Fluorescent labeling of blood cells for evaluation of retinal and choroidal circulation

To develop a method of staining and tracking cells in vivo with a scanning laser ophthalmoscope (SLO) to evaluate the retinal and choroidal circulations.

Twelve pigmented male rats were used. Staining of leukocytes. Two commercially available nucleic acid stains SYTO 16 and SYTO 59 (Molecular Probes, Eugene, OR) were used to stain leukocytes. Blood (0.5 mL) was withdrawn from the subject animal and placed in a heparinized tube to which phosphate buffered solution was added (1.0 mL buffer/0.5 mL blood). Ten microliters of solution of SYTO 16 or SYTO 59 in alcohol (5 mM) was added. Staining of Erythrocytes. The lipophilic carbocyanine dye indocarbocyanine (D-307, Molecular Probes) was used to stain erythrocytes. Blood (0.1 mL) was withdrawn from the subject and handled as above; plasma and leukocytes were removed with a pipette. HEPES buffer was added to the remaining erythrocytes (1.4 mL buffer added to 0.1 mL blood). Ten microliters of D-307 in alcohol (5 mM) was added to the 1.5 mL buffered blood. The final concentration of dyes in the buffered blood mixtures was 0.03 mM. The stained cells were injected intravenously in the subject animal. The eyes were observed with SLO; the resident helium-neon laser was used to excite the SYTO 59 and the resident argon laser for SYTO 16. The He-Ne laser was used to excite the D-307-stained erythrocytes.

The movement of the leukocytes and erythrocytes in the retinal and choroidal vessels was clearly distinguishable from the surrounding tissue.

By selecting different dyes for staining leukocytes or erythrocytes and choosing appropriate laser wavelengths, the hemodynamics of blood cells can be observed and evaluated in the retina and choroid.