Effect of LPS on the permeability of the blood-brain barrier to insulin

Insulin has emerged as an important neuropeptide. Central actions of insulin appear to oppose those in the periphery. Insulin is transported across the blood-brain barrier (BBB) by a saturable transport system. The permeability of the BBB to insulin is altered by various events, but no studies exist that have examined the permeability of the BBB to insulin during infection or inflammation, states which can induce peripheral insulin resistance. We looked at the effects of lipopolysaccharide (LPS), a bacterial endotoxin and a powerful cytokine releaser, on the permeability of the BBB to human insulin in CD-1 mice. Intraperitoneal injections of LPS significantly increased the uptake by the brain of 131I-insulin and disrupted the BBB to 125I-albumin. After subtraction of the brain/serum ratio for 125I-albumin, brain/serum ratios for insulin were increased: 10.38 +/- 0.70 microl/g (LPS) vs. 3.62 +/- 0.27 microl/g (no LPS), P<0.0001, showing that LPS increased the uptake of insulin independent of BBB disruption. This increase in insulin uptake was due to enhanced saturable transport. Pretreatment with indomethacin 10 min before LPS injections enhanced BBB disruption, but not insulin transport. Pretreatment with the nitric oxide (NO) synthase inhibitor aminoguanidine had no effect on insulin or albumin uptake, but pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) enhanced insulin transport, but not BBB disruption. We conclude that LPS increases the saturable transport of insulin across the BBB independent of disruption and prostaglandins with potentiation by NO inhibition. Such increased transport could potentiate the central effects of insulin and so contribute to the peripheral insulin resistance seen with infection and inflammation.     

Effect of octanoate on blood-brain barrier permeability using L-dopa as a marker

Short-chain fatty acid injection into animals produces coma, seizures, and hyperventilation. One mechanism of coma production may be through alterations in membrane permeability characteristics. The SPG histofluorescence technique was used to evaluate changes in blood-brain barrier permeability to injected L-dopa in rats after intraperitoneal injections of the short-chain fatty acid, sodium octanoate. Diffusion of intravascular fluorescence was observed around brain capillaries in the octanoate-injected rats suggesting an alteration in capillary permeability to L-dopa. Diffusion of fluorescence around neuronal cell bodies and brain fiber tracts was also seen after treatment with octanoate. These findings suggest that octanoate may also alter neuronal membrane function.     

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.     

6-Hydroxydopamine-induced alterations in blood-brain barrier permeability

Vascular inflammation is well known for its ability to compromise the function of the blood--brain barrier (BBB). Whether inflammation on the parenchymal side of the barrier, such as that associated with Parkinson's-like dopamine (DA) neuron lesions, similarly disrupts BBB function, is unknown. We assessed BBB integrity by examining the leakage of FITC-labeled albumin or horseradish peroxidase from the vasculature into parenchyma in animals exposed to the DA neurotoxin 6-hydroxydopamine (6OHDA). Unilateral injections of 6OHDA into the striatum or the medial forebrain bundle produced increased leakage in the ipsilateral substantia nigra and striatum 10 and 34 days following 6OHDA. Microglia were markedly activated and DA neurons were reduced by the lesions. The areas of BBB leakage were associated with increased expression of P-glycoprotein and beta 3-integrin expression suggesting, respectively, a compensatory response to inflammation and possible angiogenesis. Behavioural studies revealed that domperidone, a DA antagonist that normally does not cross the BBB, attenuated apomorphine-induced stereotypic behaviour in animals with 6OHDA lesions. This suggests that drugs which normally have no effect in brain can enter following Parkinson-like lesions. These data suggest that the events associated with DA neuron loss compromise BBB function.     

