参芎葡萄糖注射液对脑缺血再灌注损伤保护作用的研究进展

脑缺血再灌注损伤主要与炎症反应、自由基过度形成、兴奋性氨基酸毒性作用及细胞内钙超载等多种机制相关.单体复方中药制剂参芎葡萄糖注射液可抑制脑缺血再灌注损伤,现将其作用机制综述如下.     

局灶性脑缺血再灌注大鼠海马CA1区神经元凋亡研究

目的 观察大鼠局灶性脑缺血再灌注(ischemia reperfusion,I/R)损伤后海马CA1区神经元凋亡、TUNEL阳性细胞变化,以及凋亡相关蛋白Bcl-2与Bax蛋白的表达情况.方法 将健康雄性SD (Sprague-Dawley)大鼠随机分为假手术组和I/R组,每组再分为缺血再灌注后3、6、12、24、48、72 h亚组.应用免疫组化方法检测再灌注后不同时间点大鼠海马CA1区神经元凋亡基因Bcl-2和Bax蛋白的表达及Bcl-2/Bax比值变化,采用原位细胞凋亡检测(TUNEL)技术检测凋亡阳性细胞数.结果 各组非缺血侧相应区域神经元胞质中Bcl-2均有微弱表达.I/R组缺血侧海马CA1区于再灌注3 h开始出现Bcl-2和Bax蛋白微弱表达,随再灌注时间延长神经元内Bcl-2表达逐渐增强,再灌注24 h后Bcl-2表达达高峰,假手术组与I/R组比较差异有统计学意义(P<0.05).结论 I/R损伤后海马CA1区神经元不仅存在变性坏死,还存在明显的细胞凋亡且细胞凋亡在大鼠I/R损伤中发挥重要作用;I/R可诱导海马CA1区细胞凋亡基因Bcl-2和Bax蛋白表达,且其表达呈一定规律.     

大鼠局灶性脑缺血再灌注后Fas表达与细胞凋亡关系的研究

目的 :本实验通过观察缺血再灌注后大鼠脑组织中 Fas的表达及细胞凋亡的情况 ;初步探讨脑缺血后 Fas表达与细胞凋亡的关系 ,以及其发生的机理。方法 :采用 TUNEL和免疫组化方法观察细胞凋亡及 Fas蛋白在脑缺血再灌注后随时间延长动态变化情况 ,并利用图像分析测定二者的免疫强度。结果 :脑缺血再灌注后神经细胞过度地表达 Fas,以缺血半暗带明显 ,且在缺血再灌注后 6小时达高峰 ,于 2 4小时开始下降。而细胞的凋亡于缺血再灌注后 6小时开始增加 ,于 2 4小时达高峰 ,于 36小时略有下降 ,凋亡细胞分布也以缺血半暗带明显。结论 :脑缺血再灌注可引起Fas的过度表达和神经细胞凋亡增加 ,而缺血后 Fas的过度表达是诱导细胞凋亡的重要的因素之一     

缬沙坦对肾性高血压大鼠模型脑缺血-再灌注后细胞凋亡的影响

目的通过观察血管紧张素Ⅱ受体阻断药缬沙坦对肾性高血压大鼠模型脑缺血。再灌注后神经功能缺损程度和神经元凋亡的影响．探讨血管紧张素Ⅱ受体阻断药的神经保护机制。方法采用Wistar雄性大鼠建立肾性高血压模型，随机分为缬沙坦大剂量组（12mg／kg）、小剂量组（6mg／kg）和缺血-再灌注组．分别于脑缺血2h-再灌注1h和脑缺血2h-再灌注24h时进行神经功能缺损程度评分，并采用TUNEL法和免疫组织化学方法检测海马组织缺血后细胞凋亡发生情况．以及Bcl-2蛋白和Bax蛋白表达水平的变化。结果缬沙坦大剂量组和小剂餐组大鼠的神经功能缺损程度评分低于缺血-再灌注组（P=0．047，0．043），Bcl-2阳性细胞数目高于缺血-再灌注组（均P=0．000），Bax阳性细胞数目低于缺血-再灌注组（均P=0．000）．且神经元凋亡数目明显减少（均P=0．000）：缬沙坦大剂量组Bcl-2阳性细胞数目明显高于小剂量组（P=0．000）．Bax阳性细胞数目明显低于小剂量组（P=0．000）。神经元凋亡数目较小剂量组明显减少（P=0．000）。结论血管紧张素Ⅱ受体阻断药缬沙坦具有明显改善肾性高血压大鼠模型局灶性脑缺血后神经功能缺损程度、减少神经元凋亡、上调脑组织Bcl-2蛋白及抑制Bax蛋白表达水平的作用．对脑缺血-再灌注损伤后的脑组织有一定的保护作用。     

