Mediators of Blood-Brain Barrier Disruption

描述(申请人提供)：脑缺血引起血脑屏障(BBB)的破坏，这可能与脑水肿、出血和梗塞体积增大有关。有大量证据表明，血脑屏障的破坏与涉及细胞因子、趋化因子和白细胞-内皮细胞相互作用的炎症过程有关。在实验性卒中模型中，抑制卒中后的炎性级联可减少血脑屏障的破坏和卒中体积。然而，由于卒中模型的内在变异性、多个变量(如动脉血气和血流)的相互依赖以及药物对不同细胞类型和细胞过程的多因素影响，与体内BBB中断相关的病理机制的分析变得复杂。我们实验室建立了一种动态的体外血脑屏障(DIV-BBB)模型，该模型概括了血脑屏障的形态、生化和生理特性。使用该模型的初步研究表明，在常氧、正常血糖条件下，血流停止(降低切应力)可立即产生非白细胞依赖的细胞因子表达，随后只有在灌流液中存在白细胞(VVBC)时，才会延迟BBB中断。 这一建议的统一假设是，缺血时剪应力的降低(与缺氧或低血糖无关)触发一连串的炎症过程，导致血脑屏障的破坏。DIV-BBB模型将被用来测试四个目标，通过评估腔内和腔外液体腔内和腔外液体间随时间停止血流的反应以及由DIVBBB组成的细胞类型(内皮细胞、白细胞和星形胶质细胞)。第一个目的将确定一氧化氮(NO)调节的、非白细胞依赖的星形胶质细胞细胞因子的产生以及白细胞内皮细胞和星形胶质细胞的白细胞依赖细胞因子的释放是随后炎症的关键先例。在目标2中，血流停止后EC表面抗原的表达将与白细胞黏附和随后的BBB破坏相关。在目标3中，将从细胞因子刺激的前列腺素合成和释放活性氧物种的角度来检测WBC-EC黏附的性质和激活的WBC表型。最后，将确定基质金属蛋白酶(基质金属蛋白酶-2、-3和-9)在炎症介导的血脑屏障破坏中的作用。这些实验应该能更好地理解微血管血流量减少和血脑屏障之间的关系，并可能导致有效的治疗方法来防止中风后血脑屏障的破坏。此外，对切应力和血脑屏障功能关系的基本了解也可应用于其他以血脑屏障生理异常为特征的神经退行性和肿瘤性疾病(如阿尔茨海默氏症、脱髓鞘疾病、脑肿瘤)。

DESCRIPTION (provided by applicant): Cerebral ischemia causes disruption of the blood-brain barrier (BBB), which may be associated with cerebral edema, hemorrhage, and increased infarct volume. There is substantial evidence linking BBB disruption to inflammatory processes involving cytokines, chemokines, and leukocyte-endothelial cell interactions. In experimental stroke models, inhibition of the inflammatory cascade after stroke reduces BBB disruption and stroke volume. However, analysis of pathologic mechanisms related to BBB disruption in vivo is complicated by inherent variability in stroke models, co-dependence of multiple variables (e.g., arterial blood gases and blood flow), and multi-factorial effects of drugs upon different cell types and cellular processes. A dynamic in vitro BBB (DIV-BBB) model has been developed in our laboratory that recapitulates morphologic, biochemical, and physiologic properties of the blood-brain barrier. Preliminary studies using this model showed that flow cessation (reduction of shear stress) under normoxic normoglycemic conditions produced immediate leukocyte-independent cytokine expression, which was followed by delayed BBB disruption only when leukocytes (VVBC) were present in the perfusate. The unifying hypothesis of this proposal is that reduction of shear stress in ischemia (independent of hypoxia or hypoglycemia) triggers a cascade of inflammatory processes leading to BBB disruption. The DIV-BBB model will be used to test four Aims by assessing the response to flow cessation over time in intra-and extraluminal fluid compartments and cell types comprising the DIVBBB (endothelium, leukocytes, and astrocytes). The first Aim will determine that nitric oxide (NO)-modulated, WBC-independent cytokine production by astrocytes and WBC-dependent cytokine release by WBC endothelium and astrocytes are critical precedents to subsequent inflammation. In Aim 2, expression of EC surface antigens after flow cessation will be correlated to leukocyte adhesion and subsequent BBB disruption. In Aim 3, the nature of WBC-EC adhesion and activated WBC phenotype will be examined in terms of cytokine-stimulated prostaglandin synthesis and release of reactive oxygen species. Finally, the role of matrix metalloproteinases (MMP-2, -3 and -9) in inflammation-mediated BBB disruption will be determined. These experiments should provide a better understanding of the relationship between microvascular blood flow reductions and blood-brain barrier, and may lead to effective therapies to prevent BBB disruption after stroke. In addition, basic understanding of the relationship of shear stress and BBB function may be applied to other neurodegenerative and neoplastic disorders characterized by abnormal BBB physiology (e.g. Alzheimer's, demyelinating diseases, brain tumors).