Name: neonatal murine cochlear explant technique as an in vitro screening tool in hearing research
Uploaded: 2017-06-08T15:00:00
Description: Watch this Scientific Journal Video about Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research at JoVE.com

Dette arbeidet ble støttet av Nasjonalt institutt for døvhet og andre kommunikasjonsforstyrrelser som gir R01DC015824 (KMS) og T32DC00038 (støtte SD), forsvarsdepartementet W81XWH-15-1-0472 (KMS), Bertarelli Foundation (KMS), Nancy Sayles Day Foundation (KMS) og Lauer Tinnitus Research Center (KMS). Vi takker Jessica E. Sagers, BA for innsiktige kommentarer på manuskriptet.

Studieprotokollen ble godkjent av Institutt for dyrepleie og brukskomite (IACUC) i Massachusetts Eye and Ear. Eksperimenter ble utført i henhold til etikkloven fra Verdens medisinske forening. 1. Forbered disseksjonen <ol><li> Forbereder kirurgisk bord <ol><li> Bruk 70% etanol til å desinfisere det kirurgiske bordet. </li><li> Plasser to nonsterile prep pads ved siden av mikroskopet. </li><li> Klargjør instrumentbrettet, inkludert varmsterilisert operasjonssaks, et skal...

<ol>
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Forskere må perfeksjonere disseksjonsteknikken før de utfører eksperimenter som involverer cochlear eksplantater. Hårceller blir ofte skadet under disseksjoner som utføres tidlig i læringskurven, og et spesielt problematisk øyeblikk for deres integritet er fjerning av tectorial membranen, som krever stabile hender, riktige verktøy og erfaring. For å spare tid og ressurser, bør en visuell kontroll utføres under disseksjonsmikroskopet og potensielt skadede områder bør noteres. I stedet for å bruke dyre prim?...

Sensorineural Hearing Loss (SNHL) reflekterer skade på det indre øret eller stigende hørselsbane. Mens hørselstap er det vanligste sensoriske underskuddet hos mennesker 1 , finnes ikke kurative terapier 2 . Selv om cochleære eller auditive hjernestammeimplantater kan gjenopprette en viss grad av hørsel hos pasienter med alvorlig til dyp SNHL, er høreapparatet fra disse enhetene fortsatt svært forskjellig fra &quot;naturlig&quot; hørsel, spesielt under forsøk på å forstå tale i støy eller å høre på musikk. Mens hårcelledegenerasjon lenge har vært ansett som den primære konsekvensen av traumatiske hørselshendelser (eksponering for høy lyd), er det voksende bevis på at synapsene som overfører informasjon fra hårceller til hørsnerven, er minst like utsatt for akustisk traume 3 , 4 , 5 </sup<html> &gt; , 6 . Siden menneskelige audiometriske terskler, gjeldende gullstandard for evaluering av hørefunksjon, forutsier ikke spesifikk cellulær skade i det indre øret, er det behov for mer raffinerte verktøy for å oppdage cellulær degenerasjon så snart som mulig og å starte tilstrekkelig behandling 7 . Lovende farmasøytiske behandlinger for hørselstap testes ofte på homogene cellekulturer in vitro , men slike systemer presiserer ikke nøyaktig det cochleære mikromiljøet. Kochleære celler er kjent for å utsette trofiske faktorer som påvirker andre celletyper i cochlea 8 , 9 , en viktig in vivo- prosess som går tapt når organet fra Corti 10 , 11 eller Spiral Ganglion Neurons (SGNs) 12 dyrkes i isolasjon eller når Molekylære markører analyseresEf &quot;&gt; 13. Imidlertid krever in vivo studier som kan være nødvendige for validering av in vitro data for å etablere nye, personlig behandling for hørselstap i jakten på&quot; presisjonsmedisin &quot;, betydelige ressurser og tid. Dette er spesielt relevant når man vurderer Hvor mye innsats er nødvendig for å perfeksjonere og utføre mellomøre eller runde membraninnsprøytninger med hørselstester og den etterfølgende disseksjonen av cochlear hele mounts. Den effektive screeningen av lovende forbindelser i organotypiske ex vivo kulturer kjent som cochlear eksplanterer gir et økonomisk og pålitelig alternativ 14 , 15 , 16 , 17 . Denne artikkelen beskriver en protokoll for å generere, vedlikeholde og evaluere behandlede cochleære eksplanteringsstoffer. Spesifikke bruksområder for denne modellen er understreket, inkludert bruken i skjermenAv potensielt terapeutiske forbindelser og den komparative evaluering av virale vektorer for genterapi. En eks vivo eksplosiv tilnærming tillater forskere å visualisere effektene av en gitt behandling på forskjellige cellepopulasjoner på stedet , forenkle identifiseringen av celletypespesifikke mekanismer og den etterfølgende forfining av målrettede terapeutiske midler. Samlet gir denne teknikken en modell for å studere cochlea ex vivo mens du opprettholder viktig kryssprut mellom de svært forskjellige celletyper som eksisterer i cochlea.

