Animal models of cochlear implants can advance our knowledge of the technological basis of treating permanent sensory neural hearing loss with electric stimulation. This protocol demonstrates acute deafening and cochlear implantation in the mouse. And the functional assessment of cochlear implant stimulation with auditory brain stem response.
Some main challenges of performing cochlear implant surgery in mice are the small size of the cochlea and the presence of the large stapedial artery near the round window niche. To measure the normal hearing status, load acoustic foam into a one milliliter syringe and inject 100 to 200 microliters of foam into the contralateral ear canal of an anesthetized eight to twelve week old C57 black 6 mouse to isolate the auditory brain stem response from the ipsilateral ear, taking care that the syringe seals closely to the ear for delivery of the foam all the way into the ear canal. Next, place a speaker 10 centimeters from the ipsilateral ear and clean the aVR electrodes with 70%ethanol.
Place the reference electrode under the skin below the pinna of the ipsilateral ear and the active electrode under the skin of the vertex. The put the ground electrode under the skin of the hind leg. Connect the headstage and preamplifier to the auditory processor via the optic fiber port.
And check the impedance of the active and reference electrodes. When the electrodes display the same impedance, close the soundproof booth and present the click stimulation. Recording the aVR in a free field condition with an appropriate complex auditory processor and software.
Standardize the click stimulus to 0.1 milliseconds single channel monophasic clicks presented at 21 hertz with a decreasing click level from 90 decibels of sound pressure level to 10 decibels of sound pressure level in 10 decibel steps in a 10 millisecond recording window. Then determine the aVR threshold as the lowest decibel level with a recognizable aVR wave response. To induce the deafening, place the mouse on its right side taking care to keep the body straight so the airways remain open.
Remove the fur behind the ipsilateral ear and disinfect the exposed skin with sequential 70%ethanol solution and a Povidone-Iodine Solution Scrubs. Move the animal under a dissecting microscope and use the 16 times objective and a scalpel to make a one to one and a half centimeter postauricular incision. Under a 25 to 40x magnification, use forceps to blunt dissect through the exposed subcutaneous fat layer and retract the sternocleidomastoid muscle to reveal the timpanic bulla periosteum.
The facial nerve wraps around the posterior dorsal edge of the sternocleidomastoid muscle and runs rostrally along the ear canal toward the pinna. Using the facial nerve as a landmark for identification of the auditory bulla gently place a self-retaining retractor tool into the incision for access to the bulla. Remove the tissue overlying the mediodorsal area of the bulla to allow clear visualization of the ridge between the bulla and the mastoid process and gentle rotate a 30 guage needle into the bulla to generate a hole on the posterior superior side of the ridge.
Pinch small bone pieces with fine tipped forceps to widen the bullastomy until the middle ear cavity is exposed. And extend the hole dorsally toward the mastoid process until the round window niche is clear of overlying bone. The most critical step is the preparation of the window niche.
Be sure to take your time as so not to damage the stapedial artery. Taking care not to damage the stapedial artery, extend the bullastomy in the anterior superior direction to visualize the stapes, the middle ear bone connected to the oval window. Then remove the stapes to expose the oval window.
To apply the ototoxic agent, use a blunted 30 guage needle to gently perforate the round and oval window membranes, confirming that the perilymph runs out. And use a one milliliter syringe equipped with a 30 gauge needle to slowly perfuse 0.2 milliliters of 5%weight by volume neomycin dissolved in PBS through the oval window. When the entire volume of agent has been delivered profuse the solution into the round window taking care not to damage the window bone structures.
And place one square millimeter pieces of spongostan soaked in neomycin within the round window and oval window niche. Then remove the retractor and close the incision, recording a post-deafening acoustic aVR as demonstrated after 30 minutes. To insert the cochlear implant electrode array, place the retractor tool back into the incision to re-access the bulla and insert the electrode array into the scala tympani at a depth such that the fourth platinum ring of the array is located just inside the round window.
The insertion of the electrode array can also be tricky. So take your time to find a good insertion angle using one forcep tool to lead wire and another to insert the array. Coil the lead wire inside the bulla and glue the wire to the tissue above the bulla.
After carefully removing the retractor and closing the insertion with tissue glue, use a scalepel to make a 0.5 millimeter incision in the neck, perpendicular to the line between where the active and reference aVR electrodes will be. Place the ground ball in the insertion and connect the electrode array board to the animal stimulator platform. To perform and electric aVR, place the electrodes as previously demonstrated and open the animal stimulator platform software.
Then define the electric pulse stimulation paradigm and present the electric pulse trains recording the evoked electric aVR responses continuously via the TBT Headstage, preamplifier, and auditory processor. Pre and post surgical hearing thresholds serve as a functional readout of the deafening procedure. Topical application of 5%neomycin to the oval and round windows significantly increases the clicked evoked hearing thresholds.
Electric stimulation of an intracochlear electrode post deafening can reliably generate electric aVR activity. In some cases, cochlear implant stimulation activates the facial nerve and produces a high amplitude wave with either a short latency characterized by a rapid amplification of Wave III around three milliseconds and likely a direct response of the facial nerve, or a long latency which appears around five to six milliseconds and is likely to be a non auditory myogenic response, invoked indirectly by the facial nerve. In this study we have demonstrated that the cochlear implant mouse model is feasible also the cochlear implant is small and the surgery:challenging.
While attempting this procedure, it's important to consider a deafening protocol to eliminate any electrophonic responses in the aVR recordings and to mimic the hair cell loss found in most C.I.users. In summary, the growing number of genetic models of human deafness and the biomedical tools available in mice makes us an attractive animal model for the tool research including for cochlear implants.
