【作者】 孙静；

【导师】 熊利泽； 董海龙；

【作者基本信息】 第四军医大学 ， 麻醉学， 2008， 硕士

【摘要】 近来研究发现,在脑缺血后,再灌注前,反复行数次短暂再灌注/缺血能明显减轻脑的梗死容积,减少再灌注损伤,具有脑保护作用,此现象被称为“缺血后处理”(Ischemic Postconditioning, IPost)。IPost作为减轻脑缺血再灌注损伤的新型干预措施,如何实施才能达到有效脑保护效应,目前仍未可知。线粒体敏感性转化孔(Mitochondrial Permeability Transition Pore, mPTP)是线粒体内、外膜之间的非特异性孔道,在再灌注早期开放,可以诱导细胞的凋亡。心肌IPost的机制研究提示,mPTP在IPost对心肌的保护作用中扮演重要的角色,可能是IPost保护机制的共同通路,然而,mPTP在脑IPost的作用如何,目前仍需进一步的探索和研究。因此,本研究正是利用大鼠中动脉阻闭模型(Middle Cerebral Artery Occlusion, MCAO),探寻和筛选脑IPost保护作用的有效时间,并且以mPTP为切入点,初步探讨脑IPost的保护机制。第一部分脑IPost保护作用的时间方案筛选及与缺血预处理比较方法1.缺血后处理对大鼠局灶性脑缺血再灌注损伤保护作用的时间方案筛选健康雄性SD大鼠60只随机分为6组:对照组(缺血90min)和IPost不同时间点组(缺血90min后,分别在15s/30s/1min/2min/5min进行再灌注/缺血处理,反复三次)。所有动物再灌注24h行神经功能障碍评分。24h后处死动物,TTC染色以确定梗死容积。2.缺血后处理与缺血预处理脑保护效应的比较30只SD大鼠随机分为3组:对照组,IPost组和缺血预处理组(Ischemic Pretconditioning, IPC)组(缺血20min,再灌注24h后行MCAO 90min)。所有动物再灌注24h行神经功能障碍评分。24h处死动物,TTC染色确定梗死容积。结果术后所有动物都存活。缺血再灌注后大鼠均表现一定的神经功能障碍。1.缺血再灌注24h后,IPost-15s/30s/1min/2min组神经功能障碍评分与对照组相比显著降低,尤以IPost-15s组低于其它各组,而IPost-5min组与对照组相比较无统计学意义。再灌注24h脑梗死容积百分比:IPost-15s组,IPost-30s组,IPost-1min组和IPost-2min组分别明显小于对照组,而IPost-5min组与对照组相比较差异无统计学意义。IPost-15s组脑梗死容积显著小于其它各组。最后确定IPost-15s组做为后续实验时间。2. IPost组和IPC组神经功能障碍评分显著低于对照组。IPost组和IPC组脑梗死容积小于对照组。但IPost组脑梗死容积大于IPC组。第二部分mPTP在脑缺血后处理保护机制中的作用研究方法1.mPTP开放剂苍术苷最低有效剂量的筛选再灌注前10min侧脑室注射不同浓度的mPTP开放剂苍术苷(Atractyloside, Atr),以确定可以促使mPTP开放的最低有效剂量。30只SD大鼠随机分为3组:对照组,Atr-1组(4mmol/L,15μl),Atr-2组(2mmol/L,15μl);再灌注24h行Garcia神经功能行为学评分。24h后处死动物,TTC染色以确定梗死容积。2.在体实验确定mPTP在脑缺血后处理保护效应中的作用利用侧脑室注射mPTP的关闭剂环孢菌素A(Cyclosporin A, CsA)和开放剂Atr来确定mPTP在脑IPost保护中的作用。70只SD大鼠随机分为7组。溶剂组(100%乙醇),对照组,对照组+CsA(2μmol/L,15μl再灌注前10min给予),对照组+Atr(再灌注前15min给予);IPost组,IPost+CsA(2μmol/L,15μl再灌注前10min给予),IPost+Atr(再灌注前15min给予)。所有动物分别再灌注24h、48h和72h行Garcia神经功能行为学评分。72h后处死动物,TTC染色确定脑梗死容积。3.透射电镜下观察脑缺血后处理保护效应中线粒体的超微结构实验分组同实验2,待模型制作成功后,再灌注72h后取损伤侧海马组织,通过透射电镜观察脑组织线粒体的超微结构。4.离体实验近一步证实mPTP在脑缺血后处理保护效应中的作用利用分光光度计检测mPTP开放和关闭的程度。70只SD大鼠随机分为7组。90min单纯缺血组,其余分组同实验2。所有动物于再灌注15min后提取线粒体,利用分光光度计在520nm波长下所测得吸光度值来检测mPTP开放和关闭的程度。结果1.缺血再灌注24h后,Atr-1组神经功能行为学评分与对照组相比有显著统计学差异。Atr-2组神经功能行为学评分显著大于Atr-1组。再灌注24h脑梗死容积百分比:Atr-1组明显大于对照组,而Atr-2组脑梗死容积百分比显著低于Atr-1组。最后确定Atr为2mmol/L,15μl作为后续实验剂量。2.在再灌注的各个时间点中,对照组+CsA和IPost组神经功能行为学评分显著高于对照组。IPost+Atr神经功能行为学评分与IPost组相比具有统计学意义。而IPost+CsA神经功能行为学评分与IPost组相比无统计学意义。再灌注72h后,对照组+CsA和IPost组的脑梗死容积显著低于对照组。IPost+Atr脑梗死容积显著大于IPost组。而IPost+CsA脑梗死容积与IPost组相比无统计学意义。对照组和溶剂组之间无统计学差异。3.通过透射电镜可以观察到正常线粒体呈杆状,结构完整,外膜平滑光整,线粒体嵴排列整齐,并且清晰可见;对照组和加入Atr组的线粒体结构破坏严重,线粒体肿胀,线粒体嵴消失,内部空泡化。而IPost组和加入CsA组线粒体的内部结构基本完整,线粒体嵴排列整齐,线粒体膜清晰可见。4.与单纯缺血组相比,其它各组吸光度的改变值明显升高;加入CsA组的吸光度改变值明显低于对照组;加入Atr组的吸光度改变值明显高于IPost组。但IPost+CsA与IPost组相比,吸光度的改变值无统计学意义。结论1.缺血后处理对大鼠局灶性脑缺血再灌注损伤的保护作用时间为15s再灌注/缺血,反复三次。缺血后处理的脑保护效应略弱于缺血预处理。2.脑缺血后处理可以抑制脑的缺血再灌注损伤,改善线粒体的结构和功能,最终起到脑的保护作用。3.脑缺血后处理的部分机制可能是通过再灌注早期抑制线粒体通透性转化孔的开放,改善了线粒体的结构和功能,最终起到脑的保护作用。更多还原

【Abstract】 Background: It has been reported that several brief episodes of reperfusion/ischemia before reperfusion, termed as ischemic postconditioning (IPost), could induce neuroprotection from ischemia/reperfusion (I/R) injury. IPost is a new intervention measure, which can alleviate cerebral I/R injury. IPost with a series of mechanical interruptions of reperfusion reduces I/R injury. However, the effective time of IPost agaist focal cerebral I/R injury is still unclear.Mitochondrial permeability transition pore (mPTP), as a nonspecific channel, remains closed during the ischemic period and only opens in the first few minutes of reperfusion. The open of mPTP can lead to mitochondrial swelling and efflux of cytochrome c, which induces apoptosis in the setting of I/R injury. IPost is taking reperfusion as point of cut-in to against I/R injury and relationship between IPost and mPTP in neuroprective as not been reported previously. Thus, the object