However, as indicated by studies with macrophage depletion, CCL2 requires the contribution of infiltrated macrophages within the hurt nerve trunk to sustain the allodynia in a prolonged model of neuropathic pain, such as the pSNL in mice. of ensheathed nociceptors to sustain mechanical allodynia. Introduction Neuropathic pain, which is usually defined as pain caused by a lesion or disease of the somatosensory nervous system1, encompasses a large variety of conditions2. Lesions of the peripheral nervous system can cause lifelong neuropathic pain. Following peripheral nerve injury, local infiltration of inflammatory cells, a hallmark of Wallerian degeneration, occurs3C5, Estradiol dipropionate (17-Beta-Estradiol-3,17-Dipropionate) and is associated with the development of neuropathic pain. Even though infiltration of macrophages into the damaged nerve trunk is known to induce mechanical allodynia in mice with sciatic nerve injury6C9, the precise pathway by which inflammatory cells cause persistent allodynia is only partially defined. A series of mediators have been reported to contribute to macrophage infiltration in the damaged nerve10. Notably, inhibition of the chemokine (CCC motif) ligand 2 (CCL2) has been shown to attenuate neuroinflammation and allodynia7,8,11. Oxidative stress contributes to neuropathic pain, since antioxidants attenuate mechanical hypersensitivity in mouse models, including chronic constriction of the sciatic nerve12 and spinal nerve ligation13. The transient receptor potential Estradiol dipropionate (17-Beta-Estradiol-3,17-Dipropionate) ankyrin 1 (TRPA1) channel is highly expressed by a subpopulation of main sensory neurons14,15 that contain and release the proinflammatory neuropeptides material P (SP) and calcitonin gene-related peptide (CGRP)15. TRPA1 is usually activated by a series of exogenous brokers, including allyl isothiocyanate (AITC)16,17, and is typically sensitive to the redox state of the milieu18. Notably, a series of reactive oxygen, nitrogen or carbonyl species, including hydrogen peroxide (H2O2), activate TRPA1, resulting in nociceptor activation or sensitization19C24. TRPA1 has been shown to mediate mechanical hypersensitivity in different models of inflammatory and neuropathic pain, including those evoked by peripheral nerve injury25C29. Recent findings in mice with trigeminal nerve injury (constriction of the infraorbital nerve, CION) show that macrophages, recruited by a CCL2-dependent process, increase H2O2 levels within the site of nerve injury30. The producing oxidative stress and the ensuing increases in reactive carbonyl species were proposed to mediate prolonged mechanical allodynia by gating TRPA1 Estradiol dipropionate (17-Beta-Estradiol-3,17-Dipropionate) in trigeminal nerve fibers30. Thus, TRPA1, expressed by main sensory neurons, appears to be the target of the macrophage-dependent oxidative burst required to promote neuropathic pain. Here, we surprisingly found that pharmacological blockade or genetic deletion of TRPA1 not only induced the expected inhibition of mechanical allodynia, but also suppressed macrophage infiltration and H2O2 generation in the hurt nerve. The current study was undertaken to identify the cellular and RAF1 molecular mechanisms responsible for this TRPA1-mediated macrophage infiltration and generation of oxidative stress. By using pharmacological and genetic approaches to disrupt TRPA1, including conditional deletion in Schwann cells, we found that Schwann cells that ensheath the hurt sciatic nerve axons express TRPA1. Macrophages, which are recruited by CCL2, generate a NADPH oxidase-2 (NOX2)-dependent oxidative burst that targets Schwann cell TRPA1. TRPA1, via NOX1, produces sustained oxidative stress that maintains, in a spatially confined manner, macrophage infiltration into the hurt nerve, and which activates TRPA1 on nociceptor nerve fibers to produce allodynia. Results TRPA1 mediates neuroinflammation In C57BL/6 mice pSNL, but not sham surgery (Fig.?1a), induced prolonged (3C20 days) mechanical allodynia (Fig.?1b) accompanied by macrophage (F4/80+ cells) recruitment (Fig.?1c, e and Supplementary Fig.?1) and oxidative stress (H2O2) generation (Fig.?1d) within the injured nerve. (Fig.?1f), but not or (Supplementary Fig.?2a), deletion prevented mechanical allodynia. or deletion (Supplementary Fig.?2c). As previously reported28,30,31 in comparable models, at day 10 after pSNL (all.