Sensitive blood-retinal barrier breakdown quantitation using Evans blue.
OBJECTIVE
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.
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Department of Surgical Research, Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
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D000818:Animals; D001813:Blood-Retinal Barrier; D019559:Capillary Leak Syndrome / Q000175:diagnosis* / Q000209:etiology; D002199:Capillary Permeability / Q000187:drug effects; D004396:Coloring Agents; D003921:Diabetes Mellitus, Experimental / Q000150:complications; D016228:Endothelial Growth Factors / Q000494:pharmacology; D005070:Evans Blue; D008222:Lymphokines / Q000494:pharmacology; D008297:Male; D009929:Organ Size; D051381:Rats; D017207:Rats, Sprague-Dawley; D012171:Retinal Vessels / Q000187:drug effects / Q000473:pathology*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D004396:Coloring Agents; D016228:Endothelial Growth Factors; D008222:Lymphokines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D005070:Evans Blue