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			<td>Ampicillin, Sodium Salt</td>
			<td>Invitrogen</td>
			<td>11593-027</td>
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			<td>anti-CtBP2 antibody, mouse(IgG1)</td>
			<td>BD Transduction Laboratories</td>
			<td>612044</td>
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			<td>anti-Myo7A antibody, rabbit</td>
			<td>Proteus Biosciences</td>
			<td>25-6790</td>
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			<td>anti-NF-H antibody, chicken</td>
			<td>EMD Millipore</td>
			<td>AB5539</td>
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			<td>anti-PSD95 antibody, mouse(IgG2a)</td>
			<td>Antibodies Inc.</td>
			<td>75-028</td>
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			<td>anti-TuJ1 antibody, mouse</td>
			<td>BioLegend</td>
			<td>801202</td>
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			<td>Cell-Tak Cell and Tissue Adhesive, 5 mg</td>
			<td>Corning</td>
			<td>354241</td>
			<td></td>
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			<td>CELLSTAR 15 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile</td>
			<td>Greiner Bio-One</td>
			<td>188161</td>
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			<td>CELLSTAR Cell Culture Dish, 100x20 mm</td>
			<td>Greiner Bio-One</td>
			<td>664160</td>
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			<td>CELLSTAR Cell Culture Dish, 35x10 mm, four inner rings</td>
			<td>Greiner Bio-One</td>
			<td>627170</td>
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			<td>CELLSTAR Cell Culture Dish, 60x15 mm</td>
			<td>Greiner Bio-One</td>
			<td>628160</td>
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			<td>CELLSTAR 50 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
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			<td>Clear Nail Polish</td>
			<td>Electron Microscopy Sciences</td>
			<td>72180</td>
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			<td>Clear Wall Glass Bottom Dishes (Glass 40mm), PELCO&#174;, Sleeve/20, 50x7 mm</td>
			<td>Ted Pella Inc.</td>
			<td>14027-20</td>
			<td></td>
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			<td>Coverslips, Round, Glass, 10 mm diameter, Thickness #1, 0.13-0.16mm</td>
			<td>Ted Pella Inc.</td>
			<td>260368</td>
			<td></td>
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			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
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			<td>Distilled water, 500 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>15230-162&#160;</td>
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			<td>DMEM, high glucose, pyruvate, no glutamine, 500 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>10313-039</td>
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			<td>Dumont #4 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11241-30</td>
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			<td>Dumont #55 Forceps (Dumostar)</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
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			<td>Ethyl alcohol, Pure, 200 proof, anhydrous, &#8805;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>459836-1L</td>
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			<td>Fetal Bovine Serum, qualified, USDA-approved regions, 500 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>10437-028&#160;</td>
			<td>Aliquot in 50 ml tubes and store in -20&#176;C freezer</td>
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			<td>Glutamate - GlutaMAX supplement, 100 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050-061</td>
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			<td>goat anti-chicken-647 secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21469</td>
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			<td>goat anti-mouse(IgG)-568 secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11004</td>
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			<td>goat anti-mouse(IgG1)-568 secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21124</td>
			<td></td>
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			<td>goat anti-mouse(IgG2a)-488 secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21131</td>
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			<td>goat anti-rabbit-488 secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37116</td>
			<td></td>
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			<td>H2O, sterile, EmbryoMax Ultra Pure Water, 500ml</td>
			<td>EMD Millipore</td>
			<td>TMS-006-B</td>
			<td></td>
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			<td>HBSS, calcium, magnesium, no phenol red, 500 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>14025-092</td>
			<td></td>
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			<td>Instrument Tray with Lid Stainless Steel</td>
			<td>Mountainside Medical</td>
			<td>TechMed4255</td>
			<td></td>
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			<td>Micro (dissecting) knife &#8211; angled 30&#176;</td>
			<td>Fine Science Tools</td>
			<td>10056-12</td>
			<td></td>
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			<td>Microscope slides, VistaVision, color-coded, 75 x 25 mm (3 x 1&#34;), 1 mm thick, white, pack of 72</td>
			<td>VWR</td>
			<td>16004-382</td>
			<td></td>
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			<td>N-2 Supplement (100X), 5 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502-048</td>
			<td></td>
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neonatal murine cochlear explant technique as an in vitro screening tool in hearing research

