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="https://cloudfront-jove-com.remotexs.ntu.edu.sg/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="https://cloudfront-jove-com.remotexs.ntu.edu.sg/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 rats25,26 and also in humans27,28,29, where they effectively decreased the severity of sleep apnea. In addition, hypoglossal nerve stimulation is now a surgical treatment for obstructive sleep apnea22,23,30.
Our experimental protocol aims to guide the user in the analysis of both mechanical and viscoelastic properties of the tongue muscle. The assessment of these parameters has significant translational value, allowing correlation of modern imaging results with functional data. In particular, this technique can be relevant to several fields including speech therapy31, sleep medicine10,25 and the pathogenesis of obstructive sleep apnea15. The described stimulation protocol and parameters can be easily modified to obtain other physiological information such as the force-frequency relationship, fatigability and fatigue recovery capacity, as well as kinetics of muscle contraction in response to stimulation.
When used without the stimulation-evoked force measurements, the passive force/deformation protocol we describe is suitable for repeated analysis over time, since it does not require any of the surgical procedures used for nerve stimulation and force measurements, except for the minimally invasive suturing of the tongue. In this condition, the physiological length of the tongue calculated by MRI can be used as reference (Table 2, parameter d) in place of the measured L0. For this purpose, the use of injectable anesthesia will eliminate the need for tracheal intubation.
A limitation of the current approach resides in the inability to distinguish between protrusive and retrusive components of the tongue contraction. The stimulatory hook electrodes described in this protocol are placed proximal to the bifurcation of the nerve, causing the whole tongue is stimulated to be stimulated. Other groups have described the use of silicon nerve cuff electrodes in rats19,32 or rabbits21, which allow selective access and stimulation of one or both branches of the hypoglossal nerves. Additionally, the use of injectable anesthetics instead of isoflurane inhalation would provide better access to the submandibular region where the hypoglossal nerves branch.
With practice, this protocol is applicable to smaller animals like mice, where the vast majority of disease models are currently available. In particular, a series of mouse models in which tongue physiology is altered are known33,34,35,36. Scaling down will require the utilization of smaller surgical tools, electrodes and tracheal cannulas (if needed), and optionally with a force transducer designed for lower force ranges.

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">
	<tbody>
		<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>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">LED Lighting and Magnification Kit</td>
			<td style="width:99px;">Kent Scientific</td>
			<td style="width:89px;">SURGI- 5003</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<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>
			<td style="width:187px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Isoflurane</td>
			<td style="width:99px;">Butler Schein Animal Health Supply</td>
			<td style="width:89px;">29405</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:203px;">Alcohol Prep</td>
			<td style="width:99px;">Webcol</td>
			<td style="width:89px;">6818</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:203px;">Cotton-tipped applicators</td>
			<td style="width:99px;">MediChoice</td>
			<td style="width:89px;">WOD1002</td>
			<td style="width:187px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:203px;">Hair clipper</td>
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			<td style="width:89px;"></td>
			<td style="width:187px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:203px;">Hair remover lotion</td>
			<td style="width:99px;">Nair</td>
			<td style="width:89px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:203px;">Medical tape</td>
			<td style="width:99px;">Transpore</td>
			<td style="width:89px;">3M</td>
			<td style="width:187px;"></td>
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		<tr height="20">
			<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>
			<td style="width:187px;"></td>
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		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Operating Scissors</td>
			<td style="width:99px;">World Precision Instruments</td>
			<td style="width:89px;">503717-12</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="34">
			<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>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Microdissecting Forceps, Angled, Serrated, 10.2cm, SS</td>
			<td style="width:99px;">World Precision Instruments</td>
			<td style="width:89px;">504479</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">Suture Tying Forceps</td>
			<td style="width:99px;">Fine Science Tools</td>
			<td style="width:89px;">18025-10</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">Blunt Micro Hook</td>
			<td style="width:99px;">Fine Science Tools</td>
			<td style="width:89px;">10062-12</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">Microhemostat</td>
			<td style="width:99px;">Fine Science Tools</td>
			<td style="width:89px;">12075-14</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">Thermal cautery</td>
			<td style="width:99px;">WPI</td>
			<td style="width:89px;">501292</td>
			<td style="width:187px;">Disposable cauteries are available at lower cost</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:203px;">IV 14g x 3.25&#34; cannula</td>
			<td style="width:99px;">BD</td>
			<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>
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		<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 repositioning the stimulating electrodes or avoiding the contact between the electrode terminations and the surrounding muscle tissue. (C) Example of sine wave displacement from L0 (25% of L0) used to determine the viscoelastic properties of the tongue. The average lengthening (blue square) and shortening (green square) forces can be used to calculate the Peak-To-Peak force24. The red square indicates the range considered for the analysis (excluding the first and the last sine waves). (D) Representation of the force/displacement relationship as Lissajous loops, obtained increasing the displacement from 5% to 50% L0. The area inside each loop corresponds to the energy loss, defined as the difference between the work done on the muscle to lengthen it and the work done by the muscle as it shortens after the stretch24. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56006/56006fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Expected values for twitch and tetanic forces in a 3 month-old Zucker rat (average body weight 400 g for lean and 700 g for obese Zucker rats) are shown in Figure 3A. The tetanic force developed following stimulation should quickly peak (black arrowhead) and then slowly decrease until the stimulation stops. Figure 3B shows examples of unsuccessful contractions in which the force generation did not reach a plateau level before the end of the stimulation (blue line - e.g. if tongue length was set to a value lower than L0, or if the animal&#39;s upper body is not entirely stabilized), or in which the stimulus triggered the contraction of the neck muscles (green line). In most of the cases, careful repositioning of the electrodes may improve the contraction. If the neck muscles are visibly contracting during the nerve stimulation, try to isolate any contact point between electrodes and muscle tissue either by cauterizing the muscle or by moving the electrodes away from it. Forces can be normalized by the volume of the tongue measured by MRI if this is available. An alternative to the use of the volumetric data is to normalize against the weight of the tongue after dissection.
An example of Lissajous work loops obtained stretching the tongue from 5% to 50% of L0 is shown in Figures 3C and 3D. Figure 3C shows the length and force traces separately, which when combined as in Figure 3D generate the typical Lissajous loop traces. It is important to ensure that the tongue remains wet between each cycle by adding a few drops of PBS. Increasing displacement from L0 corresponds to higher passive (work done on the muscle during lengthening) and active (work done by the muscle during negative) forces. The analysis of these traces may be complicated. A curve fitting of maximum average force and/or center of mass for each cycle can be used to describe the viscoelastic properties of the tongue. A more detailed analysis of the Lissajous work-loops has been described by D.A. Syme24 using the rat diaphragm.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protocol</td>
			<td>Pulse Voltage (V)</td>
			<td>Pulse frequency (Hz)</td>
			<td>Pulse width (ms)</td>
			<td>Pulses per train</td>
			<td>Train duration (ms)</td>
		</tr>
		<tr>
			<td>Twitch</td>
			<td>10</td>
			<td>2500</td>
			<td>0.2</td>
			<td>1</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td>Tetanus</td>
			<td>10</td>
			<td>120</td>
			<td>0.2</td>
			<td>96</td>
			<td>800</td>
		</tr>
	</tbody>
</table>
Table 1: Suggested Contraction Stimulation Parameters. 
Isometric twitch contraction is obtained by bilateral nerve stimulation with a single electrical pulse of 10V, lasting 0.2 ms. After 20 s, a tetanic isometric contraction is obtained by stimulating bilaterally with a train of pulses (10 V, 120 Hz frequency, for 800 ms). The twitch/tetanus stimulation is repeated three times with 3 - 5 min interval between each cycle. The highest twitch and highest tetanic forces from the three repetitions are considered for the analysis.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Total deviation (d) from L0 (%)</td>
			<td>Sine amplitude (%L0)</td>
			<td>Sine frequency (Hz)</td>
			<td>Number of cycles</td>
			<td>Time between cycles (s)</td>
		</tr>
		<tr>
			<td>d</td>
			<td>d/2</td>
			<td>2</td>
			<td>10</td>
			<td>30</td>
		</tr>
	</tbody>
</table>
Table 2: Lissajous Curve Parameters (Sinusoidal Deviation from L0). 
The application of the Lissajous force/displacement protocol begins 3 - 5 min after the twitch/tetanus stimulation. Each cycle lasts 5 s (10 repetitions at 2 Hz frequency) and is performed 30 s after the previous one. The % displacement from L0 (d) increases at each cycle, from an arbitrary minimum of 5% up to 50% of L0. Higher excursions can be achieved with other force transducer models (Table 3).

