Involvement of spinal neurokinins, excitatory amino acids, proinflammatory cytokines, nitric oxide and prostanoids in pain facilitation induced by Phoneutria nigriventer spider venom

Brain Research 1021 (2004) 101 – 111 www.elsevier.com/locate/brainres

Research report

Involvement of spinal neurokinins, excitatory amino acids, proinflammatory cytokines, nitric oxide and prostanoids in pain facilitation induced by Phoneutria nigriventer spider venom Eliane Maria Zancheta,b, Ingrid Longoa, Yara Curya,* a

Laborato´rio de Fisiopatologia, Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900, Sa˜o Paulo, SP, Brazil Departamento de Fisiologia, Universidade Federal de Santa Maria, Campus Universita´rio, 97105-900, Santa Maria, RS, Brazil

b

Accepted 27 June 2004 Available online 28 July 2004

Abstract The major local symptom of Phoneutria nigriventer envenomation is an intense pain, which can be controlled by infiltration with local anesthetics or by systemic treatment with opioid analgesics. Previous work showed that intraplantar (i.pl) injection of Phoneutria nigriventer venom in rats induces hyperalgesia, mediated peripherally by tachykinin and glutamate receptors. The present study examined the spinal mechanisms involved in pain-enhancing effect of this venom. Intraplantar injection of venom into rat hind paw induced hyperalgesia. This phenomenon was inhibited by intrathecal (i.t.) injection of tachykinin NK1 (GR 82334) or NK2 (GR 94800) receptor antagonists, a calcitonin gene-related peptide (CGRP) receptor antagonist (CGRP8–37) and N-methyl-d-aspartate (NMDA; MK 801 and AP-5), non-NMDA ionotropic (CNQX), or metabotropic (AIDA and MPEP) glutamate receptor antagonists, suggesting the involvement of spinal neurokinins and excitatory amino acids. The role of proinflammatory cytokines, nitric oxide (NO), and prostanoids in spinally mediated pain facilitation was also investigated. Pharmacological blockade of tumour necrosis factor-a (TNFa) or interleukin-1h (IL-1h) reduced the hyperalgesic response to venom. Intrathecal injection of L-N6-(1-iminoethyl)lysine (L-NIL), but not of 7-nitroindazole (7-NI), inhibited hyperalgesia induced by the venom, indicating that NO, generated by the activity of the inducible form of nitric oxide synthase, also mediates this phenomenon. Furthermore, indomethacin, an inhibitor of cyclooxigenases (COX), or celecoxib, a selective inhibitor of COX-2, abolished venom-induced hyperalgesia, suggesting the involvement of spinal prostanoids in this effect. These data indicate that the spinal mechanisms of pain facilitation induced by Phoneutria nigriventer venom involves a plethora of mediators that may cooperate in the genesis of venom-induced central sensitization. D 2004 Elsevier B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation: pharmacology Keywords: Phoneutria nigriventer venom; Hyperalgesia; Spinal cord; Neurotransmitter; Proinflammatory cytokine; Prostanoid

1. Introduction The South American spider Phoneutria nigriventer (armed spider) is responsible for most cases of human araneism in Brazil [58]. Systemic effects observed in human envenomation by this spider include spastic paralysis, * Corresponding author. Tel.: +55 11 3726 7222; fax: +55 11 3726 1505. E-mail address: [email protected] (Y. Cury). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.06.041

tremors, autonomic dysfunction, pulmonary edema, cold sudoresis and priapism [5]. The major local clinical manifestation of this envenomation is an intense and radiating local pain which may be followed by local swelling and erythema [5]. The therapeutic approach for pain treatment consists of local infiltration with local anesthetics, and, when more than two infiltrations are necessary, systemic treatment with opioid analgesics is recommended [2,5]. The first experimental investigation to

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examine the mechanisms whereby Phoneutria nigriventer venom induces hyperalgesia has demonstrated that the hypernociceptive effect is mediated, at least partially, by stimulation of capsaicin-sensitive neurons, with activation of peripheral tachykinin NK1 and NK2 receptors and both the N-methyl-d-aspartate (NMDA) and a-amino-3hydroxy-5-methylisoxazole-4-propionic acid (AMPA) peripheral receptors [85]. Despite the clear involvement of neurotransmitters in the nociceptive effect of the venom in the periphery, no information is available about the potential contribution of these transmitters, released in the spinal cord, to the pain-enhancing effect of venom. The dorsal horn of the spinal cord is an important site involved in the integration and modulation of synaptic transfer of sensory input from the periphery to the central nervous system. Various neurotransmitters mediate the transmission of this information [36,56,74]. Peptides, such as substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP), as well as amino acids, such as glutamate, act on specific receptors and play a role in dorsal horn hyperexcitability, contributing to the generation of wind up and central sensitization [74,78]. Activation of these receptors results in the release of nitric oxide (NO), cyclooxygenase products and kinase activation. In addition to these neurotransmitters, spinal glial cells and proinflammatory cytokines have been shown to induce central sensitization, modulating pain behaviors during peripheral inflammation [29,63,64,70,71,73]. Based on these data, the aim of the present study was to characterize the spinal mechanisms involved in the hyperalgesic response induced by Phoneutria nigriventer spider venom, determining the involvement of tachykinin NK1 and NK2 receptors, CGRP receptors, ionotropic and metabotropic glutamate receptors, glial cells, cytokines, nitric oxide, and prostanoids in the pain-enhancing effect of the venom.

