Effects of endothelin-1 on blood-brain barrier permeability during focal cerebral ischemia in rats

The purpose of this study was to determine whether the transport of small hydrophilic molecules across the blood-brain barrier (BBB) during focal cerebral ischemia could be altered by a topical application of endothelin-1 (ET-1) in the ischemic cortex (IC). Forty minutes after middle cerebral artery (MCA) occlusion, patches of 10 nM ET-1 (low-endothelin group), 100 nM ET-1 (high-endothelin group), or normal saline (control group) were placed on the IC of rats for a 20-min period. One hour after MCA occlusion, transfer coefficient (Ki) of [14C-alpha-]aminoisobutyric acid (14C-AIB) or regional cerebral blood flow (rCBF) was determined. Vital signs were not significantly different among the experimental groups. In the control group (n=8), the Ki of the IC was significantly higher than that of the contralateral cortex (CC; 11.9+/-5.8 vs 5.0+/-1.9 microl/g per minute). In the low-endothelin group (n=8), the Ki of the IC was still significantly higher than that of the CC (9.4+/-5.2 vs 5.3+/-2.5 microl/g per minute). However, in the High-endothelin group (n=8), the Ki of the IC was not different from that of the CC (6.9+/-2.1 vs 5.6+/-2.3 microl/g per minute) and 42% lower than that of the control group. The rCBF was not affected by 100 nM of ET-1 [control (n=6): IC 53+/-18 ml/100 g per minute, CC 94+/-23 ml/100 g per minute; high-endothelin (n=6): IC 49+/-15 ml/100 g per minute, CC 98+/-24 ml/100 g per minute]. Our data suggest that the application of endothelin-1 in the IC could reduce the transfer coefficient of small hydrophilic molecules across the BBB during focal ischemia.     

Local cerebral glucose utilization in the neocortical areas of the rat brain

Summary The neocortex of the rat brain can be subdivided into regions of different local cerebral glucose utilization (LCGU). However, only a few neocortical areas can be delineated by differences in mean LCGUs between neighbouring areas. These area borders correspond exactly with cytoarchitectonically defined borders found in adjacent Nissl-stained preparations. On the other hand, nearly all of the architectonically defined area borders are also recognizable in the LCGU pictures, if differences in laminar distribution patterns of LCGU are taken into account. Furthermore, interareal differences in mean LCGU mainly reflect changes in layer IV, whereas layers II–III and V–VI show nearly identical LCGU values in all neocortical areas of the rat brain. The primary sensory areas exhibit the highest LCGU in layer IV, while the primary motor cortex shows a high LCGU in layer V.As the cytoarchitectonically defined pattern of the cortex is generally corroborated by the regional and laminar LCGU distribution, anatomical, metabolic and functional aspects of cortical architecture are associated.     

Local cerebral glucose utilization in the homozygous Brattleboro rat

The [14C]deoxyglucose technique was used to measure brain glucose utilization in homozygous male Brattleboro and age-matched Long-Evans control rats. Brattleboro homozygotes had significantly higher daily water intakes and plasma osmolalities and significantly lower body weights than controls. Glucose utilization for the brain as a whole and for 46 discrete brain structures was not significantly different for the two strains. Our results indicate that vasopressin is not essential for the maintenance of overall brain glucose utilization in resting, awake rats.     

Modifications of local cerebral glucose utilization during circadian food-anticipatory activity

