Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

This technical report describes a variation of the modified Bergström technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

Welcome to the Ruhr-University Bochum, Faculty of Sports Science. Today, we will demonstrate the collection of skeletal muscle biopsies from the superior compartment of the human musculus tibialis anterior. While biopsy techniques are effective at collecting muscle tissue, the fibers are often structurally damaged in such a way that evaluating fiber mechanics is difficult.

We present a variation of a modified Bergstrom technique that limits fiber damage and maximizes fiber length so that the mechanical evaluation of human fibers can be conducted. The demonstration will be conducted by team physician, Dr.Markus de Marees and research scientists, Dr.Daniel Hahn and Anthony Hessel. The participant and physician first meet to review and discuss the procedure.

If satisfied, the participant signs the voluntary informed consent contract. Instruct the subject to lay supine and keep their limb as relaxed as possible. Dorsiflex the participant's foot so that the tibialis anterior is slack.

Use an ultrasound probe to visualize the transverse plane of the superficial compartment within the tibialis anterior muscle belly, identify a biopsy area with no major arteries or nerves. Finally, record the depth of the central aponeurosis so that the collection needle is never driven past this point. Rotate the ultrasound probe to access the sagittal plane of the identified target area.

Save an image of the muscle volume with fascicles visible. The surgery tools are sterilized and prepared before the procedure and are fully described within the written protocol. The Bergstrom needle has a window close to the tip.

Within this needle is a smaller hollow trocar, which moves up and down the shaft and cuts the muscle when passing over the needle window. Within the trocar is a smaller ramrod that is used to push the biopsy out of the trocar. To pull the muscle into the needle window, a suction hose is attached to the top of the needle that sucks air out of the needle and pulls the muscle through the needle window by negative pressure.

Instruct the participant to lay supine on the procedure table and relax their leg muscles. Replicate the slight ankle dorsiflexion used during ultrasound imaging, and then fix the ankle joint to minimize rotation during the procedure. Shave and disinfect the target incision area.

Next administer a superficial injection of 1.5 CCs of 2%xylocitin with epinephrine. This functions as a local anesthetic and vessel constrictor. The affect time is 20 to 30 minutes.

Remove the patient's line of sight to the biopsy location with a screen. At the previously marked biopsy site make a one centimeter proximal to distal incision with a sterile scalpel that cuts through the skin and fascia, exposing the underlying muscle tissue. Position the biopsy needle over the incision and orient it perpendicular to the skin.

Push the biopsy needle one half to one centimeter into the muscle. There may be some light resistance to the needle movement, but any extra resistance is abnormal and often indicates an inapt incision. Next orient the needle to a distal to proximal position, push the needle another one to two centimeters into the muscle.

The exact orientation and travel depth of the needle will be participant specific. And thus based off the ultrasound examination. Attach the syringe hose to the biopsy needle and create negative pressure in the needle.

This will draw the muscle tissue through the needle window. Forcefully push the trocar over the needle window to excise the muscle. Remove the needle from the leg and pass to a technician for tissue processing.

Use wound closure tape or adhesive to primarily close the wound and apply dressings to protect the wound from infection or mechanical abrasion. Instruct the participant to stand up and walk around. They will be sore, but able to walk around immediately.

To remove the tissue from the biopsy window, first expose the tissue, then submerged the needle tip into a vile filled with physiological buffer and vigorously move the needle until the muscle floats out of the needle into the solution. Process muscle tissue for mechanical evaluation using standard methods. We recently collected 22 biopsies from participants using this protocol.

The average collection yield was 100 to 200 milligrams of usable muscle tissue. Each biopsy yielded 150 to 300 undamaged fibers of three to five millimeters in length. Fiber bundles were chemically skinned with glycerol and then activated in a high calcium solution at an average sarcomere length of 2.7 microns.

Biopsies that are properly stored are viable for mechanical testing for several months. This biopsy procedure takes one hour. Complications from biopsies are very rare, but most likely is an infection of the incision site.

A major challenge in human muscle physiology and biomechanics is to distinguish between the various adaptations of muscle performance to exercise and disease. To appropriately identify fiber level adaptations to exercise or disease, biopsies are appropriate technique. However, care must be taken to protect against muscle damage during tissue extraction.

The protocol demonstrated here provides a high yield of muscle tissue for these purposes.