Name: preparation of the rat vocal fold for neuromuscular analyses
Uploaded: 2020-05-15T14:45:00
Description: Watch this Scientific Journal Video about Preparation of the Rat Vocal Fold for Neuromuscular Analyses at JoVE.com

Denne forskning blev støttet af tilskud F31DC017053-01A1 (Lenell, PI) og K23DC014517 (Johnson, PI) fra National Institute on Døvhed og andre kommunikationsforstyrrelser fra National Institutes of Health.

Denne undersøgelse blev udført i overensstemmelse med den institutionelle Animal Care and Use Committee of New York University School of Medicine.
1. Dissekere rotte strubehovedet
<ol><li>Aflive rotte i henhold til den institutionelt godkendte protokol. Barber ventral hals fra underkæben til manubrium og vatpind med alkohol for at forhindre pelsforurening i vævsprøverne.</li>
	<li>Under en dissekering omfang med 10x forstørrelse punktafgifter hele strubehovedet ved at skabe en midline ...

<ol>
	<li>Connor, N. P., Suzuki, T., Lee, K., Sewall, G. K., Heisey, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+junction+changes+in+aged+rat+thyroarytenoid+muscle.">Neuromuscular junction changes in aged rat thyroarytenoid muscle.</a> Annals of Otology, Rhinology and Laryngology. 111 (7), 579-586 (2002).</li><li>Suzuki, T., 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=Age-Related+Alterations+in+Myosin+Heavy+Chaing+Isoforms+in+Rat+Intrinsic+Laryngeal+Muscles.">Age-Related Alterations in Myosin Heavy Chaing Isoforms in Rat Intrinsic Laryngeal Muscles.</a> Annals of Otology, Rhinology and Laryngology. 111 (11), 962 (2002).</li><li>Johnson, A. M., Grant, L. M., Schallert, T., Ciucci, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+Rat+50-kHz+Ultrasonic+Vocalizations+During+Dopamine+Denervation+and+Aging:+Relevance+to+Neurodegeneration.">Changes in Rat 50-kHz Ultrasonic Vocalizations During Dopamine Denervation and Aging: Relevance to Neurodegeneration.</a> Current Neuropharmacology. 13 (2), 211-219 (2015).</li><li>Wright, J. M., Gourdon, J. C., Clarke, P. B. <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+multiple+call+categories+within+the+rich+repertoire+of+adult+rat+50-kHz+ultrasonic+vocalizations:+effects+of+amphetamine+and+social+context.">Identification of multiple call categories within the rich repertoire of adult rat 50-kHz ultrasonic vocalizations: effects of amphetamine and social context.</a> Psychopharmacology. 211 (1), 1-13 (2010).</li><li>Bowers, J. M., Perez-Pouchoulen, M., Edwards, N. S., McCarthy, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxp2+mediates+sex+differences+in+ultrasonic+vocalization+by+rat+pups+and+directs+order+of+maternal+retrieval.">Foxp2 mediates sex differences in ultrasonic vocalization by rat pups and directs order of maternal retrieval.</a> Journal of Neuroscience. 33 (8), 3276-3283 (2013).</li><li>Basken, J. N., Connor, N. P., Ciucci, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+aging+on+ultrasonic+vocalizations+and+laryngeal+sensorimotor+neurons+in+rats.">Effect of aging on ultrasonic vocalizations and laryngeal sensorimotor neurons in rats.</a> Experimental Brain Research. 219 (3), 351-361 (2012).</li><li>Ciucci, M. R., 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=Reduction+of+dopamine+synaptic+activity:+degradation+of+50-kHz+ultrasonic+vocalization+in+rats.">Reduction of dopamine synaptic activity: degradation of 50-kHz ultrasonic vocalization in rats.</a> Behavioral Neuroscience. 123 (2), 328-336 (2009).</li><li>Ciucci, M. R., Vinney, L., Wahoske, E. J., Connor, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+translational+approach+to+vocalization+deficits+and+neural+recovery+after+behavioral+treatment+in+Parkinson+disease.">A translational approach to vocalization deficits and neural recovery after behavioral treatment in Parkinson disease.</a> Journal of Communication Disorders. 43 (4), 319-326 (2010).