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).

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 &#177; 2%) and temperature-controlled (23 &#177; 2 &#176;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...

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

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J., Afoyalan, O., Kioussi, C., Dietrich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+roll+the+eyes+and+snap+a+bite+-+function,+development+and+evolution+of+craniofacial+muscles.">To roll the eyes and snap a bite - function, development and evolution of craniofacial muscles.</a> Seminars In Cell &amp; Developmental Biology. 91, 31-44 (2019).</li><li>Vyas, B., Nandkishore, N., Sambasivan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+cranial+mesoderm:+developmental+trajectory+and+evolutionary+origin.">Vertebrate cranial mesoderm: developmental trajectory and evolutionary origin.</a> Cellular and Molecular Life Sciences: CMLS. 77 (10), 1933-1945 (2020).</li><li>Sambasivan, R., Kuratani, S., Tajbakhsh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+eye+on+the+head:+the+development+and+evolution+of+craniofacial+muscles.">An eye on the head: the development and evolution of craniofacial muscles.</a> Development. 138 (12), 2401-2415 (2011).</li><li>Cheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Head+muscle+fibro-adipogenic+progenitors+account+for+the+tilted+regeneration+towards+fibrosis.">Head muscle fibro-adipogenic progenitors account for the tilted regeneration towards fibrosis.</a> Biochemical and Biophysical Research Communications. 589, 131-138 (2022).</li><li>Pavlath, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+among+muscle+precursor+cells+in+adult+skeletal+muscles+with+differing+regenerative+capacities.">Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities.</a> Developmental Dynamics. 212 (4), 495-508 (1998).</li><li>Camacho-Alonso, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+lingual+musculature+in+rats+using+myoblasts+over+porcine+bladder+acellular+matrix.">Regeneration of lingual musculature in rats using myoblasts over porcine bladder acellular matrix.</a> Oral Diseases. 27 (6), 1580-1589 (2021).</li><li>LeBoff, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinician's+guide+to+prevention+and+treatment+of+osteoporosis.">The clinician's guide to prevention and treatment of osteoporosis.</a> Osteoporosis International. 33 (10), 2049-2102 (2022).</li><li>Julien, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+contribution+of+skeletal+muscle+mesenchymal+progenitors+to+bone+repair.">Direct contribution of skeletal muscle mesenchymal progenitors to bone repair.</a> Nature Communications. 12 (1), 2860 (2021).</li><li>Mahdy, M. A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycerol-induced+injury+as+a+new+model+of+muscle+regeneration.">Glycerol-induced injury as a new model of muscle regeneration.</a> Cell and Tissue Research. 374 (2), 233-241 (2018).</li><li>Guardiola, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+acute+skeletal+muscle+regeneration+by+cardiotoxin+injection.">Induction of acute skeletal muscle regeneration by cardiotoxin injection.</a> Journal of Visualized Experiments: JoVE. (119), 54515 (2017).</li><li>Larouche, J. A., Wallace, E. C., Spence, B. D., Buras, E., Aguilar, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+mapping+of+immune+and+stem+cell+dysregulation+after+volumetric+muscle+loss.">Spatiotemporal mapping of immune and stem cell dysregulation after volumetric muscle loss.</a> JCI Insight. 8 (7), e162835 (2023).</li><li>Anderson, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+a+critical+size+threshold+for+volumetric+muscle+loss+in+the+mouse+quadriceps.">Determination of a critical size threshold for volumetric muscle loss in the mouse quadriceps.</a> Tissue Engineering. Part C, Methods. 25 (2), 59-70 (2019).</li><li>Lukjanenko, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+disrupts+muscle+stem+cell+function+by+impairing+matricellular+WISP1+secretion+from+fibro-adipogenic+progenitors.">Aging disrupts muscle stem cell function by impairing matricellular WISP1 secretion from fibro-adipogenic progenitors.</a> Cell Stem Cell. 24 (3), 433-446.e7 (2019).</li><li>Dong, J., Dong, Y., Chen, Z., Mitch, W. E., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathway+to+muscle+fibrosis+depends+on+myostatin+stimulating+the+differentiation+of+fibro/adipogenic+progenitor+cells+in+chronic+kidney+disease.">The pathway to muscle fibrosis depends on myostatin stimulating the differentiation of fibro/adipogenic progenitor cells in chronic kidney disease.</a> Kidney International. 91 (1), 119-128 (2017).</li><li>Lemos, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nilotinib+reduces+muscle+fibrosis+in+chronic+muscle+injury+by+promoting+TNF-mediated+apoptosis+of+fibro/adipogenic+progenitors.">Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors.</a> Nature Medicine. 21 (7), 786-794 (2015).</li><li>Joe, A. W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+injury+activates+resident+fibro/adipogenic+progenitors+that+facilitate+myogenesis.">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nature Cell Biology. 12 (2), 153-163 (2010).</li><li>Biferali, B., Proietti, D., Mozzetta, C., Madaro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro-adipogenic+progenitors+cross-talk+in+skeletal+muscle:+the+social+network.">Fibro-adipogenic progenitors cross-talk in skeletal muscle: the social network.</a> Frontiers In Physiology. 10, 1074 (2019).</li></ol>

