To investigate the role of TRPA1 on radicular pain behaviors, the TRPA1 antagonist HC-030031 (10, 25, and 50 μg, i.t.) was administered twice daily on postoperative days 3, 7, 10, and 14. PWMT was observed at baseline (before injection), 0.5, 1, 2, 4, and 6 hours after administration. The dosage of HC-030031 was selected based on a previous study and on our pilot experiments.9 I.t. administration of HC-030031 attenuated CCD-induced mechanical allodynia dose-dependently. There was no significant difference of PWMT in HC-030031 (10 μg, i.t.) group and HC-030031 (25 μg, i.t.) group compared with the DMSO group. The HC-030031 (50 μg, i.t.) group had significantly increased PWMT compared with DMSO groups at 0.5, 1, 2, and 4 hours after administration (P = 0.011 HC 50 μg, i.t. vs DMSO at 4 hours after administration, Fig. 4A; P = 0.008 HC 50 μg, i.t. vs DMSO at 4 hours after administration, Fig. 4B; P = 0.022 HC 50 μg, i.t. vs DMSO at 0.5 hours after administration, P = 0.014 HC 50 μg, i.t. vs DMSO at 4 hours after administration Fig. 4C; P = 0.028 HC 50 μg, i.t. vs DMSO at 4 hours after administration, Fig. 4D).
To investigate the effect of i.t. administration HC-030031 on TRPA1 and p-NR2B expression, Western blots were used to detect changes in TRPA1 and p-NR2B in the spinal cord. The lumbar enlargement was quickly dissected under deep anesthesia at 2 hours after i.t. administration of 50 μg HC-030031 on the seventh and 14th days after CCD. The protein level of TRPA1 is shown in Fig. 5, A and B. I.t. administration of 50 μg of HC-030031 significantly decreased the TRPA1 protein level in the spinal cord (P = 0.001 D7-HC 50 μg, i.t. vs D7-DMSO, P = 0.002 D14-HC 50 μg, i.t. vs D14-DMSO; Fig. 5, A and B).
Our study showed that the CCD induced significant mechanical allodynia and upregulation of TRPA1 and p-NR2B protein levels in the spinal cord. This neuropathic pain model induced by SURGIFLO™ could produce a different CCD from the metal rods used in previous rat models of CCD.23,24 This modification of CCD may closely mimic a subset of clinical radicular pain resulting from conditions such as foraminal stenosis and herniated intervertebral disk. Though it was not widely used in neuropathic pain studies, our experiments demonstrated that this model could induce obvious mechanical allodynia on the ipsilateral side after CCD. These findings were in accordance with a previous study by Gu et al.22
Our present study focused on investigation of spinal TRPA1 in neuropathic pain conditions caused by CCD. TRPA1 on peripheral terminals in transduction of noxious stimuli has been well studied,2,5,25–27 but the importance of spinal TRPA1 in modulation of hypersensitivity associated with neuropathic pain conditions still remains poorly understood.28 That the spinal TRPA1 channel contributes to central pain facilitation is supported by studies showing that spinal administration of TRPA1 antagonists could reduce mechanical hypersensitivity in various induced pain hypersensitivity conditions.15 Only secondary mechanical hypersensitivity was attenuated by spinal administration of a TRPA1 antagonist; primary mechanical hypersensitivity was not influenced by spinal administration of a TRPA1 antagonist. These results were found in various pain models, such as formalin-induced, capsaicin-induced, and mustard oil-induced pain models.19 A recent study demonstrated that TRPA1 is expressed on peripheral terminals and on central endings of primary afferent nociceptive nerve fibers within the spinal dorsal horn.28–30 In our present study, we demonstrated that CCD could induce upregulation of TRPA1 protein levels. These results are in accordance with our behavioral data, which indicate that TRPA1 may play an important role in CCD-induced neuropathic pain.
Our study demonstrated upregulation of p-NR2B protein levels in the spinal cord after CCD. Accumulating evidence has suggested that activation of the central glutaminergic system, especially N-methyl-D-aspartate receptor (NMDA) receptors, plays a central role in maintenance of neuropathic pain.31 Tyrosine phosphorylation of NR2B subunits is important for NMDA receptor activation and contributes to nociceptor activity-induced spinal plasticity and development of central sensitization.32–34 It has been reported that tyrosine phosphorylation of NR2B in the dorsal horn is involved in the development of neuropathic35 and inflammatory pain.32 These data and our study suggest that tyrosine phosphorylation of NR2B should be involved in hyperalgesia in the spinal dorsal horn.
