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

Published: December 29th, 2023



1State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, 2Department of Oral and Maxillofacial Surgery, West China School of Stomatology, Sichuan University
* These authors contributed equally

The goal of this protocol is to establish an orofacial muscle fibrosis model. Comparison of the histology between mice masseter and tibialis anterior muscle after freezing injury confirmed masseter muscle fibrosis. This model will facilitate further investigation into the mechanism underlying orofacial muscle fibrosis.

Orofacial muscle constitutes a subset of skeletal muscle tissue, with a distinct evolutionary trajectory and development origin. Unlike the somite-derived limb muscles, the orofacial muscles originate from the branchial arches, with exclusive contributions from the cranial neural crest. A recent study has revealed that regeneration is also different in the orofacial muscle group. However, the underlying regulatory mechanism remains to be uncovered. Current skeletal muscle regeneration models mainly focus on the limb and trunk muscle. In this protocol, dry ice was used to induce freezing injury in the mouse masseter muscle and tibialis anterior muscle to create an orofacial muscle fibrosis model. The temporal dynamics of muscle satellite cells and fibro-adipogenic progenitors were different between the two muscles, leading to impaired myofiber regeneration and excessive extracellular matrix deposition. With the help of this model, a deeper investigation into muscle regeneration in the orofacial area could be carried out to develop therapeutic approaches for patients with orofacial diseases.

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 s....

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All animal procedures in this study were reviewed and approved by the Ethical Committee of the West China School of Stomatology, Sichuan University (WCHSIRB-D-2020-114). Male C57BL/6 mice (5 weeks old) were raised in a humidity-controlled (53 ± 2%) and temperature-controlled (23 ± 2 °C) facility and were on a 12 h light/dark cycle. See Table of Materials for details related to all materials, reagents, and instruments used in this protocol.

1. Freezing inju.......

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HE and Sirius Red staining (Figure 4 and Supplemental Figure S1) revealed complete muscle regeneration of TA in this freezing-injury model. In contrast, MAS exhibited impaired myofiber regeneration and excessive extracellular matrix deposition. The histology of intact MAS and TA muscle is shown in Figure 4A,B, where myofibers are in alignment and the fibrotic area only appeared in the interstitial space and amon.......

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There are a variety of injury models for studying skeletal muscle regeneration, including the use of physical, chemical, and surgical stimuli10,11,12,13,14,15,16. Cardiotoxin and barium chloride are the two most widely used chemicals to initiate muscle regeneration10<.......

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This study was supported by grants from the Sichuan Provincial Health and Wellness Committee (Grant Number: 21PJ063) and the National Natural Science Foundation of China (Grant Number: 82001031).


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NameCompanyCatalog NumberComments
1 mL syringeShifeng Medical Apparatus and Instrument (Chengdu, Sichuan, China)1-ml syringe/
AcetoneChron ChemicalsAceton/
Adhesion microscope slidesCitotest Scientific188105/
Animal depilatoryPhygene ScientificPH1877/
BSA (bovine serum albumin)Solarbio Life SciencesA8010/
DAPISolarbio Life SciencesC0065/
Donkey anti-goat Alexa Fluor 488Abcamab1501291:200
donkey serumSolarbio Life SciencesSL050/
Dry IceSinrro Technology (Chengdu, Sichuan, China)rice-shaped dry ice/
IFKine Red Donkey anti-rabbitAbbkine Scientific CompanyA244211:200
Insulation barrels (big)ThermosD600/
Insulation barrels (small)Polar Ware250B/
IsofluraneRWD Life Technology Company (Shenzhen, Guangdong, China)R510-22/
Liquid nitrogenSinrro Technology (Chengdu, Sichuan, China)//
M.O.M kitVector LaboratoriesBMK-2202
Mice  Dashuo Biological Technology Company(Chengdu, Sichuan, China)5 weeks old/
mounting mediumSolarbio Life SciencesS2100/
Nertral balsamSolarbio Life SciencesG8590/
Pax7Developmental Studies Hybridoma Bank Pax71:5
PdgfraR&D systemsAF10621:40
Sirus Red Staining KitSolarbio Life SciencesG1472/
Surgical instruments (forceps, scissors, needle holder, scalpel, and suture)Zhuoyue Medical Instrument (Suqian, Jiangsu, China)//
Tissue-tek OCTSakura4583/
TritonShanghai Scigrace BiotechABIO-Biofroxx-0006A/
ZoletilVirbacZoletil 50/

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