The Impact Of Route Of Administration And Form Of Iron On The Retina

Purpose:Iron overload has been implicated in the pathogenesis of neurodegenerative diseases including age-related macular degeneration.However,the mechanisms of iron-induced retinal toxicity are incompletely understood.The retina is protected from the systemic circulation by the blood retinal barrier(BRB).It is important to determine conditions under which serum iron could cross the BRB into the neurosensory retina(NSR).Retinal vascular endothelial cells(rVECs)are an essential component of the BRB,and the localization of the only known cellular iron exporter,ferroportin(Fpn),to the abluminal membrane of these cells,leads to the hypothesis to test in this thesis that Fpn may play a crucial role in the import of iron across the BRB.Besides,a mouse model of ocular siderosis is observed and analyzed to shed light on mechanisms of iron-induced retinopathy in hereditary iron overload mouse models.Methods:1.Wild-type(WT)and retina-specific hepcidin knockout(RS-Hepc KO)mice were given intraperitoneal iron dextran(IP FeDex).In vivo retina imaging was performed for the observation of retinal morphology and autofluorescence.Blood and tissues were analyzed for iron levels.Quantitative PCR was used to measure levels of m RNAs encoding iron regulatory and photoreceptor(PR)-specific genes.Ferritin and albumin were localized by immunofluorescence in the retina.2.A mouse model with knockout of Fpn within theVECs in both the retina and the brain was developed through tail vein injection of AAV9-Ple261(CLDN5)-icre to both experimental Fpn^f/fand control Fpn^+/+mice at P21.Mice were aged to 9 months and changes in iron distribution in the retina and the brain were observed.In vivo retina imaging and quantitative serum iron detection were used for model validation.Eyes and brains were collected for immunofluorescence.3.Fe²⁺,Fe³⁺or saline were injected into the vitreous of wild-type mice.Pretreatment with Ferrostatin-1(Fer-1)was used to investigate if iron-induced retinal toxicity resulted from ferroptosis.Color and autofluorescence retinal imaging and optical coherence tomography were performed on day 2 and 7 post-injection.Eyes were collected for quantitative PCR,western blot analysis and immunofluorescence.Results:1.IP FeDex in both WT and RS-Hepc KO mice induced significantly high levels of iron in the liver,serum,rVECs and RPE,but not NSR.Exposed to IP FeDex-induced systemic iron overload for a short or long term,the BRB of young adult and aged WT mice remained intact.Retinal degeneration didn't occur.2.Deletion of Fpn from the retinal and brainVECs leads to ferritin-L accumulation in the retinal and brainVECs.This occurred despite lower serum iron levels in the experimental mice.This occurred despite decreased serum iron levels in the experimental mice.3.Intravitreal(IVT)Fe²⁺injection induced upregulation of oxidative stress markers in the NSR of WT mice,but IVT Fe²⁺injection didn't.IVT Fe²⁺induced photoreceptor(PR)autofluorescence and DNA fragmentation on day 2,PR death and RPE autofluorescence on day 7.4.rd10 mutant mice aged 6 weeks,with almost total loss of PRs,were given IVT Fe³⁺or Fe²⁺injection,neither induced RPE autofluorescence.Conclusions:1.Iron accumulated in BRB but not NSR after high-dose IP FeDex injections.BRB can shield NSR from iron delivered in this manner,while this ability is not dependent on NSR hepcidin production.2.Fpn onVECs may be essential for iron transport into the retina and brain.3.IVT Fe²⁺causes PR oxidative stress,PR death and RPE autofluorescence.4.IVT Fe²⁺-induced RPE autofluorescence in WT mice resulted from phagocytosed,oxidized outer segments.