The general goal of these studies is to examine the role of astrocyte endothelial interactions in restoring the blood brain barrier after injury. The studies will test the hypothesis that the following sequence of events occurs: 1) endothelial cells attract new astrocytes to forming vessels, 2) astrocytes stimulate the endothelium to alter the matrix proteins they produce resulting in the formation of tight junctions, 3) astrocytes signal the endothelium to increase glucose and amino acid transport and production of gamma glutamyl transpeptidase (GGTP). By combining in vivo studies on the regeneration of the barrier after injury with in vitro studies on individual cell types alone, in conditioned medium from other cell types or in co-culture, we will be able to isolate events, derive quantitative data and correlate the significance of observations found in vitro with those seen in vivo. Using a freeze- injury model we have previously described the time of endothelial and astrocyte replication, appearance and differentiation in the new vessel will be defined. The proposed in vivo studies will concentrate on the time of appearance of pericytes and the type of matrix proteins appearing in the endothelial basement membrane at various times after injury. In vitro studies will examine 1) the effects of astrocyte on endothelial nutrient uptake, production of GGTP, tight junction formation and production of matrix macromolecules and 2) the effect of endothelium on astrocyte replication, migration and process formation. We will characterize the types of molecules involved in the cell interactions as to whether thy are arachidonate metabolites or peptides. Arachidonate metabolites will be identified by chromatography and/or RIA. Peptides will be isolated by chromatography and electrophoresis and the amino acid sequence determined. Synthetic peptides will be produced and used to generate antibodies for confirmation of in vivo relevance. By gaining an understanding of the molecules that regulate the formation of the """"""""blood brain barrier"""""""", we can potentially regulate this process to more rapidly restore barrier function after injury.
