This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. It is possible to isolate individual brain microvessels intact from recently sacrificed macaques. Using confocal microscopy, we demonstrate imaging of such vessels with a focus on tight junctions. Using similar techniques, combined with detection of other cell types, cytokines and chemokines, allows us to visualize the effects of virus and viral-infected cells on the tight junctions of the blood-brain barrier (BBB) in real time. Brain microvessels stained with phalloidin-488 (to stain the extensive actin network of endothelial vessels) and zo-1 (a tight junction marker) demonstrate a zipper of tight junction protein extending the length of the vessels. The use of multiple color imaging allows us to colocalize activation markers (such as VCAM-1) or signal transduction markers (activated NFKB) and their relationship with down regulation of tight junction proteins in microvessel derived from encephalitic brains. In vessels derived from macaques with encephalitis, we noticed that the staining of zo-1 was lacking from some areas of vessel and not from others: of the same vessels. These areas are rich in VCAM-1 expression, and are thus ideal for adhesion and emigration of mononuclear cells through the BBB.
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