A Rat Model for Choroidal Neovascularization Using Subretinal Lipid Hydroperoxide Injection

Abstract

The purpose of this study was to develop and characterize a rat model of choroidal neovascularization (CNV) as occurs in age-related macular degeneration. The lipid hydroperoxide 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (HpODE) is found in submacular Bruch's membrane in aged humans and has been reported to generate neovascularization in a rabbit model. Three weeks after a single subretinal injection of 30 g of HpODE, eyes of Sprague-Dawley rats were harvested. Follow-up fluorescein angiography was done on other animals until 5 weeks postinjection. Histological studies, immunohistochemical staining, and flatmount choroids for CNV measurements were performed. In addition, we used murine neuronal, bovine endothelial, and human ARPE19 cells for testing the in vitro effects of HpODE. CNV developed in 85.7% of HpODE-injected eyes. The neovascular areas were significantly greater in HpODE-injected eyes compared with those in control eyes (P ‫؍‬ 0.023). The CNV had maximum dye leakage at 3 weeks, which subsided by the 5th week. Histologically, CNV extended from the choriocapillaris into the subretinal space. ED1-positive macrophages were recruited to the site. In vitro assays demonstrated that only 30 ng/ml HpODE induced cell proliferation and migration of endothelial cells. HpODE-induced CNV was highly reproducible, and its natural course seems to be ideal for evaluating therapeutic modalities. Because HpODE has been isolated from aged humans, the HpODE-induced rat model seems to be a relevant experimental model for CNV in age-related macular degeneration. Age-related macular degeneration (AMD) is the leading cause of central vision loss for populations older than 65 years in developed countries.1 There are two major types of AMD: dry and wet (or exudative). One type of dry AMD is geographic atrophy, which is the atrophy of the retinal pigment epithelium (RPE) in a well defined area centered on the fovea. Wet AMD progresses faster and results in more severe visual loss. In wet AMD, choroidal neovascularization (CNV) is associated with many pathological changes such as subretinal hemorrhage, serous retinal detachment, fibrovascular and serous pigment epithelium detachment, and subretinal scar formation. Both forms of AMD have characteristic extracellular lesions (drusen and basal linear and laminar deposits) that have an impact on RPE and photoreceptor viability and health. 2,3An experimental model is essential to investigate the nature and treatment of CNV. The first experimental CNV model of Ryan and his colleagues was reported in 1979. 4 They attempted to generate CNV using three different methods in monkeys. Their hypothesis was that disruption of Bruch's membrane induces neovascularization from choroid. According to their report, mechanical/enzymatic disruption of Bruch's membrane (BrM) via transscleral and transvitreal injections resulted in a low incidence of CNV (8.9 and 12.5%). Laser-induced disruption of BrM eventually produced CNV at a higher rate (80%), and the authors hypothesized that subretinal scar ...

Retinal energy demands control vascular supply of the retina in development and disease: The role of neuronal lipid and glucose metabolism

“…Despite evidence converging on the role of energy deregulation, surprisingly little is known about the energy signals that may govern AMD. Few suitable animal models replicate all aspects of AMD, but several transgenic mice present some characteristic signs of the disease (Table 1) (Ambati et al, 2003; Baba et al, 2010; Cao et al, 2010; Cashman et al, 2011; Chen et al, 2007; Combadière et al, 2007; Hahn et al, 2004; Heckenlively et al, 2003; Imamura et al, 2006; Jo et al, 2011; Luhmann et al, 2009; Lyzogubov et al, 2011; Malek et al, 2005; Markovets et al, 2011; Shen et al, 2006; Takada et al, 1994; Tuo et al, 2007; Zeiss, 2010; Zhao et al, 2011b). …”

Cholesterol in the retina: The best is yet to come

“…Compounds capable of these side effects are linoleate hydroperoxide and 7-ketocholesterol, by-products of lipoproteins found in atherosclerotic plaque (Upston et al, 2002) and also in aging BrM (Moreira et al, 2009; Spaide et al, 1999) and drusen (I. Rodriguez and C.A. Curcio, work in progress) and confirmed as deleterious in vivo (Amaral et al, 2013; Baba et al, 2010). Thus RPE-secreted apoB/apoE lipoproteins are a source of peroxidizable lipids immediately subjacent to the RPE, in the compartment where choroidal neovascularization occurs.…”

7-ketocholesterol accumulates in ocular tissues as a consequence of aging and is present in high levels in drusen

“…This response includes aggregation of macrophages and VEGF induction, events that do not occur following implantation of native Ch. Thus 7KCh, along with linoleate hydroperoxide (Baba et al, 2010; Spaide et al, 1999), is a good candidate for instigating downstream sequelae of Ch-rich lipoprotein deposition in Bruch’s membrane and drusen (Curcio et al, 2011; Spaide et al, 2003)…”