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J. T. Flynn, Q. Wen; Immunofluorescence Detection of Complement Factor C3 in Murine Oxygen Injury Retinopathy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4469.
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Quan Wen and John T. FlynnDepartment of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, NY 10032
To evaluate whether Complement Factor C3 was present in the retinal neovasculature of new born mice with oxygen injury retinopathy (OIR). This murine retinopathy is the animal model resembling Retinopathy of Prematurity in the human.
Litters of newborn mice were divided in half after birth. At seven days of life (DOL) one half of the litter were placed with nursing mothers into an oxygen incubator maintained at 75 +/- 2% oxygen until postnatal day (P)12. The oxygen exposed mice then were removed from the incubator and maintained in room air until P17. The control half of the litter were raised in room air throughout. Both the oxygen exposed mice and room air mice were sacrificed at DOL 17. The eyes were enucleated and fixed in 4% paraformaldehyde at 4C overnight. After extraction and washing , the retinas were permeabilized with 0.1% Triton and stained with Alexa Fluor 488-conjugated Griffonia Simplicifolia isolectin-B4, followed by blocking with PBS containing 1% Bovine Serum Albumin (BSA) and 5% donkey serum. The retinas were then incubated with rat monoclonal antibody against mouse C3 and Rhodamine (TRITC) conjugated donkey anti-rat IgG. Finally, the retinas were flat-mounted on glass slides with cover slips. Images were obtained with a Zeiss Axioplan 2 microscope.
By P17, retinal neovascularization was present in all oxygen exposed experimental mice. There were extensive C3 punctate immunostainings throughout the retinas, most prominently in the neovascular tufts and large damaged vessels. No positive C3 staining was found in control subjects. This is the first report of the Complement System involvement in OIR.
Complement system is involved in the pathogenesis of murine OIR. Additional studies including Western blot quantification, thin section immunostaining and confocal microscopy are underway to elucidate this finding.
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