局灶性脑缺血预处理上调大鼠梗死区周围巢蛋白的表达

目的 观察局灶性脑缺血预处理(IP)对巢蛋白(NESTIN)表达的影响,探讨NESTIN与脑缺血耐受(BIT)的关系及可能的内源性神经保护机制.方法 45只SD雄性大鼠随机分为脑缺血预处理(CIP)组、大脑中动脉阻塞(MCAO)组、假手术(sham)组.采用TTC染色测定脑梗死体积,光镜下观察脑组织病理改变,免疫组织化...     

大鼠短暂性局灶性脑缺血模型的改进

目的:改进大鼠短暂性(<2小时)局灶性脑缺血模型的制作方法。方法:采用雄性SD大鼠24只,分实验组和对照组,实验组又分三组,用线栓法使大脑中动脉(MCA)分别缺血15min、30min、60min,每组6只;假手术对照组6只。所有实验动物均采用乙醚麻醉,在手术过程中用5-0丝线结扎颈外动脉,在结扎的近侧剪断颈外动脉或其分支-枕动脉,经颈外动脉断端或枕动脉将4-0单丝尼龙线插入颈内动脉直至大脑中动脉起始处。假手术对照组动物线栓插入颅内但不达大脑中动脉起始处。术后即可观察动物行走时是否转圈。结果:所有实验组动物都及时观察到动物行走时出现转圈的表现,符合实验的入组标准。假手术对照组动物术后没有观察到转圈的表现。结论:短暂性局灶性脑缺血模型制作方法经改进更易于制作、可行性更强、可信度更高且有利于进一步推广运用。     

大鼠局灶脑缺血诱导巢蛋白的反应模式

目的:观察巢蛋白(nestin)在局灶脑缺血区的反应模式,以探讨其在脑梗塞灶修复过程中的作用。方法:采用局灶脑缺血模型和免疫组织化学染色方法,探查36只成年雄性SD大鼠的大脑不同区域的巢蛋白阳性细胞的形态、反应时程和分布形式。结果:假手术大鼠的巢蛋白阳性反应存在小血管、微血管和室管膜上皮,在第三脑室底壁内有许多细的阳性纤维,胞体不明显。在脑缺血3 d组,大量巢蛋白阳性细胞分布于缺血侧大脑半球,以大脑皮质缺血区、视前区、尾壳核及第三脑室底部最为显著。缺血区周围的大脑皮质浅层和视前区皮质的阳性细胞呈纤维状,而皮质深层的阳性细胞则呈星状。 巢蛋白阳性细胞的形态和数量变化在脑缺血1周时最显著。阳性细胞显示高度的肥大和增生性变化,其数量亦明显增加,以大脑皮质缺血区和尾壳核区最显著。皮质缺血区和尾壳核区的巢蛋白阳性反应持续到脑缺血6周。随后,其反应稍有减弱。结论:局灶脑缺血诱导反应型星形胶质细胞重表达巢蛋白,提示其可能参与脑缺血性损伤的修复过程。     

大鼠局灶性脑缺血实验指标及评价

缺血性脑血管疾病是一个复杂的病理生理过程，评价脑缺血的指标各种各样，在实验中选取何种指标评价是试验成功的关键，本就脑缺血大鼠在记忆、形态、微循环等指标的研究进展作一综述与评价。     

缺血后适应对大鼠局灶性脑缺血再灌注后谷氨酸的影响

目的:研究缺血后适应对大鼠局灶性脑缺血再灌注后谷氨酸(Glu)浓度变化的影响。方法:建立大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)模型,将36只雄性SD大鼠随机分为假手术(Sham)组、脑缺血再灌注(I/R)组和缺血后适应(I Postcond)组,每组12只。应用高效液相色谱检测缺血脑组织匀浆Glu浓度。结果:局灶脑缺血再灌注6 h后,I Postcond组Glu浓度相较I/R组明显降低(P<0.01),局灶脑缺血再灌注24 h后,I Postcond组Glu浓度较I/R组低,但两者差异亦无显著性(P>0.05)。结论:本研究表明,I Postcond能够减轻MCAO大鼠模型中I/R损伤。细胞间隙Glu清除的加速可能是其重要保护机制之一。     

