Joshua L. Dunaief

Purpose: Dense deposit disease (DDD) is caused by dysregulation of the alternative pathway of the complement cascade and characterized by electron-dense deposits in the kidney glomerular basement membrane (GBM) and drusen in Bruch's membrane (BrM). Complement factor H (fH) and factor properdin (fP) regulate complement activation; fH inhibits alternative pathway (AP) activation, whereas fP promotes it. We report pathologic changes in eyes of an fH and fP double-mutant mouse, which we previously showed have dense deposits in the GBM and early mortality from nephropathy. Methods: fH^m/m, fP^−/−, and fH^m/m/fP^−/− mice were generated on a C57BL/6–129J background. Fundus imaging at 8 weeks of age was followed by analysis via light and electron microscopy. Retinal function was assessed by electroretinography (ERG). Complement levels and localization were tested by immunohistochemistry and ELISA. Retinas of fH^m/m/fP^−/− mice treated with intraperitoneal injections of an anti-C5 antibody were compared to those of age- and genotype-matched mice injected with an isotype control antibody. Results: fH^m/m/fP^−/− mice suffered early-onset retinal hypopigmented spots detected using in vivo retinal photography, and histologic examination showed basal laminar deposits (BLamD), degeneration of the photoreceptors, and RPE vacuolization. ERG showed diminished retinal function. The anti-C5 antibody was retina-protective. Conclusions: This unique mouse represents a new model of complement-mediated rapid-onset DDD, and could be useful in exploring the pathologic changes associated with BLamD in age-related macular degeneration.

The complement system is involved in the pathogenesis of age-related macular degeneration (AMD). Because activated microglia are also associated with AMD, we studied the relationship between complement anaphylatoxin receptors and microglial recruitment. We assessed the effect of anaphylatoxin C3a receptor (C3aR) and C5a receptor (C5aR) knockout (KO) on light damage–induced migration of microglia/macrophages into the mouse outer retina via immunofluorescence and real-time quantitative PCR. We found that the mRNA levels of C3, C5, C3aR, C5aR, and two activators of the complement alternative pathway, Cfb and Cfd, were all upregulated after light exposure. Retinal Iba1-positive microglia/macrophages express receptors for C3a and C5a. Light damage increased the number of retinal Iba1-positive cells and the mRNA levels of Iba1. Compared with the wild-type (WT) mice, these increases were attenuated in the C5aR KO mice but not in the C3aR KO mice. C5aR but not C3aR promoted the recruitment of microglia/macrophages. These divergent properties of complement anaphylatoxins in the light damage model provide a rationale for testing the differential effects of these receptors in additional retinal and neurodegeneration models.

(−)-epigallocatechin-3-gallate (EGCG), a major catechin component of green tea, is reported to delay or prevent certain forms of cancer, arthritis, cardiovascular disease, and neurodegenerative disorders. In this study, we determined if systemically administered EGCG could protect the retina against light damage (LD) in mice. BALB/cJ mice were treated with either EGCG or saline via intraperitoneal (IP) injection, and then placed under constant cool white light-emitting diode (LED) light (10,000 lux) for 5 h. Retinal structure and function were evaluated using optical coherence tomography (OCT), histology, and electroretinography (ERG) 7 days after LD. In addition, the mRNAs of several oxidative stress genes were quantified by qPCR before LD and 24 h after LD. OCT and photomicrographs of mouse retinas showed morphologic protection of photoreceptors. Mice in the EGCG group had significantly higher ERG amplitudes for all three wave types compared with mice in the saline control group, which indicated that EGCG protected retinal function. Furthermore, qPCR results showed that EGCG administration can increase the mRNA level of the antioxidant gene Sod2 before LD and 24 h after LD. The IP injection of EGCG attenuated the detrimental effects of bright light on the retinas of BALB/cJ mice by protecting the structure and function of the retina.

Purpose: High RPE iron levels have been associated with age-related macular degeneration. Mutation of the ferroxidase ceruloplasmin leads to RPE iron accumulation and degeneration in patients with aceruloplasminemia; mice lacking ceruloplasmin and its homolog hephaestin have a similar RPE degeneration. To determine whether a high iron diet (HID) could cause RPE iron accumulation, possibly contributing to RPE oxidative stress in AMD, we tested the effect of dietary iron on mouse RPE iron. Methods: Male CD1 strain mice were fed either a standard iron diet (SID) or the same diet with extra iron added (HID) for either 3 months or 10 months. Mice were analyzed with immunofluorescence and Perls' histochemical iron stain to assess iron levels. Levels of ferritin, transferrin receptor, and oxidative stress gene mRNAs were measured by quantitative PCR (qPCR) in neural retina (NR) and isolated RPE. Morphology was assessed in plastic sections. Results: Ferritin immunoreactivity demonstrated a modest increase in the RPE in 10-month HID mice. Analysis by qPCR showed changes in mRNA levels of iron-responsive genes, indicating moderately increased iron in the RPE of 10-month HID mice. However, even by age 18 months, there was no Perls' signal in the retina or RPE and no retinal degeneration. Conclusions: These findings indicate that iron absorbed from the diet can modestly increase the level of iron deposition in the wild-type mouse RPE without causing RPE or retinal degeneration. This suggests regulation of retinal iron uptake at the blood-retinal barriers.

Iron is essential for life, but excess iron can be toxic. As a potent free radical creator, iron generates hydroxyl radicals leading to significant oxidative stress. Since iron is not excreted from the body, it accumulates with age in tissues, including the retina, predisposing to age-related oxidative insult. Both hereditary and acquired retinal diseases are associated with increased iron levels. For example, retinal degenerations have been found in hereditary iron overload disorders, like aceruloplasminemia, Friedreich's ataxia, and pantothenate kinase-associated neurodegeneration. Similarly, mice with targeted mutation of the iron exporter ceruloplasmin and its homolog hephaestin showed age-related retinal iron accumulation and retinal degeneration with features resembling human age-related macular degeneration (AMD). Post mortem AMD eyes have increased levels of iron in retina compared to age-matched healthy donors. Iron accumulation in AMD is likely to result, in part, from inflammation, hypoxia, and oxidative stress, all of which can cause iron dysregulation. Fortunately, it has been demonstrated by in vitro and in vivo studies that iron in the retinal pigment epithelium (RPE) and retina is chelatable. Iron chelation protects photoreceptors and retinal pigment epithelial cells (RPE) in a variety of mouse models. This has therapeutic potential for diminishing iron-induced oxidative damage to prevent or treat AMD.