In Vivo Canine Muscle Function Assay

The overall goal of this procedure is to measure and characterize mechanical properties of hind limb muscles in the living dog using non-invasive methods. This is accomplished by first positioning the dog properly in the physiology rig. The second step of the procedure is to measure and record isometric twitch torque and Titanic tibial tarsal flexion torque.

The third step of the procedure is to perform eccentric contractions. The final step of the procedure is to measure and record isometric twitch, torque, and titanic torque during tibial tarsal extension. Ultimately, results can be obtained that show hind limb muscle strength and response to repeated eccentric contractions through a non-invasive physiologic method.

The main advantage of this technique over existing methods like ex vivo, isolated muscle assays, which are terminal studies, is that the method used here can be repeated over time because it is non-invasive and well tolerated. This method can help answer key questions in the muscle physiology field, such as the muscles response to contraction induced injury or Fatigue. The implications of this technique extend toward therapy or diagnosis of muscular dystrophies because the methods measures muscle function in dogs or other large mammals.

Such large animals are required in preclinical studies of many new therapies prior to clinical trials and patients. Though this method can provide insight into canine models, it can also be applied to other animal model systems such as non-human primates. Generally, individuals new to this method will struggle because of the care required in placing the stimulation electrodes, and the knowledge of what to expect from the torque tracings Visual demonstration of this method is critical as the electrode positioning steps are difficult to learn because of the precision required to elicit the maximal torque response from the muscles.

Under study Two protocols written in the dynamic muscle control or DMC software are used to define the stimulation and data logging parameters. The first protocol, the isometric contraction protocol, is used for both the twitch and the tetanus contractions. The second protocol called the eccentric stretch injury protocol is used for the eccentric contractions, the stimulator settings and the software sequences used for each protocol are given in the table shown here, twitch contractions are elicited with one hertz stimulation.

The Titanic and eccentric contractions are elicited with 50 hertz stimulation. In the case of the eccentric contraction, a stretch is superimposed on the contracting muscle. These experiments involve eliciting several types of muscle contractions, stimulating the peroneal nerve to elicit twitch Titanic, or eccentric contractions of a flexor muscle, and stimulating the tibial nerve to elicit twitch or Titanic contractions of an extensor muscle.

After anesthetizing the dog, check the blink reflex to be sure that the animal is deeply anesthetized. Closely monitor the carbon dioxide, pulse oximetry, and temperature. During the procedure, shave the hair in the lateral stifle where the stimulating electrodes will be placed.

Next, position the dog on the physiology table. In dorsal recumbent position the pelvic limb so that the femur and tibia make a right angle, and the tibia is parallel to the tabletop check that the force pedal is able to rotate safely. It should feel stiff when it is moved manually.

Next, power on the physiology rig and activate the pedal. The movement of the pedal is controlled by the serva motor, which is controlled by the computer. Now, gently rest the dog's paw in the foot pedal measure and record the length of the dog's paw from the center axis of rotation to the end of the lateral digit.

Then secure the dog's paw in the foot pedal using elastic self adhering wrap. The transducer in the pedal records the force of the legs muscle contractions. Set up the stimulation electrodes by connecting the shielded monopolar 24 gauge electrodes to the stimulus isolation unit.

With gloved hands, clean the dog's skin with alcohol and palpate the common peroneal nerve just distal to the lateral proximal fibula with a finger marking the area of the nerve, carefully insert the black cathode electrode under the skin and just external to the periosteum place the red anode electrode under the skin parallel and slightly distal to the cathode electrode. Both electrodes should now be internal to the common peroneal nerve just distal to the fibular head. Next, activate the stimulator at one hertz and observe the twitch response on the monitor.

Make small adjustments in the electrode positions as needed to obtain the largest twitch amplitude possible. Once the electrodes are well positioned, activate the DMC software to collect a twitch response. The next step is to elicit a Titanic contraction without moving the electrodes.

Set the stimulator as specified in the table for a titanic contraction. Then activate the isometric contraction protocol in the software to generate an isometric titanic contraction of the muscle. The next experiment, the eccentric stretch injury protocol is also done without moving the electrodes.

Set the stimulator for an eccentric contraction and initiate stimulation. In this protocol, the nerve is stimulated 10 times at 50 hertz for one second. During that one second, the flexor muscles contract isometrically for the first 800 milliseconds, and then the muscle is stretched for the final 200 milliseconds.

After one set of 10 eccentric contractions allow the muscle to rest for four minutes. Then repeat the eccentric stretch injury protocol twice more with a four minute rest between each set to stimulate the extensor muscle, move the electrodes to the tibial nerve by inserting the electrodes perpendicular to the skin and proximal to the original electrode placement used for flexion. The approximate point of placement is along a line that bisects the 90 degree angle formed by the knee.

The tibial nerve lies deeper and coddle to the common peronial nerve. Move the electrodes as needed to elicit the optimal twitch potentials. Once the electrodes are situated, elicit a maximal twitch using the isometric protocol without moving the electrodes.

Change the stimulator settings as specified for titanic contractions. Then activate and record a Titanic contraction of the tibial tarsal extensor muscle. The dynamic muscle analysis or DMA program is used to analyze torque and temporal information from the muscle contractions.

In this figure tibial nerve stimulation at one hertz activated single twitches of the tibial tarsal extensor muscles for data analysis, the positions of the three cursors are set as follows. The blue cursor is set at the point where torque rises from the resting torque. The yellow cursor is placed where the torque returns to resting torque.

That is after the relaxation phase, the white cursor is not needed and so is placed off to the right of the yellow cursor. This figure shows isometric Titanic torque stimulation of the tibial nerve with a 1.5 second train of 50 hertz pulses resulted in a tetin contraction of the tibial tarsal extensor muscle. For data analysis, the blue and yellow cursors are placed.

As for the twitch analysis in this figure, eccentric contractions were induced in the tibial tarsal flexors. Percutaneous perineal nerve stimulation activated an initial isometric contraction for 800 milliseconds, at which point a forced stretch was imposed on the leg. For this data analysis, the blue cursor is positioned at the point where isometric torque begins to rise from resting torque.

The yellow cursor is placed at the point where the eccentric stretch torque begins to rise from the isometric plateau. The white cursor is placed where the torque returns to resting torque Once mastered, this technique can be done in about one hour if it is performed properly. While attempting this procedure, it's important to remember to a printed protocol with step-by-step checkpoints Following this procedure.

Other methods like muscle biopsies can be performed in order to answer additional questions like muscle fiber type, or expression of transgene After its development. This technique paved the way for researchers in the field of muscle physiology to explore the responses to exercise in canines with muscular dystrophy. After Watching this video, you should have a good understanding of how to perform a non-invasive muscle physiology assay in canines.