铁螯合剂-DFX通过保护血脑屏障减轻大鼠短暂性局灶性脑缺血后出血性转化的研究

目的 旨在明确铁螯合剂-DFX能否减轻大鼠短暂性局灶性脑缺血后出血性转化的发生率和程度,并探讨减轻出血性转化的机制.方法 采用高血糖结合大脑中动脉线栓(MCAO)2h再灌注模型,将98只SD大鼠随机分为DFX组和对照组,分别观察其死亡率和出血性转化率、脑梗死体积、脑水肿程度、出血体积及血脑屏障通透性.结果 MCAO 2h再灌注2h,DFX组患侧半球Evans-blue漏出率明显低于对照组(P ＜0.01);MCAO缺血2h再灌注6h,DFX组出血量明显少于对照组(P ＜0.05);MCAO缺血2h再灌注22h,DFX组动物死亡率、脑梗死体积、基底节区脑肿胀比率均明显低于对照组(P＜0.05).结论 DFX通过保护血脑屏障减少了出血性转化的发生率及其伴随的脑损伤.     

诱导缺血耐受的缺血预处理动物模型的建立

目的 :建立能诱导脑缺血耐受的缺血预处理SD大鼠模型。方法 :SD大鼠分别给予生理盐水右侧颈内动脉灌注 (SI)、双侧颈总动脉夹闭 (BCAO)和双侧颈总动脉夹闭并生理盐水右侧颈内动脉灌注 (BCAO +SI)预处理 ,每次持续 3min ,间隔 7min ,反复 3次 ,2 4h后作线栓大脑中动脉栓塞 (MCAO)。观察处理对MCAO缺血对侧肢体活动、脑组织含水量和梗死体积的影响。结果 :MCAO后 2 4h和 48hBCAO +SI预处理组神经损害体征和脑含水量较SI和BCAO两组轻 ;MCAO 72hBCAO +SI组脑梗死体积也低于其他两组。结论 :BCAO +SI预处理能诱导脑组织明显缺血耐受 ,是研究缺血耐受机制比较理想的缺血预处理模型     

观察肢体缺血后处理对急性脑梗死大鼠血脑屏障的保护效应及其机制的探讨

目的探讨肢体缺血后处理对大鼠急性脑梗死后血脑屏障的保护效应及其机制。方法按实验计划建立大鼠大脑中动脉闭塞(MCAO)模型2 h后,再灌注组和肢体缺血后处理组连续5 d进行肢体缺血后处理。比较脑梗死侧的含水量、伊文思蓝(EB)的含量,应用免疫组化方法检测Eph A2、β-淀粉样蛋白(beta amyloid protein,Aβ)的表达。结果再灌注72 h、5 d右侧脑组织含水量均明显高于同时间肢体缺血后处理组;肢体缺血后处理72 h、5 d组比再灌注组同一时间Eph A2及Aβ的阳性细胞数均较低。结论肢体缺血后处理能减轻大鼠急性脑梗死血脑屏障损伤的作用,脑梗死周围区Eph A2和Aβ表达水平的降低可能为保护血脑屏障的机制。     

两次线栓法构建脑缺血耐受模型大鼠血脑屏障通透性改变与基质金属蛋白酶9的表达☆

背景：诸多研究证实,短暂性脑缺血预处理可诱导脑缺血耐受。然而,脑缺血耐受的内源性保护机制尚未明确。 目的：观察脑缺血预处理诱导脑缺血耐受大鼠再灌注不同时间窗血脑屏障通透性改变及基质金属蛋白酶9表达的变化。 方法：将Wistar大鼠随机分为3组,缺血预处理组采用线栓法阻塞大脑中动脉10 min建立局灶性缺血预处理模型,分别在缺血预处理后1,3,7,14,21 d进行再次缺血2 h;模型组不进行缺血预处理,假手术组不阻塞血管。于再灌注22 h进行神经功能检测,采用TTC染色测定脑梗死体积,通过测定渗出血管外的伊文思蓝含量来评价血脑屏障通透性的变化,免疫组织化学和原位杂交法检测基质金属蛋白酶9蛋白及mRNA的表达。 结果与结论：与模型组比较,缺血预处理组1,3,7 d亚组的神经功能评分、脑梗死体积、血脑屏障通透性、脑含水量以及基质金属蛋白酶9蛋白和mRNA表达均明显减小/降低(P < 0.05或P < 0.01),其中以3 d亚组降低最为明显。提示缺血预处理诱导了脑缺血耐受,预缺血诱导的血脑屏障通透性改变以及基质金属蛋白酶9表达减低在脑缺血耐受中发挥重要作用。     

