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P. Tababat-Khani, C.-D. Agardh, M.-L. Smith, E. Agardh; In vitro Photocoagulation of Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5606.
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Photocoagulation therapy is routinely used as treatment for diabetic macular edema and proliferative retinopathy, but the exact mechanisms involved in the therapeutic benefits are still poorly understood. Since photocoagulation affects the retinal pigment epithelium (RPE), we postulate that the tissue response induced by laser can be involved in the therapeutic outcome. The aim of this study was therefore to establish a model for photocoagulation of human RPE cells in vitro and to examine their response in terms of morphology, apoptosis, proliferation and VEGF-A gene expression.
Human RPE cells (ARPE-19) were grown to confluence on glass cover slips and exposed to photocoagulation (200 µm spot size, 300 mW, 0.1 ms). The morphological damage and regrowth were documented by conventional light microscope photography after fixation at 30 min, 2h, 6h, 12h, 24h, 48h, 72h and seven days after photocoagulation. Apoptosis and subsequent proliferation were examined using ELISA-assays. mRNA levels of VEGF-A were measured using quantitative RT-PCR.
Photocoagulation of RPE cells resulted in morphological cell damage, regrowth (at 6h - 12h) and loss of cellular nuclei at 24h. Cells invaded the wound by 24h - 48h which was completely healed after seven days. Laser induced apoptosis (4-fold increase by 48h, 8-fold increase by seven days compared to non-treated cells, p<0.001 for both time points) and a proliferative response (2-fold increase by 72h, p<0.05). VEGF-A mRNA levels decreased to less than half within 24h after laser coagulation (p=0.001).
In vitro photocoagulation resulted in instant morphological damage of RPE cells followed by apoptosis and subsequent proliferation, supporting the morphologically observed regrowth. Photocoagulation inhibited VEGF-A mRNA expression within 24h. As VEGF-A is a potent angiogenic factor, its downregulation might play a role in the regression of vascular leakage and newformed vessels seen after photocoagulation treatment for diabetic macular edema and proliferative retinopathy. Our model can serve for future studies of RPE cell reactions to photocoagulation during e.g. hyperglycemic conditions.
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