Задняя полукружных канала подход для доставки генов внутреннего уха у новорожденных мышей

The overall goal of this procedure is to access the mouse inner ear for gene delivery. This method can help deliver gene therapy to the mouse inner ear in an efficient manner. The main advantages of this technique is that it's reliable and easy to learn.

Generally individuals new to this method will struggle because of the small size of the mouse inner ear. However, with practice, most people can complete a surgical dissection within ten minutes. Begin the procedure with sterile instruments and turn on the bead sterilizer to clean instruments between animals.

Load the therapeutic virus into an ten micron diameter micropipette attached to a micro injector. The viral vector used in our experiments is AAV28-whirlin with a titer of one times ten to the 13 genome copies per milliliter. For this video, methylene blue dye was used for ease of visualization.

Clean the skin behind the ear with three betadine scrubs followed by alcohol wipe. Then, use micro scissors to make a postauricular approximately two millimeters behind the ear and divide the underlying soft tissue. Identify the facial nerve and the bulla.

The sternocleidomastoid muscle lies perpendicular to the facial nerve. The stapedial artery can be seen through the bulla which is a useful landmark. Follow the facial nerve superiorly and posteriorly to locate the posterior semi-circular canal.

Use the micro scissors to remove the muscle fibers and soft tissue overlying the posterior semi-circular canal. Then, penetrate the posterior semi-circular canal with the loaded glass micropipette on the micro injector and inject the dye into the inner ear. Typically, a total of 20 injections of 49 nano liters are delivered into the posterior semi-circular canal over 40 seconds for a total volume of approximately one microliter.

Withdraw the pipette immediately after administering the last volume. Then, close the skin incision using a 5-0 polyglactin suture. Injection of AAV8-whirlin into neonatal whirler mice as shown in this video resulted in whirlin expression shown in green in utricular hair cells.

Transduced hair cells had elongated stereocilia shown in red compared to hair cells from contralateral, non-injected ears. Posterior semi-circular canal injection of AAV8-whirlin also resulted in transduction of cochlear hair cells in the whirler mice. Transduced hair cells expressed whirlin at stereocilia tips and had elongated stereocilia compared to hair cells from contralateral, non-injected ears.

Following this procedure, other methods like ABI and the stereo testing can be performed in order to answer addition questions like whether inner ear gene delivery has an effect on auditory and vestibular functions. After it's development, this technique paved the way for researchers in the field of inner ear gene delivery to explore defects of gene delivery in the mouse inner ear.