The overall goal of this study is to describe the surgical techniques involved in a canalostomy in both adult and neonatal mice. This method first in the late 80s. Broad distribution of injected reagents in the cochlear and vestibular end organs of both adult and the neonatal mice.
The main advantage of the technique is that it causes minimal damage to hearing and vestibular function. And multiple surgeries could be easily performed, if required. Demonstrating the procedure will be Dr.Jing-Ying Guo, a resident in our department.
Begin by connecting polyimide tubing to polyethylene tubing in order to make the injection cannula. Use super glue to seal the connection with at least three applications. And sterilize the injection cannula with ethylene oxide.
Next, use a 30 gauge needle to connect the end of the polyethylene tubing to a one-CC syringe containing normal saline. Fill the cannula with normal saline by injection with the one-CC syringe to check for any leakage or blockage at the connection between the polyimide tubing and the polyethylene tubing. Evacuate a 10 microliter microsyringe with normal saline and connect it to the 30 gauge needle with the injection cannula.
Then, install the microsyringe on a microinjection pump. Set the injection speed to 0.5 microliters per minute and the volume to one microliter for fast green dye and the viral detector AAV8-GFP or two microliters for streptomycin. Finally, pull back the microsyringe to one microliter and extract the injection reagent to create an air gap between the normal saline and the injected reagent.
Begin by placing the anesthetized mouse on a preheated electric pad. Set the temperature of the electric pad to approximately 37 degrees celsius. Cover the animal's eyes with eye ointment.
Then, shave the left postauricular region with an electric animal clipper and disinfect the skin three times with 75%ethanol. Next, place the animal in the right lateral position to facilitate surgery on the left ear. Make a one to 1.5 centimeter postauricular incision three millimeters from the left retroauricular groove.
Note, when the root of the pinna is defined as the origin, and the plane parallel to the calvarium as three to nine o'clock, the posterior semicircular canal and the lateral semicircular canal are typically located about three millimeters from the root of the pinna, between two and three o'clock. Bluntly dissect the muscle covering the temporal bone with micro forceps to expose the posterior semicircular canal and lateral semicircular canal, whose margins are clearly visible as dark stripes in the temporal bone. Then, collect a small piece of muscle with micro forceps and let it dry.
Next, make a small hole in the middle portion of the posterior semicircular canal using a 26 gauge needle, such that fluid leakage through the hole indicates successful penetration of the bony wall of the posterior semicircular canal. Enlarge the hole to slightly larger than the diameter of the polyimide tubing. Then, clean the effusion surrounding the hole of the posterior semicircular canal using a cotton pellet.
Insert the tip of the polyimide tubing gently into the posterior semicircular canal, towards the crus commune, to a depth of one to two millimeters. Start the injection by pressing the run button on the pump. After the injection, wait two minutes to allow the reagent to spread.
Then, remove the injection cannula and immediately place the muscle into the hole in the posterior semicircular canal. Return the separated muscles and subcutaneous tissues together. Suture the incision using a 5-0 suture.
Disinfect the incision region with povidone iodine. Finally, after the surgery is complete, position the animal in the right lateral position on an electric pad preheated to 37 degrees celsius for recovery. The following steps differ in the neonate compared to the adult mice.
On the sedated neonate, lying on an ice-filled platform, begin by making a three millimeter postauricular incision from two millimeters posterior to the auricular crease. Then, gently make an opening in the soft posterior semicircular canal using a 26 gauge needle. Insert the cannula into the posterior semicircular canal without enlarging the opening.
After injection, use the piece of muscle to cover, rather than plug, the opening, as the latter can lead to a fracture of the soft posterior semicircular canal. Close the skin using a 6-0 suture. Here are stereoscopic images of the inner ears of adult and neonatal mice, as fast green dye was administered via canalostomy.
Samples were collected immediately after surgery. Further, these confocal images show whole mounts of the cochlea and utricle of adult mice, prepared 30 days after AAV8-GFP injection via canalostomy. In the cochlea, GFP is expressed in most inner hair cells.
And, at the focal plane of the cuticular plate of the utricle, numerous hair cells express GFP. Lastly, these confocal images of a traumatized utricle were obtained 30 days after AAV8-GFP injection via canalostomy. GFP-positive, myosin 7A-positive cells are transduced hair cells, whereas GFP-positive, myosin 7A-negative cells are transduced supporting cells.
While attempting this procedure, it's important to ensure that the tip of the cannula is well-inserted in the semicircular canal and that the cannula is not bent or blocked. After injection in adult mice, a small piece of dry muscle is recommended to be used to seal the hole in the semicircular canal. Check for any liquid leaking from the hole to ensure it is completely sealed.
Our study shows that canalostomy is an effective and safe approach for drug delivery into the inner ear of mice. Once mastered, this technique can be completed in 25 minutes if it is performed properly. After watching this video, you should have a good understanding for how to position the semicircular canals and deliver viral vectors, siRNA, stem cells, or drugs into the inner ear of adult and neonatal mice via canalostomy.
