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术刀暴露喉部。</li>
	<li>将气管尾部切开到第三气管环，并在舌骨上做一个切口，用解剖剪刀切除整个喉部。</li>
	<li>使用显微切割工具（镊子、销和微剪刀）从喉部取出外源性喉组织（食道、甲状腺和外源性喉肌）。</li>
	<li>使用微剪刀，使用后腱肌之间的中线作为标志，将喉背侧的肛门一分为二。用微剪刀将喉部的侧壁固定以暴露声带，然后通过声带前部之间的甲状腺软骨中线将腹侧一分为二（图1）。 注意:此步骤可以是可选的;可以跳过它以保持喉部完整。喉部的两切允许通过分别使用同一喉部的右侧和左侧来进行多种免疫染色技术。</li>
	<li>在磷酸盐缓冲溶液（PBS）中冲洗每个半喉部约10秒，并用任务刮水器轻轻干燥，以减少冷冻过程中的冰晶形成。</li>
</ol>2. 固定和/或快速冷冻喉组织
注意:固定可能不适用于所有免疫染色方案。喉组织通常在解剖后立即被快速冷冻。跳过步骤2.1，无需固定即可快速冷冻喉部组织。
<ol><li>为了固定半喉，将组织置于装有4%甲醛的PBS离心管中，室温下在70rpm的轨道振荡器上1小时。将组织转移到干净的离心管中，并在PBS中冲洗3次20分钟。然后转移到干净的离心管中，并在4°C下浸没在20%蔗糖/ 5%甘油溶液中（约18小时或直到组织下沉）。 注意:甲醛是危险的，应与适当的个人防护设备一起在通风橱中使用。</li>
	<li>将所有半喉部均匀放置到充满最佳切割温度（OCT）化合物的冷冻模具中。对于半边，将声带内侧表面面向冷冻器底部的组织放置，并将声带的纵向面平行于冷冻器开口的下边缘。对于整个喉部，将后侧胪骨的组织朝向冷冻膜的底部，并且声带的纵向面平行于冷冻孔的下边缘。 注意:OCT化合物内一致的喉部方向对于大鼠声带的冷冻切除至关重要。一旦包埋并冷冻了半边，就必须解冻以改变其方向，从而引入多次解冻-冷冻循环导致组织损伤的风险。</li>
	<li>使用异戊烷（2-甲基丁烷）在被液氮包围的钢制烧杯中冷却的闪冻组织。 注:当烧杯的侧面和底部开始形成白色沉淀物时，异戊烷达到组织冷冻的最佳温度13。使用异戊烷是因为它具有比液氮更高的导热性，这有助于防止组织块在快速冷冻过程中开裂。有关OTC中冷冻组织的更详细说明，请参阅Kumar等人13。</li>
	<li>将每个模具包裹在预先标记的箔中，并放入单独的冷冻袋中以防止脱水，并立即储存在干冰上，直到转移到-80°C的冰箱中储存。</li>
</ol>3. 横截面上的冷冻切除术
<ol><li>将低温恒温器中的腔室温度设置为-20°C，该温度介于制造商手册推荐用于肌肉组织切片的温度范围（15-25°C）的中间。</li>
	<li>将低温恒温器截面厚度设置为 10 μm 厚截面。 注意:对于肌肉纤维横截面分析，10μm厚的切片是最佳的，可以对标记的肌肉纤维进行完整的染色和强大的成像强度，以进行纤维分型分析14，15，16。某些方案可能需要不同的切片厚度，具体取决于神经肌肉靶标。</li>
	<li>将组织转移到低温恒温器室，在低温恒温器标本盘（卡盘）上加入一层均匀的OCT化合物，并将嵌入的组织块放在标本盘上的OCT化合物的顶部。要获得用于甲状腺样动脉 （TA） 肌纤维分析的声带横截面，请将标本贴在卡盘上，使腹侧甲状腺软骨面向低温恒温器叶片，而类肾上腺软骨面向标本盘。 注意:重要的是要注意，这些地标在此阶段是不可见的，因为OCT化合物在冷冻时会变白和不透明。这种可见性的缺乏是为什么在闪光冻结阶段注意下颌的方向至关重要的原因。</li>
	<li>通过将标本头部推进100μm直到甲状腺软骨的腹侧部分出现来修剪OCT化合物。</li>
	<li>然后修剪并跟踪从甲状腺软骨开始的30μm切片，直到固有层，内侧TA肌肉和外侧TA肌肉暴露。 注意:从甲状腺软骨开始，每100μm一次，应跟踪并注意喉部标志，以确保切片的角度不倾斜。 图2 表示横截面上以10倍放大倍率下的两组喉部特征。</li>
	<li>一旦达到目标TA肌肉，在10μm处收集带正电荷的载玻片上的切片。</li>
	<li>将切片储存在4°C的PBS中以保持水分，直到它们准备好染色。 注意:固定组织可以在PBS中储存长达一周，具体取决于IHC靶标，而未固定的组织应立即处理。</li>
</ol>4. 纵平面冷冻切除术
<ol><li>将低温恒温器室再次设置为-20°C，将截面厚度更改为30μm。 注意:对于 NMJ 分析，30−60 μm 的组织厚度可用于捕获喉肌内的几个完整的 NMJ，而不会碎裂神经末梢或运动终板11，12，17。</li>
	<li>为了获得用于TA肌肉NMJ分析的纵向声带切片，请将标本贴在卡盘上，使会厌朝向低温恒温器叶片，气管腔朝下朝向标本盘。</li>
	<li>通过将标本头部向前推进100μm直到甲状腺软骨出现来修剪OCT化合物。</li>
	<li>修剪和跟踪从甲状腺发病到TA肌肉的内侧和外侧分裂暴露的30μm切片。 注意:建议在纵平面上使用五组喉部标志，以跟踪组织向目标TA肌的深度进展。 图3 显示了纵平面上10倍放大镜下的喉部特征。</li>
	<li>一旦达到目标TA肌肉，在30μm处收集带正电荷的载玻片上的切片。</li>
	<li>将切片储存在4°C的PBS中以保持水分，直到它们准备好染色。</li>
</ol>

