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PurposeTo investigate the in vitro effect of four vital dyes on toxicity and apoptosis in a human retinal pigment epithelial (RPE) cell line.MethodsARPE-19 cells were exposed to brilliant blue (BriB), methyl blue (MetB), acid violet (AcV) and indocyanine green (ICG). Balanced salt solution was used as control. Five different concentrations of each dye (1, 0.5, 0.25, 0.05 and 0.005 mg/mL) and two exposure times (3 and 30 min) were tested. Cell viability was determined by cell count and MTS assay and cell toxicity by LDH assay. Real-time PCR and Western blotting were used to access the apoptosis process.ResultsICG significantly reduced cell viability after 3 minutes of exposure at all concentrations (p<0.01). BriB was safe at concentrations up to 0.25 mg/mL and MetB at concentrations up to 0.5 mg/mL, while AcV was safe up to 0.05 mg/ml, after 3 minutes of exposure. Toxicity was higher, when the cells were treated for 30 minutes. Expression of Bax, cytochrome c and caspase-9 was upregulated at the mRNA and protein level after ICG exposure, while Bcl-2 was downregulated. AcV and MetB were similar to control. However, BriB resulted in upregulation of Bcl-2, an antiapoptotic protein.ConclusionsThe safest dye used on RPE cells was MetB followed by BriB and AcV. ICG was toxic at all concentrations and exposure times tested. Moreover, ICG was the only dye that induced apoptosis in ARPE-19 cells. BriB significantly increased Bcl-2 protein levels, which might protect against the apoptosis process.

Aims: Experimental retinal research has gained great importance due to the ophthalmic pharmacotherapy era. An increasing number of drugs are constantly released into the market for the treatment of retinal diseases. In this review, animal species, animal models and toxicity assays in retinal research are discussed. Methods: An extensive search of the literature was performed to review various aspects of the methods of investigation of drug toxicity. The different types of animal species, as well as single animal models available for the evaluation of safety and efficacy of retinal pharmacotherapy, were identified. In addition, a large variety of reported laboratory techniques were critically examined. Results: In vitro studies are the first-line experiments for the development of a new drug for retinal diseases, using retinal pigment epithelial cells and other cell lines. The next step involves in vivo animal studies where nonhuman primates are considered the gold standard. However, cost and legal issues make their use difficult. Mice and rats provide genetically controlled models for investigations. Pigs, dogs and cats represent good large-size animal models, while rabbits are one of the most used species for retinal toxicity evaluations. Various laboratory methods were identified, including light microscopy, electron microscopy, electroretinography and new emerging methods, such as optical coherence tomography and scanning laser ophthalmoscopy for experimental purposes. Conclusions: A great number of animal species and models are available that simulate retinal diseases and provide experimental data for further human use. Work with animal models should include properly designed toxicity assays to obtain reliable results for safety and efficacy.