A Novel Approach for Freeze-Frame Video Determination of Volumetric Blood Flow in the Rat Retina

Abstract

The goal of this study was to quantify retinal hemodynamics using the freeze-frame technique for determining volumetric retinal blood flow. Leukocytes and erythrocytes were removed from the circulation of rats, stained with fluorescent dyes, returned to the animals' circulation, and excited to fluorescence using the scanning laser ophthalmoscope. The retinal circulation was videotaped and viewed frame-by-frame to count the total number of visible stained cells passing through the field of view during a known period of time while excluding repeated appearances of cells in multiple frames. The total number of cells visualized, in conjunction with cell density data collected in a coincident in vitro study, was used to calculate in vivo volumetric blood flow for both cell types. Measurements of volumetric blood flow based on fluorescent leukocytes (34.2 +/- 5.9 nL/sec [mean +/- standard deviation]; n = 30 rats) and fluorescent erythrocytes (35.1 +/- 6.1 nL/sec; n = 30 rats) were not significantly different, indicating that the freeze-frame technique provides a blood flow measurement independent of cell type. Repeated blood flow measurements obtained from an individual rat over time showed little difference (P < .0001), demonstrating the reproducibility of the technique. The freeze-frame technique offers a promising method for calculating volumetric blood flow independent of individual cell velocity, size, or type.