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T.L. Jackson, L. Griffin, B. Vote, J. Hillenkamp, J. Marshall; A Systematic, Experimental Method of Screening Novel Retinal Vital Stains . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5530.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To develop a method of testing the potential surgical utility of novel retinal vital stains. Methods: Bovine retina was exposed to test agents at a range of concentrations. Masked observers determined the minimum dye concentration that reliably stained the retina, defined as the minimum visible concentration (MVC). Computer image analysis (CIE94 color difference equation) was used to estimate the magnitude of the color difference between stained and unstained retina. Agents that had favourable staining characteristics underwent safety testing using a retinal pigment epithelium (ARPE19) and glial (MIO–M1) cell culture model . Cells were exposed to each agent and viability was assessed with a mitochondrial enzyme (MTT) assay, and fluorescent live–dead probe (ethidium homodimer–1 / calcein–AM). Techniques were tested on the following agents: alcian blue; diethyloxadicarbocyanine; evans blue; fast green; fluorescein; janus green; methylene blue; naphthol green; neutral red; procian (reactive) yellow; rose bengal; and trypan blue. Results: For most dyes the calculated color differences increased linearly with dye concentration, although some showed a more exponential relationship. Five agents showed favourable staining characteristics: evans blue, rose bengal, naphthol green, neutral red, and trypan blue (MVC 0.02, 0.01, 0.1, 0.002, 0.01% respectively). Safety testing of these five agents did not show toxicity, except in glial cells exposed to rose bengal. Relative to the negative control (saline), these showed a 48% reduction in viability using the MTT assay (p <0.001; t = 4.71; CI 30 to 75%), and qualitative damage on fluorescence microscopy. Conclusions: There are thousands of biological stains available and many of these may be more effective or safer than those currently used for retinal surgery. This study provides a means of screening potentially useful vital stains.
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