September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Ex-vivo assessment of metabolic alterations of retinal pigment epithelial cells using Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO)
Author Affiliations & Notes
  • Natalie Blimke
    Medical Lasercenter Luebeck, Luebeck, Germany
  • Joachim Pruessner
    Medical Lasercenter Luebeck, Luebeck, Germany
  • Lars Alt
    Medical Lasercenter Luebeck, Luebeck, Germany
  • Gereon Huttmann
    Institut for Biomedical Optics, Luebeck, Germany
  • Ralf Brinkmann
    Medical Lasercenter Luebeck, Luebeck, Germany
    Institut for Biomedical Optics, Luebeck, Germany
  • Yoko Miura
    Institut for Biomedical Optics, Luebeck, Germany
    Medical Lasercenter Luebeck, Luebeck, Germany
  • Footnotes
    Commercial Relationships   Natalie Blimke, None; Joachim Pruessner, None; Lars Alt, None; Gereon Huttmann, None; Ralf Brinkmann, None; Yoko Miura, None
  • Footnotes
    Support  Heidelberg Engineering GmbH
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 253. doi:
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      Natalie Blimke, Joachim Pruessner, Lars Alt, Gereon Huttmann, Ralf Brinkmann, Yoko Miura; Ex-vivo assessment of metabolic alterations of retinal pigment epithelial cells using Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO). Invest. Ophthalmol. Vis. Sci. 2016;57(12):253.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : Fluorescence lifetime (FLT) decays of endogenous fluorophores in the fundus is becoming of more interest as a non-invasive and non-labeling indicator of retinal metabolic status. The aim of this study is to investigate changes of FLT of retinal pigment epithelial (RPE) cells in static organ culture over time and its relevance to cell metabolic status.

Methods : Porcine RPE-choroid-sclera explants were cultivated in static culture medium. At 2, 24 and 48h after the initiation of cultivation the FLT of the RPE was measured using a fluorescence lifetime imaging ophthalmoscope (FLIO: Heidelberg Engineering) with 80 MHz pulsed excitation at 473 nm and two emission spectral channels (channel 1: 498-560 nm, channel 2: 560-700nm) coupled with a time-correlated single photon counting system. A custom-made artificial eye model mimicking optical characteristics of the human eye served as a measuring chamber for the explants. RPE cell viability was examined using calcein-AM staining, and cellular glucose uptake was estimated by measuring glucose concentration of the conditioned medium using GlucCell® glucose meter.

Results : Calcein-positive living RPE cells in the culture was found in 99.4% ± 0.27 at 48h, which was not significantly different from at 2h. There was an increase in cellular glucose uptake over time, from 48.0 ± 14.2 mg/mm2/h at 2h to 56.2 ± 17.2 mg/mm2/h at 24h, and 56.3 ± 17.3 mg/mm2/h at 48h (p=0.035). In FLIO, the FLT decay curve of the RPE was well fitted to a biexponential curve. The t2 lifetime (longer component) in channel 2 was significantly longer at 24h and 48h than at 2h (2308 ± 76 ps at 2h, 2473 ± 90 ps at 24h (p=0.006), and 2430 ± 103 ps at 48h (P = 0.017)). The ratio of the amplitude of t1 and t2 (a1/a2) in channel 2 also showed a significant decrease over time, 12.4 ± 3.2 at 2h, 11.0 ± 1.7 at 24h, and 9.8 ± 1.4 at 48h (p=0.04 vs 2h).

Conclusions : Results suggest that FLIO might be useful to investigate metabolic alterations of the ex-vivo RPE. The change of cell energy metabolisms which increases glucose uptake might result in the increase of the length of long lifetime component and its amplitude. Further investigations will provide insight into its clinical use, e.g. in evaluating cell health and metabolic activity of the in-vivo RPE, or of the RPE sheet for RPE cell transplantation therapy.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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