Procedures for Rat in situ Skeletal Muscle Contractile Properties

The overall goal of this procedure is to study skeletal muscle contractile properties under physiological conditions. This is accomplished by anesthetizing an animal and surgically isolating the motor supply to the muscle of interest. The next step of the procedure is to isolate the distal end of the muscle.

The muscle is then set up in a myo graft such that the muscle origin is immobilized, and muscle length is set at a reproducible reference length. By stimulating the motor nerves, results can be obtained that show fundamental contractile properties like force velocity, force frequency, and fatigue, or unique properties associated with unusual stimulation patterns. The main advantage to this technique over other methods, like the in vitro assessment of contractile properties, is that in situ assessment allows the direct examination of muscle contractile properties at body temperature with a normal blood flow.

This method can help answer key questions in the fields of muscle mechanics, muscle metabolism, and exercise physiology, such as what are the mechanisms of peripheral skeletal muscle fatigue? What are the short term consequences of repeated activation? Chronic treatment of the animals training or hind limb suspension, for example, followed by assessment of contractile properties, allows the determination of the impact of such treatment on the contractile properties.

This insitu method can provide insight when skeletal muscle contractile properties change. Contractile properties can change acutely with repetitive activation or chronically with disease inactivity or training. Contractions can be isometric or dynamic, and considerations can be given for the length of the muscle.

Once the selected rat has been fully anesthetized, immobilize it on a plexiglass platform with masking tape. Add a drop of paraffin oil to the eyes and double check that corneal or toin flexes are absent before proceeding. Sterile technique is not a concern because this procedure is acute.

Now using scissors, cut through the shaved skin from the heel to the vertebral column, separate the skin from the underlying tissue. Keep exposed surfaces covered with isotonic saline soaked gauze cut proximally through the superficial muscle over the gastrocnemius muscle along the same line as the skin incision. While cutting through the muscle, be sure to locate and avoid the sciatic nerve.

Use the nerve's path to guide the incision, but stay well above the nerve. Next, identify the clear seam between the muscles and cut along that seam towards the knee. Be careful not to cut any blood vessels.

Excessive bleeding or even damage to the essential blood vessel will result in a muscle preparation that deteriorates quickly and has slow muscle relaxation. Now, clear the connective tissue away from the sciatic nerve so it can be slipped into the nerve cuff. Be sure to treat the nerve gently.

Stretching the nerve can lead to inex excitability, thus ending the experiment prematurely. Put a tie around the nerve cut proximal to the tie and reflect the nerve back over the muscle. Now, use a handheld rotary tool to drill a small pilot hole through the cortex almost into the medulla of the femur.

Do not drill too deeply or excessive bleeding will result. Avoid contacting soft tissues under a dissecting microscope. Locate the place where the popal nerve disappears behind the medial gastrocnemius muscle.

Gently spread out the various branches and cut the branch to the lateral gastrocnemius. The other branches will be severed when the distal part of the leg is removed. The innervation can be determined by micro stimulation.

Also cut the superficial branches using scissors. Cut or bluntly dissect the gastrocs muscle to separate it from other tissues. Pull the tendon of the plen terrace out from under the achilles tendon.

Tie it off and sever its tendon. Then separate a substantial length of the plantars from the gastrocnemius muscle and cut away the string tied to the plantars tendon. Now place a number one silk ligature around the achilles tendon and tie it in a square knot.

Do not pull too tightly or the tendon will be damaged. Use bone ros to cut the calcaneus, leaving a small piece of bone attached to the achilles tendon. Keep the cutting surfaces horizontal to be sure that the bone is cut and not just the tendon.

Locate the sous muscle along the underside of the gastrocnemius and with blunt dissection, separate the two muscles. Then cut the soleus tendon close to the distal end. Finally, separate the medial and lateral gastroc muscles.

Cut the lateral tendon so the medial gastrocnemius muscle is the only tissue attached to the achilles tendon with the gastrocnemius muscle separated from the Achilles place a tight ligature around the shank of the tibia just above the midpoint. Do not cut into the tissue. Make a second loop to prevent slipping and secure the ligature with a square knot.

Now using a small saw, cut the lower leg off midway along the tibia. Then insert a sharp probe into the medulla of the tibia. Using michel clips attached two elastics to the, these will be used to hold the skin up around the muscle to form a container for paraffin oil.

Position the rat on the myo graft base and immobilize its leg by orienting the tibial probe toward the lever and placing it in the holder. Now pull the skin up at the sides to form a container and secure the skin with elastics. Support the femur on the anterior side and set a drill bit into the femur.

Affix the drill to a crossbar. Next place the distal stump of the sciatic nerve through the nerve stimulating cuff. Tuck it close to the origin of the muscle and connect the stimulator to the nerve cuff wires.

Attach the achilles tendon to the lever. Tie it off snugly leaving some room for adjustments to be made. Then align the muscle perpendicularly to the lever.

Now fill the container formed by the skin with warmed paraffin oil and position a heat lamp over the animal to ensure that the animal's core does not exceed 38 degrees Celsius position. Foil sheets between the heat lamp and the forced transducer and between the rat's head and the heat lamp. The final step in this preparation is to adjust the length of the muscle until there is no slack in the string between the medial gastroc anus and the force transducer While the muscle is warming up.

Set up the stimulator pulse duration to 50 microseconds and 0.5 volts. Now stimulate the muscle at eight to 12 second intervals. Gradually increase the voltage until the twitch amplitude plateaus.

The plateau occurs at the maximal voltage, and unless the nerve is damaged, it should be less than 1.5 volts going forward, stimulate at double the maximal voltage to assure activation of all motor units. Next, increase the muscle length in one millimeter increments and measure the contraction amplitude at each length. Use double pulse stimulation of 200 hertz when the length that provides the maximum contraction amplitude is determined.

Set the muscle at this length. Now set the stimulator to deliver pulses at 200 hertz for 500 milliseconds to generate a completely fuse Titanic contraction. This tightens all muscle connections, including the knots on the muscle, then redetermine the reference length and the muscle will be ready for experimentation.

These contractions were obtained to illustrate the force frequency relationship. Calculating the peak active force of these contractions and plotting the active force against frequency yielded this force frequency relationship. The data can be fit into the equation presented on the screen where AF is active force, F is frequency, and A, B, C, and D are constants.

This equation makes a line that closely fits with the data. Once mastered, the surgery and setup for this technique can be completed in one hour, but don't make a competition of it While attempting this procedure. It's important to regularly check the level of anesthesia of the animal Following this procedure.

Other measures like EMG blood flow or biochemical assessment can be completed to answer other questions such as, has membrane excitability been decreased? Has energy supplies been depleted, or has blood flow been compromised?