Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Behavioral phenotypes of mice lacking purinergic P2X4 receptors in acute and chronic pain assays

Behavioral phenotypes of mice lacking purinergic P2X4 receptors in acute and chronic pain assays A growing body of evidence indicates that P2X receptors (P2XRs), a family of ligand-gated cation channels activated by extracellular ATP, play an important role in pain signaling. In contrast to the role of the P2X R subtype that has been extensively studied, the precise roles of others among the seven P2XR subtypes (P2X R-P2X R) remain to be determined because of a lack of sufficiently 1 7 powerful tools to specifically block P2XR signaling in vivo. In the present study, we investigated the behavioral phenotypes of a line of mice in which the p2rx4 gene was disrupted in a series of acute -/- and chronic pain assays. While p2rx4 mice showed no major defects in pain responses evoked by acute noxious stimuli and local tissue damage or in motor function as compared with wild-type mice, these mice displayed reduced pain responses in two models of chronic pain (inflammatory and neuropathic pain). In a model of chronic inflammatory pain developed by intraplantar injection -/- of complete Freund's adjuvant (CFA), p2rx4 mice exhibited attenuations of pain hypersensitivity to innocuous mechanical stimuli (tactile allodynia) and also of the CFA-induced swelling of the hindpaw. A most striking phenotype was observed in a test of neuropathic pain: tactile allodynia -/- caused by an injury to spinal nerve was markedly blunted in p2rx4 mice. By contrast, pain hypersensitivity to a cold stimulus (cold allodynia) after the injury was comparable in wild-type and -/- p2rx4 mice. Together, these findings reveal a predominant contribution of P2X R to nerve injury- induced tactile allodynia and, to the lesser extent, peripheral inflammation. Loss of P2X R produced no defects in acute physiological pain or tissue damaged-induced pain, highlighting the possibility of a therapeutic benefit of blocking P2X R in the treatment of chronic pain, especially tactile allodynia after nerve injury. have important roles in regulating neuronal and glial Findings The purinergic P2X receptors, of which seven subtypes functions in the nervous system under physiological and –P2X ) have been cloned, are a family of ligand- (P2X pathological conditions [1-3]. There has been much 1 7 gated cation channels activated by extracellular ATP. They recent attention paid to their roles in generating and mod- Page 1 of 7 (page number not for citation purposes) Molecular Pain 2009, 5:28 http://www.molecularpain.com/content/5/1/28 ulating pain signaling [1-3]. Various attempts to block Next, to investigate the role of P2X R in pain hypersensi- P2XRs pharmacologically or to suppress their expression tivity under chronic pain conditions, we employed two molecularly and genetically have demonstrated that these distinct models of chronic pain, namely inflammatory receptors make a major contribution to pain responses and neuropathic pain. Wild-type mice with peripheral evoked by tissue damage, chronic peripheral inflamma- inflammation induced by intraplantar injection of com- tion and nerve injury [4-6]. While the role of P2X R (and plete Freund's adjuvant (CFA), used as a model of inflam- P2X R) has been studied extensively, recent evidence matory pain, displayed decreased paw withdrawal 2+3 from our and other studies indicates that the P2X R sub- thresholds in their ipsilateral hindpaws (Fig. 2A). By con- type critically contributes to neuropathic pain, a highly trast, the peripheral inflammation-induced decrease in debilitating pain condition that commonly occurs after paw withdrawal threshold was significantly smaller in -/- nerve damage [7-13]. However, the precise role of P2X R p2rx4 mice than wild-type mice. The paw withdrawal in neuropathic pain remains to be fully determined thresholds for the contralateral hindpaws in both geno- because, in stark contrast to P2X R [14-16] and P2X R types of mice did not change (Fig. 2A). In wild-type mice, 3 7 [17,18], there is a lack of sufficiently powerful pharmaco- a marked swelling of the ipsilateral hindpaw was R signal- logical and genetic tools to selectively block P2X observed, and the weight of the hindpaw was increased on -/- ing in vivo. Recently, several groups have independently day 14 (Fig. 2B). In p2rx4 mice, however, the CFA- developed lines of mice in which the p2rx4 gene is dis- induced increase in the weight of the ipsilateral hindpaw rupted [19-21]. It is of particular importance to study the was significantly suppressed (p < 0.05, Fig. 2B). phenotypes of these mice to determine the in vivo func- tions of P2X R in pain signaling [22]. In the present study, To determine the role of P2X R in tactile allodynia under 4 4 we sought to characterize behavioral phenotypes in a wide neuropathic pain conditions, we injured the forth lumbar -/- range of assays of acute and chronic pain, including neu- spinal nerves of wild-type and p2rx4 mice; this approach ropathic pain, using a line of P2X R-deficient mice [19]. has been used to generate an animal model of neuro- pathic pain. While wild-type mice showed a decrease in To examine acute physiological pain responses, the with- paw withdrawal threshold after nerve injury (Fig. 3A), this -/- drawal responses from a noxious range of heat and effect was markedly blunted in p2rx4 mice. The attenu- ated tactile allodynia was observed until the last time mechanical stimuli applied by von Frey filaments were -/- measured in both wild-type and p2rx4 mice. In tail- and point tested (day 1, p < 0.05; day 3, 7, 10 and 14, p < paw-flick tests, the latencies for animals to flick their tails 0.001; Fig. 3A). The loss of P2X R did not change the paw and hindpaws away from radiant heat at either 30 V or 50 withdrawal threshold of the contralateral hindpaw after -/- V were not different between wild-type and p2rx4 mice nerve injury (Fig. 3A) as seen in wild-type mice (except on -/- (Fig. 1A, B). In a test of mechanical pain, p2rx4 mice day 1, p < 0.05 vs. wild-type, Fig. 3A). A reduction in pain were indistinguishable from wild-type mice in terms of behaviors is occasionally misinterpreted as a result of their paw withdrawal thresholds (Fig. 1C). Abdominal non-specific motor dysfunction, but the rotarod perform- writhing behavior in response to intraperitoneal injection ance test demonstrated no significant difference in the with acetic acid, a model of chemical-induced visceral time on the rotarod between the two genotypes (data not pain, was also comparable between the two lines of mice shown). We