Chronic Stress Induces Neuroimmune Modulated Primary Muscle Afferent Sensitization

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SUMMARY Many patients with chronic pain concomitantly present with increased levels of stress and anxiety. Multiple reports point to the immune system as a potential link between stress and chronic pain development. Stress activates immune cells and increases pro-inflammatory cytokines, which can modulate peripheral sensitization and regulate prolonged hypersensitivity through gene expression changes in primary sensory neurons. Our objective is to characterize the stress-induced neuroimmune interactions that modulate primary afferent sensitization and lead to the development of prolonged ischemic muscle pain. Macrophages are one of the key immune cells involved in these neuroimmune interactions that regulate peripheral sensitization, but the role of macrophages in development of stress-induced muscle hyperalgesia and hypersensitivity remains unknown. This proposal explores effects of stress in the peripheral nervous system in a new way -- selectively inhibiting or enhancing macrophage -mediated inflammatory responses to stress and testing its role in the development muscle pain-related behaviors. It will use and validate a never before used, loss of environmental enrichment (LOE) mouse model of stress that does not employ the potential confounder of noxious physical stimulation. Interestingly, this paradigm replicates a stressor frequently experienced during the COVID-19 pandemic - LOE due to social distancing. Our preliminary data showed gene expression changes in the dorsal root ganglia (DRG) and innervated tissue modulated response properties of muscle afferents after ischemic injury (I/R) modulated, in part, by proinflammatory cytokine signaling. LOE induced stress resulted in increased pain- related behaviors after injury and increased macrophage infiltration in skeletal muscle. We hypothesize that LOE-induced stress induces peripheral sensitization via immune cell-dependent mechanisms to modulate the development of ischemic myalgia. We will use transgenic and chemogenetic approaches in mice with LOE-related stress plus ischemic/reperfusion (I/R) injury, our ex vivo hind paw muscle afferent recording strategies and pain-related behavioral assays. Aim 1 will identify if stress modulates peripheral sensitization after injury (I/R) through macrophage infiltration by depleting macrophages in macrophage fas- induced apoptosis (MaFIA) mice exposed to LOE with or without I/R. Electrophysiological experiments will be paired with detailed profiling of the transcriptome of the DRG under stress. Aim 2 will evaluate the role of stress- induced immune alterations in peripheral sensitization after I/R by using transgenic mice expressing designer receptors exclusively activated by designer drugs (DREADDs) driven by Cre-recombinase expression from the Lysozyme-2 promotor (LysM, only expressed myelomonocytic cells, such as macrophages).Characterization of the mechanisms of stress-induced neuroimmune interactions that modulate primary afferent sensitization will elucidate the role of macrophages in development of stress-induced muscle hyperalgesia and hypersensitivity.