In Vitro Detection of UV‐Induced Damage of the Cornea, Lens and RPE

Abstract Purpose UV‐A and UV‐B radiation from sunlight is a major source of ocular oxidative damage. This paper describes in vitro methods that can be used to detect ocular damage from UV radiation. Methods Human corneal epithelial cells, lens epithelial cells, and RPE cells were cultured and Ultraviolet A/Ultraviolet B blocking filters and UVB‐only blocking filters were placed between the cells and a UV light source. Cells were irradiated with UV radiations at various energy levels, with and without filters. Cell viability after exposure was determined using the metabolic dye alamarBlue and by evaluating changes in nuclei, mitochondria, membrane permeability, and cell membranes using the fluorescent dyes Hoechst 33342, rhodamine 123, calcein AM, ethidium homodimer‐1, and annexin V. Images of the cells were taken with a Zeiss 510 confocal laser scanning microscope Results The alamarBlue assay results of UV‐exposed cells without filters showed energy level‐dependent decreases in cellular viability. However, UV treated cells with 400 nm LP filter protection showed the equivalent viability to untreated cells at all energy levels. Also, UV irradiated cells with 320 nm LP filter showed lower cell viability than the unexposed control cells, yet higher viability than UV‐exposed cells without filters in an energy level‐dependent manner. The confocal microscopy results also showed that UV radiation can cause significant dose‐dependent degradations of nuclei and mitochondria in ocular cells. The annexin V staining also showed an increased number of apoptotic cells after UV irradiation. Conclusion UV‐induced damage can be evaluated to test the effectiveness of UV‐absorbing contact lenses and intraocular lenses with bioassays that measure change in in vitro cells