of this study is to explore the effective time of IPost against focal cerebral I/R injury and the role of mPTP in the neuroprotection induced by IPost. Part 1 Neuroprotective effect of ischemic postconditioning against focal cerebral ischemia and compare the neuroprotective effects between ischemic postconditioning with ischemic preconditioningMethods1. The optimal protocol of ischemic postconditioning against focal cerebral I/R injury in rats60 male SD rats weighing 290-310g were randomized into six groups: Control group and IPost groups with different time intervals (Post-15s/30s/1min/2min/5min). Control group underwent occlusion of the middle cerebral artery occlusion (MCAO) for 90min. IPost was performed by three cycles of reperfusion/ischemia of different time intervals (15s/30s/1min/2min/5min respectively) after MCAO for 90min. The neurological defict scores (NDS) were evaluated 24h after reperfusion. Infarct volume, as a percentage of volume at normal cerebral hemisphere, was determined by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining.2. Compare the protective effect of ischemic postconditioning with that of ischemic preconditioning30 male SD rats were divided into three groups: Control group, IPost group and ischemic preconditioning (IPC). The rats in IPC group were subjected to 20min MCAO and reperfusion for 24h before 90min MCAO. The NDS was evaluated at 24h after reperfusion. The infarct volume was then assessed with TTC staining after the NDS. Results1. The NDS in Post-15s/30s/1min/2min groups at 24h after reperfusion were significantly lower than those in the Control. And there was no significant difference between the Control and the IPost-5min group. The infarct volume at 24h after reperfusion in Post-15s/30s/1min/2min groups were significant smaller than that in the Control. But there was no significant difference between the Control and IPost-5min. These results indicate the effective time of IPost is three cycles of 15s reperfusion/ischemia. It was determined to be used in the subsequent studies.2. The NDS in IPost-15s group and IPC group were better than that in the Control. The infarct volume at 24h after reperfusion in IPost-15s and IPC were significant smaller than that in the Control. But, the infarct volume in IP-15s group was larger than that in IPC.Part 2 The neuroprotective effect of ischemic postconditioning is mediated by inhibiting the mitochondrial permeability transition pore in ratsMethods1. The dose-response of Atractyloside in middle cerebral artery occlusion 30 male SD rats were divided into three groups: Control group, rats in Atractyloside-1 (Atr-1) and Atr-2 groups received intracerebroventricular injection 15μl, 4mmol/L and 2mmol/L respectively 15min before reperfusion. The NDS was evaluated at 24h after reperfusion. The infarct volume was then assessed with TTC staining after the NDS. Thus the optimal dose for subsequent experiments can be determined.2. Neuroprotective effect of ischemic postconditioning is mediated by inhibition of mPTP opening --- in vivo study70 male SD rats were divided into seven groups: Control group, IPost group and the additional groups of rats separately intracerebroventricular injection 15μl, 2μmol/L of Cyclosporin A (CsA,which inhibits mPTP opening) and 15μl, 2mmol/L Atractyloside(Atr, a mPTP opener) respectively 10min and 15min before reperfusion during Control group and IPost group. And the last group received 15μl Alcohol (the solvent of CsA) given at reperfusion. The NDS were assessed at 24, 48 and 72h after reperfusion. Then at the 72h after reperfusion, the animals were decapitated and brain infarct volumes were evaluated with TTC staining.3. Neuroprotective effect of ischemic