脑缺血再灌注大脑皮质微血管生成及胚胎肝激酶1表达与年龄的关系

目的：从Flk-1因子表达变化揭示老年脑缺血/再灌注血管再生机制。方法：SD雄性青年和老龄大鼠,随机分为青年假手术组、青年模型组、老龄假手术组、老龄模型组,模型组分为I 3h、I/R 1d、3d、6d、12d时间点。采用线栓法制备局灶性脑缺血/再灌注模型,运用免疫组化和原位杂交等技术测定脑微血管密度（MVD）、微血管场面积、Flk-1蛋白及mRNA表达。结果：老龄假手术组MVD较青年假手术增高（P<0.01）,老龄模型组I/R 1d、3d、6d、12d MVD、血管场面积较青年模型组降低（P<0.05,P<0.01）。青年模型组MVD于I 3h开始增加,I/R 6d达峰值,持续至I/R 12d;老龄模型组MVD自I 3h持续下降至I/R 12d。青年模型组I/R 1d血管场面积明显增加,I/R 1d～6d逐渐下降,至I/R 12d又明显增高;老龄模型组血管场面积于I/R 1d达峰值,后逐步下降。老龄假手术组Flk-1表达较青年假手术组增强（P<0.01）,老龄模型组I 3h Flk-1表达较青年模型组增强（P<0.01）,I/R 3d、6d、12d Flk-1表达较青年模型组减弱（P<0.01）。老龄模型组Flk-1表达逐步增强至I/R 1d达峰值,此后表达迅速减弱。青年模型组Flk-1表达峰值出现较老龄模型组延迟,I/R 3d表达最强,至I/R 12d表达仍能维持一个较高水平。老龄模型组I/R 3d、6d、12d Flk-1 mRNA表达水平较青年模型组降低（P<0.01）。青年和老龄模型组Flk-1 mRNA均随再灌注时间延长而呈现出表达逐步增强的趋势,均于I/R 1d达到峰值。老龄大鼠Flk-1 mRNA表达于I/R 1d后迅速减弱,而青年大鼠至I/R 12d仍可维持一个相对高的表达水平。结论：老年脑缺血/再灌注损伤后脑微血管生成能力明显减弱,其机制可能与促血管生长因子Flk-1的蛋白及其mRNA表达减弱有关。Flk-1表达在血管生成中具有重要作用,增龄因素可能是导致Flk-1因子表达减弱的主要原因之一。     

老龄大鼠脑缺血/再灌注微血管生成的变化意义

目的探讨老年脑缺血/再灌注(I/R)脑微血管生成的意义。方法SD雄性青年和老龄大鼠,随机分为青年假手术组、青年模型组、老龄假手术组、老龄模型组;模型组分为脑缺血3h、I/R1d、3d、6d、12d时间点。采用线栓法制备局灶性脑I/R模型,测定脑微血管密度(MVD)、血管场面积、神经细胞凋亡数以及脑组织病理改变等。结果老龄模型组I/R6d、12d MVD较老龄假手术组降低(P<0.05,P<0.01),I/R 1d血管场面积较老龄假手术组增高(P<0.05);与青年组同时间点分别比较,老龄假手术组MVD增高(P<0.01),老龄模型组I/R 1d、3d、6d、12d大鼠MVD、血管场面积均降低(P<0.05,P<0.01)。老龄模型组MVD自脑缺血3h持续下降至I/R 12d;血管场面积于I/R 1d达峰值,后逐步下降。老龄模型组I/R 1d、3d、6d、12d大鼠神经细胞凋亡数较其假手术组增多(P<0.05,P<0.01);与青年组同时间点分别比较,老龄假手术组及模型组I/R3d、6d神经细胞凋亡数增多(P<0.01)。老龄模型组神经细胞凋亡数I/R 3d达峰值,I/R 3~12d逐渐减少。脑组织病理改变方面...     

阿斯匹林干预对沙鼠脑缺血再灌注后NF-κB、MCP-1表达的影响

目的 通过研究阿斯匹林干预对沙鼠脑缺血再灌注后NF-κB、MCP-1表达的影响，旨在进一步探讨阿斯匹林在脑缺血再灌注损伤中的作用。方法 将50只健康蒙古沙鼠随机分为正常对照组、假手术组、脑缺血再灌注组（I／R组）、阿斯匹林干预组；夹闭双侧颈总动脉10min后松夹，建立沙鼠全脑缺血再灌注模型；用免疫组化法检测缺血脑组织NF-κBp65、MCP-1的表达。结果缺血再灌注后6h～7d NF-κBp65的表达量显著高于正常对照组及假手术组（P〈0．01），且出现MCP-1阳性表达；同I／R组比较，阿斯匹林干预组在缺血再灌注后6h、1d、3d、7d NF-κBp65与MCP-1表达量均下降（P〈0．05）。结论 阿斯匹林能够下调NF-κB、MCP-1的表达而减轻脑缺血再灌注损伤。     

