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Existing preclinical models fail to replicate this clinical phenotype; therefore, we aimed to develop animal models mimicking referred pain phenomenon and investigate whether referred pain leads to gene plasticity at the referred sites. We modeled masticatory myalgia by stimulation of either the masseter (MM) or temporal muscle (TM) in mice. MM and TM were stimulated with a single high-dose injection of Collagenase-type II (Col), repetitive low-dose Col injections, repetitive gentle MM stimulation, or single or repetitive forceful mouth opening. Referred pain was assessed by measuring mechanical hypersensitivity in the periorbital area (representing headache-like behavior) and another masticatory muscle. Stimulation of the MM, whether through single or repetitive Col injections or mouth opening, produced inconsistent, short-lasting (1-2 days) headache-like behavior in both males and females. In contrast, stimulation of the TM, using different paradigms, triggered mechanical hypersensitivity in both the MM and the periorbital area. Referred headache-like behavior lasted longer in females compared to males, while referred myalgia in the MM was pronouncer in males. The referred pain in the MM and periorbital areas triggered by TM stimulation was associated with significant gene plasticity in the MM and dura mater. Transcriptional changes in the MM following Col injection into the TM resembled those observed after direct MM injections. Presented data imply that referred pain modeled by TM stimulation could be accounted by nociceptive signaling from multiple local sites involved in this referred pain network.<\/jats:p>","DOI":"10.1101\/2025.03.26.645551","type":"posted-content","created":{"date-parts":[[2025,3,31]],"date-time":"2025-03-31T13:22:25Z","timestamp":1743427345000},"source":"Crossref","is-referenced-by-count":0,"title":["Modeling Masticatory Myalgia to Headache-like Referred Pain Triggers Local Gene Plasticity at Referred Pain Sites"],"prefix":"10.64898","author":[{"given":"Anahit H.","family":"Hovhannisyan","sequence":"first","affiliation":[{"name":"Center for Pain Therapeutics and Addiction Research, School of Dentistry, University of Texas Health San Antonio, Texas, 78229, USA"},{"name":"Department of Endodontics, School of Dentistry, University of Texas Health San Antonio, Texas, 78229, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jessica","family":"Aldape","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Physiology, School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer M.","family":"Mecklenburg","sequence":"additional","affiliation":[{"name":"Center for Pain Therapeutics and Addiction Research, School of Dentistry, University of Texas Health San Antonio, Texas, 78229, USA"},{"name":"Department of Endodontics, School of Dentistry, University of Texas Health San Antonio, Texas, 78229, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jessie","family":"Alfaro","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Physiology, School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229"},{"name":"Integrated Biomedical Sciences (IBMS) Program, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229 USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yi","family":"Zou","sequence":"additional","affiliation":[{"name":"Departments of Molecular Medicine, The Long School of Medicine, UTHSCSA, San Antonio, Texas 78229"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhao","family":"Lai","sequence":"additional","affiliation":[{"name":"Departments of Molecular Medicine, The Long School of Medicine, UTHSCSA, San Antonio, Texas 78229"},{"name":"Greehey Children\u2019s Cancer Research Institute, UTHSCSA, San Antonio, Texas 78229"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wei","family":"Guo","sequence":"additional","affiliation":[{"name":"Department of Neural and Pain Sciences, School of Dentistry, & Program in Neuroscience, University of Maryland, Baltimore, MD 21201, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiale","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Neural and Pain Sciences, School of Dentistry, & Program in Neuroscience, University of Maryland, Baltimore, MD 21201, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Malin","family":"Ernberg","sequence":"additional","affiliation":[{"name":"Division of Oral Rehabilitation, Department of Dental Medicine Karolinska Institutet, Huddinge, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M. 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