硫酸镁对大鼠脑缺血-再灌注损伤的脑保护研究

目的：观察ＭｇＳＯ４作为非竞争性ＮＭＤＡ－Ｒ拮抗剂对大鼠脑缺血－再灌注的保护作用。方法：用线栓法制作７２只Ｗｉｓｔａｒ大鼠的大脑中动脉栓塞模型,在术后２小时给予再灌注,分别在再灌注同时及其２小时后给予不同剂量ＭｇＳＯ４治疗,术后进行皮层脑电图描记,测定血Ｍｇ＾２＋浓度,进行运动功能评估,测定脑组织含水量,观察病理组织学变化,并计算凋亡细胞数。结果：ＭｇＳＯ４能减少扩展性抑制波产生数目（Ｐ〈０．０５）;１０％,ＭｇＳＯ４组疗效优于５％ＭｇＳＯ４组,并明显改善大鼠运动功能,且早期给药效果更佳（Ｐ〈０．０５）;ＭｇＳＯ４能减轻脑组织含水量;但在短期内不能减少凋亡细胞数。结论：通过电生理、病理研究和临床评估证明ＭｇＳＯ４对大鼠脑缺血－再灌注损伤确有保护作用。     

动员自体骨髓干细胞对大鼠脑缺血／再灌注损伤及细胞凋亡的影响

目的 探讨应用G-CSF动员自体骨髓干细胞对大鼠脑缺血／再灌注损伤及细胞凋亡的影响。方法 应用线栓法制备大鼠局灶性大脑中动脉栓塞／再灌注(MCAO／R)模型，应用粒细胞集落刺激因子(G-GSF)刺激自体骨髓干细胞分裂增殖，并用5-溴脱氧尿核苷(Brdu)标记。观察大鼠神经病学评分，HE染色和免疫组化检测脑缺血区病理改变及CD34和Brdu阳性细胞，原位末端标记法(TUNEL法)观察细胞凋亡。结果 模型动员组大鼠脑缺血／再灌注后24h，大量炎症细胞浸润。再灌注后48h，缺血区可见CD34和Brdu阳性细胞；72h后CD34阳性细胞消失，而Brdu阳性细胞持续存在；模型未动员组缺血区无CD34和很少Brdu阳性细胞表达。48h缺血区新生毛细血管密度明显高于对照组。再灌注后24h细胞凋亡显著，1周时达高峰；与模型非动员组比较，模型动员组48h后细胞凋亡改善明显。结论 自体骨髓干细胞经G-CSF动员后可向大鼠脑缺血区趋化并可分化为神经元前体细胞，显著促进脑缺血区血管再生，降低脑神经功能评分，降低细胞凋亡率。     

