We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.
The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include 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.
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'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.
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). The described procedure is terminal and does not require the utilization of aseptic conditions or pharmaceutical grade products.
1. Surgical Procedures
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'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. Please click here to view a larger version of this figure.
2. Experimental Setup
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. Please click here to view a larger version of this figure.
Note: Position the animal supine on the heated tray of the apparatus, using the following securing measures to avoid movement during the experiment.
3. Optimal Length (L0) and Maximal Isometric Force Determination
4. Viscoelastic Properties Determination (Lissajous Curves)
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. Please click here to view a larger version of this figure.
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'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.
Protocol | Pulse Voltage (V) | Pulse frequency (Hz) | Pulse width (ms) | Pulses per train | Train duration (ms) |
Twitch | 10 | 2500 | 0.2 | 1 | 0.2 |
Tetanus | 10 | 120 | 0.2 | 96 | 800 |
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.
Total deviation (d) from L0 (%) | Sine amplitude (%L0) | Sine frequency (Hz) | Number of cycles | Time between cycles (s) |
d | d/2 | 2 | 10 | 30 |
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).
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 authors have nothing to disclose.
This investigation was supported by two National Institutes of Health Grants: HL089447 (“Obesity and OSA: Understanding the Importance of Tongue Fat & Metabolic Function”) and HL094307 (“Understanding the Relationship Between Obesity and Tongue Fat”)
SurgiSuite (heated Surgical tray) | Kent Scientific | SurgiSuite-LG | Includes heated platform |
LED Lighting and Magnification Kit | Kent Scientific | SURGI- 5003 | |
RC2 Rodent Circuit Controller | VetEquip | 922100 | |
Isoflurane | Butler Schein Animal Health Supply | 29405 | |
Alcohol Prep | Webcol | 6818 | |
Cotton-tipped applicators | MediChoice | WOD1002 | |
Hair clipper | Conair | ||
Hair remover lotion | Nair | ||
Medical tape | Transpore | 3M | |
D-PBS | Corning | 21-030-CM | |
Operating Scissors | World Precision Instruments | 503717-12 | |
Hemostatic Forceps | Merit | 97-458 | Any tissue forceps can be used instead |
Microdissecting Forceps, Angled, Serrated, 10.2cm, SS | World Precision Instruments | 504479 | |
Suture Tying Forceps | Fine Science Tools | 18025-10 | |
Blunt Micro Hook | Fine Science Tools | 10062-12 | |
Microhemostat | Fine Science Tools | 12075-14 | |
Thermal cautery | WPI | 501292 | Disposable cauteries are available at lower cost |
IV 14g x 3.25" cannula | BD | B-D382268H | For tracheal cannulation |
Braided silk non-absorbable suture size 4-0 | Harvard Apparatus | SP104 | For stabilization of the tracheal cannula |
Braided non-absorbable silk 5/0 suture | Surgik LC, USA | ESILRC15387550 | For suturing the tongue |
Plastic-coated metal twist-tie (or electrical wire) | For securing the rat's nose to the platform | ||
Camera stick | |||
3 way-swivel and Trilene 9Kg test monofilament line | Berkley | For securing the jaw and maintaining the mouth open | |
Camera stick with adjustable angle | For supporting the 3 way-swivel and maintaining the mouth open. | ||
in situ Muscle Test System | Aurora Scientific | 809C | This system is designed for mice and was modified by extending the platform. Alternatively the rat-specific 806D system can be used. |
Dual-Mode Muscle lever (force transducer) | Aurora Scientific | 305C-LR | 309C offers higher excursion capabilities than 305C-LR. Link for more information and specifications: http://aurorascientific.com/products/muscle-physiology/dual-mode-muscle-levers/ |
Needle Electrodes (surgical steel, 29 gauge) | AD Instruments | MLA1204 | 300C is recommended for use in mice. |
Magnetic Stands | World Precision Instruments | M10 | Used for making the bipolar stimulating hook electrodes |
Kite Manual Micromanipulator | World Precision Instruments | KITE-R and KITE-L | Require a steel plate |
Stackable Double Binding Post with Banana Jack x BNC Jack | McMaster Carr | 6704K13 | |
Carbon fiber composites digital caliper | VWR | 36934-152 |