The overall goal of this procedure is to isolate healthy, intact muscles to study in vitro contractile function. This is accomplished by first isolating the intact extensor digitorum longus or EDL and the soleus or sole muscles from the mouse. The second step is to tie the ligaments and tendons of the EDL and sole muscles with surgical suture.
Next, mount the isolated muscles on the force transducer and in the contractile chamber. The final step is to stimulate the muscle with field electric pulses to induce contractions. Ultimately, the resulting forces are transmitted to the force transducer and recorded various physiological and pharmacological manipulations can be performed to compare their effects on muscle contractility.
The main advantage of this technique over existing method to evaluate muscle function, like in situ contractility measurement, treat male performance, et cetera, is that this method removes the neuronal and the vascular components away from the skeleton muscle, allowing direct assessment of the intrinsic property of the contracting muscle. Generally individuals new to this method, the wheel struggle because the dissection of intact inhales muscles require spread dexterity and patience Demonstrated. This procedure will be one, a technician in my laboratory and a keyhole park, a postdoc fellow in Dr.Jma laboratory.
Visual demonstration of this method is critical as a muscle dissection and mounting onto the contractile chamber are difficult to run. Thus, this video helps to understand the anatomical structure of the muscle and to appreciate the scale required for the mounting of the isolated muscle. In this procedure, sacrifice a wild type C 57 black six mouse, or a disease mouse model by cervical dislocation and place it at the lateral position.
To dissect the EDL, make a superficial skin incision. Cut open the fascia between the anterior tibias and the posterior muscle group. Locate the proximal origin where the ligament is connected to the lateral condyle of the tibia and to the superior, three fourths of the anterior fibular surface.
Next, cut the ligament with delicate ophthalmic scissors as distal from the muscle as possible to release the proximal origin of the EDL muscle. After that, hold the ligament with a pair of blunt forceps and pull it slowly to free the EDL. It might be necessary to cut some of the paramecium surrounding the EDL and other muscles around the EDL.
This step must be performed delicately to avoid damaging the EDL muscle. Then at the insertion region where the four distal tendons insert into the middle and distal phalanges of digits two to five, cut the tendons as far from the muscles as possible. Transfer the isolated EDL muscle into a dissection dish containing calcium free isotonic tyro solution.
The EDL muscle is a fast glycolytic muscle with pale pink white color, a length of about 10 to 13 millimeters and a weight of eight to 11 milligrams. Next, tie a surgical knot tightly at both ends of the isolated EDL muscle with six hot size suture as distal from the muscle as possible and slightly above the midpoint lengthwise of the ligament or the tendon. Then transfer the EDL muscle to the oxygen saturated tissue bath chamber.
Mount the muscle on the sample groove of the force transducer and the stationary hook on the bottom of the bath chamber. Repeat the isolation and mounting process for the EDL muscle from the other leg to dissect the sole at the posterior lateral side of the leg. Push aside the gastro muscle that normally covers it.
The sole is a slow oxidative muscle with dark red color, about one millimeter shorter than the EDL, but weighs slightly more than the EDL. At the proximal origin. Cut the ligament that connects to the proximal half of the posterior tibia along the sole line and to the proximal third of the posterior fibula.
Next at the distal insertion, cut the calcaneal tendon that inserts into the posterior calcaneus. Carefully free up the soul afterwards. Properly mount the sole muscle in the bath chamber.
Once the muscles are mounted in individual tissue bath chambers, start recording the data. Remember to zero the baseline of the force transducer. This will facilitate observation of baseline changes and simplify the comparison of muscle contractions across different experimental conditions.
Then stimulate the isolated muscles with square wave pulses. Use electrical currents ranging from 60 to 300 milliamps individual square wave pulse durations between 0.3 and one millisecond and stimulatory train durations of 350, 500 or 1000 milliseconds. The next step is to select a frequency of stimulation capable of producing maximal titanic force when the isolated muscle is stretched at its optimal length at room temperature.
