沙土鼠全脑适应性再灌注的脑保护机制研究

目的动态研究不同灌注时间和灌注量对全脑组织缺血损害的恢复程度和即早基因c- fos的表达,以探明适应性再灌注的脑保护作用机制.方法沙土鼠随机分7组,实验组夹闭两侧颈总动脉,造成沙土鼠全脑缺血模型,夹闭10 min后,不同开放时间段(4 min,8 min,10 min,15 min,30 min)开放不同脑血流量(1/4,1/2,一次性全开放)和单纯血液稀释后分别观察缺血海马CA区c-fos蛋白的表达,及缺血区大脑半球的改善状况.结果开放15 min,1/2脑血流量时c-fos蛋白表达最高(P<0.05),海马CA区缺血损害改善最明显,开放15 min,全脑血流量一次性开放时海马CA区损害最严重.结论①在适应性灌注流量中,脑缺血海马区c-fos的表达和神经元凋亡呈反相作用关系;②低流量灌注有明显改善脑缺血的作用;③夹闭10 min后,开放1/2脑血流量,持续15 min的效果比一次性再灌注开放效果要好.     

沙土鼠全脑梯度再灌注的脑保护实验研究

目的 动态研究不同灌注时间和灌注量全脑组织缺血损害的恢复程度和即早基因的表达，以探明梯度再灌注的脑保护作用机制。方法 将沙土鼠随机分为4组：实验组夹闭两侧颈总动脉，造成沙土鼠全脑缺血模型，夹闭10 min后开放10 min，开放不同脑血流量(1／4开放组，1／2开放组，一次性全开放组)，分别检测3组及单纯血液稀释组缺血海马CA区C-fos蛋白的表达，以及缺血区大脑半球的改善状况。结果 开放10 min、1／2脑血流量时C—fos蛋白表达最高(P＜0．05)，海马CA区缺血损害改善最明显；开放10 min、全脑血流量一次性开放时海马CA区损害最严重。结论 ①在梯度再灌注流量中，脑缺血海马区C-fos的表达和神经元凋亡呈反相作用关系；②低流量灌注有明显改善脑缺血的作用；⑧夹闭10 min后，开放1／2脑血流量，持续10 min的效果比一次性再灌注开放效果要好。     

大蒜素对全脑缺血再灌注损伤大鼠海马凋亡相关蛋白表达的影响

目的观察大蒜素对全脑缺血再灌注损伤大鼠海马凋亡相关蛋白表达的影响,进一步探讨大蒜素的脑保护机制。方法雄性Wistar大鼠30只,随机分为5组:假手术组(Sham组);缺血再灌注组(IR组);缺血再灌注+大蒜素10 mg/kg(All-10 mg)、20 mg/kg(All-20 mg)、30 mg/kg(All-30 mg)组,夹闭两侧颈总动脉8 min再灌注24 h建立大鼠全脑缺血再灌注模型,取海马组织硫堇染色观察海马组织学改变及存活神经元密度;免疫组织化学染色测定海马CA1区Bcl-2、Bax蛋白的表达。结果与Sham组相比,IR组海马CA1区神经元密度明显降低,Bax蛋白表达明显增加(均P0.05);与IR组相比,大蒜素处理各组海马CA1区神经元密度明显增多,Bcl-2蛋白表达明显增加,Bax蛋白表达明显减少,其中以All-20 mg组变化最显著(均P0.05)。结论大蒜素可以通过影响凋亡相关蛋白的表达而抑制细胞凋亡,从而发挥脑保护作用。     

胰岛素对脑缺血后鼠海马CA1区神经元凋亡及记忆影响

目的　观察胰岛素对全脑缺血后海马CA1区神经元凋亡及大鼠学习记忆力改变的影响 ,探讨胰岛素对全脑缺血后海马CA1区神经元产生中枢直接保护作用的机理。方法　利用 4 VO法制作大鼠全脑缺血模型。造成脑缺血 15min后行再灌注 ,于再灌注后即刻经脑室注入 1U胰岛素 ,利用免疫组化及原位标记法分别于全脑缺血后 1、3d观察海马CA1区Bcl 2、Bcl xl蛋白表达及神经元凋亡的情况 ;缺血后 8周 ,利用“Y”型迷宫测试大鼠的学习记忆功能。结果　全脑缺血后 3d ,缺血组大鼠海马CA1区Bcl 2、Bcl xl蛋白的表达呈阴性 ,海马CA1区原位标记阳性细胞计数为 14 3.5± 11.6。治疗组大鼠海马CA1区Bcl 2、Bcl xl蛋白呈阳性表达 ,海马CA1区原位标记阳性细胞计数为 75 .6±6 .7。全脑缺血后 8周 ,治疗组大鼠学习记忆力明显好于缺血组。结论　全脑缺血后脑室内注入胰岛素可促进海马CA1区Bcl 2、Bcl xl蛋白表达 ,减少神经元的凋亡 ,进而减轻脑缺血后大鼠的学习记忆力损害 ,这可能是其对全脑缺血后海马CA1区神经元产生中枢直接保护作用主要机理之一     

