Comparison of Indocyanine Green and Trypan Blue Related Toxicity in RPE 19 Cell Culture With Light Exposure

Abstract

Abstract: : Purpose: Peeling the transparent internal limiting membrane (ILM) during vitrectomy is a crucial step in macular pucker and macular hole surgery. The visualization of ILM and overlying structures can be improved by intra–operative staining. Indocyanine green (ICG) and trypan blue (TB) are commonly used. The aim of this study was to evaluate and compare the safety of these dyes in RPE 19 cell culture in different clinically relevant concentration. Methods: 100% confluent human RPE19 cell cultures were grown in 24–well plates directly on plastic or cover slips. After phase contrast microscopy the medium was removed and 0.3 ml of ICG (0.5, 0.25, 0.125, 0.05, 0.0 mg/ml) or TB (0.6, 0.30, 0.15, 0.06, 0.0 mg/ml) was added for 5 minutes, the cells were washed twice with balanced salt solution and were exposed to the endoillumination, 8 mm above the cells, for a period of 10 minutes with an irradiance of 4.6 mW/cm. As a solvent we used Glucose 5% for ICG and 1xPBS solution for TB. We examined the probes for morphology, live/dead assay, and analysed apoptosis–related cell death by mean of Western–blotting for soluble protein expression and Tunnel staining. Results:Morphological changes like edema and shrinkage were found in both groups, if higher concentrations were used. In live /dead assay the number of dead cells in the observation field (containing 2500 cells in average) was counted for statistical evaluation. We found a dose–dependent cell death in both groups. In clinically used concentrations of 0.125 and 0.05 ICG the number of the dead cells was 10.4 (SD 3.5) and 6.5 (SD 4.8), respectively. Interestingly similar results were observed in comparable samples of TB. Here, the number of the dead cells for TB 0.15 and 0.06 mg/ml was 10.5 (SD 4.8) and 6.2 (SD 1.1), respectively. No apoptosis–related gene expression or Tunnel labelling was determined in any of the samples Conclusions: In presence of the light a similar toxicity was observed for ICG and TB in comparable clinical relevant concentrations. Due to a different affinity of both substances to ILM and epiretinal structures, the selective and indication–dependent use of ICG or TB should be considered.

Keywords: retinal pigment epithelium • drug toxicity/drug effects • vitreoretinal surgery