Name: preparation of the rat vocal fold for neuromuscular analyses
Uploaded: 2020-05-15T14:45:00.000+00:00
Duration: 7 min 17 s
Description: Watch this Scientific Journal Video about Preparation of the Rat Vocal Fold for Neuromuscular Analyses at JoVE.com

This research was supported by grants F31DC017053-01A1 (Lenell, PI) and K23DC014517 (Johnson, PI) from the National Institute on Deafness and other Communication Disorders of the National Institutes of Health.

This study was performed in compliance with the Institutional Animal Care and Use Committee of New York University School of Medicine.
1. Dissect rat larynx
<ol>
	<li>Euthanize rat according to the institutionally approved protocol. Shave the ventral neck from the mandible to manubrium and swab with alcohol to prevent fur contamination in the tissue specimens.</li>
	<li>Under a dissecting scope with 10x magnification excise the entire larynx by creating a midline neck incision with a scalpel until the trachea is exposed.</li>
	<li>Separate the ventral extrinsic laryngeal muscles at the midline to expose the larynx using forceps and dissecting scissors or a scalpel.</li>
	<li>Sever the trachea caudal to the third tracheal ring and make an incision rostral to the hyoid bone to excise the whole larynx using dissecting scissors.</li>
	<li>Remove the extrinsic laryngeal tissues (esophagus, thyroid gland, and extrinsic laryngeal muscles) from the larynx using microdissection tools (tweezers, pins, and microscissors) under magnification.</li>
	<li>With microscissors, bisect the larynx dorsally between the arytenoids using the midline between the posterior cricoarytenoid muscles as a landmark. Pin lateral walls of the larynx to expose the vocal folds and then bisect ventrally through the midline of the thyroid cartilage between the anterior commissure of the vocal folds with microscissors (Figure 1). 
	NOTE: This step can be optional; it can be skipped to keep the larynx whole. Bisection of larynges allow multiple immunostaining techniques by separately using the right and left sides of the same larynx.</li>
	<li>Rinse each hemi-larynx in phosphate buffered solution (PBS) for ~10 s and delicately dry with a task wiper to reduce ice crystal formation during freezing.</li>
</ol>
2. Fix and/or flash-freeze laryngeal tissue
NOTE: Fixation may not be ideal for all immunostaining protocols. Often laryngeal tissues are flash-frozen fresh immediately following dissection. Skip step 2.1 to flash-freeze laryngeal tissue without fixation.
<ol>
	<li>To fix hemi-larynges place tissues in centrifuge tube filled with 4% formaldehyde in PBS for 1 h at room temperature on an orbital shaker at 70 rpm. Transfer tissues to a clean centrifuge tube and rinse 3x for 20 min in PBS. Then transfer to a clean centrifuge tube and submerge in a 20% sucrose/5% glycerol solution (~18 h or until tissue sinks) at 4 &#176;C. 
	CAUTION: Formaldehyde is hazardous and should be used in a fume hood along with appropriate personal protective equipment.</li>
	<li>Place all hemi-larynges in a uniform position into a cryo-mold filled with optimal cutting temperature (OCT) compound. For a hemilarynx, place the tissue with the medial surface of the vocal fold facing the bottom of the cryomold and the longitudinal aspect of the vocal fold parallel to the lower edge of the cryomold opening. For whole larynges, place the tissue with the posterior cricoarytenoids facing the bottom of the cryomold and the longitudinal aspect of the vocal fold parallel to the lower edge of the cryomold opening. 
	NOTE: Consistent laryngeal orientation within OCT compound is critical for cryosectioning of the rat vocal fold. Once the hemilarynx is embedded and frozen, it must be thawed to change its orientation, thereby introducing risks of tissue damage from multiple thaw-freeze cycles.</li>
	<li>Flash-freeze tissues using isopentane (2-methylbutane) chilled in a steel beaker surrounded by liquid nitrogen. 
	NOTE: The isopentane reaches optimal temperature for tissue freezing when white precipitates start to form on the sides and bottom of the beaker13. Isopentane is used because it has a higher thermal conductivity than liquid nitrogen, which helps prevent cracking of the tissue block during rapid freezing. For a more detailed description of freezing tissue in OTC refer to Kumar et al.13.</li>