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

Nanyang Technological University

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JoVE Journal

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8.6K 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

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

A Probing Device for Quantitatively Measuring the Mechanical Properties of Soft Tissues during Arthroscopy

Probing in arthroscopic surgery is performed by pulling or pushing the soft tissue, which provides feedback for understanding the condition of the soft tissue. However, the output is only qualitative based on the &#8220;surgeon&#8217;s feeling&#8221;. Herein is described a probing device developed to address this issue by measuring the resistance of soft tissues quantitatively with a tri-axial force sensor. Under both conditions (i.e., pull- and push-probing certain tissues mimicking the acetabular labrum and cartilage), this probing device is found to be useful for measuring some mechanical properties in joints during arthroscopy.

Probing during arthroscopy surgery is normally done to assess the condition of the soft tissue, but this approach has always been subjective and qualitative. This report describes a probing device that can measure the resistance of the soft tissue quantitatively with a tri-axial force sensor during arthroscopy.

Probing in arthroscopic surgery is performed by pulling or pushing the soft tissue, which provides feedback for understanding the condition of the soft ...

Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular matrix (ECM), the protein meshwork that binds cells and tissues together. How the elastic properties of an ECM network relate to its composition, and whether the relaxation properties of the ECM play a physiological role, are questions that have yet to be fully addressed. Part of the challenge lies in the complex architecture of most ECM systems and the difficulty in isolating ECM components without compromising their structure. One exception is the zonule, an ECM system found in the eye of vertebrates. The zonule comprises fibers hundreds to thousands of micrometers in length that span the cell-free space between the lens and the eyewall. In this report, we describe a mechanical technique that takes advantage of the highly organized structure of the zonule to quantify its viscoelastic properties and to determine the contribution of individual protein components. The method involves dissection of a fixed eye to expose the lens and the zonule and employs a pull-up technique that stretches the zonular fibers equally while their tension is monitored. The technique is relatively inexpensive yet sensitive enough to detect alterations in viscoelastic properties of zonular fibers in mice lacking specific zonular proteins or with aging. Although the method presented here is designed primarily for studying ocular development and disease, it could also serve as an experimental model for exploring broader questions regarding the viscoelastic properties of elastic ECM&#39;s and the role of external factors such as ionic concentration, temperature, and interactions with signaling molecules.

The protocol describes a method for the study of extracellular matrix viscoelasticity and its dependence on protein composition or environmental factors. The matrix system targeted is the mouse zonule. The performance of the method is demonstrated by comparing the viscoelastic behavior of wild-type zonular fibers with those lacking microfibril-associated glycoprotein-1.

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...