Dimethylthiourea Protects Retinal Photoreceptors Against Blue Light Stress in Bovine and Primate Primary Cultures

Abstract

Purpose: : Light induced photoreceptor damage is believed to be associated with age related macular degeneration (AMD). Dimethylthiourea (DMTU), an antioxidant and free radical scavenger has been shown to protect retinal photoreceptors from damage caused by light stress in in–vivo animal models. The primary objective of this study was to investigate the effect of DMTU on blue light stress in bovine and primate primary retinal cell cultures.

Methods: : Primary retinal cell cultures enriched in photoreceptors were prepared from primate and bovine retinas. A week old cultures were pretreated with DMTU at various concentrations (0.01–1000 µM) for 24 hrs. DMTU was replenished before subjecting the cultures to blue actinic light (420 nm, 300 lux, 0.4mW/cm² and 900 lux, 1.1mW/cm²) for various durations (1–24 hrs) of time. Controls were shielded from light exposure. Evidence of protection with DMTU in bovine cultures was obtained using Sytox Green assay. Immunocytochemistry on both bovine and primate cultures was performed using recoverin fluorescent antibody labeling of the photoreceptors. The effect of light stress and protection with DMTU were determined by quantifying the number of live photoreceptors using fluorescence imaging techniques. TUNEL labeling was used to detect apoptotic nuclei after light exposure.

Results: : Blue actinic light exposure induced 60–90% photoreceptor cell death in various light exposure paradigms tested. DMTU protected photoreceptors against blue light stress in a concentration–dependent fashion in both bovine and primate cell cultures. The EC₅₀ in both bovine and primate models were around 100–300 µM. TUNEL staining of photoreceptor nuclei increased under the influence of blue light. DMTU reduced the number of TUNEL positive cells subjected to light stress.

Conclusions: : DMTU protection of photoreceptors confirms that blue light causes oxidative stress in retinal photoreceptors culture model. Agreement of the in–vitro results with published in–vivo observations demonstrates the utility of photoreceptor culture model for the discovery of novel retinal protective molecules for AMD.

Keywords: age-related macular degeneration • radiation damage: light/UV • photoreceptors