purpose. The purpose of this study was to develop and characterize a quantitative assay of blood–retinal barrier (BRB) function in mice and to determine the effect of several purported vasopermeability factors on the BRB.

methods. Adult C57BL/6J mice were treated with three regimens of increasingly extensive retinal cryopexy and subsequently were given an intraperitoneal injection of 1 μCi/g body weight of [3 H] mannitol. At several time points, the amount of radioactivity per milligram tissue was compared in retina, lung, and kidney. Time points that maximize signal-to-background differential in the retina were identified, and the ratio of counts per minute (CPM) per milligram retina to CPM per milligram lung (retina-to-lung leakage ratio, RLLR) or kidney (retina-to-renal leakage ratio, RRLR) were calculated. This technique was then used to compare the amount of BRB breakdown that occurs after intravitreous injection of vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1, prostaglandin (PG) E 1, PGE 2, interleukin (IL)-1β, or tumor necrosis factor (TNF)-α.

results. Twenty-four hours after retinal cryopexy, there was a higher level of radioactivity in treated than in control retinas, and the signal-to-background difference was optimal when measurements were obtained 1 hour after injection of [3 H] mannitol. In untreated mice, the RLLR was 0.30±0.02 and the RRLR was 0.22±0.01. Twenty-four hours after one 5-second application of retinal cryopexy, the RLLR was 0.73±0.20 and the RRLR was 0.71±0.23. With increasing amounts of cryopexy, there was an increase in the RLLR and RRLR, so that after two 10-second applications, the RLLR was 1.66±0.31 and the RRLR was 1.47±0.20. Intravitreous injection of VEGF, IGF-1, PGE 1, PGE 2, IL-1β, or TNF-α each caused significant increases in the RLLR and RRLR, but there were some differences in potency and time course. VEGF caused prominent BRB breakdown at 6 hours that returned to near normal by 24 hours. IL-1β also caused relatively rapid breakdown of the BRB, but its effect was more prolonged than that caused by VEGF. There was delayed, but substantial breakdown of the BRB after injection of TNF-α. IGF-1, PGE 2, and PGE 1 caused less severe, relatively delayed, and more prolonged BRB breakdown.

conclusions. Measurement of the RLLR or RRLR after intraperitoneal injection of [3 H] mannitol in mice provides a quantitative assessment of BRB function that is normalized and can therefore be compared from assay to assay. Comparison of the extent and duration of BRB breakdown after intravitreous injection of vasoactive substances shows that agents can be grouped by resultant extent and time course of leakage. Additional studies are needed to determine whether this grouping has its basis in shared mechanisms of BRB disruption.