Mice were first anesthetized by intraperitoneal injection of midozolam (5 mg/kg), medetomidin (0.5 mg/kg), and fentanyl (0.05 mg/kg). Pupil dilation was achieved with topical application of tropicamide 1% for 1 minute followed by application of phenylephrine 2.5% for 1 minute. Animals were kept on a heating pad at 37°C and eyes were moisturized with Lacrinorm gel (Bausch & Lomb, Zug, Switzerland). Syringes were mounted on a M3301R micromanipulator (WPI, Hertfordshire, England). To place the eye in a proper orientation and have access to the desired injection site, the eyelid was pulled toward the forehead using a surgical suture while a second one was used to attach the sclera slightly above the superior limbus and pull the eye downward in order to expose the central region of the superior sclera (
Supplementary Fig. S2B). Spring scissors with a cutting edge of 2.5 mm were used to cut and remove most of the scleral tissue from the injection site (
Supplementary Fig. S2C). A hole was made 2 mm posterior to the superior limbus using a sterile 30-gauge (G) needle. The needle was inserted 1.5 mm into the vitreous (
Supplementary Fig. S2D). Using a surgical microscope, the position of the needles within the eye was visualized (
Supplementary Fig. S2A). The needle opening was oriented toward the temporal retina facing neither the optic nerve nor the lens. Part of the vitreous was aspirated by pulling the plunger of the syringe up to a maximum of 50 μL on the graduation scale prior to removing the needle from the eye, a procedure that in the following is denoted vitreous aspiration (
Supplementary Fig. S2E). Whether this procedure leads to a removal of a small portion of the vitreous or results only in a vitreous tap is a matter for further investigations. Hemorrhages on the retina were rarely observed. We excluded animals with clear retinal hemorrhages from the study, but included those that showed mild bleeding around the injection site at the sclera. Through the previously made hole, a 33-G blunt-end needle (Hamilton, Bonaduz, Switzerland) mounted onto a 5-μL glass syringe (Hamilton) was inserted 1.5 mm into the eye (
Supplementary Fig. S2F). Adeno-associated virus suspension or PBS (1.5 μL) was intravitreally injected into the eye. After vitreous aspiration, 12 eyes were injected using each of the two viruses (ssAAV2/8_CMV_GFP and scAAV2/8_mCMV_GFP). In addition, 66 eyes were injected using ssAAV2/8_CMV_GFP applying two previously published techniques to inject into the vitreous but without prior vitreous aspiration. Injections were performed at 0.1 μL/s, and after the injection, the needle was left in the eye for 2 minutes in order to prevent reflux of the viral suspension. The needle and surgical sutures were gently removed from the eye and the animal was kept warm for 20 minutes prior to administration of antagonists of the anesthetics (a mixture of atipamezole [0.75 mg/kg], flumazenil [0.2 mg/kg], and naloxone [0.12 mg/kg]). Detailed information about the equipment used during the procedure is provided in
Supplementary Table S1.