The aim of this procedure is to determine the force generated and fatigueability of isolated skeletal muscle as a way to quantitate the effects of genetic modification or therapy. First, the extensor digitorum longest muscle is dissected from the mouse hind limb. Then the muscle is mounted in a muscle strip myo.
The next step is to ascertain the voltage for stimulation and pretension of the muscle at which maximal twitch tension is achieved. The final step is to determine the force frequency relationship for the muscle, as well as the onset and degree of fatigue with low frequency to tanic stimulation. Ultimately, results can be obtained that show measurable changes in my mechanical properties of the muscle through myography.
The main advantage of this technique over existing methods like exercise testing, is that myography allows for a quantitative assessment of muscle strength and function. We first adopted this method when we realized that quantitative measures for assessing muscle function are needed as new cell and bioengineering based therapies for muscle disease are tested in preclinical animal models. This method can help answer key questions in the muscle biology and muscular dystrophy field, such as whether specific therapies or genetic modifications alter muscle function.
Visual demonstration of this method is critical as the steps needed to properly handle the muscle are difficult to master. Generally individuals new to this method will struggle because rapid, a traumatic dissection of the hindin muscles takes practice and acquired skill. Demonstrating the procedure will be when we gong a research scientist and SAB ler a graduate student in our laboratories.
Following euthanasia of a mouse, arrange the carcass ventral side up on a dissection tray and pin the leg to the tray under a dissection microscope. Cut open the skin and carefully open the fascia. Next, peel the tibialis anterior muscle from the ankle upwards to expose the extensor digitorum longus or EDL muscle.
Use drops of lactated ringer solution to keep the muscle moist during the harvest. Then dissect out the EDL preserving as much tendon as possible on each end, and being careful not to touch the EDL muscle fibers themselves. Put the EDL muscle into a Petri dish containing lactated ringer solution.
Now tie sutures to each of the muscle tendons. These studies use a tissue bath to hold the muscle and a force transducer to measure muscle tension. In addition, a square pulse electrical stimulator and data acquisition platform are used to elicit record and analyze my mechanical responses.
The next step is to fill the myo graft bath with 6.5 milliliters of Krebs, hence satellite solution. Warm the bath to 25 degrees Celsius bubble the Krebs solution with 95%oxygen, 5%carbon dioxide after bubbling for 15 minutes. Use sutures on the tendons to transfer the EDL muscle to the bath.
Secure the tendons between the clamps of the myo graft. Be careful not to clamp the muscle itself. Maintain the myo graft bath at 25 degrees Celsius.
To begin the tension analysis, adjust the initial muscle length so that it is equal to the inci muscle length. Using a stimulus duration of 0.5 milliseconds. Gradually increase the voltage to determine the stimulus required to obtain maximal twitch tension.
Then set the stimulus 20%higher to achieve a supra maximal stimulus, which is usually about 40 volts. The next step is to gradually stretch the muscle until there is no further increase in twitch tension. This length is considered the optimal muscle length.
Allow the muscle to equilibrate for three minutes, then keeping the muscle at its optimal length. Deliver a supra maximal square stimulus and record the output record the twitch tension curve. In this figure.
The maximal twitch tension is labeled pt. The contraction time is labeled ct. The half relaxation time is labeled HRT.
The bar represents one second following a three minute rest period. Measure the Titanic tension by applying a strain of super maximal stimuli for 300 milliseconds at 150 hertz at optimal length. This procedure generates a tetanus tension curve.
The maximal Titanic tension is labeled po. The half relaxation Titanic tension is labeled HRTT. The bar represents one second After a three minute rest period.
Begin the force frequency analysis by applying 300 millisecond trains of super maximal stimuli at thirty sixty one hundred, one hundred and forty, and 160 hertz. Allow three minutes of rest between each train of stimuli after a three minute rest period. Begin the fatigue analysis by applying trains of short taai 60 hertz for 300 milliseconds every three seconds for 10 minutes by 10 minutes.
The Titanic force should decline to a plateau level of around 15%of the initial value. After finishing the force recordings with the muscle at optimal length, measure the muscle diameter using the ocular on the microscope. Following this, remove the sutures and weigh the muscle to determine muscle mass.
The last step is to weigh the mouse to assess body mass. These measurements are used for the calculations listed in the accompanying article. This figure shows the increase in tension generated with increasing stimulus frequencies shown.
Here is the muscle's response to a pulse train at 30 hertz. This figure shows the greater force solicited at a pulse train of 140 hertz. This graph shows the force frequency relationship plotted as the percent maximal force versus the stimulation frequency.
The shape of the force frequency curve is characteristic of muscle strength and can be used to make comparisons between muscles from different animals. This figure demonstrates muscle fatigue over the course of 10 minutes of low frequency stimulation. These figures show the muscle's response to the stimulus train at the indicated time points.
This plot shows the low frequency muscle fatigue as a plus of percent maximal force versus time. The shape of the low frequency fatigue curve is characteristic of muscle strength and can be used to make comparisons between muscles from different animals. Once mastered, this technique can be done in 30 minutes Following this procedure.
Other methods like immunohistochemistry, western blot, and quantitative PCR can be performed in order to answer additional questions like whether and wear specific myogenic proteins and stem cell markers are being expressed in the treated Muscle while attempting this procedure. It's important to remember to have all of your equipment ready to minimize the time between muscle harvest and analysis. After watching this video, you should have a good understanding of how to dissect the muscle hind limb, mount the muscle in a muscle strip myo, such as the one we have shown you from Danish myo technology and measure changes in my mechanical properties.
