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 ...

The overall goal of this procedure is to assess the strength and viscoelastic properties of the tongue in anesthetized rats. This method can help answer key questions in the field of sleep disordered breathing, about obstructive sleep apnea. The main advantages of this technique are the fields quantitative data regarding muscle performance and with appropriate training, it can be easily undertaken using the curb pen that's commonly found in most muscle mechanics laboratories.Those method can provide insight into the physiology and mechanics of the rat tongue. It can also be applied to other systems including, mouse models. We first had the idea for this methodology when we were evaluating how fat accumulation at the base of the tongue can alter the mechanical properties of this muscle.After confirming the appropriate level of sedation by a lack of response to toe pinch, use a pair of operating scissors to resect an oval area of skin over the neck of the rat and remove the skin. Blunt dissect through any fat tissue, the sublingual and submaxillary glands and the muscle layer surrounding the trachea. Using electrocautery, resect the digastric muscle proximal to the hyoid bone in the possible contact sites around the nerve.Then, add a drop of mineral oil onto the nerve to protect it from dehydration. Use a pair of fine forceps to dissect the hypoglossal nerve from the surrounding flesh. Using a hook to make sure that at least three millimeters of the nerve are available for connecting to the bipolar electrode.Using micro hemastatic forceps, crush the hypoglossal nerve as distally from the hyoid bone as possible, to avoid retrograde propagation of the stimulus with five seconds of gentle pressure. When the hemastatic grasps the nerve, a brief twitch at the base of the tongue should be visible. Then, repeat the identification and nerve disruption on the other side.To intubate the trachea, first, gently blunt dissect the smooth muscle surrounding the trachea, to expose the tracheal tissue. Next, use a pair of suture tying forceps to carefully lift the trachea and pass a four zero suture underneath the trachea, close to the thorax. Tie an initial loose knot between the sixth and seventh ring from the larynx and slide the stem of a mineral oil lubricated transfer pipette under the trachea.When the pipette is in place, make a small incision between the fourth and fifth rings from the larynx. With the canella connected to the anesthesia, insert the canella gently into the incision for a depth of approximately, five rings. Secure the canella to the suture to create an airtight seal and use suture tying forceps to carefully grasp the tongue.Then, pass a PBS soaked 20 centimeter long silk black braided, five zero suture medially through the tip of the tongue and tie an approximately four centimeters wide loop in the suture. To determine the isometric force and viscoelastic properties of the tongue, place the animal in the supine position in the heated tray of the incisu muscle test system and use a plastic coated metal twist tie to secure the nose of the animal to the tray. Insert a three way swivel tied to a nine kilogram text monofilament line, to maintain the mouth in the open position, and connect the suture loop, to the force transducer.Tape the limbs and the abdomen to immobilize the animal during the stimulations and connect a custom made stimulating hook by polar electrode to each hypoglossal nerve. Use a stackable double binding post to convert the two banana terminals of each electrode into a single BNC connector. Then, use a BNC splitter to connect the electrodes to the stimulator mode of the apparatus.After confirming that the neck and tongue are aligned with the lever of the force transducer, gradually adjust the length of the tongue and apply twitch stimulations until a maximal force is obtained. Once the maximal twitch force is obtained, use a vernier caliper to measure the tongue length from the insertion at the hyoid bone to the tip of the tongue. To measure isometric force, apply a twitch, follow 20 seconds later by a tetanic stimulation, recording the maximal twitch and tetanic forces.To determine the viscoelastic properties of the tongue, without changing the position of the animal, three to five minutes after the isometric force determination, apply cycles of passive stretches with increasing displacement from the optimal length without stimulating the hypoglossal nerves. Here, the expected values for the twitch and tetanic forces in a three month old Zucker rat, are shown. The tetanic force developed following stimulation, should quickly peak.Followed by a slow decrease until the stimulation stops. In this graph, unsuccessful contractions in which the fourth generation did not reach a plateau level before the end of the stimulation, or in which the stimulus triggered contraction of the neck muscles, can be observed. These representative Lissajous work loops of length and force traces, were obtained by stretching the tongue from 5%to 50%of the optimal length.The loops can then be combined to generate typical Lissajous loop traces. Once mastered, this technique can completed in approximately one and a half hours if it is performed properly. After watching this video, you should have a good understanding of how to expose and isolate the hypoglossal nerves and how to determine the mechanical viscoelastic properties of the tongue.Thanks for watching and good luck with your experiments.

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