Neuroprotection of human and rodent retinal ganglion cells by a hybrid antioxidant-nitric oxide donor small molecule, SA-2

PURPOSE: Current treatments of glaucoma are aimed at lowering intraocular pressure (IOP), which is a key driver of retinal ganglion cell (RGC) death. Another contributing factor to RGC death is exposure to reactive oxygen species (ROS). At present, there is no FDA-approved neuroprotective treatment to prevent glaucomatous optic neuropathy and loss of RGCs. Our novel hybrid molecule, SA-2, contains both a nitric oxide (NO) donating group to lower IOP and a ROS scavenging group to protect RGCs. We hypothesize that SA-2 will inhibit the death of RGCs in an in vitro and an ex vivo neurotrophic factor deprivation model. METHODS: Retinal punches from human explants (n=4 donors/experiments) were isolated and treated with either SA-2 [1 mM] or vehicle and maintained without neurotrophic factors for 7 days ex vivo. In each experiment, 4 baseline retinal explants were collected on day 0. At the end of the experiment, explants were immunostained with RBPMS and Brn-3a (RGC-specific markers) and cell survival was analyzed. In three biological replicates, primary RGCs were isolated from rat pups and treated with either SA-2 (1 mM, 100 µM) or vehicle with or without neurotrophic factors for 48 h. Active caspase 3 and 7 assay was performed and apoptotic cell counts were analyzed. In another set of experiments, rat retinal explants were isolated and incubated with tert-Butyl hydroperoxide (TBHP) along with either SA-2 [1 mM] or vehicle for 2 h (n=2-4 explants/group). Production of superoxide by mitochondria was assessed using MitoSOX reagent according to manufacturer instructions. All cell counts were performed in a masked manner using ImageJ Software. One-way ANOVA or nonparametric Kruskal-Wallis was used for statistical analysis by GraphPad Prism 9 Software. RESULTS: In ex vivo human retinal explants, there was a significant increase in RGC survival by 39% in the SA-2 treated group compared to the vehicle group at day 7 (p< 0.0001). In rodent primary RGCs, SA-2 mediated a significant decrease in apoptotic cells by 30% (p< 0.01) and a 67% (p< 0.05) decrease in dead cell count. In rodent retinal explants, there was a significant decrease (by 59%, p< 0.0001) in the production of superoxide by mitochondria in the TBHP and SA-2 treated group, compared to the TBHP vehicle group. CONCLUSION: SA-2 was shown to be effective at preserving retinal ganglion cell survival in human retinal explants, rat retinal explants and primary rat RGCs by preventing apoptosis and protecting the cells from oxidative stress.