This frequency is typically around 100 hertz for EDL and 60 hertz. For soul, stretch the muscle slowly and wait for 30 seconds, then stimulate it at 100 hertz or 60 hertz. After that, stretch it again.
Wait for another 30 seconds and stimulate it again upon stretching. If the force does not increase any further, it normally means that maximal force has been achieved. Meanwhile, upon repetitive cycles of stretching and stimulation, force decrease suggests that the muscles have been overstretched In this situation, release the muscle to the previous stretch level and stimulate the muscle again until the maximal force is observed.
Now, obtain the force versus frequency relationship. Stimulate the muscle with frequencies from one to 140 hertz with increments of five to 10 hertz and a periodicity of 30 to 60 seconds. When performing the experiments at 25 degrees Celsius, when the experiments are performed at 37 degrees Celsius, extend the FF to higher frequencies of up to 300 hertz for diaphragm muscle, 180 to 200 hertz for soul and 220 to 250 hertz for EDL.
Next, by comparing the amount of force generated by each frequency of stimulation, identify the frequency that generates maximal titanic force and approximately one half maximal titanic force. Tmax provides important information of contractile machinery modulation. While the half Tmax provides information more pertinent to the calcium regulation and the ECC process.
The muscle is then stimulated with tmax frequency at an interval of one minute for 30 minutes, a process termed equilibration to study the contribution of sarcoplasmic reticulum. Calcium release to muscle contractility after equilibration fatigue the muscles by delivering one half Tmax stimulation at an interval of two seconds for five minutes. We used 500 milliseconds train duration here, and thus the duty cycle is 25%Then recover the muscle at one half Tmax at an interval of one minute.
For 30 minutes or until the force is stable, an additional fatiguing protocol can be performed. Now using tmax fatiguing at Tmax is believed to accurately test the status of the contractile machinery. Then repeat the procedures for obtaining FF so that the phenotypic differences between different strains, disease models, or drug treatments can be observed.
By analyzing the FF before and after fatigue to probe the contribution of extracellular calcium entry to muscle contractility, the bathing solution can be changed to a solution containing no calcium, but 0.1 millimolar EGTA. Alternatively, different blockers of store operated channels can be applied in the bathing solution. At the end of the experiment, measure the experimental length of the muscles, then weigh them individually with an analytical balance, flash, freeze the muscles in liquid nitrogen and store them at minus 80 degrees Celsius for biochemical analyses.
Now, calibrate the force transducer. Convert the recorded muscle force in millivolts to gram force based on the calibration results. Then normalize it to the physiological cross-sectional area.
This figure shows the contractile forces induced by five hertz, 20 hertz, and maximal titanic force here shows a trace of contractile, force of a damaged muscle. Here is a representative example showing the individual contractions of a force versus frequency relationship in EDL and the plotted curve resulting from the ff. This is a typical fast decline fatiguing profile of the EDL muscle and a slow decline fatiguing profile of the sole muscle.
Fatiguing stimulation leads to the appearance of mechanical alternates In a trick, a knockout muscle with disturbed calcium handling properties Once mastered, this technique can be done in two hours. If it is performed properly, the entire experiment will last longer and will depend on the number of interventions performed. While you are attempting this procedure, it is very important to isolate and mount the DL muscle as quick as possible because the low oxygen in vitro environment may cause irreversible damage to the fast to each muscle.
Following this procedure, gene analysis, western blotting immunohistochemistry and the structural studies can be performed on the same experimental muscles in order to answer additional questions. For example, expression level and localization of a particular protein or components of the muscle proteins regulating contractility or calcium signaling, as well as morphological or ultra structural alterations in muscles resulting from a specific conditions After its development. This technique paved the way for researchers in the field of muscle physiology to explore genetic defects affecting skelet muscle function in various models, including mice, rat and hamsters.
