June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Two Photon, Label-free Imaging of Mitochondrial Dysfunction in Retinal Ganglion Cells
Author Affiliations & Notes
  • Jasmine Sum-Yee Yung
    Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong
  • Heather Kayew Mak
    Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong
  • Heidi Ng
    Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong
  • Xu Cao
    Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong
  • Christopher Kai-Shun Leung
    Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong
  • Footnotes
    Commercial Relationships   Jasmine Yung, None; Heather Mak, None; Heidi Ng, None; Xu Cao, None; Christopher Leung, Alcon (R), Alcon (C), Allergan (R), Allergan (C), Carl Zeiss (F), Carl Zeiss Meditec (P), Glaukos (F), Global Vision (R), Lumenis (R), Merck (R), Novartis (R), Oculus (F), Optovue (F), Santen (R), Tomey (F), Tomey (R), Topcon (F)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1611. doi:
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      Jasmine Sum-Yee Yung, Heather Kayew Mak, Heidi Ng, Xu Cao, Christopher Kai-Shun Leung; Two Photon, Label-free Imaging of Mitochondrial Dysfunction in Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1611.

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

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Abstract

Purpose : While imaging and measurement of the retinal nerve fiber layer thickness is a prevailing method to detect and monitor optic nerve degeneration in clinical practice, the lack of quantitative intravital imaging precludes the characterization of metabolic function of retinal ganglion cells (RGCs). Mitochondrial nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent electron carriers in the electron transport chain which play a pivotal role in maintaining the metabolic function of neurons. With two-photon imaging, we measured the levels of NADH and FAD, quantified the redox ratio FAD/(FAD+NADH), and investigated their changes in RGCs after axon injury.

Methods : NADH and FAD were excited at 720-740nm and 900nm, and detected at 420-510nm and 520-620nm, respectively. The distribution of mitochondrial NADH in HeLa and cultured RGCs isolated from P6 SD rats RGCs was confirmed with colocalization with mitochondrial markers Rh123 and TMRM. Changes of the NADH and FAD levels were quantified in rat RGCs before and after laser-induced axonal injury (n=5) and in retinal explants after optic nerve crush (n=6). Redox ratio of individual RGC was calculated by the ratio of fluorescent intensity of FAD/(FAD+NADH). Apoptosis was detected by AnnexinV and propidium iodide.

Results : Compartment specific NADH colocalized with mitochondrial markers in HeLa cells and RGCs. After laser-induced injury, a 0.51 ± 0.16-fold decrease in NADH fluorescence and a 0.58 ± 0.32-fold increase in NADH-FAD redox ratio was detected at 30min (Fig 1) which preceded progressive retraction of proximal axon and induction of apoptosis. In the retinal explants, the average NADH level decreased by 0.27 ± 0.2-fold while FAD level increased by 1.73 ± 0.64-fold 3 days after optic nerve crush compared with controls. Areas with redox ratio above 0.6 were PI-positive.

Conclusions : Two-photon redox imaging of NADH and FAD facilitates non-invasive, label-free interrogation of mitochondrial function in RGCs. An increase in the redox ratio likely represents an early biomarker of metabolic stress in RGCs. Tracking the FAD/FAD+NADH redox ratio can provide a novel approach for early detection and monitoring of RGC degeneration.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Fig 1. Redox imaging of a P6 rat RGC after laser-induced injury. An increase in redox ratio FAD/(FAD+NADH) preceded the progressive axonal retraction after laser-induced injury.

Fig 1. Redox imaging of a P6 rat RGC after laser-induced injury. An increase in redox ratio FAD/(FAD+NADH) preceded the progressive axonal retraction after laser-induced injury.

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