Neuroprotection of Rodent Retinal Ganglion Cells using Hybrid Molecule SA-10 and its PLGA Nanosuspension

Abstract

Purpose : Oxidative stress has been shown to be associated with glaucomatous retinal ganglion cell (RGC) degeneration. In this study, we aimed to promote RGC survival by treatment with SA-10, a hybrid molecule with nitric oxide donating and sulfone reactive oxygen species (ROS) scavenging moieties in vitro, ex vivo, and after dosing in vivo following ischemia/reperfusion (I/R) injury in mice.

Methods : Endothelin-3 (ET-3) [100 or 400 nM] was used to induce oxidative stress in vitro in isolated rat primary RGCs (n=3 experiments) and ex vivo in C57BL/6J mice retinal explants (n=2-3 explants/group). Primary RGCs or retinal explants were pretreated with vehicle (DPBS) or SA-10 [10 μM] for 30 minutes, following which ET-3 treatment was carried out for 1 hour. CellROX™ Green was then used to stain for ROS produced by the cells, and the integrated density was analyzed.

In another set of experiments, acute I/R injury was induced in C57BL/6J mice (n=5-8 mice/group) through pressure elevation to 120 mmHg for 1 hour by cannulation of the anterior chamber with a reservoir of normal saline. The mice were pre-treated topically with a modified nanosuspension (1% SA-10-NPs) and post-treated after I/R injury for a total of 14 days, 3 times a week. Pattern electroretinogram (PERG) and pattern visual evoked potential (PVEP) were measured at baseline and post-treatment. Retinal flat mounts were collected and stained with RBPMS (an RGC-specific marker) for cell survival analysis.

Analysis of Variance (ANOVA) or nonparametric Kruskal-Wallis was performed for all experiments.

Results : In primary RGCs and mice retinal explants, ET-3-mediated ROS production decreased by 25.9% (p<0.01) and 14.7% (p<0.0001), respectively, following SA-10 treatment compared to the vehicle. I/R injury (vehicle NP-treated) produced a 52.1% decline (p<0.01) in PERG and a 17.9% decreasing trend in PVEP amplitudes as compared to the sham. SA-10-NPs prevented this decline by an increasing trend of 33.5% and 14%, respectively. I/R injury (vehicle NP-treated) caused a 33% decrease (p<0.01) in RGC survival in the inner retina in comparison with the sham control, which was alleviated with the SA-10-NP treatment by 33.4% (p<0.001).

Conclusions : SA-10 effectively protected RGCs in vitro and ex vivo from ET-3-mediated oxidative stress. Similarly, SA-10-NPs enhanced RGC survival and function following ischemia-induced damage in the mice I/R model.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.