2.7. Intravitreal Injections

All experiments using mice were approved by the Vanderbilt University Institutional Animal Care and Use Committee and were performed in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Eight-week old, male C57BL/6N mice (Charles River Laboratories; Wilmington, MA, USA) were anesthetized by isoflurane (Butler Animal Health Supply; Minneapolis, MN, USA) inhalation. Before intravitreal injection, 0.5% proparacaine (Allergan, Troy Hills, NJ, USA) was topically applied to the cornea. The globe was penetrated approximately 0.5mm posterior to the ora serrata, using a 30-gauge needle with a 19° bevel and a 10μL syringe (Hamilton Co., Reno, NV, USA). The needle was advanced to the posterior vitreous at a steep angle to avoid contact with the lens. The injection bolus was delivered near the trunk of the hyaloid artery proximal to the posterior pole of the retina. After injection, a topical antibiotic suspension (Vigamox; Alcon, Fort Worth, TX, USA) was applied. Mice were given a 1μl injection of vehicle (0.1% DMSO in PBS), GW0742 (1μM), 50ng/ml TNFα plus vehicle, 50ng/ml TNFα plus 1μM GSK0660, or 50ng/ml recombinant CCL8 plus 50ng/ml recombinant CXCL10.