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Applicable species: newborn/adult mice, rats, NHP, cats/dogs/pig/sheep[12-29,12-36]. Dose range: neonatal P1 mice 1×10^10 vg; adult mice 1×10^10-7×10^10 vg; rats 1×10^12 vg; NHP rhesus GLP low dose 1.7×10^13 GC, high dose 5×10^13 GC[12-29,12-36]. Injection volume: mice 1-10 μL; rats 50-100 μL; NHP 1-2 mL[12-29,12-36]. Needle: Mouse 33 G with Hamilton+ peristaltic pump; NHP 21-27 G Quincke lumbar puncture needle, C-arm fluoroscopy/CT guidance [12-36]. Speed: 1 μL/min for mice; 10 μL/min for rats (100 μL CSF reserve volume is drawn first); NHP can push 1 mL within 30±5 s (approximately 2 mL/min) [12-29,12-36]. Anesthesia: neonatal P1 5% isoflurane induction and 2% maintenance; rat ketamine + xylazine + acepromazine IP; NHP isoflurane lateral head flexion [12-29]. Operational points: The key risk is brainstem puncture (needle tip is too low/too deep → respiratory and cardiac arrest), and large animals must be guided by images; in rats, the needle hole is sealed with fibrinogen + thrombin; after surgery, Trendelenburg is inverted to increase brain coverage [12-30,12-36]. Coverage: ICM brain coverage is significantly wider than that of lumbar puncture LP - extensive cortex, hippocampus, cerebellum, brainstem; cervical/thoracic > lumbar segment (opposite to LP); high transduction of meningeal lymphatic vessels; peak expression at 3-4 weeks (scAAV9) [12-30,12-37]. Safety: brainstem puncture (most serious immediate risk), CSF leakage, DRG lesions, high-dose cerebellar toxicity, CSF lymphocytosis [12-36,12-38].