Background: The use of vital dyes in "chromovitrectomy" allows the easier removal of less recognizable structures like epiretinal membranes (EM) or the inner limiting membrane (ILM). In recent years numerous studies demonstrated the use of indocyanine green (ICG), trypan blue (TB) and patent blue (PB) for this indication. Reports of the possible risk of these dyes, i. e. especially ICG, in respect of reduced visual acuity results, possible visual field defects or alterations of the retinal pigment epithelium (RPE) resulted in limitations in their application.

Material and methods: Human RPE cells and choroidal endothelial cells were cultured in monolayers on semipermeable membranes representing an in vitro model of the outer blood-retinal barrier. By measurement of the transepithelial electrical resistance (TER) the stable barrier function was determined. Two different models representing an air-filled and a fluid-filled eye were tested on the one hand by addition of the dye to the culture medium and, on the other, by direct application on the cell monolayer. In these two models ICG (5 mg/ml, 0.5 mg/ml, 0.125 mg/ml), TB (1.5 mg/ml, 0.15 mg/ml) and PB (2.4 mg/ml, 0.24 mg/ml) were applied for three minutes and the influence on the barrier function was determined. RPE cell growth was also tested in these two models after the application of ICG, TB and PB. Finally, monolayers of RPE cells were evaluated by transmission electron microscopy (TEM).

Results: After application of TB, PB and the lowest concentration of ICG of 0.125 mg/ml, the TER remained stable in both models. In contrast, ICG in a concentration of 5 mg/ml and 0.5 mg/ml caused a significant TER decrease in the model of the air-filled eye, whereas no influence on the function of the outer blood-retinal barrier was noted in the model of the fluid-filled eye. RPE cell growth rates were not influenced by the addition of the vital dyes, with the exception of ICG in a concentration of 5 mg/ml in the model of the air-filled eye, resulting in a temporary reduction of the cell count. In good correspondence to these results also in TEM intercellular blisters were noted after application of 5 mg/mL ICG for 3 minutes in the model of the air-filled eye. However, damage to the RPE cells themselves was not obvious. No pathological changes in the TEM were noted after application of TB and PB.

Conclusions: The use of PB and TB at the posterior eye segment seems to be safe concerning damage to the PRE and its barrier function. In contrast, ICG in higher concentrations and with longer application times may cause a toxic effect on RPE morphology and function.