大鼠局灶性脑缺血MAP2表达及毛冬青黄酮的保护作用

目的：探讨大鼠局灶性脑缺血神经细胞MAP2表达，研究毛冬青黄酮对脑缺血的保护作用：方法：选用颈外动脉插入线栓法制备大鼠大脑中动脉梗塞模型，加只雄性Wistar大鼠，随机分成正常对照组、假手术组、盐水-缺血3h、6h、24h组，毛冬青黄酮-缺血3h、6h、24h组。本研究以MAP2染色为指标，评价大鼠局灶性脑缺血损伤情况以及毛冬青黄酮对大鼠局灶性脑缺血MAP2表达的影响。MAP2在大鼠神经元中表达丰富，对兴奋性氨基酸、生长因子等反应敏感，是细胞内信号传导的重要环节，参与经元的生长和损伤后修复过程。MAP2对缺血敏感，可显示缺血时神经元损伤情况。     

针刺对局灶性脑缺血模型大鼠神经元的保护作用

目的：研究探讨针刺对局灶性脑缺血模型大鼠神经元的保护作用。方法：应用栓线法栓塞大鼠大脑中动脉（MCAO）造成局灶性脑缺血模型，分为三组：假手术对照组、模型组、电针组。观察电针对局灶性脑缺血大鼠神经体征的促恢复作用，电针对局灶性脑缺血大鼠海马CAI段神经元的影响，采用免疫组织化学法检测局灶性脑缺血大鼠神经元凋亡相关基因bcl-2、bax蛋白的表达。     

大鼠局灶性脑缺血实验指标及评价

缺血性脑血管疾病是一个复杂的病理生理过程 ,评价脑缺血的指标各种各样 ,在实险中选取何种指标评价是试验成功的关键 ,本文就脑缺血大鼠在记忆、形态、微循环等指标的研究进展作一综述与评价     

龟板对局灶性脑缺血再灌注后Nestin表达的影响

目的 :探讨补肾中药龟板对局灶性脑缺血再灌注后神经干细胞Nestin表达的作用。方法 :采用大脑中动脉线栓法造成局灶性脑缺血再灌注模型 ,应用免疫组织化学技术检测神经干细胞巢蛋白 (Nestin)的表达 ,观察龟板对脑缺血后神经干细胞巢蛋白表达的影响。结果 :脑缺血再灌注后 7d ,龟板组神经病学评分明显低于缺血对照组 :龟板组缺血侧室管膜、室管膜下区、皮层和纹状体Nestin阳性细胞数显著多于缺血对照组 (P <0 .0 5 )。结论 :补肾中药龟板能上调脑缺血再灌注后Nestin的表达 ,这可能是其治疗缺血性脑血管病的机理之一。     

MK-801的浓度与局灶性脑缺血大鼠内源性神经干细胞增殖的关系

目的 研究兴奋性氨基酸（NMDA）受体拮抗剂MK-801的浓度与局灶性脑缺血大鼠神经干细胞增殖的关系。方法 将60只大鼠随机分为正常对照组（6只）、假手术对照组（6只）、手术对照组（12只）和MK-801不同荆量组（0．2mg／kg、0．4mg／kg、0．6mg／kg、0．8mg／kg、1．0mg／kg、1．2mg／kg）（36只）。除正常对照组外，其它组均需制作大鼠大脑中动脉缺血（middle cerebral artery occlusion，MCAO）动物模型，在模型制作前30分钟按照不同剂量组腹腔注射MK一801，假手术组和手术对照组腹腔注射等量的生理盐水。在模型建立后第7天，通过免疫组化技术标记大鼠梗塞灶附近大脑皮质的Nestin阳性细胞数目。结果 MK-801浓度为0．4mg／kg时，可以明显抑制内源性神经干细胞的增殖，0．8mg／kg剂量以上有显著的抑制作用，并且随着浓度升高抑制作用呈递增趋势，在1．2mg／kg以上有明显的全身副作用。结论 NMDA受体拮抗剂MK-801在局灶性脑缺血后，对大鼠内源性神经干细胞的增殖有抑制作用，并且随浓度的增加抑制作用也随之加强。     

