Alterations In Blood Retinal Barrier Function In Cultured Fetal Human RPE After Exposure To Indocyanine Green

Abstract

Purpose: : Indocyanine green (ICG) is frequently used for epiretinal membrane peeling, yet its effect on the RPE remains controversial. To assess outer blood retinal barrier (BRB) function after exposure to clinically relevant ICG concentrations, we measured changes in transepithelial resistance (TER) in cultured human RPE.

Methods: : Fetal human RPE were isolated from donor eyes at 16-24 weeks of gestation and cultured using the Hu& Bok method. 2x10E5 cells per cm² were seeded on PE transwell culture inserts (Corning) and grown for a minimum of 4-6 weeks till TER values had stabilized. 2mg ICG dry power (PULSION, Germany) was dissolved in 0.1ml distilled water, and then further diluted in Hank’s balanced salt solution (HBSS, PAA, Austria). 50% of the apical culture medium was then replaced with diluted ICG to yield a final concentration of 0.05mg/ml. Cells were incubated at 37C in a 5% CO₂ incubator for 3 minutes. Thereafter ICG was washed out 5 times with HBSS and then replaced with culture media. TER values were recorded prior to, and at 1.5h, 3h, 1, 3& 7days post ICG exposure. HBSS alone served as a vehicle control and 0.5% trypsin/ 0.2mM EDTA as a positive control. Experiments were performed in triplicate. Data were analyzed using Student’s T-Test.

Results: : At 4-6 weeks post confluence, fetal human RPE had grown into pigmented, uniformly hexagonal monolayers. TER values before treatment ranged from 600 to 1000 ohm*cm², thereby suggesting well polarized RPE monolayers with good outer BRB function. ICG treated cultures showed a statistically significant decrease in TER values until 1 day post exposure when compared to vehicle controls. TER at 1.5h& 3h dropped to a mean of 127 and 141 ohm*cm², respectively. At 7 days TER values had returned to levels prior to exposure, except for Trypsin/EDTA cultures.

Conclusions: : Preliminary data suggest a 3 minutes exposure of 0.05mg/ml ICG to induce a transient, yet reversible breakdown in outer BRB function. Future experiments will include a range of clinically relevant ICG concentrations and the effect of an endoillumination probe.

Keywords: drug toxicity/drug effects • electrophysiology: non-clinical • retinal pigment epithelium