July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
In vivo analysis reveals a switch in metabolism and oxidative state of zebrafish eyes during development
Author Affiliations & Notes
  • Andrew W Browne
    Ophthalmology, University of California Irvine, Irvine, California, United States
    Biomedical Engineering, University of California Irvine, Irvine, California, United States
  • Yuntian Xue
    Biomedical Engineering, University of California Irvine, Irvine, California, United States
  • Tej Kalakuntla
    Biomedical Engineering, University of California Irvine, Irvine, California, United States
  • William Tang
    Biomedical Engineering, University of California Irvine, Irvine, California, United States
  • Leonel Malacrida
    Biomedical Engineering, University of California Irvine, Irvine, California, United States
    Facultad de Medicina, 2Área de Investigación Respiratoria, Departamento de Fisiopatología, Hospital de Clínicas, Facultad de Medicina, Uruguay
  • Thomas F Schilling
    Developmental Biology, University of California Irvine, Irvine, California, United States
  • Irene Vorontsova
    Cell Physiology and Biophysics, University of California, Irvine, California, United States
    Developmental Biology, University of California Irvine, Irvine, California, United States
  • Footnotes
    Commercial Relationships   Andrew Browne, None; Yuntian Xue, None; Tej Kalakuntla, None; William Tang, None; Leonel Malacrida, None; Thomas Schilling, None; Irene Vorontsova, None
  • Footnotes
    Support  KL2 TR001416
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 6085. doi:https://doi.org/
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      Andrew W Browne, Yuntian Xue, Tej Kalakuntla, William Tang, Leonel Malacrida, Thomas F Schilling, Irene Vorontsova; In vivo analysis reveals a switch in metabolism and oxidative state of zebrafish eyes during development. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6085. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Metabolic and oxidative states fluctuate during normal organogenesis and tissue maturation, disruption of which can lead to disease. However, methods for observing these fluctuations in vivo have been lacking. We employed Fluorescence Lifetime Imaging Microscopy (FLIM) using non-invasive, 2-photon excitation to study intrinsic oxidative versus glycolytic metabolic and oxidative states in the eyes of living zebrafish (Danio rerio) embryos. At 2 dpf the developing lens is a ball of secondary fiber cells surrounded by the lens epithelium. At 3 dpf lens nuclear fiber cells have compacted and matured, lost their organelles, and all 5 major retinal cell types are formed.

Methods : Zebrafish “crystal” fish, which are triple mutants for nacrew2/w2;albb4/b4 and roya9/a9, lack melanin, melanophores and iridophores, resulting in a transparent retinal pigment epithelium that allows optical access for in vivo imaging. Embryos were anesthetized, mounted in agarose and eyes were imaged using FLIM with 740 nm excitation. Optical slices of the eye were collected along the optical axis and data was analyzed using the phasor plot approach. To assess the free-to-bound NADH ratio (indicating metabolic state), and existence of long lifetime species (LLS, which indicates oxidative stress) from lens and retina we used a three components analysis. Masking enabled signal isolation from each anatomical region.

Results : An increase in free NADH and decrease in the oxidative state of oleic acid (decreased LLS ratio), was observed at 3 dpf compared to 2 dpf in both the lens and retina. Structural changes on high resolution imaging mirror published histology.

Conclusions : The shift from primarily oxidative phosphorylation to glycolytic metabolism in the lens likely reflects the degradation of mitochondria in mature lens fiber cells, which cannot undergo oxidative phosphorylation. A shift to glycolytic metabolism in the retina may reflect the high metabolic demand of newly formed photoreceptors at 3 dpf. The decrease in oxidation at 3 dpf compared to 2 dpf is consistent with previously published HPLC analysis of whole zebrafish embryos at these stages. This is the first non-invasive analysis of live zebrafish eyes revealing dramatic changes in metabolism and oxidative states during key developmental stages.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

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