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" /> 
Figure 1: Surgical Procedures. 
(A) Surgical plan demarcation. The red dashed line indicates the area of the incision. Two black lines indicate the position of the jaw, while the bottom black line marks the position of the thorax. The blue line indicates the hyoid bone. (B) Exposure of the posterior belly of the digastric muscle (arrows) after blunt dissection of fat tissue, the sublingual and submaxillary glands. (C) Resection of the posterior belly of the digastric muscle (dashed green line) to expose the hypoglossal nerve (white arrow). (D) The hypoglossal nerve (white arrow) is cleared from the surrounding fascia. (E) The trachea is exposed by gently pulling apart the smooth muscle around it (the green arrows indicate the direction of the force applied), and lifted (F) to prepare for intubation. The star indicates the tongue&#39;s insertion at the hyoid bone. The green dashed line marks the point of incision for the intubation. The white arrow indicates the loose knot prepared to secure the cannula in place once inserted. (G) Incision of the trachea for cannulation. (H) The tracheal cannula is inserted and secured in place with a square knot. (I) Application of the suture to the tongue. <a href="http://cloudfront.jove.com/files/ftp_upload/56006/56006fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Anesthesia and preparation for surgery
	<ol>
		<li>Place the rat in the induction chamber with 4% isoflurane anesthesia.</li>
		<li>Lay the animal supine over a heat source and maintain anesthesia with 1 - 3% isoflurane from a nosecone, ensuring a respiratory rate of at least 70 breaths per minute.</li>
		<li>Shave the neck area with an air-clipper and hair removal cream. When using the hair removal cream, spread the cream with a cotton-tipped applicator until the fur is removed. Prolonged exposure to hair removal cream can cause irritation of the skin.</li>
		<li>Clean the surgical field with 70% alcohol (Figure 1A).</li>
		<li>Check the animal&#39;s breathing pattern and reflexes (lack of a toe pinch reflex) before proceeding with the initial incision.&#160;&#160;The continuous monitoring and assessment of the depth of anesthesia is essential (record respiratory rate and lack of reflexes every 10 min); the animal needs to be under a deep, surgical plane of anesthesia during the entire procedure.</li>
	</ol>
	</li>
	<li>Hypoglossal Nerves Identification
	<ol>
		<li>Open the surgical field with a pair of operating scissors by resecting and removing the skin as indicated by the red-dotted line in Figure 1A.</li>
		<li>Blunt dissect through any fat tissue, the sublingual and submaxillary glands and the muscle layer surrounding the trachea. 
		NOTE: Be conservative during the dissection. Use blunt dissection whenever possible and be careful not to damage the blood vessels of the neck.</li>
		<li>Identify the posterior belly of the digastric muscle (PD)&#160; lateral to the trachea (Figure 1B - white arrows). The hypoglossal nerve runs under this muscle (Figure 1C - white arrow). With the electrocautery, resect the PD proximal to the hyoid bone (Figure 1C - dashed line). 
		NOTE: Do not let the nerve or the tissue around it become dry. Add a drop of mineral oil on the nerve to protect it. To avoid muscle contraction due to field propagation of the stimulus from the electrodes, carefully cauterize the muscle tissue in the possible contact sites around the nerve.</li>
		<li>Using a pair of fine forceps, dissect the hypoglossal nerve from the surrounding fascia. With the help of a hook, make sure that at least 3 mm of the nerve are available for the connection of the bipolar electrode (Figure 1D).</li>
		<li>Using micro hemostatic forceps, crush the hypoglossal nerve as distally from the hyoid bone (Figure 1D - white arrow) as possible to avoid retrograde propagation of the stimulus. Crush the nerve by applying pressure on it with the micro hemostatic forceps for 5 s. When the hemostat grasps the nerve, a brief twitch at the base of the tongue should be visible.</li>
		<li>Repeat the procedure for the other side.</li>
	</ol>
	</li>
	<li>Tracheal intubation
	<ol>
		<li>Expose the trachea by gently separating the surrounding smooth muscles by blunt dissection (Figure 1E - as indicated by the green arrows). 
		NOTE: Be conservative and use preferentially blunt dissection. Compare the diameter of the cannula with that of the trachea. The cannula should fit correctly in the trachea and should remain stably inserted after being sutured as described in steps 1.3.2 through 1.3.5.</li>
		<li>Gently lift the trachea using a pair of suture-tying forceps and pass a 4-0 suture underneath it, close to the thorax. Tie an initial loose knot between the 6th and the 7th ring from the larynx (Figure 1F - white arrow). This knot will be closed once the cannula is inserted in order to hold it in place.</li>
		<li>Slide the stem of a transfer pipette (lubricate the pipette with mineral oil to facilitate the insertion) under the trachea to lift it and facilitate the insertion of the cannula (IV 14 g x 3.25&#34; cannula).</li>
		<li>Cut a small incision between the 4th and 5th rings from the larynx (Figure 1G - white arrow). After the first incision, the opening will tend to spread, therefore be conservative when making the initial cut in order to avoid the complete resection of the trachea. 
		NOTE: Before performing the incision of the trachea, make sure the cannula is ready and the anesthesia delivery is on.</li>
		<li>Start the anesthesia administration through the cannula and insert it gently into the incision, advancing gently inside the trachea the length of approximately 5 rings. Secure the cannula in place with the suture, to create an airtight seal (Figure 1H). 
		NOTE: Ensure that no fluid enters the nicked trachea in order to avoid suffocation. Once the trachea is incised, the animal will be breathing room air. It is therefore important to quickly insert the cannula so that the delivery of the anesthesia is maintained. When/if moving the animal, monitor the correct position of the cannula in the trachea.</li>
		<li>Gently remove the animal from the nosecone. 
		NOTE: The use of injectable anesthetics instead of isoflurane may simplify the procedure by eliminating the need for tracheal intubation and clearing the experimental workspace from excess tubing.</li>
	</ol>
	</li>
	<li>Tongue suturing
	<ol>
		<li>Soak a 20 cm long piece of 5/0 suture (we suggest Silk, black braided) in PBS.</li>
		<li>While holding the mouth of the rat open, gently hold the tip of the tongue with the suture-tying forceps.</li>
