脑缺血再灌流后海马区细胞凋亡的研究

目的:观察脑缺血再灌流后海马区细胞凋亡。方法:钳夹沙土鼠的双侧颈总动脉制造脑缺血模型,应用Tunel法染色。结果:短暂性前脑缺血不同时间再灌流后海马CA_1区锥体细胞发生凋亡,CA_2、CA_3区锥体细胞层、水平层、放射层、分子层及齿状回部位未见细胞凋亡。结论:短暂性脑缺血可导致CA_1区锥体细胞凋亡。     

脑缺血再灌流后HSP70蛋白的表达及其与凋亡的关系

目的 :研究脑缺血再灌流后 HSP70蛋白的表达及其与凋亡的关系。方法 :应用免疫组化的方法观察大鼠局灶性脑缺血再灌流后脑组织 HSP70蛋白的表达和细胞凋亡的分布。结果 :缺血 2 h再灌流 0 h即可见 HSP70表达和凋亡细胞 ,HSP70至 2 4h达高峰 ,凋亡细胞数于再灌流后 2 4～ 48h达高峰。结论 :脑缺血诱导 HSP70蛋白的表达和细胞凋亡 ,脑缺血后 HSP70蛋白的表达可能参与抑制细胞凋亡     

短暂脑缺血再灌注后大鼠大脑皮质ATP含量的动态变化

目的　探讨短暂脑缺血再灌注后大鼠大脑皮质ATP含量的动态变化及其与神经功能恢复之间的关系。方法　采用线栓法建立大鼠大脑中动脉闭塞 (MCAO)模型 ,缺血 10min后于再灌注后 0h、1h、3h、6h、12h、2 4h和 72h应用毛细血管电泳法分别测定额顶叶皮质的ATP含量 ,观察其变化规律。结果　缺血 10min后额顶叶皮质ATP的含量急剧下降至对照组的 2 0 %。再灌注后ATP的含量逐渐恢复 ,于再灌注后 1h、3h、6h和 12h恢复至对照组的 70 .5 %、6 5 .7%、84 .8%和 86 .9%。再灌注后 2 4hATP含量再次下降 ,再灌注后2 4h和 72hATP含量仅为对照组的 5 0 % ,与对照组相比差异均有显著性 (P <0 0 1,P <0 0 5 )。缺血 10min再灌注后大鼠肢体功能可逐渐恢复 ,但再灌注后 2 4h起出现不愿活动和进食等表现。结论　短暂脑缺血再灌注后大鼠额顶叶皮质存在细胞能量系统功能恢复滞后的现象。同时 ,随着再灌注的进行还出现了继发性细胞能量系统功能衰竭的现象 ,这可能与脑缺血再灌注后的神经功能延迟恢复有关     

流式细胞仪检测大鼠局灶性脑缺血再灌流后细胞凋亡的研究

本研究采用流式细胞仪（ＦＣＭ）检测大鼠局灶性脑缺血再灌流后凋亡细胞ＤＮＡ断裂百分率。结果表明：随再灌流时间的增加，ＤＮＡ断裂百分率逐渐增加，再灌流１ｄ时达高峰，再灌流３～７ｄ逐渐下降。结果提示：细胞凋亡在局灶性脑缺血再灌流损伤中呈动态过程且系神经细胞死亡的重要形式。     

不同剂量巴曲酶对大鼠局灶性脑缺血再灌流后细胞凋亡的影响

目的　探讨不同剂量巴曲酶对大鼠局灶脑缺血再灌流的保护作用。方法　采用线栓法制备大脑中动脉缺血再灌流模型。利用流式细胞仪检测细胞凋亡。结果　与对照组相比,巴曲酶组DNA断裂百分率明显降低(P<0.01),随着用药剂量增加DNA断裂百分率呈下降趋势。结论　巴曲酶对脑缺血有保护作用,其保护作用与剂量有关。     

bFGF对脑缺血再灌流后HSP70及凋亡的影响

目的：研究外源性碱性成纤维细胞生长因子（bFGF)对脑缺血再灌流后HSP70表达与细胞凋亡的影响,方法：线栓法制成大鼠大脑中动脉闭塞及再灌流模型,用免疫组化及原位末端标记的方法观察大鼠大脑中动脉闭塞2h后再通1－72h脑组织HSP70表达与凋亡细胞的分布,侧脑室注射外源性bFGF,并观察对它们的影响。结果：bFGF组与生理盐水组相比于6－48h各时间点的HSP70表达增高（P＜0．05）,凋亡细胞数明显减少（P＜0．05,P＜0．01）。结论：外源性bFGF可能诱导脑缺血再灌流后HSP70表达和抑制细胞凋亡。     

