普罗布考对大鼠脑出血后MMP-9表达及血-脑屏障通透性的影响

目的讨论普罗布考对大鼠脑出血后基质金属蛋白酶-9(MMP-9)的表达及血-脑屏障通透性的影响。方法采用自体动脉血注入大鼠尾壳核建立脑出血模型,将7 5只大鼠分成对照组、脑出血组、普罗布考组。各实验组分12h、1d、3d、5d、7d五个时间点,每个时间点5只大鼠。分别采用逆转录聚合酶链法(RT-PCR)、蛋白印迹法(Western blot)、伊文思兰染色法测定不同时间点的MMP-9 mRNA、MMP-9蛋白的表达和血-脑屏障通透性的变化。结果 MMP-9 mRNA、MMP-9蛋白的表达和血-脑屏障通透性在脑出血后1d开始升高,3d达到高峰,5d⁷d开始下降。给予普罗布考治疗后,在1d、3d、5d、7d时间点MMP-9蛋白和血-脑屏障通透性明显下调,与脑出血组相比差异有统计学意义(P<0.01),而MMP-9 mRNA无明显变化(P>0.01)。12h时间点MMP-9蛋白和血-脑屏障通透性在普罗布考组与脑出血组差异无统计学意义(P>0.01)。结论普罗布考可以减轻大鼠脑出血后的MMP-9蛋白的高表达,降低血-脑屏障的通透性,从而减轻脑水肿程度。     

缺氧预处理对创伤性脑损伤大鼠 Claudin5 表达及血脑屏障通透性的影响

目的探讨缺氧预处理对创伤性脑损伤大鼠脑组织紧密连接蛋白Claudin-5的表达及血-脑屏障通透性的影响。方法204只大鼠随机分为创伤性脑损伤组（T组）96例,缺氧预处理后脑损伤组（H组）96例及对照组12例。T组行自由落体撞击法建立大鼠创伤性脑损伤模型,H组给予3d缺氧预处理后,同法致脑损伤,两组大鼠于伤后1h、4h、8h、12h、24h、3d、7d、14d断头处死。采用干湿重法测脑组织含水量：Real—timePCR和Westernblot检测各组大鼠挫伤区周围脑组织Claudin-5mRNA及蛋白表达变化;IgG法检测血一脑屏障通透性变化。结果T组和H组伤后1hClaudin-5mRNA及蛋白表达开始降低,8～12h降至最低点,1d开始上升,直至伤后14d渐趋于对照组水平;其中H组各时间点Claudin-5mRNA及蛋白表达均高于T组。T组各时间点血一脑屏障通透性及脑组织含水量均明显高于H组（P〈0．05）,且两组均高于对照组（P〈0．05）。结论缺氧预处理可在创伤性脑损伤早期上调紧密连接蛋白Claudin-5的表达,维持血-脑屏障完整性,减轻脑水肿。     

Diazoxide preconditioning attenuates global cerebral ischemia-induced blood-brain barrier permeability

Brain edema formation due to blood-brain barrier (BBB) disruption is a major consequence of cerebral ischemia. Previously, we demonstrated that targeting mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) protects neuronal tissues in vivo and in vitro, however, the effects of mitoK(ATP) openers on cerebral endothelial cells and on BBB functions have never been examined. We investigated the effects of mitoK(ATP) channel opener diazoxide on BBB functions during ischemia/reperfusion injury (I/R). Rats were treated with 6, 20 or 40 mg/kg diazoxide ip for 3 days then exposed to global cerebral ischemia for 30 min. BBB permeability was assessed by administering Evan's-blue (EB) and Na-fluorescein (NaF) at the beginning of the 30 min reperfusion. I/R increased BBB permeability for the large molecular weight EB (ng/mg) in the cortex (control: 146 +/- 12, n = 7; I/R: 1049 +/- 152, n = 11) which was significantly attenuated in diazoxide-treated rats (575 +/- 99, n = 9; 582 +/- 104, n = 8; 20 and 40 mg/kg doses). Diazoxide pretreatment also significantly inhibited the extravasation of the low molecular weight NaF. Edema formation in the cortex was also decreased after diazoxide pretreatment. In cultured cerebral endothelial cells, diazoxide depolarized the mitochondrial membrane, suggesting a direct diazoxide effect on the endothelial mitochondria. Our results demonstrate that preconditioning of cerebral endothelium with diazoxide protects the BBB against ischemic stress.     

