大鼠脑缺血再灌注后梗死体积的动态变化

目的观察大鼠大脑中动脉缺血再灌注后梗死灶体积的变化规律。方法线栓法制作大鼠局灶性脑缺血再灌注模型,观察脑缺血2h再灌注3h、24h、3d、7d、14d及21d后的神经功能缺损评分及2％氯化三苯基四氮唑(TTC)标记的梗死体积。结果缺血2h再灌注3h组已经出现较明显的梗死灶(梗死体积占前脑体积14．4％),再灌注24h组梗死体积最大(24．3％),显著大于再灌注3h、7d、14d、21d组(P<0．05)。再灌注3d组梗死灶仍较大 (23．8％),再灌注7d组梗死体积缩小(5．0％),再灌注14d组梗死灶进一步缩小(1．2％),再灌注21d组梗死灶基本消失(0．2％)。大鼠神经功能缺损评分与梗死体积之间呈显著相关(r=0．61,P<0．01)。结论脑缺血再灌注后梗死体积于24h达最大,21d时基本消失。脑缺血再灌注后神经功能缺损评分与梗死体积之间显著相关。     

缝隙连接对大鼠脑缺血再灌注后血脑屏障通透性的影响

目的 探讨缝隙连接(GJ)在脑缺血再灌注血脑屏障通透性变化中的作用及其可能机制.方法 ①应用激光共聚焦显微镜技术观察GJ蛋白Cx43在缺血再灌注半暗带区脑毛细血管周终足上含量及分布情况的变化.②将Wistar大鼠随机分成假手术组,手术组.线栓法制备大鼠大脑中动脉缺血再灌注模型.通过荧光分光光度定量的方法测定不同时间点脑组织中的伊文蓝含量来观察血脑屏障的通透性的改变.③取伊文蓝漏出最多的时间点,增设辛醇干预组和DMSO溶剂对照组,与相同时间点手术对照组比较,观察辛醇对血脑屏障通透性的影响.结果 缺血2 h再灌注3 h脑组织伊文蓝含量开始增加,再灌注24 h达高峰.激光共聚焦显微镜发现GJ蛋白Cx43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2 h再灌注24 h半暗带内,终足上的Cx43分布发生变化,聚集成较大斑块.在缺血2 h再灌注24 h组术前给予辛醇干预,脑组织伊文蓝含量[(4.924±0.296)μg/g]低于手术对照组[(5.543±0.506)μg/g],二者差异有统计学意义(P＜0.05).结论 GJ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GJ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.     

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.     

栀子苷预处理对脑缺血再灌注大鼠血脑屏障的保护作用

目的观察栀子苷预处理对脑缺血再灌注血脑屏障(blood brain barrier,BBB)损伤的保护作用。方法采用线栓法建立大鼠大脑中动脉缺血再灌注(middle cerebral artery occlusion/reperfusion,MCAO/R)损伤模型,设假手术组、模型组以及栀子苷预处理组。通过紫外分光光度计检测脑组织伊文思兰(Evans blue,EB)含量,观察大鼠BBB通透性改变。用免疫组化染色法观察缺血侧额叶皮质区胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、基质金属蛋白酶-9(matrix metallopeptidase-9,MMP-9)及基质金属蛋白酶-2(matrix metallopeptidase-2,MMP-2)的阳性表达。用Western blot法检测水通道蛋白-4(Aquaporin-4,AQP4)的表达。结果 (1)再灌24 h后,大鼠脑组织EB含量明显增加,栀子苷预处理组较之于模型组EB含量降低(P<0.05);(2)模型组GFAP、MMP-9及MMP-2阳性表达与假手术组相比显著增加(P<0.01),而栀子苷预处理明显降低GFAP和MMP-9及MMP-2的表达(P<0.01;P<0.05);(3)缺血再灌注损伤后AQP4的表达显著提高,栀子苷预处理下调AQP4的表达(P<0.05)。结论栀子苷预处理对大鼠脑缺血再灌注引起的BBB损伤具有保护作用。     

大鼠脑缺血/再灌注后血脑屏障通透性的改变及转移生长因子β1的表达

目的：探讨大鼠脑缺血再灌注后血脑屏障（BBB）通透性的改变以及转移生长因子β1（TGF－β1）在脑组织中的表达。方法：采用线栓法制备大鼠局灶性脑缺血再灌注模型，通过测定脑组织中伊文氏蓝（EB）含量及免疫组化法来观察TGF－β1的表达。结果：缺血2h再灌注3h,BBB 通透性开始增加，24h达高峰，72h后明显减弱。同时，TGF－β1在缺血再灌注3h开始表达，24h达高峰，持续至72h,72h后逐渐减弱。结论：脑缺血后BBB的破坏与TGF－β1的表达密切相关，提示TGF－β1参与了BBB内皮细胞破坏的修复过程。     