脑缺血再灌流后脑组织一氧化氮水平变化及其与细胞凋亡的关系

目的 探讨一氧化氮（NO）在局灶性脑缺血再灌流损伤的作用。方法 采用大鼠线栓法大脑中动脉闭塞（MCAO）模型,观察脑缺血2h再灌流后2,6,18,24及48h NO含量动态变化,同时应用流式细胞仪检测DNA断裂百分率,并分析二者之间的关系。结果 脑缺血2h再灌流后6h NO水平开始升高,随再灌流时间的延长,其升高更明显,再灌注18h后DNA断裂百分率明显升高,其变化趋势与NO相似,结论 上述结果提示,脑缺血再灌流后过量产生的NO与再灌流后神经细胞凋亡的发生有关。     

脑缺血再灌流大鼠海马中一氧化氮与自由基的含量变化

目的　探讨脑缺血再灌流损伤中一氧化氮与自由基所起的作用及二者间的相互关系。方法　通过暂时夹闭大鼠两侧颈总动脉 ,造成大鼠脑缺血再灌流模型。取各组大鼠海马组织 ,测定其一氧化氮和丙二醛的含量 ,观察二者在脑缺血再灌流不同时间的动态变化。结果　脑缺血再灌流组大鼠与假手术组相比 :大鼠海马组织中一氧化氮含量明显升高 (P <0 0 1) ,其中在灌流 6h时的一氧化氮含量最高 ;大鼠海马组织中丙二醛含量也显著升高 (P <0 0 1)。结论　在急性脑缺血再灌流损伤过程中 ,一氧化氮既具有保护作用又具有细胞毒性作用。它主要通过介导自由基的大量产生 ,发挥其细胞毒性作用     

缺血预处理对大鼠脑缺血再灌流线粒体膜流动性及ATP含量的影响

目的方法：在大鼠及缺血再灌流损伤模型上观察,缺血预处置对大鼠脑组织线粒体膜流动性及ＡＴＰ含量的影响。结果：发现ＩＰＣ可以使脑ＩＲ组织线粒体膜流动性及ＡＴＰ含量下降程度减少（与对照组比较）。结论：ＩＰＣ对大鼠脑ＩＲ组织有保护作用。其保护机理可能与ＦＩＦＯ－ＡＴＰ酶,蛋白激酶（ＰＫＣ）及氧自由基清除系统有关。     

Changes in neuronal apoptosis and apoptosis modulatory factors in cerebral ischemia/reperfusion