In experiment 2, i.t. administration of HC-030031 dose-dependently attenuated CCD-induced hyperalgesia. The i.t. administration of 10 and 25 μg HC-030031 could not depress CCD-induced mechanical allodynia. Compared with a previous study in mice,9 these 2 doses may not be enough for i.t. injection to attenuate hyperalgesia. The i.t. administration of 50 μg HC-030031 downregulates expression TRPA1 and p-NR2B in the spinal cord. Because HC-030031 is a TRPA1 channel antagonist, decreased expression of spinal TRPA1 was not surprising. How HC-030031 modulates NMDA receptor activity is not as clear. Spinal TRPA1 could be active in various induced pain hypersensitivity conditions,15 and several studies demonstrated that the glutaminergic system is involved in transmission of the nociceptive stimulus induced for activation of the TRPA1 channel in the spinal cord.36 These results suggested that spinal TRPA1 participates in the enhancement of glutamatergic transmission of nociceptive signals leading to increased hypersensitivity. HC-030031, the TRPA1 channel antagonist, attenuates activity of NMDA receptors through downregulation of enhanced glutamatergic transmission. Spinal administration of a TRPA1 channel antagonist failed to attenuate mechanical pain hypersensitivity induced by direct chemical activation of NMDA.15 In cinnamaldehyde- (a TRPA1 agonist) induced pain hypersensitivity, the behavior was reduced by the coinjection (i.t.) of camphor (a TRPA1 antagonist) or MK-801 (a NMDA receptor antagonist).36 These results and our findings suggest that the effect of HC-030031 may be partially a result of depression of NMDA in the spinal cord, but the precise mechanism needs further investigation.
The present study demonstrated that CCD-induced hyperalgesia was associated with enhancement of TRPA1 and tyrosine phosphorylation of NR2B in the spinal cord. We determined that the TRPA1 antagonist HC-030031 attenuated hyperalgesia induced by CCD and upregulated TRPA1 and tyrosine phosphorylation of NR2B in the spinal cord. We provide evidence that the antihyperalgesic effect of HC-030031 may depend on its ability to modulate activation of spinal cord NMDA receptors. Our study suggests that the TRPA1 channel may be an effective novel option for treatment of neuropathic pain.
1. Patapoutian A, Tate S, Woolf CJ. Transient receptor potential channels: targeting pain at the source. Nat Rev Drug Discov. 2009;8:55–68
2. Moran MM, McAlexander MA, Bíró T, Szallasi A. Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov. 2011;10:601–20
3. Andersson DA, Gentry C, Moss S, Bevan S. Transient receptor potential A1 is a sensory receptor for multiple products of oxidative stress. J Neurosci. 2008;28:2485–94
4. Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus MJ, Earley TJ, Patapoutian A. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron. 2004;41:849–57
5. Jordt SE, Bautista DM, Chuang HH, McKemy DD, Zygmunt PM, Högestätt ED, Meng ID, Julius D. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature. 2004;427:260–5
6. Macpherson LJ, Xiao B, Kwan KY, Petrus MJ, Dubin AE, Hwang S, Cravatt B, Corey DP, Patapoutian A. An ion channel essential for sensing chemical damage. J Neurosci. 2007;27:11412–5
7. McNamara CR, Mandel-Brehm J, Bautista DM, Siemens J, Deranian KL, Zhao M, Hayward NJ, Chong JA, Julius D, Moran MM, Fanger CM. TRPA1 mediates formalin-induced pain. Proc Natl Acad Sci U S A. 2007;104:13525–30
8. Trevisani M, Siemens J, Materazzi S, Bautista DM, Nassini R, Campi B, Imamachi N, Andrè E, Patacchini R, Cottrell GS, Gatti R, Basbaum AI, Bunnett NW, Julius D, Geppetti P. 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1. Proc Natl Acad Sci U S A. 2007;104:13519–24
9. da Costa DS, Meotti FC, Andrade EL, Leal PC, Motta EM, Calixto JB. The involvement of the transient receptor potential A1 (TRPA1) in the maintenance of mechanical and cold hyperalgesia in persistent inflammation. Pain. 2010;148:431–7
10. Eid SR, Crown ED, Moore EL, Liang HA, Choong KC, Dima S, Henze DA, Kane SA, Urban MO. HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity. Mol Pain. 2008;4:48
11. Kerstein PC, del Camino D, Moran MM, Stucky CL. Pharmacological blockade of TRPA1 inhibits mechanical firing in nociceptors. Mol Pain. 2009;5:19
12. Kwan KY, Glazer JM, Corey DP, Rice FL, Stucky CL. TRPA1 modulates mechanotransduction in cutaneous sensory neurons. J Neurosci. 2009;29:4808–19
13. Petrus M, Peier AM, Bandell M, Hwang SW, Huynh T, Olney N, Jegla T, Patapoutian A. A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition. Mol Pain. 2007;3:40