2.3. Evaluation of hyperalgesia The animals were injected with either 0.1 ml of sterile saline solution (0.15 M NaCl; control group) or 0.1 ml of saline solution containing 1 Ag of Phoneutria nigriventer venom into the subplantar surface of one hind paw. The contralateral paw was not injected. Pain threshold was measured at different time intervals after venom or saline injection, using an Ugo-Basile pressure apparatus as described by Randall and Selitto [52]. Briefly, a force (in g) of increasing magnitude was applied to the paw and when the animals reacted by withdrawing the paw, the force needed to induce such response was recorded and represented the pain threshold. To reduce stress, rats were habituated to the apparatus 1 day before the experiments. 2.4. Acute lumbar puncture Lumbar puncture was carried out using the technique described by Papir-Kricheli et al. [51]. Each animal was lightly anesthetized by inhalation of 2.5% halothane and the dorsal fur was shaved. With the rat spinal column arched, a 27-gauge hypodermic needle was directly inserted into the subarachnoid space between lumbars (L)5 and L6, localized using two reference points (hip joint and the largest interspinous space). All drugs were slowly injected in a volume of 50 Al, using a 50-Al Hamilton microsyringe [6]. Immediately upon insertion, noted by a change in resistance, a characteristic tail flick was observed, indicating subarachnoid entry. The injection procedure, from the beginning of the anesthetic inhalation until withdrawal of the needle, took 3 min. Animals regained consciousness 1 min after discontinuing the anesthesia. Pilot studies (n=2/group at each time point) revealed that 50 Al of Brilliant Blue 6B (Pontamine Sky Blue 6B, American Tokyo Kasel, Portland, OR) injected at L5/L6 remained restricted to lumbosacral cord when examined 0, 1 or 4 h after injection.

2. Material and methods 2.5. Drug treatments 2.1. Venom Lyophilized crude venom of Phoneutria nigriventer was collected by electrical stimulation and supplied by Laborato´ rio de Artro´podes, Instituto Butantan (Sa˜ o Paulo, Brazil). Samples were kept at 20 8C until use. 2.2. Animals Male Wistar rats, weighing between 170 and 180 g, were used. All procedures involving the use of animals were in accordance with the guidelines for the ethical use of conscious animals in pain research published, by the International Association for the Study of Pain [86], and were approved by the Ethical Committee for the Use of Animals of Instituto Butantan (CEUAIB, Protocol Number 022/2000).

To characterize the spinal mediators involved in hyperalgesia induced by the peripheral administration of Phoneutria nigriventer venom, all drugs were injected by intrathecal (i.t.) route. To determine the involvement of glial cells, fluorocitrate (1 nmol), a glial metabolic inhibitor, was injected 30 min before the intraplantar (i.pl) injection of venom. In order to investigate the participation of neurokinin receptors, the tachykinin NK1 receptor antagonist, pGlu-AlaAsp-Pro-Asn-Lys-Phe-Tyr-Pro(spiro-g-lactam)Leu-TrpNH2 (GR82334), the tachykinin NK2 receptor antagonist, Phenyl-CO-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH 2 (GR94800; 10 Amol) or the CGRP receptor antagonist (CGRP8–37; 20 nmol) were administered 15 min before the intraplantar injection of venom. To examine the involvement of excitatory amino acid receptors, dizocilpine (MK801, 100