		<li>Pass the suture medially through the tip of the tongue (Figure 1I - white arrow) paying attention to keep the tongue moist with saline to avoid potential tissue damage.</li>
		<li>Tie a loop approximately 4 cm-wide (the diameter can change based on the distance between the animal and the force transducer). 
		NOTE: Ensure the suture and the tongue are wet to facilitate the insertion and avoid unnecessary lesions of the tissue.</li>
	</ol>
	</li>
</ol>
2. Experimental Setup
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56006/56006fig2.jpg" /> 
Figure 2: Positioning and Securing the Animal. 
(A) The mouse is positioned on the experimental platform. The jaw is secured and the mouth maintained open by the application of vertical tension (green arrow). The suture loop is connected to the force transducer (white arrow). (B) The electrodes are connected. (C) Each electrode, mounted on a micromanipulator, is stably connected to the nerve. The inlay shows the termination of the hook electrode. (D) The optimal length of the tongue is measured with a Vernier caliper, from the insertion at the level of the hyoid bone to the tip of the tongue. In this picture, the electrodes were removed for clarity. <a href="http://cloudfront.jove.com/files/ftp_upload/56006/56006fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Note: Position the animal supine on the heated tray of the apparatus, using the following securing measures to avoid movement during the experiment.
<ol>
	<li>Secure the nose of the rat to the tray with a plastic-coated metallic twist-tie directly connected to the heated platform (Figure 2A, 2B).</li>
	<li>Maintain the rat&#39;s mouth opened by applying vertical tension (use a 3 way-swivel tied to 9 kg test monofilament line and hold in place with a camera stick) (Figure 2A - green arrow). 
	NOTE: An alternative approach to stabilizing the rat is using a rodent mouth gag or a custom modification of it. It is essential that the mouth is maintained open and stable to avoid movements of the neck which could affect the quality of the measurements.</li>
	<li>Connect the suture loop to the force transducer (Figure 2A - white arrow).</li>
	<li>Using surgical tape, immobilize the limbs and the abdomen of the rat to limit movements during the stimulations.</li>
	<li>Apply the two stimulating hook bipolar electrodes to the dissected hypoglossal nerves, proximally to the crush site (Figure 2C).
	<ol>
		<li>Use stimulating bipolar electrodes custom made from a pair of surgical-grade steel 29 gauge needle electrodes, held together at a distance of 2 mm from each other using a plastic support (here, we used two 1-mL serological pipettes taped together).</li>
		<li>Bend the tips of the needle electrodes to a hook shape small enough to form a stable contact with the nerve (~1 mm diameter) (Figure 2C - inlay).</li>
		<li>Connect the two wires of each electrode to a stackable double binding post with banana jack x BNC jack, and then connect to the stimulator module of an in situ Muscle Test System (Table 3) using a BNC splitter. 
		NOTE: Ensure the animal is stable and that the neck and tongue are aligned to the force transducer lever. Continue monitoring the respiratory rate and ensure that the restraints do not interfere with the respiration.</li>
	</ol>
	</li>
</ol>
3. Optimal Length (L0) and Maximal Isometric Force Determination
<ol>
	<li>Adjust the length of the tongue gradually and apply isometric twitch stimulations until a maximal force is obtained.
	<ol>
		<li>Define L0 as the length at which the isometric twitch force (Table 1) is maximal, and record for the subsequent normalization of the force measurements, as it changes for every animal. Using a Vernier caliper, measure the tongue length from the insertion at the level of the hyoid bone (Figure 2D) to the tip of the tongue. 
		NOTE: The correct positioning of the electrodes is essential to ensure optimal contact with the nerves. Optimal positioning should exert similar and additive forces when stimulating one of the two nerves or both, respectively.</li>
	</ol>
	</li>
	<li>Before starting the experiment, connect both the electrodes to the stimulator and administer a test tetanic stimulation (Table 1). During this contraction, monitor the animal to ensure that the position is stable, and check for unwanted visible contractions of the neck muscles.</li>
	<li>Measure isometric force by applying a twitch followed after 20 s by a tetanic stimulation. Repeat the stimulations 3 times allowing 4 min recovery between stimulation cycles (a cycle includes one twitch and one tetanic stimulation), and record the maximal twitch and tetanic forces.</li>
	<li>Maintain the tongue wet at all times by applying a few drops of PBS between contractions. 
	NOTE: If neck muscles contract, carefully change the positioning of the electrodes so that they do not touch any surrounding muscle tissue and, if needed, cauterize the tissue in contact with the electrodes.</li>
</ol>
4. Viscoelastic Properties Determination (Lissajous Curves)
<ol>
	<li>Allow the tongue to rest for 3 - 5 min after the isometric force determination. Perform the Lissajous curves starting at L0, therefore the position of the animal and of the force transducer should never be changed.</li>
	<li>Without stimulating the hypoglossal nerves, apply cycles of passive stretches (Table 2) with increasing displacement (d) i.e. 5, 10, 15, 20, 25, 30, 35, 40, 50% of L0 (higher amplitudes may require a dual mode controller with higher excursion values than the 305C-LR). For example, an amplitude of 40% L0 will cause the deformation of the tongue from +20%L0 to -20%L0.
	<ol>
		<li>Use fixed 2 Hz sine frequency with 10 repetitions in each cycle, for a total duration of 5 s every cycle. Changes of the sine frequency have been used in the past with tissues such as diaphragm24 and could be adopted to modify the experimental protocol.</li>
		<li>Allow 30 s between each cycle and ensure the tongue is wet during the whole procedure. 
		NOTE: Keep the tongue wet during the procedure adding a few drops of PBS. Ensure that the restraint holding the jaw remains stably in place during the stretches, particularly when the displacement reaches 40% or higher.</li>
	</ol>
	</li>
	<li>Immediately following the completion of the experiment, while the animal is still under a deep surgical plane of anesthesia, euthanize the animal according to the approved IACUC animal protocol.&#160;&#160;Dissect the tongue for further processing (i.e. frozen sectioning or storage for biochemical analyses).</li>
</ol>

