**Study Title:** Investigation of the Relationship Between Clinical Outcomes and Pain Mediators in the Treatment of Masticatory Muscle Disorders Associated with Myospasm Using Onabotulinum Toxin A

**Study Importance:** Temporomandibular disorders (TMD) are a major cause of chronic orofacial pain, affecting 5-12% of the population. Masticatory muscle disorders (MMD) are a common subgroup of TMD, ranging from localized myalgia to fibromyalgia. Myospasm is characterized by sudden pain, malocclusion, and limited jaw movement, while myalgia includes localized, myofascial, and referred pain patterns. The etiology of MMD is complex, involving neuromuscular dysfunction, inflammation, and increased acetylcholine activity at the neuromuscular junction. Various mediators, including CGRP, substance P, and inflammatory cytokines, play a role in sensitization and pain perception.

**Objective:** This study aims to evaluate the effectiveness of onabotulinum toxin A (BTX-A) in patients with MMD who have not responded to conventional non-invasive treatments. This exploratory study investigates whether BTX-A is associated with reductions in pain and inflammatory cytokines and neuropeptides.

**Methodology:**

• **Study Design:** Prospective observational clinical study.
• **Participants:** Patients diagnosed with MMD based on DC/TMD criteria, who have not improved with conventional treatments.
• **Exclusion Criteria:**1) pregnancy or lactation; 2) use of oral contraceptives; 3) history of radiotherapy, active chemotherapy, or trauma in the maxillofacial region; 4)uncontrolled metabolic or systemic diseases; 5)active infections; allergic tendencies; significant tooth loss; 6) rheumatic diseases or other TMJ-defined disorders; 7) use of antidepressants or anti-inflammatory agents within the past week; 8) neuromuscular disorders (e.g., myasthenia gravis, Eaton-Lambert syndrome); 9) planned surgical procedures in the near future, 10) individuals undergoing concomitant therapies.
• **Intervention:** BTX-A will be injected into the masseter and temporalis muscles (30 and 15 units per side, respectively) following a standardized protocol.
• **Data Collection:**
• Before (T0) and 28 days after (T1) treatment.
• Clinical assessments include maximum mouth opening (MMO), pain levels (VAS), pain pressure threshold and oral health impact profile (OHIP-14).
• Blood and saliva samples will be analyzed for IL-1, IL-6, TNF-α, CGRP, and NGF using ELISA.
• **Statistical Analysis:** Dependent t-test or Wilcoxon signed-rank test will be used to compare pre- and post-treatment values. Correlations between biomarker levels and pain reduction will be analyzed using Spearman correlation.

**Expected Outcomes:**

• Significant reduction in pain and improvement in MMO.
• Decreased levels of inflammatory and neuropeptide biomarkers.
• Evaluation of saliva as a non-invasive medium for biomarker analysis, potentially guiding future diagnostic and monitoring strategies.

**Significance:** This study provides insights into the pathophysiology of MMD and the efficacy of BTX-A in pain management, potentially offering an alternative therapeutic approach for patients resistant to conventional treatments.

Inclusion Criteria: Patients diagnosed with MMD based on RDC/TMD criteria, who have not improved with conventional treatments.

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Exclusion Criteria:Conditions such as pregnancy, metabolic disorders, trauma, systemic diseases, and medication use that could interfere with results

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