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