BACKGROUND: The high concentration of glutamate release is the main cause for neuronal cell death. The relationship between glutamate level and apoptosis during ischemia/reperfusion injury is still unclear. OBJECTIVE: To observe the neuronal apoptosis at 24 and 72 hours following cerebral ischemia/reperfusion in rats, and analyze the possible influencing factors. DESIGN: A randomized controlled animal experiment. SETTING: School of Medicine, Southern Yangtze University. MATERIALS: Totally 30 male adult Sprague Dawley (SD) rats of clean grade, weighing 240–290 g, were obtained from Shanghai Experimental Animal Center, Chinese Academy of Sciences. The rats were randomly divided into sham-operated group (n=10) and model group (n=20). Each group was observed at 24 and 72 hours after ischemia/reperfusion, 5 rats at each time point in the sham-operated group, whereas 12 at 24 hours and 8 at 72 hours in the model group. Kits for determining apoptosis and Bcl-2 were bought from Wuhan Boster Biological Technology, Co., Ltd.; Kit for calcineurin from Nanjing Jiancheng Bioengineering Institute. METHODS: The experiment was carried out in the Functional Scientific Research Room of Southern Yangtze University from June to October in 2006. ① Right middle cerebral artery was occluded by inserting a thread through internal carotid artery (ICA). The surgical process for the sham-operated rats was the same as that in the model group except a nylon suture inserted the ICA. According to Longa five-degree standard, the neurological deficit evaluation of rats was evaluated after surgery, and grades 1–3 were taken as successful model establishment. The blood was recirculated by withdrawing the nylon filament under anesthesia at 2 hours after ischemia in successful rat models. ②After reperfusion, the brain tissue was quickly removed at 24 or 72 hours and the slices were obtained from optic chiasma to funnel manubrium. The changes of the number of apoptotic cells were observed using the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling method. The expressions of Bcl-2 protein were determined with immunohistochemical staining. The activity of calcineurin was determined by the inorganic phosphorus method. The content of excitatory amino acid was detected by high performance liquid chromatography. MAIN OUTCOME MEASURES: ① Glutanate content in brain tissue; ② Conditions of apoptosis; ③ Calcineurin activity in brain tissue; ④ Bcl-2 expression in brain tissue. RESULTS: Totally 30 SD rats were used, 5 died and the other 25 were involved in the analysis of results. ① Changes of apoptosis: There were 0–3 apoptotic cells in the sham-operated group. In the model group, the numbers of apoptotic cells were obviously increased at 24 and 72 hours of reperfusion (P < 0.01), and it was markedly reduced at 72 hours as compared with 24 hours (P < 0.01). ② Changes of glutanate content: The glutamate contents at 24 and 72 hours of reperfusion in the model group were obviously higher than those in the sham-operated group (P < 0.01); In the model group, it was obviously increased at 24 hours as compared with 72 hours (P < 0.01). ③ Changes of Bcl-2 protein: In the model group, the Bcl-2 protein expression had no obvious changes at 24 hours of reperfusion, and it was obviously enhanced at 72 hours, which was obviously different from that in the sham-operated group and that at 24 hours (P < 0.01). ④ Changes of calcinerin activity: In the model group, the activity of calcineurin in brain tissue had no obvious changes at 24 hours of reperfusion; The activity of calcineurin at 72 hours was obviously higher than that in the sham-operated group and that at 24 hours (P < 0.01). CONCLUSION: The brain cyto-apoptosis action at different time points following reperfusion incompletely depends on the glutamate levels, while it depends on the interaction of some apoptosis related factors, such as amino acid, calcineurin, and Bcl-2, etc.     

大鼠脑缺血再灌注后三磷酸腺苷含量和细胞凋亡的变化及药物的影响

目的探讨大鼠脑缺血再灌注后脑ATP含量和脑梗死体积、细胞凋亡的变化及桂哌齐特的影响。方法雄性SD大鼠79只,随机分为假手术组、模型组和桂哌齐特处理组,应用线栓法建立大脑中动脉缺血再灌注模型,采用HPLC法、TUNEL法及TTC染色分别测定脑组织ATP的含量、凋亡细胞数目及脑梗死体积。结果大鼠缺血20、60min和再灌注15、60min脑ATP含量较基础水平降低,再灌注15、60min处理组脑ATP含量较模型组升高;再灌注22h、70h处理组凋亡细胞数目低于模型组,脑梗死体积缩小。结论大鼠局灶性缺血再灌注早期脑ATP含量均降低,梗死灶周边凋亡细胞数目明显增多;桂哌齐特显著阻止脑梗死范围进一步扩大与其改善再灌注后脑组织能量代谢状态抑制细胞凋亡可能有关。     

大鼠全脑缺血再灌注损伤后调控基因Bcl-2、Bax的表达及与细胞凋亡的关系

目的:本研究旨在探讨Bcl-2及Bax蛋白在大鼠全脑缺血再灌注损伤中的变化及与细胞凋亡的关系。方法:雄性Wistar大鼠56只,随机分为假手术组、缺血15分钟再灌注1、6、12、24、48、72小时组。采用大鼠四条血管阻断方法制备大鼠全脑缺血再灌注模型。采用TUNEL法观察不同再灌注时间组海马CAl区细胞凋亡的变化。采用免疫组化法观察Bcl-2及Bax蛋白表达水平的变化。结果:脑缺血损伤后随再灌注时间延长凋亡细胞逐渐增多,至再灌注48小时达到高峰,72小时后减少。Bcl-2表达至再灌注12小时达高峰,再灌注24~72小时组逐渐减弱。Bax表达至48小时达高峰,再灌注72小时减少。结论:Bcl-2于再灌注早期表达增强,Bax于再灌注中期表达增强,Bcl-2/Bax比例失衡可能是大鼠全脑缺血再灌注后神经细胞凋亡的机制之一。     

