Approximately 12% of the US population have temporomandibular disorders (TMD) and are predominately women in their childbearing years. Although greater than 60% of those affected by TMD have jaw muscle pain as their main complaint, research has not provided much insight into the biological basis of the persistent muscle pain found in these patients. In the past decade, the role of glial cells has gained attention for their role in neuron-glia signaling and in modulating persistent pain. However, there are only a few studies of glial cell involvement in orofacial pain and these have focused on trigeminal nerve injury and on neuropathic pain or odontogenic pain. We have developed a model in the mouse using repetitive acidic saline injections into the masseter muscle to cause persistent jaw muscle pain without significant damage to the muscle. In preliminary data, we present evidence obtained with this model in support of a glial role in mediating and/or maintaining persistent jaw muscle pain. Based on this data we propose the following hypothesis: Glial cell activation after a unilateral repetitive acidic saline injection into the masseter muscle is part of a cascade of events that elicit gender-specific, bilateral primary afferent neuron neuroplastic changes in the trigeminal system and result in persistent jaw muscle pain. To test this hypothesis, we propose three specific aims. (1) Correlate the temporal and spatial activation of glia within the trigeminal ganglion and sensory nuclear complex with neuroplastic changes in neuropeptide expression in the trigeminal ganglia and with behavioral measures of jaw muscle pain;(2). Determine the effects of minocycline and propentofylline on the activation of microglia and astrocytes within the trigeminal sensory nuclear complex, on neuroplastic changes in neuropeptide expression in the trigeminal ganglia and on behavioral measures of jaw muscle pain;and (3) Correlate the temporal and spatial expression of pro-inflammatory cytokines and metalloproteinases within the trigeminal sensory nuclear complex with glial activation, neuroplastic changes in neuropeptide expression in the trigeminal ganglia and jaw muscle pain. The results of these studies should provide new data on trigeminal primary afferent and segmental cellular responses to mild, repetitive pain stimuli which induce a persistent jaw muscle pain and on gender-based differences in these responses. The research proposed in this R21 application will provide a foundation for future investigations into the role of glial cells in masticatory muscle pain. Future studies will include investigation into the basis for gender differences, mechanisms of glial cell activation after masticatory muscle injury and testing of potential targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Although greater than 60% of those affected by temporomandibular disorders have jaw muscle pain as their main complaint, research has not provided much insight into the biological basis of the persistent muscle pain found in these patients. The research proposed in this application will examine the role of glia cells in causing and maintaining jaw muscle pain in an animal model. The knowledge gained from this research could lead to the identification of new potential therapeutic targets in the treatment of jaw muscle pain.