This article describes a surgical technique for delivering a therapeutic gene to restore hearing in a mouse model with genetically induced hearing loss. The method involves precise manipulation of the inner ear structures to enable viral vector injection.
Take an anesthetized mouse pup with genetically induced hearing loss.
In this condition, hair cells in the inner ear lack glutamate transporters, preventing glutamate from being packaged into vesicles and released.
This disrupts the transmission of sound signals to the auditory nerve.
Shave the mouse behind its left ear, disinfect, and apply eye ointment.
Extend the neck and make an incision.
Retract the adipose tissue and muscles to expose the temporal bone.
Perforate the tympanic bulla, part of the temporal bone, and widen the opening to expose the round window membrane or RWM, a thin barrier to the inner ear.
Puncture the RWM and inject the adeno-associated viral vector carrying the therapeutic gene.
The viral particles diffuse and enter hair cells.
Inside hair cells, viral particles deliver their genetic material to the nucleus, triggering glutamate transporter production.
This allows glutamate packaging and release, restoring sound signaling to the auditory nerve.
After confirming anesthesia by toe pinch, shave the left postauricular region and disinfect the skin of a postnatal day 10 to 12 mouse with 70% ethanol and povidone iodine. Then cover the eyes with a protective ophthalmic ointment, and position the animal with the neck extended on a heating pad. When the surgical preparation is complete, incise the subcutaneous tissue with small scissors to expose the postauricular muscle. After retracting the adipose tissue to the posterior side of the incision, separate the muscles to the right and left sides, perpendicular to the incision.
To expose the temporal bone. Use a 25-gauge needle to perforate the tympanic bulla. Peeling the bone back with forceps as necessary to expand the hole, and to allow access to the basal turn of the cochlea.
Then, widen the hole sufficiently to allow visualization of the stapedial artery and the round window membrane. Puncture the round window membrane gently in the center with a borosilicate capillary pipette. Dry the round window membrane with a sterile filter paper. Then wait five to ten minutes for the fluid efflux to stabilize.
At this step, take care when holding and advancing the pipette, so to make the round window membrane perforation as small as possible. Depending on the microscope use, the pipette can be held by hand or by micromanipulator. As you can see here, we hold the pipette by hand, as the microscope is too close to the mouse to allow for a micromanipulator.
Now, draw one to two microliters of the virus into a pulled glass pipette with a 15-micrometer diameter tip and micro-inject the virus into the scala tympani through the same hole previously made in the round window membrane.
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