In vivo study of leukocyte-endothelium interaction in endotoxin-induced uveitis

To analyze leukocyte-endothelium interaction in iris venules of living rats and to quantify changes of leukocyte dynamics in endotoxin-induced uveitis (EIU).

Lewis rats received an intraperitoneal injection of 100 micrograms of lipopolysaccharide (LPS; Salmonella typhimurium). Using intravital fluorescence microscopy, the iris vessels were examined, 2, 4, 6, 10, 14, 24, and 72 hours after LPS injection. A setup for intravital fluorescence microscopy of iris venules in the rat is described. Images are recorded with a video camera and stored on S-VHS videotape for off-line analysis. For contrast enhancement, erythrocytes and plasma were stained with fluorescein isothiocyanate (FITC) and FITC-hydroxyethylstarch, respectively. Rhodamine 6G was used for intravital staining of leukocytes. Resolution and magnification (x850) of the system facilitates observation of individual cells in the bloodstream in real time. Leukocytes were either flowing in the center stream, rolling along the endothelium, or firmly adherent. Image analysis provided data on microvascular leukocyte flux and leukocyte velocity.

The percentage of leukocytes rolling on postcapillary venular endothelium increased significantly (P < 0.05) 4 hours after endotoxin administration, as did the number of firmly adherent cells. Leukocyte-endothelium interaction reached its maximum 6 to 10 hours before an increase of inflammatory cells in the aqueous humor. The response to endotoxin was reversible, subsiding to near-normal values after 72 hours.

Intravital fluorescence microscopy provides data on microvascular parameters, including the number of rolling and sticking leukocytes on vascular endothelium. Inflammation of the anterior uvea was characterized with regard to leukocyte recruitment from blood to the vessel wall.