Food-anticipatory activity that animals express before a daily timed meal is considered as the behavioral output of a feeding-entrainable oscillator whose functional neuroanatomy is still unknown. In order to identify the possible brain areas involved in that timing mechanism, we investigated local cerebral metabolic rate for glucose during food-anticipatory activity produced either by a 4-h daily access to food starting 4 h after light onset or by a hypocaloric feeding provided at the same time. Local cerebral metabolic rate for glucose measured by the labeled 2-[(14)C]-deoxyglucose technique was quantified in 40 structures. In both groups of food-restricted rats, three brain regions (the nucleus of the solitary tract, the cerebellar cortex and the medial preoptic area) showed a decrease in local cerebral metabolic rate for glucose, compared with control ad libitum animals. In addition, only one structure, the paraventricular thalamic nucleus, was affected by temporal restricted feeding, and not by hypocaloric feeding, compared with ad libitum rats. By contrast, three brain regions, i.e. the intergeniculate leaflets, the paraventricular hypothalamic and the arcuate nuclei, showed specifically metabolic decreases during anticipation of hypocaloric feeding, and not during anticipation of temporal restricted feeding, compared with the ad libitum group. Expression of food-anticipatory activity appears to be regulated by an integrated neural circuit of brainstem and hypothalamic pathways, with hypocaloric feeding involving more extensive forebrain areas than temporal restricted feeding.     

Changes in the permeability of the blood-brain barrier following sodium dodecyl sulphate administration in the rat

 The blood-brain barrier (BBB) arises from epithelial-like tight junctions that virtually cement adjoining capillary endothelium together in the brain microvascolature. Several experimental manipulations have been shown able to increase the permeability of brain capillaries, by altering endothelial cell membrane integrity or activating specific biochemical pathways involved in regulation of BBB functionality. Because of its amphiphilic nature, sodium dodecyl sulphate (an anionic surfactant widely used as solubilizer or stabilizer in several pharmaceutical preparations; SDS) may enter into interactions with the major membrane components, which are lipids and proteins. The aim of the present study was to determine the effect of an intracarotid infusion of SDS (25, 50 and 100 μg/kg; infusion rate: 3 ml/min for 30 s) on the functionality of the BBB in the rat. An extensive, dose-dependent Evans blue extravasation was observed, in the ipsilateral brain hemisphere, 15 min following SDS infusion. These results were confirmed by the significant increase in [¹⁴C]α-aminoisobutyric acid ([¹⁴C]AIB) transport (evaluated by calculating a unidirectional transfer constant, K _i, for the tracer from blood to brain) measured in several ipsilateral brain regions 2 min after SDS infusion; this SDS-elicited BBB opening to [¹⁴C]AIB proved to be reversible. Since the BBB is created by the plasma membrane and tight junctions of the endothelial cells, the change in BBB permeability caused by SDS might be explained as a nonspecific surfactant-membrane interaction. Furthermore, SDS might affect the functional characteristics of brain vascular endothelial cells by an interaction with specific BBB proteins and/or biochemical pathways. In conclusion, one can suggest that intracarotid infusion of SDS might provide a useful clinical approach for the intentional introduction of different substances into the brain. On the other hand, these findings should call attention to possible dangerous consequences of using SDS as solubilizer in drug excipients. Received: 14 June 1996 / Accepted: 3 February 1997     

A quantitative immunocytochemical analysis of total surface area of blood-brain barrier in developing rat brain

A mouse monoclonal antibody which specifically reacts with putative blood-brain barrier (BBB) competent endothelial cells of rat cerebral capillaries was used to identify barrier competent cells in the central nervous system (CNS). The development of the cerebral capillaries and the BBB was examined and quantified, from day 6 to day 40 postpartum, using immunocytochemical and unbiased stereological techniques.There was a progressive increase in capillary formation postnatally, with collateral branching observed with progressive age. BBB development was confined to individual endothelial cells located at the periphery of the cortex until day 10 postpartum. Antibody binding progressively increased postnatally, contributing 30% of the total capillary surface area by day 20. There was a rapid elevation of reactivity from day 20 to day 40, with a mean of 83% by day 40.The BBB constitutes minimal amounts of brain vascular capillaries before day 10 of life in the rat. There is a slower increase in BBB than in total capillaries between days 10 and 20. There is a reversal of this trend between days 20 and 40.     