Neovasculature and blood-brain barrier in ischemic brain infarct

Summary In a 53-year-old man with a progressive mental deterioration and myoclonic jerks, brain biopsy failed to show any significant light microscopical findings. Electron microscopy revealed membranebound vacuolar inclusions in many neuronal nuclei as the only prominent finding. Hamsters intracerebrally inoculated with the biopsy material demonstrated typical spongiform changes in the gray structures of the brain when sacrificed on the 309th and 332nd days post inoculation, characteristic of experimental Creutzfeldt-Jakob disease (CJD). These intranuclear vacuolar inclusions, originally reported in experimental Creutzfeldt-Jakob disease in this laboratory, may be a valuable electron microscopic feature in some CJD cases and may play an important role in supporting the diagnosis of CJD.Supported in part by the NIH grants AG 03106 and NS 12674     

黄体酮对脑梗死大鼠脑部MMP-9、TIMP-1表达及血脑屏障通透性的影响

目的观察黄体酮对脑梗死大鼠脑组织MMP-9、TI MP-1表达及血脑屏障通透性的影响。方法制作SD大鼠永久性局灶性脑缺血模型(permanent middle cerebral arteryocclusion,pMCAO),将大鼠随机分成假手术组、模型组、HBC溶剂治疗组和黄体酮治疗组。模型组、HBC溶剂治疗组和黄体酮治疗组,其中后3组设24h、48h、7d 3个时间点,每组每个时间点12只。应用干湿重法检测脑组织含水量;免疫组织化学法研究脑组织MMP-9、TI MP-1的表达情况。结果模型组MMP-9、TI MP-1的表达及脑水含量在各个时间点与HBC溶剂治疗组相比差异无统计学意义(P0.05),但在黄体酮治疗组脑组织中的表达较模型组和HBC治疗组明显降低(P0.05)。结论黄体酮可能通过调节MMP-9/TI MP-1表达,降低血脑屏障通透性,减轻脑缺血后脑水肿程度。     

高血糖对缺血再灌注大鼠脑组织中MMP-9及对血脑屏障的影响

目的 探讨高血糖对脑缺血后脑组织中MMP-9表达的影响,从而研究高血糖加重脑损伤的机制.方法 采用链脲佐菌素腹腔注射复制高血糖模型;线栓法制作大脑中动脉栓塞模型;应用免疫组化技术观察MMP-9蛋白表达,并测血脑屏障的通透性.结果 高血糖组缺血再灌注24h、48h的MMP-9 表达明显增加,其阳性细胞数与血糖正常组对比有明显差异,且血脑屏障通透性比正常对照组明显增加.结论 高血糖可诱导脑缺血再灌注后脑组织内的MMP-9 表达,增加血脑屏障的通透性,从而加重脑组织的损伤.     

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。     

MMP-9 and EBA immunoreactivity after papaverine mediated opening of the blood-brain barrier

Papaverine is a non-specific vasodilatory drug, which was found to unexpectedly cause opening of the blood-brain barrier (BBB). We studied the expression of two endothelial cell related markers, endothelial barrier antigen (EBA) as a marker of BBB intactness, and matrix metalloproteinase-9 (MMP-9), an extracellular matrix molecule, the activation of which results in BBB opening. Immunoreactivity was quantified after intra-carotid injection of papaverine in rats. BBB opening was identified at 1 h, but had been reversed by 24 h. Reduction in EBA-immunoreactivity occurred at 3 h, whereas MMP-9 was not observed until 24 h. The activation of MMP-9 thus occurred much later than BBB opening, which suggests a different role for MMP-9 when selective and reversible opening of the BBB is produced by pharmacological manipulation.     

溶血磷脂酸对大鼠uPA蛋白的表达及其对血脑屏障的影响

目的 研究溶血磷脂酸对uPA蛋白表达及其对血脑屏障的影响.方法 在立体定向仪下向大鼠右侧尾壳核注射50uL溶血磷脂酸, 免疫组化检测不同时间点注射部位邻近脑组织uPA蛋白表达,伊文思蓝(EB)作为示踪剂定量测定相应时间点BBB通透性.结果 注射溶血磷脂酸6 h后尾壳核uPA蛋白表达开始增高,在24 h达高峰, 48 h以后逐渐下降,与对照组相同时间点比较差异有显著性(P<0.05),同侧基底节区BBB对EB的通透性6 h开始增加,24 h通透性达最大,到48 h通透性渐减,与对照组相同时问点比较差异有显著性(P<0.01).结论 溶血磷脂酸可能促进脑微血管内皮细胞及其周围星形胶质细胞uPA蛋白表达的增加,降解基底膜,破坏血脑屏障,从而促进脑水肿的发生.     

