Measuring Temporal and Spatial Variability of Red Blood Cell Velocity in Human Retinal Vessels

Abstract

Purpose: The retinal circulation regulates blood flow through various internal and external factors; however, it is unclear how locally these factors act within the retinal microcirculation. We measured the temporal and spatial variability of blood velocity in small retinal vessels using a dual-beam adaptive optics scanning laser ophthalmoscope.

Methods: In young healthy subjects (n = 3), temporal blood velocity variability was measured in a local vascular region consisting of an arteriole, capillary, and venule repeatedly over 2 days. Data consisted of 10 imaging periods separated into two sessions: (1) five 6-minute image acquisition periods with 30-minute breaks, and (2) five 6-minute image acquisition periods with 10-minute breaks. In another group of young healthy subjects (n = 5), spatial distribution of velocity variability was measured by imaging three capillary segments during three 2-minute conditions: (1) baseline imaging condition (no flicker), (2) full-field flicker, and (3) no flicker condition again.

Results: Blood velocities were measurable in all subjects with a reliability of about 2%. The coefficient of variation (CV) was used as an estimate of the physiological variability of each vessel. Over 2 days, the average CV in arterioles was 7% (±2%); in capillaries, it was 19% (±6%); and, in venules, it was 8% (±2%). During flicker stimulation, the average capillary CV was 16% during baseline, 15% during flicker stimulation, and 18% after flicker stimulation.

Conclusions: Capillaries in the human retina exhibit spatial and temporal variations in blood velocity. This inherent variation in blood velocity places limits on studying the vascular regulation of individual capillaries, and the study presented here serves as a foundation for future endeavors.

Figure 1.

A representation of the targeted vascular region in a subject. (A) An OCTA image of the superficial vascular plexus superimposed on a subject's SLO image taken prior to AO imaging. The red square indicates the region within which blood velocity was measured. (B) A vascular flow image obtained by calculating the variation in pixel intensity across all frames throughout the video. Color-coded arrows mark the selected capillaries measured for Experiment 2. Labels A and V correspond to the terminal arteriole and venule visible in the image. Each capillary segment selected was supplied by the same arteriole. (C–E) The RBCs in the capillary segments indicated by the red arrows (C) and green arrows (D) are traveling to the venule above the arteriole. The RBCs in the capillary segment indicated by the blue arrow (E) are traveling to the venule to the right of the arteriole. Three-second velocity measurements of the three capillary segments were acquired in the same video. Individual capillaries can have missing data because small eye movements could move one capillary, but not another, out of the frame (compare D and E). If we could not detect RBCs in a capillary at any time, the data were not plotted, and the missing data are not included in the CV computation. Scale bar: 50 µm.

Figure 2.

A control experiment verifying the reproducibility of this technique for measuring RBC velocity in an individual capillary by making measurements over two non-overlapping sections of an unbranched capillary. (A) A flow map identifying the region measured. The two dashed red lines indicate the capillary segments selected for measurements. (B) The average of five RBC velocity measurements for two segments of the selected capillary under different flicker conditions, as in Experiment 2 (before, during, and after exposure to 10-Hz full-field flicker). The RBC velocities of the capillary segments differed on average 2% (±1%) from each other across conditions. Scale bar: 50 µm.

Figure 3.

Repeated RBC velocity measurements of an arteriole (A, D), capillary (B, E), and venule (C, F), as well as corresponding CV for one subject plotted for each session on day 2 of imaging. RBC velocity measurements in an arteriole (A), capillary (B), and venule (C) with 30-minute breaks in between acquisition periods (A–C) and 10-minute breaks (D–F). The number of measurements made varied somewhat between acquisitions due to eye blinks or eye motion. The x-axis indicates the sequence number of the 3-second videos taken during each acquisition period, and the y-axis indicates the average RBC velocities measured. The CV is also shown for each acquisition period separated by the appropriate break.

Figure 4.

Box plots of the CV of blood velocity for arterioles, capillaries, and venules across all subjects for each imaging session in Experiment 1. The median CV is indicated by the line within the box and the mean by an “×.” The filled vertical dots indicate outliers. The filled portions show the 25% to 75% interquartile range.