大鼠短暂脑缺血后海马区c-fos mRNA和FOS蛋白表达的时间规律

目的 探讨大鼠脑缺血-再灌注后脑海马区c-fos mRNA和FOS蛋白表达规律。方法 采用左侧大脑中动脉插入丝线结扎(LMCAO)方法制作大鼠脑缺血-再灌注缺血模型，分别按照30min至7d的不同灌注时间取材，应用原位杂交方法标记各实验组大鼠脑组织海马区c-for mRNA表达数量；应用免疫组化方法标记各实验组大鼠脑组织中海马区FOS阳性神经元数量。结果 c-fos mRNA阳性细胞在MCAO缺血再灌注1h开始表达，2h达高峰，至2d时c-fos mRNA阳性细胞表达基本消失；c-fos免疫活性细胞从2h开始表达，4h达高峰，4d时表达基本消失。脑缺血—再灌注后c-fos mRNA与FOS蛋白在海马区表达具有一定的时间上规律性。结论 本研究证明了大鼠MCAO缺血—再灌注后可诱发FOS蛋白和c-fos mRNA表达增加，且基因和蛋白的表达均具有一定的时间规律性和相关性。     

脑缺血后脑室内注射胰岛素减轻大鼠学习记忆力损害的实验研究

目的探讨经脑室直接注射胰岛素对全脑缺血后大鼠学习记忆力的影响.方法以4-VO法建立大鼠全脑缺血再灌注模型.全脑缺血20min后行再灌注,于再灌注即刻经脑室注入1U胰岛素,缺血再灌注后8周利用三等分Y型迷宫箱测试大鼠的学习记忆功能,并对大鼠海马CA1区神经元的病理改变进行观察.结果经Y型迷宫测试,对照组、治疗组及缺血组大鼠达9/10正确反应所需电击数分别为5.6±1.2,8.6±1.3,19.6±2.6;对照组、治疗组及缺血组大鼠海马CA1区神经元计数分别为176.5±10.6,112.4±11.7,10.5±0.4.结论全脑缺血后脑室内注射胰岛素可明显减少大鼠海马CA1区神经元的死亡,进而减轻大鼠学习记忆力损害.     

大鼠全脑缺血再灌注损伤后海马CA1区神经元酪氨酸羟化酶和多巴胺-β-羟化酶表达的变化

目的　研究大鼠全脑缺血再灌注损伤后酪氨酸羟化酶 (TH)和多巴胺 β 羟化酶 (DβH)表达的改变及意义。方法　利用改良四血管闭塞法 ,建立大鼠全脑缺血模型。于缺血再灌注后 1,3,5d断头取脑 ,行免疫组化染色 ,在光镜下观察海马CA1区神经元TH及DβH表达的变化 ,并计数海马CA1区正常神经元。结果　全脑缺血再灌注后 1d ,TH及DβH的表达呈阴性 ,海马CA1区神经元未见显著的病理形态学改变 ;全脑缺血再灌注后 3d ,TH及DβH呈阳性表达 ,海马CA1区可见部分神经元死亡 ;全脑缺血再灌注后 5d ,TH及DβH呈明显阳性表达 ,海马CA1区可见大部分神经元死亡。结论　全脑缺血再灌注后 ,由于TH及DβH异常表达 ,使得儿茶酚胺生物合成增加 ,这可能是短暂性脑缺血损伤的机理之一。     

磷酸化cAMP反应元件结合蛋白在全脑缺血大鼠海马中的表达

目的观察转录因子磷酸化cAMP反应元件结合蛋白(pCREB)在大鼠短暂性全脑缺血再灌注后海马中的表达，并探讨其表达的意义。方法采用四动脉阻断法制备大鼠全脑缺血模型，缺血15min。Western印迹检测海马组织pCREB的表达，免疫组化观察其在大鼠海马CA1区及齿状回的表达。结果Western印迹显示，缺血再灌注3h，pCREB的表达至高峰，并随再灌注时间延长呈现下降趋势。免疫组化显示缺血再灌注3h海马CA1区pCREB表达至高峰，并迅速下降，于24h降至假手术组水平；而在齿状回其表达较强且持久。结论脑缺血再灌注促进pCREB的表达，pCREB的表达可能参与了缺血性脑损伤的内源性保护机制。     

