Name: in vivo evaluation of the mechanical and viscoelastic properties of the rat tongue
Uploaded: 2017-07-06T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue at JoVE.com

This investigation was supported by two National Institutes of Health Grants: HL089447 ("Obesity and OSA: Understanding the Importance of Tongue Fat &amp; Metabolic Function") and HL094307 ("Understanding the Relationship Between Obesity and Tongue Fat")

All the procedures including animal subjects have been approved by Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania (Protocol number 805822).&#160;The described procedure is terminal and does not require the utilization of aseptic conditions or pharmaceutical grade products.
1. Surgical Procedures
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56006/56006fig1.jpg" /> 
Figu...

<ol>
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P., Konopacki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+contractile+properties+of+tongue+muscles+in+old+rats.">Alterations in contractile properties of tongue muscles in old rats.</a> Ann Otol Rhinol Laryngol. 114 (10), 799-803 (2005).</li><li>Schwarz, E. C., Thompson, J. M., Connor, N. P., Behan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Aging+on+Hypoglossal+Motoneurons+in+Rats.">The Effects of Aging on Hypoglossal Motoneurons in Rats.</a> Dysphagia. 24 (1), 40 (2008).</li><li>Pae, E. -. K., Wu, J., Nguyen, D., Monti, R., Harper, R. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-situ+mechanical+characteristics+of+the+tongue+are+not+altered+in+the+obese+Zucker+rat.">In-situ mechanical characteristics of the tongue are not altered in the obese Zucker rat.</a> Sleep. 32 (7), 957 (2009).</li><li>Brennick, M. J., 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=Tongue+fat+infiltration+in+obese+versus+lean+Zucker+rats.">Tongue fat infiltration in obese versus lean Zucker rats.</a> Sleep. 37 (6), 1095-1102 (2014).</li><li>Stedman, H. H., Sweeney, H. L., Shrager, J. B., Maguire, H. C., Panettieri, R. A., Petrof, B., Narusawa, M., Leferovich, J. M., Sladky, J. T., Kelly, A. 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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=The+effect+of+intramuscular+fat+on+skeletal+muscle+mechanics:+implications+for+the+elderly+and+obese.">The effect of intramuscular fat on skeletal muscle mechanics: implications for the elderly and obese.</a> J R Soc Interface. 12 (109), 20150365 (2015).</li><li>Kim, A. M., 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=Tongue+fat+and+its+relationship+to+obstructive+sleep+apnea.">Tongue fat and its relationship to obstructive sleep apnea.</a> Sleep. 37 (10), 1639-1648 (2014).</li><li>Kim, A. 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Changes in tongue metabolism and/or composition, e.g. tongue fat infiltration as a consequence of obesity, are predicted to cause quantifiable changes of the parameters assessed by our protocol. The quantification of tongue force is of great interest since an imbalance between protrusive and retrusive activity or overall tongue weakening may result in the occlusion of the upper airway15. Exercise techniques aiming to increase tongue strength have been successfully applied in rats<sup clas...

The tongue provides essential support for mastication, deglutition, taste-sensing and speaking. The presence of extrinsic and intrinsic musculature, with distinct innervation and anatomy/function, accounts for the uniqueness of this muscular hydrostat. Recent advances in imaging techniques have provided a more detailed view of its complex anatomy1. Decreased tongue functionality, tongue atrophy, dysphagia, and speech impediments are also common manifestations of myopathic conditions such as Parkinson2, Amyotrophic Lateral Sclerosis (ALS)3, Myotonic Dystrophy (MD)4 and other myopathies.
Changes in muscle composition associated with common disease states affect the mechanical and viscoelastic properties of the tongue. For example, functional analysis of tongue force has uncovered changes in contractile properties associated with aging5,6, hypoxia7,8 and obesity9,10. In the case of muscular dystrophy, increased fibrosis leads to higher muscle stiffness, which translates to lower compliance to deformation when a Lissajous deformation protocol is applied11. Conversely, changes in muscle fat content, like those documented in obese patients, alter both metabolic12 and mechanical properties of skeletal muscle13,14 and are predicted to increase muscle compliance to deformation. Increased tongue fat also correlates with the development of obstructive sleep apnea (OSA) in humans17 by increasing tongue volume to the point of partial upper airway occlusion (apnea)15,16. Similarly to humans, tongue fat infiltration has been documented in obese Zucker rats10, suggesting that this model is a valuable tool for studying the effects of fat infiltration on tongue physiology.
Measuring tongue force requires delicate surgical techniques to isolate and bilaterally stimulate the hypoglossal nerves17,18. Such techniques have been previously described in rats5,17,19,20, rabbits21 and humans22,23, yet with limited visual aids to the investigator. Due to its highly technical nature, the availability of a detailed protocol would significantly improve the accessibility and reproducibility of this technique. The goal of our experimental paradigm is to illustrate a valid and reliable technique for measuring strength and viscoelastic properties of the tongue in a rat model. To accomplish this, the rat is anesthetized, the hypoglossal nerves are exposed and the trachea is cannulated to ensure free access to the animal&#39;s tongue. A suture loop then connects the tip of the tongue to a force transducer, capable of controlling both force and length, while two bipolar hook electrodes stimulate the hypoglossal nerves to induce contraction of the tongue. After the force measurement is completed, the length-controlling capabilities of the force transducer are used to rapidly change the length of the tongue, according to a sine-wave protocol with fixed amplitude (Lissajous curves), duration and frequency, allowing one to derive its viscoelastic properties11,24. The protocol will guide the investigator through the dissection steps, the positioning of the animal on the experimental platform, placement of electrodes, and finally to the acquisition and analyses of the force and viscoelasticity data.

