Name: dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection
Uploaded: 2012-03-28T12:00:00
Description: Watch this Scientific Journal Video about Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection at JoVE.com

Dette arbeidet ble støttet av Divisjon for egenutført forskning ved National Institute on døvhet og andre kommunikasjonstjenester Disorders. Ytterligere støtte ble gitt av NIDCD 5R01 DC007613.

1. Utricle Dissection og kultur <ol><li> Avlive en voksen (4 uker eller eldre) mus ved hjelp av en godkjent protokoll og halshugge. </li><li> Klipp den eksterne øregangen på begge sider av hodet og dra huden frem mot nesen. Halvere hodet fra rygg til front og fjerne hjernen fra begge sider for å avsløre benete labyrinten. </li><li> Trim skallen unna benete sneglehuset og overføre benete labyrinten (inkludert Bulla) til en vev kultur hette utstyrt med en dissekere mikroskop (Merk: Adenovirus arbeid skal utføres i...

<ol>
	<li>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=The+adult+mouse+utricle+as+an+in+vitro+preparation+for+studies+of+ototoxic-drug-induced+sensory+hair+cell+death.">The adult mouse utricle as an in vitro preparation for studies of ototoxic-drug-induced sensory hair cell death.</a> Brain Res. , (2006).</li><li>Schmitt, N. C., Rubel, E. W., Nathanson, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin-induced+hair+cell+death+requires+STAT1+and+is+attenuated+by+epigallocatechin+gallate.">Cisplatin-induced hair cell death requires STAT1 and is attenuated by epigallocatechin gallate.</a> J. Neurosci. 29, 3843-3851 (2009).</li><li>Ou, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+FDA-approved+drugs+and+bioactives+that+protect+hair+cells+in+the+zebrafish+(Danio+rerio)+lateral+line+and+mouse+(Mus+musculus)+utricle.">Identification of FDA-approved drugs and bioactives that protect hair cells in the zebrafish (Danio rerio) lateral line and mouse (Mus musculus) utricle.</a> J. Assoc. Res. Otolaryngol. 10, 191-203 (2009).</li><li>Chiu, 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=Using+the+zebrafish+lateral+line+to+screen+for+ototoxicity.">Using the zebrafish lateral line to screen for ototoxicity.</a> J. Assoc. Res. Otolaryngol. 9, 178-190 (2008).</li><li>Sugahara, K., Rubel, E. W., 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=JNK+signaling+in+neomycin-induced+vestibular+hair+cell+death.">JNK signaling in neomycin-induced vestibular hair cell death.</a> Hear Res. , 221-221 (2006).</li><li>Matsui, J. I., Cotanche, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+hair+cell+death+and+regeneration:+two+halves+of+the+same+equation.">Sensory hair cell death and regeneration: two halves of the same equation.</a> Curr. Opin. Otolaryngol. Head Neck Surg. 12, 418-4125 (2004).</li><li>Ryals, B. M., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+cell+regeneration+after+acoustic+trauma+in+adult+Coturnix+quail.">Hair cell regeneration after acoustic trauma in adult Coturnix quail.</a> Science. 240, 1774-176 (1988).</li><li>Corwin, J. T., Cotanche, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+sensory+hair+cells+after+acoustic+trauma.">Regeneration of sensory hair cells after acoustic trauma.</a> Science. 240, 1772-174 (1988).</li><li>Shang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supporting+cell+division+is+not+required+for+regeneration+of+auditory+hair+cells+after+ototoxic+injury+in+vitro.">Supporting cell division is not required for regeneration of auditory hair cells after ototoxic injury in vitro.</a> J. Assoc. Res. Otolaryngol. 11, 203-222 (2010).</li><li>Stone, J. S., Cotanche, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+cell+regeneration+in+the+avian+auditory+epithelium.">Hair cell regeneration in the avian auditory epithelium.</a> Int. J. Dev. Biol. 51, 6-7 (2007).