Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

The overall goal of this procedure is to measure the mechanical properties of skeletal muscles isolated from mice. This is accomplished by first dissecting the extensor digitorum longus or EDL muscle, as well as the diaphragm muscle from the animal. After preparing a diaphragm strip pin the EDL and the diaphragm strip in a dish of oxygenated ringer solution and then tie suture loops to both ends of the muscles.

Next, the EDL or diaphragm strip is secured in an organ bath between two electrodes attached to a force transducer or lever at one end and adjusted to its optimal length based on twitch force. The final step is to stimulate the muscle using a number of mechanical protocols and measure force production. Ultimately, calculation of force production, resistance to damage force frequency relationship, and other properties are used to show the strength and integrity and other properties of the muscle, and if applicable, the effects of a treatment or genetic modification.

This technique can be used to test potential therapies for the muscular dystrophies because most of these therapies aim to regain muscle strength and reduce muscle fragility. Although we are not the first to utilize this technique, we routinely use this to study a number of different mouse models and assess their muscle function, and also to characterize and evaluate multiple therapies for the muscular dystrophies. In addition to Dr.Catherine Morewood, we will demonstrate this procedure using two members of our physiological assessment core, Dr.Min Liu, a research associate, and Mr.Ian, a research specialist.

The mice used in this procedure are anesthetized using ketamine and xylazine to ensure that they experience no pain or distress, but the muscles remain well oxygenated by the circulation. After confirming by a toe pinch that the animal is sufficiently anesthetized immobilize its high limbs using medical tape and remove the skin of the lower anterior hind limb to expose the muscles of this area, keep the muscles moist with the application of PBS at regular intervals. Under a dissecting scope, make a small incision lateral to the knee in order to expose the proximal tendon of the extensor digitorum longus or EDL muscle.

There are two tendons in this region and both should be cut to enable removal of the EDL at the medial ankle. Cut the tendon of the tibials anterior or TA muscle to expose the distal tendons of the EDL, which extend along the metatarsals. Lift the TA muscle out of the way.

Being careful not to cut or touch the EDL underneath. Return to the distal tendons of the EDL and snip each one. Next, grasp the tendons and gently pull the EDL away from the rest of the limb.

It should release from the proximal end freely. Remove the EDL muscle and place in a dissecting dish filled with chilled oxygenated wringers. Pin the muscle through the tendons at approximately resting length, which is the length found in vivo.

The length is too short when the muscle is flad in the dish and too long if the muscle is pulling on the dissecting pins. Tie sutures to the tendons as close as possible to the muscle, but not touching the muscle. Pin the muscle at approximately resting length using the sutures.

To begin this procedure. Make an incision in the skin of the euthanized animal. To expose the abdominal and chest cavity.

Open the abdominal cavity and cut the body wall just below the ribs using bone scissors and starting above the diaphragm insertion. Cut around the entire rib cage following the line of the ribs and cut through the spine. Cut blood vessels running through the center of the diaphragm so that the diaphragm can be removed easily.

Remove the diaphragm from the mouse and place in a dissecting dish filled with oxygenated ringers. Gently agitate the diaphragm in the dish to wash away blood. Refresh ringer solution as needed.

Next, cut a small strip of the diaphragm from the central tendon to the ribs along the orientation of the fibers in the central portion of the lateral hemi diaphragms. The strip should be between two to four millimeters wide. Tie sutures to the central tendon, tie sutures to each of the laterally protruding rib ends, and then tie these together to make a large loop using bone scissors.

Cut the rib on either side of the strip, leaving approximately one to two millimeters, overhang of rib on either side of the diaphragm. Strip force generation assessment of the isolated skeletal muscles is performed by an in vitro muscle test system to mount the muscles in the mechanic's bath, grasp the sutures and use these to attach the muscle to a rigid post on one end and to a force transducer on the other end. A good approximation is the resting muscle length.

As shown previously, the bath is filled with oxygenated ringers solution maintained at 22 degrees Celsius. To prolong muscle stability for testing muscles should rest for five minutes in this bath prior to functional testing, so that muscle temperature is equated to 22 degrees Celsius to establish supra maximal stimulation conditions. After a muscle is placed in the bath, use single 0.5 millisecond stimulation pulses to generate a twitch and monitor force output.

