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Yoko Miura, Eric Beck, Gereon Huttmann, Sayon Roy, Ralf Brinkmann; Fluorescence lifetime imaging microscopy on retinal microvascular endothelial cells under high glucose conditions. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5943.
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© ARVO (1962-2015); The Authors (2016-present)
High glucose-induced metabolic change in retinal endothelial cells has been suggested to precede the morphological alterations in the pathogenesis of diabetic retinopathy. Fluorescence lifetime imaging microscopy (FLIM) is a new imaging technology, which could be useful to detect cell metabolic changes by measuring the fluorescence lifetime (FLT) of nicotinamide adenine dinucleotide (NADH) autofluorescence. This method utilizes the difference in the FLT of free- and protein-bound-NADH. In this study, we conducted in-vitro NADH FLIM on retinal microvascular endothelial cells under high glucose condition, comparing with the different cell biological status assessed with other experimental methods.
Cultured human retinal microvascular endothelial cells (HRMEC) were used in the study. Cells were incubated in normal (4.5 mM) or high (30 mM) concentration of glucose for 60 hrs, followed by examination with FLIM combined with two-photon microscopy (TPM; λex=730nm). Amount of intracellular reactive oxygen species (ROS) was estimated with 2‘,7’- dichlorodihydrofluorescein diacetate (DCFH-DA)-assay, and the protein expression of vascular cell adhesion molecule-1 (VCAM-1) was investigated with Wwestern blotting.
High glucose condition significantly increased intracellular ROS (30%) and VCAM-1 protein (150%) expression. FLIM results demonstrated the significant increase of the fluorescence lifetime of cultured HRMEC (τ1: 684 ps to 693 ps, τ2: 3163 ps to 3294 ps, τm: 1087 ps to 1126 ps) and the significant decrease in the ratio of the amplitude of shorter and longer lifetime component, which is suggested to indicate the ratio of the amount of free- to protein-bound NADH (a1/a2; whole cells: 7.64 to 6.84, mitochondria: 4.37 to 3.94, nucleus: 12.08 to 8.09) in the cells under high glucose conditions.
The FLIM results suggest that metabolic changes could be documented with TPM-FLIM in living HRMEC undergoing high glucose-induced oxidative stress. TPM-FLIM might be a useful method to detect intracellular oxidative stress and related metabolic changes in retinal vascular cells.
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