Laser-Induced CNV Mouse Model

To induce CNV in C57BL/6 mice, laser-induced ruptures of the Burch's membrane were performed with an 532 nm green laser photocoagulator mounted on a slit-lamp (Nidek Inc.; San Jose, CA). Four lesions were created in both the left and right eyes of each mouse. Laser parameters used were 100-μm spot size, 0.1-s duration, and 0.3 Watts. On day-3 post-laser treatment, mice were divided into two groups and received systemic injections of AS-Eng shRNA–lipid at a concentration of 0.5 mg/kg in PBS. Eighteen hours after the probe-injection, the mice were sacrificed and the eyes were fixed in neutral buffer formalin (NBF) for 30 min. The anterior segments and lenses were removed while submerging the eye in NBF solution. The choroid-Bruch’s membrane-RPE complex was dissected as previously described^{40 (link),41 (link)} and tissues were stained and analyzed for IBA1 and IB4, as described above.