血管紧张素-(1-7)对大鼠局灶性脑缺血再灌注损伤的神经保护作用

目的 探讨血管紧张素-(1-7)[Ang-(1-7)]对大鼠局灶性脑缺血再灌注损伤的保护作用.方法 对Sprague-Dawley(SD)大鼠制备大脑中动脉梗死(MCAO)模型和假手术模型,并于再灌注24 h和48 h以微型渗透泵从侧脑室给予Ang-(1-7)(100 pmol,0.5 μL/h)或人工脑脊液(aCSF)(0.5 μL/h),由此分组为假手术组(假手术+aCSF)、Ang-(1-7)治疗组[MCAO+Ang-(1-7)]和aCSF治疗组(MCAO+aCSF).检测实验大鼠神经功能评分、再灌注48 h后脑水肿以及再灌注24 h后脑梗死体积,并以试剂盒测定再灌注24 h和48 h后缺血脑组织中超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,以原位末端标记法(TUNEL)检测再灌注48 h后脑梗死灶周围组织神经细胞凋亡数.结果 Ang-(1-7)治疗MCAO模型大鼠,能显著改善神经功能评分(P<0.05)、缩小脑梗死体积(P<0.05)、降低组织MDA含量(P<0.05)、提高组织SOD活性(P<0.01),并明显减少脑梗死灶周围组织神经细胞凋亡数(P<0.01),但对脑组织含水量无明显影响作用.结论 Ang-(1-7)可能通过抗氧化应急反应、减轻神经细胞凋亡程度等实现治疗缺血再灌注损伤、神经保护作用.     

松龄血脉康预处理对脑缺血再灌注大鼠全脑及血清IL-6表达的影响

目的探讨松龄血脉康预处理对脑缺血再灌注大鼠全脑及血清白细胞介素-6(Interleukin-6,IL-6)表达的影响。方法 60只雄性SD大鼠,随机分为松龄血脉康(SL-xmk)预处理组、假手术组、对照组。SL-xmk预处理组采用SL-xmk悬浮液(937.5mg/kg)对大鼠进行为期8w的预防性灌胃处理(n=20),假手术组(n=20)、生理盐水对照组(n=20)采用等容量生理盐水预防性灌胃处理。在预处理时程终点采用线栓法制作大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)2h再灌注24h模型。观察SL-xmk对预处理MCAO大鼠神经功能缺损评分、脑水含量和脑梗死体积的影响。采用ELISA法检测全脑及血清IL-6含量。结果 SL-xmk预处理后全脑和血清IL-6表达显著下降,缺血脑组织含水量和脑梗死体积也明显降低。结论松龄血脉康预处理能显著抑制脑缺血再灌注大鼠脑组织和血清IL-6的表达,减轻神经功能缺损,降低脑组织水含量,缩小梗死体积。     