MgSO_4对大鼠脑缺血-再灌注损伤的保护作用

目的验证MgSO4作为NMDA受体非竞争性拮抗剂对大鼠脑缺血再灌注后的保护作用及其与凋亡的关系。方法：用线栓法制作Wistar大鼠大脑中动脉栓塞模型．术后2小时予以再通．分别于再通的同时及再通后2小时给予不同剂量MgSO4治疗。术后21小时进行运动功能评估．测定脑组织含水量,观察病理组织形念学变化．并计算每视野下凋亡细胞数量。结果：早期大剂MgSO4组大鼠运动功能改善明显．并可减轻脑组织含水量．但早期给予MgSO4在短期内也不能减少凋亡细胞数目。结论：MgSO4作为兴奋性氨基酸受体（EAAs-R）非竞争性拮抗剂对大鼠脑缺血-再灌注有保护作用,但在短期内其作用与抑制细胞凋亡发生无关     

缺血后处理对大鼠脑缺血—再灌注中细胞凋亡的作用及其机制研究

目的观察缺血后处理(IP)对大鼠脑缺血-再灌注(I/R)中细胞凋亡的影响,并探寻其机制。方法开颅永久性阻断大脑中动脉+临时夹闭双侧颈总动脉法制作模型。将大鼠随机分为空白对照组(Control组)12只、假手术组(Sham组)、I/R组、IP组各48只、I/R+caspase-3抑制剂组(I/R+Z-DEVD-FMK组)、I/R+溶剂组(I/R+DMSO组)各12只。通过免疫荧光及Western blot法检测细胞凋亡数、细胞色素c(cyt-c)释放及caspase-3活性。结果 IP组较I/R组TUNEL阳性细胞数减少(P<0.05)。Sham组、IP组和I/R组均有细胞呈cyt-c/TUNEL双阳性,但cyt-c阳性不全伴有TUNEL阳性。I/R组与IP组cyt-c呈双峰样释放(再灌注3 h和48 h),在48 h时IP组较I/R组降低(P<0.05)。I/R组caspase-3活性在再灌注3 h时开始升高,12 h和24 h时最高。各相应时点IP组较I/R组的caspase-3活性降低(P<0.01和P<0.05)。I/R+Z-DEVD-FMK组cyt-c后期释放量小于I/R+DMSO组(P<0.01),但完整细胞数多于I/R+DMSO组(P<0.01)。结论 IP可以抑制凋亡;cyt-c参与凋亡,并与caspase-3形成相互作用的反馈回路。IP对该反馈回路具有调节作用。     

MgSO_4对大鼠脑缺血-再灌注损伤的保护作用

目的验证MgSO4作为NMDA受体非竞争性拮抗剂对大鼠脑缺血再灌注后的保护作用及其与凋亡的关系。方法：用线栓法制作Wistar大鼠大脑中动脉栓塞模型．术后2小时予以再通．分别于再通的同时及再通后2小时给予不同剂量MgSO4治疗。术后21小时进行运动功能评估．测定脑组织含水量，观察病理组织形念学变化．并计算每视野下凋亡细胞数量。结果：早期大剂MgSO4组大鼠运动功能改善明显．并可减轻脑组织含水量．但早期给予MgSO4在短期内也不能减少凋亡细胞数目。结论：MgSO4作为兴奋性氨基酸受体（EAAs-R）非竞争性拮抗剂对大鼠脑缺血-再灌注有保护作用，但在短期内其作用与抑制细胞凋亡发生无关     

