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Applicable species: only mice (adults and newborns), rats are generally not recommended (high risk of orbital anatomy) [12-9]. Dose range: equivalent to tail vein IV, commonly used is 1×10^11-2×10^12 vg/animal; literature on neonatal rat AAV9 uses 1.5×10^14 vg/kg[12-10]. Injection volume: 100-150 μL/eye for adult mice (the upper limit for a single injection is 200 μL or 1% of body weight, whichever is smaller); neonatal mice ≤ 10 μL [12-9]. Needle: 27-32 G (29-30 G is commonly used) [12-9]. Speed: Inject slowly for 10-30 s, wait for 2-3 s before withdrawing the needle [12-9]. Anesthesia: General anesthesia is required (isoflurane inhalation is preferred) [12-9]. Operational points: After anesthesia, the head is tilted towards the operator, and the neck is gently pressed to make the retroorbital venous plexus congested with blood. The needle is inserted from the medial canthus along the orbital wall at 45° to the dorsal side for 2-3 mm, and blood is sucked back to confirm the insertion of the plexus; the same eye should be used ≤ 2 times, and the two eyes should be alternated with an interval of ≥ 2 days [12-9]. Coverage: equivalent to system IV (liver, heart, skeletal muscle, BBB); off-target includes occasional inflammation of periocular tissues and Harderian glands. Limitations: There is a risk of eyeball damage, retroorbital hematoma, and keratitis, and some institutions have restricted non-essential use [12-9].