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pmol) or (F)-2-amino-5-phosphonopentanoic acid ((F)-AP5, 40 pmol), a noncompetitive and competitive NMDA receptor antagonist, respectively, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 nmol), an non-NMDA ionotropic receptor antagonist, MPEP or AIDA (300 nmol), a metabotropic mGlu5 and mGlu1 receptor antagonists, respectively, were administered immediately or 20 min (metabotropic receptor antagonists) before the i.pl. injection of venom. To evaluate the participation of prostanoids, indomethacin (5.5 mmol), a nonselective cyclooxygenase, or celecoxib (5.2 mmol), a type 2 cyclooxygenase inhibitor was injected 10 min before venom. The contribution of nitric oxide was investigated by treating animals with L-N6-(1-iminoethyl)lysine (L-NIL) or 7-nitroindazole (7-NI; 1.1 Amol), inhibitors of inducible and neuronal nitric oxide synthase, respectively, 15 min before the venom. To investigate the mediation by cytokines, animals were treated with an anti-TNFa antibody (1 Ag) or with an anti-interleukin-1 antibody (10 Ag), 20 min before venom injection. The doses of the drugs used were based on previous investigations [6,7,15,17,23,29,37,41, 48,50,68,71,79]. However, to assure the efficacy of the pharmacological interventions used, the agonists of tachykinin NK1, H2N-(CH2)4-CO-Phe-Phe-Pro-NmeLeu-Met-NH2 (GR73632, 1 nmol) and tachykinin NK2, Lys-Asp-Ser-PheVal-Gly-R-g-lactam-Leu-Met-NH2 (GR64349, 100 nmol) receptors, as well as glutamate (1 ng), were injected by i.t. route and used as positive controls. 2.6. Drugs GR73632, GR64349, GR82334, GR94800, CGRP8–37, MK 801, (F)-AP-5, CNQX, glutamate, indomethacin, 7-NI, L-NIL and fluorocitrate were purchased from Sigma (USA). Celecoxib was purchased from Pharmacia Brasil (Brazil). Anti-IL-1h and Anti-TNFa were purchased from R&D Systems (USA). AIDA and MPEP were purchased from Tocris (USA). Halothane was purchased from Crista´lia (Brazil). 2.7. Statistical analysis Statistical analyses of data were performed by analysis of variance (ANOVA), followed by Tukey’s contrast analysis. Differences with Pb0.05 were considered statistically significant [20].

3. Results 3.1. Effect of neurokinin receptor antagonists on Phoneutria nigriventer venom-induced hyperalgesia 3.1.1. Effects of tachykinin receptor antagonists Intraplantar injection of Phoneutria nigriventer venom (1 Ag/paw) evoked a significant decrease in pain threshold, as compared to pre-injection values (Fig. 1A,B). The peak of

Fig. 1. The antagonists of neurokinin (A, B) and CGRP (C) receptors inhibited hyperalgesia induced by P. nigriventer venom. GR82334, GR94800 (10 Amol/50 Al), or CGRP8–37 (20 nmol/50 Al) was injected i.t. 15 min before venom (ven, 1 Ag/paw). Saline, injected by intraplantar or intrathecal route, was used as control. Decrease in pain threshold was determined in rat hind paw before and at different time intervals after venom injection. Sensitivity to pain was measured as the threshold response to pressure and expressed as g. Each point represents the meanFS.E.M. of six animals. *Significantly different from mean values before venom injection, **significantly different from mean values for Saline+ven group ( Pb0.05).

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hyperalgesic response occurred 4 h after venom injection, decreasing thereafter (Fig. 1A). Injection of saline (vehicle control) by intraplantar or intrathecal route did not modify the pain threshold of animals, as compared to basal values (Fig. 1A). Venom-induced hyperalgesia was significantly inhibited by intrathecal injection of tachykinin NK1 (GR 82334) or NK2 (GR 94800) receptor antagonists. GR 82334 abolished hyperalgesia only at 2 h after venom injection (Fig. 1A), whereas GR 94800 reduced this phenomenon at 1 and 2 h (Fig. 1B). The antagonists per se did not alter the pain threshold of animals (Fig. 1A, B). 3.1.2. Effect of CGRP receptor antagonist Intraplantar injection of Phoneutria nigriventer venom caused a significant increase in sensitivity to pressure pain (Fig. 1C). Pretreatment with intrathecal CGRP8–37 inhibited the hyperalgesic response induced by the venom (Fig. 1C). The antagonist per se did not modify the pain threshold of animals (Fig. 1C). 3.2. Inhibition of hyperalgesia by excitatory amino acid receptor antagonists Intraplantar injection of Phoneutria nigriventer venom caused a significant increase in sensitivity to pressure pain (Fig. 2). Intrathecal injection of MK801, a noncompetitive NMDA receptor antagonist, and AP5, a competitive NMDA antagonist, inhibited the hyperalgesic response induced by Phoneutria nigriventer venom (Fig. 2 A). The non-NMDA ionotropic receptor antagonist, CNQX, signifi-

Fig. 2. Effect of antagonists of glutamate receptors on hyperalgesia induced by P. nigriventer venom. MK801 (100 pmol/50 Al) or AP5 (40 pmol/50 Al) (Panel A), CNQX (10 nmol/50 Al, Panel B), AIDA or MPEP (300 nmol/50 Al, Panel C) were administered i.t. immediately or 20 min (AIDA, MPEP) before venom (1 Ag/paw). Saline, injected by intraplantar route, or vehicle, injected by intrathecal route, was used as control. Decrease in pain threshold was determined in rat hind paw before and at different time intervals after venom injection. Sensitivity to pain was measured as the threshold response to pressure and expressed as g. Each point represents the meanFS.E.M. of six animals. *Significantly different from mean values before venom injection, **significantly different from mean values for Vehicle + venom group ( Pb0.05).