MSK-1和CREB参与低氧预适应降低小鼠缺血性脑损伤

目的 探讨丝裂原和应激激活蛋白激酶1(MSK-1)及cAMP反应元件结合蛋白(CREB)在低氧预适应(HPC)保护小鼠缺血脑组织中的作用.方法 将健康成年雄性BALB/c小鼠随机分为4组:常氧假手术组(NS)、HPC假手术组(HS)、常氧缺血组(NI)和HPC缺血组(HI).利用已建整体HPC和大脑中动脉阻塞(MCAO)小鼠模型,并结合神经行为学评价、氯化三苯基四氮唑(TTC)染色和蛋白印迹(Western blot)等技术,观察HPC对小鼠脑缺血损伤的影响,检测小鼠脑皮层不同缺血区域内MSK-1和CREB磷酸化水平和蛋白表达量的变化.结果 HPC可明显改善小鼠脑缺血后神经行为学表现,减小MCAO致脑缺血皮层梗死体积和水肿率;MCAO致小鼠脑局部皮层缺血后,其缺血核心区内MSK-1和CREB磷酸化水平明显下降(P<0.05);HPC可明显增加缺血半影区内MSK-1和CREB磷酸化水平(P<0.05),而MSK-1和CREB总蛋白表达量在缺血核心区和半影区内均无明显改变.结论 HPC可能通过提高缺血半影区内MSK-1及其底物CREB的磷酸化水平来降低小鼠缺血性脑损伤.     

大鼠脑缺血预适应对脑线粒体功能的影响

观察缺血预适应对大鼠大脑中动脉梗塞的保护作用 ,以及对大鼠脑线粒体功能的影响。观察大鼠双侧颈总动脉缺血预适应后 ,短暂缺血作用对脑梗塞面积、神经症状评分、倾斜板停留时间的影响 ,并测定线粒体复合酶活性、膜肿胀、膜电位、膜流动性的变化。与大脑中动脉梗塞 (MCAO)组比较 ,预适应可以显著降低大脑梗塞面积 (P <0 0 0 1) ;明显改善神经症状评分 ;延长在倾斜板上停留时间 (P <0 0 0 1) ;并可增强线粒体复合酶Ⅰ、Ⅲ、Ⅳ活性 ;线粒体膜肿胀降低 (P <0 0 0 1) ,流动性好于MCAO组 (P <0 0 1) ;膜电位无显著性变化。结果表明 ,缺血预适应对脑有保护作用 ;线粒体的膜受到了保护     

大鼠局灶性脑缺血/再灌注后缺血脑区缓激肽含量的变化

目的研究大鼠局灶性脑缺血/再灌注后缺血脑区缓激肽含量的变化。方法用线栓法制作SD大鼠大脑中动脉阻塞的脑缺血/再灌注动物模型,用免疫细胞化学法结合图像分析技术,检测缺血脑区缓激肽样免疫反应阳性物的平均光密度(A)值作为缓激肽的相对含量,比较局灶性脑缺血3h组、假手术对照组、正常对照组和再灌注30min、2h、4h、16h组缓激肽的相对含量。结果缺血脑区缓激肽样免疫反应阳性物的A值于脑缺血3h/再灌注2h后明显增高(P<0.05),随后下降至增高前水平。结论大鼠局灶性脑缺血/再灌注后,缺血脑区缓激肽含量于再灌注2h后明显增高,其可能在脑缺血后脑水肿的发生中起着重要作用。     

