IN-VIVO CELL TRACKING BY SCANNING LASER OPHTHALMOSCOPY - QUANTIFICATION OF LEUKOCYTE KINETICS

PURPOSE. To image peripheral blood leukocyte traffic in the normal ret inal and choroidal vasculature and to quantify the differences in the circulation dynamics between normal and concanavalin A (ConA)-activate d leukocytes. METHODS. Normal or ConA-activated splenocytes were fluor escently labeled in vitro with 6-carboxyfluorescein diacetate (CFDA) a nd reinfused in vivo where they were tracked in the retinal and choroi dal circulations of syngeneic rats by means of a scanning laser ophtha lmoscope (SLO). Simultaneous digital and video images were captured fo r as long as 30 minutes, and the initial lj seconds of image sequences and leukocyte dynamics were analyzed from digitized images by recordi ng the velocity of trafficking cells and the number of stationary cell s that accumulated with time, using a customized software package. RES ULTS. Mean velocity (+/-SD) was 29.8 +/- 15.3 mm/sec in the retinal ar teries, 14.7 +/- 7.2 mm/sec in the retinal veins, and 3.0 +/- 3.6 mm/s ec in the retinal capillaries. Mean velocity in the choroidal vessels was 6.1 +/- 6.0 mm/sec. No significant difference in leukocyte velocit y was found between activated and normal leukocytes in any of the vess el systems. However, activated leukocytes were observed to accumulate more within the choroidal vasculature (P < 0.001) and the retinal capi llaries (P < 0.001) than in control animals, but not in larger retinal vessels. CONCLUSIONS. A technique to measure the kinetics of circulat ing leukocytes in vivo has been developed. Although leukocyte activati on itself is insufficient to cause slowing of leukocyte velocity, the data indicate that leukocyte adherence to endothelium can be induced i n the absence of local or systemic activating stimuli.