An experimental method for testing novel retinal vital stains Timothy L. Jackson a,d, * , Lewis Griffin b,d , Brendan Vote a , Jost Hillenkamp a,c , John Marshall a a Academic Department of Ophthalmology, The Rayne Institute, St Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK b Imaging Sciences, King’s College London, London, UK c Eye Hospital, University of Regensburg, Regensburg, Germany d Vitrocetinal Unit, Moorfields Eye Hospital, London EC1V 2PD, UK Received 10 November 2004; accepted in revised form 7 March 2005 Available online 31 May 2005 Abstract There is uncertainty surrounding the safety of the vital stains currently used to assist macular surgery, and there may be other agents that are more suitable. This study aimed to validate a method of screening retinal vital stains for their potential surgical utility. 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 colour difference equation) was used to estimate the magnitude of the colour difference between stained and unstained retina. Agents that had favourable staining characteristics underwent safety testing using a retinal pigment epithelium and glial 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). Frozen sections were used to determine which retinal layers were stained. Techniques were tested on the following agents: alcian blue; diethyloxadicarbocyanine; Evan’s blue; fast green; fluorescein; Janus green; methylene blue; naphthol green; neutral red; procian (reactive) yellow; rose bengal; and trypan blue. For most dyes, the results of image analysis showed that colour differences increased linearly with dye concentration, although some displayed a more exponential relationship. Five agents showed favourable staining characteristics: Evan’s 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; tZ4.71; CI 30–75%), and qualitative damage on fluorescence microscopy. Frozen sections showed that some agents produced diffuse staining of all retinal layers, others produced selective inner retinal staining. 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. q 2005 Elsevier Ltd. All rights reserved. Keywords: vital stain; retina; macula; toxicity; retinal pigment epithelium; Mu ¨ller cell; glia 1. Introduction Indocyanine green (ICG) assisted macular surgery is well described. By staining the retinal internal limiting mem- brane (ILM), ICG makes an otherwise optically transparent structure much easier to visualise and remove. There has, however, been debate regarding its safety (Kampik and Sternberg, 2003; Sebag, 2004; Jackson, in press). Some recent clinical studies reported favourable results (Wein- berger et al., 2002; Kwok et al., 2003; Kwok and Lai, 2003; Slaughter and Lee, 2004); others suggested that it caused a functional visual loss (Gandorfer et al., 2001; Engelbrecht et al., 2002; Haritoglou et al., 2002; Uemura et al., 2003). Experimental studies have shown possible damage in human retinal pigment epithelium (RPE) (Sippy et al., 2001; Stalmans et al., 2002; Yam et al., 2003; Ho et al., 2003; Jackson et al., 2004a), retinal ganglion cells (Iriyama et al., 2004), and Mu ¨ ller cells (Jackson et al., 2004a). In vivo animal experiments also suggested a toxic effect (Enaida et al., 2002; Maia et al., 2004), but only when using prolonged contact times or concentrations that were higher than those used clinically. More recently, infracyanine green has been advocated as an alternative vital stain (Stalmans et al., 2003). This agent is closely related to ICG but is theoretically safer, as it comes with a glucose diluent that results in a more iso-osmotic solution. Experimental Experimental Eye Research 81 (2005) 446–454 www.elsevier.com/locate/yexer 0014-4835/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.03.004 * Corresponding author. E-mail address: tim.jackson@nhs.net (T.L. Jackson).