Laser–Induced Injury to ARPE–19 Cells: Molecular and Cytoprotective Evaluation by Preindcution of the Heat Shock Response

Abstract

Abstract: : Purpose: To improve care for laser–induced retinal injury, a better understanding of this injury is required along with new approaches to treatment Methods: ARPE–19 cells derived from human retinal pigmented epithelium, were cultured to 95% confluence in either 35 mm dia 6–well or 16 mm 24–well multiplates. A carbon dioxide laser (10.6 um, beam dia 200 um) with output at 2W was scanned over the monolayer with a 95% overlap raster (0.16mm scan spacing) over a 6–15s scan rate for16 mm dia wells and a 20–30s range for 35 mm dia wells. For induction of the 70 kDa heat shock protein (HSP70), cells were exposed to 55^oC for 3s by dipping into heated saline (0.9%), laser irradiated for 8s at 2W or treated for 6h with 17–(Allylamino)–17–demethoxygeldanamycin (17AAG; 0.1 ug/ml) a known heat shock inducer. A 6 h incubation period for development of the heat shock response was followed by lethal injury for untreated cells by heating in saline at 55^oC for 9s or laser irradiated for 11s at 2W. Western blotting, gene expression analysis with microarrays, and RT–PCR were used to evaluate genes and proteins in common between injury and treatment processes. Cell viability was assessed by MTT staining or cell counting. Results: A uniform injury occurred after heated saline and laser exposure with scanning of the beam across the monolayer thus enabling comparison of thermal injury induced by heat or laser exposure by biochemical analysis. Pretreatment with heat for 3s at 55^oC, 8s at 2W laser exposure, or 17–AAG for induction of a HSP70 response prevented death from 9s/55,^oC saline or 11s/2W laser exposure. Immunocytochemical detection of western blots and genomic analysis with microarrays confirmed that HSP70 expression was elevated in both heated saline and laser treatments, suggesting that thermal effects occurred in both processes. Also, HSP70 was elevated in both thermal and pharmacologic treatments. Conclusions: Induction of HSP70 by heated saline, laser exposure, and 17AAG prevented lethal injury from both heated saline and laser exposure. Gene expression analysis indicated minor differences between heated saline and laser exposure, suggesting that both processes are related to thermal effects and that survival is related to HSP70 and related gene induction. This approach can also be applied to other types of lasers for understanding their effects on the retina and for developing treatments.

Keywords: laser • candidate gene analysis • pharmacology