脑缺血再灌注心肌损伤老龄大鼠神经肽的变化

目的 从内皮素（ET）,降钙素基因相关肽（CGRP）,神经肽Y（NPY）,神经降压素（NT（的变化方面研究老龄大鼠脑缺血再灌注心肌损伤的机制。方法 青年（5月龄）和老龄（20月龄以上（大鼠均为分为模型组和正常对照组,观察大鼠全脑缺血30min 再灌注60 min后心肌组织病理形态和血浆,脑组织中ET,CGRP,NPY含量。结果 脑缺血再灌注心肌组织出现明显的病理改变,老龄大鼠较青年大鼠严重,青年对照组和青年模型组,老龄对照组脑组织中NPY低于青年对照组和老龄西药组,结论 脑缺血再灌注心肌损伤与以ET占优势的CGRP与ET的平衡失调有关,由于老龄大鼠ET与CGRP间平衡失调和NPY的增龄变化使脑缺血再灌注后这些病理改变较青年大鼠更为明显。     

大鼠局灶性脑缺血再灌注血脑屏障超微结构及紧密连接蛋白Occludin变化的研究

目的研究大鼠局灶性脑缺血再灌注模型血脑屏障(BBB)超微结构和Occludin的变化,探讨BBB的结构改变及Occludin的表达异常在再灌注损伤中的作用。方法雄性Wistar大鼠,随机分成假手术组、缺血2h再灌注3h、12h、24h、72h组,应用透射电镜、RT-PCR、免疫组化和Western Blot等方法观察再灌注后不同时相缺血区皮质BBB的超微结构,Occludin mRNA和蛋白水平的变化。结果局灶性脑缺血再灌注后,缺血区皮质BBB的超微结构受损,Occludin mRNA和蛋白表达水平下调。上述变化开始于再灌注后3h,再灌注24h达到高峰,72h开始减弱。结论脑缺血再灌注过程中,BBB的超微结构损伤及Occludin的表达下降加重了缺血再灌注损伤。     

增强MRI评价脑缺血再灌注后大鼠血脑屏障的通透性

目的：通过增强MRI来研究脑缺血再灌注后大鼠血脑屏障（BBB）通透性 的改变。方法：采用线栓法制备大鼠局灶性脑缺血再灌注模型。脑缺血4h后再灌注。用1．5T超导型MR机进行大鼠脑冠状位扫描，随后经股静脉注入Gd-DTPA,迅速行同层间歇扫描（每隔10min扫描1次），持续100min。双双侧对称的脑实质为兴趣区，测得兴趣区信号强度（SI），求其增强率。结果：脑缺血4h,再灌注50min时，缺血侧纹状体增强度与对侧相比差别具有非常显著性意义（P＜0．01）。再灌注70min时，缺血侧大脑皮层增强率较对侧增高（P＜0．05）。缺血侧侧脑室在再灌注10min时可见明显强化。结论：纹状体区BBB双大脑皮层更易受到再灌注损伤，血脑屏障较早受到破坏。增强MRI是一种敏感的在活体条件下检测BBB损伤的理想方法。     

钙拮抗剂对大鼠脑缺血后血脑屏障通透性的影响

目的 研究钙离子拮抗剂对大鼠脑缺血再灌注后血脑屏障(BBB)通透性和脑梗死灶体积的影响. 方法 插线法制作大鼠脑缺血再灌注模型.缺血2 h后再灌注.将150只大鼠按随机数字表法分尼莫地平组和对照组,每组分再灌注6h、12h、24 h、48h、72 h五个时间段,再灌注后尼莫地平组和对照组立即分别腹腔注射尼莫地平和生理盐水2 mg/kg.每12小时注射一次,用甲酰胺荧光法及透射电镜观察不同时段BBB通透性破坏的情况,TTC染色后计算梗死灶体积百分比.结果 大鼠脑缺血再灌注后BBB通透性和梗死灶体积百分比随时间延长逐渐增加.且BBB通透性的增加呈现两个高峰,第一个高峰在再灌注后12 h,第二个高峰在再灌注后48 h.尼莫地平组BBB通透性及脑梗死灶体积百分比的增加均较对照组明显,差异有统计学意义(P<0.05). 结论 脑缺血再灌注增加BBB的通透性和脑梗死灶体积百分比.再灌注后给予尼莫地平可加重这些病理变化.     