Edema and neuronal apoptosis in the hippocampus and cortex of elderly rats following transient cerebral ischemia/reperfusion injury

BACKGROUND： Previous studies of cerebral ischemia have used young animals, with an ischemic time greater than 5 minutes （safe time limit）. Despite an increased understanding of neuronal apoptosis, it remains uncertain whether brief cerebral ischemic events of 5 minutes or less damage brain tissue in elderly rodents. OBJECTIVE： To investigate the effects of transient cerebral ischemia （5 minutes）/reperfusion injury on brain cortical and hippocampal edema, aquaporin-4 （AQP-4） expression, and neuronal apoptosis in aged rats, and to compare ischemic sensitivity between cortex and hippocampus. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Institute of Cerebrovascular Disease, Qingdao University Medical School from April 2008 to March 2009. MATERIALS： Rabbit anti-AQP-4 polyclonal antibody, TUNEL kit, and SABC immunohistochemistry kit were purchased from Wuhan Boster Bioengineering, China. METHODS： A total of 160 healthy, male, aged 19-21 months, Wistar rats were randomly assigned to 4 groups： sham-surgery, and ischemia 1-, 3-, and 5-minute groups, with 40 rats in each group. The global cerebral ischemia model was established using the Pusinelli four-vessel occlusion, and the three cerebral ischemia groups were subdivided into reperfusion 12-hour, 1-, 2-, 3-, and 7-day subgroups, with 8 rats in each subgroup. The sham-surgery group was subjected to exposure of the first cervical bilateral alar foramina and bilateral common carotid arteries. MAIN OUTCOME MEASURES： The dry-wet weight assay was used to measure brain water content and histopathology of the cortex and hippocampus was observed following hematoxylin-eosin staining. In addition, cortical and hippocampal AQP-4 expression was detected by streptavidin-biotin complex immunohistochemistry, and neuronal apoptosis was detected by the TUNEL method. RESULTS： There was no significant difference in brain water content or AQP-4 expression in the cortex and hippocampus between ischemia 1- and 3-minute groups and the sham-surgery group or brain water content or AQP-4 expression in the cortex between ischemia 5-minute group and sham-surgery group （P 〉 0.05）. However, brain water content and AQP-4 expression in the hippocampus after 5 minutes of cerebral ischemia were significantly increased compared with the sham-surgery group （P 〈 0.05 or P 〈 0.01）. Several TUNEL-positive cells were observed in the cortex and hippocampus of the sham-surgery group and ischemia 1-minute group, as well as in the cortex of the ischemia 3-minute group. In addition, the number of apoptotic neurons in the hippocampus of ischemia 3-minute group and in the cortex and hippocampus of ischemia 5-minute group was significantly increased （P 〈 0.05 or P 〈 0.01 ）. Neuronal apoptosis was increased after 12 hours of ischemia/reperfusion, and it reached a peak by 2 days （P 〈 0.01）. CONCLUSION： Transient cerebral ischemia （5 minutes） resulted in increased hippocampal edema, AQP-4 expression, and neuronal apoptosis. Moreover, cerebral ischemia had a greater effect on neuronal apoptosis than brain edema or AQP-4 expression, and the hippocampus was more sensitive than the cortex.     

