inducing freezing injury to establish an orofacial muscle fibrosis mouse model

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.

Inducing Freezing Injury to Establish an Orofacial Muscle Fibrosis Mouse Model

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-cooled 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-cooled 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.

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 ...