脑缺血再灌注后大鼠血脑屏障通透性的免疫组织化学研究

目的:研究脑缺血再灌注后大鼠血脑屏障(BBB)通透性改变。方法:采用线栓法大鼠大脑中动脉闭塞的局灶性脑缺血模型,缺血1h后再灌注,分别于再灌注后0h、3h、5h、12h、及24h,采用免疫组织化学SABC法,观察内源性免疫球蛋白G(IgG)在脑组织中的表达。结果:再灌注0h、3h时,脑组织中未见IgG的表达。再灌注5h时,缺血侧大脑半球纹状体有局灶性的IgG表达。再灌注12h时,缺血侧纹状体及新皮层可见有广泛的IgG的表达。再灌注24h时,表达更加明显。结论:缺血1h再灌注3~5h时,BBB开始受损开放,通透性增加。纹状体的BBB较新皮层更易受损。再灌注12h、24h,外渗的血清蛋白累积增加。     

间质干细胞与血脑屏障通透性的研究进展

间质干细胞可通过脑实质、血液和脑脊液等移植方式进入脑组织,修复损伤组织,促进神经功能恢复,是治疗中枢神经系统疾病的理想种子细胞;但血脑屏障存在着特殊结构,研究间质干细胞与血脑屏障通透性的相关性,使更多间质干细胞穿过血脑屏障。综述了间质干细胞与血脑屏障通透性的最新研究进展。     

颈淋巴阻滞对大鼠血脑屏障通透性的影响

目的: 探讨大鼠颈淋巴阻滞后血脑屏障(BBB)通透性的变化。方法: 手术组大鼠采用Casley-Smith法阻滞颈淋巴,对照组大鼠不摘除颈淋巴结和不结扎淋巴管,只将它们分离暴露。每组于术后1、3、5、7、14、21 d,通过观察血清中S-100B的浓度、电镜观察镧离子示踪剂和BBB超微结构变化来确定BBB通透性的改变。结果: 各组血清中S100B浓度变化无显著差异,提示BBB没有破坏;电镜观察:对照组与手术组的超微结构变化相同,各组术后各时点血管腔外没有发现镧离子。结论: 颈淋巴阻滞对BBB通透性没有显著影响。     

Changes in cerebral endothelial barrier antigen, without alteration of permeability for intravenously injected leptin in diet-induced obesity in rats

Leptin, a potent anorectic, 16-kDa, adipose tissue-derived protein, predominantly acts in hypothalamic nuclei, signaling obesity and modulating ingestive behavior. To reach this brain area, leptin, probably has to cross the blood-brain barrier (BBB). In some cases of obesity, enhanced leptin levels in the blood do not result in anorectic effects, probably due to an altered leptin transport across the BBB. Therefore, we investigated the BBB in lean and diet-induced obese Lewis rats. To obtain information about the presence of microvessels with barrier dysfunction we examined three brain areas (hypothalamus, cortex, hippocampus) using a monoclonal antibody which detects intact microvessels of the BBB (anti-endothelial barrier antigen, anti-EBA). The results showed a significantly reduced EBA staining in the brain sections of the obese animals, except the hippocampus, compared to the control group. In a second step we injected I125-labeled leptin intravenously (i.v.) in permanent i.v.-cannulated, unrestrained Lewis rats (lean and obese). We measured the radioactivity in the cerebrospinal fluid after puncture of the cisterna magna, in the blood and brain tissue 90 min after injection. The leptin content in the cerebrospinal fluid and brain was not reduced in obese compared to lean rats, thus showing a similar transport capacity of the BBB in both experimental groups. Therefore, the results of the in vivo investigations do not indicate an impairment of the BBB in diet-induced obesity, despite the immunohistological findings. Further functional and morphological studies are necessary to evaluate the specific role of other organs and distinct forms of leptin (free and protein-bound) in the pathogenesis of diet-induced obesity.     