further tested whether P2X R deficiency also (Fig. 1D). To determine the role of P2X R in tissue injury- suppresses nerve injury-induced hypersensitivity to cold induced acute and persistent pain, we assessed the pain stimulation, a phenomenon known as cold allodynia. In response following the injection of formalin into the striking contrast to the lack of nerve injury-induced tactile -/- hindpaw. In wild-type mice, injection of formalin elicited allodynia in p2rx4 mice (Fig. 3A), both wild-type and -/- biphasic biting and licking behaviors: the first phase p2rx4 mice showed markedly enhanced responsiveness started immediately after the injection and lasted for 5 to a cold stimulus evoked by applying acetone to the ipsi- min, and the second phase lasted for 60 min (Fig. 1E). lateral hindpaw after nerve injury (Fig. 3B). There was no Neither the pattern nor the magnitude of biphasic behav- difference in the behavioral response to acetone in the -/- iors was altered in p2rx4 mice (Fig. 1E). The amount of contralateral hindpaw between the two lines of mice -/- swelling of the formalin-injected hindpaw, as indicated by (wild-type, 3.2 ± 0.3; p2rx4 , 3.2 ± 0.4). These results an increase in the weight of the hindpaw 60 min after the indicate that P2X R deficiency leads to a striking reduc- injection, was not significantly different between the two tion in the tactile allodynia caused by nerve injury and, to lines of mice (contralateral hindpaw: wild-type 0.137 ± the lesser extent, peripheral inflammation, without any -/- 0.008 g, p2rx4 0.130 ± 0.004 g; ipsilateral: wild-type defects in motor function. -/- 0.173 ± 0.005 g, p2rx4 0.181 ± 0.005 g). These results indicate that pain signaling elicited by acute noxious stim- By using P2X R-deficient mice, we revealed that P2X R is 4 4 -/- uli and tissue damage is intact in p2rx4 mice. not required for all forms of pain responses, but rather is crucial for specialized pain states, namely chronic inflam- Page 2 of 7 (page number not for citation purposes) Molecular Pain 2009, 5:28 http://www.molecularpain.com/content/5/1/28 -/- Figure 1 Acute thermal and mechanical pain and chemical-induced pain in p2rx4 mice -/- Acute thermal and mechanical pain and chemical-induced pain in p2rx4 mice. (A) Tail-flick and (B) paw-flick tests. -/- Values represent the latency (sec) for animals to flick their tail or paw away from the heat source (wild-type, n = 5; p2rx4 , n = 4). (C) Mechanical pain test. Values indicate the threshold (g) to elicit paw withdrawal behavior in response to mechanical -/- stimuli (wild-type, n = 5; p2rx4 , n = 4). (D) Visceral pain in response to acetic acid (0.8%). Values represent the numbers of -/- abdominal stretches (writhes) (wild-type, n = 6; p2rx4 , n = 5). (E) Formalin test. Mice were injected intraplantarly with forma- lin (5%, 20 μL). Values represent the duration (sec) of licking and biting responses for each 5-min interval (E), from 0 to 5 min -/- (1st phase) and for 10–60 min (2nd phase) (inset) (wild-type, n = 8; p2rx4 , n = 6). All data are presented as means ± SEM. Page 3 of 7 (page number not for citation purposes) Molecular Pain 2009, 5:28 http://www.molecularpain.com/content/5/1/28 -/- Figure 2 Reduction in the amount of peripheral inflammation-induced pain in p2rx4 mice -/- Reduction in the amount of peripheral inflammation-induced pain in p2rx4 mice. (A) Paw withdrawal thresholds -/- (left panel, ipsilateral hindpaw; right panel, contralateral hindpaw) of wild-type (n = 8) and p2rx4 mice (n = 8) before (0) and 1, 3, 7, 10 and 14 days after intraplantar injection of CFA (0.01 mg/20 μL). *p < 0.05, **p < 0.01, ***p < 0.001 vs. wild-type mice. (B) Change in the weight (g) of the ipsilateral and contralateral hindpa0w 14 days after the injection of CFA (wild-type, n = 6; -/- p2rx4 , n = 6). *p < 0.05 vs. wild-type mice. All data are presented as means ± SEM. matory and, in particular, neuropathic pain. Our present tile allodynia without affecting cold allodynia [25]. Our results showing the striking reduction of tactile allodynia previous study has implicated Lyn (microglial SFK) as a -/- after an injury to spinal nerves in p2rx4 mice together critical kinase causing P2X R upregulation [13]. It is thus with our previous findings in rats [7] and recent findings conceivable that P2X R-dependent microglial signaling in -/- in a line of p2rx4 mice after an injury to the sciatic nerve the dorsal horn may participate predominantly in allody- [22] provide compelling evidence for an essential role of nia evoked by mechanical rather than cold stimulation P2X R in nerve injury-induced tactile allodynia. To eluci- after peripheral nerve injury. date the mechanisms underlying the blunted neuropathic -/- allodynia in p2rx4 mice requires further investigations, Our present study showed a reduction in the level of CFA- -/- but it may involve a lack of microglial P2X Rs in the spinal induced inflammatory pain in p2rx4 mice. In contrast to cord, expression of which is markedly upregulated after the neuropathic pain model, neither the upregulation of nerve injury [7,22]. Stimulation of P2X R in microglia P2X R expression [7] nor activation of Lyn tyrosine kinase 4 4 induces release of brain-derived neurotrophic factor [13] has been demonstrated in spinal microglia following [9,22,23], a factor that is crucial for producing aberrant peripheral inflammation caused by CFA. The mechanisms excitability of dorsal horn neurons [9,24]. Therefore, the underlying the reduction in the amount of inflammatory -/- attenuated neuropathic allodynia in p2rx4 mice may be pain remain unknown, but the phenotype may be related -/- associated with a reduction in the amount of pathologi- to a reduction in hindpaw inflammation in p2rx4 mice. cally altered neurotransmission in dorsal horn neurons This notion is supported by evidence of expression of caused by microglia-derived BDNF [22]. functional P2X Rs in peripheral inflammatory cells, including macrophages [21,26]. An interesting finding in the present study was that nerve -/- injury-induced cold allodynia was retained in p2rx4 In conclusion, by employing a series of acute and chronic mice, indicating that P2X R may have distinct roles in pain tests using mice lacking P2X R, we demonstrated 4 4 mechanical and cold hypersensitivities after nerve injury. that the loss of P2X R leads to a marked reduction in the In a recent study it was shown that inhibition of Src-fam- degree of tactile allodynia caused by nerve injury and, to a ily kinases (SFKs), whose activity is enhanced in spinal lesser extent, in peripheral inflammation, without any microglia after nerve injury, results in suppression of