Retinal ultrastructure of murine models of dry age-related macular degeneration (AMD)

Abstract

Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness worldwide in the elderly population. The pathology of dry AMD consists of macular degeneration of photoreceptors and the RPE, lipofuscin (A2E) accumulation, and drusen formation. Mice have been widely used for generating models that simulate human AMD features for investigating the pathogenesis, treatment and prevention of the disease. Although the mouse has no macula, focal atrophy of photoreceptors and RPE, lipofuscin accumulation, and increased A2E can develop in aged mouse eyes. However, drusen are rarely seen in mice because of their simpler Bruch's membrane and different process of lipofuscin extrusion compared with humans. Thus, analyzing basal deposits at the ultrastructural level and understanding the ultrastructural pathologic differences between various mouse AMD models are critical to comprehending the significance of research findings and response to possible therapeutic options for dry AMD. Based on the multifactorial pathogenesis of AMD, murine dry AMD models can be classified into three groups. First, genetically engineered mice that target genes related to juvenile macular dystrophies are the most common models, and they include abcr(-/-) (Stargardt disease), transgenic ELOVL4 (Stargardt-3 dominant inheritary disease), Efemp1(R345W/R345W) (Doyne honeycomb retinal dystrophy), and Timp3(S156C/S156C) (Sorsby fundus dystrophy) mice. Other murine models target genes relevant to AMD, including inflammatory genes such as Cfh(-/-), Ccl2(-/-), Ccr2(-/-), Cx3cr1(-/-), and Ccl2(-/-)/cx3cr1(-/-), oxidative stress associated genes such as Sod1(-/-) and Sod2 knockdown, metabolic pathway genes such as neprilysin(-/-) (amyloid beta), transgenic mcd/mcd (cathepsin D), Cp(-/-)/Heph(-/Y) (ferroxidase ceruloplasmin/hepaestin, iron metabolism), and transgenic ApoE4 on high fat and high cholesterol diet (lipid metabolism). Second, mice have also been immunologically manipulated by immunization with carboxyethylpyrrole (CEP), an oxidative fragment of DHA found in drusen, and found to present with dry AMD features. Third, natural mouse strains such as arrd2/arrd2 (Mdm gene mutation) and the senescence accelerated mice (SAM) spontaneously develop features of dry AMD like photoreceptor atrophy and thickening of Bruch's membrane. All the aforementioned models develop retinal lesions with various features that simulate dry AMD lesions: focal photoreceptor degeneration, abnormal RPE with increased lipofuscin, basal infolding, decreased melanosomes and degeneration. However, Bruch's membrane changes are less common. Most mice develop retinal lesions at an older age (6-24 months, depending on the models), while the Ccl2(-/-)/cx3cr1(-/-) mice develop lesions by 4-6 weeks. Although murine models present various degrees of retinal and/or RPE degeneration, classical drusen is extremely rare. Using electron microscopy, small drusenoid deposits are found between RPE and Bruch's membrane in a few models including Efemp1(R345W/R345W), Ccl2(-/-)/cx3cr1(-/-), neprilysin(-/-), transgenic mcd/mcd, and ApoE4 transgenic mice on a high fat diet. High A2E levels are measured in the retinas of abcr(-/-), transgenic ELOVL4, and Ccl2(-/-)/cx3cr1(-/-) mice. In summary, murine models provide useful tools for studying AMD pathogenesis and evaluating novel therapies for this disease. This review compares the major dry AMD murine models and discusses retinal pathology at the ultrastructural level.

Figure 1. A comparison of the mouse and human retina and AMD-like pathology

a) A normal human fundus, b) light microscope histologic picture of the normal human maculae, c) the fundus of a patient with dry AMD with drusen and scarring in the maculae, d) light microscope histologic picture of a patient with dry AMD, e) A normal mouse fundus, f) light microscope histologic picture of the normal mouse retina, g) fundus picture of a 3 month-old Ccl2^−/−/Cx3cr1^−/− mouse with drusenoid deposits and a retinal scar, h) light microscope histologic picture of a 3 month-old Ccl2^−/−/Cx3cr1^−/− mouse with RPE hyperpigmentation and hypertrophy and photoreceptor outer segment disorganization and atrophy

Figure 2

Transmission electron micrographs illustrated outer retinal degeneration in a 21-days old Ccl2^−/−/Cx3cr1^−/− mouse. Comparing with WT mice (A1–2), Ccl2^−/−/Cx3cr1^−/− mice revealed disorganization and migration of photoreceptors (open arrow in B1), rich lipofuscin (arrows in B2) and more basal infoldings (bi in B2) in RPE, as well as higher electron-dense of elastic layer in BM (asterisk in B2). Scale bars, 10 μm (A1, B1); 500 nm (A2, B2). (ONL, outer nuclear layer or photoreceptor nuclei; IS, inner segments of photoreceptors; OS, outer segments of photoreceptors; RPE, retinal pigment epithelial cells; BM, Bruch’s membrane)

Figure 3

Diagrams of classic ultrastructural changes in the photoreceptor, RPE, Bruch’s membrane and choroid in different mouse AMD models (IS, photoreceptor inner segment; OS, photoreceptor outer segment; RPE, retinal pigment epithelial cells; BM, Bruch’s membrane; Cr, Choroid; BlamD, basal laminar deposits; CNV, choroidal neovascularization)