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

Dette arbejde blev støttet af midler fra den NIDCD Division af murene forskning /NIH (DC000082-02 til W.W.C.), samt DC000081 til avanceret billedbehandling kerne. Vi er taknemmelige for NIDCD Dyrefacilitet personale til pasning af vores dyr.

Alle dyr procedurer blev godkendt af Animal Care og brug Udvalget på National Institute on døvhed og anden kommunikation lidelser (NIDCD ASP1378-15).
1. procedure Setup og forberedelse
<ol><li>Sterilisere alle instrumenter af ethylenoxid i begyndelsen af eksperimentet. Mellem dyrene, rense instrumenter ved hjælp af perle sterilisation.</li>
	<li>Indlæse den løsning, der indeholder viral genterapi i en mikropipette på mikro-injektor. Den virale vektor, der anvendes i denne undersøgelse...

<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>

Flere kirurgiske metoder er blevet beskrevet for at få adgang til gnaver indre ører. Cochleostomy og runde vindue tilgange er oftest bruges til at få adgang til cochlea, bageste halvrunde kanalen og endolymphatic sac tilgange anvendes typisk til at få adgang til de vestibulære organer1. I en nylig undersøgelse viste vi, at bageste halvrunde kanalen injektioner af viral genterapi resulterede i høj effektivitet af hår celle signaltransduktion i både de vestibulære organer og cochlea<sup cl...

Indre øre genterapi er en rivende udvikling inden for undersøgelse. Det har været anvendt i forskellige dyr modeller til at bekæmpe ototoksicitet, støj traumer og arvelige hearing tab1. Flere nyere undersøgelser har vist funktionel genopretning af hørelse og balance funktioner i mutant-mus efter indre øre gen terapi levering2,3,4,5,6, 7. et af vigtigste faktorer ved afgørelsen af succesen af indre øre genterapi er den kirurgiske metode bruges til at få adgang til det indre øre. Ideelt, den kirurgiske tilgang ville være let at udføre, de anatomiske landemærker ville være konsekvent og let at identificere, og den resulterende transduktion af målrettede celletyper ville være høj.
I en nylig undersøgelse viste vi, at når viral genterapi var injiceres gennem den bageste halvrunde kanalen whirler mutant musen (en model af høretab og vestibulær dysfunktion), effektiv transduktion af sensoriske hår celler blev set i den vestibulære organer så godt som i cochlea5. Den høje effektivitet af sensoriske hår celle transduktion resulteret i forbedring af auditive og vestibulære funktioner i disse muterede mus.
I denne artikel beskriver vi i detaljer den bageste halvrunde kanal tilgang til at få adgang til neonatal mus indre øre.

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		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5-0 vicryl sutures</td>
			<td style="width:113px;">Ethicon</td>
			<td style="width:111px;"></td>
			<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>
		</tr>
		<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

Indre øre genterapi tilbyder meget lovende som en potentiel behandling af høretab og svimmelhed. En af de kritiske faktorer for succes af indre øre genterapi er at finde en leveringsmetode, hvilket resulterer i konsekvent transduktion effektiviteten af målrettede celletyper og minimere tab af hørelse. I denne undersøgelse beskriver vi bageste halvrunde kanalen som en levedygtig metode for indre øre gen levering i neonatal mus. Vi viser, at genet levering gennem den bageste halvrunde kanalen er stand til at perfuse det hele indre øre. Nem anatomiske identifikation af den bageste halvrunde kanalen, samt minimal manipulation af den tidsmæssige knoglen kræves, gør denne kirurgiske tilgang en attraktiv mulighed for indre øre gen levering.

Injektion af AAV8-whirlin genterapi til neonatal whirler mus gennem den bageste halvrunde kanalen resulterede i whirlin udtryk (grøn) i utricular hårceller (figur 2), med den samlede infektion effektivitet på 53,1% (SD 38.1, n = 28)5 . Transduced hårceller havde aflange stereocilia (red) i forhold til hår celler fra kontralaterale ikke injiceres ører (5,35 ± 2.11 µm vs 3,20 ± 0,34 µm, henholdsvis)5.</p...

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

I denne undersøgelse beskriver vi bageste halvrunde kanalen som en pålidelig metode til indre øre gen levering i neonatal mus. Vi viser, at genet levering gennem den bageste halvrunde kanalen er stand til at perfuse det hele indre øre.

Bageste halvrunde kanalen tilgang til indre øre gen levering i Neonatal mus

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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