tac- defects in motor function. By contrasting the roles of Page 4 of 7 (page number not for citation purposes) Molecular Pain 2009, 5:28 http://www.molecularpain.com/content/5/1/28 -/- Figure 3 Nerve injury-induced tactile allodynia is markedly blunted in p2rx4 mice -/- Nerve injury-induced tactile allodynia is markedly blunted in p2rx4 mice. Paw withdrawal thresholds (left panel, -/- ipsilateral hindpaw; right panel, contralateral hindpaw) of wild-type (n = 5) and p2rx4 mice (n = 6) before (0) and 1, 3, 7, 10 and 14 days after spinal nerve injury. (B) Cold allodynia evoked by applying acetone to the plantar surface of the ipsilateral -/- hindpaw 7 days after nerve injury (wild-type, n = 5; p2rx4 , n = 5). *p < 0.05, ***p < 0.001 vs. wild-type mice. All data are pre- sented as means ± SEM. P2X R in distinct types of nerve injury-induced pain (Ugo Basile, Italy) to the tail and the plantar surface of hypersensitivities, namely tactile and cold allodynia, the hindpaw, respectively [27,28]. The intensity of the heat present study demonstrates a predominant contribution stimulus was adjusted to 30 or 50 V, and the latency of the of P2X R to tactile allodynia rather than cold allodynia. paw withdrawal response (sec) was measured. The sensi- The fact that acute physiological pain and tissue damage- tivity to mechanical stimulus was assessed using von Frey -/- induced pain were normal in p2rx4 mice highlights the filaments (0.02~2.0 g, Stoelting, Wood Dale, Illinois, possibility of a therapeutic benefit of blocking P2X R in USA), and the mechanical stimulus producing the 50% the treatment of chronic pain, especially tactile allodynia paw withdrawal threshold was determined using the up- after nerve injury. down method [29,30]. In the tests of formalin-induced pain, mice were injected intraplantarly with formalin Methods (5%, 20 μL), and then the duration of the licking and bit- Animals ing responses to the injected hindpaw was recorded at 5 All experimental procedures were performed under the min intervals for 60 min after the injection (formalin guidelines of Kyushu University. Male mice lacking P2X R pain) [31,32]. For the measurement of hindpaw swelling -/- (p2rx4 ) that were backcrossed to C57BL/6J (Clea Japan) by formalin, the weights of the hind feet amputated at the for more than 10 generations were kindly provided by ankle were measured 60 min and 14 days after the injec- Prof. Joji Ando (The University of Tokyo) [19], and we tion of formalin and CFA, respectively [32]. In the chemi- used C57BL/6J as the corresponding control mice. All cal visceral pain test, mice were injected intraperitoneally mice were used at age of 9~11-week-old (at the start of with acetic acid (0.8%), and the number of abdominal each experiment). Mice were housed in groups of 2~3 per writhes was counted for 5 min starting from 5 min after cage at a temperature of 22 ± 1°C with a 12-h light-dark the injection [32]. Motor coordination was assessed using cycle (light on 8:30 to 20:30), and fed food and water ad the rotarod performance test [31]. For the inflammatory libitum. pain model, CFA (0.01 mg/20 μL) was injected into the plantar surface of the left hindpaw [13]. For the neuro- Behavioral assays for acute and chronic pain pathic pain model, the left L4 spinal nerve of mice was Noxious heat-evoked tail and hindpaw withdrawal transected under isoflurane (2%) anesthesia [33-35]. To responses were detected by the application of radiant heat assess the tactile allodynia, mice were placed individually Page 5 of 7 (page number not for citation purposes) Molecular Pain 2009, 5:28 http://www.molecularpain.com/content/5/1/28 6. Tsuda M, Inoue K: Chapter 9 – P2X Receptors in Sensory Neu- in an opaque plastic cylinder which was placed on a wire rons. In The Nociceptive Membrane Volume 57. Edited by: Oh U. San mesh and habituated for 1 hr to allow acclimatization to Diego: Academic Press; 2006:277-310. the new environment. After that, calibrated von Frey fila- 7. Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, Inoue K: P2X4 receptors induced in spinal micro- ments (0.02–2.0 g, Stoelting) were applied to the plantar glia gate tactile allodynia after nerve injury. Nature 2003, surface of the hindpaw from below the mesh floor, and 424:778-783. 8. Tsuda M, Inoue K, Salter MW: Neuropathic pain and spinal the 50% paw withdrawal threshold was determined. To microglia: a big problem from molecules in "small" glia. assess cold allodynia [36], a drop (50 μL) of acetone was Trends Neurosci 2005, 28:101-107. placed against the centre of the plantar surface of the 9. Coull JA, Beggs S, Boudreau D, Boivin D, Tsuda M, Inoue K, Gravel C, Salter MW, De Koninck Y: BDNF from microglia causes the hindpaw and a stopwatch was started. The mouse's shift in neuronal anion gradient underlying neuropathic pain. response was monitored in the first 20 sec after acetone Nature 2005, 438:1017-1021. application. If the mouse did not withdraw, flick or stamp 10. Trang T, Beggs S, Salter MW: Purinoceptors in microglia and neuropathic pain. Pflugers Arch 2006, 452:645-652. its hindpaw within this 20-sec period then no response 11. Scholz J, Woolf CJ: The neuropathic pain triad: neurons, was recorded for that trial (0). However, if within this 20 immune cells and glia. Nat Neurosci 2007, 10:1361-1368. 12. Tsuda M, Toyomitsu E, Komatsu T, Masuda T, Kunifusa E, Nasu-Tada sec period the animal responded to the cooling effect of K, Koizumi S, Yamamoto K, Ando J, Inoue K: Fibronectin/integrin the acetone, then the animal's response was assessed for system is involved in P2X(4) receptor upregulation in the an additional 20 sec (a total of 40 sec from initial applica- spinal cord and neuropathic pain after nerve injury. Glia 2008, 56:579-585. tion). Responses to acetone were graded according to the 13. Tsuda M, Tozaki-Saitoh H, Masuda T, Toyomitsu E, Tezuka T, following four-point scale: 0, no response; 1, quick with- Yamamoto T, Inoue K: Lyn tyrosine kinase is required for P2X(4) receptor upregulation and neuropathic pain after drawal, flick or stamp of the paw; 2, prolonged with- peripheral nerve injury. Glia 2008, 56:50-58. drawal or repeated flicking (more than 2 times) of the 14. Cockayne DA, Hamilton SG, Zhu QM, Dunn PM, Zhong Y, Novako- paw; 3, repeated flicking of the paw with licking directed vic S, Malmberg AB, Cain G, Berson A, Kassotakis L, et al.: Urinary bladder hyporeflexia and reduced pain-related behaviour in at the plantar surface of the hindpaw. Acetone was applied P2X3-deficient mice. Nature 2000, 407:1011-1015. alternately five times to each hindpaw and the responses 15. Souslova V, Cesare P, Ding Y, Akopian AN, Stanfa L, Suzuki R, Car- were scored categorically. Cumulative scores were then penter K, Dickenson A, Boyce S, Hill R, et al.: Warm-coding defi- cits and aberrant inflammatory pain in mice lacking P2X3 generated for each mouse. receptors. Nature 2000, 407:1015-1017. 