Our objective is to develop an in vitro model of the blood brain barrier through which major hypotheses of vasogenic cerebral edema may be tested. Cerebral edema accompanies many different pathological insults to the CNS and is a prime determining factor of the ultimate morbidity or mortality associated with many of these insults. Several hypotheses have been proposed to explain the disruption of the blood brain barrier which precedes the formation of vasogenic cerebral edema. Arachidonic acid metabolism is a central issue in most of these hypotheses, but precise characterization of the metabolites involved, their sites of origin and/or action, and the mechanisms of their actions remains largely undetermined. Uniquely, mouse cerebral endothelium and vascular smooth muscle and rat C6 glioma cells are available in the laboratory for in vitro exploration of these hypotheses. Preliminary studies suggest that some cerebral endothelial properties are maintained in culture (i.e, impermeability to certain macromolecules), while others require co-culture of cerebral endothelium with C6 glioma cells for their expression (i.e., Gamma-GTP activity and polarity to amino acid transport). Further studies to characterize this in vitro blood brain barrier model will include: 1) permeability studies with small versus large M.W. and low versus high octanol/water partition co-efficient solutes, 2) ultrastructural probes for analysis of vesicular transport, 3) measurement of transendothelial electrical resistance, and 4) more extensive co-culture studies to assess intercellular interactions required for blood brain barrier differentiation and maintenance. Cell type specific studies of arachidonic acid metabolism utilizing primarily thin layer chromatography, high performance liquid chromatography, and radioimmunoassay will characterize sites of specific eicosanoid metabolism. Preliminary studies reveal differences between endothelium and smooth muscle. Knowledge of cerebrovascular eicosanoid metabolism will then be utilized to assess its effects on the in vitro model of the blood brain barrier developed earlier.
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