脑衰蛋白反应调节蛋白-2参与低氧预适应减轻小鼠脑缺血损伤

目的 研究参与低氧预适应(HPC)的经典型蛋白激酶C II(cPKC II)相互作用的脑衰蛋白反应调节蛋白-2(CRMP-2) 对缺血脑是否有保护作用。方法 成年雄性BALB/c小鼠随机分为常氧(Nor)、HPC、常氧假手术(Nor+sham)、HPC假手术(HPC+sham)、常氧缺血(Nor+I)和HPC缺血(HPC+I)等6组(每组n=6)。应用小鼠整体HPC和脑中动脉梗死(MCAO)致脑局部缺血模型,结合免疫沉淀、双向凝胶电泳和质谱等技术,分离和鉴定与cPKC II相互作用蛋白;利用聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白印迹(Western blot)技术,分析CRMP-2磷酸化和蛋白降解水平在脑HPC和缺血中的变化。结果 与Nor组比,HPC鼠脑皮层组织内有10种与cPKC? II相互作用蛋白的表达发生了明显变化,其中CRMP-2在膜相关蛋白组分中的表达量升高,而在胞质蛋白组分中的表达量降低。在脑缺血模型中,与Nor+Sham组相比,Nor+I组小鼠脑皮层缺血核心区(Ic)CRMP-2磷酸化水平明显降低(p<0.05, n=6);与Nor+I组相比,HPC+I组小鼠脑皮层Ic区内CRMP-2磷酸化水平明显增高(p<0.05, n=6)。脑缺血可导致CRMP-2发生水解并伴随着大量55-ku水解片段(BDP)的出现,但与Nor+I组相比,HPC+I组小鼠脑皮层缺血半影区(P)内CRMP-2水解片段减少,水解率明显降低(p<0.05, n=6)。结论 CRMP-2参与了 HPC缓解小鼠脑缺血Ic区内CRMP-2磷酸化水平的降低和减少P区内CRMP-2水解片段从而减轻缺血脑组织的损伤。     

TNFR1 mediates increased neuronal membrane EAAT3 expression after in vivo cerebral ischemic preconditioning

A short ischemic event (ischemic preconditioning) can result in subsequent resistance to severe ischemic injury (ischemic tolerance). Glutamate is released after ischemia and produces cell death. It has been described that after ischemic preconditioning, the release of glutamate is reduced. We have shown that an in vitro model of ischemic preconditioning produces upregulation of glutamate transporters which mediates brain tolerance. We have now decided to investigate whether ischemic preconditioning-induced glutamate transporter upregulation takes also place in vivo, its cellular localization and the mechanisms by which this upregulation is controlled. A period of 10 min of temporary middle cerebral artery occlusion was used as a model of ischemic preconditioning in rat. EAAT1, EAAT2 and EAAT3 glutamate transporters were found in brain from control animals. Ischemic preconditioning produced an up-regulation of EAAT2 and EAAT3 but not of EAAT1 expression. Ischemic preconditioning-induced increase in EAAT3 expression was reduced by the TNF-alpha converting enzyme inhibitor BB1101. Intracerebral administration of either anti-TNF-alpha antibody or of a TNFR1 antisense oligodeoxynucleotide also inhibited ischemic preconditioning-induced EAAT3 up-regulation. Immunohistochemical studies suggest that, whereas the expression of EAAT3 is located in both neuronal cytoplasm and plasma membrane, ischemic preconditioning-induced up-regulation of EAAT3 is mainly localized at the plasma membrane level. In summary, these results demonstrate that in vivo ischemic preconditioning increases the expression of EAAT2 and EAAT3 glutamate transporters the upregulation of the latter being at least partly mediated by TNF-alpha converting enzyme/TNF-alpha/TNFR1 pathway.     

Thinning,movement, and volume loss of residual cortical tissue occurs after stroke in the adult rat as identified by histological and magnetic resonance imaging analysis

