AAV-9.RSV.AP and AAV-9.CMV.eGFP injection were delivered to young (2- to 3-week-old), adult (3-month-old), and old (12-month-old) C57BL/6J mice. AAV-9.CMV.∆R4–23/∆C injection was delivered to 3-month-old adult mdx3cv mice. HEPES-buffered saline was used as vehicle control in a subset of animals.
Mice were given 1% atropine eyedrops 3 h before they were anesthetized with an intraperitoneal injection of a mixture of 75 mg/kg ketamine and 13.6 mg/kg xylazine. Following general anesthesia, 2.5% phenylephrine hydrochloride eyedrops were applied. One drop of 1% proparacaine hydrochloride was administered as local anesthesia, followed by 2.5% hydroxypropyl methylcellulose. Subretinal injection was performed according to a previous publication with modifications [29 (link)]. Briefly, an aperture within the pupil area was made through the superior cornea with a 30-gauge needle. A 33-gauge blunt needle mounted on a 10-μl syringe was introduced through the corneal opening, avoiding the lens and penetrating the neuroretina to reach the posterior subretinal space. The NanoFil™ sub-microliter injection system (WPI, Sarasota, FL) was used to inject 1 µl of AAV vector in 30 s. The injection was considered successful when retinal blebs occupied more than half of the retina. Evaluation was performed only in mice that were successfully injected. Following subretinal injection, 1% atropine eyedrops and antibiotic ophthalmic ointment were administered daily for three days. Seven days after injection, the mice were anesthetized with ketamine and xylazine as described previously, and their eyes were examined under microscope. Eyes that exhibited any sign of surgical complications, including anterior or posterior synechia, cataract, vitreous and retinal hemorrhage, and unresolved retinal detachment, were excluded from the study. Such signs were observed in 20% to 30% of the eyes.
Mice were given 1% atropine eyedrops 3 h before they were anesthetized with an intraperitoneal injection of a mixture of 75 mg/kg ketamine and 13.6 mg/kg xylazine. Following general anesthesia, 2.5% phenylephrine hydrochloride eyedrops were applied. One drop of 1% proparacaine hydrochloride was administered as local anesthesia, followed by 2.5% hydroxypropyl methylcellulose. Subretinal injection was performed according to a previous publication with modifications [29 (link)]. Briefly, an aperture within the pupil area was made through the superior cornea with a 30-gauge needle. A 33-gauge blunt needle mounted on a 10-μl syringe was introduced through the corneal opening, avoiding the lens and penetrating the neuroretina to reach the posterior subretinal space. The NanoFil™ sub-microliter injection system (WPI, Sarasota, FL) was used to inject 1 µl of AAV vector in 30 s. The injection was considered successful when retinal blebs occupied more than half of the retina. Evaluation was performed only in mice that were successfully injected. Following subretinal injection, 1% atropine eyedrops and antibiotic ophthalmic ointment were administered daily for three days. Seven days after injection, the mice were anesthetized with ketamine and xylazine as described previously, and their eyes were examined under microscope. Eyes that exhibited any sign of surgical complications, including anterior or posterior synechia, cataract, vitreous and retinal hemorrhage, and unresolved retinal detachment, were excluded from the study. Such signs were observed in 20% to 30% of the eyes.
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