Effects of Indocyanine green on cultured retinal ganglion cells in-vitro

Cultured rat retinal ganglion cells (RGC-5) were exposed to different concentrations of ICG (0.25, 0.5, 1.0, 1.25, & 5 mg/ml) and at various time intervals (1, 5, 15, 30, & 60 minutes). Changes in structural morphology were identified using phase contrast bright field microscopy. Cell viability was quantified using the neutral red assay and cell death was characterized using Annexin-V staining.Significant morphologic changes were observed at the 15 and 60 min intervals for all concentrations, where a reduction in cell size and loss of normal spindle shape was noted. A dose dependent decrease in cell viability was observed with increasing concentration of ICG as well as increasing exposure intervals. Compared to control, 48-74% reduction in neutral red uptake at all concentrations for exposures 5 min or greater (p < 0.001). Even at 1 min exposure, a dose dependent decline was observed in cell viability, with a 28-48% decline for doses above 1.25 mg/ml (p = 0.007). Staining with Annexin-V, demonstrated a similar dose and time dependent increase in number of cells exhibiting early apoptosis. A greater than two-fold increase in Annexin-V expression for all doses at exposures greater than 1 min was noted.ICG dye exhibits toxicity to retinal ganglion cells at clinically relevant doses following 1 min exposure.Indocyanine green (ICG) is commonly used to stain the internal limiting membrane (ILM) [1,2] during macular surgery for the treatment of idiopathic macular holes [3-5] and diffuse diabetic macular edema [6]. However, the safety of intravitreal use of ICG is not well established. Adverse effects such as visual field defects [7-9] and atrophy of the retinal ganglion cell layer [10] subsequent to ICG assisted membrane peeling have been reported. The underlying cause of ICG-related adverse effects has been proposed to be due to the osmolarity of the solution [11] or photochemical damage [12].The retinal ganglion cell (RGC) layer is the first to come in contact with ICG d