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

• Published in: Journal of immunological methods 2000-06, Vol.240 (1), p.23-37
• Main Authors: Becker, M.D, Nobiling, R, Planck, S.R, Rosenbaum, J.T
• Format: Article
• Language: English
• Subjects: Angiography; Angiography - methods; Animals; Biological and medical sciences; Cell Movement; Fluorescence microscopy; Image Processing, Computer-Assisted; Imaging techniques; Investigative techniques of ocular function and vision; Investigative techniques, diagnostic techniques (general aspects); Iris; Iris - blood supply; Iritis; Leukocytes; Leukocytes - immunology; Lipopolysaccharides - pharmacology; Male; Medical sciences; Mice; Mice, Inbred BALB C; Microcirculation; Microscopy, Video - methods; Rhodamine; Uveitis - chemically induced; Uveitis - pathology; Vascular endothelium
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(00)00165-4

The process of inflammation is accompanied by an alteration of leukocyte–endothelial dynamics. Reciprocal changes in the endothelium and the white cell 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 been under-utilized for this purpose. Iris vasculature can be videophotographed without the artifact of trauma. We used rhodamine 6G in vivo staining of leukocytes from BALB/c mice in a model of inflammation induced by intravitreally injected endotoxin. Digital video technology was used to record observations 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 increase in leukocyte arrest within 4 h (601±119 cells per mm 2 vs. 2±1 cells per mm 2 in control animals). Although shear stress differs minimally between iris arterioles and venules, both rolling and arrest occurred preferentially in venules indicating that shear stress is not the dominant factor for determining cell adhesion. Compared to previous reports on intravital microscopy, our methodology includes refinements or advantages in visualizing cells that have transmigrated as well as the avoidance of surgical trauma. The resolution and quantifiable nature of this technique are such that the methodology can be applied to repetitive observation of leukocyte–endothelial dynamics during an immune response.