Selv om det har vært bemerkelsesverdige fremskritt i å høre forskning de siste tiårene, er det fortsatt ingen kur for Sensorineural Hearing Loss (SNHL), en tilstand som vanligvis involverer skade på eller tap av de delikate mekanosensoriske strukturer i det indre øret. Sofistikerte in vitro- og ex vivo- analyser har vist seg de siste årene, noe som muliggjør screening av et økende antall potensielt terapeutiske forbindelser samtidig som ressursene minimeres og akselerert arbeid for å utvikle botemidler for SNHL. Selv om homogene kulturer av bestemte celletyper fortsetter å spille en viktig rolle i dagens forskning, bygger mange forskere på mer komplekse organotypiske kulturer av murine indre ører, også kjent som cochleære eksplanteringsstoffer. Bevaringen av organiserte cellulære strukturer i det indre øre muliggjør in situ- evaluering av ulike komponenter i den cochleære infrastrukturen, inkludert indre og ytre hårceller, spiralganglionneuroner, neurItes og støttende celler. Her presenterer vi forberedelse, kultur, behandling og immunostaining av neonatal murine cochlear eksplanterende stoffer. Den forsiktige forberedelsen av disse eksplanterer muliggjør identifisering av mekanismer som bidrar til SNHL og utgjør et verdifullt verktøy for høreforskningsfellesskapet.

Mens mange protokoller fokuserer på organ av Corti-eksplanter, forsøker denne teknikken å bevare anatomien til hele cochlear-svinget, inkludert SGN-ene. Dette gir forskere muligheten til å analysere effektene av en gitt behandling på neuritter og somata av SGN i tillegg til organs Corti. Å utføre en disseksjon som bevarer en del av modiolus, som beskrevet her, er mer teknisk utfordrende enn å eksplantere Corti alene. Nevit og SGN-området er imidlertid viktig, fordi signifikante ...

Watch this Scientific Journal Video about Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research at JoVE.com

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

Målet med denne protokollen er å demonstrere forberedelse, kultur, behandling og immunostaining av neonatale murine cochleære eksplanteringsstoffer. Teknikken kan benyttes som et in vitro- screeningsverktøy i høreforskning.

Neonatal Murine Cochlear Explanting Technique som en<em&gt; I Vitro</em&gt; Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

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