Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

This technique allows the combination of the intramuscular injection of the snake venom cardiotoxin, the intramuscular injection of self-delivering siRNA, thereby allowing the analysis of regeneration of skeletal muscle. The application of self-delivering siRNA is an elegant way to investigate the functional loss of single genes during muscle regeneration, thereby avoiding transgenic animals. After confirming a lack of response to toe pinch, shave the injection area of the cranial lower leg from the knee to the paw, and remove any loose hair.

Place the mouse in the supine condition on a heading pad covered with a sterile surgical cloth, and use 70%ethanol to disinfect the injection area of the cranial lower leg. Next, load an insulin syringe equipped with a 29-gauge needle, with 50 microliters of 20 micromolar cardiotoxin and pierce the skin fully into the muscle, just distal to the knee. When the needle is in place, inject the cardiotoxin over a 10-to 20-second delivery period along the full length of the muscle, while moving the needle back and forth, to allow an even distribution of the cardiotoxin, thereby injuring the entire tibialis anterior muscle.

Then return the mouse to its cage on a heating pad with monitoring until full recumbency. Three days after the surgery, disinfect the injection area as just demonstrated. Inject up to 50 microliters of siRNA directed against the target gene of interest into the tibialis anterior muscle of the anesthetized mouse, as just demonstrated.

Then return the mouse to its cage, with monitoring until full recumbency. Before collecting the injured muscle tissue, wrap foil around a pencil and seal the pencil with tape, such that the bottom of the mold provides an even, closed surface. When the mold is ready, disinfect the whole animal with 70%ethanol, and use extra sharp scissors to remove the skin at the ankle.

Before harvesting the muscle, use fine forceps to pinch the closed forceps through the fascia next to the tibia bone at the ankle of the injured leg, and move the forceps toward the knee to tear the fascia, exposing the tibialis anterior muscle. To isolate the tibialis anterior muscle, expose the distal tendon, and grab the tendon with fine forceps. Use spring scissors to cut the tendon, and grasp the muscle at the tendon to pull the muscle toward the knee.

To allow the analysis of the midbelly region, use straight scissors to cut the tibialis anterior muscle at the midbelly region into two halves of equal size, and fill the pencil mold halfway with freezing solution. Insert the two halves of the tibialis anterior muscle into the freezing mold in an upright position, with the midbelly region facing the bottom of the mold. Use forceps to transfer the freezing mold halfway into liquid nitrogen.

When the freezing medium changes from transparent to white and becomes solid, transfer the freezing mold to a 80 degree Celsius freezer, or to dry ice for future processing. In control muscles, the architecture of the tissue remains intact, as observed by the localization of the nuclei in the periphery of the myofibers, and the lack of accumulation of mononucleated cells within the interstitial space. Seven days after cardiotoxin mediated injury, new myofibers are formed as marked by centrally located nuclei, and an accumulation of mononucleated cells consisting mostly of satellite cells, but also nonmyogenic cells like immune cells.

Under resting conditions, satellite cells as marked by Pax7 staining in red, are located underneath the basal lamina, shown in green here. Three days after injury, the number of satellite cells increases, and the satellite cells are no longer located under the basal lamina. At day 10 after injury, the number of satellite is still increased.

To further analyze the regeneration process, newly formed myofibers can be stained with antibodies directed against developmental myosin. Two days after injection with siRNA, satellite cells can be analyzed for the presence of the fluorescently labeled siRNA. In this representative experiment, about 75%of the satellite cells in the regenerating muscle were positive for the fluorescently labeled siRNA.

Furthermore, about 74%of all of the regenerating myofibers were also positive for the fluorescently labeled siRNA, suggesting that either 74%of the regenerating myofibers took up the siRNA, that siRNA-positive satellite cells had fused together to form new myofibers, or that fusion with already existing regenerating myofibers had occurred. The most critical step is to achieve a complete injury of the tibialis anterior muscle. In addition to histological analysis, parameters such as the force generated can be recorded to analyze regeneration of skeletal muscle in a functional manner.