In vivo cell tracking by scanning laser ophthalmoscopy: quantification of leukocyte kinetics

To image peripheral blood leukocyte traffic in the normal retinal and choroidal vasculature and to quantify the differences in the circulation dynamics between normal and concanavalin A (ConA)-activated leukocytes.

Normal or ConA-activated splenocytes were fluorescently labeled in vitro with 6-carboxyfluorescein diacetate (CFDA) and reinfused in vivo where they were tracked in the retinal and choroidal circulations of syngeneic rats by means of a scanning laser ophthalmoscope (SLO). Simultaneous digital and video images were captured for as long as 30 minutes, and the initial 15 seconds of image sequences and leukocyte dynamics were analyzed from digitized images by recording the velocity of trafficking cells and the number of stationary cells that accumulated with time, using a customized software package.

Mean velocity (+/-SD) was 29.8 +/- 15.3 mm/sec in the retinal arteries, 14.7 +/- 7.2 mm/sec in the retinal veins, and 3.0 +/- 3.6 mm/sec in the retinal capillaries. Mean velocity in the choroidal vessels was 6.1 +/- 6.0 mm/sec. No significant difference in leukocyte velocity was found between activated and normal leukocytes in any of the vessel systems. However, activated leukocytes were observed to accumulate more within the choroidal vasculature (P < 0.001) and the retinal capillaries (P < 0.001) than in control animals, but not in larger retinal vessels.

A technique to measure the kinetics of circulating leukocytes in vivo has been developed. Although leukocyte activation itself is insufficient to cause slowing of leukocyte velocity, the data indicate that leukocyte adherence to endothelium can be induced in the absence of local or systemic activating stimuli.