Fig. 3. The cytokine-neutralizing antibodies partially reverse hyperalgesia induced by P. nigriventer venom. Anti-IL-1h (10 Ag/50 Al) or anti-TNF-a (1 Ag/50 Al) was injected intrathecally 20 min before venom (1 Ag/paw). Saline, injected by intraplantar or intrathecal route, was used as control. Decrease in pain threshold was determined in rat hind paw before and at different time intervals after venom injection. Sensitivity to pain was measured as the threshold response to pressure and expressed as g. Each point represents the meanFS.E.M. of six animals. *Significantly different from mean values before venom injection. **Significantly different from mean values for Saline+venom group ( Pb0.05).

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cantly reduced this response only at the 1 h after venom injection (Fig. 2B). The metabotropic mGlu5 receptor antagonist, MPEP, inhibited hyperalgesia at 1 and 2 h after venom injection, whereas the mGlu1 receptor antagonist, AIDA, blocked this response only at 1 h (Fig. 2C). All antagonists did not modify per se the pain threshold of animals (Fig. 2). 3.3. Inhibitory effect of cytokine-neutralizing antibodies on hyperalgesia induced by Phoneutria nigriventer venom Intraplantar injection of Phoneutria nigriventer venom caused a significant increase in sensitivity to pressure pain (Fig. 3). Intrathecal administration of anti-TNFa antibodies blocked the hyperalgesic response up to 2 h after venom injection and partially reversed this response at 4 h (Fig. 3). Interleukin-1h neutralizing antibodies significantly inhibited the mechanical hyperalgesia induced by venom (Fig. 3). The cytokine-neutralizing antibodies did not alter per se the pain threshold of animals (Fig. 3). 3.4. Effect of nitric oxide synthase inhibitors on hyperalgesia induced by Phoneutria nigriventer venom Intraplantar injection of Phoneutria nigriventer venom caused a significant increase in sensitivity to pressure pain (Fig. 4). This hyperalgesic response was significantly

Fig. 4. The nitric oxide synthase inhibitor L-N6-(1-iminoethil)lisina, but not 7-nitroindazole, partially reduce the hyperalgesia induced by P. nigriventer venom. 7-Nitroindazole (7-NI) or L-N6-(1-iminoethyl)lysine (L-NIL; 1.11 Amol/50 Al) was injected intrathecally 10 min before venom (1 Ag/paw). Saline, injected by intraplantar route, or vehicle, injected by intrathecal route was used as control. Decrease in pain threshold was determined in rat hind paw before and at different time intervals after venom injection. Sensitivity to pain was measured as the threshold response to pressure and expressed as g. Each point represents the meanFS.E.M. of six animals. *Significantly different from mean values before venom injection. **Significantly different from mean values for Vehicle + venom group ( Pb0.05).

Fig. 5. The eicosanoid synthesis inhibitors indomethacin (A) and celecoxib (B) block hyperalgesia induced by P. nigriventer venom. Indomethacin (5.5 mmol/50 Al) or celecoxib (5.2 mmol/50 Al) was injected intrathecally 10 min before venom (1 Ag/paw). Saline, injected by intraplantar route, or vehicle, injected by intrathecal route, was used as control. Decrease in pain threshold was determined in rat hind paw before and at different time intervals after venom injection. Sensitivity to pain was measured as the threshold response to pressure and expressed as g. Each point represents the meanFS.E.M. of six animals. *Significantly different from mean values before venom injection. **Significantly different from mean values for Vehicle + venom group ( Pb0.05).

inhibited by the intrathecal injection of L-NIL, an iNOS inhibitor at 1 and 2 h (Fig. 4). On the other hand, the administration of 7-NI, a nNOS inhibitor, did not modify this response (Fig. 4). Both NOS inhibitors did not modify per se the pain threshold of the animals (Fig. 4).

Fig. 6. Fluorocitrate, a glial metabolic inhibitor, did not modify the hyperalgesic response induced by P. nigriventer venom. Fluorocitrate (1 nmol/50 Al) was injected intrathecally 30 min before the venom (1 Ag/paw). Saline, injected by intraplantar or intrathecal route was used as control. Decrease in pain threshold was determined in rat hind paw before and at different time intervals after venom injection. Sensitivity to pain was measured as the threshold response to pressure and expressed as g. Each point represents the meanFS.E.M. of six animals. *Significantly different from mean values before venom injection.

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3.5. Effect of prostanoid synthesis inhibitors on hyperalgesia induced by Phoneutria nigriventer venom

of venom (Fig. 5). Both inhibitors did not alter per se the pain threshold of the animals (Fig. 5).