乌司他丁对脑缺血再灌注损伤大鼠血脑屏障通透性和MMP-9活性的影响

目的 观察乌司他丁对大鼠局灶脑缺血再灌注损伤后血脑屏障(BBB)通透性和基质金属蛋白酶-9(MMP-9)活性的影响.方法 采用栓线法制备大鼠局灶性脑缺血再灌注损伤模型,腹腔注射乌司他丁,于脑缺血再灌注后6h、24h、48h、72h处死大鼠.通过测定损伤侧脑组织中伊文思蓝(EB)含鼍来观察BBB通透性的改变,用明胶酶谱法检测同侧脑部MMP-9活性变化.结果 脑缺血再灌注损伤后,大鼠EB含量增加,24h最明显;MMP-9活性明显升高,48h达高峰.乌司他丁处理组EB含量及MMP-9活性水平明显低于脑缺血再灌注组(P<0.05).结论 乌司他丁可抑制脑缺血再灌注后大鼠MMP-9的活性,减轻BBB通透性的破坏.     

人工合成E-选择素对大鼠局灶性脑缺血再灌注损伤后脑组织一氧化氮合酶表达的影响

目的 探讨人工合成E-选择素对大鼠局灶性脑缺血/再灌注（I/R）损伤后脑组织一氧化氮合酶（NOS）及血清一氧化氮（NO）含量的影响.方法 采用改良的Zea Longa法建立脑I/R损伤模型.66只雄性SD大鼠随机分为对照组、模型组和人工合成E-选择素治疗组（治疗组）.治疗组大鼠采用股静脉注射人工合成E-选择素10 mg·kg-1.应用硝酸盐还原法测定血清中NO含量和免疫组化法检测缺血区脑组织神经型一氧化氮合酶（nNOS）、诱导型一氧化氮合酶（iNOS）阳性细胞数.结果 ①NO：以对照组NO含量为正常生理数据,模型组脑缺血2h/再灌注2～24h NO含量呈上升趋势,24 h时达高峰,72 h有所降低但仍高于对照组,各时间点与对照组比较明显增高（P＜0.01）;治疗组NO变化趋势同模型组,NO含量较模型组减少（P＜0.05）,较对照组增多（P＜0.01）.②NOS：以对照组nNOS、iNOS阳性细胞数为正常生理数据,模型组nNOS阳性细胞在脑缺血2h/再灌注2h后开始表达,12h达高峰,至24h开始降低,各时间点与对照组比较明显增高（P＜0.01）;模型组iNOS阳性细胞在脑缺血2h/再灌注2h开始出现,并持续增多,随时间延长呈上升趋势,24h达高峰,至72 h出现下降,各时间点与对照组比较明显增多（P＜0.01）;治疗组各时间点nNOS、iNOS阳性细胞变化趋势同模型组,但较模型组减少（P＜0.05）,较对照组增多（P＜0.01）.结论 大鼠脑I/R损伤后脑组织NOS活性表达增多,NO浓度升高导致脑组织损伤;人工合成E-选择素通过降低NOS表达,减少NO释放、减轻炎症反应和脑I/R损伤,起脑保护作用.     

单胺类神经递质和神经肽Y在老龄大鼠脑缺血再灌注心脏组织损伤中的意义

目的 从多巴胺（DA）、去甲肾上腺素（NE）、肾上腺素（E）、神经肽Y（NPY）方面揭示脑缺血心肌损伤的机制。方法 观察青年（5－6月龄）和老龄（20月龄以上）大鼠全脑缺血心肌损伤乳酸脱氢酶（LDH）、肌酸磷酸激酶（CPK）活性及NPY、DA、NE、E的含量。结果 青年模型组血清中CPK和LDH水平明显高于青年对照组和老龄模型组，老龄模型组高于老龄对照组。青年模型组和老龄模型组血中NE分别高于青年和老龄对照组；老龄模型组血中NE、E水平和下丘脑NE水平高于青年模型组，老龄模型组下丘脑中NE和E水平低于老龄对照组。脑组织中NPY老龄对照组低于青年对照组和老龄模型组。结论 在鼠脑缺血再灌注心肌损伤与交感－肾上腺系统兴奋性增强有关，老龄大鼠尤为明显。     

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.     

