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Jessica Gomez, Zhichun Jiang, Jane Hu, Marcia Lloyd, Samer Habib, Steven Nusinowitz, Roxana A. Radu; The Cfh-/-Abca4 -/- Double Knockout Mouse Model: Investigating The Development Of Retinal Disease And Complement Regulation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1631.
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Chronic inflammation has been shown to underlie the pathogenesis of age-related macular degeneration with one of the main contributors being activation of complement. Activation can be induced by the accumulation of photo-oxidized forms of A2E, a bis-retinoid pigment that aggregates as lipofuscin in retinal pigmented epithelial (RPE) cells. Autofluorescent subretinal deposits, thought to be evidence of A2E-lipofuscin accumulation in the RPE, have been shown to form in certain mouse models including albino Abca4-/- and Cfh -/- mutants. In the current study, we generated Cfh-/-Abca4 -/- double-knockout (DKO) mice to investigate the pathogenic effects of losing both proteins.
Cfh-/-Abca4-/- (DKO), Cfh-/-, Abca4-/- and 129/Sv wild-type (WT) mouse strains were used in this study. All strains were pigmented and on the rpe65-Leu450 background. Retinas and RPE-containing eyecups were collected from age-matched mice at different times. Levels of the negative complement-regulatory protein (CRRY, CFH, CD59a, CD59b, DAF1, DAF2) mRNA’s were measured by qRT-PCR. A2E-lipofuscin pigments were quantified by HPLC. Retina structure was evaluated in vivo by optical coherence tomography (OCT) and fundus photography. Retina histology was assessed by light microscopy. Autofluorescence was quantified by confocal microscopy, and Bruch’s membrane thickness was measured by electron microscopy.
Fundus photography showed subretinal depigmentation in DKO mice beginning at eight months. The DKO strain showed significantly higher A2E-precursors in the RPE compared to the other strains at eight months. Consistently, RPE autofluorescence was also higher in the DKO mice at eight months. Bruch's membrane was significantly thickened in the DKO mice compared to the other strains at 17-20 months. By OCT, retina thickness was decreased by 10% in DKO versus WT mice at 13 months. Surprisingly, two-month-old DKO mice showed significantly increased complement regulatory protein mRNA levels (approximately two-fold increased CRRY and CD59a, and five-fold increased CD59b) compared to age-matched WT mice, suggesting a compensatory mechanisms due to loss of Cfh.
These data suggest that the combined loss of Cfh and Abca4 leads to significant lipofuscin accumulation in the RPE compared to the single-mutant models. Decreased retinal thickness by OCT suggests ongoing retinal degeneration in the DKO mice. Increased expression of several complement regulatory genes may indicate abnormal complement activation as a mechanism for the retinal and RPE pathology in DKO mice.
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