Ferrous but not ferric iron sulfate kills photoreceptors and induces photoreceptor-dependent RPE autofluorescence

Iron has been implicated in the pathogenesis of retinal degenerative diseases, including ocular siderosis. However, the mechanisms of iron-induced retinal toxicity are incompletely understood. Previous work shows that intravitreal injection of Fe(2+) leads to photoreceptor (PR) oxidative stress, resulting in PR death within 14 days, and cones are more susceptible than rods to iron-induced oxidative damage. In order to further investigate the mechanism of intravitreal iron-induced retinal toxicity and shed light on mechanisms of iron-induced retinopathy in other mouse models, Fe(2+), Fe(3+), or saline were injected into the vitreous of adult wild-type mice. Pre-treatment with Ferrostatin-1 was used to investigate whether iron-induced retinal toxicity resulted from ferroptosis. Color and autofluorescence in vivo retinal imaging and optical coherence tomography were performed on day 2 and day 7 post-injection. Eyes were collected for quantitative PCR and Western analysis on day 1 and for immunofluorescence on both day 2 and 7. In vivo imaging and immunofluorescence revealed that Fe(2+), but not Fe(3+), induced PR oxidative damage and autofluorescence on day 2, resulting in PR death and retinal pigment epithelial cell (RPE) autofluorescence on day 7. Quantitative PCR and Western analysis on day 1 indicated that both Fe(2+) and Fe(3+) induced iron accumulation in the retina. However, only Fe(2+) elevated levels of oxidative stress markers and components of ferroptosis in the retina, and killed PRs. Ferrostatin-1 failed to protect the retina from Fe(2+)-induced oxidative damage. To investigate the mechanism of Fe(2+)-induced RPE autofluorescence, rd10 mutant mice aged 6 weeks, with almost total loss of PRs, were given intravitreal Fe(2+) or Fe(3+) injections: neither induced RPE autofluorescence. This result suggests Fe(2+)-induced RPE autofluorescence in wild-type mice resulted from phagocytosed, oxidized outer segments. Together these data suggest that intraretinal Fe(2+) causes PR oxidative stress, leading to PR death and RPE autofluorescence.