A Search for Preconditioning Laser Irradiances Capable of Producing a Protective Adaptive Response in RPE Cells

Abstract

Purpose:: To search for photochemical preconditioning conditions (laser irradiances) in RPE cells that lead to protection against subsequent photochemical damage.

Methods:: Pigmented hTERT-RPE1 cells were used to determine an ED₅₀ irradiance threshold for damage by a photochemical laser exposure (0.3 mm, 100 s, 413 nm). Binary (yes/no) damage endpoints, based on a fluorescent dye assay (calcein-AM and ethidium homodimer1) one hr post-exposure, were used as input to the Probit computer program. This threshold value served as a reference point for damaging exposures (challenge) in preconditioning experiments. Sublethal preconditioning exposures (2.5 mm, 100s, 413 nm) were applied to RPE cells at predetermined time intervals prior to a suprathreshold challenge exposure (0.3 mm, 100 s, 413 nm). Two types of damage assessment were used to determine if preconditioning caused a protective adaptive response.

Results:: Threshold ED₅₀ values for 0.3 mm and 2.5 mm laser exposures (100 s at 413 nm) were 2.3 fold different from each other. The use of a large beam for preconditioning and a small beam for challenging, along with meticulous alignment procedures, reduced uncertainty regarding challenge exposures occurring outside of preconditioned regions in culture dishes. Using both a Probit and a Fractional Damage Area approach to damage assessment, no measurable protective effect was found over a broad range of preconditioning (more than 4 orders of magnitude) and challenging (3 fold at, or above ED₅₀) irradiances.

Conclusions:: Preconditioning with photochemical laser exposure is either insufficient in producing a measurable protective response against photochemical damage in pigmented RPE cells, or the effective range of preconditioning laser irradiances is very narrow or extraordinarily low.

Keywords: laser • oxidation/oxidative or free radical damage • cell survival