16. Jarvis MF, Burgard EC, McGaraughty S, Honore P, Lynch K, Brennan Statistical Analysis TJ, Subieta A, Van Biesen T, Cartmell J, Bianchi B, et al.: A-317491, a novel potent and selective non-nucleotide antagonist of The statistical analyses of the results were evaluated by P2X3 and P2X2/3 receptors, reduces chronic inflammatory using the Student’s t test or the Mann-Whitney U test. and neuropathic pain in the rat. Proc Natl Acad Sci USA 2002, 99:17179-17184. 17. Chessell IP, Hatcher JP, Bountra C, Michel AD, Hughes JP, Green P, Competing interests Egerton J, Murfin M, Richardson J, Peck WL, et al.: Disruption of the The authors declare that they have no competing interests. P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain. Pain 2005, 114:386-396. 18. Honore P, Donnelly-Roberts D, Namovic MT, Hsieh G, Zhu CZ, Authors' contributions Mikusa JP, Hernandez G, Zhong C, Gauvin DM, Chandran P, et al.: A- 740003 [N-(1-{[(cyanoimino)(5-quinolinylamino) MT designed, performed and supervised the experiments, methyl]amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphe- analyzed the data, and wrote the manuscript; KK and TI nyl)acetamide], a novel and selective P2X7 receptor antag- performed the experiments; KN, HST. analyzed the data; onist, dose-dependently reduces neuropathic pain in the rat. J Pharmacol Exp Ther 2006, 319:1376-1385. KI coordinated the project, helped to interpret the data, 19. Yamamoto K, Sokabe T, Matsumoto T, Yoshimura K, Shibata M, and edited the manuscript. All authors have read and Ohura N, Fukuda T, Sato T, Sekine K, Kato S, et al.: Impaired flow- approved the final manuscript. dependent control of vascular tone and remodeling in P2X4- deficient mice. Nat Med 2006, 12:133-137. 20. Sim JA, Chaumont S, Jo J, Ulmann L, Young MT, Cho K, Buell G, Acknowledgements North RA, Rassendren F: Altered hippocampal synaptic poten- We thank Dr. Joji Ando (The University of Tokyo) for providing P2X R- tiation in P2X4 knock-out mice. J Neurosci 2006, 26:9006-9009. 21. Brone B, Moechars D, Marrannes R, Mercken M, Meert T: P2X cur- knockout mice. This work was supported by grants from the Ministry of rents in peritoneal macrophages of wild type and P2X4 -/- Education, Culture, Sports, Science and Technology of Japan (to M.T., K.I.). mice. Immunol Lett 2007, 113:83-89. 22. Ulmann L, Hatcher JP, Hughes JP, Chaumont S, Green PJ, Conquet F, Buell GN, Reeve AJ, Chessell IP, Rassendren F: Up-regulation of References P2X4 receptors in spinal microglia after peripheral nerve 1. Fields RD, Burnstock G: Purinergic signalling in neuron-glia injury mediates BDNF release and neuropathic pain. J Neuro- interactions. Nat Rev Neurosci 2006, 7:423-436. sci 2008, 28:11263-11268. 2. Khakh BS, North RA: P2X receptors as cell-surface ATP sen- 23. Trang T, Beggs S, Wan X, Salter MW: P2X4-receptor-mediated sors in health and disease. Nature 2006, 442:527-532. synthesis and release of brain-derived neurotrophic factor in 3. Burnstock G: Purinergic signalling and disorders of the central microglia is dependent on calcium and p38-mitogen-acti- nervous system. Nat Rev Drug Discov 2008, 7:575-590. vated protein kinase activation. J Neurosci 2009, 29:3518-3528. 4. Burnstock G: Purinergic P2 receptors as targets for novel 24. Keller AF, Beggs S, Salter MW, De Koninck Y: Transformation of analgesics. Pharmacol Ther 2006, 110:433-454. the output of spinal lamina I neurons after nerve injury and 5. Nakatsuka T, Gu JG: P2X purinoceptors and sensory transmis- microglia stimulation underlying neuropathic pain. Mol Pain sion. Pflugers Arch 2006, 452:598-607. 2007, 3:27. Page 6 of 7 (page number not for citation purposes) Molecular Pain 2009, 5:28 http://www.molecularpain.com/content/5/1/28 25. Katsura H, Obata K, Mizushima T, Sakurai J, Kobayashi K, Yamanaka H, Dai Y, Fukuoka T, Sakagami M, Noguchi K: Activation of Src- family kinases in spinal microglia contributes to mechanical hypersensitivity after nerve injury. J Neurosci 2006, 26:8680-8690. 26. Bowler JW, Bailey RJ, North RA, Surprenant A: P2X4, P2Y1 and P2Y2 receptors on rat alveolar macrophages. Br J Pharmacol 2003, 140:567-575. 27. Tsuda M, Ueno S, Inoue K: In vivo pathway of thermal hyperal- gesia by intrathecal administration of alpha,beta-methylene ATP in mouse spinal cord: involvement of the glutamate- NMDA receptor system. Br J Pharmacol 1999, 127:449-456. 28. Tsuda M, Koizumi S, Kita A, Shigemoto Y, Ueno S, Inoue K: Mechan- ical allodynia caused by intraplantar injection of P2X recep- tor agonist in rats: involvement of heteromeric P2X2/3 receptor signaling in capsaicin-insensitive primary afferent neurons. J Neurosci 2000, 20:RC90. 29. Dixon WJ: Efficient analysis of experimental observations. Annu Rev Pharmacol Toxicol 1980, 20:441-462. 30. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL: Quantita- tive assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994, 53:55-63. 31. Tsuda M, Ueno S, Inoue K: Evidence for the involvement of spi- nal endogenous ATP and P2X receptors in nociceptive responses caused by formalin and capsaicin in mice. Br J Phar- macol 1999, 128:1497-1504. 32. Tsuda M, Ishii S, Masuda T, Hasegawa S, Nakamura K, Nagata K, Yamashita T, Furue H, Tozaki-Saitoh H, Yoshimura M, et al.: Reduced pain behaviors and extracellular signal-related pro- tein kinase activation in primary sensory neurons by periph- eral tissue injury in mice lacking platelet-activating factor receptor. J Neurochem 2007, 102:1658-1668. 33. Kim SH, Chung JM: An experimental model for peripheral neu- ropathy produced by segmental spinal nerve ligation in the rat. Pain 1992, 50:355-363. 34. Tozaki-Saitoh H, Tsuda M, Miyata H, Ueda K, Kohsaka S, Inoue K: P2Y12 receptors in spinal microglia are required for neuro- pathic pain after peripheral nerve injury. J Neurosci 2008, 28:4949-4956. 35. Rigaud M, Gemes G, Barabas ME, Chernoff DI, Abram SE, Stucky CL, Hogan QH: Species and strain differences in rodent sciatic nerve anatomy: implications for studies of neuropathic pain. Pain 2008, 136:188-201. 36. Flatters SJ, Bennett GJ: Ethosuximide reverses paclitaxel- and vincristine-induced painful peripheral neuropathy. Pain 2004, 109:150-161. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 7 of 7 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Pain Springer Journals

Behavioral phenotypes of mice lacking purinergic P2X4 receptors in acute and chronic pain assays

Loading next page...