	<li>Wrap each mold in prelabeled foil and place in an individual freezer bag to prevent dehydration and immediately store on dry ice until transferred for storage in a -80 &#176;C freezer.</li>
</ol>
3. Cryosection hemilarynx in cross-sectional plane
<ol>
	<li>Set chamber temperature in the cryostat to -20 &#176;C, which is in the middle of the temperature range (15&#8722;25 &#176;C) recommended for muscle tissue sectioning by the manufacturer&#8217;s manual.</li>
	<li>Set cryostat section thickness to 10 &#181;m thick sections. 
	NOTE: For muscle fiber cross-sectional analysis, 10 &#181;m thick sections are optimal to allow for complete staining and robust imaging intensity of the labeled muscle fibers for fiber typing analysis14,15,16. Some protocols may require different section thickness depending on neuromuscular targets.</li>
	<li>Transfer tissues to the cryostat chamber, add a uniform layer of OCT compound on the cryostat specimen disk (chuck), and place the embedded tissue block on top of the OCT compound on the specimen disk. To obtain cross-sections of the vocal fold for thyroarytenoid (TA) muscle fiber analysis, affix the specimen to the chuck so that the ventral thyroid cartilage faces the cryostat blade and the arytenoid cartilage faces the specimen disk. 
	NOTE: It is critical to note that these landmarks are not visible at this stage, due to the OCT compound becoming white and opaque when frozen. This lack of visibility is why it is critical to note the orientation of the hemilarynx during the flash freezing stage.</li>
	<li>Trim OCT compound by advancing the specimen head by 100 &#181;m until the ventral portion of the thyroid cartilage appears.</li>
	<li>Then trim and track 30 &#181;m sections from the onset of the thyroid cartilage until the lamina propria, medial TA muscles, and lateral TA muscle are exposed. 
	NOTE: Laryngeal landmarks should be tracked and noted from the onset of the thyroid cartilage every 100 &#181;m to ensure the angle of sectioning is not oblique. Figure 2 represents the two sets of laryngeal landmarks in the cross-sectional plane at 10x magnification.</li>
	<li>Once the target TA muscle is reached, collect sections on positively charged slides at 10 &#181;m.</li>
	<li>Store sections in PBS at 4 &#176;C to retain moisture until they are ready to be stained. 
	NOTE: Fixed tissue can be stored in PBS up to one week depending on IHC target whereas unfixed tissue should be immediately processed.</li>
</ol>
4. Cryosection hemilarynx in longitudinal plane
<ol>
	<li>With the cryostat chamber again set to -20 &#176;C, change the section thickness to 30 &#181;m. 
	NOTE: For NMJ analysis, a tissue thickness between 30&#8722;60 &#181;m can be used to capture several complete NMJs within the laryngeal muscles without fragmentation of either the nerve terminal or motor endplate11,12,17.</li>
	<li>To obtain longitudinal vocal fold sections for NMJ analysis of the TA muscle, affix the specimens to the chuck so that the epiglottis is oriented towards the cryostat blade and the tracheal lumen faces down towards the specimen disk.</li>
	<li>Trim the OCT compound by advancing the specimen head by 100 &#181;m until the thyroid cartilage appears.</li>
	<li>Trim and track sections of 30 &#181;m from the onset of the thyroid until the lamina propria and medial and lateral divisions of the TA muscle are exposed. 
	NOTE: Five sets of laryngeal landmarks in the longitudinal plane are recommended to track tissue depth progression towards the target TA muscle. Figure 3 represents the laryngeal landmarks in the longitudinal plane at 10x magnification.</li>
	<li>Once the target TA muscle is reached, collect sections on positively charged slides at 30 &#181;m.</li>
	<li>Store sections in PBS at 4 &#176;C to retain moisture until they are ready to be stained.</li>
</ol>

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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>

The authors have nothing to disclose.

