The overall aim of this procedure is to accurately determine the mechanical properties of mouse limb muscles. This is accomplished by measuring the contractile and the passive properties of the extensor digitorum longus muscle ex evo, or by measuring the contractile properties of the tibialis anterior muscle in situ, you ultimately results can be obtained that allow a better understanding of the physiological changes in various muscle diseases and whether experimental interventions enhance muscle function. This method provide a complete evaluation of the contractile and the passive property of the skeletal muscle, which are two inseparable aspect of the mechanical function of skeletal muscle necessary for body movement.
Therefore, visual demonstration of the muscle dissection procedure is critical because it require a lot of skills and manipulation. Also, the main advantage of the NC two evaluation of the contractile property of the large muscles over existing methods like the in vitro SA, is that institute approach does not disturb normal blood flow and oxygenation. A major concern in muscle physiology study is the oxygenation of the target muscle.
In an in vitro SA for large muscle, such as the TA muscle oxygen diffusion may not reach the center Of the muscle. The implications of this technique extend to a therapy of muscle diseases because the accurate measurement of the mechanical function of skeletal muscle can also be used to evaluate the progression of muscle diseases and determine the therapeutic efficacy of novel gene cell and pharmacological interventions. All animal procedures presented here have been approved by the institutional animal care induced committee.
This is a non survival surgery to set up the muscle test system for the ex vivo experiments. Begin by assembling the tissue bath by securing the oxy tube to the water jacket tissue bath. Attach the assembled bath to the muscle mounting apparatus and place the needle valve into the bath drainage.
Connect the gas line and the water circulation lines to the bath. Allow 30 degrees Celsius water to circulate in the jacket chamber and five PSI of gas to flow through the oxy tube. Fill the bath with ringer's buffer and equilibrate it with a steady gas flow.
Turn on the instruments and load the DMC software to dissect the EDL muscle arter arter anesthetizing and placing the mouse on a heating pad. Shave the hind limb and check for sedation by performing a toe pinch. Position the mouse on the dissection board and peel off the leg skin.
Use two dressmaker pins to secure the hind limb in the foot and in the gracilis muscle. Place a heating lamp above the mouse to maintain the core body temperature at 37 degrees Celsius and continuously super fuse all exposed muscles with warm ringers buffer under a stereo microscope, dissect the skin to expose the distal TA tendon and the extensor ligament, and gently remove the fascia covering the TA muscle. Cut the extensor ligament to release the distal TA tendon.
Cut the distal TA tendon and use it to remove the TA muscle with a thin piece of ringer's buffer soaked cotton. Stop the bleeding caused by rupture of the TA muscle vasculature. Next, using a bread silk suture tie a double square knot followed by a loop knot at the distal muscle tendon junction of the EDL muscle.
Then make an incision in the biceps fems muscle to expose the proximal tendon and tie the same set of knots at the proximal muscle tendon junction. Use the same suture line to make a double square knot. To secure the lever arm hook to either the proximal or the distal knots.
To gently dissect the EDL muscle from the hind limb. First, cut the proximal tendon superior to the suture knots and cut the vasculature beneath the muscle. Then cut the distal tendon inferior to the suture knot.
Cover the exposed hind limb with a piece of ringers soaked cotton. Finally, attach the hook to the lever arm and align the muscles vertically between the electrodes. Secure the distal suture line to the fixed post.
Submerge the muscle in ringer's buffer, and adjust the resting tension to 1.0 grams. Allow the muscle to equilibrate for at least 10 minutes. To measure the contractile properties of the EDL muscle, set the parameters for each of the following protocols in the DMC software as presented here.
For the first protocol, stabilize the EDL muscle by stimulating it three times at 150 hertz with a 62nd rest between stimulations. For the second protocol, allow the muscles to relax for two minutes, and then determine the optimal length or LN by stimulating the EDL muscle at different resting tensions. Use the digital caliper to measure the optimal length of muscle between the distal and proximal knots.
Allow the muscle to relax for two minutes. For protocol three, adjust the resting tension to L naught and measure the muscle force at single twitch stimulation. Determine the Twitch tension or pt the time to peak tension or TPT and the half relaxation time of the pt.
Allow the muscle to relax for two minutes. To carry out the fourth protocol, adjust the resting tension to LN and measure the testic muscle force generated at different stimulation frequencies. Measure the P NAUGH where Muscle Force reaches the maximal titanic force.
