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

<p>Abstract</p> <p>Background</p> <p>Indocyanine green (ICG) dye is commonly used to stain the inner limiting membrane during macular surgery. There are reports documenting the toxicity of ICG on retinal pigment epithelial cells, with conflicting results in retinal ganglion cells. In the present study, we evaluated the effect of ICG on retinal ganglion cells in vitro.</p> <p>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.</p> <p>Findings</p> <p>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.</p> <p>Conclusion</p> <p>ICG dye exhibits toxicity to retinal ganglion cells at clinically relevant doses following 1 min exposure.</p

Osmolarity and spectrophotometric property of brilliant blue green define the degree of toxicity on retinal pigment epithelial cells exposed to surgical endoilluminator

Sankarathi Balaiya, Kumar Sambhav, William B Cook, Kakarla V Chalam Department of Ophthalmology, University of Florida College of Medicine, Jacksonville, FL, USA Objective: To evaluate the effect of varying concentrations of brilliant blue green (BBG) and their different biochemical characteristics on retinal pigment epithelial (RPE) cells under xenon light source illumination at varying distances to identify safe parameters for intraoperative use. Methods: Human retinal RPE cells (ARPE-19) were exposed to two concentrations (0.25 and 0.50 mg/mL) of BBG and illuminated with a xenon surgical illuminator at varying distances (10&nbsp;and 25 mm), intensity levels, and time intervals (1, 5, and 15 minutes). Additionally, the effect of osmolarity was examined by diluting BBG in different concentrations of glucose. Cytotoxicity of BBG and osmolarity effects on cell viability were evaluated using a WST-1 assay. Light absorption and emission characteristic of BBG in different solvents were measured using a plate reader at different wavelengths. Lastly, the activity of caspase-3 was also studied. Results: Cell viability of ARPE-19 cells was 77.4%&plusmn;12.7%, 78.7%&plusmn;17.0%, and 65.0%&plusmn;19.7% at 1, 5, and 15 minutes to exposure of high illumination xenon light at 10 mm (P&lt;0.05) compared to controls. At both distances of illumination (10 and 25 mm), similar cell viabilities were seen between 1 and 5 minutes of exposure. However, there was a decline in viability when the illumination was carried out to 15 minutes in all groups (P&lt;0.05). There was no significant reduction in cell viability in presence or absence of xenon light in different osmolar solutions concentrations of glucose (P&gt;0.05). Maximal light absorption of BBG was noted between 540 and 680 nm. Activated caspase-3 level was not significant in both the concentrations of BBG (P&gt;0.05). Conclusion: Our findings suggest that BBG at 0.25 mg/mL during vitreoretinal surgery is safe and not toxic to RPE cells up to 5 minutes under focal high illumination (10 mm) and up to 15&nbsp;minutes under medium diffuse illumination (25 mm). BBG was safe to be mixed with isotonic glucose solution at the concentration range of 2.5%&ndash;10%, regardless of the illumination status. Keywords: brilliant blue green dye, endoilluminator, vitrectomy, human retinal RPE cells, internal limiting membrane pee