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. 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, 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. 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. 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: neither induced RPE autofluorescence. This result suggests Fe2+-induced RPE autofluorescence in wild-type mice resulted from phagocytosed, oxidized outer segments. Together these data suggest that intraretinal Fe2+ causes PR oxidative stress, leading to PR death and RPE autofluorescence.

Purpose: Diabetic retinopathy (DR) is characterized by increased oxidative and nitrosative stress, both of which lead to neurotoxicity and vascular permeability. Previous studies on a variety of organs indicate that hydrogen-rich saline not only has considerable antioxidant and anti-inflammatory properties, but also suppresses oxidative stress-induced injury. In the present study, we assessed the effects of hydrogen-rich saline on neurovascular dysfunction and oxidative stress in an animal model (rat) of DR. Materials and Methods: Male Sprague-Dawley rats with streptozotocin (STZ)-induced diabetes mellitus (DM) were injected intraperitoneally with 5 ml/kg hydrogen-saturated (experimental) or plain (control) saline daily for one month. Visual function and blood–retinal barrier (BRB) integrity were evaluated by electroretinography (ERG) and bovine serum albumin (BSA)-fluorescence, respectively. Histological changes in the inner retina were assessed by light microscopy. Biomarkers of oxidative stress, including 4-hydroxynonenal (4-HNE) and 8-hydroxy-2-deoxyguanosine (8-OH-dG), and antioxidant enzymes, including superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione transferase, were evaluated by ELISA. Synaptophysin and brain-derived neurotrophic factor (BDNF) levels were measured by immunoblotting. Results: STZ-diabetic rats were marked by clearly reduced b-wave amplitudes and oscillatory potentials, DM-related BRB breakdown and histological changes in the inner retina, all of which were suppressed following treatment with hydrogen-rich saline. Furthermore, hydrogen-rich saline reduced oxidative stress, increased antioxidant enzyme activities and preserved synaptophysin and BDNF levels in the diabetic rat retina. Conclusions: Based on its inhibition of oxidative stress and up-regulation of anti-oxidative enzymes, we conclude that hydrogen-rich saline is a potentially valuable therapeutic modality for the treatment of DR.

Background: The development of alternative anti-angiogenesis therapy for choroidal neovascularization (CNV) remains a great challenge. Nanoparticle systems have emerged as a new form of drug delivery in ocular diseases. Here, we report the construction and characterization of arginine-glycine-aspartic acid (RGD)-conjugated polyethyleneimine (PEI) as a vehicle to load antioxidant salvianolic acid A (SAA) for targeted anti-angiogenesis therapy of CNV. In this study, PEI was consecutively modified with polyethylene glycol (PEG) conjugated RGD segments, 3-(4′-hydroxyphenyl) propionic acid-Osu (HPAO), and fluorescein isothiocyanate (FI), followed by acetylation of the remaining PEI surface amines to generate the multifunctional PEI vehicle PEI.NHAc-FI-HPAO-(PEG-RGD) (for short, RGD-PEI). The formed RGD-PEI was utilized as an effective vehicle platform to load SAA. Results: We showed that RGD-PEI/SAA complexes displayed desirable water dispersibility, low cytotoxicity, and sustainable release of SAA under different pH conditions. It could be specifically taken up by retinal pigment epithelium (RPE) cells which highly expressed ɑ_vβ₅ integrin receptors in vitro and selectively accumulated in CNV lesions in vivo. Moreover, the complexes displayed specific therapeutic efficacy in a mouse model of laser induced CNV, and the slow elimination of the complexes in the vitreous cavity was verified by SPECT imaging after ¹³¹I radiolabeling. The histological examinations further confirmed the biocompatibility of RGD-PEI/SAA. Conclusions: The results suggest that the designed RGD-PEI/SAA complexes may be a potential alternative anti-angiogenesis therapy for posterior ocular neovascular diseases. Graphic

Purpose: To investigate whether caffeic acid phenethyl ester (CAPE) has a protective effect on retinal ischemia/reperfusion (I/R) injury in rats, and to determine the possible antioxidant mechanisms. Methods: Seventy-six female Wistar rats were randomized evenly into Sham, I/R injury model (M group), model plus vehicle (MV), and model plus CAPE (MC) groups. Retinal ischemia/reperfusion injury was induced by increasing the intraocular pressure to 110 mmHg for 60 min. Rats in the MV and MC groups were injected with vehicle and CAPE (10 µmol/kg i.p.), respectively, before reperfusion and once a day for one or seven days after I/R. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in the retinal tissues were determined 24 hr after I/R. Retinal cells apoptosis was detected 24 hr after I/R injury by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling staining. On day 7 after reperfusion, the electroretinogram (ERG) was recorded, and the retinal histology was examined and quantified using light microscopy. Results: CAPE significantly decreased the MDA levels and increased the activities of SOD, GSH-Px, and CAT in the retina compared with the ischemia group (p < 0.05). CAPE attenuated the I/R-induced apoptosis of retinal cells in the inner nuclear and ganglion cells of the rat retina. CAPE also suppressed the I/R-induced reduction in the a- and b-wave amplitudes of the ERG (p < 0.05). The thickness of the entire retina, inner nuclear layer, and inner plexiform layer and the number of cells in the ganglion cell layer in the MC group were significantly greater than those in the M group (p < 0.05). Conclusions: CAPE can protect the rat retina from I/R injury by enhancing the antioxidation ability and inhibiting the apoptosis of retinal cells, which suggests that CAPE is potentially useful for treating I/R-induced eye disorders.

Purpose: Iron supplementation therapy is used for iron-deficiency anemia but has been associated with macular degeneration in a 43-year-old patient. Iron entry into the neurosensory retina (NSR) can be toxic. It is important to determine conditions under which serum iron might cross the blood retinal barrier (BRB) into the NSR. Herein, an established mouse model of systemic iron overload using high-dose intraperitoneal iron dextran (IP FeDex) was studied. In addition, because the NSR expresses the iron regulatory hormone hepcidin, which could limit iron influx into the NSR, we gave retina-specific hepcidin knockout (RS-HepcKO) mice IP FeDex to test this possibility. Methods: Wild-type (WT) and RS-HepcKO mice were given IP FeDex. In vivo retina imaging was performed. Blood and tissues were analyzed for iron levels. Quantitative PCR was used to measure levels of mRNAs encoding iron regulatory and photoreceptor-specific genes. Ferritin and albumin were localized in the retina by immunofluorescence. Results: IP FeDex in both WT and RS-HepcKO mice induced high levels of iron in the liver, serum, retinal vascular endothelial cells (rVECs), and RPE, but not the NSR. The BRB remained intact. Retinal degeneration did not occur. Conclusions: Following injection of high-dose IP FeDex, iron accumulated in the BRB, but not the NSR. Thus, the BRB can shield the NSR from iron delivered in this manner. This ability is not dependent on NSR hepcidin production.