Project Summary Persistent pain secondary to tissue and nerve injury involves key roles for DRG neurons, macrophages, and spinal microglia. Therapies targeting single components of this tripartite pain axis have not proven clinically efficacious, suggesting a strategy is needed that addresses all three components in concert. We have exciting new findings that suggest one such therapeutic strategy may be to target the subset of cellular membrane cholesterol rich lipid rafts that contain Toll-like receptor 4 (TLR4-rafts). Membrane lipid rafts are a fundamental organizational nidus in all cells for numerous membrane channels, receptors and enzymes that regulate reactivity and excitability. The TLR4-raft subtype is special as these are specifically localized in the three key cellular elements regulating the excitability of nociceptive signaling. Following tissue inflammation or nerve injury, TLR4rafts transform from small diffuse labile membrane structures into markedly enlarged and persistent complexes supporting the formation of homo and hetero dimers of several excitatory channels and receptors, underpinning the transition from an acute to a persistent pain phenotype and to establishing a “primed state” after an initial system activation. Specific disruption of these neuraxial TLR4-rafts has profound effects upon injury induced pain behavior. While we have gained insight into mechanisms by which TLR4-rafts regulate microglial function, we know little about the mechanisms by which TLR4-rafts regulate excitability of DRG neurons and macrophages. To address this knowledge gap, we propose three specific aims: i) Define time dependent changes in TLR4-rafts and pain behavior induced by trauma, inflammatory and neuropathic pain, and the effects of targeted TLR4-raft disruption on pain behavior in these models; ii) Determine baseline molecular architecture of DRG neuronal and macrophage TLR4-rafts and then how this architecture and function is altered with tissue trauma, inflammation and nerve injury; and iii) Define role of DRG neuronal TLR4-raft localized receptors in regulating excitatory phenotype in mouse, rat and human DRG neurons and effects of TLR4-raft disruption in modifying this excitatory phenotype. This is a large multi-PI / multi-center study that defines mechanisms by which neuraxial TLR4-rafts affect multiple pain-related signaling processes, integrated pain behavior and the molecular anatomy underpinning this signaling and the degree to which these mechanisms are conserved between rodents and humans, across differing pain phenotypes. This work will have a sustained impact on understanding the neurobiology of a largely unexplored cellular element regulating post injury afferent excitability. In sum, this proposal centers on the unifying thesis that neuraxial TLR4-rafts are a common facilitatory element of the persistent pain facilitated states observed following tissue and nerve injury and that this effect is mediated by TLR4 rafts not onl...