牛磺酸对沙土鼠短暂性前脑缺血作用研究

目的 观察牛磺酸对沙土鼠短暂性前脑缺血的保护作用。方法 结扎沙土鼠双侧颈动脉建立前脑缺血模型,缺血5 min后进行再灌注。在缺血1 h后经静脉给予2.5 mg/kg、5 mg/kg、15 mg/kg及50 mg/kg牛磺酸。再灌注7 d时,对沙土鼠的神经功能行为缺损进行评分,并留取全脑,观察海马CA1区锥体细胞的死亡情况。结果 2.5 mg/kg牛磺酸虽然改善前脑缺血5 min再灌注7 d时沙土鼠的行为,降低海马CA1区锥体细胞的死亡,但与缺血对照组相比,无统计学意义,5 mg/kg、15 mg/kg及50 mg/kg牛磺酸可明显改善前脑缺血沙土鼠的行为缺损（P分别为0.001、0.006和0.037）,降低海马CA1区锥体细胞的死亡（P均＜0.001）,其中,5 mg/kg、15 mg/kg和50 mg/kg牛磺酸组的锥体细胞的死亡低于2.5 mg/kg牛磺酸组（P分别为0.001、0.001和0.005）。结论 牛磺酸能以剂量依赖方式降低沙土鼠前脑缺血损伤,提示牛磺酸可能是一种有效的短暂性缺血的大脑保护剂。     

亚低温对大鼠全脑缺血再灌注损伤后海马CA1区c-Jun蛋白表达的影响

目的研究亚低温对大鼠全脑缺血再灌注损伤后海马CA1区神经元的保护作用,并探讨其可能的机制。方法采用四血管阻断法建立大鼠全脑缺血模型。SD大鼠,随机分为假手术组(SH组)、常温组(IR组)和亚低温组(HIR组)。各组在全脑缺血15min后分别再灌注6h、12h、1d、3d,采用苏木素-伊红(HE)染色观察各时间点海马CA1区细胞形态学变化和TUNEL法检测海马CA1区神经元凋亡,免疫印迹检测c-Jun蛋白表达。结果(1)HE染色结果 IR组和HIR组于全脑缺血再灌注后6h,HE染色未见明显改变,IR组缺血再灌注1d时CA1区出现严重改变,3d时损伤最严重,出现细胞数目减少,细胞胞体缩小、胞核固缩深染,损伤严重,排列紊乱,核膜不清,核仁消失。而HIR组海马存活的锥体细胞数较之IR组12h、1d、3d时间点均明显增加(P<0.05)。(2)TUNEL标记IR组于缺血再灌注后6h在海马CA1区阳性细胞开始增多,缺血再灌注1 d时阳性细胞数最多。而HIR组各时间点阳性细胞数均较IR组明显减少(P<0.01)。(3)免疫印迹结果全脑缺血再灌注后6h c-Jun蛋白在IR组海马CA1区表达开始增加,12h达高峰,持续到3d;HIR组在各时间点的表达均弱于IR组(P<0.01)。结论亚低温通过减少海马CA1区c-Jun的表达,抑制海马CA1区神经元的凋亡,可能是亚低温脑保护作用的机制之一。     

Activation of mitogen-activated protein kinases after transient forebrain ischemia in gerbil hippocampus.

We investigated the expression, activation, and distribution of c-Jun N-terminal kinases (JNKs), p38 mitogen-activated protein kinases (p38s) and extracellular signal-regulated kinases (ERKs) using Western blotting and immunohistochemistry in gerbil hippocampus after transient forebrain ischemia to clarify the role of these kinases in delayed neuronal death (DND) in the CA1 subfield. Immunoblot analysis demonstrated that activities of JNK, p38, and ERK in whole hippocampus were increased after 5 min of global ischemia. We used an immunohistochemical study to elucidate the temporal and spatial expression of these kinases after transient global ischemia. The immunohistochemical study showed that active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and then gradually reduced and disappeared in the hippocampal CA1 region. On the other hand, in CA3 neurons, active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and peaked at 6 hr of reperfusion and then gradually reduced but was continuously detected 72 hr after ischemia. Active ERK immunoreactivity was observed transiently in CA3 fibers and dentate gyrus. Pretreatment with SB203580, a p38 inhibitor, but not with PD98059, an ERK kinase 1/2 inhibitor, reduced ischemic cell death in the CA1 region after transient global ischemia by inhibiting the activity of p38. These findings indicate that the p38 pathway may play an important role in DND during brain ischemia in gerbil. Components of the pathway are important target molecules for clarifying the mechanism of neuronal death.     

