Digital video-imaging of leukocyte migration in the iris: intravital microscopy in a physiological model during the onset of endotoxin-induced uveitis

The process of inflammation is accompanied by an alteration of leukocyte-en dothelial dynamics. Reciprocal changes in the endothelium and the white cel l permit the leukocyte to relinquish its normal free-flowing state in order to roll, arrest, and emigrate through the endothelium. Although intravital microscopy is an established method to observe this process, the eye has b een under-utilized for this purpose. Iris vasculature can be videophotograp hed without the artifact of trauma. We used rhodamine 6G in vivo staining o f leukocytes from BALB/c mice in a model of inflammation induced by intravi treally injected endotoxin, Digital video technology was used to record obs ervations at baseline, 2 h, and 4 h after the endotoxin injection. Off-line analysis of microhemodynamic parameters established that the percentage of venules exhibiting rolling increased significantly from 4% at baseline to 34% at 2 h and 82% at 4 h after endotoxin injection. We found a marked incr ease in leukocyte arrest within 4 h (601+/-119 cells per mm(2) vs. 2+/-1 ce lls per mm2 in control animals). Although shear stress differs minimally be tween iris arterioles and venules, both rolling and arrest occurred prefere ntially in venules indicating that shear stress is not the dominant factor for determining cell adhesion. Compared to previous reports on intravital m icroscopy, our methodology includes refinements or advantages in visualizin g cells that have transmigrated as well as the avoidance of surgical trauma . The resolution and quantifiable nature of this technique are such that th e methodology can be applied to repetitive observation of leukocyte-endothe lial dynamics during an immune response. (C) 2000 Elsevier Science B.V. All rights reserved.