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>
	<li>Chien, W. W., Monzack, E. L., McDougald, D. S., Cunningham, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+sensorineural+hearing+loss.">Gene therapy for sensorineural hearing loss.</a> Ear Hear. 36, 1-7 (2015).</li><li>Askew, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tmc+gene+therapy+restores+auditory+function+in+deaf+mice.">Tmc gene therapy restores auditory function in deaf mice.</a> Sci Transl Med. 7, 295 (2015).</li><li>Akil, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+hearing+in+the+VGLUT3+knockout+mouse+using+virally+mediated+gene+therapy.">Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.</a> Neuron. 75, 283-293 (2012).</li><li>Pan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+restores+auditory+and+vestibular+function+in+a+mouse+model+of+Usher+syndrome+type+1c.">Gene therapy restores auditory and vestibular function in a mouse model of Usher syndrome type 1c.</a> Nat Biotechnol. 35, 264-272 (2017).</li><li>Isgrig, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Therapy+Restores+Balance+and+Auditory+Functions+in+a+Mouse+Model+of+Usher+Syndrome.">Gene Therapy Restores Balance and Auditory Functions in a Mouse Model of Usher Syndrome.</a> Mol Ther. 25, 780-791 (2017).</li><li>Lentz, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+hearing+and+vestibular+function+by+antisense+oligonucleotides+in+a+mouse+model+of+human+deafness.">Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness.</a> Nat Med. 19, 345-350 (2013).</li><li>Shibata, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Interference+Prevents+Autosomal-Dominant+Hearing+Loss.">RNA Interference Prevents Autosomal-Dominant Hearing Loss.</a> Am J Hum Genet. 98, 1101-1113 (2016).</li><li>Van Sluyters, R. C., Obernier, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+care+and+use+of+mammals+in+neuroscience+and+behavioral+research.">Guidelines for the care and use of mammals in neuroscience and behavioral research.</a> Contemp Top Lab Anim. 43, 48 (2004).</li><li>Chien, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Therapy+Restores+Hair+Cell+Stereocilia+Morphology+in+Inner+Ears+of+Deaf+Whirler+Mice.">Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice.</a> Mol Ther. 24, 17-25 (2016).</li><li>Hirose, K., Hartsock, J. J., Johnson, S., Santi, P., Salt, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+lipopolysaccharide+compromises+the+blood-labyrinth+barrier+and+increases+entry+of+serum+fluorescein+into+the+perilymph.">Systemic lipopolysaccharide compromises the blood-labyrinth barrier and increases entry of serum fluorescein into the perilymph.</a> J Assoc Res Otolaryngol. 15, 707-719 (2014).</li><li>Okada, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+targeting+mouse+vestibule+using+adenovirus+and+adeno-associated+virus+vectors.">Gene transfer targeting mouse vestibule using adenovirus and adeno-associated virus vectors.</a> Otol Neurotol. 33, 655-659 (2012).</li><li>Suzuki, J., Hashimoto, K., Xiao, R., Vandenberghe, L. H., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+gene+therapy+with+ancestral+AAV+in+adult+mice:+complete+transduction+of+inner+hair+cells+without+cochlear+dysfunction.">Cochlear gene therapy with ancestral AAV in adult mice: complete transduction of inner hair cells without cochlear dysfunction.</a> Sci Rep. 7, 45524 (2017).</li></ol>

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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nanoliter2000 microinjector</td>
			<td style="width:113px;">World Precision Instruments</td>
			<td style="width:111px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:113px;">Mastex</td>
			<td>Model 500/600</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5-0 vicryl sutures</td>
			<td style="width:113px;">Ethicon</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AAV8-whirlin</td>
			<td style="width:113px;">Vector Biolabs</td>
			<td style="width:111px;"></td>
			<td></td>
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		<tr height="42">
			<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 ...

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