A Comparative Study on the Mitigation of Oxidative Stress and Excitotoxicity by a Hydrogen Sulfide-Releasing Compound in Bovine Retina, Ex Vivo

Abstract

Purpose : We have evidence that exogenously administered hydrogen sulfide (H₂S)-releasing compounds can attenuate oxidative stress (Rai et al., IOVS,64(8):3247, 2023.) and excitatory amino acid neurotransmission in bovine isolated retina (Opere CA et al., JOPT, 34:10, 2018). In the present study, we compared the neuroprotective effect of a H₂S-releasing compound, diallyltetrasulfide (DATTS) against both hydrogen peroxide (H₂O₂)- and N-methyl-D-aspartate (NMDA)-induced damage in bovine isolated retina, ex vivo.

Methods : Freshly isolated bovine eyeballs were incubated in oxygenated Kreb’s buffer solution (37^oC) for 0.5 or 1 h followed by an intravitreal injection of H₂O₂ (10^-5M) or NMDA (10^-4 M) in the absence and presence of DATTS (10^-12 M to 10^-6M). After an incubation period (0.5 or 1 h), eyeballs were dissected, and retinas were isolated and fixed in formalin for morphological (TUNEL) and antioxidant (glutathione, GSH) assays.

Results : Treatment with H₂O₂ (10^-5 M) and NMDA (10^-4M) induced significant (p < 0.05) loss in RGC count by 38.78±5.1 (n=3) % and 36.57±4.54% (n=3), respectively in comparison to controls. DATTS (10^-10M) significantly (p<0.05) reversed both H₂O₂ (10^-5 M) and NMDA (10^-4M)-induced RGC loss from 61.21± 5.52% (n=3) and 78.41±5.24 % (n=3) to 91.25 ± 0.84% (n=3) and 111.71± 3.11% (n=3) respectively, thereby increasing RGC survival. H₂O₂ (10^-5 M) induced a decrease in endogenous GSH content from 40.88 ± 0.80 µmoles/gram to 34.50 ± 2.10 µmoles/g. DATTS (10^-10M and 10^-8M) significantly (p<0.001) reversed the H₂O₂-induced inhibitory response by 215% and 167%, respectively. Conversely, NMDA (10^-4M) significantly (p<0.001) enhanced GSH levels by 612.35 ± 56.81% (n=3). In this scenario, DATTS (10^-8M) reversed the NMDA-induced response to 34.97±7.88 µmoles/g (close to control level, 29.42 ± 7.29), thereby achieving a 192.8±9.28% reduction in GSH content.

Conclusions : We conclude that a H₂S-releasing compound, DATTS can protect retinal neurons from both peroxide-induced oxidative stress and NMDA-induced excitotoxicity, ex vivo.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.