缺血预处理对全脑缺血大鼠脑组织超微结构及细胞凋亡的影响

目的：为探讨脑缺血预处理对全脑缺血大鼠脑组织超微结构及细胞凋亡的影响。方法：本文以Wistar大鼠为受试对象,筛选后144只大鼠随机分为正常对照组(24只)、假手术组(24只)、脑缺血预处理对照组(32只)、脑缺血组(32只),脑缺血预处理组(32只)5组和术后12、24、48、72h四个时间点。采用“4-动脉阻断”方法建立大鼠全脑缺血模型,脑缺血后大脑皮层、海马CA1区HE染色观察组织形态学变化、电镜观察组织超微结构,原位末端标记(TUNEL染色)法检测神经元凋亡。结果：正常组、假手术组、脑缺血预处理对照组大脑皮层、海马CA1区无形态学改变,未见有细胞凋亡。脑缺血组大脑皮层、海马CA1区神经元缺失明显,12h、24h、48h、72h神经元凋亡逐渐加重。与脑缺血组相比,脑缺血预处理组大脑皮层、海马CA1区神经元损伤小,水肿程度轻,神经元形态恢复快,凋亡细胞数目少。结论：脑缺血预处理对全脑缺血大鼠大脑皮层、海马CA1区神经元具有保护作用,可以抑制全脑缺血大鼠大脑皮层、海马CA1区神经元凋亡。     

鼠急性全脑缺血再灌注后葛根素对海马CA1区Bcl-2、Bax表达的影响

目的　观察全脑缺血再灌注后葛根素对神经细胞凋亡相关基因Bcl 2、Bax表达的影响。方法　采用免疫组织化学法、原位末端标记法检测大鼠全脑缺血再灌注不同时间内海马CA1区Bcl 2和Bax蛋白表达水平及凋亡细胞数的变化。结果　①脑缺血再灌注后 ,海马CA1区Bcl 2蛋白的表达随再灌注时间不同而变化 ,缺血 10min后再灌注 6h达高峰 ;葛根素治疗组Bcl 2蛋白的表达于相应的时间点明显增加 ;②Bax蛋白表达在再灌注 2 4h达高峰 ,葛根素治疗组Bax蛋白表达在相应时间点则下降 ;③神经细胞凋亡数随再灌注时间延长而增加 ,葛根素可减少神经细胞凋亡数。结论　在全脑缺血再灌注后细胞凋亡中 ,Bcl 2、Bax发挥重要作用 ;葛根素通过上调Bcl 2蛋白的表达 ,下调Bax蛋白的表达抑制细胞凋亡发挥神经保护作用     

脑缺血再灌注损伤大鼠海马神经元凋亡及c—fos蛋白表达研究

目的：观察脑缺血再灌注损伤大鼠海马神经元凋亡及c-fos蛋白的表达。方法：采用大脑中动脉阻断法制备大鼠脑缺血再灌注模型,取海马组织,用免疫组织化学方法检测海马神经元c-fos蛋白表达及TUNEL法观察细胞凋亡情况。结果：脑缺血再灌注损伤大鼠与对照组比较海马区c-fos蛋白阳性细胞表达增强（P〈0．05）,凋亡细胞表达增多（P〈0．05）。结论：脑缺血再灌注损伤大鼠海马区c-fos蛋白阳性细胞表达增强,凋亡细胞表达增多。     

5,6-二羟乙基黄芩苷对大鼠急性脑缺血再灌注所致海马神经细胞凋亡的保护作用

目的考察5,6-二羟乙基黄芩苷对脑缺血再灌注所致神经元损伤的保护作用,并探讨其可能的作用机制。方法采用大鼠双侧颈总动脉阻断合并降压法导致全脑缺血再灌注损伤模型,用HE染色法观察大鼠海马CA1区神经元形态变化;TUNEL法测定海马CA1区神经细胞凋亡的数量;免疫组织化学法检测大鼠海马CA1区Bcl-2、Bax与Caspase-3蛋白的表达。结果 5,6-二羟乙基黄芩苷各给药组能够剂量依赖性地减少TUNEL阳性凋亡细胞的数量,下调促凋亡蛋白Bax及Caspase-3的表达,上调抑凋亡蛋白Bcl-2的表达,并不同程度地改善了大鼠海马CA1区神经元的病理改变。结论 5,6-二羟乙基黄芩苷对脑缺血再灌注损伤具有显著的保护作用,其作用机制可能与抗神经元凋亡有关。     