 
/lp/springer-journals/behavioral-phenotypes-of-mice-lacking-purinergic-p2x4-receptors-in-9sx9jkDBRS

References (41)

Publisher
Springer Journals
Copyright
Copyright © 2009 by Tsuda et al; licensee BioMed Central Ltd.
Subject
Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology
eISSN
1744-8069
DOI
10.1186/1744-8069-5-28
pmid
19515262
Publisher site
See Article on Publisher Site

Abstract

A growing body of evidence indicates that P2X receptors (P2XRs), a family of ligand-gated cation channels activated by extracellular ATP, play an important role in pain signaling. In contrast to the role of the P2X R subtype that has been extensively studied, the precise roles of others among the seven P2XR subtypes (P2X R-P2X R) remain to be determined because of a lack of sufficiently 1 7 powerful tools to specifically block P2XR signaling in vivo. In the present study, we investigated the behavioral phenotypes of a line of mice in which the p2rx4 gene was disrupted in a series of acute -/- and chronic pain assays. While p2rx4 mice showed no major defects in pain responses evoked by acute noxious stimuli and local tissue damage or in motor function as compared with wild-type mice, these mice displayed reduced pain responses in two models of chronic pain (inflammatory and neuropathic pain). In a model of chronic inflammatory pain developed by intraplantar injection -/- of complete Freund's adjuvant (CFA), p2rx4 mice exhibited attenuations of pain hypersensitivity to innocuous mechanical stimuli (tactile allodynia) and also of the CFA-induced swelling of the hindpaw. A most striking phenotype was observed in a test of neuropathic pain: tactile allodynia -/- caused by an injury to spinal nerve was markedly blunted in p2rx4 mice. By contrast, pain hypersensitivity to a cold stimulus (cold allodynia) after the injury was comparable in wild-type and -/- p2rx4 mice. Together, these findings reveal a predominant contribution of P2X R to nerve injury- induced tactile allodynia and, to the lesser extent, peripheral inflammation. Loss of P2X R produced no defects in acute physiological pain or tissue damaged-induced pain, highlighting the possibility of a therapeutic benefit of blocking P2X R in the treatment of chronic pain, especially tactile allodynia after nerve injury. have important roles in regulating neuronal and glial Findings The purinergic P2X receptors, of which seven subtypes functions in the nervous system under physiological and –P2X ) have been cloned, are a family of ligand- (P2X pathological conditions [1-3]. There has been much 1 7 gated cation channels activated by extracellular ATP. They recent attention paid to their roles in generating and mod- Page 1 of 7 (page number not for citation purposes) Molecular Pain 2009, 5:28 http://www.molecularpain.com/content/5/1/28 ulating pain signaling [1-3]. Various attempts to block Next, to investigate the role of P2X R in pain hypersensi- P2XRs pharmacologically or to suppress their expression tivity under chronic pain conditions, we employed two molecularly and genetically have demonstrated that these distinct models of chronic pain, namely inflammatory receptors make a major contribution to pain responses and neuropathic pain. Wild-type mice with peripheral evoked by tissue damage, chronic peripheral inflamma- inflammation induced by intraplantar injection of com- tion and nerve injury [4-6]. While the role of P2X R (and plete Freund's adjuvant (CFA), used as a model of inflam- P2X R) has been studied extensively, recent evidence matory pain, displayed decreased paw withdrawal 2+3 from our and other studies indicates that the P2X R sub- thresholds in their ipsilateral hindpaws (Fig. 2A). By con- type critically contributes to neuropathic pain, a highly trast, the peripheral inflammation-induced decrease in debilitating pain condition that commonly occurs after paw withdrawal threshold was significantly smaller in -/- nerve damage [7-13]. However, the precise role of P2X R p2rx4 mice than wild-type mice. The paw withdrawal in neuropathic pain remains to be fully determined thresholds for the contralateral hindpaws in both geno- because, in stark contrast to P2X R [14-16] and P2X R types of mice did not change (Fig. 2A). In wild-type mice, 3 7 [17,18], there is a lack of sufficiently powerful pharmaco- a marked swelling of the ipsilateral hindpaw was R signal- logical and genetic tools to selectively block P2X observed, and the weight of the hindpaw was increased on -/- ing in vivo. Recently, several groups have independently day 14 (Fig. 2B). In p2rx4 mice, however, the CFA- developed lines of mice in which the p2rx4 gene is dis- induced increase in the weight of the ipsilateral hindpaw rupted [19-21]. It is of particular importance to study the was significantly suppressed (p < 0.05, Fig. 2B). phenotypes of these mice to determine the in vivo func- tions of P2X R in pain signaling [22]. In the present study, To determine the role of P2X R in tactile allodynia under 4 4 we sought to characterize behavioral phenotypes in a wide neuropathic pain conditions, we injured the forth lumbar -/- range of assays of acute and chronic pain, including neu- spinal nerves of wild-type and p2rx4 mice; this approach ropathic pain, using a line of P2X R-deficient mice [19]. has been used to generate an animal model of neuro- pathic pain. While wild-type mice showed a decrease in To examine acute physiological pain responses, the with- paw withdrawal threshold after nerve injury (Fig. 3A), this -/- drawal responses from a noxious range of heat and effect was markedly blunted in p2rx4 mice. The attenu- ated tactile allodynia was observed until the last time mechanical stimuli applied by von Frey filaments were -/- measured in both wild-type and p2rx4 mice. In tail- and point tested (day 1, p < 0.05; day 3, 7, 10 and 14, p < paw-flick tests, the latencies for animals to flick their tails 0.001; Fig. 3A). The loss of P2X R did not change the paw and hindpaws away from radiant heat at either 30 V or 50 withdrawal threshold of the contralateral hindpaw after -/- V were not different between wild-type and p2rx4 mice nerve injury (Fig. 3A) as seen in wild-type mice (except on -/- (Fig. 1A, B). In a test of mechanical pain, p2rx4 mice day 1, p < 0.05 vs. wild-type, Fig. 3A). A reduction in pain were indistinguishable from wild-type mice in terms of behaviors is occasionally misinterpreted as a result of their paw withdrawal thresholds (Fig. 1C). Abdominal non-specific motor dysfunction, but the rotarod perform- writhing behavior in response to intraperitoneal injection ance test demonstrated no significant difference in the with acetic acid, a model of chemical-induced visceral