显著改善线粒体ATP合成量 ,以亚低温治疗 2 4h升高最明显 (99.0± 8.8vs 4 5 .0± 6 .1,P <0 .0 1) ,37℃缺氧缺血组大鼠脑线粒体肿胀 ,嵴模糊 ,断裂明显 ,基质电子密度增高或出现部分透明区 ,少数线粒体空泡变性 ,而亚低温治疗后大部分线粒体结构保持完整 ;72h亚低温还可显著降低脑细胞凋亡发生率 (2 5 .3± 1.5vs 6 .4±1.7,P <0 .0 1) ,而对细胞坏死无明显改善 (13.0± 1.4vs 11.2± 0 .7,P >0 .0 5 )。结论　亚低温治疗可通过保护缺氧缺血脑损伤后的线粒体功能而特异性地减轻脑细胞凋亡更多还原

【Abstract】 Purpose To investigate the effects of moderate hypothermia on mitochondria function and neuronal apoptosis after hypoxia ischemia brain damgage in neonatal rats. Methods Seven day old rats were subjected to permanent unilateral carotid artery ligation and exposed to hypoxia (8% oxygen in nitrogen)for 2 h,and the rats were randomly divided into normothermia group (IN,rectal temperature:37℃)and moderate hypothermia group(IH,rectal temperature:33℃).Sham operated rat pups from the same litter used as a control group (CN and IN group).0,2,6,24,48,72 h after hypoxia ischemia(HI)insult,the ipsilateral hemisphere mitochondria were isolated for adenosine triphosphate (ATP) synthetic content assay using bioluminescence method based on luciferin luciferase reaction and mitochondria ultrastructure alteration observed by electron microscopy.Coronal sections from each group were used for evaluating neuronal death pattern by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining (TUNEL) combined with HE and Nissl neuronal staining. Results Compared with sham control group,mitochondria ATP synthetic content was decreased from 2 h after HI insult in HI-37℃ group,and a significant decrease being seen at 6 h(76.6±1.6 vs 122.5±6.7, P <0.05),reached the deepest point at 24 h which amount for 36% of control group (45.0±6.1 vs 122.5±6.7, P <0.01),and after that there was a slowly increase up to 72 h (86.8±3.1 vs 122.5±6.7, P <0.05).In contrast to HI-37℃,moderate hypothermia treatment showed a significant increase of ATP synthetic content at 6 h (102±10.9 vs 76.6±1.6, P < 0.05 ) and 24 h (99.0±8.8 vs 45.0±6.1, P <0.01).Most of mitochondria in hippocampus CA 1 area developed a considerable degree of swelling and disruption of cristal membrane even vacuolar degeneration,or increased electron density of matrix at 72 h of reflow in HI-37℃ group,whereas the majority of mitochondria were intact or mild injury in HI-33℃ group.A time dependent increase in the number of TUNEL positive neuron was noted in the hemisphere ipsilateral to carotid ligation,and moderate hypothermia could substantially decrease the apoptotic neuron ratio after HI insult,there was a significant difference between these two groups (25.3±1.5 vs 6.4±1.7, P <0.01) at 72 h after HI,but there was no significant amelioration of necrosis neuron ratio (11.3±0.7 vs 13.0±1.4, P >0.05). Conclusions These findings indicate the specific inhibition of apoptosis after cerebral hypoxia ischemia by moderate hypothermia and this inhibition effect could be through protect the mitochondria function (energy synthesis) and subsequent steps leading to neuronal apoptosis.更多还原