高压氧对全脑缺血大鼠海马神经元凋亡及代谢的影响

目的 探讨高压氧时全脑缺血大鼠模型海马神经元凋亡及代谢的影响.方法 成年雄性SD大鼠45只,随机分为假手术组、全脑缺血未治疗组和高压氧治疗组.采用改良的Pulsineli法制作全脑缺血大鼠模型.治疗组给予高压氧(2ATA)lh/d,连续5d;应用Nissl染色检测大鼠海马神经元数,免疫组化检测大鼠海马caspase-3蛋白表达,电镜观察其神经元超微结构.结果 高压氧治疗组与未治疗组相比大鼠海马CAl区神经元计数明显增加,差异有显著性(P＜0.05).高压氧治疗组大鼠海马CAl区caspase-3蛋白表达阳性细胞计数明显低于未治疗组,差异有显著性(P＜0.05).结论 严重全脑缺血损伤急性期应用高压氧可抑制凋亡相关基因的表达,改善神脑组织的能量代谢,具有神经保护作用.     

纳洛酮对大鼠脑缺血再灌注损伤后神经细胞凋亡的影响

目的：研究纳洛酮在大鼠局灶性脑缺血再灌注损伤中的保护机制。方法：腔内线栓法制备大鼠大脑中动脉闭塞模型。16只雄性Wistar大鼠随机分为缺血组及纳洛酮治疗组，采用TUNEL法检测各组大鼠脑组织于缺血再灌注各2．5h后神经细胞凋亡情况。结果：大鼠脑缺血再灌注2．5h后凋亡的神经细胞主要分布在梗死灶边缘，纳洛酮治疗组细胞凋亡数量低于缺血组(P＜0．05)。结论：纳洛酮具有减少大鼠脑组织缺血再灌注损伤后神经细胞凋亡的作用。纳洛酮具有抗脑缺血再灌注损伤的保护作用。     

神经生长因子对大鼠全脑缺血再灌注小脑神经细胞凋亡的影响

目的观察神经生长因子（nerve growth factor,NGF）对大鼠全脑缺血再灌注小脑皮层神经细胞凋亡的影响。方法采用闭塞大鼠四动脉建立全脑缺血模型,应用电镜及末端转移酶介导的缺口末端标记法（TUNEL染色法）观察模型组和NGF治疗组动物脑缺血30min再灌注7d小脑皮层神经细胞凋亡的情况。结果模型组小脑皮层神经细胞有明显形态学损伤,蒲肯野细胞凋亡明显。NGF组神经细胞结构损伤明显减轻,凋亡细胞明显减少。结论NGF对大鼠全脑缺血再灌注小脑皮层神经细胞凋亡有明显的保护作用。     

龙眼参多糖对大鼠局灶性脑缺血再灌注损伤细胞凋亡的影响

目的：研究龙眼参（LYS）多糖对局灶性脑缺血再灌注损伤大鼠细胞凋亡的影响。方法：180只Wistar♂性大鼠随机分为脑缺血再灌注组，LYS多糖高、中、低剂量组，阳性对照组，假手术组。给药组术前连续灌胃7d，于末次给药60min后行手术，采用大脑中动脉局灶性缺血再灌注模型，观察缺血2h再灌注24h后，LYS多糖对各组大鼠脑梗死体积、脑含水量及脑组织中Bcb2、Bax蛋白表达的影响，并在光镜下观察各组大鼠皮层区细胞损伤程度。结果：与脑缺血再灌注模型组相比，LYS多糖能够降低大鼠脑梗死体积和脑含水量，增加脑组织中Bcl-2蛋白表达，减少Bax蛋白表达（P〈0．01或P〈0．05），并减轻皮层神经细胞水肿。结论：龙眼参多糖对大鼠急性脑缺血再灌注损伤具有保护作用，其机制可能与其上调Bcl-2蛋白表达，下调Bax蛋白表达，通过调节凋亡相关基因表达抑制凋亡有关。     