Preparing rat vocal folds for neuromuscular analysis can present with various challenges. Not only are laryngeal muscles small and surrounded by cartilage, thereby making it difficult to directly extract target muscle, high variability was also found between animals in the depth of laryngeal anatomical landmarks. For muscle the cross-section plane protocol, complete vocal fold sections appeared between 21&#8722;85 sections (10 &#181;m per section) after the initial appearance of the ventral thyroid cartilage, which is quite a bit fewer than the 63&#8722;126 sections (35 &#181;m per section) in the longitudinal plane for NMJ analysis protocols (Table 1).
Variability was noted in distances between laryngeal landmarks despite the uniform embedding, orienting, and sectioning of tissues for each type of protocol. Furthermore, differences in body weight did not account for variability in the wide ranges of tissue depth from one set of laryngeal landmarks to the next. This variability in distance between laryngeal landmarks may be due to individual differences in laryngeal anatomy across animals, small differences in orientation of larynges in the cryomold within the OCT compound at the time of dissection, or how the specimens were placed on the specimen disk within the cryostat when sectioning (i.e., the amount of OCT compound placed on the specimen disk prior to mounting or slight differences in angle of placement).
With an understanding that these slight differences in specimen preparation can lead to substantial variability in depth of laryngeal tissue landmarks, it is critical that novice investigators have a reference map from which to work. Outlined study protocols defining methods to identify the muscle(s) of interest and prevent protocol pitfalls&#8212;such as the ones outlined in this document&#8212;can improve reproducibility and prevent unwanted tissue loss.
Although this study focused on the TA muscle, this methodology is applicable for other intrinsic laryngeal muscles as well. For example, sectioning in the longitudinal vocal fold plane yields longitudinal muscle fiber sections of the alar, lateral TA, medial TA, lateral cricoarytenoid, and superior cricoarytenoid muscles, and cross-sections of the posterior cricoarytenoid muscles. Sectioning in the cross-sectional vocal fold plane yields cross-sections of the alar, lateral TA, medial TA, lateral cricoarytenoid, superior cricoarytenoid, and cricothyroid muscles, as well as longitudinal sections of the posterior cricoarytenoid muscles. Additionally, although this study did not include female rats, differences between male and female rats in laryngeal landmark appearance are not expected because sexual dimorphism within the rat larynx is muscle specific and not related to laryngeal framework anatomy16,18.
The variability in distance between laryngeal landmarks can make cryosectioning the rat vocal fold difficult for novice investigators. This study demonstrated that despite consistency in how the rat larynges were frozen, embedded, and cryosectioned, the distance between laryngeal landmarks varied greatly; animal weight did not account for this variability. This study provides detailed procedures with associated images on how to appropriately prepare laryngeal muscle tissue and identify laryngeal landmarks for neuromuscular histological investigation of the rat vocal fold.

The rat larynx is a well-established model to investigate structural and functional neuromuscular laryngeal adaptations to development, aging, disease, and pharmacological agents1,2,3,4,5. Consistency of histological methods is critical to this line of work, as there are multiple intricacies involved in muscle preparation and analysis as well as challenges associated with laryngeal size, shape, and topography of the muscles encapsulated within the laryngeal cartilages1,6,7,8,9,10,11. Due to the small size of the rat intrinsic laryngeal muscles, systematic embedding, freezing, and cryosectioning are critical to achieve consistent and accurate results. For example, when sectioning the rat vocal fold in the coronal plane, the neuromuscular junctions (NMJs) of four of the intrinsic laryngeal muscles are located within less than 1.8 mm of tissue depth11. Therefore, precise monitoring of laryngeal muscle anatomy during cryosectioning is imperative to accurately identify the section(s) of interest and prevent oversectioning of tissue. Oversectioning of the target muscle can result in inaccurate identification of number and topography of NMJs11 or can result in overall reductions in sample size if the target muscle is discarded due to landmark orientation confusion12. As novel models for the study of laryngeal muscle and their respective adaptations are developed, standard operating procedures are essential to ensure results are precise, reliable, and reproducible across studies.
The objective of this article is to detail preparation of the rat vocal fold for optimal longitudinal and cross-sectional analysis. Detailed methods used regularly in our laboratory are described to identify target muscle landmarks during cryosectioning. Although similar methods are used in several laboratories, greater detail is provided here than in the literature to ensure reliable and accurate replication when implemented by novice investigators. The goal of this tutorial is to provide a standard methodology for immunohistochemical (IHC) evaluation of the rat vocal fold to improve consistency across laboratories and investigations.

