Effect of Vital Dyes on Retinal Pigmented Epithelial Cell Viability and Apoptosis: Implications for Chromovitrectomy

Abstract

Purpose: To investigate the in vitro effect of vital dyes on toxicity and apoptosis in a human retinal pigment epithelial cell line. Methods: ARPE-19 cells were exposed to brilliant blue (BBG), Evans Blue (EB), bromophenol blue (BroB), indocyanine green (ICG), infracyanine green (IfCG), light green (LG), fast green (FG), indigo carmine (IC) and Congo red (CR). Balanced salt solution was used as the control. Five different concentrations and 2 exposure times were tested. Cell viability was determined by the MTS (1-solution methyl thiazolyl tetrazolium) assay and apoptosis by Bax expression on Western blot. Results: All dyes significantly reduced cell viability after 3 min of exposure at all concentrations (p < 0.01), except for BBG that was safe at concentrations up to 0.25 mg/ml and CR up to 0.05 mg/ml, while LG was safe at all concentrations. Toxicity was higher after 30 min of exposure. Expression of Bax was upregulated after all dye exposures, except BBG; ICG had the highest Bax expression (p < 0.01). Conclusions: Overall the safest dye was BBG followed by LG, IfCG, FG, CR, IC, BroB, EB and ICG. ICG was toxic at all concentrations and exposure times tested. Moreover, BBG was the only dye that did not induce apoptosis in ARPE-19 cells.

The synthetic progesterone Norgestrel is neuroprotective in stressed photoreceptor‐like cells and retinal explants, mediating its effects via basic fibroblast growth factor, protein kinase A and glycogen synthase kinase 3β signalling

“…661W cells were serum‐starved over 24 h and treated with 20 μ m Norgestrel. Cell viability was then compared to non‐serum‐starved control (100% viability) using the MTS assay, an assay frequently used in ocular studies as a measure of cellular proliferation and viability (Penha et al ., ; Sobolewska et al ., ). Norgestrel significantly increased cellular viability after serum deprivation (Fig.…”

A Short Review on the Safety of Bromphenol Blue for Dye-Assisted Vitreoretinal Interventions

“…The results are relatively heterogenous. While Haritoglou et al [4] did not observe any undesired effects at dye concentrations of 0.2 and 2 mg/mL and exposure times of 10 min, the results of Penha et al [5] indicated reduced cell viability at all concentrations tested (0.005 to 1.0 mg/mL) and at a dye contact time down to 3 min. Cell viability in the experiments by Balaiya et al [6] revealed toxicity at longer exposure times (30 min) of 0.5 mg/mL bromphenol blue concentrations, and apoptosis was observed already after 5 min of dye exposure.…”

“…The following observations were especially worrying: 1. In RPE cell culture experiments, undesired effects have been observed at concentrations below that presently available (1.3 mg/mL) and at relatively short exposure times (3-5 min) [5,6]. 2.…”

Progesterone receptor signalling in retinal photoreceptor neuroprotection

“…With this in mind, the ability of Norgestrel to rescue 661W cells from apoptoticinduced, serum starved cell death was investigated using the MTS assay for cellular proliferation and viability. This is a well-practised method in ocular studies (Okumichi et al 2008;Guerin et al 2011;Penha et al 2013;Sobolewska et al 2013). Norgestrel significantly inhibited apoptotic cell death by approximately 10% (*p < 0.05) (Fig.…”