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P., 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+muscle+plasticity+following+hypoglossal+nerve+stimulation+in+aged+rats.">Tongue muscle plasticity following hypoglossal nerve stimulation in aged rats.</a> Muscle &amp; nerve. 47 (2), 230-240 (2013).</li><li>Brady, J. P., 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=AlphaB-crystallin+in+lens+development+and+muscle+integrity:+a+gene+knockout+approach.">AlphaB-crystallin in lens development and muscle integrity: a gene knockout approach.</a> Invest Ophthalmol Vis Sci. 42 (12), 2924-2934 (2001).</li><li>Spassov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+myosin+heavy+chain+isoforms+in+the+masticatory+muscles+of+dystrophin-deficient+mice.">Differential expression of myosin heavy chain isoforms in the masticatory muscles of dystrophin-deficient mice.</a> Eur J Orthod. , ciq113 (2010).</li><li>Lever, T. E., 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=Videofluoroscopic+Validation+of+a+Translational+Murine+Model+of+Presbyphagia.">Videofluoroscopic Validation of a Translational Murine Model of Presbyphagia.</a> Dysphagia. 30 (3), 328-342 (2015).</li><li>Gantois, I., 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=Ablation+of+D1+dopamine+receptor-expressing+cells+generates+mice+with+seizures,+dystonia,+hyperactivity,+and+impaired+oral+behavior.">Ablation of D1 dopamine receptor-expressing cells generates mice with seizures, dystonia, hyperactivity, and impaired oral behavior.</a> Proc Natl Acad Sci. 104 (10), 4182-4187 (2007).</li></ol>