postconditioning --- the ultrastructure of mitochondria changes detected by transmission electron microscopeExperimental groups as described in experiment 2. After performing MCAO,the ultrastructure of mitochondria of the hippocampus of injured hemisphere was detected by transmission electron microscope at 72h after reperfusion.4. Neuroprotective effect of ischemic postconditioning is mediated by inhibition of mPTP opening --- in vitro study70 male SD rats were divided into seven groups: Ischemic group: 90 min of ischemia and no reperfusion. The additional groups as described in experiment 2. All animals underwent 90 min of ischemia and 15min of reperfusion except the ischemic group. Then at the 15min after reperfusion, the animals were decapitated and brain mitochondria were isolated. The absorbance at 520nm (A520) of isolated mitochondria was measured by spectrophotometer. Opening of the mPTP was determined by Ca2+-induced mitochondrial swelling. The decrease in light scattering closely parallels the percentage of the mitochondrial population undergoing permeability transition.Results1. At 24h after reperfusion,the NDS in Atr-1 group was significantly lower than that in the Control. The NDS in Atr-2 group was significantly highly than that in the Atr-1 group.The infarct volume at 24h after reperfusion, Atr-1 group was significant larger than that in the Control. Atr-2 group was significant small than that in the Atr-1 group. These results indicate the optimal dose of Atr is 2mmol/L, 15μl. It was determined to be used in the subsequent studies.2. All the rats survived until 72h after reperfusion. At 24, 48 and 72h after reperfusion, the NDS in Control+CsA and IPost group were significantly higher than that in the Control. The NDS in IPost+Atr group was significantly lower than that in IPost. But, there was no significant difference between in IPost and IPost+CsA.The infarct volume at 72h after reperfusion in Control+CsA and IPost were significantly smaller than that in the Control. The infarct volume of IPost+Atr was significantly larger than that in the IPost. But, there was no significant difference between in IPost and IPost+CsA. And there was no significant difference between in Alcohol and Control. These results indicate mPTP activation aggravates cerebral tissue injury.3. The electron micrographs indicated that the shapes of mitocbondria were round and had numerous transversae cristae which exhibited parallel alignment. The outer and inner mitochondrial membranes were clearly distinguishable. This was a normal mitochondrial structure. Mitochondria swelling, loss of mitochondria crests and vesicle mitochondria were observed in the Control and administration of Atr. In the IPost and Administration of CsA, the structure of mitochondrial was nearly normal. But mitochondria crest were slightly solution.4. The charge of A520 of ischemic group decreased compared with rest six groups, which confirms that mPTP remains closed during the ischemia period and only open in the first few minutes of reperfusion. The charge of A520 of administration of CsA and IPost were significantly lower than that in the Control, and the absorbance of administration of Atr was significantly higher than that in the IPost. However, there was no significant difference between in IPost and IPost+CsA.Conclusions1. The optimal protocol of IPost against focal cerebral ischemia was three cycles of 15s reperfusion/ischemia, and the neuroptrotection of IPC is superior to that of IPost.2. IPost produced neuroprotection against focal cerebral ischemia and improves function of mitochondrial.3. The improvement of mitochondrial function was probably mediated by inhibiting mPTP opening during reperfusion.更多还原