SUMMARY For a long time, it has been known that there is a strong link between chronic pain and high levels of stress and anxiety. High levels of stress are a risk factor for developing chronic post-surgical pain. It remains unclear if and how stress can facilitate the transition from acute to chronic pain. In the periphery, pain is perceived by primary sensory neurons, whose cell bodies reside in the dorsal root ganglia, where each sensory neuron is enveloped by satellite glial cells, a non-neuronal support cell. Previous studies show that peripheral injury as well as stress may induce satellite glial cell (SGC) activation, a phenomenon characterized by increased expression of glial fibrillary acidic protein, changes in gene expression, and release of pro-nociceptive signaling molecules. SGC activation has been linked to the development of mechanical hyperalgesia and primary afferent sensitization in a variety of rodent models. Our objective is to characterize the how stress and/or peripheral injury-induced satellite glial cell activation may modulate primary afferent sensitization and lead to the development of prolonged ischemic muscle pain. To explore this, the proposed research will use novel mouse model of stress based on loss of environmental enrichment (LEE). This model does not employ the potential confounder of noxious physical stimulation. Our preliminary data shows that LEE induced stress results in mechanical hyperalgesia before injury and increased pain-related behaviors after an ischemia with reperfusion injury (I/R). We hypothesize that LEE+(I/R)-induced satellite glial cell activation induces peripheral sensitization via increased expression and release of pro-nociceptive signaling molecules that may modulate the development of chronic pain after injury. We will use RNA sequencing, bioinformatics, animal behavior and ex-vivo electrophysiology to characterize how SGC modulate pain perception after I/R in the context of stress. Aim 1 Characterize the transcriptomic changes in I/R induced SGC activation and the signaling molecules that mediate muscle afferent sensitivity in the presence or absence of stress. Transcriptomic changes in activated satellite glial cells after injury, stress, or a combination of both have never been thoroughly characterized. The proposed RNAseq experiments and bioinformatics analysis on these data will allow us to better characterize how gene expression changes in SGCs mediates pain development. Aim 2 will explore how SGC activation impacts the function of muscle primary sensory neurons. We will use a combination of behavior, ex-vivo electrophysiology, and calcium imaging to determine the changes in muscle primary afferent function that are mediated by SGC activation after stress and/or injury. The data from these studies will become the cornerstone of a research program focused on how stress facilitates the transition from acute to chronic pain and the specific role that SGCs play in this process and wil...