Laser-induced choroidal neovascularization (CNV) mice model

To induce choroidal neovascularization in mice, laser coagulation was used as described (27). All animals used in this model were between 6 and 8 wk of age and of female gender to exclude any variance caused by gender. Briefly, mice were anesthetized with a mixture of ketamine (40 mg/kg body weight) and xylazine (20 mg/kg body weight). Pupils were dilated with tropicamide eye drops (Mydriadicum Stulln; Pharma Stulln GmbH). To keep eyes moist, hydroxypropyl methylcellulose (Methocel 2%; OmniVision GmbH) was applied on both eyes before placing mice on warm platform. Photocoagulation was performed using the image-guided laser system (Micron IV; Phoenix Research Laboratories) with the following parameters: laser wavelength, 532 nm; burn duration, 70 ms; energy, 280 mW for mixed-background Tpc2^−/− and litter-matched WT mice and 230 mW for C57BL/6J mice. Four laser burns per eye were injected on both eyes in a distance of two optic discs from the optic disc border.

Angiographic analysis of CNV leakage was performed on day 7 and day 14 post laser coagulation. Mice were anesthetized and pupils dilated as described above. Mice received a subcutaneous injection of fluorescein sodium solution (7.5 mg/kg) to visualize vessels. Fundus fluorescein angiography (FFA) was performed 5–10 min after the injection with the retinal imaging microscope (Phoenix Research Laboratories). Leakage areas were quantified using ImageJ64 software (National Institutes of Health).

For experiments with pharmacological inhibitors, mice were anesthetized and pupils dilated as described above. Anesthetized mice were subjected to a single intravitreal injection with 1 µl of 10 µM tetrandrine or 2 mM Ned-19 into right eyes, respectively. Left eyes were used for vehicle controls. Laser injury was performed immediately after intravitreal injection and CNV leakage was assessed on day 14 after laser treatment as described above.