P2X3-mediated peripheral sensitization of neuropathic pain in resiniferatoxin-induced neuropathy Yu-Lin Hsieh a, b , Hao Chiang a , June-Horng Lue a, ⁎, Sung-Tsang Hsieh a, c, d, ⁎⁎ a Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan b Department of Anatomy, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan c Department of Neurology, National Taiwan University Hospital, Taipei 10002, Taiwan d Graduate Institute of Brain and Mind Science, College of Medicine, National Taiwan University, Taipei 10051, Taiwan abstract article info Article history: Received 17 December 2011 Revised 10 February 2012 Accepted 20 February 2012 Available online 28 February 2012 Keywords: Purinergic receptor P2X3 Mechanical allodynia Peripheral sensitization Small-fiber neuropathy Resiniferatoxin Calcitonin gene-related peptide Activating transcription factor-3 Patients suffering from sensory neuropathy due to skin denervation frequently have paradoxical manifes- tations of reduced nociception and neuropathic pain. However, there is a lack of satisfactory animal models to investigate these phenomena and underlying mechanisms. We developed a mouse system of neuropa- thy induced by resiniferatoxin (RTX), a capsaicin analog, and examined the functional significance of P2X3 receptor in neuropathic pain. From day 7 of RTX neuropathy, mice displayed mechanical allodynia (p b 0.0001) and thermal hypoalgesia (p b 0.0001). After RTX treatment, dorsal root ganglion (DRG) neurons of the peripherin type were depleted (p = 0.012), while neurofilament (+) DRG neurons were not affected (p = 0.62). In addition, RTX caused a shift in neuronal profiles of DRG: (1) increased in P2X3 receptor (p = 0.0002) and ATF3 (p = 0.0006) but (2) reduced TRPV1 (p = 0.036) and CGRP (p = 0.015). The number of P2X3(+)/ATF3(+) neurons was linearly correlated with mechanical thresholds (p = 0.0017). The peripheral expression of P2X3 receptor in dermal nerves was accordingly increased (p = 0.016), and an intraplantar injection of the P2X3 antagonists, A-317491 and TNP–ATP, relieved mechanical allodynia in a dose-dependent manner. In conclusion, RTX-induced sensory neuropathy with upregulation of P2X3 re- ceptor for peripheral sensitization of mechanical allodynia, which provides a new therapeutic target for neuropathic pain after skin denervation. Crown Copyright © 2012 Published by Elsevier Inc. All rights reserved. Introduction Neuropathic pain develops after peripheral nerve degeneration, which presumably reduces nociceptive input. In human peripheral nerve diseases affecting small-diameter nociceptive nerves, some pa- tients could have paradoxical symptoms: neuropathic pain but with reduced sensitivities to noxious stimuli due to skin denervation (Baron et al., 2009; Cheng et al., 2009; Obrosova, 2009; Polydefkis et al., 2004). Similar phenomena are observed after capsaicin- induced skin denervation (Gibbons et al., 2010; Rage et al., 2010). Pe- ripheral sensory nerves are axonal extensions of dorsal root ganglion (DRG) neurons which consist of large-diameter and small-diameter neurons with corresponding sizes of nerve fibers. It is, however, not clear whether pure injury to small neurons could result in such a par- adoxical combination of symptoms: reduced nociception and neuro- pathic pain. There is also a lack of experimental systems to test this hypothesis. We previously established a neuropathy system induced by resiniferatoxin (RTX), a capsaicin analog. Skin denervation with degeneration of unmyelinated nerves is the predominant feature of RTX-induced neuropathy (Hsieh et al., 2008), which can serve as a prototype of pure small-fiber neuropathy and provide a good oppor- tunity to examine underlying molecular mechanisms of clinical pre- sentations: neuropathic pain and reduced nociception. Nerve injury causes a cascade of responses in neuronal cell bodies, for example, the upregulation of transcription factors, which leads to the generation of effector molecules responsible for maladaptive be- haviors of neuropathic pain. Activating transcription factor-3 (ATF3) is a member of the ATF/CREB transcription factor superfamily (Hai and Hartman, 2001) and upregulated in DRG neurons after nerve in- jury, for example, spinal nerve ligation (Fukuoka et al., 2012). These findings raise the possibility of exploring whether or not ATF3 is upregulated in RTX-induced neuropathy, which only selectively de- pletes cutaneous nerves (Avelino and Cruz, 2000; Hsieh et al., 2008; Neubert et al., 2003). If so, will the pattern of ATF3 expression in Experimental Neurology 235 (2012) 316–325 Abbreviations: ATF3, Activating transcription factor-3; CGRP, Calcitonin gene- related peptide; DRG, Dorsal root ganglion; RTX, Resiniferatoxin; TRPV1, Transient re- ceptor potential vanilloid subtype 1. ⁎ Correspondence to: J.-H. Lue, Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Rm. 609, 1 Jen-Ri Road, Sec. 1, Taipei 10051, Taiwan. Fax: + 886 2 23915292. ⁎⁎ Correspondence to: S.-T. Hsieh, Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Rm. 638, 1 Jen-Ri Road, Sec. 1, Taipei 10051, Taiwan. Fax: + 886 2 23915292. E-mail addresses: thomas@ntu.edu.tw (J.-H. Lue), shsieh@ntu.edu.tw (S.-T. Hsieh). 0014-4886/$ – see front matter. Crown Copyright © 2012 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2012.02.013 Contents lists available at SciVerse ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr