The overall goal of this work is to describe and compare two micro-surgery approaches for local drug delivery to the inner ear in terms of impact on hearing parameters and cochlear cytoarchitecture markers. These procedures can help us to answer key questions in the auditory field, especially in the development of animal pro-chemical studies. The main advantage of these techniques is that they are minimally invasive.
And this allows the substance to reach didactically the inner ear. The application of these techniques extends towards therapy of hearing loss, because its microsurgical procedures are suitable for dry delivery in the middle ear. Peaceful demonstration of this method is critical, because bullostomy and transtympanic injection are difficult to learn and they require an anatomical knowledge and surgical training.
To begin, place an ear plug in the contralateral ear of an anesthetized mouse to obtain predominately mono-aural responses. Then, place the mouse in prone position on a heating pad within a sound-attenuating chamber. Place the positive stainless steel subdermal needle electrode in the scalp between the ears over the vertex of the skull.
Place the reference electrode in the parotid region below the pinna. Then place the ground electrode in the back, tail, or hind limb region. Check the electrical impedance in the positive and negative electrodes.
Ensure that the impedance is less than three kiloohms. Place the pre-filled speaker at a fixed distance facing the selected ear, with the center of the speaker aligned with the external auditory canal. Ensure that there are no obstacles between the speaker and the ear, and that the pinna is completely opened.
Connect the digital-to-analog workstation output to the selected speaker. Free-field speakers are used here. Connect an amplifier with low noise and a good signal-to-noise ratio to the analog-to-digital input.
Then begin the process to deliver acoustic stimuli into the external auditory canal using preset stimuli or new signals designed with the appropriate software. Present the stimuli beginning with high intensity and reducing the intensity in five to 10 decibel sound pressure level steps. Record the electrical response in the first 10 milliseconds after stimulation, as evoked ABR responses appear at six to eight milliseconds.
Use the software to determine the ABR threshold as described in the written portion of the protocol. After administering anesthesia according to approved protocols, check that a surgical plane of anesthesia has been reached by the absence of a reaction to a toe pinch and the presentation of regular breathing. Use clippers to remove the fur at the ventral surface of the neck and protect the eyes from corneal keratitis with a hydroxy-propyl-methyl-cellulose based gel.
Place the mouse in the sterile surgical area in a decubitus supine position. Clean the skin with a povidone-iodine based antiseptic solution and cover with a sterile drape. After using a scalpel to make a two-centimeter longitudinal incision from the mandible to the clavicle, look through the surgical microscope and identify the submandibular glands.
Separate both submandibular glands with forceps, retract them, and localize the origin of the digastric muscle in the facial nerve. Next, make an incision in the origin of the digastric muscle with a scissors, and retract it ventrally, exposing the underlying inferior medial aspect of the tympanic bulla. Then, use a 27-gauge needle to drill into the bulla and create an opening.
Clean the blood from the drilled area with an absorbable gelatin sponge. Locate the stapedial artery in the round window membrane coddled to it. Using a 34-gauge catheter and a glass micro-syringe, slowly inject three to five microliters of vehicle solution through the bullostomy and directly onto the round window niche to fill it.
Seal the bullostomy with one to two drops of tissue adhesive. Return the submandibular glands to their initial position, and close the skin incisions with 5-0 silk surgical suture. Apply povidone-iodine or chlorhexidine-based antiseptic around the incision to avoid wound infection.
Keep the mouse on a heating pad until it has regained sufficient consciousness to maintain sternal recumbency. Return the animal to the home cage only after it is fully recovered. Place the mouse in the lateral decubitus position and prepare in a septic surgical area below the external auditory meatus as previously shown.
After making a 0.5 centimeter longitudinal incision in the vertical part of the outer ear canal, locate the tympanic membrane with the aid of the surgical microscope. Identify the upper pars flaccida in the inferior pars tensa, which is divided into anterior and posterior sections by the handle of the malleus. Then, make a small myringostomy in the caudal portion of the pars flaccida, and an additional incision in the pars tensa of the tympanic membrane to allow air evacuation during injection.
Use a glass micro-syringe connected to a 34-gauge catheter to gently inject 10 to 15 microliters of vehicle through the pars flaccida, close to the round window niche, until the middle ear is clearly full. Close the skin incisions with a 5-0 silk surgical suture and clean as previously described. This graph shows ABR thresholds in male 8-week old C57BL/6J mice in response to click stimuli before surgery and seven, 14, and 28 days after microsurgery.
Bullostomy data are shown in orange, transtympanic injection data are shown in blue, and data from non-operated animals are shown in gray. This graph shows the ABR thresholds in response to tone-burst stimuli in the range of eight to 40 kilohertz over the same time period following microsurgery. Again, no significant differences were observed between bullostomy and trans-tympanic approaches.
Morphological studies demonstrate that the scala media does not show alterations following vehicle delivery by bullostomy or transtympanic injection, indicating that either procedure alone does not result in damage. While attempting the bullostomy procedure, it is important to remember to review the critical and anatomical structure surrounding the middle and inner ear of rodents. Following these procedures, other techniques could be performed.
For example, Berlin sampling for pharmacokinetical studies After its development, these techniques pave the way for researchers in the field of auditory neuroscience to explore potential local therapies in animal models, mainly in rodents. After watching this video, you should have a good understanding of how to administer drugs in the middle ear and how to assess hearing in rodents.