实验性糖尿病大鼠内皮屏障抗原表达和血脑屏障通透性变化

目的 研究糖尿病对血脑屏障(BBB)通透性和内皮屏障抗原(EBA)表达的影响.方法 雄性SD大鼠20只,随机分为正常对照组和糖尿病组.用链脲霉素(streptozotocin,50 mg/kg)静脉一次性注射制备糖尿病模型.4个月后取两组大鼠前额叶皮质,用免疫组织化学的方法检测微血管内皮细胞EBA及微血管周围纤维蛋白原(fibrinogen)的表达.结果 免疫组织化学染色显示,糖尿病组EBA阳性微血管数(11.30±1.15)与正常对照组(17.20±1.03)比较明显减少(P＜0.05).糖尿病组微血管周围纤维蛋白原呈弱阳性表达(5.80±1.98),正常对照组为阴性表达.结论 糖尿病可引起EBA表达减少,BBB通透性增加;EBA表达减少可能是糖尿病引起BBB通透性增加的一个重要因素.     

Effect of insulin-induced hypoglycemia on blood-brain barrier permeability

The effects of hypoglycemia on cerebrovascular permeability to the Evans blue-albumin complex were studied in rats injected with 50 IU/kg, i.v. crystalline zinc insulin. One group of hypoglycemic animals was warmed to keep their body temperatures close to 37 degrees C, and the rats in the other group were allowed to become hypothermic by hypoglycemia. The arterial blood pressures of the hypoglycemic rats were continuously monitored during the coma and a significant rise in pressure was observed in most animals at the end of the coma. When glucose was administered i.v. to five animals of each group, this elevated pressure returned to normal values within 0.5 min and the animals slowly recovered normal behavior. At termination of the coma, most brains in the hypothermic hypoglycemic group showed an intensive and extensive staining by Evans blue; whereas only two brains in the normothermic hypoglycemic group showed any noticeable extravasation of Evans blue-albumin. Arterial PO2, PCO2, and pH were determined and no significant difference was found between values from animals in hypoglycemic coma and the controls. Four animals were surface-cooled and were used to examine the effects of hypothermia on blood-brain barrier permeability. These brains did not show any macroscopically evident Evans blue-albumin extravasation. The results indicated that prolonged, severe hypoglycemia with hypothermia caused a profound blood-brain barrier dysfunction whereas normothermic hypoglycemia resulted in few cases of any noticeable increase in blood-brain barrier permeability.     

Role of ischemic blood-brain barrier on amyloid plaques development in Alzheimer's disease brain

This review demonstrated that ischemic brain injury induces chronic changes in blood-brain barrier in the gray and white matter. This insufficiency of blood-brain barrier may allow entry of uncellular blood components such as different fragments of amyloid precursor protein and cellular blood components like leukocytes and platelets into the brain parenchyma. These blood components may have chronic harmful effects on the ischemic neuronal cells, axons and myelin and can intensify and finish the neuropathology in ischemic brain parenchyma. Pathological accumulation of different toxic fragments of amyloid precursor protein in extracellular space and myelinated axons appears after ischemic blood-brain barrier injury and seem to be concomitant with, but independent of neuronal ischemic cytoplasmic injury. It seems that ischemic blood-brain barrier disturbances may play an important, both direct and indirect role in the pathogenesis of extra- and intracellular space in gray and white matter lesions following ischemic episode. This neuropathology appears to have similar character and distribution as in sporadic Alzheimer's disease. This review presented chronic micro-blood-brain barrier openings in ischemic gray and white matter lesions that probably would act as seeds of future Alzheimer's amyloid plaques.     

溶血磷脂酸对血脑屏障及AQP4蛋白表达的影响

目的 研究溶血磷脂酸对大鼠血脑屏障通透性及水孔蛋白-4(aqaporin-4,AQP4)表达的影响.方法 在立体定向仪下向大鼠右侧尾壳核注射50uL溶血磷脂酸,在不同时间点对注射部位邻近脑组织AQP4蛋白进行免疫组化检测;用伊文斯蓝(Evans blue, EB)作为示踪剂定量测定不同时间点血脑屏障(Blood-brain barrier,BBB)通透性.结果 注射溶血磷脂酸后6 h尾壳核AQP4蛋白表达开始增高,在第2 d达到高峰,并维持到第3 d,以后逐渐下降. 表达增高的部位主要为微血管内皮细胞及其周围的胶质细胞.与对照组各时间点相比,均有显著性差异;LPA注射后6 h同侧尾壳核区BBB对EB通透性开始增加,24 h达最大,到48 h逐渐减低,与对照组相同时间点比差异有显著性(p<0.05).结论 溶血磷脂酸可以促进脑微血管内皮细胞及其周围胶质细胞AQP4蛋白表达的增加,引起血脑屏障通透性的增加,参与脑水肿的发生.     

