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). ? ?
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