Tumor necrosis factor-alpha (TNF-a) is a cytokine released in response to inflammatory events in the body. The increased levels of TNF-a in the central nervous system observed in conditions such as multiple sclerosis, bacterial meningitis, viral infections and brain ischemia suggest that the cytokine may be involved in the pathogenesis of these diseases. A major cellular target for TNF-a is the endothelium, where increases in coagulant activity, cell adhesiveness and vascular permeability are observed. Indeed, exogenous administration of TNF-a produces significant increases in blood-brain barrier (BBB) permeability. Therefore, understanding the mechanisms through which TNF-a produces changes in the permeability of brain microvessel endothelial cells that form the BBB, may provide insight into the cause and effective treatment of inflammatory events within the central nervous system. The proposed studies will examine the effects of TNF-a on BBB permeability on two distinct levels. First, primary cultured bovine brain microvessel endothelial cells (BBMEC) will be used as an in vitro model of the BBB to examine the cellular factors involved in the permeability effects of TNF-a. The hypothesis to be examined is that inhibition of actin stress filament formation in the cells will prevent increases in BBMEC monolayer permeability observed with TNF-a.
The specific aims for the in vitro studies will be to evaluate the effects that 1) inhibition of Rho-mediated GTP binding proteins, 2) alterations in cyclic nucleotide signaling pathways, and 3) inhibition of arachidonic acid metabolism has on TNF-alpha-induced changes in actin filament formation and BBMEC monolayer permeability. Secondly, the effects of TNF-a on BBB will be evaluated in vivo. The hypothesis for the in vivo studies is that changes in BBB permeability observed under inflammatory conditions in the brain are directly correlated to the amount of TNF-a released. For these studies, microdialysis probes will be implanted into the cortex of rats.
The specific aims for the in vivo portion of the proposal are to 1) determine the dose-response relationship between cortically administered TNF-a and increases in BBB permeability, and 2) correlate the endogenous release of TNF-a following cortical injection of bacterial toxin or cerebral blood flow occlusion with changes in BBB permeability. Microdialysis probes will be used to deliver exogenous TNF-a to specific sites in the cortex as well as sampling endogenously released TNF-a. Together, the proposed studies will provide a better understanding of the mechanisms involved in TNF-a effects on BBB permeability.
