Eyes were enucleated 3 days after laser induction of CNV. The anterior segment and lens were removed, and the retina was carefully separated from the underlying choroid and sclera. The retina was sonicated in 100 μL mammalian cell lysis buffer (5× buffer Tris-EDTA, 5× NaCl, 5× lauryl sulfate, 5× deoxycholic acid, 5× Igepal CA 630, (protease inhibitor enzyme; Sigma-Aldrich, St. Louis, MO) and kept on ice for 30 minutes. The lysate was cleared of debris by centrifugation at 13,000 rpm for 30 minutes at 4°C. The supernatant was assayed for VEGF with an enzyme-linked immunosorbent assay (ELISA) kit (mouse VEGF Quantikine kit; R&D Minneapolis, MN). The choroid and sclera were prepared as a lysate in a similar way to the retina lysate preparation. Total protein was measured with a commercial assay (DC protein assay; Bio-Rad, Hercules, CA). Duplicate measurements were performed for all samples, standards, and controls.
To determine the effect of pitavastatin on VEGF formation in the posterior segment of the eye, we administered CMC or pitavastatin by oral gavage for 3 days without prior laser induction of CNV. VEGF measurements were performed in retina and choroid-sclera lysates.