time on the rotarod between the two genotypes (data not pain, was also comparable between the two lines of mice shown). We further tested whether P2X R deficiency also (Fig. 1D). To determine the role of P2X R in tissue injury- suppresses nerve injury-induced hypersensitivity to cold induced acute and persistent pain, we assessed the pain stimulation, a phenomenon known as cold allodynia. In response following the injection of formalin into the striking contrast to the lack of nerve injury-induced tactile -/- hindpaw. In wild-type mice, injection of formalin elicited allodynia in p2rx4 mice (Fig. 3A), both wild-type and -/- biphasic biting and licking behaviors: the first phase p2rx4 mice showed markedly enhanced responsiveness started immediately after the injection and lasted for 5 to a cold stimulus evoked by applying acetone to the ipsi- min, and the second phase lasted for 60 min (Fig. 1E). lateral hindpaw after nerve injury (Fig. 3B). There was no Neither the pattern nor the magnitude of biphasic behav- difference in the behavioral response to acetone in the -/- iors was altered in p2rx4 mice (Fig. 1E). The amount of contralateral hindpaw between the two lines of mice -/- swelling of the formalin-injected hindpaw, as indicated by (wild-type, 3.2 ± 0.3; p2rx4 , 3.2 ± 0.4). These results an increase in the weight of the hindpaw 60 min after the indicate that P2X R deficiency leads to a striking reduc- injection, was not significantly different between the two tion in the tactile allodynia caused by nerve injury and, to lines of mice (contralateral hindpaw: wild-type 0.137 ± the lesser extent, peripheral inflammation, without any -/- 0.008 g, p2rx4 0.130 ± 0.004 g; ipsilateral: wild-type defects in motor function. -/- 0.173 ± 0.005 g, p2rx4 0.181 ± 0.005 g). These results indicate that pain signaling elicited by acute noxious stim- By using P2X R-deficient mice, we revealed that P2X R is 4 4 -/- uli and tissue damage is intact in p2rx4 mice. not required for all forms of pain responses, but rather is crucial for specialized pain states, namely chronic inflam- Page 2 of 7 (page number not for citation purposes) Molecular Pain 2009, 5:28 http://www.molecularpain.com/content/5/1/28 -/- Figure 1 Acute thermal and mechanical pain and chemical-induced pain in p2rx4 mice -/- Acute thermal and mechanical pain and chemical-induced pain in p2rx4 mice. (A) Tail-flick and (B) paw-flick tests. -/- Values represent the latency (sec) for animals to flick their tail or paw away from the heat source (wild-type, n = 5; p2rx4 , n = 4). (C) Mechanical pain test. Values indicate the threshold (g) to elicit paw withdrawal behavior in response to mechanical -/- stimuli (wild-type, n = 5; p2rx4 , n = 4). (D) Visceral pain in response to acetic acid (0.8%). Values represent the numbers of -/- abdominal stretches (writhes) (wild-type, n = 6; p2rx4 , n = 5). (E) Formalin test. Mice were injected intraplantarly with forma- lin (5%, 20 μL). Values represent the duration (sec) of licking and biting responses for each 5-min interval (E), from 0 to 5 min -/- (1st phase) and for 10–60 min (2nd phase) (inset) (wild-type, n = 8; p2rx4 , n = 6). All data are presented as means ± SEM. Page 3 of 7 (page number not for citation purposes) Molecular Pain 2009, 5:28 http://www.molecularpain.com/content/5/1/28 -/- Figure 2 Reduction in the amount of peripheral inflammation-induced pain in p2rx4 mice -/- Reduction in the amount of peripheral inflammation-induced pain in p2rx4 mice. (A) Paw withdrawal thresholds -/- (left panel, ipsilateral hindpaw; right panel, contralateral hindpaw) of wild-type (n = 8) and p2rx4 mice (n = 8) before (0) and 1, 3, 7, 10 and 14 days after intraplantar injection of CFA (0.01 mg/20 μL). *p < 0.05, **p < 0.01, ***p < 0.001 vs. wild-type mice. (B) Change in the weight (g) of the ipsilateral and contralateral hindpa0w 14 days after the injection of CFA (wild-type, n = 6; -/- p2rx4 , n = 6). *p < 0.05 vs. wild-type mice. All data are presented as means ± SEM. matory and, in particular, neuropathic pain. Our present tile allodynia without affecting cold allodynia [25]. Our results showing the striking reduction of tactile allodynia previous study has implicated Lyn (microglial SFK) as a -/- after an injury to spinal nerves in p2rx4 mice together critical kinase causing P2X R upregulation [13]. It is thus with our previous findings in rats [7] and recent findings conceivable that P2X R-dependent microglial signaling in -/- in a line of p2rx4 mice after an injury to the sciatic nerve the dorsal horn may participate predominantly in allody- [22] provide compelling evidence for an essential role of nia evoked by mechanical rather than cold stimulation P2X R in nerve injury-induced tactile allodynia. To eluci- after peripheral nerve injury. date the mechanisms underlying the blunted neuropathic -/- allodynia in p2rx4 mice requires further investigations, Our present study showed a reduction in the level of CFA- -/- but it may involve a lack of microglial P2X Rs in the spinal induced inflammatory pain in p2rx4 mice. In contrast to cord, expression of which is markedly upregulated after the neuropathic pain model, neither the upregulation of nerve injury [7,22]. Stimulation of P2X R in microglia P2X R expression [7] nor activation of Lyn tyrosine kinase 4 4 induces release of brain-derived neurotrophic factor [13] has been demonstrated in spinal microglia following [9,22,23], a factor that is crucial for producing aberrant peripheral inflammation caused by CFA. The mechanisms excitability of dorsal horn neurons [9,24]. Therefore, the underlying the reduction in the amount of inflammatory -/- attenuated neuropathic allodynia in p2rx4 mice may be pain remain unknown, but the phenotype may be related -/- associated with a reduction in the amount of pathologi- to a reduction in hindpaw inflammation in p2rx4 mice. cally altered neurotransmission in dorsal horn neurons This notion is supported by evidence of expression of caused by microglia-derived BDNF [22]. functional P2X Rs in peripheral inflammatory cells, including macrophages [21,26]. An interesting finding in the present study was that nerve -/- injury-induced cold allodynia was retained in p2rx4 In conclusion, by employing a series of acute and chronic mice, indicating that P2X R may have distinct roles in pain tests using mice lacking P2X R, we demonstrated 4 4 mechanical and cold hypersensitivities after nerve injury. that the loss of P2X R leads to a marked reduction in the In a recent study it was shown that inhibition of Src-fam- degree of tactile allodynia caused by nerve injury and, to a ily kinases (SFKs), whose activity is enhanced in spinal lesser extent, in