Increased blood-brain barrier permeability in scrapie-infected mice

The present investigation was designed to study the ultrastructural integrity of the blood-brain barrier (BBB) in the cerebral microvasculature of scrapie-infected mice showing clinical illness. Cerebral microvessels from either IM, VM, or C57BL/6J mice, terminally affected with various strains of scrapie agent showed a focal leakage of horseradish peroxidase (HRP) in all agent-strain and mouse-strain combinations. This leakage was most pronounced in and near the primary site of agent inoculation, but was also observed in microvessels scattered throughout the brain. Cytochemical studies also revealed a redistribution of plasmalemma-bound alkaline phosphatase in the endothelial cells. In control mice, the enzymatic activity was mainly concentrated in the luminal plasmalemma, while in the scrapie-infected mice the activity also appeared in the abluminal side in the majority of microvessels. Our observations are evidence that the BBB of the mouse is altered in some way by the scrapie agent. Such an alteration may have important implications for human disease, since the scrapie agent is related to the group of "slow" viral infections, including kuru and Creutzfeldt-Jakob disease. Scrapie may also serve as an important model for the study of senile dementia of the Alzheimer type (SDAT).     

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.     

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.     

Influence of antioxidants on blood-brain barrier permeability during adrenaline-induced hypertension

We have examined the effect of antioxidants (vitamin E, and selenium) on the blood-brain barrier permeability during adreneline-induced acute hypertension in the female rats. The rats supplemented with nontoxic doses of sodium selenite in drinking water for three months or vitamin E was given intraperitoneally before adrenaline-induced acute hypertension. Evans-blue was used as a blood-brain barrier tracer. Mean values for Evans-blue dye were found to be 0.28 +/- 0.04 microg/g tissue in control animals and 1.0 +/- 0.2 microg tissue after adrenaline-induced acute hypertension (p < .01). Rats pretreated with selenium or vitamin E also showed macroscopic leakage of Evans-blue albumin after adrenaline injection i.e., there was no significant difference in protein extravasation between untreated and treated animals (p > .5). The mean value for Evans-blue dye was found to be 1.0 +/- 0.2 microg/g tissue in acute hypertension group, 0.9 +/- 0.2 microg/g tissue in selenium pretreated animals and 1.0 +/- 0.2 micrg/g tissue vitamin E injected animals after acute hypertension. The results show that antioxidants did not influence the blood-brain barrier breakdown during adrenaline-induced acute hypertension.     

Increased blood-brain barrier permeability with thymidine phosphorylase deficiency

Mitochondrial neurogastrointestinal encephalomyopathy is an autosomal recessive multisystemic disorder caused by thymidine phosphorylase deficiency. Whereas the pathomechanism of the secondary mitochondrial dysfunction has been extensively studied, that of the leukoencephalopathy has not been elucidated. We hypothesized that the white matter hyperintensities on T2-weighted magnetic resonance images reflect disturbance of blood-brain barrier function. Albumin immunohistochemistry disclosed quantitative (p < 0.01) and qualitative differences between the mitochondrial neurogastrointestinal encephalomyopathy and control brains, indicating that loss of thymidine phosphorylase function impairs the integrity of the blood-brain barrier.     

Adenosine receptor signaling modulates permeability of the blood-brain barrier

The blood-brain barrier (BBB) is comprised of specialized endothelial cells that form the capillary microvasculature of the CNS and is essential for brain function. It also poses the greatest impediment in the treatment of many CNS diseases because it commonly blocks entry of therapeutic compounds. Here we report that adenosine receptor (AR) signaling modulates BBB permeability in vivo. A(1) and A(2A) AR activation facilitated the entry of intravenously administered macromolecules, including large dextrans and antibodies to β-amyloid, into murine brains. Additionally, treatment with an FDA-approved selective A(2A) agonist, Lexiscan, also increased BBB permeability in murine models. These changes in BBB permeability are dose-dependent and temporally discrete. Transgenic mice lacking A(1) or A(2A) ARs showed diminished dextran entry into the brain after AR agonism. Following treatment with a broad-spectrum AR agonist, intravenously administered anti-β-amyloid antibody was observed to enter the CNS and bind β-amyloid plaques in a transgenic mouse model of Alzheimer's disease (AD). Selective AR activation resulted in cellular changes in vitro including decreased transendothelial electrical resistance, increased actinomyosin stress fiber formation, and alterations in tight junction molecules. These results suggest that AR signaling can be used to modulate BBB permeability in vivo to facilitate the entry of potentially therapeutic compounds into the CNS. AR signaling at brain endothelial cells represents a novel endogenous mechanism of modulating BBB permeability. We anticipate these results will aid in drug design, drug delivery and treatment options for neurological diseases such as AD, Parkinson's disease, multiple sclerosis and cancers of the CNS.     