Measure the time to peak tension and half relaxation time for the p naugh. Begin protocol five by allowing the muscle to relax for five minutes, adjust the resting tension to L Naugh and apply 10 cycles of eccentric contractions with two minutes rest between cycles. Calculate the relative force loss of the PN after each cycle of eccentric contraction.
Finally, detach the EDL muscle from the apparatus and cut the tendons at the suture site. Determine the muscle wet weight and calculate the muscle cross-sectional area. To measure the passive properties of the EDL muscle, dissect the contralateral EDL muscle and attach it to the apparatus as demonstrated earlier.
In this video, subject the muscle to a six step stretching protocol where the muscle is strained to 160%L naugh at 10%increments. Analyze the stress strain profile to evaluate the viscous property of the EDL muscle stretch and hold the muscle at 10%L naught and measure the stress relaxation rate at the following timeframes. Begin by heating the thermo controlled animal stage to 37 degrees Celsius with circulatory water.
Turn on the and load the DMC software. After exposing the TA muscle on the anesthetized mouse's hind limb, use a bread silk suture to tie a double square knot around the patella ligament. Then tie a double square knot followed by a loop knot at the muscle tendon junction or MTJ of the distal ta tendon.
Use the same suture to make an approximate 10 millimeter loop. Position the animal prone and expose the biceps fems muscle. Expose the sciatic nerve and tie a double square knot around its proximal end.
Cut the nerve superior to the knot and carefully dissect around five millimeters of its length towards the knee. Do not stretch or damage the nerve. Prepare the contralateral ta muscle as demonstrated earlier in this video and cover one of the hind limbs with a piece of ringer's buffer soaked cotton.
Next, position the animal prone on the animal platform and use the patella ligament suture lines around the knee pin to tie a double square knot to secure the knee. Then after pinning the feet on the SIL guard block, secure the animal platform onto the thermo controlled stage and position the heat lamp to maintain the animal core body temperature at 37 degrees Celsius. After attaching the electrode to the platform, attach the sciatic nerve to the electrode, then cut the TA tendon of the uncovered hind limb distal to the distal knot and attach the suture loop to the lever arm hook.
Cover the exposed hind limb muscle with warm ringers buffer soaked cotton to record contractile measurements of the TA muscle. Use the parameters and protocol in the DMC software demonstrated earlier in this video for the EDL muscle and use the software to analyze the data. After taking measurements of the TA muscle, detach the distal TA tendon suture loop from the lever arm hook and measure the contractile properties of the contralateral TA muscle as demonstrated After euthanizing the animal, remove the TA muscles and determine their wet weight.
To calculate the cross-sectional area, this table shows the morphometric properties of the EDL muscle in normal black 10 and dystrophin deficient or MDX mice at four to six months of age. Shown here a representative contractile and passive properties of the EDL muscle from black 10 and MDX mice, including Twitch force specific maximal titanic force time to peak tension and half relaxation time of the absolute maximal titanic force. The time to peak tension and half relaxation time can also be calculated from the absolute twitch force.
The stress strain profile and stress relaxation rate are used to describe the passive properties of the EDL muscle. Absence of dystrophin has a significant impact on the contractile and passive properties of the EDL muscle. Specific Twitch and Titanic forces are significantly reduced in the M-D-X-E-D-L muscle.
For example, the time to peak tension is significantly faster while the half relaxation time is significantly slower in the M-D-X-E-D-L muscle. In addition, the stress strain profile suggests that stiffness is significantly increased in the M-D-X-E-D-L muscle. The M-D-X-E-D-L muscle also yields significantly higher resistance force before reaching the peak stress while the post peak stresses decline much faster.
Furthermore, the stress relaxation rate was significantly higher in the M-D-X-E-D-L muscle compared to that of the black 10 EDL muscle. Once the dissection procedure are mastered, the ex vivo analysis of the contractile and the passive property of the EDL muscle can be done in 40 minutes. And the in situ evaluation of the contractile property of both TA muscles can be done in 60 minutes After its development.
This technique paved the way for researchers in evaluating the contractile and the passive properties of skeletal muscle in the field of muscle disease to help determine the efficiency of treatment Following this procedure. Other method, like the histopathology analysis of skeletal muscle can be performed in order to answer additional question. For example, the effect of the experimental treatment on the histopathology of skeletal muscle.