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。     

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。     

促红细胞生成素对大鼠脑缺血再灌注后血脑屏障通透性和ZO-1表达的影响

目的研究促红细胞生成素(erythropoietin,EPO)预处理对大鼠脑缺血再灌注后血脑屏障通透性和紧密连接蛋白ZO-1的影响。方法采用大鼠大脑中动脉栓塞(MCAO)模型,伊文思蓝渗透性实验检测血脑屏障通透性,免疫组化法和Western blot法测定ZO-1蛋白表达,RT-PCR法测定ZO-1 mRNA表达。结果 EPO预处理能够明显减轻脑缺血再灌注后血脑屏障的通透性(P<0.01),并显著上调缺血脑组织中ZO-1蛋白及mRNA的表达水平(P<0.01)。结论 EPO通过上调脑缺血再灌注后脑组织中ZO-1的表达,降低BBB通透性,这可能是EPO保护脑缺血再灌注后神经组织的机制之一。     

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。     

缺血预处理对大鼠局灶性脑缺血的保护作用

目的 探讨缺血预处理对大鼠局灶性脑缺血的保护作用及其机制。方法 将30只雄性SD大鼠随机分为假手术组、缺血组和缺血预处理组,每组10只。使用改良线栓法制作大鼠局灶性脑缺血模型。采用Longa-Bederson（LB）评分法评估缺血预处理对大鼠局灶性脑缺血后神经功能的影响,采用TTC染色方法分析大鼠海马区梗死面积,采用TUNEL染色法分析大鼠海马区细胞凋亡,使用气相色谱方法分析缺血预处理后大鼠海马区域丙酮酸含量。结果 缺血预处理组大鼠LB评分[（1.67±0.21）分}明显优于缺血组[（3.17±0.31）分;P<0.05]。缺血预处理组大鼠海马区梗死面积[（154±8.1）mm2]明显少于缺血组[（221.20±5.86）mm2;P<0.05]。缺血预处理组大鼠海马区域丙酮酸含量[（3.70±0.20）μmol/g]明显高于缺血组[（2.58±0.17）μmol/g;P<0.05]。结论 缺血预处理对大鼠局灶性脑缺血具有显著神经保护作用,其机制可能与明显减少大鼠脑组织梗死、增加海马丙酮酸含量、减少细胞凋亡有关。     

Intraluminal increase of superoxide anion following transient focal cerebral ischemia in rats

Using a modification of Karnovsky's Mn²⁺/diaminobenzizine (DAB) technique, we examined the production of superoxide anion (O⁻₂) in the vascular lumen following transient occlusion and reperfusion of the left middle cerebral artery (MCA) in Sprague–Dawley rats. The MCA was occluded for 2 h using an intraluminal suture method. Zero, 15, 30, and 60 min after reperfusion, animals were perfused transcardially with buffer containing Mn²⁺ and DAB, and brain samples were prepared for light and electron microscopic examination. The amber reaction deposits of O⁻₂ were observable to the naked eye along the major cerebral vessels of the ischemic hemisphere after each reperfusion period. Upon microscopic examination the deposits were revealed to be within arterial, capillary, and venular lumen. The amount of reaction deposits in the ischemic hemisphere corresponded to the duration of reperfusion. The formation of O⁻₂ was suppressed when the perfusate contained superoxide dismutase and when either Mn²⁺ or DAB was omitted, confirming that the reaction products produced are due to the enhanced production of O⁻₂. These results show that there is a progressive increase in intraluminal O⁻₂ during reperfusion following an ischemic insult which may participate in the aggravation of cerebral damage.     

低氧预适应对小鼠急性脑缺血性损伤保护作用的研究

目的 探讨低氧预适应对小鼠急性脑梗死致脑缺血性损伤的保护作用.方法 将Blb/c近交系小鼠按照随机数字表法分为正常对照组(N组),不做任何处理;假手术组(C组),仅行冷光源照射,不注射玫瑰红;急性脑梗死组(CI组),光化学法诱导制作小鼠脑皮层梗死模型;低氧预适应+急性脑梗死组(HP+CI组),低氧预适应后光化学法诱导制作小鼠脑皮层梗死模型.利用行为学、免疫荧光和激光共聚焦等实验方法.分别行神经功能评定、脑梗死体积测定、细胞凋亡检测.结果 (1)脑梗死体积比较:N组、C组未发现梗死灶,CI和HP+CI组均见明显缺血梗死灶,其中HP+CI组脑梗死体积较CI组明显减小,差异有统计学意义(P<0.05);(2)神经功能评分比较:N组、C组无神经功能缺损症状,CI组和HP+CI组出现明显神经功能缺损症状,神经功能评分明显降低,其中HP+CI组神经功能评分比CI组明显增加,差异有统计学意义(P<0.05);(3)细胞凋亡比较:N组、C组小鼠大脑皮层偶见TUNEL阳性细胞,CI组和HP+CI组TUNEL阳性细胞数明显增多,其中HP+CI组TUNEL阴性细胞数较CI组减少,差异有统计学意义(P<0.05).结论 低氧预适应可能通过减小小鼠急性脑梗死体积、减少细胞凋亡两个方面发挥脑缺血性损伤的保护作用.     

