Stitutes of Health, Bethesda, USA) and normalized to -actin24. The uncropped scan on the blot is reported within the Supplementary Fig. 7. Reside animal imaging. Macrophage localization in vivo was obtained by NIR imaging with the fluorescent label macrophage mice by utilizing PhotonImager (Biospace Laboratory, Paris, France)83. Mouse thioglycollate-elicited peritoneal macrophages had been harvested (up to 250 106 cells per ml) and incubated for 15 min at RT with VivoTrack 680 (PerkinElmer, Inc., Waltham, USA), dissolved in sterile PBS, washed, centrifuged (400 g, ten min) and diluted to a final concentration of 5 106 cells 40 l-1. Retro-orbital vein injection (40 l) of labeled macrophages was performed in pSNLsham C57BL6 mice at day 9 just after surgery. Twenty-four h later,NATURE COMMUNICATIONS | eight:| DOI: ten.1038s41467-017-01739-2 | www.nature.comnaturecommunicationsNATURE COMMUNICATIONS | DOI: 10.1038s41467-017-01739-ARTICLE21. Taylor-Clark, T. E., Ghatta, S., Bettner, W. Undem, B. J. Nitrooleic acid, an endogenous solution of nitrative pressure, activates nociceptive sensory nerves by means of the direct activation of TRPA1. Mol. Pharmacol. 75, 82029 (2009). 22. Trevisani, M. et al. Cangrelor (tetrasodium) custom synthesis 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation by way of activation of your irritant receptor TRPA1. Proc. Natl Acad. Sci. USA 104, 135193524 (2007). 23. Bautista, D. M. et al. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 124, 1269282 (2006). 24. Trevisan, G. et al. Novel therapeutic method to prevent chemotherapy-induced persistent sensory neuropathy by TRPA1 blockade. Cancer Res. 73, 3120131 (2013). 25. Obata, K. et al. TRPA1 induced in sensory neurons contributes to cold hyperalgesia just after inflammation and nerve injury. J. Clin. Invest. 115, 2393401 (2005). 26. Katsura, H. et al. Antisense knock down of TRPA1, but not TRPM8, alleviates cold hyperalgesia following spinal nerve ligation in rats. Exp. Neurol. 200, 11223 (2006). 27. Caspani, O., Zurborg, S., Labuz, D. Heppenstall, P. A. The contribution of TRPM8 and TRPA1 channels to cold allodynia and neuropathic discomfort. PLoS One particular 4, e7383 (2009). 28. Eid, S. R. et al. HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity. Mol. Discomfort four, 48 (2008). 29. McNamara, C. R. et al. TRPA1 mediates formalin-induced discomfort. Proc. Natl Acad. Sci. USA 104, 135253530 (2007). 30. Trevisan, G. et al. TRPA1 mediates trigeminal neuropathic pain in mice downstream of monocytesmacrophages and oxidative strain. Brain 139, 1361377 (2016). 31. Kim, H. K. et al. Reactive oxygen species (ROS) play a vital part in a rat model of neuropathic discomfort. Pain 111, 11624 (2004). 32. Liu, T., van Rooijen, N. Tracey, D. J. Depletion of macrophages reduces axonal degeneration and hyperalgesia following nerve injury. Discomfort 86, 252 (2000). 33. Zhu, X., Fujita, M., Snyder, L. A. Okada, H. Systemic delivery of neutralizing antibody targeting CCL2 for glioma therapy. J. Neurooncol. 104, 832 (2011). 34. Perkins, N. M. Tracey, D. J. Hyperalgesia 5-Methoxy-2-benzimidazolethiol Purity & Documentation because of nerve injury: function of neutrophils. Neuroscience 101, 74557 (2000). 35. Hackel, D. et al. The connection of monocytes and reactive oxygen species in pain. PLoS A single eight, 2013 (2013). 36. Szolcsanyi, J., Szallasi, A., Szallasi, Z., Joo, F. Blumberg, P. M. Resiniferatoxin: an ultrapotent selective modulator of capsaicin-sensitive key afferent neurons. J. Pharmacol. Exp. Ther. 255, 923.