黄体酮对局灶性脑缺血再灌注大鼠水通道蛋白-4表达及血脑屏障通透性的影响

目的研究黄体酮对大鼠局灶性脑缺血再灌注后脑组织内水通道蛋白-4(AQP4)的表达及血脑屏障通透性的影响。方法健康雄性SD大鼠96只,随机分为4大组:假手术组、手术组、溶剂治疗组和黄体酮治疗组,建立大脑中动脉栓塞再灌注(MCAO/R)模型,分别在缺血2h再灌注6h、1d、3d、5d 4个时间点将大鼠麻醉后断头取脑,采用Western blot法、伊文氏蓝(EB)渗出量及干湿重法分别测定脑组织AQP4的表达、血脑屏障的通透性及脑含水量。结果手术组AQP4的表达、EB的含量及脑组织含水量明显高于假手术组;黄体酮组AQP4的表达、EB的含量及脑组织含水量较手术组及溶剂组明显降低,差异有统计学意义。结论黄体酮可以降低缺血再灌注大鼠脑组织AQP4的表达,从而降低血脑屏障的通透性,减轻脑水肿。     

新型AMPA受体拮抗剂对脑梗死再灌注损伤的保护作用

目的探讨新型AMPA受体拮抗剂他仑帕奈(Talampanel,TAL)对缺血性脑卒中的脑保护作用及其可能机制。方法实验大鼠分为假手术组(Sham组)、模型组(MCAO组)、TAL组,根据TAL组给药时间的不同,分TAL0 h、TAL1 h、TAL3 h、TAL5 h 4个亚组。利用线栓法建立大脑右侧中动脉脑梗死模型。Z-Longa评分; TTC染色比较各组大鼠脑梗死体积; HE染色观察海马CA_1区神经元的形态变化;免疫组织化学方法观察脑梗死周围区caspase-3的表达及梗死皮质区神经元数量。结果与MCAO组相比,TAL各组海马CA_1脑水肿和神经元病理损害程度明显减轻,神经功能损伤评分及脑梗死体积明显降低(P 0. 05),梗死周围区caspase-3阳性细胞数明显减小(P 0. 05),梗死皮质区存活神经元数量明显增加(P 0. 05),而TAL5 h组较TAL0 h、TAL1 h、TAL3 h给药组相比,脑保护作用减弱(P 0. 05)。结论他仑帕奈可通过减少缺血再灌注大鼠神经功能损伤评分,缩小脑梗死体积,减少caspase-3阳性细胞表达,对脑缺血再灌注损伤具有很好的保护作用,对脑保护作用具有一定的时间依赖性,随着时间延长,脑保护作用会减弱。     

白藜芦醇对大鼠缺血再灌注损伤后脑组织的保护作用

目的探讨白藜芦醇对大鼠缺血再灌注损伤后脑组织的保护作用及其机制。方法利用线栓法制作大鼠脑缺血再灌注损伤模型。72只SD大鼠按随机数字表法随机平均分为假手术组、对照组、白藜芦醇高剂量组和白藜芦醇低剂量组。缺血2h再灌注24h后,分别测定动物的神经损伤功能评分、脑组织梗死体积,缺血再灌注损伤的脑组织中髓过氧化物酶(MPO)的活性、伊文思兰的含量、肿瘤坏死因子-α(TNF-α)的含量及基质金属蛋白酶-9(MMP-9)的表达水平。结果白藜芦醇治疗组神经功能损伤评分均较对照组明显降低(P<0.05),脑梗死体积明显缩小(P<0.05),MPO的活性、伊文思兰的含量、TNF-α的含量及MMP-9表达水平均也明显低于对照组(P<0.05)。结论白藜芦醇可能通过降低炎症反应和血脑屏障通透性对大鼠脑缺血再灌注损伤的脑组织起神经保护作用;其抗炎作用可能与其降低TNF-α的含量有关,而降低血脑屏障通透性则可能与MMP-9的表达下调有关。     