peripheral inflammation, without any microglia after nerve injury, results in suppression of tac- defects in motor function. By contrasting the roles of Page 4 of 7 (page number not for citation purposes) Molecular Pain 2009, 5:28 http://www.molecularpain.com/content/5/1/28 -/- Figure 3 Nerve injury-induced tactile allodynia is markedly blunted in p2rx4 mice -/- Nerve injury-induced tactile allodynia is markedly blunted in p2rx4 mice. Paw withdrawal thresholds (left panel, -/- ipsilateral hindpaw; right panel, contralateral hindpaw) of wild-type (n = 5) and p2rx4 mice (n = 6) before (0) and 1, 3, 7, 10 and 14 days after spinal nerve injury. (B) Cold allodynia evoked by applying acetone to the plantar surface of the ipsilateral -/- hindpaw 7 days after nerve injury (wild-type, n = 5; p2rx4 , n = 5). *p < 0.05, ***p < 0.001 vs. wild-type mice. All data are pre- sented as means ± SEM. P2X R in distinct types of nerve injury-induced pain (Ugo Basile, Italy) to the tail and the plantar surface of hypersensitivities, namely tactile and cold allodynia, the hindpaw, respectively [27,28]. The intensity of the heat present study demonstrates a predominant contribution stimulus was adjusted to 30 or 50 V, and the latency of the of P2X R to tactile allodynia rather than cold allodynia. paw withdrawal response (sec) was measured. The sensi- The fact that acute physiological pain and tissue damage- tivity to mechanical stimulus was assessed using von Frey -/- induced pain were normal in p2rx4 mice highlights the filaments (0.02~2.0 g, Stoelting, Wood Dale, Illinois, possibility of a therapeutic benefit of blocking P2X R in USA), and the mechanical stimulus producing the 50% the treatment of chronic pain, especially tactile allodynia paw withdrawal threshold was determined using the up- after nerve injury. down method [29,30]. In the tests of formalin-induced pain, mice were injected intraplantarly with formalin Methods (5%, 20 μL), and then the duration of the licking and bit- Animals ing responses to the injected hindpaw was recorded at 5 All experimental procedures were performed under the min intervals for 60 min after the injection (formalin guidelines of Kyushu University. Male mice lacking P2X R pain) [31,32]. For the measurement of hindpaw swelling -/- (p2rx4 ) that were backcrossed to C57BL/6J (Clea Japan) by formalin, the weights of the hind feet amputated at the for more than 10 generations were kindly provided by ankle were measured 60 min and 14 days after the injec- Prof. Joji Ando (The University of Tokyo) [19], and we tion of formalin and CFA, respectively [32]. In the chemi- used C57BL/6J as the corresponding control mice. All cal visceral pain test, mice were injected intraperitoneally mice were used at age of 9~11-week-old (at the start of with acetic acid (0.8%), and the number of abdominal each experiment). Mice were housed in groups of 2~3 per writhes was counted for 5 min starting from 5 min after cage at a temperature of 22 ± 1°C with a 12-h light-dark the injection [32]. Motor coordination was assessed using cycle (light on 8:30 to 20:30), and fed food and water ad the rotarod performance test [31]. For the inflammatory libitum. pain model, CFA (0.01 mg/20 μL) was injected into the plantar surface of the left hindpaw [13]. For the neuro- Behavioral assays for acute and chronic pain pathic pain model, the left L4 spinal nerve of mice was Noxious heat-evoked tail and hindpaw withdrawal transected under isoflurane (2%) anesthesia [33-35]. To responses were detected by the application of radiant heat assess the tactile allodynia, mice were placed individually Page 5 of 7 (page number not for citation purposes) Molecular Pain 2009, 5:28 http://www.molecularpain.com/content/5/1/28 6. Tsuda M, Inoue K: Chapter 9 – P2X Receptors in Sensory Neu- in an opaque plastic cylinder which was placed on a wire rons. In The Nociceptive Membrane Volume 57. Edited by: Oh U. San mesh and habituated for 1 hr to allow acclimatization to Diego: Academic Press; 2006:277-310. the new environment. After that, calibrated von Frey fila- 7. Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, Inoue K: P2X4 receptors induced in spinal micro- ments (0.02–2.0 g, Stoelting) were applied to the plantar glia gate tactile allodynia after nerve injury. Nature 2003, surface of the hindpaw from below the mesh floor, and 424:778-783. 8. Tsuda M, Inoue K, Salter MW: Neuropathic pain and spinal the 50% paw withdrawal threshold was determined. To microglia: a big problem from molecules in "small" glia. assess cold allodynia [36], a drop (50 μL) of acetone was Trends Neurosci 2005, 28:101-107. placed against the centre of the plantar surface of the 9. Coull JA, Beggs S, Boudreau D, Boivin D, Tsuda M, Inoue K, Gravel C, Salter MW, De Koninck Y: BDNF from microglia causes the hindpaw and a stopwatch was started. The mouse's shift in neuronal anion gradient underlying neuropathic pain. response was monitored in the first 20 sec after acetone Nature 2005, 438:1017-1021. application. If the mouse did not withdraw, flick or stamp 10. Trang T, Beggs S, Salter MW: Purinoceptors in microglia and neuropathic pain. Pflugers Arch 2006, 452:645-652. its hindpaw within this 20-sec period then no response 11. Scholz J, Woolf CJ: The neuropathic pain triad: neurons, was recorded for that trial (0). However, if within this 20 immune cells and glia. Nat Neurosci 2007, 10:1361-1368. 12. Tsuda M, Toyomitsu E, Komatsu T, Masuda T, Kunifusa E, Nasu-Tada sec period the animal responded to the cooling effect of K, Koizumi S, Yamamoto K, Ando J, Inoue K: Fibronectin/integrin the acetone, then the animal's response was assessed for system is involved in P2X(4) receptor upregulation in the an additional 20 sec (a total of 40 sec from initial applica- spinal cord and neuropathic pain after nerve injury. Glia 2008, 56:579-585. tion). Responses to acetone were graded according to the 13. Tsuda M, Tozaki-Saitoh H, Masuda T, Toyomitsu E, Tezuka T, following four-point scale: 0, no response; 1, quick with- Yamamoto T, Inoue K: Lyn tyrosine kinase is required for P2X(4) receptor upregulation and neuropathic pain after drawal, flick or stamp of the paw; 2, prolonged with- peripheral nerve injury. Glia 2008, 56:50-58. drawal or repeated flicking (more than 2 times) of the 14. Cockayne DA, Hamilton SG, Zhu QM, Dunn PM, Zhong Y, Novako- paw; 3, repeated flicking of the paw with licking directed vic S, Malmberg AB, Cain G, Berson A, Kassotakis L, et al.: Urinary bladder hyporeflexia and reduced pain-related behaviour in at the plantar surface of the hindpaw. Acetone was applied P2X3-deficient mice. Nature 2000, 407:1011-1015. alternately five times to each hindpaw and the responses 15. Souslova V, Cesare P, Ding Y, Akopian AN, Stanfa L, Suzuki R, Car- were scored categorically. Cumulative scores were then penter K, Dickenson A, Boyce S, Hill R, et al.: Warm-coding defi- cits and aberrant inflammatory pain in mice lacking P2X3 generated for each mouse. receptors. Nature 2000, 407:1015-1017. 