Lipid Hydroperoxide Stimulates Subretinal Choroidal Neovascularization in the Rabbit

doi:10.1006/exer.2001.1121

Experimental Eye Research

Volume 74, Issue 2, February 2002, Pages 301-308

https://doi.org/10.1006/exer.2001.1121 Get rights and content

Abstract

The authors sought to evaluate the effect of linoleic acid hydroperoxide (18:2/LHP) in promoting choroidal neovascularization (CNV). Albino male rabbits received a subretinal injection of various amounts of LHP (ranging from 5 to 200 μ g) dissolved in 50 μ l of sodium borate buffer. Control eyes received the buffer only. Eyes were examined up to 4 weeks later with indirect ophthalmoscopy, fundus photography and fluorescein angiography. Animals were killed on days 3, 7, 14 or 28, and eyes examined by light and transmission electron microscopy. In eyes injected with LHP of 150–200 μ g, exposed areas turned white as observed ophthalmoscopically and showed both severe retinal and choroidal atrophy histologically. Neither fluorescein leakage nor CNV was found in these eyes or in controls. In 33 eyes injected with LHP of 100 μ g or less, prominent fluorescein leakage was seen in three (9%) and less prominent focal leakage in five (15%). In 11 (46%) of the 24 eyes injected with 12.5–50 μ g LHP, CNV was found histologically. Subretinal injection of LHP is capable of inducing CNV.

References (17)

W.R. Green et al.
Age-related macular degeneration histopathologic studies. The 1993 Lorenz E Zimmerman Lecture
Ophthalmology
(1993)
C. Schwesinger et al.
Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial growth factor in the retinal pigment epithelium
Am. J. Pathol.
(2001)
K. Akco et al.
Comparative effects of linoleic acid and linoleic acid hydroperoxide on growth and morphology of bovine retinal pigment epithelial cells in vitro
Curr. Eye Res.
(1996)
D. Armstrong et al.
Synthesis of lipid and cholesterol hydroperoxide standards
D. Armstrong et al.
Lipid hydroperoxide stimulates retinal neovascularization in rabbit retina through expression of tumor necrosis factor-α, vascular endothelium growth factor and platelet-derived growth factor
Angiogenesis
(1998)
D. Armstrong et al.
Dose dependent mechanisms of lipid hydroperoxide induced retinal pathology
J. Baffi et al.
Choroidal neovascularization in the rat induced by adenovirus mediated expression of vascular endothelial growth factor
Invest. Ophthalmol. Vis. Sci.
(2000)
M. De La Paz et al.
Region and age-dependent variation in susceptibility of the human retina to lipid peroxidation
Invest. Ophthalmol. Vis. Sci.
(1992)

There are more references available in the full text version of this article.

