Characterization of the blue light toxicity spectrum on A2E-loaded RPE cells in sunlight normalized conditions

Abstract

Purpose: Among the identified risk factors of AMD, sunlight is known to induce cumulative damage to the retina. The portion of the visible spectrum at higher energy, between 400 nm and 500 nm, the blue light, is incriminated. However, the identification of the precise spectrum of retinal toxicity induced by sunlight, in physiological irradiances conditions, remains to be investigated. The present study that screened in vitro toxicity by 10 nm examined this question to improve ophthalmic devices that provide blue light protection.

Methods: Our AMD in vitro model was a primary culture of retinal pigment epithelium cells incubated for 6 hours with 0, 12.5, 20 or 40 µM of A2E. These 96 wells plate cultures were exposed to 10 nm bandwidths centered from 390 to 520 nm in 10 nm increments (one additional bandwidth at 630 nm) for 18 hours. Light irradiances were normalized with respect to the natural sunlight reaching the retina after filtering by the different eye ocular structures. Control cells were maintained in the dark during the experiment. Six hours after light exposure, viability, necrosis and Caspase-3/7 activity were assessed using the Apotox-Glo Triplex Assay.

Results: When A2E was added to the culture medium, retinal pigment epithelium cells presented fluorescent bodies within the cytoplasm that showed a similar spectrum to that of A2E. Exposition to the 10 nm bandwidths induced morphological changes associated to a loss in cell viability. Quantification of the cell viability revealed that light toxicity was higher in the blue range of the spectrum with maxima in a specific 30 to 40 nm bandwidth. Increasing A2E concentrations increased light toxicity. In parallel to the loss of cell viability, caspase-3/7 activity indicative of cell apoptosis was highly induced by the same wavelengths whereas necrosis was not significantly different to cells maintained in the dark.

Conclusions: Our results describe for the first time the precise spectrum of light toxicity in physiological conditions of illumination on an in vitro model of AMD. Under these conditions, we showed that a narrow bandwidth of the blue spectrum represents the greatest phototoxic risk to RPE cells. This provides new information for the definition of protective filters for patients with AMD.

Keywords: 582 ipofuscin • 634 oxidation/oxidative or free radical damage • 701 retinal pigment epithelium