<table border="0" cellpadding="0" cellspacing="0" width="577">
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		<tr height="20">
			<td height="20" style="height:20px;width:203px;">SurgiSuite (heated Surgical tray)</td>
			<td style="width:99px;">Kent Scientific</td>
			<td style="width:89px;">SurgiSuite-LG</td>
			<td style="width:187px;">Includes heated platform</td>
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			<td height="34" style="height:34px;width:203px;">LED Lighting and Magnification Kit</td>
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			<td style="width:89px;">SURGI- 5003</td>
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			<td height="20" style="height:20px;width:203px;">RC2 Rodent Circuit Controller</td>
			<td style="width:99px;">VetEquip</td>
			<td style="width:89px;">922100</td>
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			<td height="51" style="height:51px;width:203px;">Isoflurane</td>
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			<td style="width:89px;">29405</td>
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			<td height="20" style="height:20px;width:203px;">Alcohol Prep</td>
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			<td style="width:89px;">6818</td>
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			<td height="20" style="height:20px;width:203px;">Cotton-tipped applicators</td>
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			<td height="20" style="height:20px;width:203px;">Hair remover lotion</td>
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			<td height="20" style="height:20px;width:203px;">D-PBS</td>
			<td style="width:99px;">Corning</td>
			<td style="width:89px;">21-030-CM</td>
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			<td height="34" style="height:34px;width:203px;">Hemostatic Forceps</td>
			<td style="width:99px;">Merit</td>
			<td style="width:89px;">97-458</td>
			<td style="width:187px;">Any tissue forceps can be used instead</td>
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		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Microdissecting Forceps, Angled, Serrated, 10.2cm, SS</td>
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			<td style="width:89px;">504479</td>
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			<td style="width:89px;">12075-14</td>
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			<td height="34" style="height:34px;width:203px;">Thermal cautery</td>
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			<td style="width:187px;">Disposable cauteries are available at lower cost</td>
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			<td height="20" style="height:20px;width:203px;">IV 14g x 3.25&#34; cannula</td>
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			<td style="width:89px;">B-D382268H</td>
			<td style="width:187px;">For tracheal cannulation</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">Braided silk non-absorbable suture size 4-0</td>
			<td style="width:99px;">Harvard Apparatus</td>
			<td style="width:89px;">SP104</td>
			<td style="width:187px;">For stabilization of the tracheal cannula</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">Braided non-absorbable silk 5/0 suture</td>
			<td style="width:99px;">Surgik LC, USA</td>
			<td style="width:89px;">ESILRC15387550</td>
			<td style="width:187px;">For suturing the tongue</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">Plastic-coated metal twist-tie (or electrical wire)</td>
			<td style="width:99px;"></td>
			<td style="width:89px;"></td>
			<td style="width:187px;">For securing the rat&#39;s nose to the platform</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:203px;">Camera stick</td>
			<td style="width:99px;"></td>
			<td style="width:89px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">3 way-swivel and Trilene 9Kg test monofilament line</td>
			<td style="width:99px;">Berkley</td>
			<td style="width:89px;"></td>
			<td style="width:187px;">For securing the jaw and maintaining the mouth open</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Camera stick with adjustable angle</td>
			<td style="width:99px;"></td>
			<td style="width:89px;"></td>
			<td style="width:187px;">For supporting the 3 way-swivel and maintaining the mouth open.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:203px;">in situ Muscle Test System</td>
			<td style="width:99px;">Aurora Scientific</td>
			<td style="width:89px;">809C</td>
			<td style="width:187px;">This system is designed for mice and was modified by extending the platform. Alternatively the rat-specific 806D system can be used.</td>
		</tr>
		<tr height="119">
			<td height="119" style="height:119px;width:203px;">Dual-Mode Muscle lever (force transducer)</td>
			<td style="width:99px;">Aurora Scientific</td>
			<td style="width:89px;">305C-LR</td>
			<td style="width:187px;">309C offers higher excursion capabilities than 305C-LR. Link for more information and specifications: http://aurorascientific.com/products/muscle-physiology/dual-mode-muscle-levers/</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">Needle Electrodes (surgical steel, 29 gauge)</td>
			<td style="width:99px;">AD Instruments</td>
			<td style="width:89px;">MLA1204</td>
			<td style="width:187px;">300C is recommended for use in mice.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Magnetic Stands</td>
			<td style="width:99px;">World Precision Instruments</td>
			<td style="width:89px;">M10</td>
			<td style="width:187px;">Used for making the bipolar stimulating hook electrodes</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Kite Manual Micromanipulator</td>
			<td style="width:99px;">World Precision Instruments</td>
			<td style="width:89px;">KITE-R and KITE-L</td>
			<td style="width:187px;">Require a steel plate</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">Stackable Double Binding Post with Banana Jack x BNC Jack</td>
			<td style="width:99px;">McMaster Carr</td>
			<td style="width:89px;">6704K13</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:203px;">Carbon fiber composites digital caliper</td>
			<td style="width:99px;">VWR</td>
			<td style="width:89px;">36934-152</td>
			<td style="width:187px;"></td>
		</tr>
	</tbody>
</table>

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.

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56006/56006fig3.jpg" /> 
Figure 3: Representative Results. 
(A) Examples of successful twitch and tetanic force traces. The corresponding stimulation is represented by the red trace. (B) Example of unsuccessful tongue tetanic contractions due to submaximal contraction (blue trace) and indirect stimulation of the neck muscles. Both conditions may be improved by reposition...

Watch this Scientific Journal Video about In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue at JoVE.com

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.

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 ...

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