Abstract
Purpose: :
Obtain in vitro laser damage thresholds for several exposure conditions and make comparisons to existing in vivo data.
Methods: :
Previously described artificially pigmented hTERT–RPE1 cells were used to determine irradiance thresholds for exposure to lasers of four wavelengths (532 nm, 514 nm, 458 nm, or 413 nm) for durations of 0.1 s, 1 s, 10 s, and 100 s. Threshold endpoint was based on a fluorescent dye assay (cellular loss of calcein AM and appearance of ethidium homodimer1) after one hr post–exposure recovery in culture incubator. Binary data (yes/no) for a broad range of laser irradiances for each wavelength/time parameter were assessed for an ED50 using Probit analysis.
Results: :
When threshold radiant exposure values were plotted with respect to exposure duration, straight lines extended over the 0.1 s to 10 s range for all wavelengths tested, indicating a photothermal damage mechanism. The lines for the two shortest laser wavelengths leveled off between 10 s and 100 s exposure durations (principle of reciprocity), indicating a photochemical damage mechanism. These trends have similarity to trends found in the literature for animal studies. Effects of varying intracellular melanosome density on absolute threshold and trends with respect to wavelength and exposure duration were also studied.
Conclusions: :
Our in vitro system of analysis serves as a good laser–damage model, providing data with similarities to data taken from in vivo ocular studies.
Keywords: laser • apoptosis/cell death • retinal pigment epithelium