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Kyle Carver, Naheed W Khan, Cheng-mao Lin, Dongli Yang; Generation and Phenotyping of P2x7r and Sod1 Double-Knockout Mice. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6546.
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© ARVO (1962-2015); The Authors (2016-present)
Chronic oxidative stress (OS) contributes to AMD. In mice, chronic OS induced by Cu, Zn-superoxide dismutase (Sod1) knockout (KO) leads to features typical of age-related macular degeneration (AMD) in humans. However, the underlying mechanisms of AMD-like pathology in Sod1 KO mice are not fully understood. The P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed in the retinal pigment epithelium (RPE) and neural retina. It mediates multiple critical signaling pathways in the RPE and AMD. We hypothesized that P2X7R deficiency has a protective role in Sod1 KO mice. The purpose of this study was to generate and phenotype P2x7r and Sod1 double-knockout (DKO) mice with particular emphasis on microparticle (MP) release, drusen-like deposit formation, and retinal function.
P2x7r KO and Sod1 KO mice were obtained from Jackson Laboratories, and cross-bred to generate all colonies: wild type (WT), P2x7r KO, Sod1 KO, and P2x7r/Sod1 DKO. MPs were isolated from RPE/choroid complex and quantified by flow cytometry (FC). Drusen-like deposits were examined by fundus imaging and transmission electron microscopy (TEM). Retinal function was examined using the electroretinogram (ERG).
We generated P2x7r/Sod1 DKO and detected MPs isolated from RPE/choroid complex by FC. MP counts were increased in Sod1 KO mice which were attenuated in P2x7r/Sod1 DKO mice. WT, P2x7r KO, and P2x7r/Sod1 DKO littermates had limited evidence of drusen-like deposits in fundus images while drusen-like deposits were present in Sod1 KO mice. The number of drusen per eye was increased in Sod1 KO mice compared to all other groups. TEM showed that Sod1 KO mice developed basal laminar and linear deposits with MP size vesicles present near the deposits. Dark-adapted ERG a- and b-waves were reduced in Sod1 KO mice but were within normal range in the P2x7r/Sod1 DKO mice.
The results indicate that P2X7R deficiency protects against the release of MPs, formation of drusen, and loss of retinal function induced by OS in Sod1 KO mice, suggesting that P2X7R could be critical to MP release and drusen formation under chronic OS.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Drusen-like deposits observed in Sod1 knockout (KO) (A) are attenuated in P2x7r/Sod1 double KO (B) mice. Transmission electron microscopy of basal laminar deposits (C, black arrows) and basal linear deposits (D, blue arrow) in Sod1 KO mice with microparticle sized vesicles present (C, red arrows).
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