CO对局灶性缺血脑组织血脑屏障通透性的影响

目的:研究一氧化碳及其限速酶(血红素氧合酶-1)对局灶性缺血脑组织血脑屏障通透性的影响。方法:将SD大鼠随机分为3组(n=6),使用血红素氧合酶诱导剂及抑制剂腹腔注射,用等量生理盐水腹腔注射作为对照组,12h后复制MCAO模型。梗塞后24h后检测血液中一氧化碳浓度、血脑屏障通透性。结果:诱导剂组一氧化碳浓度明显高于对照组(P＜0.01),血脑屏障通透性明显低于对照组(P＜0.05),而抑制剂组一氧化碳浓度明显低于对照组(P＜0.01),血脑屏障通透性明显高于对照组(P＜0.05)。血红素氧合酶诱导剂、抑制剂对非梗塞侧的血脑屏障通透性没有影响(P＞0.05)。结论:一氧化碳作为一种信使分子,脑缺血时浓度升高具有保护血脑屏障的作用。     

脑缺血再灌注后大鼠血脑屏障通透性的荧光定量分析

目的：通过敏感的荧光方法,定量分析脑缺血再灌注后大鼠血脑屏障（BBB）的通透性。方法：采用线栓法制备大鼠大脑中动脉闭塞（MCAO）模型，缺血2 h后再灌注，采用伊文思蓝（EB）荧光定量的方法，分别观察了再灌注2 h、3 h、9 h、24 h和48 h时BBB的通透性。结果：再灌注3 h时，缺血侧纹状体EB的含量开始增加（与假手术组相比，P<0.05）。再灌注9 h时，缺血侧新皮层EB的含量开始增加（P<0.01）。而再灌注2 h时纹状体EB的含量和再灌注2 h、3 h时新皮层EB的含量与假手术组相比差别无显著（P>0.05）。结论：脑缺血2 h再灌注2-3 h时，纹状体的BBB通透性开始增加。再灌注9 h时，新皮层的BBB通透性增加。纹状体BBB较新皮层更易受损。EB荧光的方法可精确定量地反映BBB通透性，是研究BBB通透性理想的方法。     

Enhanced delivery of [I]glial cell line-derived neurotrophic factor to the rat CNS following osmotic blood-brain barrier modification

Subsequent to osmotic (mannitol-induced) blood-brain barrier (BBB) opening, [¹²⁵I]glial cell line-derived neurotrophic factor (GDNF) was detected throughout the ventricular system, associated with the ependymal cell layer and extracellular matrix and to some extent penetrated into the cerebral cortex, subcortical gray matter, substantia nigra, septum, eye and optic nerve at 1 and 24 h following an intracarotid administration. Our study indicates that osmotic opening of the BBB allows for extensive distribution of GDNF throughout the central nervous system (CNS).     

当归补血汤对大鼠局灶性脑缺血再灌注血-脑屏障和大脑皮质神经元的影响

目的:探讨当归补血汤对大鼠局灶性脑缺血再灌注后大脑皮质神经元的治疗作用。方法:将90只雄性SD大鼠随机分为正常对照组、模型组、当归补血汤(高、中、低剂量)组。观察各组大鼠行为学缺陷、从股静脉注入伊文斯蓝溶液观察血-脑屏障通透性变化、用Nissl染色法观察大脑皮质神经元的形态和数量。结果:与模型组相比较,当归补血汤治疗组各剂量均能改善大鼠神经功能(P<0.05)、减少脑组织中伊文斯蓝含量(P<0.05)、增加大脑皮质神经元数量(P<0.05),并以中剂量组效果最好。结论:当归补血汤对缺血再灌注引发的大脑皮质神经元死亡有保护作用,这可能与其降低血-脑屏障通透性有关。     

壮通饮对缺氧/复氧诱导的HUVEC/U251细胞屏障损伤的影响

目的:观察壮通饮(ZTY)对缺氧/复氧诱导的人脐静脉血管内皮细胞(HUVEC)和人星形细胞瘤U251细胞损伤模型的作用,探讨壮药保护血脑屏障的相关机制.方法:将8只SD大鼠随机分为4组:空白组(蒸馏水)及低、中、高剂量(3.74、7.48和14.96 g/kg)ZTY含药血清组,每天1次,连续7 d灌胃饲养,获取含药血...     

