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Svenja Rebecca Sonntag, Alessa Hutfilz, Britta Lewke, Dirk Theisen-Kunde, Ralf Brinkmann, Salvatore Grisanti, Yoko Miura; Extensive impact of selective retina treatment on RPE metabolism: insights from fluorescence lifetime measurement. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6079.
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© ARVO (1962-2015); The Authors (2016-present)
Aim of this study was to investigate whether laser irradiation on retinal pigment epithelium (RPE) has an influence on the fluorescence lifetime (FLT) of RPE cells in peripheral untreated areas.
Porcine RPE-choroid-sclera explants were treated by selective retina treatment (SRT; wavelength: 527 nm, beam diameter: 200 µm). Two rows of laser spots with a pulse energy of 110 µJ were applied on the RPE. At 24h and 72h after irradiation the FLT of the RPE at three different zones, which were defined with the distance from the row of laser spots (zone 1: 400-1200 µm, zone 2: 1200-2000 µm, zone 3: 2000-2800 µm, area size of each zone: 800 x 4000 µm), was measured with fluorescence lifetime imaging ophthalmoscopy (FLIO: excitation wavelength: 473 nm, emission channels: channel (Ch1) 1: 498-560 nm, Ch2: 560-720 nm). The cell defect directly after irradiation was examined with calcein-AM viability test, and cell morphology was observed with actin staining using FITC-phalloidin.
The diameter (mean ± standard deviation) of RPE defect through the irradiation of SRT was 169.0 ± 9.30 µm. F-actin staining could confirm the wound healing during the observation time period (72h). Autofluorescence intensity images showed no visible differences, whereas the FLIO images clearly revealed a significant elongation of FLT (τm: mean FLT) in the peripheral zones after 24h, which was apparent in Ch1 (τm= 328.4 ± 49.1 ps, 319.2 ± 54.9 ps, and 302.1 ± 54.9 ps at zone 1, 2, and 3, respectively, compared to the baseline 261.1 ± 44.8 ps). After 72h the significance was seen still in the nearest zone to the laser spots (zone 1) (p= 325.2 ± 58.9 ps compared to the baseline 267.6 ± 44.5 ps).The alterations in FLT were significant in Ch1, which might be associated with the molecular state of a fluorescent metabolic cofactor, flavin adenine dinucleotide (FAD), as its fluorescence peak lies in Ch1 and its protein-binding state may change its FLT under metabolic alteration.
FLIO may be able to show the metabolic activation of the RPE through laser treatment. Our results suggest that SRT might influence the function of the RPE cells in wider area than the irradiated sites.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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