【作者】 韩冬；

【导师】 冯加纯；

【作者基本信息】 吉林大学 ， 神经内科， 2006， 硕士

【摘要】 目的:观察脑缺血再灌注后血脑屏障通透性的变化规律,探讨干预缝隙连接功能对血脑屏障通透性变化的影响并分析其可能机制。方法:①将体重250g～300g的雄性Wistar大鼠随机分成假手术组和缺血再灌注组。线栓法制备大鼠大脑中动脉缺血再灌注模型。分别于缺血2h再灌注1h、3h、6h、12h、24h、48h、72h,采用荧光分光光度法测定脑组织伊文思蓝含量,来观察血脑屏障的通透性的改变。②应用激光共聚焦显微镜技术观察缝隙连接蛋白Cx43在缺血再灌注半暗带毛细血管周终足上量及分布情况的变化。③取伊文思蓝漏出最多的时间点,设立假手术组、缺血再灌注组、辛醇干预组和DMSO溶剂对照组,分析脑组织内伊文思蓝含量以观察辛醇对血脑屏障通透性的影响。结果缺血2h再灌注3h脑组织伊文思蓝含量开始增加,再灌注24h达高峰。激光共聚焦显微镜发现缝隙连接蛋白Cx43在脑内广泛分布,在脑内毛细血管周围的星形胶质细胞终足上分布密集。在缺血再灌注后半暗带内,终足上的Cx43分布变化、聚集成较大斑块。在缺血2h再灌注24h给予辛醇干预,脑组织伊文思蓝含量低于手术组,二者有显著性差异。结论血脑屏障的通透性在缺血再灌注3h开始上升,再灌注24h达高峰,再灌注72h有所下降但仍高于正常水平。缝隙连接在脑缺血再灌注后半暗带终足上分布变化明显,可能加重了血脑屏障通透性的增加。辛醇阻断缝隙连接可以降低脑缺血再灌注血脑屏障的通透性,从而起到减轻脑水肿的作用。更多还原

【Abstract】 Brain edema is a common complication associated with focalischemia in stroke and increases the risk of brain herniation anddeath. Experimentally, reperfusion following focal cerebralischemia exacerbates the level of brain edema. Changes inblood-brain barrier permeability are responsible for the brain edemaassociated with reperfusion after cerebral ischemia. Astrocytes areintermediately positioned between neurons and brain vessels, bothin contact with their stellate extensions, and therefore occupy a keysignaling position between these two important players. Astrocytesare in contact with endothelial cells of capillary vessels, which formthe blood-brain barrier where important transports take place. Workin astrocyte-endothelial co-cultures has demonstrated that calciumsignals can be communicated between astrocytes and endothelialcells in a bidirectional way, making use of both paracrine ATPsignaling and gap junctions. Gap junctions are ubiquitousthroughout the central nervous system. Gap junctions are the sites ofdirect cell-to-cell communication, facilitating the exchange ofchemical and electrical signals between cells. In this experiment, weinvestigate the influence of gap junction blockade on blood-brainbarrier (BBB) permeability at different time points of reperfusionafter middle cerebral artery occlusion (MCAO) and discuss thepossible mechanism of the gap junctional influence on the change inpermeability of BBB in ischemia.In the test laser scanning confocal microscope(LSCM) wasused to investigate the change of connexin(Cx)43 levels anddistribution. The MCAO/R model was induced using intraluminalsuture technique first described by Longa with a little modification.To identify the influence of the function of astrocytic gap junctionon focal ischemia stroke, octanol, the specific blocker for gapjunctions was used in an intervention study. A total of 60 Wistar ratswere divided into 4 groups: the sham-operation group, control group,octanol-treatment group and DMSO vehicle control group. Controlgroup were divided further into senven subgroups, I2hR1h(ischemia for 2 hours and reperfusion for 1 hour),I2hR3h, I2hR6h,I2hR12h, I2hR24h, I2hR48h and I2hR72h. There were 6 rats in eachgroup. Praxiology changes and neurologic impairment wereevaluated by Longa five-grade scoring standard. We observed thechanges in permeability of BBB by measuring the amount of EB inbrain. Evans Blue(EB, 2% in saline , 4ml/kg) was injected throughfemoral vien 1 hour before killing the rats. After the animals weredecapitated, the brains were removed carefully and cut into 3mmslices. Coronal blocks were next divided into right and lefthemispheres. Samples were weighed and placed in 50%trichloroacetic acid solution. Following homogenization andcentrifugation, the extracted dye was diluted with (1:3), and itsfluorescence was determined (excitation at 620 nm and emission at680 nm) with a luminescence spectrometer. Calculations were basedon external standards in the same solvent (100～1000ng/ml). Thetissue content of EB was quantified from a linear standard curvederived from known amounts of the dye and was expressed pergram of tissue. Octanol-treatment group and DMSO vehicle controlgroup were done at the point of the peak of permeability of BBB.With the same method ,we measure the amount of EB in brain. Tocompare the amount of EB with the same point of group’s, weinvestigate the influence of octanol on BBB permeability.After operation, animals of all groups except the sham-operatedone displayed obvious manifestation of neurologic impairment.Cx43 disribute extensively between cells in the brain, especiallyaround the vessels. The Cx43 expression formed into bigger plagueand remained linear disposition in the penumbra after reperfusionsubsequent to cerebral ischemia. At 3h of reperfusion after cerebralischemia for 2h, the permeability of BBB began to increase, reachedthe peak at 24h of reperfusion and was still elevated at 72h, andafterwards began to decrease. Octanol group was done at 24h ofreperfusion after cerebral ischemia. The amount of EB of octanolgroup was significantly lower than of corresponding operationcontrol group.From the study we can conclude that the permeability of BBBafter reperfusion subsequent to cerebral ischemia is graduallyincreased in 24h. Cx43 expression is concentrated around vessels inbrain. The Cx43 form into bigger plague and the function maybestrengthen after reperfusion. Gap junction might aggravate thedisruption of BBB. Octanol, the specific blocker of gap junctions,could effectively prevent the permeability of BBB from increasingand has a protective effect on BBB.更多还原