Abstract
Purpose :
Decreased metabolic activity is an essential factor in degenerative cellular changes. Fluorescence lifetime (FLT) measurement was introduced as a novel diagnostic tool to detect metabolic changes in the fundus. Serum deprivation has been shown to lead to age-related macular degeneration causing AMD-like changes in cultured retinal pigment epithelial (RPE) cells. In this study, we aim to show that the fluorescence lifetime imaging microscopy (FLIM) can be helpful in detecting early metabolic changes in RPE cells under serum-deprivation.
Methods :
ARPE-19 cells were cultured in Dulbecco’s modified Eagle’s Medium with normal (10%) or deprived (1%) fetal calf serum (FCS). The mitochondrial respiratory function was assesd using the Seahorse XFe24 Analyzer. Furthermore, we assessed the both groups using FLIM, where FLT of ARPE-19 cells was measured over 14 days in two separate channels; one detecting primarily autofluorescence of nicotinamide adenin dinucleotide (NADH), and another channel, detecting autofluorescence of flavin adenin dinucleotide (FAD) and flavin mononucleotide (FMN) in cells.
Results :
Mitostress assay revealed that serum-deprivation significantly reduced maximal respiration of ARPE-19 cells compared with those under normal serum condition. In FLIM, the mean lifetime τm of RPE cells in the NADH channel was significantly shorter under serum-deprived conditions compared to normal serum conditions after 8 days, and this difference persisted until 14 days: 1072.9 ± 39.4 ps (serum-deprived) vs. 1152.3 ± 25.3 ps (normal serum conditions) on day 14 (p<0.05). In contrast, the FAD channel showed a significantly prolonged τm under serum deprivation: 1117.6 ± 23 ps (serum deprived, day 14) vs. 890.3 ± 36.9 ps (normal serum conditions, day 14).
Conclusions :
Serum-deprivation was shown to have significant impacts on mitochondrial function and cellular FLT in RPE cells. FLIM results indicated that the metabolic states of both NADH and FAD/FMN were significantly altered. The recently introduced fluorescence lifetime imaging opthalmoscopy (FLIO) uses the same excitation and detection wavelength range as the FAD/FMN channel in FLIM used in the current study. These results suggest that FLIO may detect the changes in the state of FAD/FMN due to altered mitochondrial function of RPE and retinal cells in degenerative retinal disease such as AMD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.