orofacial muscle tissue collection after freezing injury induction in mouse 

Establishing an Orofacial Muscle Fibrosis Model in Mice via a Freezing Injury

Orofacial muscles are critical in daily physiological activities such as mastication, speech, respiration, and facial expression1. In congenital orofacial deformities, however, these muscles exhibit atrophic and fibrotic alterations, leading to impaired body health and social cognition2. Facial reconstructive surgery remains the first-line treatment, but up to 30-70% of postoperative patients still suffer from muscle loss and muscle dysfunction3,4 The failure of orofacial muscle regeneration has been attributed to intrinsic factors, which cannot be corrected by surgery alone. 
The emergence of orofacial muscles is an evolutionary novelty, accompanying the complex vertebrate head and chambered heart5,6. Unlike their somite-derived limb counterparts, orofacial muscles originate from the branchial arch7. These phylogenetic and ontogenetical characters may predispose them to distinct regenerative behaviors8. It has been reported that the masseter (MAS) muscle developed severe fibrosis at the time when the tibialis anterior (TA) muscle fully regenerated after exposure to the same extent of injury1,9. However, the underlying mechanism of regeneration remains poorly understood. 
In this study, a freezing injury model of the mice masseter muscle was established to facilitate the investigation into orofacial muscle regeneration. We chose 14 days after injury as the time point for assessing fibrosis phenotype as it was the earliest time point where discernible divergence was detectable between two muscles. Complete regeneration of the MAS after injury requires at least 40 weeks1. Consistently, this study revealed a remarkable deposition of collagen following freezing injury of MAS compared to the regular regeneration of the TA at 14 days post injury. With the help of this model, further mechanistic studies of muscle atrophy and fibrosis can be carried out, which will in turn help the development of potential therapeutic avenues to promote orofacial muscle regeneration after surgery.

Author Spotlight: Exploring Orofacial Muscle Regeneration &#8211; Insights and Innovations

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 characteristics and mechanism of orofacial muscle regeneration largely unexplored. The scope of this research is to create and 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 half-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 us a power for two 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.

Orofacial Muscle Tissue Collection After Freezing Injury Induction in Mouse 

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 glands 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 muscle showed complete regeneration after 14 days with morphology similar to intact control.In contrast, masseter muscle exhibited impaired myofiber regeneration and 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 PDGFRalpha-positive FAPs was observed.

Research

JoVE Journal

Biology

Orofacial muscle constitutes a subset of skeletal muscle tissue, with a distinct evolutionary trajectory and development origin. Unlike the somite-derived ...