The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

The overall goal of this procedure is to establish an inner ear injury model by strategically delivering ototoxic agents directly to the round window niche of the mouse cochlea. This is accomplished by first dissecting the subcutaneous fat layer and gently withdrawing the muscle body to reveal the tympanic bulla periosteum. The second step is to uncap the bullah by gradually removing bone fragments until the round window niche is exposed.

Next, the visible fluid of the middle ear and round window niche is dried completely allowing for optimal visualization of the round window niche. The final step is to fill the round window niche with the selected ototoxic agent without disrupting the STA pedial artery, followed by appropriate repetitions according to specifications of the agent. Ultimately, functional procedures, including auditory brainstem response and morphological evaluation are used to show the selective preservation or degeneration of the cochlear micro architecture.

The main advantage of this technique, our existing measure, is that this procedure allow for excellent exposure of the wrong window niche and the memory to reach a selective auto Toxic aging can be directly applied. Advantages of this technique include preservation of inner ear, micro architecture, avoidance of systemic toxicity, speedy onset of effect, selective degeneration of certain cochlear cell types and reproducible results. This technique can be applied with few alterations between other rodent species, including rats, Guinea pigs, and gerbils.

Now I will demonstrate the procedure in my laboratory. All aspects of animal research were conducted in accordance with the guidelines of the appropriate institutional animal care and use committee. All vertebrae experimental procedures described here were approved under the guidelines of the Medical University of South Carolina's Institutional Animal Care and use Committee.

After anesthetizing the animal according to approved procedures, administer preoperative analgesic 0.1 milligrams per kilogram. Buprenorphine is used here. Check that the animal has reached a surgical plane of anesthesia.

There should be no response to a toe pinch, and the writing reflex should be absent in mice as seen here. Prera 0.2 milliliters of an aqueous solution containing the selected ototoxic agent into a one milliliter tuberculin syringe fitted with a blunted 28 gauge half inch needle, expel any air within the syringe as bubbles can prevent proper application of the agent. Use electrical clippers to remove fur in the area, extending from the acephalic crease, roly to the shoulder girdle coddly.

Extend hair removal from the dorsal sagittal midline to the mandibular angle laterally. Use a disposable razor to assure a cleanly shaved preparation. Sterilize the skin of the prepared area according to institutional protocols.

Here Betadine alternated with ethanol is applied topically in a circular manner for two minutes. Place a small heating pad under the body to maintain the animal's body temperature. At 36 to 38 degrees Celsius, secure the head of the animal via a head holder apparatus.

Gently tighten a small clamp over the dorsum of the animal's snout to hold it in place. Confirm that the head holder is rigidly connected to the center of a U-shaped articulating arm. Secure a one centimeter wide rod to the left arm of the U to be used as a left side headrest to allow access to the right side.

Once firmly secured in the head holder, rotate the mouse to a left lateral decubitus position. Position the body carefully on the flat operating surface to ensure it will be stable throughout the procedure and avoid undo torsional stress on the cervical vertebrae. Position an operating microscope capable of Forex 10 x and 20 x magnification over the surgical field.

Confirm that the microscope can maintain its position in a hands-free manner so that both hands can be used for surgery while working under microscopic magnification. Use sharp scissors or a scalpel blade to create a one to 1.5 centimeter postauricular Incision approximately six to eight millimeters coddle to the auricular cephalic crease. Avoid cutting deeply to preserve the underlying vascular structures.

Blunt dissect through the subcutaneous fat layer. If dissecting in a ventral to medial direction, take care to avoid the external jugular vein. If bleeding occurs, use absorbable gelatin, sponges, or cotton pellets to stem the flow.

Once the fat layer is properly divided, expose the cervical musculature. An important landmark is a small nerve branch of cranial nerve 11 that wraps around the posterior edge of the C CTO mastoid. To extend roly toward the pinna, use a self retaining retractor to gently retract the CTO mastoiditis muscle body in a posterior direction.

Gently divide the transparent fascia, enveloping the muscle body. Also gently retract the parotid and external jugular vein in the opposite direction. With good retraction of the CTO Masto muscle body, the shiny dome of the tympanic bulla periosteum is visible at the coddle aspect of the bulla, the insertion of a deeper cervical muscle.

The Sterno Mastoiditis will come into view. Place a self retaining retractor to hold the CTO mastoid apart using two-handed technique, gently used bipolar diathermy to divide the bull periosteum and expose the underlying bone. Then use forceps or an otologic cured to gently elevate and push the periosteum in a peripheral direction to fully expose the bull dome.

Now use a belt-driven dental hand drill with a one or two millimeter tapered tip to carefully drill a two millimeter pilot hole through the bullah bone. Drill between the coddle margin of the dome and the visibly opaque line extending across the rostral aspect of the bullah. Next, carefully drill a second pilot hole nearby To facilitate un roofing of the bullah bone using a pair of jewelers tip forceps, uncapped the bullah bone in a dorsal and coddle direction.

Take care not to puncture the EDL artery which lies directly beneath the bullock cap. Minimize the amount of bone removed to prevent excessive fluid entry to the middle ear before application of the agent. Use paper wick to remove all visible fluid in the middle ear and round window niche until the dry bone is visible.

This step is crucial to the success of the protocol. While viewing the area under maximum magnification, use a fine caliber needle on a one milliliter tuberculin syringe to apply one drop of Ototoxic agent directly to the round window niche filling it completely. Allow the agent to rest in the round window niche for 10 minutes.

In the early stages, watch closely for the replacement of a small light reflection at the base of the dry niche with a dull and hazy fluid meniscus. This indicates that the niche is filling properly. When 10 minutes have elapsed completely wick out the agent and replace it with a new application of the same agent.

The number of applications depends on the agent used. Exposure time typically ranges between 30 to 60 minutes. Following the final application, leave the bull uncapped and use forceps to close the soft tissue over the surgical site.

Use four oh non-absorbable monofilament suture to close the skin over the surgical site. Allow the animal to recover from anesthesia in a clean home cage, which should be equipped with bedding and soft food. Monitor the animals daily until sacrifice and cochlear tissue harvesting adult C-B-A-C-A-J.

Mice of both genders were exposed to the ototoxic agent Hept ethanol. The around window diffusion. Significant increases in a BR thresholds were observed in hept treated mice from postoperative day one to day 14 control animals receiving sham surgery with delivery of saline instead of hept.

Ethanol did not demonstrate significant threshold shifts at any tested frequency. Immuno staining against an inwardly rectifying potassium channel shown in green served as an indirect method for visualizing the damage and recovery of cochlear structures. Staining intensity was markedly decreased within theria vais.

On postoperative day one, evidence of disrupted nuclear integrity and chromosomal condensation, typical of cellular apoptosis was also seen on nuclear counter staining shown in red. When potassium channel staining intensity was quantified in the areas of stratis treated ears demonstrated an initial trough followed by a significant shift back toward control intensity seven days after hept exposure with recovery continuing thereafter. Once master, this procedure can be done within 30 to 60 minutes if it perform properly.

It's important to remember that additional exposure or replenishing may help to avoid unwanted dilution of the auto toxic agent by blood condensation or interstitial fluid. However, caution should be used with this approach as it tends to increase the risk of inadvertently injuring the cedal artery or introducing interstitial fluid to the round window niche. This minimally invasive technique allows for detailed study of delicate biochemical processes and has been tantamount in furthering our research aimed at understanding the cochlear regenerative potential in vivo.