Characterization of the Natural History of Neovascularization and Basal Deposits in a Non–invasive Murine Model of Age–related Macular Degeneration

Abstract

Abstract: : Purpose: 1) Assess formation and origin of non laser–induced neovascularization (NV) in a murine model of age–related macular degeneration (AMD). 2) Localize proteins implicated in human AMD within murine NV and its associated basal deposits. Methods: Human APOE targeted replacement (TR) mouse lines, each expressing one of the three common human APOE isoforms (E2, E3, E4) were created and maintained as described previously (Malek et al., ARVO 2003). Aged mice (average 90 wks) were fed either a fat/cholesterol and sodium cholate–enriched diet for 9 wks or normal chow. Fluorescein angiography (FA) was performed at wk 4 and 8 of diet. At wk 9 mice were euthanized, eyes were enucleated, post–mortem fundus pictures were taken, and eyes were processed for light and electron microscopy (EM). Progression and origin of NV was scrutinized in 1 µm serial sections. Proteins of interest included growth factors, endothelial markers, macrophage markers and cytokines. The histological stain oil red O was used to demonstrate neutral lipid distribution. Results: APOE TR mice express APOE isoforms at physiological levels in patterns similar to wild–type mice and humans. Histologically, retinas of APOE mice on normal chow were unremarkable. APOE2 mice on the cholate diet showed sub–retinal pigment epithelial (RPE) deposits, and photoreceptor and RPE loss. EM analysis identified sub–RPE deposits as basal laminar deposits, which were oil red O positive. APOE3 retinas had a less severely disrupted RPE. Only the APOE4 retinas showed NV (33%) and RPE changes including atrophy, hyper– and hypo–pigmentation. FA at 4 wks revealed regions of hyperfluorescence, corresponding to NV by histology. In some eyes, the number of hyperfluorescent spots increased at 8 wks. Serial sections through NV revealed both a retinal and potential choroidal component. Growth factors and endothelial markers were localized in cryosections with NV. Conclusions: APOE2 and especially APOE4 isoforms, when combined with other AMD–associated risks (advanced age, high fat diet), results in increased degenerative changes in the retina/choroid analogous to that seen in dry AMD (lipid–rich basal deposits) and wet AMD (NV). NV seen in these mice is analogous to that seen in laser induced models. Furthermore, presence of macrophages suggests inflammatory factors may play a role in development of NV in these mice. Knowledge of the natural history of the disease in these mice strengthens this as a potential experimental model of AMD.

Keywords: age-related macular degeneration • neovascularization • growth factors/growth factor receptors