Intraplantar injection of Phoneutria nigriventer venom caused a significant increase in sensitivity to pressure pain (Fig. 5). Intrathecal administration of indomethacin, a nonselective COX1 and COX2 inhibitor, or celecoxib, a COX2 selective inhibitor, blocked the pain-enhancing effect

3.6. Lack of effect of fluorocitrate on hyperalgesia induced by Phoneutria nigriventer venom Intraplantar injection of Phoneutria nigriventer venom caused a significant increase in sensitivity to pressure pain.

Fig. 7. Effect of tachykinin and excitatory amino acid receptor antagonists on hyperalgesia induced by tachykinin NK1 or NK2 receptors and glutamate. GR82334 or GR94800 (10 Amol/50 Al), or saline (control group) was injected intrathecally 15 min before GR73632 (1 nmol/50 Al) or GR64349 (100 nmol/50 Al, Panel A). MK801 (100 pmol/50 Al), AP5 (40 pmol/50 Al), CNQX (10 nmol/50 Al), AIDA or MPEP (300 nmol/50 Al, Panel B) or vehicle (control group) were administered intrathecally immediately or 20 min (AIDA, MPEP) before glutamate (1 ng/50 Al). Sensitivity to pain was measured as the threshold response to pressure and expressed as g. Data represent decrease in pain threshold 1 h after injection of tachykinin NK1 or NK2 receptor agonists (Panel A) or glutamate (Panel B) injection. Each point represents the meanFS.E.M. of six animals. *Significantly different from mean values for GR72632 or GR64349 (Panel A) or Glutamate (Panel B) group ( Pb0.05).

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Pretreatment of animals with the glial cell metabolic inhibitor, fluorocitrate, did not modify the hyperalgesic response induced by venom (Fig. 6). The drug, per se, did not alter the pain threshold of animals (Fig. 6). 3.7. Effect of pharmacological treatment on hyperalgesia induced by intrathecal administration of tachykinin or glutamate receptor agonists To determine the efficacy of the pharmacological treatments, the effect of the tachykinin receptor antagonists were evaluated against the hyperalgesic effect induced by tachykinin receptor agonists. The injection of tachykinin NK1 (GR73632) or NK2 (GR64349) agonists into the spinal cord induced a maximal decrease in pain threshold at 1 h after administration of agonists (Fig. 7A). The hyperalgesic response was abolished by the respective tachykinin NK1 or NK2 receptor antagonist, but was not modified by the noncorresponding antagonist (Fig. 7A). To assure the efficacy of the dose of glutamate receptor antagonists, glutamate was used as positive control. The intrathecal injection of glutamate induced a decrease in the pain threshold of animals, which peaked at 1 h after injection (Fig. 7B). Intrathecal administration of either ionotropic (AP5, MK 801 or CNQX) or metabotropic (AIDA or MPEP) glutamate receptor antagonists significantly inhibited the hyperalgesic response caused by glutamate (Fig. 7B).

4. Discussion A previous work demonstrated that intraplantar injection of Phoneutria nigriventer spider venom in rats induces hyperalgesia mediated mainly by activation of peripheral tachykinin NK1 and NK2 receptors and peripheral NMDA and AMPA receptors [85]. Our present results extended the characterization of mechanisms involved in the painenhancing effect of venom, providing the first investigation of the spinal mediators responsible for it. The dose of venom used (1 Ag/paw) was based on previous work, which showed that it reduced the pain threshold of animals by 50% [85]. The present results provide evidence of the role of spinal tachykinin receptors as mediators of the pain-enhancing effect of Phoneutria nigriventer venom, inasmuch as intrathecal administration of antagonists of tachykinin NK1 and NK2 receptors reduced venom-induced hyperalgesia. These results corroborate several data indicating that tachykinin NK1 and NK2 receptors are involved in hypernociceptive states induced by peripheral inflammation [8,10,32,47,57]. It is important to point out that the inhibitory effect of both tachykinin NK1 and NK2 receptor antagonists on Phoneutria nigriventer venom-induced hyperalgesia was observed only in the earlier periods (1 to 2 h) of the hypernociceptive response, suggesting that other receptors and neurotransmitters con-