3-Aminobenzamide reduces brain infarction and neutrophil infiltration after transient focal cerebral ischemia in mice

Poly(ADP-ribose) polymerase (PARP) was shown to be detrimental in cerebral ischemia but the mechanisms whereby PARP is deleterious have yet to be determined. They may include a role in neutrophil infiltration known to aggravate ischemic damage. In this context, we investigated the effect of 3-aminobenzamide (3-AB), a PARP inhibitor, on brain damage and neutrophil infiltration after transient focal cerebral ischemia in mice. Ischemia was induced in male Swiss mice, anaesthetized with chloral hydrate (400 mg/kg, i.p.), by a 15-min-occlusion of the left middle cerebral artery using an intraluminal suture. Treatments with 3-AB were first administered intraperitoneally 15 min before reperfusion and endpoints measured at 24 h. Among the range of dosages studied (20-320 mg/kg), 40 mg/kg gave the maximal neuroprotection with a 30% decrease in the infarct volume and tended to improve the neurological score evaluated by a grip test. The same dosage was, however, devoid of effect when injection was delayed 2 or 6 h after reperfusion. Myeloperoxidase (MPO) activity used as an index of neutrophil infiltration showed that infiltration peaked 48 h after reperfusion in our model. At this time point, 3-AB (40 mg/kg given 15 min before reperfusion) markedly reduced the neutrophil infiltration, as evidenced by a 72%-decrease in MPO activity, and was still neuroprotective. Our results confirm that 3-AB reduces brain damage. Moreover, for the first time, a quantitative study shows that 3-AB decreases neutrophil infiltration elicited by cerebral ischemia.     

短暂脑缺血发作特点与脑梗死的关系研究

目的:探讨短暂脑缺血发作(TIA)发作持续时间、发作次数及临床特征与脑梗死的关系。方法:对173例TIA患者临床资料进行回顾性分析,记录其发作持续时间、发作次数及临床特征。发作后均在24小时内行头颅MRI或CT检查,部分于发作48小时后复查,记录影像学异常(责任病灶)的例数。结果:TIA发作≤30分钟、>30分钟患者发生脑梗死比例分别为16/112、22/61,组间比较差异具有显著意义(P<0.01);TIA发作≤3次、>3次TIA患者发生脑梗死比例分别为19/129、19/44,组间比较差异具有显著意义(P<0.01);既往有、无卒中病史者发生脑梗死比例分别为15/43、23/130,组间比较差异具有显著意义(P<0.05);既往有、无高血压病史者发生脑梗死比例为28/102、10/71,组间比较差异具有显著意义(P<0.05);表现为偏肢无力和(或)麻木、非偏肢无力麻木者发生脑梗死的比例分别为33/82、5/91,组间比较差异具有显著意义(P<0.01)。结论:局灶神经功能障碍持续时间>30分钟、发作次数>3次、既往有卒中、高血压病史及表现为偏肢无力和(或)麻木的TIA患者发生脑梗死的比率明显增高。     

Effects of tetrahydrobiopterin on cerebral infarction after transient focal ischemia in rats

Abstract

Objectives: The present study investigated the effects of tetrahydrobiopterin (BH4) on cerebral infarction after transient focal ischemia in rats.

Methods: Focal ischemia (1·5 hours) was created in male Sprague-Dawley rats (250-280 g) by middle cerebral artery occlusion. Some rats were treated with 20 mg/kg tetrahydrobiopterin by intraperitoneal injection 30 minutes before reperfusion. At 2, 6, and 12 hours of reperfusion, the brains were harvested for the nitric oxide synthase (NOS) activity and nitric oxide (NO) level assays. At 12 hours of reperfusion, the brains were harvested for infarct size measurement.

Results: NOS activity and NO level were all augmented after reperfusion. BH4 treatment significantly further increased NOS activity and NO level. Cerebral infarct size was significantly bigger in BH4 treatment group compared to that in no treatment group.

Conclusions: The data indicate that BH4 enhances cerebral infarction after transient focal ischemia in rats, through NOS and NO pathway.     