<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&#039;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&amp;trade; 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>NOTE: For all supplies, these are examples of equipment to purchase. The exact model is not necessary to complete our methods.</td><td></td><td></td><td></td></tr></tbody></table>

preparation of the rat vocal fold for neuromuscular analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

The representative results were part of an ongoing investigation of the effects of vocal exercise on the laryngeal neuromuscular system. Twenty-nine male Fischer 344/brown Norway rats (12 9-month-old, 17 24-month-old) were weighed and euthanized with CO2 inhalation followed by a bilateral thoracotomy.
The procedures followed the outlined protocol to label NMJs and fiber size of the lateral and medial TA muscles. The distance between laryngeal landmarks was tracked in both longitudinal and cross-sectional planes using laryngeal muscles and surrounding cartilages to determine progression during cryosectioning (Table 1). Tracking commenced at the first appearance of the thyroid cartilage in both directional planes. Figure 2 illustrates the appearance of laryngeal landmarks during cross-sectional cryosectioning in temporal order with the thyroid (Figure 2a,b) appearing prior to the medial TA muscle and to the lamina propria (Figure 2c,d). Figure 3 illustrates the appearance of laryngeal landmarks during longitudinal cryosectioning in temporal order with the alar muscle (Figure 3a,b) appearing prior to the medial TA muscle (Figure 3c,d) and to the lamina propria (Figure 3e,f).
In both directional planes, distances among landmarks greatly varied for individual animals.
Weight and laryngeal landmark appearances had weak to moderate correlations for young rats and weak correlations for aged rats (Table 2 and Table 3). Distances among landmarks within each plane were moderately to strongly correlated for both age groups, but weakly correlated between the two dissection planes. Therefore, variability in landmark appearance could not be accounted for by weight or individual variations in laryngeal size.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61327/61327fig01.jpg" /> 
Figure 1: A rat larynx dorsally bisected between the arytenoid cartilages (ArC). 
The right side of the hemi-larynx is annotated with landmarks in the longitudinal plane (LZ1-LZ5) corresponding to the five longitudinal landmarks in Table 1. The left side of the hemi-larynx is annotated with landmarks in the cross-sectional plane (CZ1 and CZ2) that correspond to the beginning of the lateral TA muscle and full cross-section of the vocal fold respectively. VF = vocal fold, CrC = cricoid cartilage, AlC = alar cartilage, and T1 = first tracheal ring. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61327/61327fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61327/61327fig02.jpg" /> 
Figure 2: Two cross-sections imaged at 10x magnification in brightfield (right) and in the fluorescent 488 channel (left) following immunostaining for laminin to outline muscle fibers. 
The sections (from top to bottom) show the progression during cryosectioning in temporal order with the thyroid (a,b) appearing prior to the medial TA muscle and to the lamina propria of the vocal fold (c,d). ThC = thyroid cartilage, LTA = lateral thyroarytenoid, and MTA = medial thyroarytenoid. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61327/61327fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61327/61327fig03.jpg" /> 
Figure 3: Three longitudinal sections imaged at 10x magnification in brightfield (right) and in the fluorescent 488 channel (left) following immunostaining for neuromuscular junctions. 
The sections (from top to bottom) show the progression during cryosectioning in temporal order with the alar muscle (a,b) appearing prior to the medial TA muscle (c,d) and to the lamina propria (e,f) of the vocal fold. AlC = alar cartilage, ThC = thyroid cartilage, ArC = arytenoid cartilage, LTA = lateral thyroarytenoid, MTA = medial thyroarytenoid, and SCA = superior cricoarytenoid. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61327/61327fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Longitudinal landmarks</td>
			<td>Mean (standard deviation) in &#956;m</td>
			<td>Range in &#956;m</td>
		</tr>
		<tr>