Gradually increase current until the force attains a maximum but steady level, increase current to 10%more than this level for the remaining experiments to establish optimum length, first, make sure the muscle is not slack, but not taught either. A good approximation is the resting muscle length as shown previously. Using isometric twitch stimulations adjust the length of the muscle gradually until a maximal force is obtained.

Rest the muscle about 10 seconds between each contraction. Optimal muscle length is achieved when twitch force is maximal record muscle length using vernier calipers. For the EDL, it is the length between the myo tendonous junctions.

For the diaphragm, it is the length between the central tendon myo tendonous junction and the muscle insertion to the rib. To determine the maximum isometric Titanic force stimulate the muscle set at optimal muscle length for a period of 500 milliseconds with a series of 0.5 millisecond pulses at super maximal stimulation and at fusion frequency. The plateau for EDL muscles is typically achieved with 120 hertz and for diaphragm muscles with 100 hertz stimulate three times at the respective frequency with rest periods of five minutes between stimulation bouts for every muscle following a five minute rest.

After performing isometric contractions, begin the procedure for eccentric contractions. Stimulate muscle at 80 hertz isometrically for the initial 500 milliseconds, followed by a 10%optimal muscle length stretch in the final 200 millisecond stimulation. Repeat the stimulation pattern with five minute rests in between.

For the desired number of eccentric contractions, measure the force for each contraction in the time period prior to the stretch. Calculate the drop in force between the first and last contraction at the completion of the functional test. Shorten the muscle length and gently remove the muscle from the transducer and post.

Return the muscle to a dissecting dish with ringers. Remove sutures from the muscles for the EDL muscle. Block the muscle twice and then weigh it before subsequent processing.

This will be important for calculating the cross-sectional area and specific force for the diaphragm muscle soak in 0.1%pros and orange, a membrane and permeant dye for 15 to 20 minutes. This will provide an index of dissection damage, dissect the diaphragm muscle away from the bony insertion as well as the central tendon. This is necessary to provide an accurate weight of the muscle blot before weighing and subsequent preparation.

The cross-sectional area or CSA is calculated using the following formula where L over LO is the fiber to muscle length ratio and 1.06 is the density of muscle expected values for the isometric forces in wild type C 57 and muscular dystrophy or M-D-X-E-D-L muscles from 12 week old animals are shown in this figure. Because MDX muscles exhibit compensatory hypertrophy total to titanic force can be higher in MDX muscle compared to age-matched wild type controls. A more appropriate measure of functional output is specific force where force is normalized for cross-sectional area.

To calculate this value specific force depends on the inherent functional capacity of the muscle, where the weakness in dystrophic muscles is more apparent when both absolute force and cross-sectional area are accounted for. It was observed in this study that EDL muscles from MDX mice generate approximately 20 to 25%lower specific forces than those of age matched wild type mice from 10 to 26 weeks of age. For the diaphragm only specific force is relevant for comparisons because the preparation is a piece of the muscle dependent upon the dissection.

A comparison of specific force between C 57 wild type muscle indicated by the blue line and MDX diaphragm muscle indicated by the red bars reflects the progressive pathology in this tissue. Isometric force output diminishes with age so that by six months of age diaphragm muscles from MDX mice produce no more than half of the functional output of diaphragm strips from age-matched wild type controls. An example of eccentric contractions from diaphragm testing is shown in this figure.

With each subsequent eccentric contraction force output diminishes in diaphragm strips from both wild type C 57 shown in blue and MDX mice shown in red. However, the loss of force is more dramatic in muscle samples from the MDX mouse, presumably from the absence of dystrophin and its associated proteins Once mastered. This technique can be performed in about one hour for a single muscle if it is performed properly following this technique.

Other methods such as western blotting or other biochemical measurements can be used to answer additional questions such as whether the treatment altered protein levels or normalized muscle integrity. After Watching this video, you should have a good understanding of how to dissect the EDL and diaphragm from the mouse and how to measure and analyze force production before and after eccentric contractions.