Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

This technique replicates in vivo biomechanical stretch testing of the brachial plexus in a piglet, serving as a highly relevant clinical large neonatal animal model. These methods can not only help in understanding stretch injury mechanisms but can also report the injury threshold values for functional and structural deficits within the neonatal brachial plexus. Demonstrating the procedure will be Rachel Magee, a graduate student from my lab.

After confirming a lack of palpebral and withdrawal reflexes, place the anesthetized pig in the supine position on the operating table, with the upper limb in abduction to expose to axillary region. Place a drape over the animal and use a number 10 scalpel blade to make incisions over the marked skin. Midline incision is overlying the trachea down to the upper third of the sternum, exposing the brachial plexus complex on both sides of the spine.

To expose one side of the animal's brachial plexus, a superior incision is made from the upper end of the midline incision, corresponding to C3, to the upper arm, and an inferior incision is made from the lower end of the midline incision, corresponding to T3, to the upper arm. Use forceps on each side of the incision to separate tissue from the suprasternal notch along the edge of the clavicle to the upper arm while sparing the cephalic and basilic veins. Using scissors, forceps, and blunt dissection, release the superior flap to access the cervical region of the brachial plexus, and the inferior flap to access the thoracic region of the brachial plexus.

Perform blunt dissection on superficial muscles to expose the brachial plexus. Then examine the plexus carefully to locate bifurcations of the divisions and identify the brachial plexus regions below the bifurcations, closer to the arm, as the cord and the nerve and the regions above the bifurcations, closer to the spine, as the root or trunk. To set up the biomechanical testing device, attach the base of the device to a cart and use large C-clamps to attach the electromechanical actuator to the base.

Attach a 200-newton load cell to the actuator and screw in a clamp with padded plexiglass to prevent stress concentration at the clamping site. Using a tripod, attach a camera that can record up to 100 frames per second at a 658 by 492-pixel resolution and attach USB cables from the camera, actuator, and load cell to the computer to integrate and synchronize all of the components of the setup. Then plug the computer, actuator, and load cell into a power source.

To calibrate the load cell before recording the applied loads, use the adjustable handle to set the actuator at a 90-degree angle such that it is aligned vertically and check the angle with a protractor. Open the load cell software and click Start to show a live readout of the voltage. Next hang zero to 1, 000-gram weights from the clamp in 100-gram increments, recording the measured voltages at each load.

When the voltages have been recorded for all 10 weights, calculate the slope and intercept to determine the linear equation of the voltages and weights. For biomechanical testing of the isolated brachial plexus nerve, use fine scissors to cut the nerve and use a custom clamp to clamp the cut side of the nerve. Label the clamped nerve segment with black acrylic paint and place a one-centimeter ruler flat within the animal to set the scale for the data analysis.

In the camera software, place the camera field of view directly over the tested segments to allow monitoring of the motion and/or displacement of the markers and to determine the actual tissue strain at a specific time point. Record the baseline measurements such as the height at which the nerve inserts into the body from the table, the height of the clamp from the table, the angle of the actuator, and the full length of the tissue. Open the programming software and click Run.

Enter the file name and displacement and click Initialize and TARE. Click Start to stretch the brachial plexus segment. The tissue will be pulled at an assigned rate of 500 millimeters per minute until complete failure occurs in any segment of the nerve tissue.

Then save a video file, the applied tensile load, the displacement of the tissue, and the duration of the test, and record the failure site as the segment at which the tissue ruptures. In this representative test of four brachial plexus segments, the obtained failure load was 8.3 newtons and the average strain failure was 35%for the neonatal nerve tissue samples when subjected to stretch. Some regions of the nerve underwent a higher strain than others, indicating a nonuniform injury along the length of the nerve.

The camera data allowed identification of the location of failure in this experiment as proximal to the foramen. It is important to ensure that first, the animal is deeply anesthetized and shows no sign of pain or discomfort, second, to calibrate the load cell, and third, to clamp the tissue firmly. This in vivo animal model can be used to study functional and histological changes in the brachial plexus tissue after varying degrees of stretches and to study complicated birthing scenarios.