</li><li>Nagai, H., Ota, F., Konopacki, R., Connor, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discoordination+of+laryngeal+and+respiratory+movements+in+aged+rats.">Discoordination of laryngeal and respiratory movements in aged rats.</a> American Journal of Otolaryngology. 26 (6), 377-382 (2005).</li><li>Ma, S. T., Maier, E. Y., Ahrens, A. M., Schallert, T., Duvauchelle, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+intravenous+cocaine+experience:+development+and+escalation+of+pre-drug+anticipatory+50-kHz+ultrasonic+vocalizations+in+rats.">Repeated intravenous cocaine experience: development and escalation of pre-drug anticipatory 50-kHz ultrasonic vocalizations in rats.</a> Behavioural Brain Research. 212 (1), 109-114 (2010).</li><li>Inagi, K., Schultz, E., Ford, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+anatomic+study+of+the+rat+larynx:+establishing+the+rat+model+for+neuromuscular+function.">An anatomic study of the rat larynx: establishing the rat model for neuromuscular function.</a> Otolaryngology and Head and Neck Surgery. 118 (1), 74-81 (1998).</li><li>Lenell, C., Newkirk, B., Johnson, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laryngeal+Neuromuscular+Response+to+Short-+and+Long-Term+Vocalization+Training+in+Young+Male+Rats.">Laryngeal Neuromuscular Response to Short- and Long-Term Vocalization Training in Young Male Rats.</a> Journal of Speech, Language, and Hearing Research. 62 (2), 247-256 (2019).</li><li>Kumar, A., Accorsi, A., Rhee, Y., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do's+and+don'ts+in+the+preparation+of+muscle+cryosections+for+histological+analysis.">Do's and don'ts in the preparation of muscle cryosections for histological analysis.</a> Journal of Visualized Experiments. (99), e52793 (2015).</li><li>McMullen, C. A., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+morphological+evidence+of+age-related+denervation+in+rat+laryngeal+muscles.">Functional and morphological evidence of age-related denervation in rat laryngeal muscles.</a> Journals of Gerontology. Series A: Biological Sciences and Medical Sciences. 64 (4), 435-442 (2009).</li><li>McMullen, C. A., 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=Chronic+stimulation-induced+changes+in+the+rodent+thyroarytenoid+muscle.">Chronic stimulation-induced changes in the rodent thyroarytenoid muscle.</a> Journal of Speech, Language, and Hearing Research. 54 (3), 845-853 (2011).</li><li>Lenell, C., Johnson, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+laryngeal+muscle+fibers+and+ultrasonic+vocalizations+in+the+adult+rat.">Sexual dimorphism in laryngeal muscle fibers and ultrasonic vocalizations in the adult rat.</a> Laryngoscope. 127 (8), 270-276 (2017).</li><li>Johnson, A. M., Ciucci, M. R., Connor, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vocal+training+mitigates+age-related+changes+within+the+vocal+mechanism+in+old+rats.">Vocal training mitigates age-related changes within the vocal mechanism in old rats.</a> Journals of Gerontology. Series A: Biological Sciences and Medical Sciences. 68 (12), 1458-1468 (2013).</li><li>Feng, X., Zhang, T., Ralston, E., Ludlow, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+neuromuscular+junctions+of+laryngeal+and+limb+muscles+in+rats.">Differences in neuromuscular junctions of laryngeal and limb muscles in rats.</a> Laryngoscope. 122 (5), 1093-1098 (2012).</li></ol>

Forberedelse rotte vokal folder til neuromuskulær analyse kan præsentere med forskellige udfordringer. Ikke alene er larynx muskler små og omgivet af brusk, hvilket gør det vanskeligt at direkte udtrække målmuskel, høj variation blev også fundet mellem dyr i dybden af larynx anatomiske vartegn. For muskel tværsnit plan protokol, komplet vokal fold sektioner dukkede op mellem 21−85 sektioner (10 μm per sektion) efter den første forekomst af den ventrale skjoldbruskkirtlen brusk, som er en hel del færre end d...