The authors have nothing to disclose.

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><tbody><tr><td>SurgiSuite (heated Surgical tray)</td><td>Kent Scientific</td><td>SurgiSuite-LG</td><td>Includes heated platform</td></tr><tr><td>LED Lighting and Magnification Kit</td><td>Kent Scientific</td><td>SURGI- 5003</td><td></td></tr><tr><td>RC&lt;sup&gt;2&lt;/sup&gt; Rodent Circuit Controller</td><td>VetEquip</td><td>922100</td><td></td></tr><tr><td>Isoflurane</td><td>Butler Schein Animal Health Supply</td><td>29405</td><td></td></tr><tr><td>Alcohol Prep</td><td>Webcol</td><td>6818</td><td></td></tr><tr><td>Cotton-tipped applicators</td><td>MediChoice</td><td>WOD1002</td><td></td></tr><tr><td>Hair clipper</td><td>Conair</td><td></td><td></td></tr><tr><td>Hair remover lotion</td><td>Nair</td><td></td><td></td></tr><tr><td>Medical tape</td><td>Transpore</td><td>3M</td><td></td></tr><tr><td>D-PBS</td><td>Corning</td><td>21-030-CM</td><td></td></tr><tr><td>Operating Scissors</td><td>World Precision Instruments</td><td>503717-12</td><td></td></tr><tr><td>Hemostatic Forceps</td><td>Merit</td><td>97-458</td><td>Any tissue forceps can be used instead</td></tr><tr><td>Microdissecting Forceps, Angled, Serrated, 10.2 cm, SS</td><td>World Precision Instruments</td><td>504479</td><td></td></tr><tr><td>Suture Tying Forceps</td><td>Fine Science Tools</td><td>18025-10</td><td></td></tr><tr><td>Blunt Micro Hook</td><td>Fine Science Tools</td><td>10062-12</td><td></td></tr><tr><td>Microhemostat</td><td>Fine Science Tools</td><td>12075-14</td><td></td></tr><tr><td>Thermal cautery</td><td>WPI</td><td>501292</td><td>Disposable cauteries are available at lower cost</td></tr><tr><td>IV 14g x 3.25&quot; cannula</td><td>BD</td><td>B-D382268H</td><td>For tracheal cannulation</td></tr><tr><td>Braided silk non-absorbable suture size 4-0</td><td>Harvard Apparatus</td><td>SP104</td><td>For stabilization of the tracheal cannula</td></tr><tr><td>Braided non-absorbable silk 5/0 suture</td><td>Surgik LC, USA</td><td>ESILRC15387550</td><td>For suturing the tongue</td></tr><tr><td>Plastic-coated metal twist-tie (or electrical wire)</td><td></td><td></td><td>For securing the rat&#039;s nose to the platform</td></tr><tr><td>Camera stick</td><td></td><td></td><td></td></tr><tr><td>3 way-swivel and Trilene 9 Kg test monofilament line</td><td>Berkley</td><td></td><td>For securing the jaw and maintaining the mouth open</td></tr><tr><td>Camera stick with adjustable angle</td><td></td><td></td><td>For supporting the 3 way-swivel and maintaining the mouth open.</td></tr><tr><td>&lt;em&gt;in situ&lt;/em&gt; Muscle Test System</td><td>Aurora Scientific</td><td>809C</td><td>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><td>Dual-Mode Muscle lever (force transducer)</td><td>Aurora Scientific</td><td>305C-LR</td><td>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><td>Needle Electrodes (surgical steel, 29 gauge)</td><td>AD Instruments</td><td>MLA1204</td><td>300C is recommended for use in mice.</td></tr><tr><td>Magnetic Stands</td><td>World Precision Instruments</td><td>M10</td><td>Used for making the bipolar stimulating hook electrodes</td></tr><tr><td>Kite Manual Micromanipulator</td><td>World Precision Instruments</td><td>KITE-R and KITE-L</td><td>Require a steel plate</td></tr><tr><td>Stackable Double Binding Post with Banana Jack x BNC Jack</td><td>McMaster Carr</td><td>6704K13</td><td></td></tr><tr><td>Carbon fiber composites digital caliper</td><td>VWR</td><td>36934-152</td><td></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 ...