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Evolution of oxidative stress, inflammation and neovascularization in the choroid and retina in a subretinal lipid induced age-related macular degeneration model
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Tamai et al. (2002) have also reported similar events when HpODE was injected subretinally in rabbits. In the subretinal space, HpODE triggered an inflammatory cascade that led to infiltration and accumulation inflammatory cells, amplifying the series of pathological events leading to angiogenesis and CNV formation (Tamai et al., 2002; Armstrong et al., 1998). Microglia/macrophage activation and accumulation has been reported in human tissues both in early and advanced forms of AMD (Cherepanoff et al., 2010; McLeod et al., 2016).
Oxidative stress, inflammation and neovascularization are the key pathological events that are implicated in human age-related macular degeneration (AMD). There are a limited number of animal models available for evaluating and developing new therapies. Most models represent late exudative or neovascular AMD (nAMD) but there is a relative paucity of models that mimic early events in AMD. The purpose of this study is to characterize the evolution of oxidative stress, inflammation, retinal degeneration and neovascularization in a rat model of AMD, created by subretinal injection of human lipid hydroperoxide (HpODE) that found in the sub-macular region in aged and AMD patients. Subretinal HpODE induced retinal pigment epithelium (RPE) and retinal degeneration resulting in loss of RPE cells, photoreceptors and retinal thinning. RPE degeneration and atrophy were detected by day 5, followed by neural tissue degeneration at day 12 with robust TUNEL positive cells. Western blot analysis confirmed an increase in pro-apoptotic Bak protein at day 12 in retinal tissues. Oxidative damage biomarkers (4-hydroxynonenal, malondialdehyde, 8-hydroxy-2′-deoxyguanosine, and nitrotyrosine) increased in retinal tissue from days 5–12. Müller glial activation was observed in the HpODE injected area at day 5 followed by its remodeling and migration in the outer retina by day 20. RT-qPCR analysis further indicated upregulation of pro-inflammatory genes (TNF-α, IL-1β and IL-6) both in retinal and RPE/choroidal tissue as early as day 2 and persisted until day 12. Upregulation of oxidative stress markers such as NADPH oxidase (NOX and DOUX family) was detected early in retinal tissue by day 2 followed by its upregulation in choroidal tissue at day 5. Neovascularization was demonstrated from day 12 to day 20 post HpODE injection in choroidal tissue. The results from this study indicate that subretinal HpODE induces advanced AMD phenotypes comprising many aspects of both dry/early and late) and neovascular/late AMD as observed in humans. Within 3 weeks via oxidative damage, upregulation of reactive oxygen species and pro-inflammatory genes, pro-apoptotic Bak and pro-angiogenic VEGF upregulation occurs leading to CNV formation. This experimental model of subretinal HpODE is an appropriate model for the study of AMD and provides an important platform for translational and basic research in developing new therapies particularly for early/dry AMD where currently no viable therapies are available.
Modulation of inflammatory processes by thermal stimulating and RPE regenerative laser therapies in age related macular degeneration mouse models
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Inflammatory processes play a major role within the multifactorial pathogenesis of age-related macular degeneration (AMD). Neuroretina sparing laser therapies, thermal stimulation of the retina (TSR) and selective retina therapy (SRT), are known to reduce AMD-like pathology in vitro and in vivo. We investigated the effect of TSR and SRT on inflammatory processes in AMD mouse models.
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The inflammatory gene expression profile of both knock out models compared to healthy BL/6J mice suggests a regulation of pro- and anti-inflammatory processes especially concerning T-cell activity and immune cell recruitment. TSR resulted in downregulation of several pro-inflammatory cell-mediators both in ApoE -/- and NRF2-/- mice compared to treatment naïve litter mates one day after treatment. In contrast, SRT induced pro-inflammatory cell-mediators connected with necrosis one day after treatment as expected following laser-induced selective RPE cell death. Seven days after laser treatment, both findings were reversed.
Both TSR and SRT influence inflammatory processes in AMD mouse models. However, they act conversely. TSR leads to anti-inflammatory processes shortly after laser therapy and induces immune-cell recruitment one week after treatment. SRT leads to a quick inflammatory response to laser induced RPE necrotic processes. One week after SRT inflammation is inhibited. It remains unclear, if and to what extent this might play a role in a therapeutic or preventive approach of both laser modalities on AMD pathology.
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7-Ketocholesterol (7KCh), an oxidized form of cholesterol, is present at a high level in drusen and has been believed to play a role in the pathogenesis of age-related macular degeneration (AMD). Therefore, we developed a rat model to study the direct impact of 7KCh on retina. We delivered 7KCh to the rat retina by intravitreal injection using hydroxypropyl-β-cyclodextrin as a vehicle. We observed that 7KCh mainly deposited in the retinal pigment epithelial (RPE) cells and induced marked photoreceptor apoptosis. Transmission electron microscope examination demonstrated cytoplasmic vacuoles in RPE cells and the microvilli detached from the outer segment after 7KCh treatment. In vitro experiments also revealed that RPE cells could take up 7KCh in culture. Moreover, 7KCh up-regulated IL-1βmRNA, TNF-αmRNA, IL-6 mRNA, and IL-1β secretion of RPE. U0126, a MEK1/2 inhibitor, down regulated the expression of these inflammation factors. Our findings may help elucidate the potential role of 7KCh in the pathogenesis of AMD.
Animal models of age related macular degeneration
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Citation Excerpt :
Lipid deposition in Bruch’s membrane is a characteristic component of the pathogenesis of AMD (Wang et al., 2009) and oxidized lipids have been detected in aged Bruch’s membrane (Spaide et al., 1999). Subretinal injection of one such oxidized lipid (13(S)-Hydroperoxy-9Z,11E-octadecadienoic acid (HpODE) led to CNV in a rabbit model (Tamai et al., 2002). Baba et al. extended subretinal injection of HpODE to a rat model, finding photoreceptor degeneration, lipid laden macrophages and RPE cells, and resultant CNV (Baba et al., 2010).
Age related macular degeneration (AMD) is the leading cause of vision loss of those over the age of 65 in the industrialized world. The prevalence and need to develop effective treatments for AMD has lead to the development of multiple animal models. AMD is a complex and heterogeneous disease that involves the interaction of both genetic and environmental factors with the unique anatomy of the human macula. Models in mice, rats, rabbits, pigs and non-human primates have recreated many of the histological features of AMD and provided much insight into the underlying pathological mechanisms of this disease. In spite of the large number of models developed, no one model yet recapitulates all of the features of human AMD. However, these models have helped reveal the roles of chronic oxidative damage, inflammation and immune dysregulation, and lipid metabolism in the development of AMD. Models for induced choroidal neovascularization have served as the backbone for testing new therapies. This article will review the diversity of animal models that exist for AMD as well as their strengths and limitations.
Animal models of choroidal and retinal neovascularization
2010, Progress in Retinal and Eye Research
There have been numerous types of animal models of choroidal neovascularization (CNV) and retinal neovascularization (RNV). Understanding the pathobiology of CNV and RNV is important when evaluating and utilizing these models. Both CNV and RNV are dynamic processes. A break or defect in Bruchs’ membrane is necessary for CNV to develop. This may be induced with a laser, mechanically via surgery, or in the setting of transgenic mice. Some of the transgenic mouse models spontaneously develop RNV and/or retinal angiomatous proliferation (RAP)-like lesions. The pathogenesis of RNV is well-known and is generally related to ischemic retinopathy. Models of oxygen-induced retinopathy (OIR) closely resemble retinopathy of prematurity (ROP). The streptozotocin (STZ) rat model develops features similar to diabetic retinopathy. This review summarizes general categories and specific examples of animal models of CNV and RNV. There are no perfect models of CNV or RNV and individual investigators are encouraged to choose the model that best suits their needs.
A rat model for choroidal neovascularization using subretinal lipid hydroperoxide injection
2010, American Journal of Pathology
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 μg 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.

