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