Differential autophagic effects of vital dyes in retinal pigment epithelial ARPE-19 and photoreceptor 661W cells.

Indocyanine green (ICG) and brilliant blue G (BBG) are commonly used vital dyes to remove internal limiting membrane (ILM) in vitreoretinal surgery. The vital dyes have shown cytotoxic effects in ocular cells. Autophagy is a stress responsive pathway for either protecting cells or promoting cell death. However, the role of autophagy in ocular cells in response to the vital dyes remains unknown. In this study, we found that ICG and BBG reduced cell viability in both human retinal pigment epithelial ARPE-19 and mouse photoreceptor 661W cells. ICG and BBG induced lipidated GFP-LC3-II and LC3-II in ARPE-19 and 661W cells. Combination treatment with the autophagy inhibitor chloroquine indicated that ICG and BBG reduced autophagic flux in ARPE-19 cells, whereas the vital dyes induced autophagic flux in 661W cells. Moreover, genetic and pharmacological ablation of autophagy enhanced vital dyes-induced cytotoxicity in ocular cells. Dietary supplements, including resveratrol, lutein, and CoQ10, induced autophagy and diminished the cytotoxic effects of ICG and BBG in ocular cells. These results suggest that autophagy may protect ARPE-19 and 661W cells from vital dyes-induced damage.

Protective effect of a laser-induced sub-lethal temperature rise on RPE cells from oxidative stress.

Recently introduced new technologies that enable temperature-controlled laser irradiation on the RPE allowed us to investigate temperature-resolved RPE cell responses. In this study we aimed primarily to establish an experimental setup that can realize laser irradiation on RPE cell culture with the similar temperature distribution as in the clinical application, with a precise time/temperature history. With this setup, we conducted investigations to elucidate the temperature-dependent RPE cell biochemical responses and the effect of transient hyperthermia on the responses of RPE cells to the secondary-exposed oxidative stress. Porcine RPE cells cultivated in a culture dish (inner diameter = 30 mm) with culture medium were used, on which laser radiation (λ = 1940 nm, spot diameter = 30 mm) over 10 s was applied as a heat source. The irradiation provides a radially decreasing temperature profile which is close to a Gaussian shape with the highest temperature in the center. Power setting for irradiation was determined such that the peak temperature (Tmax) in the center of the laser spot at the cells reaches from 40 °C to 58 °C (40, 43, 46, 50, 58 °C). Cell viability was investigated with ethidium homodimer III staining at the time points of 3 and 24 h following laser irradiation. Twenty four hours after laser irradiation the cells were exposed to hydrogen peroxide (H2O2) for 5 h, followed by the measurement of intracellular glutathione, intracellular 4-hydroxynonenal (HNE) protein adducts, and secreted vascular endothelial growth factor (VEGF). The mean temperature threshold for RPE cell death after 3 h was found to be around 52 °C, and for 24 h around 50 °C with the current irradiation setting. A sub-lethal preconditioning on Tmax = 43 °C significantly induced the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, and decreased H2O2-induced increase of intracellular 4-HNE protein adducts. Although sub-lethal hyperthermia (Tmax = 40 °C, 43 °C, and 46 °C) caused a slight increase of VEGF secretion in 6 h directly following irradiation, secondary exposed H2O2-induced VEGF secretion was significantly reduced in the sub-lethally preheated groups, where the largest effect was seen following the irradiation with Tmax = 43 °C. In summary, the current results suggest that sub-lethal thermal laser irradiation on the RPE at Tmax = 43 °C for 10 s enhances cell defense system against oxidative stress, with increasing the GSH/GSSG ratio. Together with the results that the decreased amount of H2O2-induced 4-HNE in sub-lethally preheated RPE cells was accompanied by the lower secretion of VEGF, it is also strongly suggested that the sub-lethal hyperthermia may modify RPE cell functionality to protect RPE cells from oxidative stress and associated functional decrease, which are considered to play a significant role in the pathogenesis of age-related macular degeneration and other chorioretinal degenerative diseases.

Selective retina therapy (SRT) of chronic subfoveal fluid after surgery of rhegmatogenous retinal detachment: three case reports.

BACKGROUND: Shallow subfoveal fluid accumulation after successful surgery for retinal detachment can be the reason for compromised visual acuity. To date, therapeutical options to tackle this problem have not been established. Selective retina therapy (SRT) is a new laser technology that uses a train of mus-laser pulses to selectively damage retinal pigment epithelial (RPE) cells while sparing retinal structures. METHODS: We treated three patients with chronic subfoveal fluid accumulation after retinal detachment surgery. The median period between retinal surgery and SRT treatment was 7 months. For SRT, we used a prototype frequency-doubled, Q-switched Nd:YLF laser (lambda = 527 nm). Each laser exposition contained 30 pulses (t = 1,7 micros, 100 Hz, E = 100-400 microJ). Two of the three patients were treated subfoveally. OCT III (optical coherence tomography) examinations were performed to evaluate changes in subretinal fluid accumulation. RESULTS: In all three patients, we observed complete resolution of subfoveal fluid within 1-5 months. Follow-up has been 16 months to 2 years. Visual acuity improved in all patients. In one patient, cystoid macular edema developed 3 months after treatment. Additional SRT treatments were not necessary. CONCLUSION: SRT is a safe treatment. Visual acuity improved after SRT, even in subfoveal irradiations. SRT is an option to support subretinal fluid reabsorption. In this situation where no other therapeutical options are established, SRT may be a beneficial treatment for chronic subfoveal fluid accumulation after retinal detachment surgery.