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作者没有什么可透露的。

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）。
尽管每种方案的组织嵌入，定向和切片均匀，但喉标志物之间的距离仍存在变异性。此外，体重的差异没有解释从一组喉标志物到下一组喉标志物的组织深度范围的变异性。喉部标志物之间距离的这种差异可能是由于动物之间喉部解剖结构的个体差异，解剖时OCT化合物内冷冻剂中喉部方向的微小差异，或者切片时如何将标本放置在低温恒温器内的标本盘上（即，安装前放置在标本盘上的OCT化合物量或放置角度的微小差异）。
认识到标本制备中的这些微小差异会导致喉组织标志物深度的实质性差异，因此新手研究人员必须有一个参考地图来工作。概述的研究方案定义了识别感兴趣的肌肉和防止方案陷阱的方法 - 例如本文档中概述的那些 - 可以提高可重复性并防止不必要的组织损失。
虽然这项研究的重点是TA肌肉，但这种方法也适用于其他内在喉肌。例如，在纵向声带平面上进行切片可产生鼻翼、外侧 TA、内侧 TA、外侧膈肌和上膈肌的纵向肌纤维切片，以及后侧腱肌的横截面。在横截面声带平面上进行剖面可产生鼻翼、外侧 TA、内侧蝽蚣肌、上胭脂肌和胫骨肌的横截面，以及后胫骨肌的纵向截面。此外，尽管本研究不包括雌性大鼠，但雄性和雌性大鼠在喉部标志性外观方面的差异预计不会，因为大鼠喉部内的性二态性是肌肉特异性的，与喉骨框架解剖学无关16，18。
喉部标志物之间距离的变异性可能使新手研究者难以冷冻切除大鼠声带。该研究表明，尽管大鼠喉部的冷冻，嵌入和冷冻切除方式一致，但喉部标志物之间的距离差异很大;动物体重没有解释这种变异性。本研究提供了详细的程序和相关图像，说明如何适当地制备喉部肌肉组织，并确定喉部标志物，以进行大鼠声带的神经肌肉组织学研究。

