PURPOSE. To investigate the cellular mechanisms involved in the cell death of human retinal pigment epithelial (hRPE) cells after their exposure to laser injury. METHODS. Cultured human hRPE cells were irradiated for different lengths of time and at different levels of energy using diode laser photocoagulation coupled with an intraocular laser probe. Apoptosis was determined by TUNEL staining and annexin-V labeling of phosphatidylserine exposure. Ceramide levels were quantified by the diacylglycerol kinase assay using thin-layer chromatography. RESULTS. Laser irradiation caused areas of apoptosis in the hRPE cells. These areas were detected around the ablated and necrotic laser scar and developed several hours after the laser irradiation. Laser irradiation concomitantly induced an increase in the intracellular production of ceramide, a lipid second messenger. CONCLUSIONS. The results demonstrate that laser irradiation induces apoptosis in hRPE cells and suggest that the underlying signaling mechanism involves ceramide generation.
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