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tributed to the development of later stages of the painenhancing effect of venom. In addition, the present results also indicate a distinct role for both tachykinin receptors, since the NK2 tachykinin receptor antagonist inhibited the hyperalgesic effect at 1 and 2 h after venom injection, whereas the NK1 tachykinin receptor antagonist blocked this response only at 2 h after venom injection. Several reports have shown a distinct role for NK1 and NK2 receptors in nociceptive transmission during injury. In the dorsal horn, NK2 receptors seem to have a more prominent role than NK1 receptors for intense and brief nociceptive stimuli, particularly during the development of hypernociceptive states [18,45,61]. Furthermore, a distinct release of tachykinin NK1 and NK2 receptor agonists, SP and NKA, respectively [54], has been demonstrated following noxious peripheral stimuli. SP release is focal and short-lasting, whereas neurokinin A release is diffuse through the dorsal horn and more long-lasting than SP [13,14]. Results presented herein also suggest that CGRP is involved in the spinal mediation of hyperalgesia induced by Phoneutria nigriventer venom, since intrathecal injection of the CGRP receptor antagonist inhibited this phenomenon. The contribution of CGRP, released in the dorsal horn, to hypernociceptive states observed during peripheral inflammation has also been demonstrated [34,35,46,83]. Since the mechanisms responsible for CGRP action may involve the release of SP and excitatory amino acids in the dorsal horn [34,49], an interaction between CGRP and SP in the mediation of Phoneutria nigriventer venom-induced hyperalgesia can be presently suggested. In addition to neurokinins, excitatory amino acids, especially glutamate, participate in nociceptive mechanisms in the dorsal horn of the spinal cord [9,24,48,55,60,75]. In the present study, the involvement of NMDA and nonNMDA receptors in the hyperalgesic effect induced by Phoneutria nigriventer venom was evidenced by results showing that ionotropic and metabotropic glutamate receptor antagonists interfered with the pain-enhancing effect of venom. The involvement of NMDA receptors seems to be more prominent than non-NMDA ionotropic receptors, since MK 801 and AP5 fully inhibited the hyperalgesic effect of venom, whereas CNQX reversed this effect only at 1 h after venom injection. These results corroborate several pieces of evidence, which indicate that activation of NMDA receptors is important for generating and maintaining dorsal horn neuron hyperexcitability and hyperalgesia [9,24,48,55]. In addition to glutamate ionotropic receptors, a significant role was also observed for metabotropic mGlu1 and mGlu5 receptors, which are predominantly involved in the earlier stages of the hypernociceptive response induced by venom. This is because AIDA and MPEP, mGlu1 and mGlu5 receptor antagonists, respectively, inhibited venominduced hyperalgesia at 1 and 2 h after venom injection. A role has been demonstrated for spinal metabotropic mGlu1 and mGlu5 receptors in nociceptive processing and central sensitization [16,19,82], although some investigations have

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not confirmed the contribution of mGlu5 receptors to inflammatory hyperalgesia [68,84]. Despite these controversial reports, the present investigation further contributes to establish an important role for glutamate metabotropic receptors in the spinal mechanisms responsible for the development of peripheral hypernociceptive states. An interaction between metabotropic and ionotropic glutamate receptors in the spinal cord has also been proposed [4,33], which might also occur in Phoneutria nigriventer venominduced hyperalgesia and contribute to central sensitization caused by intraplantar venom injection. Data presented herein indicate that peripherally administered Phoneutria nigriventer venom triggers the spinal release of neurokinins and glutamate. In primary sensory afferent neurons, glutamate and substance P coexist, being released by the same stimulus [3,11]. A possible cooperative action between these neurotransmitters in the development of venom-induced hyperalgesia should be considered, since enhanced spinal excitability induced by SP could be mediated by an increase in the release of excitatory amino acids [34,38] and by activation of NMDA receptors [53,67]. The spinal release of neurokinins and glutamate by i.pl. injection of Phoneutria nigriventer venom could induce the activation of spinal glial cells, since several investigations have indicated that these neurotransmitters are able to activate these cells [72]. However, our results indicate that Phoneutria nigriventer venom facilitates pain responses by mechanisms independent of glial activation, since the intrathecal administration of fluorocitrate, a glial metabolic inhibitor, did not modify the hyperalgesic effect of this venom. Results described herein also showed that spinal inflammatory cytokines are involved in the genesis of the painenhancing effect of Phoneutria nigriventer venom, since intrathecal administration of neutralizing antibodies against TNF and IL-1 partially reversed venom-induced hyperalgesia. The involvement of both cytokines was observed from 1 h after venom injection onwards, suggesting that these cytokines can be rapidly released in the spinal cord after peripheral inflammation. Our results are in accordance with previous reports that have demonstrated the involvement of spinal proinflammatory cytokines in exaggerated pain states [42,63,64]. A cooperative action between those cytokines in inducing the venom effect may be presently suggested, inasmuch as a synergistic action between tumour necrosis factor-a (TNFa) and interleukin-1h (IL-1h) in promoting exaggerated pain states was evidenced by Sweitzer et al. [64]. Furthermore, an interaction between cytokines and other neurotransmitters in venom-induced hyperalgesia might also be suggested, since IL-1 increases the release of SP [12,30,31]. It is important to consider the possible source of these cytokines in the spinal cord. Spinal glial cells, which can release proinflammatory cytokines [69,72], cannot be currently considered as the source of these cytokines, since we could not detect an activation of these cells by venom. Several data have indicated that, in the spinal cord, in addition to glial cells (astrocytes, oligoden-