Rottestrubehovedet er en veletableret model til at undersøge strukturelle og funktionelle neuromuskulære larynx tilpasninger til udvikling, aldring, sygdom og farmakologiske agenser1,2,3,4,5. Konsistensen af histologiske metoder er afgørende for denne branche, da der er flere snørklede involveret i muskelforberedelse og -analyse samt udfordringer forbundet med larynx størrelse, form og topografi af musklerne indkapslet i larynx brusk1,6,7,8,9,10,11 . På grund af den lille størrelse af rotten iboende larynx muskler, systematisk indlejring, frysning, og cryosectioning er afgørende for at opnå konsekvente og præcise resultater. For eksempel, når du sektionerer rottevokalfolden i koronarplanet, er de neuromuskulære knudepunkter (NMJs) af fire af de iboende larynxmuskler placeret inden for mindre end 1,8 mm vævsdybde11. Derfor er præcis overvågning af larynx muskel anatomi under cryosectioning er afgørende for præcist at identificere de afsnit (r) af interesse og forhindre tilsyn med væv. Tilsyn med målet muskel kan resultere i unøjagtig identifikation af antal og topografi af NMJs11 eller kan resultere i samlede reduktioner i stikprøvestørrelsen, hvis målet muskel kasseres på grund af skelsættende orientering forvirring12. Som nye modeller for studiet af larynx muskel og deres respektive tilpasninger er udviklet, standard driftsprocedurer er afgørende for at sikre resultaterne er præcise, pålidelige og reproducerbare på tværs af undersøgelser.
Formålet med denne artikel er at detaljeret forberedelse af rotte vokal fold for optimal langsgående og tværsnit analyse. Detaljerede metoder, der anvendes regelmæssigt i vores laboratorium er beskrevet til at identificere mål muskel vartegn under cryosectioning. Selvom lignende metoder anvendes i flere laboratorier, gives der større detaljer her end i litteraturen for at sikre pålidelig og præcis replikation, når de implementeres af nybegyndere. Målet med denne tutorial er at give en standard metode til immunohistokemisk (IHC) evaluering af rotte vokal fold for at forbedre konsistensen på tværs af laboratorier og undersøgelser.

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			<td>35514</td>
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			<td>Fisherbrand</td>
			<td>1213101</td>
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			<td>Cryo Tongs SS</td>
			<td>Thermo Scientific</td>
			<td>11679123</td>
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			<td>Cryostat</td>
			<td>Leica Biosystems</td>
			<td>CM3050</td>
			<td></td>
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			<td>Cryostat blades</td>
			<td>C.L. Sturkey D554X50</td>
			<td>22-210-045</td>
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			<td>Disposable Base Molds 15mm x 15mm</td>
			<td>Thermo Scientific</td>
			<td>41-741</td>
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			<td>Disposable Underpads</td>
			<td>Medline</td>
			<td>23-666-062</td>
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			<td>Dissection kit</td>
			<td>Thermo Scientific</td>
			<td>9996969</td>
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			<td>DPBS - Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Gibco</td>
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			<td>Frozen Section Medium</td>
			<td>Fisher Healthcare</td>
			<td>23-730-571</td>
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			<td>Bel-Art</td>
			<td>11999054</td>
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			<td>Immunostain Moisture Chamber</td>
			<td>Ted Pella Inc</td>
			<td>NC9425474</td>
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			<td>Needle holders</td>
			<td>Assi</td>
			<td>ASSI.B148</td>
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			<td>Non-Woven Sponges, 4 Ply</td>
			<td>Quick Medical</td>
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			<td>02-217-987</td>
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			<td>Pap pen</td>
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			<td>Paraformaldehyde, 16% w/v aq. soln., methanol free</td>
			<td>Alfa Aesar</td>
			<td>50-00-0</td>
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			<td>Premium Microcentrifuge Tubes</td>
			<td>Fisherbrand</td>
			<td>5408129</td>
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			<td>Specimen Storage Bags</td>
			<td>Fisherbrand</td>
			<td>19240093</td>
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			<td>Stainless Steel Graduated Measure 32 oz/100 mL</td>
			<td>Polar Ware</td>
			<td>114231B</td>
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			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
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			<td>Kimberly-Clark Professional&#8482; 34155</td>
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			<td>Timer</td>
			<td>Fisherbrand</td>
			<td>2261840</td>
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			<td>Vannas Pattern Scissors</td>
			<td>Assi</td>
			<td>ASSI.SAS15RV</td>
			<td></td>
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		<tr>
			<td colspan="4">NOTE: For all supplies, these are examples of equipment to purchase. The exact model is not necessary to complete our methods.</td>
		</tr>
	</tbody>
</table>