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。

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		<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（表1）。跟踪在甲状腺软骨在两个方向平面上首次出现时开始。 图2 显示了横截面冷冻切除术期间喉标志物的出现，甲状腺（图2a，b）出现在内侧TA肌和固有层之前（图2c，d）。 图3 显示了纵向冷冻切除期间喉标志物以时间顺序出现，其中鼻翼肌（图3a，b）出现在内侧TA肌（图3c，d）和固有层（图3e，f）之前。
在两个方向平面上，单个动物的地标之间的距离差异很大。
年轻大鼠的体重和喉部标志性表现具有弱至中度的相关性，而老年大鼠的相关性较弱（表2 和 表3）。对于两个年龄组，每个平面内地标之间的距离具有中度至强相关性，但两个解剖平面之间的相关性较弱。因此，地标性外观的变异性不能通过体重或喉部大小的个体变化来解释。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61327/61327fig01.jpg"> 图1:大鼠喉背侧一分为二，位于类芳烯类软骨（ArC）之间。 半喉右侧用纵平面（LZ1-LZ5）中的地标注释，对应于 表1中的五个纵向标志。半喉左侧在横截面（CZ1 和 CZ2）中标注有标记，分别对应于外侧 TA 肌的开始和声带的整个横截面。VF = 声带，CrC = 环状软骨，AlC = 胫骨软骨，T1 = 第一气管环。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61327/61327fig01large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61327/61327fig02.jpg"> 图2:在对层粘连蛋白进行免疫染色后，在明场（右）和荧光488通道（左）中以10倍放大倍率成像的两个横截面，以勾勒肌肉纤维。 这些部分（从上到下）显示冷冻切除术期间按时间顺序的进展，甲状腺（a，b）出现在内侧TA肌和声带的固有层（c，d）之前。ThC = 甲状腺软骨，LTA = 外侧甲状腺样动脉，MTA = 内侧甲状腺样动脉。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61327/61327fig02large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61327/61327fig03.jpg"> 图3:在对神经肌肉接头进行免疫染色后，在明场（右）和荧光488通道（左）中以10倍放大倍率成像的三个纵向切片。 这些部分（从上到下）显示冷冻切除术期间按时间顺序的进展，其中鼻翼肌（a，b）出现在内侧TA肌（c，d）之前，并出现在声带的固有层（e，f）之前。AlC = 甲状软骨，ThC = 甲状腺软骨，ArC = 类芳烯类软骨，LTA = 外侧甲状腺样肌，MTA = 内侧甲状腺素，SCA = 上类环状动脉。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61327/61327fig03large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="2">纵向地标</td>
			<td>均值（标准偏差）（以 μm 为单位）</td>
			<td>范围（μm）</td>
		</tr>
<tr>
<td colspan="2">1.所有三个主要软骨（甲状腺，鼻翼，类肾上腺素）都随着肌肉纤维的出现而出现</td>
			<td>1,591 (665)</td>
			<td>350–2,800</td>
		</tr>
<tr>
<td colspan="2">2. 出现上颌骨样肌 （SCA）、鼻翼环状肌 （ACA） 和外侧甲状腺样肌 （LTA）</td>
			<td>2,344 (591)</td>
			<td>91–3,500</td>
		</tr>
<tr>
<td colspan="2">3. ACA和LTA肌肉完全伸展，无碎裂</td>
			<td>2,631 (532)</td>
			<td>1505–3,640</td>
		</tr>
<tr>
<td colspan="2">4.类芳氨酸软骨增大，ACA消失，内侧甲状腺素（MTA）肌出现</td>
			<td>2,948 (606)</td>
			<td>1765–4,305</td>
		</tr>
<tr>
<td colspan="2">5.目标全声带部分:LTA和MTA肌肉完全伸展，无碎片化，出现固有层</td>
			<td>3,131 (542)</td>
			<td>2205–4410</td>
		</tr>
<tr>
<td colspan="4">横截面地标</td>
		</tr>
<tr>
<td colspan="2">1. LTA肌肉出现</td>
			<td>303 (138)</td>
			<td>110–690</td>
		</tr>
<tr>
<td colspan="2">2.出现MTA肌肉，约为LTA大小的50%，并注意到明显的固有层和上皮。</td>
			<td>482 (167)</td>
			<td>210–850</td>
		</tr>
</tbody></table>表1:冷冻切除术期间从甲状腺软骨首次出现到每个喉部标志的距离（以μm为单位）（n = 29）。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td></td>
			<td colspan="2">断续器</td>
			<td colspan="5">纵的</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>断续器</td>