			<td colspan="2">1. All three major cartilages (thyroid, alar, arytenoid) appeared with emergence of muscle fibers</td>
			<td>1,591 (665)</td>
			<td>350&#8211;2,800</td>
		</tr>
		<tr>
			<td colspan="2">2. Superior cricoarytenoid (SCA), alar cricoarytenoid (ACA), and lateral thyroarytenoid (LTA) muscles appeared</td>
			<td>2,344 (591)</td>
			<td>91&#8211;3,500</td>
		</tr>
		<tr>
			<td colspan="2">3. ACA and LTA muscles extended completely without fragmentation</td>
			<td>2,631 (532)</td>
			<td>1505&#8211;3,640</td>
		</tr>
		<tr>
			<td colspan="2">4. Arytenoid cartilage enlarged, ACA disappeared, medial thyroarytenoid (MTA) muscle emerged</td>
			<td>2,948 (606)</td>
			<td>1765&#8211;4,305</td>
		</tr>
		<tr>
			<td colspan="2">5. Target full vocal fold section: LTA and MTA muscles extended completely without fragmentation and lamina propria emerged</td>
			<td>3,131 (542)</td>
			<td>2205&#8211;4410</td>
		</tr>
		<tr>
			<td colspan="4">Cross-sectional landmarks</td>
		</tr>
		<tr>
			<td colspan="2">1. LTA muscle appeared</td>
			<td>303 (138)</td>
			<td>110&#8211;690</td>
		</tr>
		<tr>
			<td colspan="2">2. MTA muscle appeared and was ~50% of the LTA size with clear lamina propria and epithelium noted.</td>
			<td>482 (167)</td>
			<td>210&#8211;850</td>
		</tr>
	</tbody>
</table>
Table 1: Distances in &#181;m from the first appearance of the thyroid cartilage to each laryngeal landmark during cryosectioning (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">CSA</td>
			<td colspan="5">Longitudinal</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>LTA</td>
			<td>MTA</td>
			<td>Cartilages</td>
			<td>Alar/SCA</td>
			<td>LTA</td>
			<td>MTA</td>
			<td>LP</td>
		</tr>
		<tr>
			<td rowspan="2">CSA</td>
			<td>LTA</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MTA</td>
			<td>0.88</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Longitudinal</td>
			<td>Cartilages</td>
			<td>0.42</td>
			<td>0.42</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alar/SCA</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>LTA</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>MTA</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>LP</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>Weight</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>
Table 2: Results of Pearson correlation between weight and the depth of laryngeal landmarks in the cross-sectional (CSA) and longitudinal planes for young male rats. LTA = lateral thyroarytenoid, MTA = medial thyroarytenoid, SCA = superior cricoarytenoid, and LP = lamina propria.
<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">CSA</td>
			<td colspan="5">Longitudinal</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>LTA</td>
			<td>MTA</td>
			<td>Cartilages</td>
			<td>Alar/SCA</td>
			<td>LTA</td>
			<td>MTA</td>
			<td>LP</td>
		</tr>
		<tr>
			<td rowspan="2">CSA</td>
			<td>LTA</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MTA</td>
			<td>0.9</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Longitudinal</td>
			<td>Cartilages</td>
			<td>0.21</td>
			<td>0.33</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alar/SCA</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>LTA</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>MTA</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>LP</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>Weight</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>
Table 3: Results of Pearson correlation between weight and the depth of laryngeal landmarks in the cross-sectional (CSA) and longitudinal planes for old male rats. LTA = lateral thyroarytenoid, MTA = medial thyroarytenoid, SCA = superior cricoarytenoid, and LP = lamina propria.

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

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

2.7K Views.  New York University School of Medicine. This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

Video: Preparation of the Rat Vocal Fold for Neuromuscular Analyses

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations.
The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training.
To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 &mu;g of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging).
Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1).
An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting ...

Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if any, therapies exist that target age-related laryngeal muscle dysfunction. Neurotrophins are involved in muscle innervation and differentiation of neuromuscular junctions (NMJs). It is thought that neurotrophins enhance neuromuscular transmission by increasing neurotransmitter release. The neuromuscular junctions (NMJs) become smaller and less abundant in aging rat laryngeal muscles, with evidence of functional denervation. We explored the effects of NTF4 for future clinical use as a therapeutic to improve function in aging human laryngeal muscles. Here, we provide the detailed protocol for systemic application and direct injection of NTF4 to investigate the ability of aging rat laryngeal muscle to remodel in response to NTF4 application. In this method, rats either received NTF4 either systemically via osmotic pump or by direct injection through the vocal folds. Laryngeal muscles were then dissected and used for histological examination of morphology and age-related denervation.

Here, we present a protocol to describe the use of neurotrophin 4 (NTF4) systemically and directly to remodel rat aging laryngeal muscles.

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if ...

Developmental Biology

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Medicine

3D Printing of Multilayered Silicone Vocal Fold Models

Manufacturing Process for Non-Adhesive Super-Soft Vocal Fold Models

This study aims to develop super-soft, non-sticky vocal fold models for voice research. The conventional manufacturing process of silicone-based vocal fold models results in models with undesirable properties, such as stickiness and reproducibility issues. Those vocal fold models are prone to rapid aging, leading to poor comparability across different measurements. In this study, we propose a modification to the manufacturing process by changing the order of layering the silicone material, which leads to the production of non-sticky and highly consistent vocal fold models. We also compare a model produced using this method with a conventionally manufactured vocal fold model that is adversely affected by its sticky surface. We detail the manufacturing process and characterize the properties of the models for potential applications. The outcomes of the study demonstrate the efficacy of the modified fabrication method, highlighting the superior qualities of our non-sticky vocal fold models.&#160;The findings contribute to the development of realistic and reliable vocal fold models for research and clinical applications.

Author Spotlight: Advancements in the Fabrication of Synthetic Vocal Fold Models for Phonetic and Robotic Applications

This study demonstrates the manufacturing of non-sticky and super-soft vocal fold models by introducing a specific way to create the vocal fold layers, providing a detailed description of the manufacturing procedure, and characterizing the properties of the models.

This study aims to develop super-soft, non-sticky vocal fold models for voice research. The conventional manufacturing process of silicone-based vocal ...

Engineering

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

Endoscopic Approach for Studying Laryngeal Dysfunction in Rodents

Minimally Invasive Murine Laryngoscopy for Close&#45;Up Imaging of Laryngeal Motion During Breathing and Swallowing

The larynx is an essential organ in mammals with three primary functions - breathing, swallowing, and vocalizing. A wide range of disorders are known to impair laryngeal function, which results in difficulty&#160;breathing (dyspnea), swallowing impairment (dysphagia), and/or voice impairment (dysphonia). Dysphagia, in particular, can lead to aspiration pneumonia and associated morbidity, recurrent hospitalization, and early mortality. Despite these serious consequences, existing treatments for laryngeal dysfunction are largely aimed at surgical and behavioral interventions that unfortunately do not typically restore normal laryngeal function, thus highlighting the urgent need for innovative solutions.
To bridge this gap, we have been developing an experimental endoscopic approach to investigate laryngeal dysfunction in murine (i.e., mouse and rat) models. However, endoscopy in rodents is quite challenging due to their small size relative to current endoscope technology, anatomical differences in the upper airway, and the necessity for anesthesia to optimally access the larynx. Here, we describe a novel transoral laryngoscopy approach that permits close-up, unobstructed video imaging of laryngeal motion in mice and rats. Critical steps in the protocol include precise anesthesia management (to prevent overdosing that abolishes swallowing and/or risks respiratory distress-related mortality) and micromanipulator control of the endoscope (for stable video recording of laryngeal motion by a single researcher for subsequent quantification).
Importantly, the protocol can be performed over time in the same animals to study the impact of various pathological conditions specifically on laryngeal function. A novel advantage of this protocol is the ability to visualize airway protection during swallowing, which is not possible in humans due to epiglottic inversion over the laryngeal inlet that obstructs the glottis from view. Rodents therefore provide a unique opportunity to specifically investigate the mechanisms of normal versus pathological laryngeal airway protection for the ultimate purpose of discovering treatments to effectively restore normal laryngeal function.