14. Wei H, Hämäläinen MM, Saarnilehto M, Koivisto A, Pertovaara A. Attenuation of mechanical hypersensitivity by an antagonist of the TRPA1 ion channel in diabetic animals. Anesthesiology. 2009;111:147–54
15. Wei H, Koivisto A, Saarnilehto M, Chapman H, Kuokkanen K, Hao B, Huang JL, Wang YX, Pertovaara A. Spinal transient receptor potential ankyrin 1 channel contributes to central pain hypersensitivity in various pathophysiological conditions in the rat. Pain. 2011;152:582–91
16. Wei H, Karimaa M, Korjamo T, Koivisto A, Pertovaara A. Transient receptor potential ankyrin 1 ion channel contributes to guarding pain and mechanical hypersensitivity in a rat model of postoperative pain. Anesthesiology. 2012;117:137–48
17. Uta D, Furue H, Pickering AE, Rashid MH, Mizuguchi-Takase H, Katafuchi T, Imoto K, Yoshimura M. TRPA1-expressing primary afferents synapse with a morphologically identified subclass of substantia gelatinosa neurons in the adult rat spinal cord. Eur J Neurosci. 2010;31:1960–73
18. Wrigley PJ, Jeong HJ, Vaughan CW. Primary afferents with TRPM8 and TRPA1 profiles target distinct subpopulations of rat superficial dorsal horn neurones. Br J Pharmacol. 2009;157:371–80
19. Wei H, Chapman H, Saarnilehto M, Kuokkanen K, Koivisto A, Pertovaara A. Roles of cutaneous versus spinal TRPA1 channels in mechanical hypersensitivity in the diabetic or mustard oil-treated non-diabetic rat. Neuropharmacology. 2010;58:578–84
20. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain. 1983;16:109–10
21. Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav. 1976;17:1031–6
22. Gu X, Yang L, Wang S, Sung B, Lim G, Mao J, Zeng Q, Yang C, Mao J. A rat model of radicular pain induced by chronic compression of lumbar dorsal root ganglion with SURGIFLO. Anesthesiology. 2008;108:113–21
23. Hu SJ, Xing JL. An experimental model for chronic compression of dorsal root ganglion produced by intervertebral foramen stenosis in the rat. Pain. 1998;77:15–23
24. Song XJ, Hu SJ, Greenquist KW, Zhang JM, LaMotte RH. Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia. J Neurophysiol. 1999;82:3347–58
25. Bandell M, Macpherson LJ, Patapoutian A. From chills to chilis: mechanisms for thermosensation and chemesthesis via thermoTRPs. Curr Opin Neurobiol. 2007;17:490–7
26. Andrade EL, Luiz AP, Ferreira J, Calixto JB. Pronociceptive response elicited by TRPA1 receptor activation in mice. Neuroscience. 2008;152:511–20
27. Roberts K, Shenoy R, Anand P. A novel human volunteer pain model using contact heat evoked potentials (CHEP) following topical skin application of transient receptor potential agonists capsaicin, menthol and cinnamaldehyde. J Clin Neurosci. 2011;18:926–32
28. Pertovaara A, Koivisto A. TRPA1 ion channel in the spinal dorsal horn as a therapeutic target in central pain hypersensitivity and cutaneous neurogenic inflammation. Eur J Pharmacol. 2011;666:1–4
29. Kosugi M, Nakatsuka T, Fujita T, Kuroda Y, Kumamoto E. Activation of TRPA1 channel facilitates excitatory synaptic transmission in substantia gelatinosa neurons of the adult rat spinal cord. J Neurosci. 2007;27:4443–51
30. Kim YS, Son JY, Kim TH, Paik SK, Dai Y, Noguchi K, Ahn DK, Bae YC. Expression of transient receptor potential ankyrin 1 (TRPA1) in the rat trigeminal sensory afferents and spinal dorsal horn. J Comp Neurol. 2010;518:687–98
31. Zhang W, Shi CX, Gu XP, Ma ZL, Zhu W. Ifenprodil induced antinociception and decreased the expression of NR2B subunits in the dorsal horn after chronic dorsal root ganglia compression in rats. Anesth Analg. 2009;108:1015–20
32. Guo W, Zou S, Guan Y, Ikeda T, Tal M, Dubner R, Ren K. Tyrosine phosphorylation of the NR2B subunit of the NMDA receptor in the spinal cord during the development and maintenance of inflammatory hyperalgesia. J Neurosci. 2002;22:6208–17
33. Yang Q, Liao ZH, Xiao YX, Lin QS, Zhu YS, Li ST. Hippocampal synaptic metaplasticity requires the activation of NR2B-containing NMDA receptors. Brain Res Bull. 2011;84:137–43
34. Jung SC, Eun SY, Kim J, Hoffman DA. Kv4.2 block of long-term potentiation is partially dependent on synaptic NMDA receptor remodeling. Brain Res Bull. 2011;84:17–21
35. Guo W, Wei F, Zou S, Robbins MT, Sugiyo S, Ikeda T, Tu JC, Worley PF, Dubner R, Ren K. Group I metabotropic glutamate receptor NMDA receptor coupling and signaling cascade mediate spinal dorsal horn NMDA receptor 2B tyrosine phosphorylation associated with inflammatory hyperalgesia. J Neurosci. 2004;24:9161–73
36. Klafke JZ, da Silva MA, Trevisan G, Rossato MF, da Silva CR, Guerra GP, Villarinho JG, Rigo FK, Dalmolin GD, Gomez MV, Rubin MA, Ferreira J. Involvement of the glutamatergic system in the nociception induced intrathecally for a TRPA1 agonist in rats. Neuroscience. 2012;222:136–46