高浓度外源ATP对脑缺血再灌注线粒体编码基因表达的影响

目的针对线粒体的半自主特性,采用高浓度外源能量物质ATP对脑缺血再灌注线粒体自身编码基因表达进行干预,探讨外源能量物质ATP对缺血再灌注神经元能量代谢环节的影响。方法采用颈动脉分流法制备大鼠脑缺血再灌注模型,将缺血时间设置为5分钟,再灌注时间为2小时,采用Fernandez-Silva的细胞器体外3H-UTP掺入法建立脑线粒体体外RNA转录体系,其中脑缺血再灌注线粒体体外RNA转录体系分为1、标准体系。2、ATP2mmol/L。应用TripureIsloationreagent法分离线粒体体外RNA转录体系中线粒体悬浮液总RNA对RNA转录体系产物进行PCR扩增及定性、定量分析。结果在ATP浓度2mmol/L的情况下CoxImRNA的相对表达量为0.42,与缺血再灌注组的CoxlmRNA相对表达量为0.68相比有明显降低(P<0.01).在实验ATP浓度2mmol/L的情况下线粒体再灌编码基因-CoxⅡmRNA的相对表达量为0.62,与再灌CoxⅡmRNA的相对表达量0.78相比有降低(P<0.05),CoxⅢmRNA缺血再灌注后相对表达量为1.05.ATP浓度2mmoI/L时相对表达量为1.03两者比较无明显差别(P>0.05).ATPase6mRNA在缺血再灌注时相对表达量为0.42.ATP浓度2mmol/L时ATPase6mRNA相对表达量为0.34.后者比前者有降低(P<0.05).ATPase8mRNA在缺血再灌注时相对表达量为0.76.ATP浓度2mmol/L时相对表达量为0.74,两者比较无明显差别(P>0.05).结论脑缺血再灌注后,过高浓度的胞外ATP会导致线粒体自身编码基因的不正常表达,对于脑缺血再灌注损伤的临床治疗,不应提倡大剂量外源ATP的应用。     

短暂脑缺血再灌流后大鼠突触蛋白-I的表达及其磷酸化水平

目的探讨短暂脑缺血再灌流后突触传递功能的改变和神经系统的可塑性。方法采用线栓法建立大鼠大脑中动脉闭塞(MCAO）模型，应用免疫组织化学的方法于缺血10mm后再灌流1h、3h、6h、12h、24h和72h分别观察尾壳核区(caudate putamen．CPU)及额顶叶皮质区(frontparietal cortex．FPC)突触蛋白Ⅰ(synapsinⅠ)及磷酸化突触蛋白Ⅰ(P—synapsinⅠ)表达的动态变化。结果缺血10min后再灌流12h内CPU区和FPC区synapsinⅠ的表达无明显变化．再灌流24h后降低，至72h恢复至对照组水平。而缺血10min再灌流后synapsinⅠ的磷酸化一直处于低下水平。结论短暂脑缺血再灌流后存在失神经及其后的神经再获现象，同时还存在着突触传递阻断的现象。     

神经节苷脂对大鼠脑缺血再灌注损伤的脑保护作用

目的探讨神经节苷脂对大鼠脑缺血再灌注损伤的脑保护作用。方法采用线栓法制作缺血再灌注大鼠模型,分别用神经节苷脂(治疗组)和生理盐水(对照组)腹腔注射。观察两组大鼠缺血90min、缺血90min再灌注24h的脑梗死面积、神经功能缺损程度、细胞凋亡数、细胞凋亡率。结果治疗组大鼠于相同时间点脑梗死面积较对照组明显减小,仅表现轻度的神经功能缺损,且神经细胞的凋亡数较对照组显著减少(均P<0.01)。结论神经节苷脂能明显减小大鼠实验性脑缺血的脑梗死面积,减轻脑缺血再灌注后神经功能缺损程度,显著减轻缺血区神经元损害,具有显著的脑保护作用。     

异丙酚预处理大鼠缺血再灌注脑皮质细胞的凋亡

The present study aimed to observe cortical expression of Bcl-2 and Bax,cysteine-dependent aspartate directed proteases-3 activity and apoptotic cell death in a rat model of middle cerebral artery occlusion pretreated with propofol.Results showed that,propofol pretreatment significantly reduced oxidative stress levels and attenuated neuronal apoptosis in the cortex of rats.Propofol pretreatment upregulated Bcl-2 expression,and downregulated Bax expression and cysteine-dependent aspartate directed proteases-3 activity.These findings indicate that propofol pretreatment inhibits cell apoptosis during focal cerebral ischemia/reperfusion injury.This neuroprotective effect is most likely achieved through the Bcl-2/Bax/cysteine-dependent aspartate directed proteases-3 pathway.