丁苯酞预处理对大鼠脑缺血再灌注损伤后脑水肿和血脑屏障的影响

目的观察丁苯酞(NBP)预处理对大鼠脑缺血再灌注损伤(CIRI)后脑水肿和血脑屏障的影响。方法雄性SD大鼠90只,随机分为假手术组(Sham组)、模型组(IR组)、NBP低剂量组(NBPⅠ组)、NBP中剂量组(NBPⅡ组)、NBP高剂量组(NBPⅢ组),每组18只。采用线栓法复制大脑中动脉栓塞(MCAO)模型,干湿重法测定脑含水量反映脑水肿程度,比色法测定脑组织伊文思蓝(EB)含量反映血脑屏障的损伤程度,实时荧光定量PCR法检测MMP-9及TIMP-1 mRNA的表达水平。结果 NBP预处理脑缺血再灌注损伤大鼠后,可明显降低脑含水量及EB含量,MMP-9表达显著减少,TIMP-1表达明显增加(P<0.01),而NBPⅡ、Ⅲ组差异无显著性。结论NBP预处理对大鼠脑缺血再灌注损伤的保护作用,可能是通过调节MMP-9/TIMP-1的表达,降低血脑屏障通透性,减轻脑水肿实现的。     

一氧化碳及一氧化氮对局灶性缺血脑组织的影响

目的 研究一氧化碳与一氧化氮对局灶性缺血脑组织脑含水量及血脑屏障通透性的影响。方法 将SD大鼠随机分为4组：为生理盐水组、Hemin组、ZnPP组及Hemin ZnPP组，分别用等量生理盐水、Hemin、ZnPP、Hemin ZnPP腹腔注射，1h后制成MCAO模型。栓塞后24h检测血浆CO及NO浓度、脑组织含水量、血脑屏障通透性。结果 与生理盐水组相比，Hemin组血浆CO浓度明显升高，NO浓度无变化，脑组织含水量下降，血脑屏障通透性减低；ZnPP组血浆CO浓度明显减低，NO浓度上升，脑组织含水量上升，血脑屏障通透性增高；同时注射Hemin ZnPP时CO浓度无变化，NO浓度升高，脑组织含水量上升，血脑屏障通透性增高。结论 NO及CO在缺血早期对脑组织均具有保护作用，两者之间存在着相互作用，CO通过HO影响NOS及NO。     

丁苯酞预处理对大鼠脑缺血再灌注损伤的神经保护作用

目的研究丁苯酞预处理对大鼠局灶性脑缺血再灌注损伤的神经保护作用。方法健康成年SD雄性大鼠48只,随机分为假手术组、缺血再灌注组、丁苯酞预处理组,每组各16只。各组均灌胃5d后,采用线栓法制作大鼠局灶性脑缺血再灌注(MCAO)模型,缺血2h、再灌注24h,进行神经功能缺损评分,TTC染色及图像分析观察脑梗死体积,免疫组化法检测脑组织caspase-3、bcl-2表达的变化。结果与缺血再灌注组相比,丁苯酞预处理组神经缺损程度改善,梗死灶体积减少,caspase-3阳性细胞数量减少,bcl-2表达上调。结论丁苯酞可减轻缺血性脑血管病的发作,具有一定的神经保护作用。     

局灶预缺血诱导脑缺血耐受的动物模型

目的　建立一种简便可靠的 SD大鼠局灶性脑缺血预处理模型。方法　将大鼠随机分为 3组 ,分别给予 10 m in大脑中动脉缺血 ( MCAO)预处理 ( PC) ;10 min PC后 2 h MCAO( PC MCAO)及假手术 ( SS)后 2 hMCAO( SS MCAO) ,再灌注 2 2 h后处死 ,观察各组神经功能缺损、梗死体积及脑含水量变化。结果　 PC MCAO组神经功能评分、梗死体积及含水量均明显低于 SS MCAO组 ,PC组无神经功能缺损及梗死灶形成。结论　 2次线栓法建立的大鼠局灶脑缺血预处理模型 ,能有效减轻 MCAO所致的神经损伤 ,操作简便 ,稳定性好 ,是一种研究局灶脑缺血耐受的有用工具。     

Matrix metalloproteinase-9 expression and blood brain barrier permeability in the rat brain after cerebral ischemia/reperfusion injury

