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

这项研究得到了美国国立卫生研究院国家耳聋和其他沟通障碍研究所的F31DC017053-01A1（Lenell，PI）和K23DC014517（Johnson，PI）的资助。

本研究是根据纽约大学医学院机构动物护理和使用委员会进行的。
1. 解剖大鼠喉部
<ol><li>根据机构批准的协议对大鼠实施安乐死。将腹颈部从下颌骨剃到手洗，并用酒精拭子擦拭，以防止组织标本中的毛皮污染。</li>
	<li>在具有10倍放大倍率的解剖镜下，通过用手术刀创建中线颈部切口来切除整个喉部，直到气管暴露。</li>
	<li>将腹侧外侧喉肌分开，使用镊子和解剖剪刀或手...

<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>

准备用于神经肌肉分析的大鼠声带可能会带来各种挑战。喉部肌肉不仅小且被软骨包围，从而难以直接提取目标肌肉，而且在喉部解剖学标志物的深度上还发现了动物之间的高变异性。对于肌肉横截面方案，在腹侧甲状腺软骨最初出现后，完整的声带切片出现在21-85个切片（每节10μm）之间，这比NMJ分析方案纵平面中的63-126个截面（每节35μm）要少得多（表1）。
?...

大鼠喉是一个成熟的模型，用于研究结构和功能神经肌肉喉对发育，衰老，疾病和药理药物的适应性1，2，3，4，5。组织学方法的一致性对于这项工作至关重要，因为肌肉制备和分析涉及多种复杂性，以及与喉软骨内封装的肌肉的喉部大小，形状和形貌相关的挑战1，6，7，8，9，10，11.由于大鼠内在喉部肌肉的尺寸很小，系统地嵌入，冷冻和冷冻切除对于获得一致和准确的结果至关重要。例如，当在冠状平面上切割大鼠声带时，四个内在喉肌的神经肌肉接头（NMJ）位于组织深度小于1.8 mm以内11。因此，在冷冻切除过程中精确监测喉部肌肉解剖结构对于准确识别感兴趣的部分并防止组织过度切除至关重要。目标肌肉的过度切除可能导致 NMJs11 的数量和形貌识别不准确，或者如果目标肌肉由于特征性方向混淆而被丢弃，则可能导致样本量总体减少12。随着喉部肌肉研究及其各自适应性的新模型的开发，标准操作程序对于确保结果在研究中精确，可靠和可重复至关重要。
本文的目的是详细准备大鼠声带，以实现最佳纵向和横截面分析。描述了我们实验室中经常使用的详细方法，以在冷冻切除过程中识别目标肌肉标志物。虽然在几个实验室中使用了类似的方法，但这里提供了比文献中更多的细节，以确保新手研究人员实施时可靠和准确的复制。本教程的目标是为大鼠声带的免疫组织化学（IHC）评估提供标准方法，以提高实验室和研究的一致性。

<table>
	<tbody>
		<tr>
			<td>2-Methylbutane Certified</td>
			<td>Fisher Chemical</td>
			<td>35514</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Foil</td>
			<td>Fisherbrand</td>
			<td>1213101</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo Tongs SS</td>
			<td>Thermo Scientific</td>
			<td>11679123</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystems</td>
			<td>CM3050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat blades</td>
			<td>C.L. Sturkey D554X50</td>
			<td>22-210-045</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Base Molds 15mm x 15mm</td>
			<td>Thermo Scientific</td>
			<td>41-741</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Underpads</td>
			<td>Medline</td>
			<td>23-666-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection kit</td>
			<td>Thermo Scientific</td>
			<td>9996969</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS - Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Gibco</td>
			<td>14190136</td>
			<td></td>
		</tr>
		<tr>
			<td>Frozen Section Medium</td>
			<td>Fisher Healthcare</td>
			<td>23-730-571</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice Bucket</td>
			<td>Bel-Art</td>
			<td>11999054</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunostain Moisture Chamber</td>
			<td>Ted Pella Inc</td>
			<td>NC9425474</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holders</td>
			<td>Assi</td>
			<td>ASSI.B148</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Woven Sponges, 4 Ply</td>
			<td>Quick Medical</td>
			<td>9023</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Troemner</td>
			<td>02-217-987</td>
			<td></td>
		</tr>
		<tr>
			<td>Pap pen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16% w/v aq. soln., methanol free</td>
			<td>Alfa Aesar</td>
			<td>50-00-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Microcentrifuge Tubes</td>
			<td>Fisherbrand</td>
			<td>5408129</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen Storage Bags</td>
			<td>Fisherbrand</td>
			<td>19240093</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Graduated Measure 32 oz/100 mL</td>
			<td>Polar Ware</td>
			<td>114231B</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Task wiper</td>
			<td>Kimberly-Clark Professional&#8482; 34155</td>
			<td>06666A</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Fisherbrand</td>
			<td>2261840</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Pattern Scissors</td>
			<td>Assi</td>
			<td>ASSI.SAS15RV</td>
			<td></td>
		</tr>
		<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

本教程的目的是描述用于组织化学神经肌肉研究的大鼠声带的准备工作。该协议概述了大鼠喉部解剖，闪光冷冻和声带冷冻切除术的程序。本研究描述了如何在纵向和横截面上冷冻切除声带。该协议的新颖之处在于冷冻切除过程中的喉部跟踪，可确保准确识别内在喉部肌肉并减少组织丢失的机会。数字显示了两个平面的渐进式冷冻切除。对29只大鼠半喉部进行了冷冻切除术，并从甲状腺软骨的出现到包括整个声带的第一部分的外观进行了跟踪。两个平面上所有动物的完整声带都可视化了。从甲状腺软骨的外观到两个平面上的完整声带的距离存在很高的变异性。体重与喉部标志物的深度无关，这表明个体变异性以及与组织准备相关的其他因素可能是切片过程中标志物外观高变异性的原因。本研究详细介绍了一种方法，并提供了用于准备大鼠声带以进行组织化学神经肌肉检查的形态学数据。由于个体变异性高，在冷冻切除过程中应密切跟踪喉部标志物，以防止过度切除组织和组织丢失。使用一致的方法，包括足够的组织准备和对大鼠喉内标志物的认识，将有助于在研究中获得一致的结果，并帮助有兴趣使用大鼠声带作为模型的新研究人员研究喉神经肌肉机制。

代表性的结果是正在进行的声乐运动对喉神经肌肉系统影响的研究的一部分。对29只雄性Fischer 344/棕色挪威大鼠（12只9个月大，17只24个月大）进行称重和安乐死，吸入CO2 ，然后进行双侧开胸术。
这些程序遵循概述的协议来标记外侧和内侧TA肌肉的NMJ和纤维大小。使用喉部肌肉和周围软骨在纵向和横截面中跟踪喉标志物之间的距离，以确定冷冻切除术期间的进展（<stron...

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

该协议描述了用于准备大鼠声带以进行组织化学神经肌肉研究的方法。

用于神经肌肉分析的大鼠声带的制备

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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Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...