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R.C. Tripathi, S. Lin, W. Philips, B.J. Tripathi, K.V. Chalam; Effects of Trypan Blue on Cell Viability and Gene Expression of Retinal Ganglion Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5419.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Because trypan blue at various concentrations is used intraoperatively for visualization of the vitreous cortex and internal limiting membrane in macular surgery and vitrectomy, we evaluated the dose/time effects of this vital dye on cell viability and gene expression of retinal ganglion cells (RGCs). Methods: Three concentrations (0.06, 0.6, and 4 mg/ml) of trypan blue were applied to confluent cultures of rat RGCs. After 1, 30, 60, 120, 360 mins the cultures were processed to assess the effects of trypan blue on cell viability and gene expression. Cell viability was measured by using the neutral red assay. Reverse transcription–polymerase chain reaction (RT–PCR) and immunostaining were used to analyze the expression of apoptosis related and cell cycle arrest gene expression, including cytochrome C, Bax and Bcl–2. Results: Compared to untreated controls, the neutral red assay showed a concentration and time dependent suppression of cell viability, with greater reduction in the 0.6 mg/ml and 4 mg/ml trypan blue treated groups. An increase in the expression of cytochrome C was found only in the group treated with 4 mg/ml for 30 mins. Immunostaining and RT–PCR showed a significant increase in Bax expression in cultures treated with either 0.6 mg/ml (p = 0.032) or 4 mg/ml (p = 0.025) trypan blue. Marked reduction of Bcl–2 expression was also noted after exposure to trypan blue for 60 mins or longer (p=0.01). Conclusions: The application of 0.06 mg/ml trypan blue for 1 minute appears to have no significant effect on cultured RGCs. Exposure to higher concentrations for a prolonged time is cytotoxic as evidenced by reduction in cell viability and the up regulated expression of apoptosis related genes.
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