BACKGROUND: The integrity of the blood brain barrier (BBB) plays an important role in the patho-physiological process of cerebral ischemia/reperfusion injury. It has been recently observed that metalloproteinase-9 (MMP-9) is closely related to cerebral ischemia/reperfusion injuryOBJECTIVE: This study was designed to observe MMP-9 expression in the rat brain after cerebral ischemia/reperfusion injury and to investigate its correlation to BBB permeability.DESIGN, TIME AND SETTING: This study, a randomized controlled animal experiment, was performed at the Institute of Neurobiology, Central South University between September 2005 and March 2006.MATERIALS: Ninety healthy male SD rats, aged 3-4 months, weighing 200-280g, were used in the present study. Rabbit anti-rat MMP-9 polyclonal antibody (Boster, Wuhan, China) and Evans blue (Sigma, USA) were also used.METHODS: All rats were randomly divided into 9 groups with 10 rats in each group: normal control group, sham-operated group, and ischemia for 2 hours followed by reperfusion for 3,6,12 hours, 1,2,4 and 7 days groups. In the ischemia/reperfusion groups, rats were subjected to ischemia/reperfusion injury by suture occlusion of the right middle cerebral artery. In the sham-operated group, rats were merely subjected to vessel dissociation. In the normal control group, rats were not modeled.MAIN OUTCOME MEASURES: BBB permeability was assessed by determining the level of effusion of Evans blue. MMP-9 expression was detected by an immunohistochemical method.RESULTS: All 90 rats were included in the final analysis. BBB permeability alteration was closely correlated to ischemia/reperfusion time. BBB permeability began to increase at ischemia/reperfusion for 3 hours, then it gradually reached a peak level at ischemia/reperfusion for 1 day, and thereafter it gradually decreased. MMP-9 expression began to increase at ischemia/reperfusion for 3 hours, then gradually reached its peak level 2 days after perfusion, and thereafter it gradually decreased.CONCLUSION: MMP-9 expression increases in rat brain tissue after focal cerebral ischemia/reperfusion injury, which correlates with increased permeability of the BBB.     

Effects of ischemic preconditioning on blood-brain barrier permeability and MMP-9 expression of ischemic brain

The study was designed to investigate the effects of ischemic preconditioning (IP) on permeability of blood-brain barrier (BBB) and expression of matrix metalloproteinase-9 (MMP-9) in subsequent ischemic hemisphere. Rats were divided into four groups, one group was used as control, and the other three groups were given three different pretreatments: the first group received a saline injection into the right internal carotid artery (SI), the second group underwent both left and right carotid arteries occlusion (BCAO), and the third group was treated with BCAO and SI simultaneously (BS). After 24 hours of pretreatments, the focal cerebral ischemia was induced by inserting a thread into the right middle cerebral artery causing occlusion (MCAO). Brain water content, BBB permeability and MMP-9 expression of ischemic hemisphere brains were measured at 24 and 48 hours after MCAO. After 24 and 48 hours MCAO, averages for brain water content were 82.92 and 83.12% in BS group, 85.19 and 85.73% in SI group and 86.06 and 85.88% in BCAO group. Evans blue content of ischemic hemispheres were 14.01 and 11.74 microg/mm(3) at 24 and 48 hours after MCAO in BS group, which were lower than the other two groups, 16.22, 15.01 and 16.61, 15.58 microg/mm(3), respectively (p<0.01). The expression levels of MMP-9 in ischemic hemisphere in BS were lower than that in other two groups (p<0.01). Therefore, ischemic preconditioning could ameliorate brain edema and BBB disruption caused by subsequent cerebral ischemia. Ischemic preconditioning could decrease MMP-9 protein and mRNA expression, which may be an important mechanism of cerebral ischemic tolerance.     