Mild postischemic hypothermia limits cerebral injury following transient focal ischemia in rat neocortex

Intraischemic mild hypothermia has been shown to attenuate cerebral infarction occurring after transient focal ischemia. In contrast, the capacity of mild hypothermia to provide a protective effect when administered postischemically has not been clearly defined for transient focal events such as occur in many types of stroke. The present study addressed this issue by investigating the influence of timing and duration of mild hypothermia on cerebral infarction in a rat model of reversible focal ischemia. Sprague-Dawley rats (n = 45) were subjected to 3 h of focal neocortical ischemia by occluding reversibly one middle cerebral artery and both carotid arteries. Mild hypothermia was established after reperfusion and maintained for brief (1 h) or prolonged (21 h) periods. Animals were sacrificed 24 or 48 h after ischemia. A significant reduction (32%) in the volume of infarction was obtained when hypothermia was established immediately after reperfusion and maintained for a prolonged (21 h) period. In contrast, immediate but brief (1 h) hypothermia did not reduce infarction volume. Delaying hypothermia until 30 min post reperfusion and maintaining it for 21 h reduced infarction volume by 22%; however, this effect did not achieve statistical significance. These findings demonstrate that mild postischemic hypothermia is capable of protecting against cerebral injury following transient focal ischemia but that prolonged hypothermia is required to achieve this effect. These findings are consistent with increasing evidence that the window of therapeutic opportunity after transient focal ischemia is rather brief and that critical mechanisms involved in this form of ischemic injury remain activated over a rather lengthy postischemic interval.     

局灶性缺血预处理对脑梗死大鼠神经生长因子表达的影响

目的:研究局灶性缺血预处理对脑梗死大鼠神经生长因子(nerve growthfactor,NGF)表达的影响,探讨缺 血预处理诱导脑缺血耐受机制。方法:SD大鼠随机分为3组。预缺血组和假手术组在大脑中动脉缺血(MCAO)前3天 分别接受10min的预缺血或假手术,MCAO 2h后再灌注22h处死;对照组两次均为假手术,比较各组神经功能评分、梗 死体积及NGF的表达。结果:预缺血组神经功能评分、梗死体积较假手术组减少(P<0．05),NGF表达明显高于其余两 组(P<0．01)。结论:局灶性缺血预处理可诱导脑缺血耐受的产生,其作用机制可能与NGF的表达改变有关。     

大鼠脑梗死后乏氧组织长时间存在与星形胶质细胞的关系

目的 探讨大鼠脑梗死后乏氧组织的长时间存在与星形胶质细胞的关系.方法 利用大鼠脑缺血1.5 h再灌注(1.5 h IR组)和持续缺血(PI组)的大脑中动脉闭塞模型,采用EF5和胶质纤维酸性蛋白(GFAP)免疫荧光双标方法观察乏氧组织和星形胶质细胞增殖活化情况,比较两组术后1、3、7、14d缺血侧大脑半球皮层的星形胶质细胞GFAP荧光强度以及与乏氧组织的关系.结果 1.5 h IR组术后1、3、7、14 d均见乏氧组织存在,PI组乏氧组织仅存在3 d.各观察时间点乏氧组织内的GFAP荧光强度均较周围区域高,差异有统计学意义(P<0.05).随着时间的延长,两组的GFAP荧光强度均不断增强,在术后7 d达到高峰,14 d时下降,差异有统计学意义(P<0.05);且各时间点的1.5 h IR组的GFAP荧光强度均高于PI组,差异有统计学意义(P<0.05).结论 脑梗死后星形胶质细胞在乏氧组织内增殖活化尤为明显,乏氧组织的长时间存在与星形胶质细胞密切相关.     

短暂局灶预缺血对脑梗死大鼠脑肝细胞生长因子表达的影响

目的:探讨短暂局灶缺血预处理对脑梗死大鼠脑肝细胞生长因子(HGF)表达的影响及其意义。方法:采取线栓法建立局灶脑缺血模型,预处理组大鼠经10分钟短暂预缺血处理.24h后二次处理造成大脑中动脉闭塞,脑梗死组只在第二次处理造成大脑中动脉闭塞。单纯短暂缺血组只在第一次处理时缺血10分钟。各组均在末次处理24小时后用RT-PCR法检测HGF的表达。结果:单纯短暂预缺血组脑缺血边缘区几乎无HGF的表达.预处理组脑梗死边缘区HGF的表达较单纯脑梗死组HGF的表达增强。结论:短暂局灶缺血预处理引起脑梗死边缘区HGF的高表达可能与缺血耐受的产生有一定的关系,对脑缺血损伤组织的保护及修复可能有重要意义。