April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Dendritic degeneration as an early marker of neuronal degeneration in retinal explants
Author Affiliations & Notes
  • Kate Binley
    School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom
  • Wai Siene Ng
    School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom
  • Bing Song
    School of Dentistry, Cardiff University, Cardiff, United Kingdom
  • James E Morgan
    School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom
    Opthalmology, University Hospital of Wales, Cardiff, United Kingdom
  • Footnotes
    Commercial Relationships Kate Binley, None; Wai Siene Ng, None; Bing Song, None; James Morgan, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 1727. doi:https://doi.org/
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    • Get Citation

      Kate Binley, Wai Siene Ng, Bing Song, James E Morgan; Dendritic degeneration as an early marker of neuronal degeneration in retinal explants. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1727. doi: https://doi.org/.

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

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Abstract

Purpose: To determine the relationship between retinal ganglion cell degeneration and retinal ganglion cell loss in a retinal explant model.

Methods: Adult C57Bl/6 mice retinas were prepared and cultured as wholemounts for up to 14 days in Neurobasal-A culture medium and maintained at 37°C in 95/5% O2/CO2. Retinas were fixed and cryosectioned for nuclear staining, immunohistochemistry, and TUNEL labelling. Neuronal viability was quantified by calcein-AM staining. Single retinal ganglion cells were labelled diolistically (DiI and DiO) using a hand-held gene gun (Bio-Rad) fired at 100 psi. All cells were imaged confocally and their dendritic architecture analysed by Sholl analysis. The pan-caspase inhibitor OPH001-01M was applied to explants over 2 days to determine the extent to which neuronal degeneration could be inhibited.

Results: Nuclear staining demonstrated a loss of 30% of retinal ganglion cells over 14 days (p<0.1). Immunohistochemistry confirmed persistent Thy-1 expression in the retinal ganglion cell layer during this time. The ganglion cell layer showed a small increase in apoptotic markers over 14 days: 2.5% active caspase-3 (2±1 cells/mm) and 5% TUNEL (5±3% ganglion cell layer). Ganglion cell viability decreased by 35% over 14 days as measured by calcein-AM staining (100±30 viable cells/mm2 after 14 days). Sholl analysis revealed substantial dendrite loss in retinal ganglion cells within 24 hours with a reduction in the area under the Sholl plot of 30% (p<0.05). Following the addition of the pan-caspase inhibitor immediately after retinal dissection neuronal degeneration was prevented on the basis of Sholl plots (20% increased area under the curve relative to control).

Conclusions: In the retinal explant model dendritic degeneration may be an early marker of retinal ganglion cell loss. Significantly these changes are seen over a week before substantial cell loss can be detected. The explant model has potential as a sensitive assay for neuroprotective interventions.

Keywords: 688 retina • 694 retinal culture • 695 retinal degenerations: cell biology  
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