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^f1: Address correspondence to: Kazushi Tamai, Department of Ophthalmology, Nagoya Higashi-Shimin Hospital, 1-2-23 Wakamizu, Chikusa-ku, Nagoya, 464-0071, Japan. E-mail: [email protected]

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Regular ArticleLipid Hydroperoxide Stimulates Subretinal Choroidal Neovascularization in the Rabbit

Abstract

Ophthalmology

Am. J. Pathol.

Comparative effects of linoleic acid and linoleic acid hydroperoxide on growth and morphology of bovine retinal pigment epithelial cells in vitro

Curr. Eye Res.

Synthesis of lipid and cholesterol hydroperoxide standards

Lipid hydroperoxide stimulates retinal neovascularization in rabbit retina through expression of tumor necrosis factor-α, vascular endothelium growth factor and platelet-derived growth factor

Angiogenesis

Dose dependent mechanisms of lipid hydroperoxide induced retinal pathology

Choroidal neovascularization in the rat induced by adenovirus mediated expression of vascular endothelial growth factor

Invest. Ophthalmol. Vis. Sci.

Region and age-dependent variation in susceptibility of the human retina to lipid peroxidation

Invest. Ophthalmol. Vis. Sci.

Regular Article
Lipid Hydroperoxide Stimulates Subretinal Choroidal Neovascularization in the Rabbit