The authors have no conflicts of interest to disclose.

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<...

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.

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	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>Shifeng Medical Apparatus and Instrument (Chengdu, Sichuan, China)</td>
			<td>1-ml syringe</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Chron Chemicals</td>
			<td>Aceton</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Adhesion microscope slides</td>
			<td>Citotest Scientific</td>
			<td>188105</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Animal depilatory</td>
			<td>Phygene Scientific</td>
			<td>PH1877</td>
			<td>/</td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumin)</td>
			<td>Solarbio Life Sciences</td>
			<td>A8010</td>
			<td>/</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Solarbio Life Sciences</td>
			<td>C0065</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Donkey anti-goat Alexa Fluor 488</td>
			<td>Abcam</td>
			<td>ab150129</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>donkey serum</td>
			<td>Solarbio Life Sciences</td>
			<td>SL050</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td>Sinrro Technology (Chengdu, Sichuan, China)</td>
			<td>rice-shaped dry ice</td>
			<td>/</td>
		</tr>
		<tr>
			<td>IFKine Red Donkey anti-rabbit</td>
			<td>Abbkine Scientific Company</td>
			<td>A24421</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Insulation barrels (big)</td>
			<td>Thermos</td>
			<td>D600</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Insulation barrels (small)</td>
			<td>Polar Ware</td>
			<td>250B</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Technology Company (Shenzhen, Guangdong, China)</td>
			<td>R510-22</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Isopentane</td>
			<td>MACKLIN</td>
			<td>M813375</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L9393</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>Sinrro Technology (Chengdu, Sichuan, China)</td>
			<td>/</td>
			<td>/</td>
		</tr>
		<tr>
			<td>M.O.M kit</td>
			<td>Vector Laboratories</td>
			<td>BMK-2202</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice&#160;</td>
			<td>&#160;Dashuo Biological Technology Company(Chengdu, Sichuan, China)</td>
			<td>5 weeks old</td>
			<td>/</td>
		</tr>
		<tr>
			<td>mounting medium</td>
			<td>Solarbio Life Sciences</td>
			<td>S2100</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Nertral balsam</td>
			<td>Solarbio Life Sciences</td>
			<td>G8590</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Pax7</td>
			<td>Developmental Studies Hybridoma Bank&#160;</td>
			<td>Pax7</td>
			<td>1:5</td>
		</tr>
		<tr>
			<td>Pdgfra</td>
			<td>R&#38;D systems</td>
			<td>AF1062</td>
			<td>1:40</td>
		</tr>
		<tr>
			<td>Sirus Red Staining Kit</td>
			<td>Solarbio Life Sciences</td>
			<td>G1472</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Surgical instruments (forceps, scissors, needle holder, scalpel, and suture)</td>
			<td>Zhuoyue Medical Instrument (Suqian, Jiangsu, China)</td>
			<td>/</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Tissue-tek OCT</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Shanghai Scigrace Biotech</td>
			<td>ABIO-Biofroxx-0006A</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac</td>
			<td>Zoletil 50</td>
			<td>/</td>
		</tr>
	</tbody>
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freezing injury in mouse masseter muscle to establish an orofacial muscle fibrosis model

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.

Author Spotlight: Exploring Orofacial Muscle Regeneration &amp;#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.

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...

Watch this Scientific Journal Video about Freezing Injury in Mouse Masseter Muscle to Establish an Orofacial Muscle Fibrosis Model at JoVE.com

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 ...

freezing-injury-mouse-masseter-muscle-to-establish-an-orofacial

Research

JoVE Journal

Biology

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.

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.

388 Views.  Sichuan University.  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.

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

Summary