松龄血脉康对大鼠局灶性脑缺血-再灌注损伤和细胞凋亡的影响

目的探讨松龄血脉康对局灶性脑缺血-再灌注损伤和细胞凋亡的作用及机制。方法线栓法制作大鼠大脑中动脉阻塞(MCAO)模型。观察松龄血脉康对脑缺血-再灌注损伤大鼠行为学、海马CA1区凋亡细胞数、细胞超微结构的影响;运用免疫组织化学法检测海马凋亡相关基因Bcl-2、Bax蛋白免疫反应阳性细胞表达。结果松龄血脉康显著降低MCAO模型大鼠神经功能缺损评分,明显减少海马组织凋亡细胞数,促进海马神经元修复,松龄血脉康组海马Bcl-2的表达明显增加、Bax的表达降低。结论松龄血脉康可减少脑缺血-再灌注损伤后神经元的凋亡而发挥脑保护作用,机制可能与增强Bcl-2的表达及抑制Bax的表达有关。     

克拉维酸通过下调炎症因子表达对抗缺血损伤发挥神经保护作用

目的：探讨克拉维酸对抗大鼠脑缺血的作用及其机制。方法：实验分8组：空白对照组,假手术组,溶剂对照组,克拉维酸对照组,全脑缺血-再灌注损伤组,以及克拉维酸低、中、高剂量保护组。采用四血管闭塞法建立动物全脑缺血模型;术后3 d通过硫堇尼氏体染色观察海马CA1区细胞形态;术后1 d通过RT-PCR及ELISA分析海马组织TNF-α、IL-1β及IL-6 mRNA及蛋白含量。结果：克拉维酸可减少全脑缺血引起的大鼠海马CA1区锥体神经元迟发性神经元死亡,可降低缺血组织中炎症因子TNF-α、IL-1β及IL-6 mRNA及蛋白表达水平。结论：克拉维酸可对抗脑缺血,发挥神经保护作用,其保护作用与抑制炎症因子表达有关。     

Alterations of interneurons of the gerbil hippocampus after transient cerebral ischemia: effect of pitavastatin.

We investigated the immunohistochemical alterations of parvalbumin (PV)-expressing interneurons in the hippocampus after transient cerebral ischemia in gerbils in comparison with neuronal nitric oxide synthase (nNOS)-expressing interneurons. We also examined the effect of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor pitavastatin against the damage of neurons and interneurons in the hippocampus after cerebral ischemia. Severe neuronal damage was observed in the hippocampal CA1 pyramidal neurons 5 and 14 days after ischemia. The PV immunoreactivity was unchanged up to 2 days after ischemia. At 5 and 14 days after ischemia, in contrast, a conspicuous reduction of PV immunoreactivity was observed in interneurons of the hippocampal CA1 sector. Furthermore, a significant decrease of PV immunoreactivity was found in interneurons of the hippocampal CA3 sector. No damage of nNOS-immunopositive interneurons was detected in the gerbil hippocampus up to 1 day after ischemia. Thereafter, a decrease of nNOS immunoreactive interneurons was found in the hippocampal CA1 sector up to 14 days after ischemia. Pitavastatin significantly prevented the neuronal cell loss in the hippocampal CA1 sector 5 days after ischemia. Our immunohistochemical study also showed that pitavastatin prevented significant decrease of PV- and nNOS-positive interneurons in the hippocampus after ischemia. Double-labeled immunostainings showed that PV immunoreactivity was not found in nNOS-immunopositive interneurons of the brain. The present study demonstrates that cerebral ischemia can cause a loss of both PV- and nNOS-immunoreactive interneurons in the hippocampal CA1 sector. Our findings also show that the damage to nNOS-immunopositive interneurons may precede the neuronal cell loss in the hippocampal CA1 sector after ischemia and nNOS-positive interneurons may play some role in the pathogenesis of cerebral ischemic diseases. Furthermore, our present study indicates that pitavastatin can prevent the damage of interneurons in the hippocampus after cerebral ischemia. Thus, our study provides valuable information for the pathogenesis after cerebral ischemia.     