醒脑静联合丁苯肽对脑缺血再灌注损伤后细胞凋亡的影响

目的 观察醒脑静、丁苯酞及二者联合分别对大鼠脑缺血再灌注损伤后神经细胞凋亡及Bcl-2和Bax表达的影响。方法 60只雄性wistar大鼠(250±20)g采用改良线栓法制作脑缺血再灌注损伤模型(Middle cerebral artery occlusion,MCAO),随机分为4组,即模型组、醒脑静组、丁苯酞组、醒脑静联合丁苯酞(联合用药)组,每组又分为6、24、72 h三个亚组; 通过原位末端转移酶标记技术(TUNEL)检测神经细胞凋亡情况,采用免疫组化法观察大鼠脑缺血再灌注各个时间点Bcl-2、Bax的表达水平。结果(1)模型组手术对侧大脑半球偶见凋亡细胞,病灶区可见大量神经细胞凋亡。丁苯酞用药组、醒脑静用药组凋亡细胞数明显减少,醒脑静联合丁苯酞组凋亡细胞数最少(P<0.05);(2)丁苯酞组及联合用药组Bcl-2阳性表达水平较模型组均有提高,联合用药组Bcl-2阳性表达水平在各时间点均最高(P<0.05); 丁苯酞组及联合用药组Bax阳性表达水平较模型组均有降低,联合用药组Bax阳性表达水平最低(P<0.05)。结论(1)醒脑静、丁苯酞及二者联合均可能通过抑制脑缺血再灌注损伤后神经细胞凋亡来实现神经细胞保护作用,其中二者联合效果最佳;(2)丁苯酞可能通过增加脑缺血再灌注损伤大鼠Bcl-2表达,减少Bax表达的方式来减少神经细胞凋亡,从而减轻脑缺血再灌注损伤;(3)醒脑静本身不能对Bcl-2、Bax的表达水平产生影响,但其可能通过增强丁苯酞作用的方式影响Bcl-2、Bax的表达,从而减轻脑缺血再灌注损伤。     

葡萄糖调节蛋白78对脑缺血再灌注后神经细胞凋亡的影响

目的 通过体内体外实验探讨内质网伴侣蛋白-葡萄糖调节蛋白(GRP)78对缺血再灌注后神经细胞凋亡的影响.方法 体内实验采用大鼠大脑中动脉线栓模型,应用免疫组织化学、Western blot和RT-PCR方法检测脑缺血再灌注后缺血周围区GRP78表达的动态变化;体外实验通过原代培养的海马神经元建立脑缺血再灌注模型,Western blot方法观察去糖去氧及恢复糖氧后GRP78表达的变化,并运用2脱氧葡萄糖诱导GRP78高表达,观察对神经细胞凋亡的影响.结果 体内实验:与假手术组相比,脑缺血再灌注后GRP78表达明显增强,再灌注12 h达高峰(mRNA:0.7367±0.0651,F-477.160,P<0.01;蛋白:0.8129±0.0748,F=39.857,P<0.01).体外实验:经2脱氧葡萄糖预处理高表达GRP78的神经细胞活力明显增强(与单纯去糖去氧组相比,细胞活力增加了39.22%±0.44%,t=46.374,P<0.01),凋亡细胞减少(与单纯去糖去氧组相比,凋亡细胞数减少16.60±1.02,t=7.530,P<0.01).结论 脑缺血再灌注启动内质网应激反应,与神经细胞凋亡有关;诱导GRP78高表达可以减轻缺血再灌注性神经元的损伤和凋亡.     

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.     

胰岛素样生长因子1对糖尿病大鼠脑缺血再灌注后神经细胞凋亡的影响

背景：胰岛素样生长因子1是近年来发现的一种神经营养因子。 目的：验证胰岛素样生长因子1对局灶性脑缺血糖尿病大鼠的神经功能、神经细胞凋亡及Bcl-2、Bax蛋白表达的影响。 设计、时间及地点：随机对照动物实验,于2006-10/2008-03在齐齐哈尔医学院实验中心完成。 材料：Wistar大鼠40只,体质量200~250 g,雌雄不限,鼠龄2.0~3.0月。 方法：线栓法制作糖尿病大鼠大脑中动脉栓塞模型,随机分成胰岛素样生长因子1组和对照组,每组12只。胰岛素样生长因子1组连续皮下注射胰岛素样生长因子1,10 µg/(kg•d),对照组注射等量的生理盐水,两组分别注射7,14 d,每时间点取6只。 主要观察指标：对两组大鼠进行神经功能缺损评分,采用TUNEL染色计数脑缺血周边区神经细胞凋亡数,免疫组织化学方法观察Bcl-2、Bax蛋白的表达。 结果：胰岛素样生长因子1组神经功能缺损评分明显低于对照组(P < 0.01); 胰岛素样生长因子1组缺血周边区的TUNEL阳性细胞数均明显少于对照组(P < 0.01); Bcl-2蛋白免疫阳性细胞明显多于对照组(P < 0.01),Bax蛋白免疫阳性细胞明显少于手术对照组(P < 0.01)。 结论：胰岛素样生长因子1可增加糖尿病脑缺血之后缺血周边区Bcl-2蛋白的表达,减少Bax蛋白的表达,拮抗神经细胞凋亡,改善神经功能。     