Protective preconditioning by transient global ischemia in the rat: components of delayed injury progression and lasting protection distinguished by comparisons of depolarization thresholds for cell loss at long survival times.

Robust ischemic preconditioning has been shown in rodent brain, but there are concerns regarding the persistence of neuron protection. This issue was examined in rat hippocampus following 4-vessel occlusion (4-VO) ischemia, using DC shifts characteristic of ischemic depolarization to reproducibly define insult severity. Preconditioning ischemia producing 2 to 3.5 mins depolarization was followed at intervals of 2, 5, or 7 days by test insults of varied duration, after which CA1 counts were obtained at 1, 2, 4, or 12 weeks. Neuron loss in naive animals increased with depolarization time longer than 4 mins regardless of postischemic survival interval. Preconditioning 2, 5, or 7 days before test insults prolonged the injury threshold evaluated at 1 week survival to 15, 9, or 6 mins, respectively, showing robust protection and a rapid decay of the protected state. However, by 2 weeks survival after preconditioning at a 2-day interval, the injury threshold dramatically regressed from 15 to 9 mins. Thereafter protection remained relatively stable through 1 month, but slight progression of neuron injury was evident at 3 months. Inflammatory responses were seen in both naive and preconditioned hippocampi throughout this interval, appropriate to the extent of neuron injury. These studies show distinct components of transient and lasting protection after ischemic preconditioning. Finally, it was found that ischemic depolarization was delayed by approximately 1 min in optimally preconditioned rat hippocampus, in contrast to previous results in the gerbil, identifying one specific mechanism by which insult severity is reduced in this model.     

Downregulation of hippocampal adenosine kinase after focal ischemia as potential endogenous neuroprotective mechanism.

The rate of ischemic brain injury varies with the brain region, requiring only hours in striatum but days in hippocampus. Such maturation implies the existence of endogenous neuroprotective mechanisms. Adenosine is an endogenous neuroprotectant regulated by adenosine kinase (ADK). To investigate, whether adenosine might play a role in protecting the hippocampus after focal ischemia, we subjected transgenic mice, which overexpress ADK in hippocampal neurons (Adk-tg mice) to transient middle cerebral artery occlusion (MCAO). Although the hippocampus of wild-type (wt) mice was consistently spared from injury after 60 mins of MCAO, hippocampal injury became evident in Adk-tg mice after only 15 mins of MCAO. To determine, whether downregulation of hippocampal ADK might qualify as candidate mechanism mediating endogenous neuroprotection, we evaluated ADK expression in wt mice after several periods of reperfusion after 15 or 60 mins of MCAO. After 60 mins of MCAO, hippocampal ADK was significantly reduced in both hemispheres after 1, 3, and 24 h of reperfusion. Reduction of ADK-immunoreactivity corresponded to a 2.2-fold increase in hippocampal adenosine at 3 h of reperfusion. Remarkably, a significant reduction of ADK immunoreactivity was also found in the ipsilateral (stroked) hippocampus after 15 mins of MCAO and 3 h of reperfusion. Thus, transient downregulation of hippocampal ADK after stroke might be a protective mechanism during maturation hippocampal cell loss.     

Global ischemia induces NMDA receptor-mediated c-fos expression in neurons resistant to injury in gerbil hippocampus.

The induction of c-fos protein-like immunoreactivity (CFPLI) was examined in the hippocampus of gerbils at several time points after transient global ischemia. c-Fos protein induction was largely confined to the dentate gyrus, CA3 and CA4 regions from 2 to 8 h after transient bilateral carotid occlusion. Little CFPLI was seen in the CA1 subfield, which is disproportionately sensitive to injury after global ischemia. c-Fos induction was completely blocked by pretreatment with MK-801 (3 mg/kg). Our results show that c-fos expression after global ischemia is NMDA receptor mediated, and mainly found in hippocampal neurons resistant to ischemic injury.     