Independence of glucocorticosteroids and blood-brain barrier amino acid permeability

The effects of abnormal levels of circulating glucocorticosteroids upon the carrier-mediated transport of tyrosine and tryptophan across the blood-brain-carrier of the adult male rat were studied. The uptake of C¹⁴ labeled tyrosine or tryptophan was measured relative to that of simultaneously injected ³H₂O, a freely diffusible internal standard. Fifteen sec after rapid intracarotid injection the rats were decapitated and brain tissue was homogenized and subjected to liquid scintillation counting. The level of circulating glucocorticosteroids was altered by intraperitoneal injection of cortisol (30 mg/kg) or corticosterone (30 mg/kg) 20 min prior to the amino acid injection. Rats were also injected with cortisol (10 mg/kg) or corticosterone (10 mg/kg) for 14 days. These rats were then injected with cortisol (10 mg/kg) or corticosterone (10 mg.kg) 90 min before the experiment. The amino acid uptake indices were analyzed statistically by the Wilcoxan Rank Sum Test. In no case was the uptake of tyrosine or tryptophan found to differ significantly (p <0·10) between the experimental and control rats. It is concluded that no correlation between levels of circulating adrenal glucocorticosteroids and transport across the blood brain barrier of the amino acid precursors of the putative neurotransmitters, norepinephrine and serotonin, can be demonstrated by this method.     

Effect of hypothermia on blood-brain barrier permeability following traumatic brain injury in chronically ethanol-treated rats

The objective of this study was to investigate the effect of hypothermia on the blood-brain barrier (BBB) disruption caused by traumatic brain injury (TBI) in chronically ethanol-treated rats. BBB permeability was measured using Evans blue (EB) dye. Arterial blood pressure levels of animals in hypothermic groups decreased significantly. The EB dye extravasation into the brain significantly increased in hypothermia and at 6 and 24 h after TBI. In ethanol-treated rats that were subjected to TBI, hypothermia led to a significant decrease in EB dye content in the brain at 24 h but not at 6 h after TBI when compared with TBI alone.     

Influence of hypoosmolality on the blood-brain barrier permeability during epileptic seizures

Changes in the blood-brain barrier permeability to macromolecules were investigated during pentylenetetrazol-induced seizures, using Evans-blue as an indicator, in water-intoxicated and nonintoxicated Wistar albino (210-250 g) adult rats of both sexes. Evans-blue albumin extravasation was judged visually and estimated quantitatively with a spectrophotometer using homogenized brain to release the dye. Hypoosmolar treatment (water intoxication) was performed by the intraperitoneal administration of distilled water to a volume of 10% of the body weight; Six groups of rats were studied. Group I: female control (n=10), Group II: male control (n=10), Group III: nonwater-intoxicated female+seizure (n=15), Group IV: nonwater-intoxicated male+seizure (n=15), Group V: water-intoxicated female+seizure (n=15), Group VI: water-intoxicated male+seizure (n=15). Approximately 2 h after the injection of water, the plasma osmolarity had decreased by 25-30 mosm. Our results revealed that in female rats, the extravasation of Evans-blue albumin was greater in the brains of water-intoxicated rats compared to nonwater-intoxicated rats after pentylenetetrazol-induced seizures. In addition, hypoosmotic female rats were shown to have a larger increase in blood-brain barrier permeability than hypoosmotic male rats after pentylenetetrazol-induced seizures. This difference between male and female rats was found to be significant (P=.005).     