in-vivo-evaluation-mechanical-viscoelastic-properties-rat

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.7K Views.  University of Pennsylvania. 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.

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

Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents

A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of humans with orofacial pain. To measure meal duration unrestrained rats are continuously kept in sound attenuated, computerized feeding modules for days to weeks to record feeding behavior. These sound-attenuated chambers are equipped with chow pellet dispensers. The dispenser has a pellet trough with a photobeam placed at the bottom of the trough and when a rodent removes a pellet from the feeder trough this beam is no longer blocked, signaling the computer to drop another pellet. The computer records the date and time when the pellets were taken from the trough and from this data the experimenter can calculate the meal parameters. When calculating meal parameters a meal was defined based on previous work and was set at 10 min (in other words when the animal does not eat for 10 min that would be the end of the animal&#39;s meal) also the minimum meal size was set at 3 pellets. The meal duration, meal number, food intake, meal size and inter-meal interval can then be calculated by the software for any time period that the operator desires. Of the feeding parameters that can be calculated meal duration has been shown to be a continuous noninvasive biological marker of orofacial nociception in male rats and mice and female rats. Meal duration measurements are quantitative, require no training or animal manipulation, require cortical participation, and do not compete with other experimentally induced behaviors. These factors distinguish this assay from other operant or reflex methods for recording orofacial nociception.

A lengthening in meal duration represents orofacial nociceptive behavior in rodents similar to the guarding behavior of humans with orofacial pain. Eating is a behavior that requires no training or animal manipulation, requires cortical participation, and is not competing with other experimentally induced behaviors, distinguishing this assay from alternative reflex or operant measurements.

A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of ...

Behavior

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

Bioengineering

Assessment of Midline Lingual Point-Pressure Somatosensation Using Von Frey Hair Monofilaments

Detection and discrimination threshold estimates for oral point pressure are assessed using Von Frey Hair monofilaments. Consistent with previously published protocols, threshold estimates are determined using a two-interval forced choice (2-IFC) paradigm with a three down/one up approach. Detection threshold estimates determine the mean force in which a participant can identify the presence of pressure. During the detection threshold procedure, the participant is instructed to choose which of two sequentially presented observation intervals contained the tactile test stimulus. If the participant performs three correct detections in a row (i.e., 3 &#39;hits&#39;), the researcher decreases the stimulus to the next lower target force level. With one incorrect detection (a &#39;miss&#39;), the researcher increases the force delivered to the next higher level. This threshold estimation approach is known as a 3-down/1-up adaptive staircase. Reponses are recorded on a paper ballot, and a participant&#39;s estimated threshold is defined as the geometric mean of five reversals. During the discrimination threshold procedure, the participant is asked to make a choice between two serially presented stimuli as to which is the &#34;harder&#34; or &#34;stronger&#34; pressure. The same scoring of &#39;hits&#39;, &#39;misses&#39;, and stopping points are used. Detection and discrimination testing for oral point pressure at tongue midline takes approximately 20 min to complete. Using these commercially-available clinical tools, individual touch sensation profiles for the midline tongue can be achieved in a relatively time and cost effective means.

This work describes a standard method to assess tactile sensation at the midline of the tongue tip. Using Von Frey Hair (VFH) monofilaments, this protocol provides estimates of detection and discrimination threshold estimates for oral point pressure (OPP).

