Abstract
Purpose:
The aim of the current study was to use an ex vivo organotypic retinal explant model to examine retinal survival mechanisms and as a screening tool for potential novel neuroprotectve agents relevant to glaucoma
Methods:
Intact retina was dissected from adult Sprague Dawley rats immediately post mortem to make 4 retinal explants per eye. Explants were placed ganglion cell side up on millipore filters with 300 μL N2/B27 culture media per well in a humidified incubator. Novel therapies were trialled by adding drugs at appropriate concentrations, or vehicle alone. At the conclusion of each experiment, explants were fixed in 4% paraformaldehyde and retinal ganglion cell density was estimated by βIII Tubulin immunohistochemistry. Live imaging of superoxide formation over 6 hrs was performed utilising dihydroethidium (DHE) and immunofluorescence microscopy. Analysis of protein expression in these explants was achieved by Western blotting.
Results:
Measurement of the retinal ganglion cell (RGC) density at different timepoints suggested that 4 days ex vivo was optimal for assessment of neuroprotection. Viable RGC at an average density of 878 cells/mm2 were observed in explanted retinas 4 days post enucleation. The effects of 8 licenced drugs on RGC survival in culture were tested. Angiotensin II blockers conferred > 50 % increase in ganglion cell survival after 4 days. Conversely, administration of Angiotensin II reduced cell survival by 40%. Western blot analysis of protein suppression suggested this effect was mediated by At1R and At2R receptors. Live retinal imaging of the RGC layer showed modulation of intracellular superoxide formation. DHE fluorescence of control explants was approximately 4 times greater than explants treated with irbesartan, an angiotensin II blocker, at 4 hrs post explant imaging, corresponding with an intracellular superoxide burst. The same imaging, undertaken with Angiotensin II treated explants, showed a 4 fold amplification of burst intensity.
Conclusions:
Our retinal explant model may be useful in screening for potentially novel neuroprotective agents in a standardised and internally controlled fashion. In addition, this model enables us to examine the mechanisms underlying neuroprotection. Angiotensin II blockers protect RGC in the explant model and are worth further investigation as a neuroprotective treatment in conditions such as glaucoma.
Keywords: 615 neuroprotection •
599 microscopy: light/fluorescence/immunohistochemistry •
694 retinal culture