drocytes, and microglia), neurons can contribute to the early production of spinal proinflammatory cytokines during injury [80,81]. Based on these data, neurons can be suggested as a possible source of TNF and IL-1 released in the spinal cord after peripheral administration of Phoneutria nigriventer venom. In addition to proinflammatory cytokines, we presently demonstrated that nitric oxide is involved in the earlier periods (at the first and second hours) of the hyperalgesic response induced by the spider venom. Nitric oxide is a key mediator of nociceptive mechanisms and spinal NO has been implicated in the central mechanisms of peripheral inflammatory hyperalgesia [25,39,50]. Results described herein also indicate that the inducible isoform of the nitric oxide synthase (iNOS) seems to be responsible for NO synthesis, since intrathecal administration of L-NIL, a relatively selective iNOS inhibitor [44], but not of 7-NI, a relatively selective neuronal NOS inhibitor [43], reduced the pain-enhancing effect of venom. The dose of 7-NI presently used was based on previous data [50], being effective in reducing carrageenin-induced changes in the dorsal horn of rats [62]. It is important to stress that iNOS activity was detected as soon as 1 h after venom injection. Some experimental studies have evidenced that the time course of induction of spinal cord iNOS expression is related to peripheral nociceptive stimuli. Guhring et al. [22] detected iNOS mRNA in the mouse spinal cord 30 min after zymosan injection, whereas Wu et al. [76] found an increase in iNOS protein in the rat spinal cord 20 min after capsaicin injection. As proposed by Wu et al. [76], the fast iNOS protein synthesis may be due to the presence of a high concentration of mRNA encoding iNOS in the spinal cord. Spinal NO can be generated as a consequence of the action of neurotransmitters or other hypernociceptive mediators, such as cytokines [27,40,66]. Based on the present results and on data from the literature, it is plausible to suggest that intraplantar administration of Phoneutria nigriventer venom causes the release of neurotransmitters and/or cytokines in the spinal cord, which in turn leads to the production of iNOS. In addition, since NO can release SP, CGRP and cytokines in the spinal cord (TNF and IL-1) [1,21,28], the ability of the NOS inhibitor to reduce venom-induced hyperalgesia may potentially reflect an inhibition release of these hypernociceptive mediators. Results presented herein also indicate that spinal prostanoids, generated by COX-2 activity, are important mediators of Phoneutria nigriventer venom-induced pain facilitation, since celecoxib blocks its hyperalgesic effect. These results corroborate previous investigations showing that peripheral inflammation increases the expression of inducible cyclooxygenase and prostaglandin E2 formation in the spinal cord, contributing to nociceptive sensitization [26,59,77]. Several lines of evidence suggest that prostaglandins induce the release of neurotransmitters, such as excitatory amino acids, SP, CGRP and NO. Conversely, glutamate, SP, CGRP, cytokines and NO increase PG release

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[65]. Based on these data, a cooperative role between spinal prostanoids and those neurotransmitters may occur and contribute to the pain enhancing effect of venom. In conclusion, data presented herein suggest a role for spinal neurokinins and glutamate receptors in the genesis of hyperalgesia induced by subcutaneous injection of Phoneutria nigriventer spider venom. In addition, spinal proinflammatory cytokines, NO and prostanoids are important mediators of this phenomenon. To our knowledge, this is the first experimental study of the central mechanisms involved in the pain facilitation phenomenon induced by Phoneutria nigriventer venom and may contribute to the further understandings of mechanisms involved in exaggerated pain associated with human envenomation by this spider, as well as to therapeutic approaches for pain control. Acknowledgements This work was supported by a grant from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP, Grant No. 00/06965-8), Brazil and by a fellowship from Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Brazil.