16. Jarvis MF, Burgard EC, McGaraughty S, Honore P, Lynch K, Brennan Statistical Analysis TJ, Subieta A, Van Biesen T, Cartmell J, Bianchi B, et al.: A-317491, a novel potent and selective non-nucleotide antagonist of The statistical analyses of the results were evaluated by P2X3 and P2X2/3 receptors, reduces chronic inflammatory using the Student’s t test or the Mann-Whitney U test. and neuropathic pain in the rat. Proc Natl Acad Sci USA 2002, 99:17179-17184. 17. Chessell IP, Hatcher JP, Bountra C, Michel AD, Hughes JP, Green P, Competing interests Egerton J, Murfin M, Richardson J, Peck WL, et al.: Disruption of the The authors declare that they have no competing interests. P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain. Pain 2005, 114:386-396. 18. Honore P, Donnelly-Roberts D, Namovic MT, Hsieh G, Zhu CZ, Authors' contributions Mikusa JP, Hernandez G, Zhong C, Gauvin DM, Chandran P, et al.: A- 740003 [N-(1-{[(cyanoimino)(5-quinolinylamino) MT designed, performed and supervised the experiments, methyl]amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphe- analyzed the data, and wrote the manuscript; KK and TI nyl)acetamide], a novel and selective P2X7 receptor antag- performed the experiments; KN, HST. analyzed the data; onist, dose-dependently reduces neuropathic pain in the rat. J Pharmacol Exp Ther 2006, 319:1376-1385. KI coordinated the project, helped to interpret the data, 19. Yamamoto K, Sokabe T, Matsumoto T, Yoshimura K, Shibata M, and edited the manuscript. All authors have read and Ohura N, Fukuda T, Sato T, Sekine K, Kato S, et al.: Impaired flow- approved the final manuscript. dependent control of vascular tone and remodeling in P2X4- deficient mice. Nat Med 2006, 12:133-137. 20. Sim JA, Chaumont S, Jo J, Ulmann L, Young MT, Cho K, Buell G, Acknowledgements North RA, Rassendren F: Altered hippocampal synaptic poten- We thank Dr. Joji Ando (The University of Tokyo) for providing P2X R- tiation in P2X4 knock-out mice. J Neurosci 2006, 26:9006-9009. 21. Brone B, Moechars D, Marrannes R, Mercken M, Meert T: P2X cur- knockout mice. This work was supported by grants from the Ministry of rents in peritoneal macrophages of wild type and P2X4 -/- Education, Culture, Sports, Science and Technology of Japan (to M.T., K.I.). mice. Immunol Lett 2007, 113:83-89. 22. Ulmann L, Hatcher JP, Hughes JP, Chaumont S, Green PJ, Conquet F, Buell GN, Reeve AJ, Chessell IP, Rassendren F: Up-regulation of References P2X4 receptors in spinal microglia after peripheral nerve 1. Fields RD, Burnstock G: Purinergic signalling in neuron-glia injury mediates BDNF release and neuropathic pain. J Neuro- interactions. Nat Rev Neurosci 2006, 7:423-436. sci 2008, 28:11263-11268. 2. Khakh BS, North RA: P2X receptors as cell-surface ATP sen- 23. Trang T, Beggs S, Wan X, Salter MW: P2X4-receptor-mediated sors in health and disease. Nature 2006, 442:527-532. synthesis and release of brain-derived neurotrophic factor in 3. Burnstock G: Purinergic signalling and disorders of the central microglia is dependent on calcium and p38-mitogen-acti- nervous system. Nat Rev Drug Discov 2008, 7:575-590. vated protein kinase activation. J Neurosci 2009, 29:3518-3528. 4. Burnstock G: Purinergic P2 receptors as targets for novel 24. Keller AF, Beggs S, Salter MW, De Koninck Y: Transformation of analgesics. Pharmacol Ther 2006, 110:433-454. the output of spinal lamina I neurons after nerve injury and 5. Nakatsuka T, Gu JG: P2X purinoceptors and sensory transmis- microglia stimulation underlying neuropathic pain. Mol Pain sion. Pflugers Arch 2006, 452:598-607. 2007, 3:27. Page 6 of 7 (page number not for citation purposes) Molecular Pain 2009, 5:28 http://www.molecularpain.com/content/5/1/28 25. Katsura H, Obata K, Mizushima T, Sakurai J, Kobayashi K, Yamanaka H, Dai Y, Fukuoka T, Sakagami M, Noguchi K: Activation of Src- family kinases in spinal microglia contributes to mechanical hypersensitivity after nerve injury. J Neurosci 2006, 26:8680-8690. 26. Bowler JW, Bailey RJ, North RA, Surprenant A: P2X4, P2Y1 and P2Y2 receptors on rat alveolar macrophages. Br J Pharmacol 2003, 140:567-575. 27. Tsuda M, Ueno S, Inoue K: In vivo pathway of thermal hyperal- gesia by intrathecal administration of alpha,beta-methylene ATP in mouse spinal cord: involvement of the glutamate- NMDA receptor system. Br J Pharmacol 1999, 127:449-456. 28. Tsuda M, Koizumi S, Kita A, Shigemoto Y, Ueno S, Inoue K: Mechan- ical allodynia caused by intraplantar injection of P2X recep- tor agonist in rats: involvement of heteromeric P2X2/3 receptor signaling in capsaicin-insensitive primary afferent neurons. J Neurosci 2000, 20:RC90. 29. Dixon WJ: Efficient analysis of experimental observations. Annu Rev Pharmacol Toxicol 1980, 20:441-462. 30. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL: Quantita- tive assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994, 53:55-63. 31. Tsuda M, Ueno S, Inoue K: Evidence for the involvement of spi- nal endogenous ATP and P2X receptors in nociceptive responses caused by formalin and capsaicin in mice. Br J Phar- macol 1999, 128:1497-1504. 32. Tsuda M, Ishii S, Masuda T, Hasegawa S, Nakamura K, Nagata K, Yamashita T, Furue H, Tozaki-Saitoh H, Yoshimura M, et al.: Reduced pain behaviors and extracellular signal-related pro- tein kinase activation in primary sensory neurons by periph- eral tissue injury in mice lacking platelet-activating factor receptor. J Neurochem 2007, 102:1658-1668. 33. Kim SH, Chung JM: An experimental model for peripheral neu- ropathy produced by segmental spinal nerve ligation in the rat. Pain 1992, 50:355-363. 34. Tozaki-Saitoh H, Tsuda M, Miyata H, Ueda K, Kohsaka S, Inoue K: P2Y12 receptors in spinal microglia are required for neuro- pathic pain after peripheral nerve injury. J Neurosci 2008, 28:4949-4956. 35. Rigaud M, Gemes G, Barabas ME, Chernoff DI, Abram SE, Stucky CL, Hogan QH: Species and strain differences in rodent sciatic nerve anatomy: implications for studies of neuropathic pain. Pain 2008, 136:188-201. 36. Flatters SJ, Bennett GJ: Ethosuximide reverses paclitaxel- and vincristine-induced painful peripheral neuropathy. Pain 2004, 109:150-161. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 7 of 7 (page number not for citation purposes)

Journal

Molecular PainSpringer Journals

Published: Jun 11, 2009

There are no references for this article.