</li><li>Lahne, M., Gale, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage-induced+activation+of+ERK1/2+in+cochlear+supporting+cells+is+a+hair+cell+death-promoting+signal+that+depends+on+extracellular+ATP+and+calcium.">Damage-induced activation of ERK1/2 in cochlear supporting cells is a hair cell death-promoting signal that depends on extracellular ATP and calcium.</a> J. Neurosci. 28, 4918-4928 (2008).</li><li>Cunningham, L. L., Cheng, A. G., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+Activation+in+Hair+Cells+of+the+Mouse+Utricle+Exposed+to+Neomycin.">Caspase Activation in Hair Cells of the Mouse Utricle Exposed to Neomycin.</a> Journal of Neuroscience. 22, 8532-8540 (2002).</li><li>Matsui, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+inhibitors+promote+vestibular+hair+cell+survival+and+function+after+aminoglycoside+treatment+in+vivo.">Caspase inhibitors promote vestibular hair cell survival and function after aminoglycoside treatment in vivo.</a> J. Neurosci. 23, 6111-6122 (2003).</li><li>Matsui, J. I., Ogilvie, J. M., Warchol, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+caspases+prevents+ototoxic+and+ongoing+hair+cell+death.">Inhibition of caspases prevents ototoxic and ongoing hair cell death.</a> J. Neurosci. 22, 1218-1227 (2002).</li><li>Nakagawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+inducing+local+inner+ear+damage.">A novel technique for inducing local inner ear damage.</a> Hear Res. 176, 1-2 (2003).</li><li>Forge, A., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+death+of+hair+cells+in+mammalian+vestibular+sensory+epithelia.">Apoptotic death of hair cells in mammalian vestibular sensory epithelia.</a> Hear Res. 139, 1-2 (2000).</li><li>Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+inhibitors+prevent+cisplatin-induced+apoptosis+of+auditory+sensory+cells.">Caspase inhibitors prevent cisplatin-induced apoptosis of auditory sensory cells.</a> Neuroreport. 9, 2609-2614 (1998).</li><li>Taleb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+inhibits+aminoglycoside-induced+hearing+loss+and+cochlear+hair+cell+death.">Hsp70 inhibits aminoglycoside-induced hearing loss and cochlear hair cell death.</a> Cell Stress Chaperones. 14, 427-437 (2009).</li><li>Taleb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+inhibits+aminoglycoside-induced+hair+cell+death+and+is+necessary+for+the+protective+effect+of+heat+shock.">Hsp70 inhibits aminoglycoside-induced hair cell death and is necessary for the protective effect of heat shock.</a> J. Assoc. Res. Otolaryngol. 9, 277-289 (2008).</li><li>Francis, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celastrol+Inhibits+Aminoglycoside-Induced+Ototoxicity+via+Heat+Shock+Protein+32+(HSP32).">Celastrol Inhibits Aminoglycoside-Induced Ototoxicity via Heat Shock Protein 32 (HSP32).</a> Cell Death and Disease. , (2011).</li><li>Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+inhibitors,+but+not+c-Jun+NH2-terminal+kinase+inhibitor+treatment,+prevent+cisplatin-induced+hearing+loss.">Caspase inhibitors, but not c-Jun NH2-terminal kinase inhibitor treatment, prevent cisplatin-induced hearing loss.</a> Cancer Res. 64, 9217-9224 (2004).</li><li>Bird, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supporting+cells+eliminate+dying+sensory+hair+cells+to+maintain+epithelial+integrity+in+the+avian+inner+ear.">Supporting cells eliminate dying sensory hair cells to maintain epithelial integrity in the avian inner ear.</a> J. Neurosci. 30, 12545-12556 (2010).</li><li>Sato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+fractalkine+receptor+CX3CR1+on+cochlear+macrophages+influences+survival+of+hair+cells+following+ototoxic+injury.">Expression of fractalkine receptor CX3CR1 on cochlear macrophages influences survival of hair cells following ototoxic injury.</a> J. Assoc. Res. Otolaryngol. 11, 223-234 (2010).</li></ol>