Inhibitory effect of acupuncture on neuronal apoptosis in rats after cerebral ischemia

BACKGROUND: Delayed neuronal death after total cerebral ischemia may accompany with apoptosis, but acupuncture may play a certain role in protecting nerve through inhibiting ischemic neuronal apoptosis. OBJECTIVE: To observe the effect of acupuncture on neuronal apoptosis in rats after cerebral ischemia and analyze its cerebral protective mechanism. DESIGN: Contrast observation among groups. SETTING: Heilongjiang University of Traditional Chinese Medicine. MATERIALS: A total of 30 male healthy Wistar rats of general grade and weighing (250±20) g were randomly divided into three groups, including sham operation group, cerebral ischemia group and acupuncture group with 10 rats in each group. Apoptosis in situ kit was provided by Baolingman Company, Germany. METHODS: The experiment was carried out in the Laboratory Center, Heilongjiang University of Traditional Chinese Medicine from May to November 2004. ① Rats in the cerebral ischemia group and the acupuncture group were used to establish total cerebral ischemic models with four vessels occlusion; in addition, models in the sham operation group were established with the same method as mentioned above. However, four vessels of rats in the sham operation were exposured and cerebral ischemia did not occur. Rats in the acupuncture group were given acupuncture treatment after operation. Needle of 40 mm in length was used to acupuncture bilateral Zusanli (St 36) and Quchi (LI 11) with the depth of 3 mm, and then bilateral acupoints were connected with KWD-808II omnipotenc impulse electro-therapeutic apparatus (frequency: 1 Hz; thin waves; voltage: 2 V) once a day for totally 30 minutes. Meanwhile, needle of 25 mm in length was used to acupuncture Baihui (Du 20) with the depth of 3 mm, and then the needle was twirled once every 5 minutes for 30 minutes in total. The course was 7 days. ② Neuronal injuries in hippocampal CA1 area after cerebral ischemia were observed with Nissl body staining method at 7 days after treatment; neuronal apoptosis was observed with TUNEL staining; manifestations of neuronal apoptosis in cerebral cortex and hippocampal CA1 area were observed with electron microscope. MAIN OUTCOME MEASURES: Neuronal injuries in hippocampal CA1 area after cerebral ischemia; neuronal apoptosis in cerebral cortex and hippocampal CA1 area after cerebral ischemia; morphological changes under electron microscope. RESULTS: Among 30 Wistar rats, 24 rats were involved in the final analysis. ① Expression of positive neurons in cerebral cortex and hippocampal CA1 area with Nissl body staining: Neuronal defect was obvious in cerebral cortex and hippocampal CA1 area in the cerebral ischemia group as compared with that in the sham operation group (P < 0.05), and neuronal defect was decreased in hippocampal CA1 area in the cerebral ischemia group as compared with that in the acupuncture group (P < 0.05). ② Expression of positive neurons in cerebral cortex and hippocampal CA1 area with TUNEL staining: Positive neurons with TUNEL staining were not observed in the sham operation group, but positive neurons were increased in the cerebral ischemia group as compared with those in the acupuncture group (P <0.05). ③ Observational results of electron microscope: Neuronal apoptosis was not found in the sham operation group; neuronal apoptosis was rarely found in the acupuncture group; neuronal apoptosis was typical in the cerebral ischemia group. CONCLUSION: Delayed neuronal death after total cerebral ischemia may accompany with apoptosis, but acupuncture may play a certain role in protecting nerve through inhibiting ischemic neuronal apoptosis.     