重组人促红细胞生成素对大鼠局灶性脑缺血再灌注损伤的保护作用

目的 探讨重组人促红细胞生成素(EPO)对大鼠局灶性脑缺血再灌注损伤所致炎性反应的保护机制。方法采用线拴法制备大鼠局灶性大脑中动脉缺血再灌注模型,应用TTC染色法、干湿重法、常规HE染色法观察EPO治疗前后再灌注24h脑梗死体积、脑组织含水量以及组织学变化,应用RT- PCR方法检测EPO治疗前后再灌注lh、3h、6h、12 h、24h、72 h缺血侧脑皮质IL-1β、TNF-α基因表达的变化。结果与假手术组相比,EPO可显著缩小缺血再灌注24h所致的脑梗死体积(P＜0.01),降低梗死侧脑组织含水量(P＜0.01),减轻病理学变化。缺血再灌注各时相点缺血侧皮层IL -lβ mRNA和TNF -α mRNA表达均显著上调(P＜0.01),12 h达高峰。EPO治疗后lh、3h、6h缺血侧皮层IL - 13 mRNA表达显著下降,与病理组相应时间点相比,分别降低了63％、55％和84％(P＜0.01)。EPO治疗后lh、3h、6h缺血侧皮层TNF -α mRNA表达亦显著下降,与病理组相应时间点相比,分别降低了75％、76％和95％（P＜0.01）。结论EPO可能通过抑制IL - 1β、TNF-α的基因表达,降低缺血再灌注的炎性反应损伤而改善脑组织的结构和功能。     

脑缺血再灌注肾脏损伤老龄大鼠的单胺类神经递质和神经肽的变化意义

目的：从多巴胺(DA)、去甲肾上腺素(NE)、肾上腺素(E)、内皮素(ET)和降钙素基因相关肽(CGRP)的变化方面揭示老龄大鼠脑缺血再灌注肾脏损伤机制。方法：青年(5月龄)和老龄(20月龄以上)大鼠均分为模型组和正常对照组，观察大鼠全脑缺血30min再灌注60min后肾脏组织形态和肌酐(Cr)、尿素氮(Bun)、DA、NE、E、ET、CGRP含量的变化。结果青年和老龄模型组大鼠肾脏组织形态和功能均出现明显的病理改变，交感-肾上腺系统兴奋性增强，老龄模型组较青年模型组严重。青年对照组血浆中CGRP高于青年模型组和老龄对照组。老龄模型组血中ET高于老龄对照组和青年模型组。结论：大鼠脑缺血再灌注肾脏损伤与交感-肾上腺系统兴奋性增强以及CGRP与ET的平衡失调有关，由于增龄的变化使老龄大鼠脑缺血再灌注肾脏损伤程度和这些病理变化尤为明显并具有一定特点。     

阿托伐他汀对大鼠脑缺血再灌注后血脑屏障通透性的影响

目的 评估阿托伐他汀对大鼠脑缺血再灌注后血脑屏障通透性的影响。方法 采用常规尼龙线栓法制备SD大鼠脑缺血再灌注模型,并将大鼠随机分为假手术组、大脑中动脉阻断再灌注(Middle cerebral artery occlusion/reperfusion,MCAO/R)(对照)组和MCAO/R阿托伐他汀(治疗)组; 对照组和治疗组分别于脑缺血2 h再灌注24 h处死; 标准湿干法测定脑组织含水量; 实时聚合酶链反应(Real-time polymerase chain reaction,RT-PCR)检测基质金属蛋白酶-2(Matrix metalloproteinases-2,MMP-2)和基质金属蛋白酶-9(Matrix metalloproteinases-9,MMP-9)的mRNA表达水平; 应用免疫组化法测定Ⅳ型胶原蛋白(Ⅳ type collagen,CoⅣ)水平; 电镜观察显示血脑屏障超微结构的改变。结果 治疗组与对照组比较,脑组织含水量减少(P<0.01); 阿托伐他汀治疗显著降低了MMP-2和MMP-9的mRNA表达水平; 治疗组脑组织CoⅣ水平高于对照组(P<0.01); 电镜观察显示治疗组血脑屏障超微结构的改变明显好于对照组。结论 阿托伐他汀可以降低脑缺血再灌注大鼠血脑屏障的通透性,从而减轻脑水肿。     

Ischemic Postconditioning Protects the Neurovascular Unit after Focal Cerebral Ischemia/Reperfusion Injury

