The Mechanism Of Oxidative Stress And Age-related Macular Degeneration Induced By Retinal Iron Overload

Objective:1.To establish an animal model of local iron overload in the retina,and explore the acute and chronic retinal phathology caused by retinal iron overload.2.To investigate the mechanism of age-related macular degeneration(AMD)induced by retinal iron ion overload.3.To investigate the effect of deuterated docosahexaenoic acid(D-DHA)on ironinduced lipid peroxidation and age-related macular degeneration.4.To investigate the effects of ferroportin and ceruloplasmin on iron metabolism in reitnal cells.Methods:1.C57BL/6 WT mice were used in this study.Mice were given an intravitreal injection of 0.5m M FAC in one eye and saline in the other eye to induce retinal iron overload.Color fundus photography,optical coherence tomography(OCT),confocal laser fundus scanning microscopy(c SLO)and electroretinogram(ERG)were employed to investigate retinal changes.TUNEL staining was used to determine cell apoptosis in the retina.To investigate the iron level in retinal cells,immunostaining of ferritin light chain and Perl’s staining were conducted.From 1 mo to 6 mo after injections,color fundus photography,OCT,c SLO and angiography were used to investigate the chronic retinal degeneration induced by iron.Toluidine blue staining was used to explore retinal morphological changes.2.The retinal iron overload mouse model was used to investigate the mechanism X of iron-induced retinal degeneration.Immunostaining showed the expression and location of the relative protein.Prussian blue staining was conducted on cryosections to evaluated retinal iron levels.High-performance liquid chromatography(HPLC)was used to analyze the contents of bisretinoid.Toluidine blue staining was employed to examine retinal morphology change.Quantitative PCR on the neural retina and isolated RPEs detected the relative m RNA expression of genes involved in retinal cell differentiation,iron regulation,oxidative stress,inflammatory response and complement pathway.3.C57BL/6 wide-type mice were fed with diets containing D-DHA or DHA for1 week,2 weeks,3 weeks,and 4 weeks,then mice were given an intravitreal injection of 0.5m M FAC vs saline.In vivo imaging systems were conducted to evaluated retinal morphology and retinal function.Immunostaining were used to evaluate the protein expression and location.HPLC was performed to analyze the contents of DHA or DDHA in the neural retina and RPE cells.Toluidine blue staining was employed to examine retinal morphology change.Quantitative PCR on the neural retina and isolated RPEs detected the relative m RNA expression of genes involved in iron metabolism,oxidative stress,inflammatory response,and complement pathway.4.Conditional knockout of Fpn in the retina and systemic knockout of Cp in wildtype mice with C57BL/6 background were used.Intraperitoneal injection of iron dextran was used to establish systemic iron overload.Immunostaining of Ft-L were used to evaluated the iron level in the retina.Quantitative PCR was used to detect the expression levels of iron regulating genes in retinal cells.Results:1.We established a new mouse model of retinal iron overload by giving an intravitreal injection of iron.In mice given an intravitreal injection of FAC,photoreceptor and RPE cells died,alongside autofluorescence accumulation in the retina.Moreover,chronic retinal iron overload-induced progressive geographic atrophy formation and development,neovascularization,and sympathetic ophthalmia.Retinal iron overload induced the activation and migration of myeloid cells into the neural retina,followed by myeloid cell infiltration in the fellow eye with saline injection.2.Iron increased all-trans-retinal dimer instead of A2 E which may responsible for iron-induced retinal autofluorescence.Iron overload increased oxidative stress lipid peroxidation products,pro-inflammatory cytokines,and complement factor C3,resulting in age-related macular degneration like pathology.3.Dietary intake of D-DHA effectively incorporated D-DHA into the neural retina and RPE cells.In mice fed with D-DHA for 4 weeks,the content of D-DHA in the retina was ≥ 50%,D-DHA completely prevented the iron-induced autofluorescence and retinal degeneration.4 weeks of D-DHA completely protected against iron-induced oxidative stress and lipid peroxidation product,CEP.4 weeks of D-DHA completely protected retina structure and function.D-DHA prevented the formation of geographic atrophy and showed a long-term protective effect.4.Conditional knockout of ferroportin in the retina failed to result in retinal iron overload.In mice with serum iron overload,systemic Cp KO and conditional retinal Fpn KO caused the iron overload in the retina,suggesting that Cp and Fpn were involved in retinal iron export.Conclusions1.Retinal iron overload induced autofluorescence accumulation in the retina,photoreceptor and RPE cells degeneration.Retinal iron overload induced chronic progressive age-related macular degeneration,including geographic atrophy and neovascularization.Iron overload induced chronic sympathetic ophthalmopathy in the fellow eye with saline injection.2.The mechanisms of iron-induced retina degeneration involve oxidative stress,lipid peroxidation products accumulation,the activation,and migration of myeloid cells,inflammation,and complement pathway activation.Our findings suggest that DHA oxidation is the core of the pathogenesis of iron-related age-related macular degeneration in the retinal iron overload mouse model.3.Dietary intake of D-DHA for 4 weeks can effectively prevent iron-induced oxidative stress and retinal degeneration,protect the retinal morphology and function.Moreover,Fpn is not the only exporter of iron in the retina,the impairment of Cp and Fpn mediated iron export resulted in iron overload in the retina.4.Iron export of retinal cells wasn’t mediated by ferroportin.The impair of ferroportin and ceruloplasmin resulted in iron overlaod in the retina.5.Herein,we generated a new mouse model,which will be useful to investigate retinal antioxidants,anti-VEGF and anti-inflammatory therapy.Our results provide preclinical evidence that dosing with D-DHA could be a viable therapeutic strategy for retinal diseases involving oxidative stress.