葛根素预处理对大鼠局灶性脑缺血再灌注损伤的保护作用及机制

目的探讨葛根素预处理对局灶性脑缺血再灌注损伤的保护作用及其可能机制。方法健康成年雄性SD大鼠120只,随机分成5组（n=24）：假手术组（S组）,脑缺血再灌注组（IR组）,Pc-24h100mg组,Pc-24h200mg组,Pc-24h400mg组,其中Pc-24h组于脑缺血前24h给予相应剂量葛根素腹腔注射行单次预处理,采用大脑中动脉线栓法建立局灶性脑缺血再灌注模型,缺血90min,再灌注24h。大鼠清醒后进行神经功能缺陷评分,再灌注24h时处死大鼠,处死后取脑组织,测定脑梗死容积比（BIVP）、脑组织含水量及MDA水平。结果葛根素预处理可以改善大鼠脑缺血再灌注损伤的神经功能缺损评分,降低BIVP、脑组织含水量及MDA水平（P均〈20.01）。其中Pc-24h400mg组减低神经功能缺损评分、BIVP及脑组织含水量明显优于Pc=24h100mg及Pc-24h200mg组（P均〈0.01）,而Pc-24h100mg及Pc-24h200mg组之间无显著差异（P均〉0.05）。结论葛根素预处理可能通过抑制脑水肿及氧化损伤来减轻大鼠局灶性脑缺血再灌注损伤。葛根素预处理的脑保护作用具有一定的量效关系。     

脑缺血后尼膜同对AQP9 mRNA表达和血脑屏障通透性的影响

目的 研究脑缺血后尼膜同对AQP9 mRNA表达和血脑屏障通透性的影响以及脑缺血后尼膜同对脑的保护作用.方法 采用阻断大鼠大脑中动脉制作脑缺血大鼠模型,通过原位杂交和图像分析方法测定梗死区AQP9 mRNA的表达水平,同时电镜进行对应部位的病理观察,并检测缺血脑组织中伊文思蓝外渗的量.结果 脑缺血后,在尼膜同组和对照组缺血组织均出现缺血性病理变化、AQP9 mRNA表达上调、血脑屏障通透性增加,其变化趋势一致,随着脑缺血时间延长其改变加重.尼膜同组AQP9 mRNA的表达变化与生理盐水对照组,虽有差别,但差异无显著性意义(P>0.05);尼膜同组血脑屏障通透性及病理变化明显低于生理盐水对照组,其差异有显著性意义(P<0.05).结论 脑缺血后,尼膜同可能阻滞血脑屏障通透性的增加,起到脑保护作用,但不影响AQP9 mR-NA的表达.

Abstract：

Objective The aim of the study was to investigate the effect of Nimotop on the expression of AQP9 mRNA and the changes of BBB penetrability, as well as the effect of Nimotop on the brain after cerebral ischemia in rats.Methods The model of cerebral ischemia was made by occluding unilateral middle cerebral artery(MCA) of the rats with the suture method. The expression of AQP9 mRNA was assessed by in situ hybridization and imaging analysis. The pathological changes of the ultrastructure were observed under TEM. The BBB permeability of ischemic brain was determined by Evans Blue (EB) extravasation method. Results In Nimotop groups, the expression of AQP9 mRNA, the increasing of BBB permeability and the ultrastructure changes of the tissues were as same as those in control groups. The changes of two groups became remarkable after cerebral ischemia. Changes of AQP9 mRNA expression in Nimotop group were as same as those in control groups(P > 0.05) ;BBB permeability and the uhrastructure changes were more slight in Nimotop group than in control group (P < 0. 05). Conclusion Nimotop could decrease the permeability of BBB and brain damage after cerebral ischemia. However, Nimotop had no effect on the expression of AQP9 mRNA.     