地塞米松预处理对氧糖剥夺大鼠血脑屏障通透性的影响

目的探讨地塞米松(dexamethasone,DEX)对氧糖剥夺(oxygen glucose deprivation,OGD)条件下大鼠血脑屏障通透性的影响。方法提取纯化大鼠脑微血管内皮细胞,建立血脑屏障OGD模型。在OGD不同时间点(0、30、60 min),以及DEX预处理OGD 60min时,应用辣根过氧化物酶渗漏实验分析血脑屏障的通透性;应用RT-PCR和western blot法分析大鼠脑微血管内皮细胞的紧密连接相关蛋白claudin-5的mRNA和蛋白的表达变化;同时应用免疫荧光方法观察细胞膜上claudin-5的变化。结果与对照组相比,OGD 30、60min,辣根过氧化物酶流量均显著升高;与OGD 60min相比,DEX预处理显著降低了辣根过氧化物酶流量。与对照组相比,OGD30、60min,claudin-5的mRNA和蛋白表达水平均显著降低;与OGD 60min相比,DEX预处理显著增加了claudin-5的mRNA和蛋白表达。免疫荧光观察claudin-5在细胞膜上的表达,与对照组相比,OGD 60min时claudin-5的表达明显减少,DEX预处理后显著增加了claudin-5的表达。结论 DEX预处理降低了OGD条件下的血脑屏障通透性,其机制之一是DEX增加紧密连接相关蛋白claudin-5的表达。     

Age-associated changes in the effects of amphetamine on the blood-brain barrier of rats

The effects of varying doses of amphetamine on the permeability of the blood-brain barrier (BBB) to albumin were studied in young and aged Fischer 344 rats. There was no change in the permeability of the BBB associated with aging as revealed by saline-treated control experiments. The changes in the permeability of the BBB did not correlate with the changes in blood pressure produced by corresponding doses of amphetamine in the young or aged animals. In the dose range reported the young and aged rats showed similar pressor responses to amphetamine. The aged rats showed a greater increase in the permeability of the BBB than the young, and this effect was seen with a lower dose of amphetamine in the aged rats. At the highest doses tested the permeability was restored to control levels in both the young and the aged rats. The enhanced vulnerability of the BBB of the aged rats may be related to a deterioration of the noradrenergic control of cerebrovascular permeability.     

人血脑屏障体外实验模型的建立及缺氧-复氧对其通透性的影响

目的建立人血脑屏障体外实验模型，并探讨缺氧-复氧对血脑屏障模型通透性的影响。 方法将分离、纯化的人脑微血管内皮细胞（HB-MVEC）在细胞插入器上培养至汇合状态，以液面试漏试验确定血脑屏障模型的形成，并通过形态学检查、跨内皮细胞电阻（TEER）测定和辣根过氧化物酶（HRP）通透率对血脑屏障模型进行鉴定；以未达汇合状态的HB-MVEC及培养至汇合状态的人脐静脉内皮细胞（HUVEC）作为对照。观察缺氧-复氧处理（缺氧2h和复氧1、2、4、8、24h）和缺氧-复氧时存在白细胞激活产物（缺氧2h后在有白细胞激活产物存在情况下复氧1h）对血脑屏障模型通透性的影响，以及前列腺素E、α1抗胰蛋白酶和丹参单体764-3对血脑屏障模型的保护作用。各项实验均观察3孔细胞。 结果HB-MVEC在细胞插入器培养至汇合状态后，液面试漏试验呈阳性；扫描电镜观察显示细胞间无间隙，透射电镜检查证实细胞间存在紧密连接。血脑屏障模型、未达汇合状态HB-MVEC和HUVEC的TEER分别为（46.0±1.3）、（30.8±1.4）、（7.5±2.1）Ω/cm^2；向细胞插入器内加入含HRP的培养基培养1h后，HRP通透率分别为0.17%±0.03%、0.26%±0.04%和0.94%±0.07%；缺氧2h和复氧1、2、4、8、24h HRP通透率分别为3.97%±0.94%、6.06%±0.75%、7.17%±0.18%、7.96%±0.47%、8.57%±0.62%、10.37%±0.78%。血脑屏障模型缺氧2h后在有白细胞激活产物的情况下复氧1h，其HRP通透率为8.87%±0.76%，明显高于无白细胞激活产物组（7.20%±0.87%）；而前列腺素E、α1抗胰蛋白酶和丹参单体764-3能够减弱这种情况下的血脑屏障模型通透性增加，3组的HRP通透率分别为7.08%±0.89%，6.01%±0.57%和5.53%±0.62%。 结论应用HB-MVEC可以构建血脑屏障体外模型，缺氧-复氧明显增加血脑屏障模型的通透性，前列腺素E、α1抗胰蛋白酶和丹参单体764-3具有保护血脑屏障     

