Reduced apoptosis and autophagy in an oxidative stress retina organ culture model through an iNOS-inhibitor

Abstract

Purpose : The overproduction of reactive oxygen species is defined as oxidative stress. This pathomechanism occurs in several retinal diseases, including glaucoma or retinal ischemia. Hydrogen peroxide (H₂O₂) can be used in vitro to simulate oxidative stress, causing a strong neurodegeneration of the inner retinal layers. Based on this model, therapeutic approaches can be tested. Hence, treatment with the nitric oxide synthase (iNOS)-inhibitor 1400W as a possible neuroprotectant was evaluated.

Methods : Oxidative stress was induced by adding 300 µM H₂O₂ on day one for three hours to porcine retinal organotypic cultures. At the same time, 500 µM iNOS-inhibitor (1400W, Merck Millipore) was added. Neuronal and glial cells were evaluated via qRT-PCR and immunohistology after four and eight days. In addition, TEM was performed. The following groups were compared: controls, H₂O₂, and H₂O₂+iNOS-inhibitor.

Results : H₂O₂-induced retinal ganglion cell (RGC) loss (4 days: p=0.0001; 8 days: p=0.005) was prevented by iNOS-inhibitor treatment (4 days: p=0005; 8 days: p=0.13). On mRNA level, a significantly increased expression of autophagic gene p62 due to H₂O₂ (p=0.037) was prevented by the iNOS-inhibitor treatment after four days (p=0.45). Likewise, a reduced expression of caspase 8 was seen after iNOS-inhibitor treatment (3.19-fold; p=0.54) in comparison to the H₂O₂-stressed group (5.37-fold; p=0.065). A late rescue of bipolar cells was noted in iNOS-inhibitor treated retinas (p=0.0001). TEM revealed that cell morphology as well as the cell compartments of RGCs were protected from oxidative stress damage by the iNOS-inhibitor.

Conclusions : Strong degeneration in porcine retinas due to H₂O₂-induced oxidative stress was prevented by treatment with the iNOS-inhibitor. A lowered apoptotic stage resulted in a cell rescue, especially of RGCs. These results indicate, a potential neuroprotective role of the iNOS-inhibitor 1400W in diseases affecting the inner retina, especially glaucoma or retinal ischemia.

This is a 2020 ARVO Annual Meeting abstract.