Effect of 2450 MHz microwave energy on the blood-brain barrier to hydrophilic molecules. D. Brain temperature and blood-brain barrier permeability to hydrophilic tracers

Measurement of temperature within the cerebral cortex, hypothalamus, cerebellum and medulla of rats sham-, heat- or microwave-exposed revealed the presence of a thermal gradient within the brain. In all groups, cerebral cortex and the cerebellum were cooler than the deeper hypothalamus and medulla. Exposure to 2450 MHz CW microwaves or ambient heat (42 +/- 2 degrees C) resulted in measurable elevation of regional brain temperature, but without alteration of temperature gradients normally observed within the brain. Exposure to 20 mW/cm2 (SAR approximately equal to 4 W/kg) for 30, 90 or 180 min induced a small, but significantly (U = 0, P less than 0.05) increased temperature of the colon, and in each region of the brain studied. Exposure to an incident power density of 65 mW/cm2 (SAR approximately equal to 13.0 W/kg) for 30 or 90 min or to ambient heat (42 +/- 2 degrees C) for 90 min resulted in a substantially greater thermal response as indicated by higher colonic and brain temperatures. Comparison of regional brain temperature with individual colonic temperatures is expressed as delta T = t degrees Cbrain--t degrees Ccolon. In general delta T values for ambient heat or microwave-exposed rats did not differ significantly from those of sham-exposed animals. Exposure to microwaves or ambient heat did not alter the general relationships between regional brain and colonic temperatures, i.e., cortical and cerebellar temperatures were always below and hypothalamic and medullary temperatures always above corresponding colonic temperatures. The plotted temperature data (brain vs colonic temperature) indicate a linear relationship between brain and colonic temperatures. Levels of sodium fluorescein (NAFl), horseradish peroxidase (HRP) and [14C]sucrose (described in preceding papers) within the brain show a high correlation (P less than 0.05) with brain temperature. Suppression of blood-brain barrier permeability to hydrophilic tracers was most pronounced at brain temperatures exceeding approximately 40 degrees C and is demonstrated to be temperature dependent.     

Effect of 2450 MHz microwave energy on the blood-brain barrier to hydrophilic molecules. D. Brain temperature and blood-brain barrier permeability to hydrophilic tracers

Measurement of temperature within the cerebral cortex, hypothalamus, cerebellum and medulla of rats sham-, heat- or microwave-exposed revealed the presence of a thermal gradient within the brain. In all groups, cerebral cortex and the cerebellum were cooler than the deeper hypothalamus and medulla. Exposure to 2450 MHz CW microwaves or ambient heat (42 ± 2 °C) resulted in measurable elevation of regional brain temperature, but without alteration of temperature gradients normally observed within the brain. Exposure to 20 mW/cm² (SAR 4 W/kg) for 30, 90 or 180 min induced a small, but significantly (U = 0, ) increased temperature of thcolon, and in each region of the brain studied. Exposure to an incident power density of 65 mW/cm² (SAR 13.0 W/kg) for 30 or 90 min or to ambient heat (42 +- 2 °C) for 90 min resulted in a substantially greater thermal response as indicated by higher colonie and brain temperatures. Comparison of regional brain temperature with individual colonie temperatures is expressed as ΔT = t °C_brain − t °C_colon. In general ΔT values for ambient heat or microwaveexposed rats did not differ significantly from those of sham-exposed animals. Exposure to microwaves or ambient heat did not alter the general relationships between regional brain and colonie temperatures, i.e., cortical and cerebellar temperatures were always below and hypothalamic and medullary temperatures always above corresponding colonie temperatures.The plotted temperature data (brain vs colonie temperature) indicate a linear relationship between brain and colonie temperatures. Levels of sodium fluorescein (NAFl), horseradish peroxidase (HRP) and [¹⁴C]sucrose (described in preceding papers) within the brain show a high correlation (P < 0.05) with brain temperature. Suppression of blood-brain barrier permeability to hydrophilic tracers was most pronounced at brain temperatures exceeding ~40 °C and is demonstrated to be temperature dependent.     

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.     

提高药物跨越血-脑屏障的研究进展

1885年，德国细菌学家Ehdich发现静脉注射苯胺染料可使动物脑以外的各脏器着色．1903年的两项经典实验确立了血一脑屏障（blood-brain barrier，BBB）的概念，随后进一步明确了它的解剖部位和结构基础。在生理状态下血-脑屏障将中枢神经系统与其他系统相隔绝，使得大部分物质与免疫细胞不能随意进出，对维持中枢神经系统内环境稳定具有决定性作用；在病理状态下血-脑屏障被破坏通透性增加，允许部分药物通过。[第一段]