Effects of Asphyxial Hypoxic Preconditioning on Apoptosis Percents and the Expression of Caspase-3, Bcl-2 after Intracranial Hemorrhage in Rats<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

安立新^＃　彭宇明^＃　王保国^*
王雅杰^▲　孙梅珍^△
＃首都医科大学2003级博士研究生
^*中国医学科学院首都医科大学附属北京天坛医院麻醉科， 北京 100050
^▲中国医学科学院首都医科大学附属北京天坛医院检验科， 北京 100050
^△北京市神经外科研究所，北京 100050

Li-xin An＃, Yu-ming Peng＃, Bao-guo Wang*, Ya-jie Wang^▲, Mei-zhen Sun^△
＃Capital University of Medical Sciences, Beijing 100054, China.
^*Department of Anesthesiology , Beijing Tiantan Hospital, Affiliate of Capital University of Medical Sciences, Chinese Academy of medical Sciences, Beijing 100050, China.
^▲Department of clinical laboratory , Beijing Tiantan Hospital, Affiliate of Capital University of Medical Sciences, Chinese Academy of medical Sciences, Beijing 100050, China.
^△Beijing Neurosurgical Institue, Beijing 100050, China.

ABSTRACT<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

Objective：To observe the effects of asphyxial hypoxic preconditioning on apoptosis percents and the expression of caspase-3, bcl-2 after intracranial hemorrhage(ICH) in rats .
　　Methods：180 male SD rats were randomly allocated into three groups: sham group (Group S, n=60) , ICH group (Group I, n=60) and preconditioning group(Group P, n=60). 40 live rats in every group were detected at 6 h, 24 h, 48 h and 72 h after ICH. The rats were anesthetized with 10% chloral hydrate 40 mg／kg celiac injection, paralyzed with vecuronium 2mg／kg and mechanically ventilated. The rats in P group were pretreated with four times of preconditioning, by being stopped ventilation for 1 min and reventilated for 5 min per circle. One hour later, endotracheal catheter was extubated. ICH model was made by injection of 50m autos blood into the caudate nucleus in group P and I, 50msaline in Group S. Apoptosis percents were detected by flow cytometer (FCM) and the expression of caspase-3, bcl-2 in cerebral tissues were detected by using immunohistochemistry techniques at 6 h, 24 h, 48 h and 72h after intracerebral hemorrhage.
　　Results：Apoptosis percents raised obviously at 24 h and peaked at 72 h in Group I and Group P. In group P apoptosis percent was 11.62±3.64 and 15.80±3.30 at 48 h and 72 h, respectively, which was much lower than that in groupI (17.76±3.49 and 21.06±2.81) (P<0.01). Caspase-3 expression raised obviously at 24 h and peaked at 72 h. The number of caspase-3 positive cells was more in group I than that in group P at 24 h, 48 h and 72 h after ICH. Whereas the number of bcl-2 positve cells in group P (18.71±1.19) was more than that in group I (14.67±2.17) at 48h (P<0.01).
　　Conclusion：Asphyxial hypoxic preconditioning enhances the expression of bcl-2, decreases the expression of caspase-3, reduces apoptosis percents, and demonstrates cerebral protective effect after ICH in rats.
　　Key words：Hypoxic preconditioning; Apoptosis; Caspase-3; Bcl-2
　　Corresponding author：Bao-guo Wang, MD; E-mail: wbgttyy@sina.com

随着对出血性脑损伤分子机制的深入了解及凋亡机制的逐步阐明, 通过抑制神经元凋亡防治脑出血(Intracranial Hemorrhage，ICH)损伤成为研究脑保护的热点。通过短暂的缺氧刺激使组织细胞在后续长期缺血缺氧中得到保护，称为缺氧预处理(Hypoxic Preconditioning, HPC)。HPC通过调动抑制凋亡基因bcl-2^[1]，增强NF-kB与DNA的结合能力^[2]和抑制Caspase-3^[3]的激活等途径抑制神经元细胞凋亡。已有研究证实停通气缺氧预处理大鼠通过调动急性被动机制提高了大鼠对缺氧的耐受力^[4]。本实验观察停通气缺氧预处理对脑出血后大鼠的凋亡百分率和bcl-2和caspase-3表达的影响, 探讨HPC的机制。

