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

Abstract

Purpose : 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 Fe²⁺ 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. This study further investigated the mechanism of intravitreal iron-induced retinal toxicity and shed light on mechanisms of iron-induced retinopathy in other mouse models.

Methods : Fe²⁺, Fe³⁺, 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. To investigate the mechanism of Fe²⁺-induced RPE autofluorescence, rd10 mutant mice aged 6 weeks, with almost total loss of PRs, were given intravitreal Fe²⁺ or Fe³⁺ injections.

Results : In vivo imaging and immunofluorescence revealed that Fe²⁺, but not Fe³⁺, 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 Fe²⁺ and Fe³⁺ induced iron accumulation in the retina. However, only Fe²⁺ elevated levels of oxidative stress markers and components of ferroptosis in the retina, and killed PRs. Ferrostatin-1 failed to protect the retina from Fe²⁺-induced oxidative damage. Neither Fe²⁺ or Fe³⁺ intravitreal injection induced RPE autofluorescence to rd10 mutant mice on day 7.

Conclusions : These data suggest that intraretinal Fe²⁺ but not Fe³⁺, causes PR oxidative stress, leading to PR death and RPE autofluorescence. Fer-1 pretreatment failed to rescue retina from Fe²⁺-induced photoreceptor death, indicating the mechanisms of cell death in this model are not exclusively ferroptosis.

This is a 2020 ARVO Annual Meeting abstract.

 

Representative in vivo color fundus photos, green autofluorescence images and OCT images 2 and 7 days post intravitreal injection.

 

Fluorescence images showing TUNEL-positive PR cells and autofluorescent cells in retinas.