Name: posterior semicircular canal approach for inner ear gene delivery in neonatal mouse
Uploaded: 2018-03-02T14:00:00
Description: Watch this Scientific Journal Video about Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse at JoVE.com

This work was supported by funds from the NIDCD Division of Intramural Research /NIH (DC000082-02 to W.W.C., as well as DC000081 to advanced imaging core). We are grateful for the NIDCD animal facility staff for caring for our animals.

All animal procedures were approved by the Animal Care and Use Committee at the National Institute on Deafness and Other Communication Disorders (NIDCD ASP1378-15).
1. Procedure Setup and Preparation
<ol>
	<li>Sterilize all instruments by ethylene oxide in the beginning of the experiment. Between animals, clean instruments using bead sterilization.</li>
	<li>Load the solution containing viral gene therapy into a micropipette on the micro-injector. The viral vector used in this study was AAV2...

<ol>
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Several surgical approaches have been described to access rodent inner ears. Cochleostomy and round window approaches are most commonly used to access the cochlea, whereas the posterior semicircular canal and endolymphatic sac approaches are typically used to access the vestibular organs1. In a recent study, we showed that posterior semicircular canal injections of viral gene therapy resulted in high efficiency of hair cell transduction in both the vestibular organs and the cochlea<sup class="xref...

Inner ear gene therapy is a rapidly developing field of investigation. It has been applied in various animals models to combat ototoxicity, noise trauma, and hereditary hearing loss1. Several recent studies have shown functional recovery of hearing and balance functions in mutant mice after inner ear gene therapy delivery2,3,4,5,6,7. One of the key factors in determining the success of inner ear gene therapy is the surgical approach used to access the inner ear. Ideally, the surgical approach would be easy to perform, the anatomic landmarks would be consistent and easy to identify, and the resulting transduction of targeted cell types would be high.
In a recent study, we showed that when viral gene therapy was injected through the posterior semicircular canal of the whirler mutant mouse (a model of hearing loss and vestibular dysfunction), efficient transduction of sensory hair cells was seen in the vestibular organs as well as in the cochlea5. The high efficiency of sensory hair cell transduction resulted in improvement of auditory and vestibular functions in these mutant mice.
In this article, we describe in detail the posterior semicircular canal approach to access the neonatal mouse inner ear.

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			<td height="42" style="height:42px;width:216px;">Operating microscope</td>
			<td style="width:113px;">Zeiss</td>
			<td style="width:111px;"></td>
			<td style="width:295px;">OPMI Pico ENT microscope. Other dissection microscopes would also work.</td>
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			<td height="42" style="height:42px;width:216px;">Micro-forcepts</td>
			<td style="width:113px;">Fine Science Tools</td>
			<td style="width:111px;">11251-10, 11295-51</td>
			<td>#5 and #55 Dumont</td>
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			<td height="42" style="height:42px;width:216px;">Micro-scissors</td>
			<td style="width:113px;">Fine Science Tools</td>
			<td style="width:111px;">15002-08</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Nanoliter2000 microinjector</td>
			<td style="width:113px;">World Precision Instruments</td>
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			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:113px;">Mastex</td>
			<td>Model 500/600</td>
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			<td height="21" style="height:21px;width:216px;">5-0 vicryl sutures</td>
			<td style="width:113px;">Ethicon</td>
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			<td height="21" style="height:21px;width:216px;">AAV8-whirlin</td>
			<td style="width:113px;">Vector Biolabs</td>
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			<td height="42" style="height:42px;width:216px;">Glass pipette</td>
			<td style="width:113px;">Sutter Instruments</td>
			<td style="width:111px;">B100-75-10</td>
			<td>Borosilicate glass</td>
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posterior semicircular canal approach for inner ear gene delivery in neonatal mouse

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. The easy anatomic identification of the posterior semicircular canal, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery.

Injection of AAV8-whirlin gene therapy into neonatal whirler mice through the posterior semicircular canal resulted in whirlin expression (green) in utricular hair cells (Figure 2), with the overall infection efficiency of 53.1% (SD 38.1, n = 28)5. Transduced hair cells had elongated stereocilia (red) compared to hair cells from contralateral non-injected ears (5.35 &#177; 2.11 &#181;m vs. 3.20 &#177; 0.34 &#181;m, respectively)<sup cl...

Watch this Scientific Journal Video about Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse at JoVE.com

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

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.

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