			<td>断续器</td>
			<td>软骨</td>
			<td>阿拉尔/爱生雅</td>
			<td>断续器</td>
			<td>断续器</td>
			<td>断续器</td>
		</tr>
<tr>
<td rowspan="2">断续器</td>
			<td>断续器</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>断续器</td>
			<td>0.88</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td rowspan="5">纵的</td>
			<td>软骨</td>
			<td>0.42</td>
			<td>0.42</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>阿拉尔/爱生雅</td>
			<td>0.57</td>
			<td>0.47</td>
			<td>0.77</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>断续器</td>
			<td>0.59</td>
			<td>0.47</td>
			<td>0.71</td>
			<td>0.98</td>
			<td>1</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>断续器</td>
			<td>0.53</td>
			<td>0.39</td>
			<td>0.72</td>
			<td>0.97</td>
			<td>0.98</td>
			<td>1</td>
			<td></td>
		</tr>
<tr>
<td>断续器</td>
			<td>0.53</td>
			<td>0.41</td>
			<td>0.76</td>
			<td>0.96</td>
			<td>0.97</td>
			<td>0.99</td>
			<td>1</td>
		</tr>
<tr>
<td></td>
			<td>重量</td>
			<td>-0.55</td>
			<td>-0.35</td>
			<td>0.08</td>
			<td>-0.45</td>
			<td>-0.46</td>
			<td>-0.46</td>
			<td>-0.41</td>
		</tr>
</tbody></table>表2:年轻雄性大鼠横截面（CSA）和纵平面中体重与喉部标志物深度之间的Pearson相关性结果。 LTA = 外侧甲状腺样肌，MTA = 内侧甲状腺样肌，SCA = 上类甲状腺，LP = 固有层。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td></td>
			<td colspan="2">断续器</td>
			<td colspan="5">纵的</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>断续器</td>
			<td>断续器</td>
			<td>软骨</td>
			<td>阿拉尔/爱生雅</td>
			<td>断续器</td>
			<td>断续器</td>
			<td>断续器</td>
		</tr>
<tr>
<td rowspan="2">断续器</td>
			<td>断续器</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>断续器</td>
			<td>0.9</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td rowspan="5">纵的</td>
			<td>软骨</td>
			<td>0.21</td>
			<td>0.33</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>阿拉尔/爱生雅</td>
			<td>0.05</td>
			<td>0.07</td>
			<td>0.73</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>断续器</td>
			<td>-0.06</td>
			<td>-0.04</td>
			<td>0.64</td>
			<td>0.96</td>
			<td>1</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>断续器</td>
			<td>-0.02</td>
			<td>-0.02</td>
			<td>0.6</td>
			<td>0.79</td>
			<td>0.84</td>
			<td>1</td>
			<td></td>
		</tr>
<tr>
<td>断续器</td>
			<td>-0.17</td>
			<td>-0.15</td>
			<td>0.52</td>
			<td>0.76</td>
			<td>0.85</td>
			<td>0.91</td>
			<td>1</td>
		</tr>
<tr>
<td></td>
			<td>重量</td>
			<td>0.23</td>
			<td>0.13</td>
			<td>-0.24</td>
			<td>-0.07</td>
			<td>-0.15</td>
			<td>-0.15</td>
			<td>-0.3</td>
		</tr>
</tbody></table>表3:老年雄性大鼠横截面（CSA）和纵平面中体重与喉部标志物深度之间的Pearson相关性结果。 LTA = 外侧甲状腺样肌，MTA = 内侧甲状腺样肌，SCA = 上类甲状腺，LP = 固有层。