Apoptosis in the repeated cerebral ischemia--behavioral & histochemical study

We have been reported that the repeated cerebral ischemia induced more severe disruption of spatial cognition than single ischemia without any other motor disturbance in 8-arm radial maze task in rats. And we have been clarified that it is corresponding with 60% of selective cell injury of the CA1 pyramidal cells in the hippocampus. Recently, characteristics of apoptosis such as internucleosomal DNA fragmentation have been found in excitotoxic neuronal death. In the present study, we investigated how necrosis and apoptosis following repeated ischemia involve to the cell death. Repeated cerebral ischemia (10 min x 2, 1 hr interval) induced significant disruption of spatial cognition not only 24 hrs but also 7 days after reperfusion. The decrease of H.E-positive neurons was found in the hippocampus CA1 area and frontal cortex within 3 days after reperfusion, while an DNA fragmentation and TUNNEL-positive neurons in the same areas were found afterward. Furthermore repeated cerebral ischemia-induced disruption of spatial cognition and apoptosis in the hippocampal CA1 area were inhibited by YM-90 K(15 mg/kg,i.p.), which is a selective AMPA/KA receptor antagonist, but not by MK-801. These results suggested that the apoptotic cell death may be occurred via non-NMDA receptor mechanism in relatively late phase of the reperfusion period and it may relate to the incidence of the disruption of spatial cognition in the rat.     

NR2A反义寡核苷酸对短暂性脑缺血/再灌注大鼠海马神经元损伤的保护作用

目的探讨NMDA受体亚单位2A(NR2A)反义寡核苷酸在短暂性脑缺血/再灌注大鼠海马神经元损伤中的保护作用,为研制和开发针对NR2A的特异性新药提供理论基础和形态学依据。方法健康♂SD大鼠随机分为正常对照组、假手术对照组和缺血/再灌注组。经生理盐水、错义寡核苷酸和反义寡核苷酸预处理后,以四血管阻断法建立短暂性全脑缺血(15min)/再灌注(1、2、3和5d)动物模型。在确定的时间点进行灌注固定、取材、石蜡包埋和组织切片(片厚8μm),然后行TUNEL反应、焦油紫染色、原位杂交染色以及免疫组织化学染色。结果短暂性脑缺血/再灌注(I/R)3d,大鼠海马CA1区出现大量的凋亡阳性细胞;I/R5d大鼠海马CA1区细胞严重受损,与对照组相比二组差异具有显著性(P<0.05)。经NR2A反义寡核苷酸预处理后,I/R3d和I/R5d海马CA1区的细胞凋亡和细胞损伤明显减轻,与对照组相比二组差异具有显著性(P<0.05)。NR2A反义寡核苷酸能抑制I/R1d NR2A mRNA表达和I/R2d蛋白质表达,与对照组相比差异具有显著性(P<0.05)。结论短暂性全脑缺血后,NR2A反义寡核苷酸能明显地减轻缺血诱导的大鼠海马CA1区细胞凋亡和细胞损伤,且这种作用与NR2A反义寡核苷酸特异性抑制NR2A及其mRNA的表达密切相关。     