缺血再灌注大鼠脑内MMP-2、MMP-9与血脑屏障的关系

目的明确脑缺血再灌注后MMP-2、MMP-9与血脑屏障的关系。方法健康雄性SD大鼠54只,线栓法制作大鼠大脑中动脉缺血再灌注模型。应用含明胶的定量酶谱技术检测再灌注3h到7d内MMP-2、MMP-9的表达,用甲酰胺浸泡法检测大鼠脑内EB含量。结果 (1)缺血再灌注24h后,大鼠缺血侧脑内MMP-9表达明显升高,48h到达顶峰,4d后显著下降,7d后水平更低。24h组和48h组与其它各组相比,有显著性差异(P<0．05)。 0MMP-2缺血再灌注48h后明显升高,7d到达高峰,7d组大鼠缺血侧脑内MMP-2水平与3h组和24h组相比,有显著性差别(P<0．01)。(2)再灌注24h大鼠缺血侧脑内EB含量最高,显著高于3h及7d组(P<0．05),但与48h无显著差别(P>0．05)。结论缺血再灌注后出现了MMP-2、MMP-9和血脑屏障的动态变化,血脑屏障损伤与MMP-9 的升高相关,而与MMP-2关系不大。     

Quantitative measurement of blood-brain barrier permeability using perfusion-CT in extra-axial brain tumors

Non-invasive assessment of vascular permeability is of main importance in the diagnosis, treatment and follow-up of intracranial tumors. Perfusion-CT is one of the imaging options available, which affords quantitative assessment of cerebral blood volume and blood-brain barrier permeability. Herein we report two cases of extra-axial tumors studied with perfusion-CT. Comparison with perfusion-MRI was available in one case. High permeability values, as measured by perfusion-CT, reflected the absence of blood-brain barrier in these extra-axial tumors.     

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.     

Ulinastatin decreases permeability of blood--brain barrier by inhibiting expression of MMP-9 and t-PA in postoperative aged rats

Tissue-type plasminogen activator (t-PA) and matrix metalloproteinase-9 (MMP-9) have been reported to play important roles in increased permeability of blood–brain barrier (BBB) under many pathological circumstances. We have showed that Ulinastatin, a broad-spectrum serine protease inhibitor, could alleviate inflammation in the hippocampus of aged rats following partial hepatectomy. In this study, we investigate the expression and potential roles of t-PA and MMP-9 in the protective effect of Ulinastatin. We found that partial hepatectomy increased Evans blue leakage in hippocampus at day 1 and 3 postoperatively. Furthermore, surgery decreased the protein levels of claudin-5, ZO-1, and NF-kB p65 while upregulating the mRNA and protein levels of t-PA and MMP-9 in brain capillaries. All these effects caused by surgery were partially reversed by administering Ulinastatin. Our study sheds light on the roles of t-PA and MMP-9 of BBB in post-surgical neuroinflammation and postoperative cognitive dysfunction. Besides, it could also help to understand the mechanism of Ulinastatin alleviating neuroinflammation.     

Changes in blood-brain barrier permeability associated with insertion of brain cannulas and microdialysis probes

Blood-brain barrier (BBB) transcapillary transport was studied after insertion of cannulas and microdialysis probes into the brains of three groups of rats. Quantitative autoradiography was used to measure changes in BBB permeability around the insertion site. In the first group, BBB function was measured with ¹⁴C-sucrose at times from immediately, and up to 28 days, after insertion of a microdialysis probe. BBB function was disrupted biphasically: a 19-fold increase in the influx constant (K₁) of sucrose occurred immediately after insertion with a second 17-fold increase at 2 days, followed by a slow decline to 5 times normal values at 28 days. In the second group, ¹⁴C-dextran (70 kDa) was used to measure BBB transcapillary transport; K₁ was increased 90-fold after probe insertion. In the 3rd group, ¹⁴C-AIB (α-aminoisobutyric acid) was used to evaluate BBB transport after insertion of a 27 gauge cannula, which was used to infuse 1 μl of saline over 5 min. The K₁ of AIB was increased 25 times control values. We conclude that BBB transcapillary transport function is disturbed in response to insertion of brain cannulas and/or microdialysis probes, that BBB dysfunction is maximal at the cannula or probe tip, varies with time after insertion, may persist for at least 28 days after insertion, and occurs over a wide molecular range of solutes. These results suggest caution when using microdialysis as a method to study normal BBB function, and suggest that microdialysis may overestimate the rate of transfer into and out of the brain.