脑缺血再灌注后脑组织c-fos基因表达与丹参的影响

采用线栓法制成大鼠大脑中动脉缺血再灌注模型，用地高辛精标记ｃ－ｆｏｓ探针进行原位杂交。结果示缺血再灌注鼠栓塞侧皮层及海马ｃ－ｆｏｓ基因表达显著增多，图像分析灰阶值为１１８．６±５．１，对侧为１５９．６±３．１（Ｐ＜０．００１）。丹参组栓塞侧皮层及海马ｃ－ｆｏｓ基因表达亦增多，灰阶为１３５．００±２．０５，对侧为１６７．００±２．００（Ｐ＜０．００１）。丹参组与缺血再灌注组比较，栓塞侧丹参组ｃ－ｆｏｓ基因表达显著低于缺血再灌组（Ｐ＜０．０５），而两组栓塞对侧比较无显著差异。本实验表明，脑缺血再灌注后脑组织ｃ－ｆｏｓ基因表达显著增多，丹参能部分抑制缺血后ｃ－ｆｏｓ基因的表达，这可能是其治疗缺血性脑血管病的机理之一。     

硫酸镁对兔全脑缺血后神经元凋亡及氨基酸影响的实验研究

目的 :观察镁对兔全脑缺血后神经元凋亡和氨基酸的影响。方法 :30只兔随机分为 3组 :对照组、缺血组和镁处理组。检测各组缺血再灌注 3d海马CA1区凋亡神经元密度及再灌注 30min海马氨基酸含量。结果 :镁处理组凋亡神经元密度显著低于缺血组 ;镁处理组海马天冬氨酸和甘氨酸含量显著低于缺血组 ,GABA含量显著高于缺血组。结论 :镁可抑制兔全脑缺血再灌注 3d海马神经元凋亡和抑制再灌注 30min海马天冬氨酸和甘氨酸的过度释放及GABA的耗竭     

硫酸镁对脑缺血再灌注大鼠脑组织Bcl-2表达的影响及神经保护作用

目的探讨硫酸镁对脑缺血再灌注大鼠脑组织抗凋亡蛋白Bcl-2表达的影响及脑保护作用。方法将32只大鼠随机分为缺血组(n=15)、硫酸镁组(n=15)和正常对照组(n=2)。采用改良的Pulsinelli法建立脑缺血再灌注大鼠模型;制模后分别给予缺血组和硫酸镁组大鼠腹腔注射生理盐水(1.5 ml/d)及硫酸镁(90 mg/kg.d)。缺血再灌注第1、3、7 d分别观察各组大鼠海马CA1区病理学改变和Bcl-2的表达水平。结果正常对照组大鼠海马CA1区神经细胞数量多,排列整齐。缺血再灌注1 d时,缺血组及硫酸镁组大鼠海马CA1区神经元均未见明显死亡;3 d时,缺血组海马CA1区神经元可见少量死亡,残存神经细胞呈较严重缺血性改变,硫酸镁组海马CA1区神经元无明显死亡;7 d时,缺血组海马CA1区神经元大部分死亡,伴有小胶质细胞增生,硫酸镁组仅见部分神经元死亡。与缺血组相比,硫酸镁组神经元受损程度较轻,坏死区较小。硫酸镁组缺血再灌注各时间点大鼠Bcl-2阳性细胞较缺血组均明显增加(均P<0.05)。结论硫酸镁能上调脑组织Bcl-2的表达,对缺血再灌注大鼠的脑组织有明显的保护作用。     

Alteration of serum/glucocorticoid regulated kinase-1 (sgk-1) gene expression in rat hippocampus after transient global ischemia

Expression of the serum/glucocorticoid regulated kinase-1 (sgk-1) gene has been reported to be induced by various stress stimuli such as hyper- or hypo-osmotic stress, UV irradiation, and heat shock stress; however, its association with global ischemia in the brain has not been studied. Using high-density oligonucleotide array analysis, we found that the sgk-1 gene was one of the genes showing alteration of expression in the rat hippocampus during 1-4 h of reperfusion after 10 min of transient global cerebral ischemia. Using TaqMan RT-PCR analysis, we confirmed an increased level of sgk-1 gene expression with statistical significance in the rat hippocampus at 2 h of reperfusion after 10 min of transient global cerebral ischemia. Using in situ hybridization (ISH) analysis, the increased level of sgk-1 gene expression was found to localize in pyramidal cells of CA2 and CA3 regions of the hippocampus after 2 h of reperfusion. These results provide an insight into the alterations of sgk-1 gene expression in the rat hippocampus after transient global cerebral ischemia.