The objective of this proposal is twofold; (1) to develop an in vitro co-culture system in which the interaction between two different cell types can be studied, and (2) to evaluate in vivo the role of the glial cell in the differentiation and maintenance of the Blood-Brain Barrier (BBB) properties in capillary endothelium of the brain. The in vivo studies will utilize a mouse freeze-injury model to study these processes in the regenerating cerebral microvessels.
The specific aims are to thoroughly investigate the temporal sequence and relationships of endothelial tight junction formation, early appearance of glial foot processes on the vascular baseement membrane, the completion of the glial sheath around the regenerating vessel, restoration of barrier function to tracers, the fate of endothelial cell Factor VIII and Thrombomodulin (TM) antigens during regeneration, and induction (or activation) or gamma-glutamyl transpeptidase (GGTP) ion the new endothelium. These studies will employ enzyme histochemistry for GGTP, immunocytochemistry for GGTP, TM and Factor VIII, ultrastructural studies and horse radish peroxidase tracer analysis for barrir function.
The Aims of the in vitro studies are to determine the mechanisms by which endothelial cells acquire GGTP in the presence of glial cells in co-culture, to test the glial cell's ability to induce other differentiated endothelial characteristics, and to delineate the extent to which glial cells can induce and/or activate GGTP in endothelium from extracranial sites. These studies will use radioimmunoassay, autoradiography, enzyme histochemistry and bioassay for GGTP, immunochemistry and bioassay for TM, as well as varied schemes of co-culture. The advantage this dual approach is that the in vivo studies will tell us what to look for in the glial/endothelial cell-cell interaction, and the in vitro studies will provide a place to test or validate the in vivo observations. The accomplishment of these aims will add significantly to our understanding of the microvascular regeneration process following a variety of clinical and pathological conditions where alterations in vascular integrity have resulted in varying degrees of brain damage. (Infarcts, trauma, anoxia, neoplasms, infection, etc.). Of equal importance will be the development and testing of in vitro co-culture schemes that allow the study of differentiated endothelial cell functions that are often lost in pure cell culture.
