This protocol is significant because it can be used to create human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. This xenograft model allows researchers to study human muscle in vivo to better understand human muscle cell biology and to develop novel models for rare or acquired muscle diseases. After obtaining a human muscle biopsy, place the specimen in a 100-by-15-millimeter Petri dish containing muscle medium, and use a stereo microscope and surgical scissors to remove any remaining fascia or fatty tissue.
Dissect the muscle biopsy into approximately seven-by-three-by-three-millimeter pieces, taking care that the fibers are arranged longitudinally within the specimen. Next, transfer the Petri dish containing the dissected muscle on ice, and place synthetic, non-absorbable sutures in a 100-by-15-millimeter Petri dish of 70%ethanol. After confirming a lack of response to toe pinch in an eight-to 12-week-old, anesthetized NOD-Rag-gamma mouse, apply ointment to the immunodeficient animal's eyes, and use a trimmer to remove the hair overlying the tibialis anterior from the ankle to the knee.
Then, apply depilatory cream to the exposed skin for one minute before disinfecting the surgical site with povidone-iodine solution and 70%ethanol. To graft the human tissue, first tape down the mouse leg, and make a straight incision over the tibialis anterior originating at the distal tendons and terminating below the knee. Use blunt dissection to separate the skin from the muscle tissue, and use scissors to make a less-than-0.5-millimeter incision through the epimysium of the tibialis anterior muscle, starting at the tendon and ending at the knee.
After cutting the distal tendon of the tibialis anterior, grasp the tendon with iris forceps, and pull the muscle up toward the knee. Next, cut the distal tendon of the extensor digitorum longus, and pull the extensor digitorum longus up toward the knee. Once the proximal tendon of the peroneus longus muscle is visible, remove the extensor digitorum longus and the tibialis anterior with the scissors.
Use a surgical wipe wet with PBS to apply slight pressure until hemostasis is achieved, and thread a suture through the proximal peroneus longus tendon, leaving an approximately 1.5-inch piece of thread on either side of the tendon. Make the first half of a two-hand surgical square knot without tightening to form a circle, and place a xenograft into the circle, tightening the loop to secure the graft tissue. Complete the other half of the square knot to suture the xenograft to the proximal tendon of the peroneus longus, and thread the suture through the distal peroneus longus tendon to repeat the square knot technique at the distal tendon.
When both ends of the graft have been secured, pull the skin over the xenografted muscle, seal the incision with surgical glue, and place two to three surgical staples over the glue. Then, place the mouse in a clean cage on a heated pad with monitoring until full recumbency. Four to six months after the surgery, place a covered beaker containing 200 milliliters of 2-methylbutane in a box of dry ice for at least 30 minutes before the xenograft harvest.
When the 2-methylbutane has cooled, confirm a lack of response to toe pinch, and remove the hair overlying the tibialis anterior from the ankle to the knee with a trimmer and depilatory cream. The sutures holding the xenograft in place should be observed through the skin. Secure the leg with tape, and use scissors and iris forceps to open the skin over the xenograft until both sutures are uncovered.
Use a scalpel to cut between the xenograft and the tibia to free one side of the xenograft before cutting between the peroneus longus muscle and the gastrocnemius muscle. Cut below the distal suture and through the distal tendon of the peroneus longus, and use the iris forceps to grasp and deflect the suture toward the knee while using scissors to cut the xenograft away from the underlying muscle tissue. Then, use scissors to cut above the proximal suture to remove the xenograft and peroneus longus, and place the harvested specimen on a small piece of cardboard.
Pin the tissue as close to the sutures as possible while gently stretching the muscle to ensure that the fiber orientation will be maintained during the snap-freezing process. When the pins are securely in place, slide the muscle up the pins so the tissue rests just above the cardboard, and snap freeze the xenograft in the pre-cooled 2-methylbutane for minus 80-degree Celsius storage. A successful xenograft demonstrates a robust regeneration of human myofibers as identified with human-specific antibodies.
Positive embryonic myosin staining within a proportion of myofibers indicates that the regeneration process is still ongoing. In contrast, poor surgical technique or an inadequate specimen may lead to a poor regeneration of the muscle fibers. Xenografts performed from a patient diagnosed with an idiopathic inflammatory myopathy exhibit moderate numbers of regenerated human myofibers at four-and six-month collections, and embryonic myosin staining persists at six months.
Inflammatory cells are also present within the xenograft, as observed by H and E staining. Individual myofiber sizes are similar between four and six months in idiopathic inflammatory myopathy xenografts. Rare fibers showing a cross-sectional area greater than 3, 500 micrometers squared are observed within the xenografts but not in the idiopathic inflammatory myopathy biopsies, indicating that some of the myofibers in the xenografts can regenerate to a cross-sectional area similar in size to healthy myofibers.
The most important parts of this procedure are identifying all of the tendons in the ankle after the initial incision is made and successfully cutting through the epimysium. The functional competency of the xenografts can be assessed by enzymatically isolating single myofibers and testing calcium transients or by performing evoked forced measurements following electrical stimulation.