This protocol describes a serial transoral laryngoscopy approach for mice and rats that permits close-up, unobstructed video imaging of the larynx during breathing and swallowing using an optimized anesthetic regimen and finely tuned endoscopic manipulation techniques.

The larynx is an essential organ in mammals with three primary functions - breathing, swallowing, and vocalizing. A wide range of disorders are known to ...

Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual documentation of vocal fold vibration is challenging, particularly in non-human mammals. As an alternative, excised larynx experiments provide the opportunity to investigate vocal fold vibration under controlled physiological and physical conditions. However, the use of a full larynx merely provides a top view of the vocal folds, excluding crucial portions of the oscillating structures from observation during their interaction with aerodynamic forces. This limitation can be overcome by utilizing a hemi-larynx setup where one half of the larynx is mid-sagittally removed, providing both a superior and a lateral view of the remaining vocal fold during self-sustained oscillation.
Here, a step-by-step guide for the anatomical preparation of hemi-laryngeal structures and their mounting on the laboratory bench is given. Exemplary phonation of the hemi-larynx preparation is documented with high-speed video data captured by two synchronized cameras (superior and lateral views), showing three-dimensional vocal fold motion and corresponding time-varying contact area. The documentation of the hemi-larynx setup in this publication will facilitate application and reliable repeatability in experimental research, providing voice scientists with the potential to better understand the biomechanics of voice production.

This paper introduces a protocol for the preparation of hemi-larynx specimens facilitating a multi-dimensional view of vocal fold vibration, in order to investigate various biophysical aspects of voice production in humans and non-human mammals.

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual ...

Biology

Quantifying Phrenic Motor Unit Function in Rodent Models of Neurodegeneration

Assessing Rat Diaphragm Motor Unit Connectivity Outcome Measures as Quantitative Biomarkers of Phrenic Motor Neuron Degeneration and Compensation

Loss of ventilatory muscle function is a consequence of motor neuron injury and neurodegeneration (e.g., cervical spinal cord injury and amyotrophic lateral sclerosis, respectively). Phrenic motor neurons are the final link between the central nervous system and muscle, and their respective motor units (groups of muscle fibers innervated by a single motor neuron) represent the smallest functional unit of the neuromuscular ventilatory system. Compound muscle action potential (CMAP), single motor unit potential (SMUP), and motor unit number estimation (MUNE) are established electrophysiological approaches that enable the longitudinal assessment of motor unit integrity in animal models over time but have mostly been applied to limb muscles. Therefore, the objectives of this study are to describe an approach in preclinical rodent studies that can be used longitudinally to quantify the phrenic MUNE, motor unit size (represented as SMUP), and CMAP, and then to demonstrate the utility of these approaches in a motor neuron loss model. Sensitive, objective, and translationally relevant biomarkers for neuronal injury, degeneration, and regeneration in motor neuron injury and diseases can significantly aid and accelerate experimental research discoveries to clinical testing.

Author Spotlight: Studying Neuromuscular Responses and Motor Neuron Plasticity in Neurodegenerative Diseases

In this study, we present an in vivo method for estimating motor unit number and size to quantify rat diaphragm motor unit connectivity. A step-by-step approach to these techniques is described.

Loss of ventilatory muscle function is a consequence of motor neuron injury and neurodegeneration (e.g., cervical spinal cord injury and amyotrophic ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

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Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

Manufacturing Process for Non-Adhesive Super-Soft Vocal Fold Models

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

Minimally Invasive Murine Laryngoscopy for Close-Up Imaging of Laryngeal Motion During Breathing and Swallowing

Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

Assessing Rat Diaphragm Motor Unit Connectivity Outcome Measures as Quantitative Biomarkers of Phrenic Motor Neuron Degeneration and Compensation