September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
A novel, safe, non-invasive method to detect dying photoreceptors in the living rat
Author Affiliations & Notes
  • Francesca Mazzoni
    Department of Biological Sciences, Center for Cancer, Genetic Diseases and Gene Regulation, Fordham University, Bronx, New York, United States
  • Claudia Müller
    Department of Biological Sciences, Center for Cancer, Genetic Diseases and Gene Regulation, Fordham University, Bronx, New York, United States
  • Silvia C Finnemann
    Department of Biological Sciences, Center for Cancer, Genetic Diseases and Gene Regulation, Fordham University, Bronx, New York, United States
  • Footnotes
    Commercial Relationships   Francesca Mazzoni, None; Claudia Müller, None; Silvia Finnemann, None
  • Footnotes
    Support  Beckman Initiative for Macular Research Grant #1306 by The Arnold and Mabel Beckman Foundation
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Francesca Mazzoni, Claudia Müller, Silvia C Finnemann; A novel, safe, non-invasive method to detect dying photoreceptors in the living rat. Invest. Ophthalmol. Vis. Sci. 201657(12):.

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

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Abstract

Purpose : Photoreceptor loss accompanies many forms of retinal degeneration. Earlier studies of animal models of degeneration or human patient tissues suggest that forms of programmed cell death/apoptosis are common mechanisms of retinal cell death in blinding diseases. Phosphatidylserine (PS) plasma membrane lipid externalization occurs early during apoptosis. Fluorescent PS-binding reagents (PSR) can mark PS exposure in live cells and tissues ex situ. We tested if PSR in eye drop formulations can detect dying photoreceptors in live experimental animals.

Methods : Experiments compared wild-type (wt) and Royal College of Surgeons (RCS) rats at postnatal day 23-24, an age at which RCS retina is reported to contain abundant apoptotic photoreceptors but intact inner retina. Commercially available fluorescent PSR were applied as eye drops to corneas of live rats or to freshly dissected retinal tissues. Following eye drop application, eyes/retinas were imaged in situ in live anesthetized rats, or rats were sacrificed and live tissue was imaged ex situ. Retina treated with PSR after dissection was imaged live within 30 min of animal sacrifice. Dissected tissues were imaged using a confocal microscope, while eyes in living rats were imaged using a Kodak In Vivo Fx-Pro small animal scanner or a Micron IV In Vivo retinal imaging microscope from Phoenix Research labs. Fluorescence signals were quantified using Multispectral FX-Pro or Image J softwares. Scotopic electroretinography (ERG) was performed on rats with and without PSR eye drop treatment.

Results : Abundant PS exposure by RCS but not wt photoreceptors was confirmed by PSR labeling of dissected retina. 24 but not 72 h following PSR eye drop treatment, RCS but not wt photoreceptors imaged ex situ showed PSR labeling indicating that topically applied PSR reached the outer retina. Both whole animal and retinal imagers detected significantly more PSR fluorescence in RCS retina than in wt retina in vivo. ERG testing 72 h after eye drop application did not show differences in light responses between PSR treated and control retina.

Conclusions : Our results establish a novel, non-invasive method detecting apoptotic cells in the living eye of experimental animals. As labeling is non-toxic and transient, this method may facilitate repeat testing of animal cohorts to establish model-specific cell death time course or to precisely time anti-death therapies.

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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