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(Investigative Ophthalmology and Visual Science. 2004;45:4197-4201.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0192
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In Vivo Three-Dimensional Evaluation of Leukocyte Behavior in Retinal Microcirculation of Mice

Shinsuke Miyahara, Junichi Kiryu, Kazuaki Miyamoto, Hideto Katsuta, Fumitaka Hirose, Hiroshi Tamura, Kunihiro Musashi, Yoshihito Honda, and Nagahisa Yoshimura

From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan.

PURPOSE. To evaluate new physiologic and three-dimensional methods for monitoring leukocyte behavior in mouse retina.

METHODS. Endotoxin-induced uveitis (EIU) was produced in mice by footpad injection of lipopolysaccharide (LPS). Leukocytes were labeled with acridine orange (AO). Leukocyte rolling in the retinal microcirculation was evaluated in vivo with AO digital fluorography. The number of migrated leukocytes was counted in flatmounted retina. The behavior of leukocyte migration was observed three-dimensionally at the time of peak migration. After leukocytes were labeled with AO, the mice were perfused with rhodamine-labeled concanavalin A lectin to stain the vascular endothelium. Leukocyte migration into the retina was then monitored three-dimensionally with confocal microscopy, and the velocity of the migration was measured.

RESULTS. Both leukocyte rolling and migration peaked at 48 hours after LPS injection. Leukocytes were seen to extravasate from the deeper capillary layers and to migrate toward the outer layer of the retina. The traveling velocity of extravasated leukocytes in retinal tissue was 2.0 ± 0.1 µm/h.

CONCLUSIONS. New methods have been demonstrated for the three-dimensional and quantitative evaluation of leukocyte behavior in mouse retina.




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