白藜芦醇预处理对局灶性脑缺血/再灌注大鼠海马CA1区神经元凋亡的保护作用和机制

目的探讨白藜芦醇(resveratrol,Res)预处理对局灶性脑缺血/再灌注大鼠海马CA1区神经元凋亡的保护作用及其机制。方法♂SD大鼠60只,随机分为假手术组(Sham)、缺血/再灌注组(I/R)、白藜芦醇预处理组(Res+I/R),每组20只。采用线栓法阻断大脑中动脉血供90 min,拔出栓线造成局部脑区缺血/再灌注损伤。Res+I/R组缺血前1 h腹腔注射白藜芦醇(30 mg·kg-1)。缺血/再灌注后第5天,TUNEL法原位标记海马CA1区凋亡的神经元细胞,免疫组化法检测海马CA1区PI3K、p-Akt及Caspase-3的表达。结果 TUNEL染色结果显示,与I/R组相比较,白藜芦醇预处理明显减少脑缺血/再灌注损伤所引起的大鼠海马CA1区神经元凋亡(P<0.05);免疫组织化学结果显示,与I/R组相比,白藜芦醇预处理使海马CA1区PI3K、p-Akt表达明显增多(P<0.05),Caspase-3表达明显减少(P<0.05)。结论缺血前1 h白藜芦醇预处理对局灶性脑缺血大鼠海马CA1区神经元凋亡具有保护作用,其主要作用机制可能是通过激活PI3K-Akt信号通路进而抑制凋亡相关蛋白Caspase-3的表达有关。     

Effect of nilvadipine on the cerebral ischemia-induced impairment of spatial memory and hippocampal apoptosis in rats

We investigated the effects of nilvadipine and amlodipine on the cerebral ischemia-induced impairment of spatial memory in 8-arm radial maze performance and hippocampal CA1 apoptosis in rats. Single cerebral ischemia impaired memory without inducing apoptosis. In these rats, neither nilvadipine nor amlodipine at 3.2 mg/kg, i.p. improved the impaired memory. On the other hand, repeated cerebral ischemia (10 min ischemia x 2, 1 h interval) impaired spatial memory and induced hippocampal apoptosis 7 days after the final occlusion/reperfusion. Moreover, repeated ischemia increased the apoptotic cell number, an effect observed after 3 days and peaked after 7 days. However, mRNA expression of the apoptosis-related early oncogene bax and CPP 32 (caspase-3) was observed after 24 h. In these rats, nilvadipine, but not amlodipine, significantly improved memory, concomitantly decreased hippocampal apoptosis, and suppressed both bax and CPP 32 expression. These results suggest that nilvadipine improved the memory impairment in repeated ischemia by reducing bax and CPP 32 expression and suppressing the induction of apoptosis in the hippocampus. Nilvadipine may have a neuroprotective effect and could be a useful pharmacotherapeutic agent for cerebrovascular dementia.     

ERK和JNK通路在沙土鼠脑缺血预处理中的表达及作用

目的探讨ERK和JNK在沙土鼠脑缺血预处理中的表达及作用。方法采用沙土鼠前脑缺血再灌注损伤模型。随机分为假手术组(SH)、预处理对照组(IC)、预处理缺血组(IP)及脑缺血再灌注组(IR);各组根据再灌注15 m in、2 h、4h、6 h、1 d、3 d、5 d及7 d又分8个亚组。在预定时间点行开阔法行为学检查、TUNEL法海马CA1/3区凋亡细胞检测、免疫组织化学SP法测定p-ERK、p-JNK在海马区的变化。结果IP可减少沙土鼠探索活动及海马CA1区凋亡锥体细胞数量(vsIR,P<0.01)各组CA1区p-ERK无表达,IR组海马CA1区p-JNK表达较强,再灌后1d最为明显,IP可明显减弱CA1区p-JNK的表达(vsIR,P<0.01),明显增强CA3区p-ERK的表达(P<0.05,P<0.01)。结论脑缺血可导致ERK及JNK在海马各亚区的差异性表达。缺血预处理可能通过抑制CA1区JNK磷酸化、增强CA3区ERK活性而保护海马细胞。     