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.     

大鼠脑缺血再灌注损伤后细胞凋亡与Survivin和IGF-1表达的关系

目的 探讨大鼠脑缺血再灌注损伤后Survivin和IGF-1因子表达与细胞凋亡的关系.方法 将80只成年健康雄性Wistar大鼠,分为Survivin组和IGF-1组,每组各40只,并随机分为对照组、假手术组和缺血1h再灌注2h、6h、12h、24h、48h、3d、7d、14d组,每组4只(n=4).应用线栓法制备大鼠脑缺血再灌注模型(MCAO),采用免疫组织化学方法检测Survivin蛋白与IGF-1蛋白在神经元凋亡中的表达情况.结果 Survivin组:假手术组Survivin阳性神经元在海马、皮质区及纹状体区呈基础表达,缺血再灌注2h表达明显增加,48h达高峰,14d表达最低,与假手术组比较有显著性差异(P＜0.05).IGF-1组:正常对照组及假手术组IGF-1阳性神经元在海马、皮质区及纹状体区呈基础表达,缺血再灌2h时IGF-1表达明显增高,48h达高峰,3～14d仍维持高表达,与正常对照组及假手术组比较有显著性差异(p＜0.05).结论 内源性Survivin与IGF-1因子可能具有在抑制神经元凋亡的作用.     

NO对实验性局灶脑缺血再灌流神经细胞凋亡影响的定量研究

目的 研究ＮＯ在局灶性脑缺血再灌流过程中对神经细胞的作用机制，特别是ＮＯ与细胞凋亡的关系。方法 利用流式细胞仪检测法，测定Ｎ－硝基－左旋－精氨酸（ＮＮＬＡ）干预后局灶性脑缺血鼠脑组织中神经细胞的凋情况。结果 在缺血再灌流期间过多或过少的ＮＯ均对缺血区凋亡峰有明显影响，小剂量ＮＮＬＡ减少神经细胞凋亡的发生率，大剂量ＮＮＬＡ增加神经细胞凋亡的发生率。结论 局灶性脑缺血的不同区域，包括正常及病变组织中的     

雷帕霉素对大鼠脑缺血再灌注后神经元损害的保护作用

目的 观察雷帕霉素对大鼠脑缺血再灌注后神经元损害的保护作用。方法 52 只雄性 SD 大鼠随机分为对照组和实验组（雷帕霉素干预）各24 只,另4 只为假手术组。采用Longa 线栓法制备 大鼠局灶性脑缺血再灌注模型,缺血3 h 后,于再灌注4 h、6 h、12 h、24 h、72 h、7 d 进行各指标观察;其 中实验组在构建脑梗模型之前进行腹腔注射雷帕霉素（ 1 mg/kg）,假手术组不插线,其余过程同对照组。 采用TTC 染色法观察脑组织变化;免疫组织化学（免疫荧光法）检测Bcl-2、Caspase-3 及自噬相关蛋白 LC-3 的变化。结果 TTC 染色假手术组未见缺血表现,缺血3 h 后可见病灶侧脑组织苍白,实验组病灶 小于对照组。实验组大鼠灌注6、12、24 h 时神经功能缺损评分均明显低于对照组（P＜ 0.05）。Caspase-3 的表达：再灌注4 h表达显著增高, 至24 h左右达高峰, 实验组再灌注6 h、24 h、7 d表达明显低于对照组, 差异有统计学意义（P＜ 0. 05）;Bcl-2 的表达：再灌注4 h 增多,24 h 达高峰,实验组再灌注4、6、12、24 h 表达明显高于对照组（P＜ 0.05）;实验组LC-3 表达明显高于对照组。结论 雷帕霉素能促进LC-3 表达, 抑制细胞凋亡;抑制Caspase-3 的表达,增加Bcl-2 的表达从而减轻缺血所致的损伤,对大鼠缺血后神经 功能的恢复有确切的疗效。