Sensitive Blood-retinal Barrier Breakdown Quantitation Using Evans Blue

Purpose: This study investigated whether a nonradioactive dye, Evans blue, can be adapted as a safe alternative to the isotope-dilution method for quantitating blood-retinal barrier breakdown.

Methods: Blood-retinal barrier breakdown was induced in rats with vascular endothelial growth factor (VEGF) or through the induction of diabetes. After allowing Evans blue to circulate in the vasculature, the dye was cleared from the bloodstream with saline, citrate, or citrate-buffered paraformaldehyde, and the efficacies of the perfusion solutions were compared. Extravasated dye was detected at 620 nm and was normalized against the time-averaged Evans blue plasma concentration, the circulation time, and also against wet and dry retina weights.

Results: Evans blue leakage from retinas treated with VEGF was 4.0-fold higher than that of contralateral untreated eyes (n = 6 rats, P: < 0.05). Retinal Evans blue leakage of eyes from 1-week diabetic animals (n = 11 retinas) was 1.7-fold higher (P: < 0.05) than that of nondiabetic controls (n = 10 retinas). Intra-animal, inter-retina weights showed significantly less variability (P: < 0.05) with the use of dry weights (11.2%, n = 74 retina pairs) than with wet weights (20.5%, n = 93 retina pairs).

Conclusions: The Evans blue dye technique can be modified to be as sensitive and quantitative as the isotope-dilution method for measuring blood-retinal barrier breakdown. The advantages of the Evans blue technique are its safety, relative simplicity, and economy.