Neurovascular and neuronal protection by E64d after focal cerebral ischemia in rats

Calpains and cathepsins are two families of proteases that play an important role in ischemic cell death. In this study, we investigated the effect of E64d, a mu-calpain and cathepsin B inhibitor, in the prevention of neuronal and endothelial apoptotic cell death after focal cerebral ischemia in rats. Rats underwent 2 hr of transient focal ischemia from middle cerebral artery occlusion (MCAO) and were sacrificed 24 hr later. E64d (5 mg/ kg intraperitoneally) was administered 30 min before MCAO. Assessment included neurological function, infarction volume, brain water content, blood-brain barrier permeability, histology, and immunohistochemistry. The E64d-treated rats had significant brain protection against ischemic damage. We observed a reduction of infarction volume, brain edema, and improved neurological scores in E64d-treated rats compared with the nontreated control. Furthermore, there was a remarkable reduction in both proteases and caspase-3 activation and apoptotic changes in both neurons and endothelial cells in E64d-treated rats. These results suggest that E64d protects the brain against ischemic/reperfusion injury by attenuating neuronal and endothelial apoptosis.     

内源性一氧化碳对局灶性脑缺血大鼠神经功能、梗死灶体积、脑含水量的影响

目的研究内源性CO浓度变化对局灶性脑缺血大鼠神经功能及脑组织含水量、梗死灶体积的影响.方法将48只S.D.大鼠随机分为2组(n=24), 一组为梗死灶体积组, 一组为脑含水量组.每一组又分为3小组, 分别为HO诱导剂、 HO抑制剂、生理盐水组(n=8).使用HO诱导剂、 HO抑制剂腹腔注射, 等量生理盐水腹腔注射作为对照组, 1 h后制成MCAO模型.栓塞后24 h观察局灶性脑缺血大鼠神经功能改变, 同时检测CO浓度、梗死灶体积、脑含水量.结果与生理盐水组相比, HO诱导剂组CO浓度明显升高(P＜0.01), 大鼠神经功能明显改善, 梗死灶体积、脑含水量明显降低, 各为(P＜0.01、 P＜0.01、 P＜0.05), 而HO抑制剂组CO浓度明显降低(P＜0.01), 大鼠神经功能缺失加剧, 梗死灶体积、脑含水量明显升高, 各为(P＜0.01、 P＜0.05、 P＜0.05).结论内源性CO是一种信使分子, 浓度升高对局灶性缺血的脑组织具有保护作用.     

Specific Role of Tight Junction Proteins Claudin-5, Occludin,and ZO-1 of the Blood–Brain Barrier in a Focal Cerebral Ischemic Insult

Blood–brain barrier (BBB) leakage plays a key role in cerebral ischemia–reperfusion injury. It is quite necessary to further explore the characteristic and mechanism of BBB leakage during stroke. We induced a focal cerebral ischemia model by transient middle cerebral artery occlusion in male rats for defining the time course of BBB permeability within 120 h following reperfusion and evaluate the specific role of tight junction (TJ) associated proteins claudin-5, occludin, and ZO-1 as well as protein kinase C delta (PKCδ) pathway in BBB leakage induced by reperfusion injury. We verified a bimodal increase in the permeability of the BBB following focal ischemia by Evans blue assay. Two peaks of BBB permeability appeared at 3 h and 72 h of reperfusion after 2 h focal ischemia, respectively. The leak at the endothelial cell was represented at the level of transmission electron microscopy. TTC staining results showed increased infarct size with time after cerebral ischemia reperfusion. The mRNA and protein expression levels of these three TJ associated proteins were significantly decreased compared with the sham-operated group within 120 h of reperfusion, corresponding to the time-dependent change of the biphasic pattern in BBB leakage. The redistribution of claudin-5, occludin, and ZO-1 in ischemia brain microvascular endothelial cells was observed at the same time points. In addition, Western blot assay revealed PKCδ level was also significantly increased in a similar biphasic pattern to above results within 120 h after cerebral ischemia–reperfusion. This study demonstrates the timing of TJ associated proteins claudin-5, occludin, and ZO-1 in light of BBB permeability associated with cerebral ischemia reperfusion, and suggests PKCδ pathway may participate in TJ barrier open and BBB leakage during reperfusion injury in a time-dependent manner.