Qualitative and quantitative analysts of monocyte transendothelial migration by confocal microscopy and three-dimensional image reconstruction

A novel for qualitative and quantitative analysis of monocyte transendothel ial migration is described. By labeling monocytes and endothelial cells wit h different fluorophores, and utilizing confocal microscopy and three-dimen sional image reconstruction, transmigrating monocytes were resolved and qua ntified within a subendothelial collagen gel. Comparison of monocyte migrat ion across endothelial monolayers derived from human brain microvessels ver sus umbilical veins revealed diapedesis across brain endothelium to be sign ificantly delayed. Inclusion of astrocytes within the sub-endothelial colla gen gel resulted in the formation of an array of astrocytic processes that simulated the glia limitans surrounding brain microvessels in situ, thus yi elding a more physiologic paradigm of the blood-brain barrier. By virtue of its unique capacity to provide information on the total number of migratin g cells, this analytic approach overcomes significant caveats associated wi th sampling only aspects of the migration process. The potential adaptabili ty of this method to computer-assisted analysis further enhances its prospe ctive use in high-throughput screening.