preparation of the rat vocal fold for neuromuscular analyses

Formålet med denne tutorial er at beskrive forberedelsen af rotte vokal fold for histokemisk neuromuskulær undersøgelse. Denne protokol skitserer procedurer for rotte larynx dissektion, flash-frysning, og cryosectioning af vokal folder. Denne undersøgelse beskriver, hvordan man cryosection vokal folder i både langsgående og tværsnit fly. En nyhed i denne protokol er larynx tracking under cryosectioning, der sikrer præcis identifikation af de iboende larynx muskler og reducerer risikoen for vævstab. Tal viser den progressive kryosectioning i begge fly. 29 rotte hemi-larynges blev cryosectioned og spores fra fremkomsten af skjoldbruskkirtlen brusk til udseendet af det første afsnit, der omfattede den fulde vokal fold. Den fulde vokalfold blev visualiseret for alle dyr i begge planer. Der var høj variation i afstanden fra udseendet af skjoldbruskkirtlen brusk til udseendet af den fulde vokal fold i begge fly. Vægten var ikke korreleret til dybden af larynx landemærker, hvilket tyder på individuelle variabilitet og andre faktorer i forbindelse med vævspræparat kan være ansvarlig for den høje variation i udseendet af landemærker under afsnit. Denne undersøgelse beskriver en metode og præsenterer morfologiske data til forberedelse af rotte vokal fold til histokemisk neuromuskulær undersøgelse. På grund af høj individuel variation bør larynx landemærker spores nøje under kryosektion for at forhindre tilsyn med væv og vævstab. Brugen af en konsekvent metode, herunder tilstrækkelig vævsforberedelse og bevidsthed om landemærker inden for rottestrubehovedet, vil hjælpe med ensartede resultater på tværs af undersøgelser og hjælpe nye forskere, der er interesseret i at bruge rottevokalfolden som model til at undersøge larynx neuromuskulære mekanismer.

De repræsentative resultater var en del af en igangværende undersøgelse af virkningerne af vokal motion på larynx neuromuskulære system. 29 hanner Fischer 344/brune norgesrotter (12 9 måneder gamle, 17 24 måneder gamle) blev vejet og aflivet med CO2-indånding efterfulgt af en bilateral thoracotomi.
Procedurerne fulgte den skitserede protokol til mærkning NMJs og fiber størrelse af de laterale og mediale TA muskler. Afstanden mellem larynx landemærker blev sporet i både l...

Watch this Scientific Journal Video about Preparation of the Rat Vocal Fold for Neuromuscular Analyses at JoVE.com

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

Denne protokol beskriver metoder, der anvendes til at forberede rotte vokal folder til histokemisk neuromuskulær undersøgelse.

Forberedelse af Rat Vocal Fold til neuromuskulære analyser

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

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