In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs.

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This technique represents a noninvasive method to assess gain or loss of muscle function in a physiological setting. It can also be used to longitudinally assess function in the same subject during disease progression or before, during or after treatment strategy. This method provides a true test of maximal strength because muscle contractions are evoked in a controlled quantifiable manner, independent of subject motivation.

All research questions that evaluate skeletal muscle can be amplified by this methodology as it provides valuable data on the primary function of muscle. Individuals typically require two to 300 experiments before perfecting a similar small animal approach. In our experience, this large animal technique requires lesser time.

Positioning the knee, placement of electrodes, subject on the gurney and height of the foot plate require practice. To begin, turn on the computer, stimulator, transducer system and the analog digital interface about 30 minutes before testing to allow stabilization of heat related material changes that can impact electrical properties. Then select the appropriate and connected data acquisition device.

Next, to optimize electrode placement, click on prepare experiment when ready to begin the study. Open the live data monitor to allow real time investigation or visualization of the contractions. Then click on Instant Sim to deliver repeat twitches.

Alternatively, push the manual trigger button on the stimulator unit to manually give one twitch. Click on run experiment when prepared to begin the experiment. Once the pig is fully anesthetized, clean both the right and left hind limbs with soap and water to remove any debris and shave the hair from the skin paying close attention to the lateral knee area which will later be used for electrode placement.

Then transport the pig to a surgical table and securely place it in the supine position with the pig toward the foot of the table and the gluteal muscles at or slightly over the end of the table. For evaluating in vivo isometric torque, place the foot on the foot plate of the force transducer, then using a flexible cohesive bandage, attach the foot to the foot plate. Hold the foot in position on the foot plate with the ankle at the neutral, then secure the foot to the plate by wrapping the cohesive bandage around the foot and foot plate in the style of a closed basket wave ankle taping.

Once the foot is secured to the foot plate, position the ankle at a right angle, defined as zero degrees or neutral for reference of degrees of planter or door deflection. After stabilizing the knee and ankle at a right angle, position the limb clamping bars close to the needed locations. And when ready, starting from the medial aspect of the limb, align the limb clamping bar at about the tibial plateau.

Then align the lateral limb clamping bar at the distal head of the femur and stabilize the bars tightly using the locking thumb screws. Next, apply 70%alcohol and using clean gauss, clean the skin around the fibular head in concentric circles starting at the center of intended electrode placement and moving outwards. Then place the sterile percutaneous needle electromyography style electrodes across the perineal nerve and implant the electrodes subdermally at a depth of approximately five to 10 millimeters.

Optimize the electrode placement using increasing current amplitudes as adjusted on the stimulator, starting at 100 milliamperes and increasing as needed. Visualize the twitch torque magnitude on the live data view and over the pig's interior compartment. The hoofs may splay and move upward as well.

Ensure that the posterior compartment via the tibia nerve is not activated during stimulation. Visually inspect and palpate posterior compartment contraction and downward movement of hoofs during stimulation. Inspect the plateau region of tetanic contraction from the live torque time tracing in the following steps for lack of antagonist muscle recruitment.

Once the electrode placement and stimulation amplitudes are optimized, illicit maximal isometric tetanic torque using the stimulation parameters described in the text manuscript. For torque-joint angle analysis, measure the maximal isometric tetanic torque across a range of ankle positions ranging from neutral to the near end ranges of planter flexion or zero to 50 degrees of planter flexion. After measurement, start loosening both locking screws of the goniometer stage to move between joint angles and ensure that both locking screws are tightened before the next contraction.

For torque frequency analysis, position the ankle at the desired joint angle then measure maximal isometric torque over a range of stimulation frequencies that induce unfused trains of twitches up to and beyond those that induce fully fused tetany. To analyze the data, open the analysis program. Then using an automated data platform, calculate the different variables in analyzing individual isometric wave forms.

During the in vivo assessment, visualization of the torque wave form is needed in real time to ensure appropriate anterior compartment activation. The wave forms should only reflect dose of reflection and have a smooth rounded appearance and an apparent tetanic plateau. Inconsistencies or perturbations of the wave form may indicate inadequate stimulation or improper electrode placement.

Shown here is twitch torque and tetanic torque time tracing with 50%max torque. Dashed bars on the ascending and descending limbs of the tetanic torque time tracing represent a range of 30 to 70%maximal torque that can determine the average rate of contraction or relaxation. Representative values for torque joint angle and torque frequency relationships for uninsured limbs are shown here.

Simultaneous isometric torque and EMG recordings were made at stimulation frequencies of 20, 60, and 100 Hertz. The number of stimulator pulses reflects the quotient of stimulation duration and time between pulses. A 20 Hertz stimulation frequency means a pulse every 50 milliseconds, therefore a 400 millisecond stimulation duration divided by 50 milliseconds between pulses equals eight pulses delivered.

Raw EMG recordings are converted via root means square analysis to visualize the total muscle activity with increasing stimulation frequency. While evaluating isometric torque, proper anatomical alignment, proper electro placement, and confirmation that the elicited contraction is to the anterior compartment only are keys to a successful procedure. Since this is a non-terminal procedure, it can be combined with several complimentary study outcomes.

For example, in the large animal model, gait and mobility assessment can be tested on the same day along with maximal strength data to better understand how neuromuscular function influences ambulation.

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Journal (Biology)

Journal (Biology)

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

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