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