Stitutes of Overall health, Bethesda, USA) and normalized to -actin24. The uncropped scan from the blot is reported inside the Supplementary Fig. 7. Live animal imaging. Macrophage localization in vivo was obtained by NIR imaging from the fluorescent label macrophage mice by using PhotonImager (Biospace Laboratory, Paris, France)83. Mouse thioglycollate-elicited peritoneal macrophages had been harvested (as much as 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, 10 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 Methylene blue Autophagy following surgery. Twenty-four h later,NATURE COMMUNICATIONS | 8:| DOI: ten.1038s41467-017-01739-2 | www.nature.comnaturecommunicationsNATURE COMMUNICATIONS | DOI: ten.1038s41467-017-01739-ARTICLE21. Taylor-Clark, T. E., Ghatta, S., Bettner, W. Undem, B. J. Nitrooleic acid, an endogenous product of nitrative tension, activates nociceptive sensory nerves by means of the direct activation of TRPA1. Mol. Pharmacol. 75, 82029 (2009). 22. Trevisani, M. et al. 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation via activation on the 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 just after 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 four, e7383 (2009). 28. Eid, S. R. et al. HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and Busulfan-D8 web neuropathy-induced mechanical hypersensitivity. Mol. Discomfort four, 48 (2008). 29. McNamara, C. R. et al. TRPA1 mediates formalin-induced pain. Proc. Natl Acad. Sci. USA 104, 135253530 (2007). 30. Trevisan, G. et al. TRPA1 mediates trigeminal neuropathic discomfort in mice downstream of monocytesmacrophages and oxidative pressure. Brain 139, 1361377 (2016). 31. Kim, H. K. et al. Reactive oxygen species (ROS) play an important part within 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 as a result 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 One particular 8, 2013 (2013). 36. Szolcsanyi, J., Szallasi, A., Szallasi, Z., Joo, F. Blumberg, P. M. Resiniferatoxin: an ultrapotent selective modulator of capsaicin-sensitive primary afferent neurons. J. Pharmacol. Exp. Ther. 255, 923.
