Freezing Injury in Mouse Masseter Muscle to Establish an Orofacial Muscle Fibrosis Model

Current skeletal muscle regeneration studies merely focus on the limb and the trunk muscle, leaving the characteristic and mechanism of orofacial muscle regeneration largely unexplored. The scope of this research is to create an easier and reliable mouse model to facilitate further investigation into muscle regeneration in the orofacial area. Recent developments review that orofacial muscles constitute a unique subset of skeletal muscle with a distinct evolutionary trajectory and developmental origin.

They also exhibit different regeneration performances compared with their limb counterparts. A deeper investigation into the orofacial muscle regeneration is warranted to figure out the mechanism underlying these marked differences. The study present a rapid and reproducible method to study orofacial muscle regeneration.

The whole freezing-injury process took 20 minutes and the harvest process took only 10 minutes for each mouse, and the mice can tolerate the injury well without any impairment of locomotion. Since our model allows for subsequent comparison between the fibrotic masseter muscle and the fully regenerated tibialis anterior muscle from the aspects of morphology, histology, and molecular regulation. It serves as a powerful tool for us to delve deeper into the underlying mechanism.

Our future research will focus on the causal effects among the alterations of the resident muscle stem cells and multiple niche cells, and correspondingly developed interventions to the process of orofacial muscle fibrosis. To begin, place the anesthetized mouse on the surgical table. Using a cotton swab, apply the depilatory paste onto the skin, covering the masseter muscle and the tibialis anterior muscle.

After waiting for one to two minutes, wash away the paste with a surgical wipe. To expose the masseter muscle, first palpate along the inferior edge of the mandible. At five millimeters from the edge, cut open the skin along the line, connecting the oral commissure to the tragus.

To perform freezing, use pre-cool forceps to hold a piece of dry ice. Place the dry ice along the long axes of the masseter muscles directly onto the surface of the muscle and hold for five seconds. Confirm that the muscle appears stiff and pale white immediately after removing the dry ice and then returns to its normal color and texture within 22 to 25 seconds.

After the muscle fully recovers, close the skin wound with at least three 7-0 sutures. For the tibialis anterior muscle, palpate along the calf to locate the anterior edge of the tibia bone. Two millimeters posterior to the edge, cut open the skin starting from the knee and ending at the ankle.

To perform freezing, use pre-cool forceps to hold a piece of dry ice. Place the dry ice along the long axes of the tibialis anterior muscles directly onto the surface and hold for five seconds. Confirm that the muscle appears stiff and pale white immediately after removing the dry ice and then returns to its normal color and texture within 22 to 25 seconds.

After suturing the skin wound, place the mouse on a heater plate maintained at approximately 38 degrees Celsius. Monitor continuously until the mouse recovers its righting reflex. Prepare embedding molds by creating cylindrical tin foils for each sample.

Fill the molds with optimum cutting temperature or OCT compound. Then prepare two insulation barrels. Fill the small barrel with isopentane and the large barrel with liquid nitrogen.

Place the small barrel inside the larger one 10 minutes before muscle dissection. Begin by preparing the mouse for dissection. Cut open the skin on the face of the mouse to expose the masseter muscle.

Remove the parotid gland to fully expose the posterior part of the masseter muscle. Dissect the masseter muscle from its anterior attachment up to its posterior attachment on the mandible. Then submerge the muscle tissue in OCT, holding it perpendicular to the compound.

Transfer the mold into the pre-chilled isopentane using a needle holder. Wait for 40 seconds until the OCT becomes white and solid. Store the fresh frozen muscle samples in labeled 24-well plates.

Section the samples immediately at 20 degrees celsius or store them at 80 degrees Celsius for long-term use. Next, cut open the skin from the ankle to the knee and remove the fascia to expose the tibialis anterior muscle. Use the tip of the microdissection forceps to isolate the tendon of the tibialis anterior and slide it up to the knee to break off the fibers attaching to the tibia, cut the tendon at the ankle.

HE and Sirius Red staining revealed that upon freezing injury the tibialis anterior muscles showed complete regeneration after 14 days, with morphology similar to intact control. In contrast, masseter muscle exhibited impaired myofiber regeneration, an excessive extracellular matrix deposition after 14 days of freezing injury. Immunohistological staining for Pax7 after 14 days of injury showed densely populated nuclei, but without proportionally increased muscle satellite cells.

A large number of centrally located myofibers were detected in the tibialis anterior muscle at 14 days post-injury. While the contour of myofibers was barely noticeable in the masseter muscle in the injured area. Instead, the infiltration of Pdgfr-alpha+FAPs was observed.