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E. Gaillard, C. Hill, t.D. Griffiths; UVC and Visible Light Damage to Re-Pigmented RPE Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):377.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The native RPE contains a monolayer of heavily pigmented cells. The melanin contained in these cells is thought to protect the cells from photochemical damage as well as damage from oxidative stress. When RPE cells are grown in cell culture, however, they rapidly lose their melanin content. We have carried out a study to determine the photoprotection afforded to RPE cells re-pigmented with melanin in tissue culture conditions when exposed to UVC and visible light. The variation in photoprotection with the age of the melanin has also been studied. Methods: Primary RPE cells were obtained from fresh calf eyes (Brown Packaging, S. Holland, IL). The cells were plated in multi-well tissue culture dishes that had been coated with Matrigel (BD Biosciences) to promote differentiation and grown at 37oC, 2% CO2 until confluent. The cultures were re-fed every three days with DMEM + 10% FBS and antibiotics. The cultures were passaged using dispase. De-pigmented cells were fed either isolated calf RPE melanin or human melanin from donor eyes of different ages. The cultures were irradiated with either an in house built UVC irradiation chamber or with a solar simulator (Full Spectrum Solutions). Melanin content was assessed by absorption spectroscopy. Results: Cells that were re-pigmented with calf melanin (as a model for young human RPE melanin) survived exposure to 20 J/m2 UVC better than cells that were re-pigmented with 70 year old human melanin (7% and 15% cell death respectively, same background values, 48 hours after exposure). Both the rates of apoptosis and necrosis after exposure to UVC were lower for the calf melanin re-pigmented cells versus human melanin re-pigmented cells (3% and 6% apoptosis, respectively, 48 hours after exposure). Matched controls grown on tissue culture plastic always sustained much more significant cell death after exposure to UVC than the counterpart cultures grown on Matrigel. Under conditions of visible light exposure, the assays suggest that older human RPE melanin actually contributes to photooxidative stress and cell death. Conclusions: RPE cells that have been re-pigmented with calf melanin survive exposure to UVC more than two times the survival of RPE cells that have been re-pigmented with older human RPE melanin. Interestingly, maintaining the cells on extracellular matrix material appears to have a protective effect against UVC insult. Human melanin appears to increase cell death and photooxidative stress to RPE cells when exposed to solar simulating radiation. These data may further our understanding of the molecular mechanisms involved in light damage to the human retina.
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