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M. L. Denton, G. D. Noojin, M. S. Foltz, L. E. Estlack, R. J. Thomas, B. A. Rockwell; Laser Protection and Sensitization of an in vitro Retinal Model Depends Upon the Order of Artificial Pigmentation and Hyperthermia Pretreatment. Invest. Ophthalmol. Vis. Sci. 2009;50(13):208.
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To study the effects of mild hyperthermia preconditioning on the susceptibility of retinal pigment epithelial (RPE) cells to laser damage at 514 nm.
An existing artificially pigmented in vitro retinal model (Denton et al., 2008, J. Biomed. Opt., 13(5):054014) was used to identify protection or enhancement of laser-induced damage 18 hr after mild hyperthermia preconditioning (PC) treatment. Control and experimental RPE cells were exposed (16 replicates of the same irradiance) to a 0.93-mm diameter laser beam (514-nm line from an argon laser) for 0.25 s. Area of damage was assessed using a live-dead fluorescence indicator method 1-hr post exposure. The hyperthermia pretreatment consisted of a 20-min ramp up from 37 ºC to 42 ºC, an additional 20 min at 42 ºC, followed by a quick ramp down to 37 ºC. In addition to the PC of RPE cells after artificial pigmentation, RPE cells and melanosome particles (MPs) were PC separately and added together 1 hr later. Finally, PC cells without pigment were pigmented with normal MPs. For each exposure, corresponding fluorescence and IR images (high-speed high-magnification) were spatially overlaid with each other to identify thermal history of cells at the boundary of death.
Combining the results of all 16 replicates, area of laser damage was summed and compared to control exposures. Cells that were PC after pigmentation had a 5.25 fold increase in damaged area. Cells PC before addition of adding normal MPs had no difference in laser-induced damage area. When cells and MPs were PC separately, but added later, they had markedly less damaged area after laser exposure. In all 4 sample types, the average temperature the cells at the boundary of death experienced was the same, indicating the integrated temperature-time product for cell death was unaltered.
The location of MPs during hyperthermia determines whether pigmented RPE cells were resistant or sensitized to subsequent laser damage at 514 nm. The PC appears to alter the photochemistry and/or the absorption properties of the MPs.
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