June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Fluorescence lifetime imaging microscopy on retinal microvascular endothelial cells under high glucose conditions
Author Affiliations & Notes
  • Yoko Miura
    Institute of Biomedical Optics, University of Luebeck, Luebeck, Germany
    Department of Ophthalmology, University Hospital Schleswig-Holstein, Luebeck, Germany
  • Eric Beck
    Institute of Biomedical Optics, University of Luebeck, Luebeck, Germany
  • Gereon Huttmann
    Institute of Biomedical Optics, University of Luebeck, Luebeck, Germany
  • Sayon Roy
    Department of Medicine and Ophthalmology, Boston University School of Medicine, Boston, MA
  • Ralf Brinkmann
    Institute of Biomedical Optics, University of Luebeck, Luebeck, Germany
    Medical Laser Center Luebeck, Luebeck, Germany
  • Footnotes
    Commercial Relationships Yoko Miura, None; Eric Beck, None; Gereon Huttmann, None; Sayon Roy, None; Ralf Brinkmann, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5943. doi:
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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)

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Abstract

Purpose: 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.

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.

Results: 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.

Conclusions: 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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