Plasticity of residual cortical tissue has been identified as an important mediator of functional post-stroke recovery. Many studies have been directed toward describing biochemical, electrophysiological, and cytoarchitectural changes in residual cortex and correlating them with functional changes. Additionally, after neonatal stroke the thickness of residual tissue can change, the tissue can move, and tissue can fill in the stroke core. The purpose of the present study was to systematically investigate and document possible gross morphological changes in peri-infarct tissue after forelimb motor cortex stroke in the adult rat. Rats received a unilateral forelimb motor cortex stroke of equivalent size by pial strip devascularization or photothrombotic occlusion and were then examined using histology or magnetic resonance imaging (MRI) at 1 h, 1, 3, 7, 14, or 31 days post-stroke. Middle cerebral artery occlusion was used as a control stroke procedure. Decreases in cortical thickness, volume, and neural density were found to extend far beyond the stroke infarct and included most of the sensorimotor regions of the stroke and intact hemispheres. Movement of residual tissue towards the infarct was observed and confirmed using anatomical markers placed in intact cortical tissue at the time of stroke induction. The results are discussed in relation to the idea that extensive time-dependent morphological changes that occur in residual tissue must be considered when evaluating plasticity-related cortical changes associated with post-stroke recovery of function.     

Neanthes japonica (Iznka) fibrinolytic enzyme reduced cerebral infarction,cerebral edema and increased antioxidation in rat models of focal cerebral ischemia

Thrombolytic agent is increasingly being used in treating acute ischemic stroke. A novel protease with strong thrombolytic activity, Neanthes japonica (Iznka) fibrinolytic enzyme (NJF) discovered in our laboratory has been reported with characteristics of direct hydrolyzing fibrin and fibrinogen. The neuroprotective effect of NJF and urokinase (UK) was tested in rat models of middle cerebral artery occlusion (MCAO). The model was successfully produced by introducing an intraluminal suture into the left middle cerebral artery (MCA). NJF (0.25, 0.5, 1 mg/kg) was injected intravenously 1 h after the onset of reperfusion. Compared with vehicle group, MCAO animals treated with NJF showed dose dependent reduction in cerebral infarction with improved neurological outcome. Meanwhile, ischemia induced cerebral edema was reduced in a dose dependent manner. Treatment with NJF at 0.5 mg/kg was almost equivalent to UK at 15,000 U/kg dosage in the reduction of cerebral infarction and cerebral edema. Biomedical assay showed that NJF treatment suppressed lipid peroxidation and restored superoxide dismutase (SOD) activities in brain tissue. These results suggest that NJF posses neuroprotective potential in rat MCAO and reperfusion model. Neuroprotection shown by NJF may be attributed to inhibition of lipid peroxidation, increase in endogenous antioxidant defense enzymes.     

Use of 2,3,5-triphenyltetrazolium chloride-stained brain tissues for immunofluorescence analyses after focal cerebral ischemia in rats

The middle cerebral artery occlusion (MCAO) model in rodents has been widely used as model for studying brain ischemic stroke. TTC (2,3,5-triphenyltetrazolium chloride) staining in fresh tissues is used to evaluate the size of the infarct in MCAO model, and TTC-stained brain tissues are considered to be possible to bring a damage to the anatomical structure of neuronal cells and unsuitable for immunofluorescence analyses of cytology, and discarded after evaluation of infarct volume. Another group of models with in vivo fixation was required to the pathological or histological analyses of the infarct brains, which lead to double the numbers of animals in researches. However, some evidences indicate that if we properly optimized staining protocol, TTC-stained brain tissues might be suitable for cytological analyses. In this work, we have optimized the immunofluorescent staining methods of TTC-stained brain slices, and found that TTC-stained brain tissues are suitable for quantitative and qualitative analyses of microglia, astrocytes and neuroblasts, the morphology of theses cell were nearly identical to the in-vivo fixed models. Our optimized-protocol provide two advantages over traditional methods one of them is providing the precise the infarct region, which reduces the differences within groups, the other one is decreasing the total number of animals in research dramatically.     

大鼠大脑中动脉超微结构的衰老性变化

目的 研究大脑中动脉超微结构的增龄变化，为探索脑血管疾病的发病机制提供参考资料。方法 应用透射电镜观察各年龄组大鼠大脑中动脉的超微结构。结果 老龄组大鼠大脑中动脉基膜增厚，内弹性膜变薄、不均质、边缘伸出分支到中膜，内皮细胞和平滑肌细胞向内弹性膜穿过，内弹性膜内出现脂质，并有分层、断裂现象，胶原纤维增多。结论 随着年龄的增加，大鼠大脑中动脉的超微结构具有明显的改变。