Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

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The goal of in vivo muscle function testing is to provide an accurate assessment of the functional status of the muscle of interest to study the effects of age, disease, injury, and reparative treatments. This method is a key component to the multi-disciplinary studies required to better understand the time course and mechanisms responsible for skeletal muscle repair and regeneration in a variety of contexts. The main advantage of this technique is that it allows repeated measures of muscle function on the same animal over time.

This provides a powerful experimental paradigm for evaluating muscle damage and recovery in an optimized physiological setting. Implications of this technique extent toward therapy of injured muscle because accurate assessment of the degree of functional muscle recovery is critical to development and evaluation of novel therapeutics for muscle regeneration. Generally, individuals new to this method will struggle because electrode placement is scalably variable from animal to animal, and it is crucial for the success of the procedure.

For these experiments, consider using 11-week-old Lewis rats. Regardless, there is a linear correlation between muscle mass and force, so be aware of the rat's size. After anesthetizing the rat, completely remove the hair on the lateral side of the leg planned for experimentation between the ankle and pelvis.

Now, position the pedal apparatus. Use two controls to adjust the foot pedal to the left-most and lowest position that the apparatus allows. From this position, the third control moves the apparatus as required for the appropriate positioning of the pedal.

Now, clean the leg with three changes of iodine and alcohol. Each application of iodine should be about 30 seconds long. Next, adjust the animal's position so that when the leg is extended, the sole is in complete contact with the foot pedal.

Then, use medical tape to secure the foot to the pedal. It is crucial that the heel is flush against the pedal with the entire foot being flat. It is also critical that the foot is securely attached.

To stabilize the leg, use the adjustable pin to lock it in place. Now, make the leg parallel with the foot pedal. First, use the coarse and fine adjustment knobs to slowly move the ankle until the foot and tibia are at a 90 degree position.

Then, continue to move the leg until the femur and tibia are at a 90 degree perpendicular angle. At this point, the animal is ready for the electrodes. In the electrode control software activate instant stim by clicking on the clearly-labelled orange button.

Now, position the first stimulation electrode just past the kneecap and just lateral to the tibia so that it is adjacent and orthogonal to the plain of the perineal nerve which runs laterally from the knee and is perpendicular to the tibia. Keep adjusting the superficial position location until spikes are seen around 0.4 Newtons. Then, insert the second needle at the boundary of the tibialis anterior and to the gastrocnemius very superficially so it is barely into the muscular layer.

Adjust the position until the electrode reads spike near 0.6 Newtons. Once adjusted, secure the electrodes in place using hobby clamps ore medical tape. Now, tweak the adjustments to find the maximal force output of the leg.

On the high-power bi-phase stimulator, find the range and adjust knobs. First, adjust the range knob to get the desired maximum amperage. The maximum amperage is the level at which three or more consecutive stimulations result in identical contractile responses.

Next, turn the adjust knob to turn the percentage of the range needed to stimulate the muscle. Make adjustments until the force in the muscle reads around 1.0 Newtons. Before proceeding, check that the electrodes are still secure.

Now in the software, stop the instant stim, then go to the live data window and click on Start Sequence. Continue to monitor the curves by going back to the control screen and clicking the Analysis button located above the orange Instant Stim button. The tetanic curve should begin to take shape around the 60 hertz stimulation.

The tetanic curve can be used to distinguish optimal results from suboptimal results. This curve usually starts to form around a frequency of 60 hertz and is fully formed at 100, and will continue to stay fully-formed at higher than 100 hertz frequencies. The ideal curve should have an uninterrupted sharp, vertical upswing at the time of stimulation followed by a flat plateau phase with minimal osculations and an uninterrupted sharp vertical decrease period at the termination of stimulation.

The key to good results is the ability to stimulate the muscle to maximum force and maintaining that force during tetanus. Normal deviations from the ideal curve can indicate that the muscle is fatigued. Deviations that result in incomplete curves are usually due to incorrect electrode placement leading to failure of maximum recruitment of muscle fibers during stimulation and thus less than maximum contraction.

After watching this video, you should have a good understanding of how to expertly perform in vivo muscle testing to better understand the impact of diverse injuries and conditions on muscle function as well as evaluating the impact of potential reparative treatments. Once mastered, this technique can be done in 30 minutes if it is performed properly. Following this procedure, other methods such as histological analysis and gene expression studies, can be performed in order to better understand myogenesis, muscle fiber and matrix formation, as well as identifying molecular mechanisms of muscle repair and regeneration.

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Journal (Bioengineering)

Journal (Bioengineering)

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

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