黄芪注射液对脑缺血/再灌注大鼠海马组织JNK-3表达的影响

目的观察黄芪注射液对脑缺血/再灌注大鼠海马组织JNK3蛋白及其mRNA表达的影响。方法采用4VO法制备脑缺血/再灌注大鼠模型。设假手术组、模型组、黄芪注射液组和黄芪注射液溶剂对照组。除假手术组外,其余各组缺血30 min后再灌注,根据再灌注不同时间点再分为0、0.5、2、6、24、72和120 h 7个亚组。黄芪注射液组于缺血前30 min腹腔注射黄芪注射液[12 g(生药)·kg-1],其中24、72、120 h组手术后每隔24 h追加给药1次,黄芪注射液溶剂对照组腹腔注射与黄芪注射液等量的无菌去离子水。分别采用HE染色观察组织形态学变化;免疫组织化学法和Western blot法检测大鼠海马组织JNK3蛋白表达的变化;RT-PCR方法检测海马组织JNK3 mRNA的表达。结果 HE染色结果显示黄芪注射液可改善脑缺血/再灌注造成的大鼠海马神经元损伤;除120 h外,模型组各时间点海马组织JNK3蛋白及mRNA表达均较假手术组增加(P<0.05);与模型组相比,黄芪注射液组除120 h外各时间点JNK3蛋白及mRNA表达均降低(P<0.05),而黄芪注射液溶剂对照组在各个时间点与模型组相比差异均无显著性(P>0.05)。结论黄芪注射液可抑制脑缺血/再灌注大鼠海马组织JNK3蛋白及其mRNA表达,从而抑制脑缺血/再灌注引起的大鼠海马神经元凋亡。     

Differential regulation of CXCL12 and PACAP mRNA expression after focal and global ischemia

Pituitary adenylate cyclase activating peptide (PACAP) and the chemokine stromal cell-derived factor (SDF-1) have been implicated in neuroprotection, neurogenesis, and regeneration. Focal ischemia is associated with rapid upregulation of PACAP in perifocal neurons and delayed induction of SDF-1 in hypoxic/ischemic tissues, the latter process being involved in the recruitment of stem cells and inflammatory cells. Here, we studied mRNA patterns of PACAP, SDF-1 and the cognate receptors PAC1 and CXCR4 by in situ hybridization in the rat hippocampus after transient global ischemia, a rat model for programmed death of CA1 pyramidal neurons. Cell death in CA1 was not associated with local induction of PACAP and SDF-1 expression or recruitment of CXCR4-expressing infiltrates. However, there was a transient, almost complete loss of SDF-1 expression in microvessels in all hippocampal regions. Granule cells transiently showed a decrease of SDF-1 and an increase of PACAP expression. While PAC1 mRNA was moderately decreased throughout the hippocampus, CXCR4 expression was selectively increased in the subgranular layer. We propose that altered PACAP and SDF-1 gene expression in granule cells plays a role in regulated neurogenesis after global ischemia. The finding that programmed neuronal death after global ischemia was not associated with SDF-1 upregulation or recruitment of CXCR4-expressing cells is in sharp contrast to SDF-1/CXCR4-mediated infiltration of infarct tissue after focal ischemia. Hence, the different modes of neuronal death after focal and global ischemia are associated with distinct SDF-1 and PACAP gene regulation patterns and distinct reorganization mechanisms.     

不同剂量美金刚对慢性脑缺血老龄大鼠认知障碍的改善作用及机制

目的：探讨美金刚（Memantine）对慢性脑缺血老龄大鼠认知障碍及海马区神经的保护作用及机制。方法：采用双侧颈总动脉结扎法（2VO）建立慢性脑缺血动物模型,将造模成功的31只12月龄雄性SD大鼠随机分为缺血组（n=7）和干预组（3.75,7.5,15,30 mg·kg^-1,n=6）,对照组（n=5）只分离血管不做结扎处理。采用水迷宫评估大鼠空间学习记忆功能,免疫荧光染色后对海马齿状回神经细胞增殖及CA1区凋亡细胞进行计数。结果：水迷宫实验显示：缺血组大鼠空间学习和记忆能力明显受损,与对照组和干预组相比有统计学差异（P<0.01）,美金刚干预后大鼠的空间学习和记忆能力得以改善（P<0.05）。免疫荧光染色显示：缺血组大鼠新生神经细胞计数明显多于对照组（P<0.01）,干预组齿状回新生神经细胞数量较对照组进一步增多（P<0.01）;缺血组大鼠凋亡细胞明显多于对照组（P<0.01）,美金刚干预后凋亡细胞数量较缺血组明显减少（P<0.05）。相关性分析表明,CA1区凋亡细胞数量与空间记忆功能呈负相关。结论：美金刚可通过促进海马神经细胞增殖、抑制细胞凋亡,改善认知功能障碍,且不同的药物剂量作用效果存在差异。