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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparing the Animal for Surgery
- Carry out surgical procedures in a clean, dedicated space. Autoclave all surgical instruments, and sterilize with a glass-bead sterilizer prior to surgery.
NOTE: In this protocol, use postnatal day 10-12 (P10-12) FVB mice. Different ages and strains of mice can be used to meet the needs of a specific project. Mice older than P40 can be challenging because the bulla bone gets harder. - Anesthetize the mice with an intraperitoneal injection of a mixture of ketamine hydrochloride (100 mg/kg), xylazine hydrochloride (10 mg/kg), and acepromazine (2 mg/kg). Begin surgical preparation only after the animal no longer responds to painful stimuli, such as toe pinch. If necessary, administer a booster dose (one-fifth of the original dose) of the anesthetic cocktail to restore the original anesthetic plane.
NOTE: Take care at this step because most of the mortality observed at this age is caused by anesthetic overdose. - Cover the animal's eyes with a protective ophthalmic ointment to keep the eyes moist during anesthesia, suppressing the animal's blink reflex.
- To prevent post-anesthesia hypothermia, position the mouse with its neck extended on a heating pad throughout the procedure until it is totally awake.
- Shave the left post-auricular region with a clipper and disinfect with 70% ethanol and povidone-iodine before surgical manipulation.
2. The Surgical Procedure and Vector Injection
- Use a post-auricular approach to expose the tympanic bulla. Incise the subcutaneous tissue with small scissors to expose the post-auricular muscle. After retracting the adipose tissue to the posterior side of the incision, separate the muscles on the right and left sides perpendicular to the incision to expose the temporal bone. Ensure that this incision is long enough to work comfortably with adequate visualization.
- Perforate the tympanic bulla with a 25 G needle and expand the hole as necessary with forceps by peeling the bone back to allow access to the basal turn of the cochlea, and then widen sufficiently to visualize the stapedial artery and the round window membrane (RWM).
- Puncture the RWM gently in the center with a borosilicate capillary pipette. Observe fluid efflux through the RWM at this point; this is normal. Wait until the efflux has stabilized (the effluxed fluid from the cochlea is dried with a sterile filter paper).
NOTE: Take care when holding and advancing the glass needle so that the RWM perforation is as small as possible. Depending on the microscope used, hold the pipettes using a micromanipulator or by hand using a pipette holder. - Prepare the pipettes for injection using a pipette puller, the same way patch clamp pipettes are prepared. Ensure that the tip diameter is large (about 15 µm in diameter) so that pipetting the virus in and out is done easily.
- Draw up 1 to 2 µl of adeno-associated viruses (AAV1-VGLUT3, AAV1-GFP, or AAV2-GFP) into an injection pipette. After the efflux is stabilized (5 to 10 min), microinject this fixed volume into the scala tympani through the same hole previously made in RWM.