Intravitreal and subretinal injections were performed as previously described [9 (link),24 (link),25 (link)]. Within the sterile surgical room, adult Balb/c mice were anesthetized by an intraperitoneal injection of 85 mg/kg ketamine and 14 mg/kg xylazine (Henry Schein Animal Health, Dublin, OH, USA). Eyes were dilated with 1% cyclopentolate (Bausch and Lomb, Rochester, NY, USA). For intravitreal injections, a very shallow puncture was made through the sclera in the dorsal hemisphere using a beveled 30-gauge needle (BD Biosciences, Franklin Lakes, NJ, USA). Through this puncture, a 33-gauge blunt-end needle was introduced into the vitreous cavity and 1.5–3 µL of material (nanospheres, saline, LPS, ST, or ST/HA-NS mixture) was injected into the vitreous space near optic nerve using a 5 µL microinjection Syringe (Hamilton, Reno, NV, USA). For subretinal injections, the needle was then advanced gently through the retina and into the subretinal space, where the nanosphere suspension was gently delivered. Visualization of a subretinal bleb during injection and subsequent fundoscopic and OCT imaging confirmed successful subretinal delivery. Triple antibiotic ointment (Taro Pharmaceuticals, Inc., Hawthorne, NY, USA) was applied to each eye after injection.
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