Detection and discrimination threshold estimates for oral point pressure are assessed using Von Frey Hair monofilaments. Consistent with previously ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Neuroscience

Assessment of Spatial Lingual Tactile Sensitivity using a Gratings Orientation Test

Individual thresholds by R-index estimates are calculated using a gratings orientation test (6 different tools of increasing grating size from 0.20-1.25 mm) to assess spatial lingual tactile sensitivity. During the experiment, the subjects are blindfolded and asked to specify the orientation of the grating (either horizontal or vertical) placed on the tongue. R-index is based on Signal Detection Theory (SDT), and it is an estimated probability of correctly identifying a target stimulus (the signal, e.g., the correct orientation) compared to an alternative stimulus (the noise, e.g., the incorrect orientation). Once the R-index values for each subject and each tool dimension are calculated, it is possible to derive the individual threshold by interpolating the two R-indices immediately below and above the established cut-off (typically 75%) based on one-sided R-index critical values. This procedure can be helpful in the medical field to study the association between oral tactile sensitivity, speech clarity, and swallowing disorders, as well as in sensory and consumer studies to explore individual variation in texture perception, food preferences, and eating behavior.

This work illustrates a standard procedure and threshold determination by the R-index to assess spatial lingual tactile sensitivity using a gratings orientation test.

Individual thresholds by R-index estimates are calculated using a gratings orientation test (6 different tools of increasing grating size from 0.20-1.25 ...

Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results by addressing the cognitive and motivational aspects of pain in rodents. This paper presents a protocol for assessing mechanical hypersensitivity following chronic constriction injury of the infraorbital nerves (CCI-ION) in rats using an orofacial operant pain system. Before CCI-ION surgery, rats were trained in an orofacial pain assessment device (OPAD) to drink sweetened condensed milk while making facial contact with the metal spiked bars and lick-tube.
In this assay, rats can choose between receiving milk as a positive reinforcer or escaping an aversive mechanical stimulus that is produced by a vertical row of small pyramid-shaped spikes on each side of the reward access hole. Following 2 weeks of training in the OPAD and before the CCI-ION surgery, baseline mechanical sensitivity data were recorded for 5 days for each rat during a 10 min testing session. During a session, the operant system automatically records the number of reward bottle activations (licks) and facial contacts, contact duration, and latency to the first lick, among other measures.
Following baseline measurements, rats underwent either CCI-ION or sham surgery. In this protocol, mechanical hypersensitivity was quantified by measuring the number of licks, latency to the first lick, the number of contacts, and the ratio of licks to facial contacts (L/F). The data showed that CCI-ION resulted in a significant decrease in the number of licks and the L/F ratio and an increase in the latency to the first lick, indicating mechanical hypersensitivity. These data support the use of operant-based pain assays to assess mechanical pain sensitivity in preclinical pain research.

This protocol describes the assessment of mechanical hypersensitivity in a rat model of neuropathic orofacial pain using an operant-based orofacial pain assessment device.

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results ...

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 &#176;C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (&#62;90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.

We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues.

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA ...

Developmental Biology

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

Biology

Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the biomechanics-related mechanisms of disease, injury, and development. The mechanical testing method is the most straightforward way for tissue characterization and is considered as verification for in vivo measurement. Among the many ex vivo mechanical testing techniques, the indentation test provides a reliable way, especially for samples that are small, hard to fix, and viscoelastic such as brain tissue. Magnetic resonance elastography (MRE) is a clinically used method to measure the biomechanical properties of soft tissues. Based on shear wave propagation in soft tissues recorded using MRE, viscoelastic properties of soft tissues can be estimated in vivo based on wave equation. Here, the viscoelastic properties of gelatin phantoms with two different concentrations were measured by MRE and indentation. The protocols of phantom fabrication, testing, and modulus estimation have been presented.

This article presents a demonstration and summary of protocols of making gelatin phantoms that mimic soft tissues, and the corresponding viscoelastic characterization using indentation and magnetic resonance elastography.

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the ...

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in the taste sensory organ. By integrating microfluidics into the intravital tongue imaging window, the &#181;Tongue provides reliable functional images of taste cells in vivo under controlled exposure to multiple tastants. In this paper, a detailed step-by-step procedure to utilize the &#181;Tongue system is presented. There are five subsections: preparing of tastant solutions, setting up of a microfluidic module, sample mounting, acquiring functional image data, and data analysis. Some tips and techniques to solve the practical issues that may arise when using the &#181;Tongue are also presented.

 &#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

The article introduces the &#181;Tongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...

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

Summary

Explore More Videos

Chapters in this video

Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

Assessment of Midline Lingual Point-Pressure Somatosensation Using Von Frey Hair Monofilaments

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Assessment of Spatial Lingual Tactile Sensitivity using a Gratings Orientation Test

Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography

µTongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo