Effect of Novel Vital Dyes on Outer Blood-Retina Barrier Function in Cultured Human Retinal Pigment Epithelium

Purpose: To assess tight junction (TJ) integrity in cultured human fetal retinal pigment epithelium (HFRPE) after exposure to clinically relevant novel vital dyes. Methods: HFRPE floater cells were harvested from RPE primary cultures of 4 donor eyes and seeded on polyester Transwell (R) for 4-6 weeks. The apical compartments of well-differentiated cultures were exposed to 0.005 mg/ml Coomassie violet R200 (CVR), methyl 2B (M2B) or Orange II. Periods of 30-300 s were chosen to mimic surgical exposure times, while 3 h was used for toxicity assays, with subsequent washout. Cell-cell junctions were studied by immunofluorescence (zonula occludens-1, ZO-1). Transepithelial electrical resistance (TER) was measured regarding blood-retina barrier (BRB) function. Results: At 4-6 weeks after confluence, HFRPE had grown into pigmented hexagonal monolayers with stable TER values (451-1,520 Omega.cm(2)). After 300-second dye treatments, a continuous ZO-1 signal was detected in all vital dye-treated groups 1.5 h after exposure, whereas trypsin controls showed patchy loss of the TJ stain. TER of CVR-, M2B- and Orange-II-treated groups had dropped 1.5 h after exposure to 148 58.4, 162 +/- 23.7 and 164 +/- 18.5 Omega.cm(2), respectively, compared to 73 +/- 44.9 Omega.cm(2) in positive controls. After 3 h of exposure to 0.005 mg/ml vital dyes in thick drops, TER maintained similar levels to those prior to exposure (90.8 +/- 4.7% of the original values, 93.8 +/- 6.5 and 91.9 +/- 3.6%, respectively), together with no difference from the vehicle controls (94.8 +/- 6.6%). TER values recovered in all groups to prior levels within 3 days. Conclusion: Novel vital dyes (CVR, M2B and Orange II) caused no outer BRB function alteration. (C) 2013 S. Karger AG, Basel