MOLECULAR MECHANISMS OF BLOOD-BRAIN BARRIER DYSFUNCTION

血脑屏障功能障碍的分子机制

• 批准号: 3398099
• 负责人: HAROLD KOENIG
• 金额: $ 9.95万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-12-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3398099
• 关键词: adrenergic agents; aspirin; astrocytes; beta adrenergic agent; blood brain barrier; brain circulation; brain edema; calcium; calmodulin; capillary; cardiovascular pharmacology; cyclic AMP; decarboxylase inhibitor; deoxyglucose; dexamethasone; eicosanoid metabolism; electrocorticography; electron microscopy; enzyme induction /repression; experimental brain lesion; fluorescent dye /probe; histochemistry /cytochemistry; ion transport; laboratory rat; mannitol; molecular pathology; neurotransmitters; ornithine decarboxylase; phase contrast microscopy; pinocytosis; polyamines; radiotracer; receptor mediated endocytosis; tissue /cell culture; vascular endothelium permeability; verapamil

This continuation application proposes to pursue the long-term goal of elucidating the molecular and cellular mechanisms, of blood- brain barrier (BBB) breakdown, and to initiate new studies of BBB regulation (stimulus-transport coupling) by vasoactive neurotransmitters. The specific aims are to continue studies on two experimental models of BBB breakdown, hyperosmolal stimulation and cold injury, using intact rat brain, isolated rat cerebral capillaries, and primary monolayer cultures of brain capillary endothelial cells. We also wish to investigate receptor-mediated brain capillary transport processes using putative physiological agonists, e.g., beta-adrenergic and cholinergic agonists, adenosine, serotonin, histamine. These studies will focus on molecular events coupling endothelial cell stimulation (hyperosmolal, injury-induced, receptor-mediated) to enhancement of ODC activity and polyamine synthesis. This will include possible involvement of: (a) guanine nucleotide regulatory (G) proteins; (b) phosphodiesteratic cleavage of phosphatidylinositol- 4,5-bisphosphate and the putative messenger roles of diacylglycerol and inositol 1,4,5-trisphosphate (IP3); (c) derivatives of the arachidonate cascade, including prostaglandins; and (d) O2-derived free radicals. Stimulus-transport coupling will be a second major topic for investigation and will emphasize Ca2+- and polyamine- sensitive processes involved in the regulation of: (a) Ca2+ fluxes and cytosolic Ca2+ levels; (b) transcytosis and paracellular transport; (c) participation of cytoskeletal elements, including F-actin-containing stress fibers. These studies have a direct impact on the basic mechanisms underlying vasogenic brain edema and its treatment. 1.6 M mannitol is infused into the right carotid artery of rats to induce reversible BBB opening. Focal cold injury is induced by applying a steel rod precooled to -186 degrees C to the rat skull. In vitro experiments are performed on capillary preparations isolated from normal and injured rat cerebra, and on primary monocell cultures of rat cerebral capillary endothelium. Transendothelial transport will be quantitated in vivo and in vitro in isolated capillaries and cultured capillary endothelium using 45Ca, horseradish, peroxidase (HRP), (3H)sucrose, and alpha(3H)aminoisobutyrate (AIB). Transport pathways will be characterized ultrastructurally with HRP. Cytosolic Ca2+ concentration will be monitored spectrofluorometrically with the Ca2+-sensitive fluorescent dye fura 2.

这种延续申请建议实现长期目标 阐明血液的分子和细胞机制 脑屏障（BBB）故障，并启动BBB的新研究 通过血管活性的调节（刺激传输耦合） 神经递质。 具体的目的是继续研究 BBB崩溃的两个实验模型，高渗刺激 使用完整的大鼠大脑，孤立的大鼠脑损伤 毛细血管和脑毛细管的主要单层培养物 内皮细胞。 我们还希望研究受体介导的 使用推定的生理学的脑毛细管运输过程 激动剂，例如β-肾上腺素能和胆碱能激动剂， 腺苷，5-羟色胺，组胺。 这些研究将重点 分子事件耦合内皮细胞刺激 （高温，损伤引起的，受体介导的）以增强 ODC活性和多胺合成。 这将包括 可能的参与：（a）鸟嘌呤核苷酸调节（G） 蛋白质； （b）磷脂酰肌醇的磷酸二酯裂解 4,5-双磷酸和二酰基甘油的推定使者角色 和肌醇1,4,5-三磷酸盐（IP3）； （c） 蛛网级联，包括前列腺素； （d）O2衍生 自由基。 刺激 - 传输耦合将是第二大专业 研究主题，并将强调Ca2+ - 和多胺 - 涉及的敏感过程：（a）Ca2+通量 和胞质Ca2+水平； （b）跨胞菌病和细胞细胞增多症 运输; （c）参与细胞骨架元素，包括 含F-肌动蛋白的应激纤维。 这些研究直接 对加工性脑水肿的基本机制的影响 它的治疗。 1.6 m甘露醇被注入正确的颈动脉 大鼠的动脉诱导可逆的BBB开口。 局灶性冷损伤 通过将钢棒预先冷却至-186摄氏度来诱导 老鼠头骨。 在毛细管上进行体外实验 从正常和受伤的大鼠脑和受伤的大鼠小脑分离的制剂，以及 大鼠脑毛细血管内皮的原发性单核细胞培养物。 跨内皮转运将在体内和体外进行定量 在孤立的毛细血管和培养的毛细血管内皮中使用 45CA，辣根，过氧化物酶（HRP），（3​​H）蔗糖和 α（3H）氨基丁酸（AIB）。 运输途径将是 用HRP表征超微结构。 胞质Ca2+ 浓度将与频谱进行监测 Ca2+ - 敏感的荧光染料Fura 2。