　　一、资料与方法
　　1. 实验动物准备
　　雄性清洁级SD大鼠180只，体重260～290g(由首都医科大学动物中心提供，许可证号SCXK11-00-0012), 随机分为三组，假手术组(S组)60只，脑出血对照组(I组)60只，缺氧预处理组(P组)60只。每组从存活大鼠中取40只在脑出血(ICH)后6h、24h、48h和72h各时间点进行检测。实验室由北京神经外科研究所提供，许可证号SYXK(京)2003-0001。饲养室恒温22℃，12小时间隔照明，标准鼠料喂养。
　　10％水合氯醛(40mg／kg)腹腔麻醉后，分离并暴露右侧股血管，从股静脉置入20号套管针连接微量输液泵以2ml／h的速度输入乳酸钠林格氏液，从股动脉置入14号静脉留置套管针连接压力转换器，使用Spacelab(型号: 90367)多功能生理监护仪监测有创动脉压和心电图。经口插入气管导管(14G套管针外管)后应用微型动物呼吸机(浙江医科大学医学仪器实验厂生产)进行纯氧机械通气(潮气量1 ml／100 g，呼吸频率60次／min,吸: 呼=1: 2)，静脉注射维库溴胺(2 mg／kg)消除大鼠自主呼吸，应用灯照法维持直肠温度37.5±0.3℃。
　　2. 模型的建立
　　P组大鼠在机械通气10分钟后关闭呼吸机并断开呼吸机接头，停通气1分钟后立即重新连接呼吸机接头恢复机械通气5分钟，此为预处理1次，反复进行4次。麻醉1小时左右腹腔内追加10％水合氯醛(10mg／kg)。I组和S组持续机械通气，不行缺氧预处理。所有大鼠(P组在预处理4次之后)机械通气1小时自主呼吸恢复，呼吸频率>50次／min，胸廓起伏超过1cm时，吸空气无缺氧时，拔出气管导管。
　　大鼠立即俯卧位固定于立体定位仪上，将微量注射仪尖端定位于前囟前0.5 mm, 中线右侧旁开3 mm处。用微型手钻在此处钻一直径0.5 mm的小孔。P组和I组从股动脉处取大鼠自体血50ml, 沿所钻小孔进针6mm(相当于大鼠尾状核部位), 按20 ml／min的速度缓慢匀速注入尾状核,留针10 min, 缓慢退针。S组注入同等剂量的生理盐水。记录血流动力学变化，观察1小时后结扎股动静脉，缝合皮肤，在4L／min吸氧箱中观察30 min后送回笼。<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

3. 凋亡百分率测定　
　　在各个时间点取5只存活大鼠在10%水合氯醛(90mg／kg)腹腔深度麻醉后断头取脑，于针道处冠切后，在尾状核血肿右侧2mm处取1mm³脑组织4块，置于70％酒精中固定留做细胞凋亡百分率的检测。
　　(1)制备单细胞悬液及流式细胞仪(Flow cytometer，FCM)检测凋亡
　　PBS浸泡10min去掉固定标本的酒精，放入0℃ PBS缓冲液约1ml，用眼科剪将脑组织剪成为0.1 mm³左右组织碎片，1ml移液器吹打15次，180钼滤网0℃ PBS冲洗过滤，收集滤液，300钼滤网再次0℃ PBS冲洗过滤，收集滤液。滤液用0℃ PBS稀释至5m1，1，000r/min离心5min，弃上清。0℃ PBS清洗2次(重悬至5m1，1,000r/min离心5 min，弃上清)。滴加PI/triton X-100 DNA染色液1.0ml 4℃染色30分钟，500钼铜网过滤，滤液上机检测。
　　(2)流式细胞仪上机检测条件
　　应用FACS Calibur型流式细胞仪：氩离子激发光波长488nm，功率300mw，测定每样本10,000个细胞，经MODFIT软件分析细胞数DNA分布图。