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 ...

preparation-of-the-rat-vocal-fold-for-neuromuscular-analyses

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.6K 次观看.  New York University School of Medicine. 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。

视频: Preparation of the Rat Vocal Fold for Neuromuscular Analyses

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

视网膜节的形态分析

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

科研

神经科学

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

连续光漂白划定单雪旺细胞在神经肌肉接头处

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

全山神经肌肉交界分析的 横向解剖阿布多米尼斯 肌肉

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

为什么量化事项:表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

测量神经肌肉接头功能

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Microstate and Omega Complexity Analyses of the Resting-state Electroencephalography

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of EEG data and have been widely used to investigate the neural mechanisms in some brain disorders. The goal of this article is to describe the protocol underlying EEG microstate and omega complexity analyses step by step. The main advantage of these two measures is that they could eliminate the reference-dependent problem inherent to traditional spectrum analysis. In addition, microstate analysis makes good use of high time resolution of resting-state EEG, and the four obtained microstate classes could match the corresponding resting-state networks respectively. The omega complexity characterizes the spatial complexity of the whole brain or specific brain regions, which has obvious advantage compared with traditional complexity measures focusing on the signal complexity in a single channel. These two EEG measures could complement each other to investigate the brain complexity from the temporal and spatial domain respectively.

This article describes the protocol underlying electroencephalography (EEG) microstate analysis and omega complexity analysis, which are two reference-free EEG measures and highly valuable to explore the neural mechanisms of brain disorders.

静止状态脑电图的微状态和欧米茄复杂性分析

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

用于下游分析的冷冻啮齿动物大脑区域集合

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

单骨骼肌肉纤维解剖，用于全山神经肌肉结的免疫荧光和形态分析

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

用于神经肌肉分析的大鼠声带的制备

摘要

探索更多视频

此视频中的章节

视网膜节的形态分析

连续光漂白划定单雪旺细胞在神经肌肉接头处

全山神经肌肉交界分析的横向解剖阿布多米尼斯肌肉

为什么量化事项:表型的表征在

测量神经肌肉接头功能

静止状态脑电图的微状态和欧米茄复杂性分析

提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备

用于下游分析的冷冻啮齿动物大脑区域集合

单骨骼肌肉纤维解剖，用于全山神经肌肉结的免疫荧光和形态分析

扩展轴突转运体内成像工具包

用于神经肌肉分析的大鼠声带的制备

摘要

探索更多视频

此视频中的章节

视网膜节的形态分析

连续光漂白划定单雪旺细胞在神经肌肉接头处

全山神经肌肉交界分析的 横向解剖阿布多米尼斯 肌肉

为什么量化事项:表型的表征在

测量神经肌肉接头功能

静止状态脑电图的微状态和欧米茄复杂性分析

提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备

用于下游分析的冷冻啮齿动物大脑区域集合

单骨骼肌肉纤维解剖，用于全山神经肌肉结的免疫荧光和形态分析

扩展轴突转运体内成像工具包

全山神经肌肉交界分析的横向解剖阿布多米尼斯肌肉