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Seth Daniel Fortmann, Valeria E Lorenc, Sean Hackett, Peter A Campochiaro; Murine Vitreous Tap (MurViTap): a novel technique to extract uncontaminated mouse vitreous humor, quantify retinal vascular permeability, and compare proteins secreted by diseased and normal retina. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5978.
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To obtain uncontaminated mouse vitreous for precise measurement of blood-retinal barrier breakdown in disease states and to compare secreted protein levels in diseased versus normal retina
MurViTap was done using the Harvard Pump Microinjection System and pulled glass micropipettes. The micropipette tip was cleaved at an inner diameter of 80-100 microns and was inserted into the vitreous cavity 1-2 mm posterior to the limbus. Gentle aspiration was used to obtain 2-4 µL of undiluted vitreous per eye. To quantify vascular permeability, vitreous was collected from P17 normoxic (n=6) and oxygen-induced ischemic retinopathy (OIR, n=5) mouse eyes, adult wild type mice (n=6), P30 Rho/VEGF transgenic mice (n=5), Tet/Opsin/VEGF transgenic mice 2 days post-doxycycline induction (n=6), and mice with choroidal neovascularization (CNV) 7 days after laser induced rupture of Bruch’s membrane in 5 locations (n=5). A mouse albumin ELISA was used to measure albumin levels in 1 µL of diluted vitreous. To compare the retinal secretome of OIR mice to age-matched controls, vitreous was collected from P15 OIR mice (n=40 eyes) and P15 wild type mice (n=40 eyes). A mouse angiogenesis protein array was used to compare the vitreous levels of 53 angiogenesis-related proteins.
Mean vitreous albumin concentration was significantly greater in OIR versus age-matched control mice (106± 10.3 µg/mL vs. 7.37±1.13 µg/mL; p<0.01). Compared to adult wild type mice (2.58±0.35 µg/mL), mean vitreous albumin concentration was significantly greater in Rho/VEGF mice (15.9±3.18 µg/mL; p<0.05), doxycycline-treated Tet/Opsin/VEGF mice (56.5±3.18 µg/mL; p<0.01), and mice with CNV in 5 locations (13.8±3.01 µg/mL; p<0.05). Eighteen secreted angiogenesis-related proteins were found to be differentially concentrated in OIR vitreous: PAI-1, IGFBP-3, IGFBP-1, IGFBP-2, NOV/CCN3, OPN, CXCL4, Endostatin, PTX-3, PIGF-2, CXCL16, DPPIV, MMP-3, MMP-9, PEDF, TIMP-1, SDF-1, and MCP-1.
Obtaining uncontaminated vitreous combined with albumin ELISA provides a precise quantitative measurement of the extent of blood-retinal barrier breakdown and combined with protein arrays allows the comparison of proteins secreted by the retina under disease versus normal conditions.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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