Sensoriske hårcellene er utsatt for dødsfall forårsaket av en rekke påkjenninger, inklusive aldring, støy traumer, og eksponering for Ototoksisk narkotika, inkludert aminoglykosidantibiotika og cytostatikum cisplatin. Hos pattedyr hår celledød resulterer i permanent hørselstap og / eller balanse forstyrrelse. In vitro modellsystemer er kritiske verktøy for studier med sikte på fastsettelse av cellulære og molekylære mekanismer bak håret celledød, samt de som tar sikte på å forebygge eller revers...

A correction was made to <a href="http://www.jove.com/video/3734">Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection</a>. There was an error with a figure, the authors middle names were omitted, and the acknowledgements were updated.

Erratum: Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Reagens</strong</td&gt;<td<strong&gt; Selskap</strong</td&gt;<td<strong&gt; Katalognummer</strong</td&gt;</tr&gt;<tr&gt;<td&gt; Medium 199</td&gt;<td&gt; Gibco / Invitrogen</td&gt;<td&gt; 12350</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM/F12</td&gt;<td&gt; Gibco / Invitrogen</td&gt;<td&gt; 11320</td&gt;</tr&gt;<tr&gt;<td&gt; Fin tang (55)</td&gt;<td&gt; Fin Science Verktøy</td&gt;<td&gt; 11255-20</td&gt;</tr&gt;<tr&gt;<td&gt; Fin tang (# 5)</td&gt;<td&gt; Fin Science Verktøy</td&gt;<td&gt; 11252-30</td&gt;</tr&gt;<tr&gt;<td&gt; Tang (# 3)</td&gt;<td&gt; Fin Science Verktøy</td&gt;<td&gt; 11231-30</td&gt;</tr&gt;<tr&gt;<td&gt; Penicillin G</td&gt;<td&gt; Sigma</td&gt;<td&gt; P3032</td&gt;</tr&gt;<tr&gt;<td&gt; Fetal bovin serum</td&gt;<td&gt; Invitrogen</td&gt;<td&gt; 10082-139</td&gt;</tr&gt;<tr&gt;<td&gt; Nunc mini-brett</td&gt;<td&gt; Fisher</td&gt;<td&gt; 12-565-68</td&gt;</tr&gt;<tr&gt;<td&gt; Adenovirus-GFP</td&gt;<td&gt; Vector Biolabs</td&gt;<td&gt; 1060 *</td&gt;</tr&gt;<tr&gt;<td&gt; Cal-Ex decalcifier</td&gt;<td&gt; Fisher</td&gt;<td&gt; CS510-1D</td&gt;</tr&gt;<tr&gt;<td&gt; Natriumborhydrid</td&gt;<td&gt; Sigma</td&gt;<td&gt; 452 882</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-Myosin 7a (polyklonale)</td&gt;<td&gt; Proteus biovitenskap</td&gt;<td&gt; 25-6790</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-Myosin 7a (monoklonalt)</td&gt;<td&gt; Utviklingsstudier hybridoma Bank</td&gt;<td&gt; MYO7A 138-1</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-calmodulin</td&gt;<td&gt; Sigma</td&gt;<td&gt; C 3545</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-Calbindin</td&gt;<td&gt; Chemicon</td&gt;<td&gt; AB1778</td&gt;</tr&gt;<tr&gt;<td&gt; Fluoromount G</td&gt;<td&gt; Sør Bioteknologi Associates</td&gt;<td&gt; 0100-01</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"<strong&gt; Tabell 1</strong&gt;. Reagenser og verktøy som brukes i utricle kultur og adenovirus infeksjon.</p&gt;<p class="jove_content"&gt; * Merk: Ad-GFP normalt ytes av Vector Biolabs på et lager titer av 1x10<sup&gt; 10</sup&gt; PFU / ml. Vi ba om en egendefinert forsterkning for å gi aksjen viruset på en titer på 1,2 × 10<sup&gt; 11</sup&gt; PFU / ml.</p&gt;

dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection

Hørselstap og balanse forstyrrelser er ofte forårsaket av død mechanosensory hårcellene, som er receptor cellene i det indre øret. Siden det er ingen celle linje som tilfredsstillende representerer mammalske hårceller, avhengig forskning på hårcellene på primære organet kulturer. Det mest preget in vitro modell system av modne mammalske hårceller benytter orgel kulturer utricles fra voksne mus (figur 1) 1-6. Den utricle er en vestibulære organ, og hårcellene i utricle er lik i både struktur og funksjon til hårcellene i det auditive organ, organ Corti. Den voksne mus utricle forberedelse representerer en moden sensorisk epitel for studier av de molekylære signaler som regulerer overlevelse, homeostase, og død av disse cellene. Mammalske cochlea hårcellene terminalt differensiert og er ikke regenerert når de går tapt. I ikke-mammaliske virveldyr, auditive eller vestibulære hår celledød etterfølges av robust regenerering som gjenoppretter hørsel og balanse funksjoner 7, 8. Håret celle gjenfødelse er mediert av gliaceller-lignende støtte celler, som kontakt med basolateral overflater av hårceller i sensorisk epitel 9, 10. Støtter celler er også viktige mediatorer av hår celle overlevelse og død 11. Vi har nylig utviklet en teknikk for infeksjon av støtte celler i dyrkede utricles med adenovirus. Bruke adenovirus type 5 (dE1/E3) å levere en transgenet inneholder GFP under kontroll av CMV arrangøren, finner vi at adenovirus spesielt og effektivt infiserer støtte celler. Støtte celle infeksjon effektivitet er ca 25-50%, og hårcellene blir ikke smittet (figur 2). Viktigere, finner vi at adenoviral infeksjon av støtte celler ikke medføre giftighet for hårcellene eller hjelpeverge celler, som blodceller i Ad-GFP infected utricles tilsvarer de i ikke-infiserte utricles (figur 3). Dermed adenovirus-mediert genuttrykk i støtte celler av dyrkede utricles gir et kraftig verktøy for å studere rollene til støtte celler som mediatorer av hår celle overlevelse, død og gjenfødelse.

Watch this Scientific Journal Video about Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection at JoVE.com

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Mechanosensory hårcellene er receptor cellene i det indre øret. Den beste-preget<em&gt; In vitro</em&gt; Modellsystem av modne mammalske hårceller benytter orgel kulturer utricles fra voksne mus. Vi presenterer den disseksjon av den voksne mus utricle, og demonstrerer vi adenovirus-mediert infeksjon av støtte celler i dyrkede utricles.

Disseksjon av Adult Mouse Utricle og Adenovirus-mediert støttende-celle Infeksjon

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear.  Since there ...

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