Recently, cerebral ischemic postconditioning (Postcond) has been shown to reduce infarction volume in cerebral ischemia/reperfusion (I/R) injury. However, it is unclear if ischemic Postcond offers more extensive neuroprotection than current therapies. The aim of this study was to investigate the neuroprotective effects of ischemic Postcond on the neurovascular unit (NVU). A middle cerebral artery occlusion rat model was used; cerebral infarct volumes, neurologic scores, and transmission electron microscopy were evaluated 24 h after reperfusion. We used Evans blue extravasation, immunohistochemistry, and Western blot analyses to evaluate the integrity of the blood brain barrier (BBB) and the distribution and expression of the tight junction (TJ)-associated proteins of claudin-5 and occludin in brain microvessel endothelium. The Postcond group showed significantly reduced infarct volumes and decreased neurologic impairment scores compared to the I/R group. Also, injuries to the cerebral microvascular endothelial cells, astrocytes, and neurons were minimized in the Postcond group. The permeability of the BBB increased in both the I/R and Postcond groups, but the Postcond group showed a significant decrease in permeability than the I/R group. Expression of both claudin-5 and occludin were higher in the Postcond groups compared to the I/R group, but expression of both proteins decreased in the I/R and Postcond groups compared to the sham group. The results of our study suggest that ischemic Postcond is an effective way to reduce injury to neurons, astrocytes, and endothelial cells, to increase protein expressions of TJ-associated proteins, and to improve BBB intergrity affected by focal I/R. Ischemic Postcond could protect the NVU from I/R injury.     

Chronic cerebral hypoperfusion and reperfusion injury of restoration of normal perfusion pressure contributes to the neuropathological changes in rat brain

Restoration of normal perfusion pressure after resection of cerebral arteriovenous malformations (AVMs) is sometimes complicated by unexplained postoperative brain swelling and/or intracranial hemorrhage, which has been termed normal perfusion pressure breakthrough (NPPB). The precise mechanism of NPPB is still unclear. In this study, we investigated the time courses of blood-brain barrier (BBB) disruption, water content, neuronal apoptosis, myeloperoxidase (MPO) activity and superoxide dismutase (SOD) activity in the brain during restoration of normal perfusion pressure in a new rat model of chronic cerebral hypoperfusion associated with AVMs. Male Sprague-Dawley rats were randomly divided into either a sham-operated group, a control group, or a model group with reperfusion assessed at 1, 12, 24 and 72 h after restoration of normal perfusion pressure. BBB disruption was judged by extravasation of Evans blue (EB) dye. We observed that EB and water content in rat brains of the model group with reperfusion were significantly increased compared with the other groups. The most predominant increase occurred at 1 h after reperfusion, and the next at 24 h after reperfusion, representing biphasic changes which are similar to the pathological processes of acute cerebral ischemia/reperfusion injury. There was no difference of the percentage of apoptotic cells in rat brains between the sham-operated group and the control group using flow cytometry. No prominent apoptotic cells were found in the model group with reperfusion at 1 h. However, the percentage of apoptotic cells increased significantly in rat brains of the model group with reperfusion at 12 h, peaked at 24 h, and decreased at 72 h after reperfusion. Apoptotic cells were confirmed with electron microscopy and terminal deoxynuleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). A significant enhancement of MPO activity in combination with reduction of SOD activity was seen at 12, 24 and 72 h in rat brains of the model group with reperfusion. Our data indicates that reperfusion after restoration of normal perfusion pressure with chronic cerebral hypoperfusion lead to secondary neuronal damage which may associate with cerebral ischemia/reperfusion injury.     

Effects of the chemokine CCL2 on blood-brain barrier permeability during ischemia-reperfusion injury.

The chemokine CCL2 is considered as one of the main effectors driving postischemic infiltration of monocytes into the brain parenchyma. New experimental data, however, suggest that CCL2 could also participate in blood-brain barrier (BBB) 'opening' during the transmigration of monocytes. The current study examines the role of CCL2 in regulating BBB permeability after ischemia in vitro. To address this issue, an in vitro BBB model (coculture of astrocytes and brain endothelial cells) was subjected to 5 h of oxygen glucose deprivation, followed by reoxgenation (in vitro ischemia/reperfusion (I/R)) for 0 to 48 h. During reperfusion, there was a biphasic enhancement of barrier permeability, with a 200-fold increase in barrier permeability to FITC-albumin at 6 h and a further period of disruption around 24 h. The latter coincided with increased secretion of CCL2 by both astrocytes and brain endothelial cells and increased levels of the CCL2 receptor, CCR2. Applying antisense oligonucleotide or neutralizing antibody to block CCL2 significantly decreased I/R-induced enhancement of BBB permeability (approximately twofold) and redistribution of tight-junction (TJ) proteins (occludin, zonula occluden-1, 2, claudin-5). Similarly, absence of CCR2 from endothelial cells caused stabilization of TJ complexes and decreased the permeability of brain endothelial barrier during in vitro I/R. These data suggest CCL2/CCR2 has an important role in regulating brain endothelial permeability and might be a potential novel therapeutic target for stroke.