大鼠脑缺血/再灌注损伤后梗死灶周围水通道蛋白4与血脑屏障通透性的动态变化

目的： 研究大鼠局灶性脑缺血/再灌注损伤对梗死灶周围脑组织水通道蛋白4（AQP4）的表达及血脑屏障通透性的影响。〖HJ1.7mm〗方法: 健康雄性Sprague-Dawley大鼠随机分为脑缺血/再灌注6 h、24 h、48 h、7 d组及相应时点的假手术组，每组各6只。采用线栓法阻塞大脑中动脉（MCAo）建立局灶性脑缺血/再灌注模型，于相应时点进行神经症状评分后断头取脑，采用免疫组织化学方法检测大鼠脑缺血/再灌注后不同时点梗死灶周围AQP4的表达及IgG的渗出以评价血脑屏障通透性的改变。结果: (1)大鼠脑缺血/再灌注损伤后神经功能缺失症状较假手术组明显（P<0.01），6-48 h呈逐渐加重趋势，72 h后有所缓解，7 d恢复正常；(2)大鼠脑缺血/再灌注6 h时AQP4表达增加不明显，至再灌注24 h后表达明显增加（P<0.05），7 d仍处于表达高峰；(3)再灌注6 h时IgG渗出不明显，再灌注24 h后开始增加（P<0.05），于再灌注48 h IgG渗出达高峰后开始下降; (4)大鼠脑缺血/再灌注损伤后血脑屏障通透性的增强与AQP4表达的增加呈显著相关(P<0.01)。结论: 大鼠脑缺血/再灌注损伤后AQP4表达增加与血脑屏障通透性增强密切相关，两者是脑缺血/再灌注损伤后脑水肿发生的重要因素。     

Adrenalectomy increases local cerebral blood flow in the rat hippocampus

The present study examined the effect of glucocorticoid manipulations on local cerebral blood flow in the hippocampus. We measured local cerebral blood flow in the hippocampus at 1-h intervals over a 1-day period in freely moving rats, by means of the H₂ clearance method, before and after sham adrenalectomy, adrenalectomy or adrenalectomy with corticosterone replacement. We also measured local cerebral blood flow in the prefrontal cortex before and after adrenalectomy. Four weeks after the adrenalectomy, hippocampal blood flow at each time of day was an average of 47% greater than before the operation, showing diurnal variation as before. After the sham adrenalectomy or adrenalectomy with corticosterone replacement, hippocampal blood flow did not change significantly with respect to either its level or its diurnal variation. Local cerebral blood flow in the prefrontal cortex increased by only 19% after adrenalectomy. The present study demonstrates that adrenalectomy causes a remarkable increase in hippocampal blood flow, probably due to a lack of corticosterone.