References [1] P. Aimar, L. Pasti, G. Carmignoto, A. Merighi, Nitric oxide-producing islet cells modulate the release of sensory neuropeptides in the rat substantia gelatinosa, J. Neurosci. 18 (1998) 10375 – 10388. [2] E. Antunes, C.M.S. Malaque, Mecanismo de ac¸a˜o do veneno de Phoneutria e aspectos clı´nicos do foneutrismo, in: J.L.C. Cardoso, F.O.S. Franc¸a, F.H. Wen, C.M.S. Ma´laque (Eds.), Animais Pec¸onhentos no Brasil, Biologia, Clı´nica e Terapeˆutica dos Acidentes, Sarvier, Sa˜o Paulo, 2003, pp. 150 – 159. [3] G. Battaglia, A. Rustioni, Coexistence of glutamate and substance P in dorsal root ganglion neurons of the rat and monkey, J. Comp. Neurol. 277 (1988) 302 – 312. [4] D. Bleakman, K.I. Rusin, P.S. Chard, S.R. Glaum, R.J. Miller, Metabotropic glutamate receptors potentiate ionotropic glutamate responses in the rat dorsal horn, Mol. Pharmacol. 42 (1992) 192 – 196. [5] F. Bucharetchi, Acidentes por Phoneutria, in: S. Schvartsman (Ed.), Plantas venenosas e animais pec¸onhentos, Sarvier, Sa˜o Paulo, 1992, pp. 196 – 201. [6] M. Chacur, J.M. Gutie´rrez, E.D. Milligan, J. Wieseler-Frank, L.R.G. Britto, S.F. Maier, L.R. Watkins, Y. Cury, Snake venom components enhance pain upon subcutaneous injection: an initial examination of spinal cord mediators, Pain (2004) (in press). [7] M. Chacur, E.D. Milligan, E.M. Sloan, J. Wieseler-Frank, R.M. Barrientos, D. Martin, S. Poole, B. Lomonte, J.M. Gutierrez, S.F. Maier, Y. Cury, L.R. Watkins, Snake venom phospholipase A2s (Asp49 and Lys49) induce mechanical allodynia upon peri-sciatic administration: involvement of spinal cord glia, proinflammatory cytokines and nitric oxide, Pain 108 (2004) 180 – 191. [8] V. Chapman, A.H. Dickenson, The effect of intrathecal administration of RP67580, a potent neurokinin 1 antagonist on nociceptive transmission in the rat spinal cord, Neurosci. Lett. 157 (1993) 149 – 152. [9] T. Coderre, R. Melzack, The contribution of excitatory amino acids to central sensitization and persistent nociception after formalin-induced tissue injury, J. Neurosci. 12 (1992) 3665 – 3670.

109

[10] M.A. Coudore-Civiale, C. Courteix, A. Eschalier, J. Fialip, Effect of tachykinin receptor antagonists in experimental neuropathic pain, Eur. J. Pharmacol. 361 (1998) 175 – 184. [11] S. De Biasi, A. Rustioni, Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 7820 – 7824. [12] M. Ding, R.P. Hart, G.M. Jonakait, Tumor necrosis factor-alpha induces substance P in sympathetic ganglia through sequential induction of interleukin-1 and leukemia inhibitory factor, J. Neurobiol. 28 (1995) 445 – 454. [13] A.W. Duggan, I.A. Hendry, C.R. Morton, W.D. Hutchison, Z.Q. Zhao, Cutaneous stimuli releasing immunoreactive substance P in the dorsal horn of the cat, Brain Res. 451 (1988) 261 – 273. [14] A.W. Duggan, P.J. Hope, B. Jarrott, H.G. Schaible, S.M. FleetwoodWalker, Release, spread and persistence of immunoreactive neurokinin A in the dorsal horn of the cat following noxious cutaneous stimulation. Studies with antibody microprobes, Neuroscience 35 (1990) 195 – 202. [15] S.H. Ferreira, B.B. Lorenzetti, Glutamate spinal retrograde sensitization of primary sensory neurons associated with nociception, Neuropharmacology 33 (1994) 1479 – 1485. [16] K. Fisher, T.J. Coderre, Comparison of nociceptive effects produced by intrathecal administration of mGluR agonists, NeuroReport 7 (1996) 2743 – 2747. [17] K. Fisher, C. Lefebvre, T.J. Coderre, Antinociceptive effects following intrathecal pretreatment with selective metabotropic glutamate receptor compounds in a rat model of neuropathic pain, Pharmacol. Biochem. Behav. 73 (2002) 411 – 418. [18] S.M. Fleetwood-Walker, R.M.C. Parker, F.E. Munro, M.R. Young, P.J. Hope, R. Mitchell, Evidence for a role of NK2 receptors in mediating brief nociceptive inputs to rat dorsal horn (laminae II–V), Eur. J. Pharmacol. 242 (1993) 173 – 181. [19] M.E. Fumdytus, K. Fisher, A. Dray, J.L. Henry, T.J. Coderre, In vivo antinociceptive activity of anti-rat mGlu1 and mGlu5 antibodies in rats, NeuroReport 9 (1999) 731 – 735. [20] S.C. Gad, C.S. Weil, Statistics for toxicologists, in: A.W. Hayer (Ed.), Principles and Methods of Toxicology, vol. 2, 1989, pp. 435 – 483 New York. [21] M.G. Garry, K.M. Hargreaves, Enhanced release of immunoreactive CGRP and substance P from spinal dorsal horn slices occurs during carrageenan inflammation, Brain Res. 582 (1992) 139 – 142. [22] H. Guhring, M. Gorig, M. Ates, O. Coste, H.U. Zeilhofer, A. Pahl, K. Rehse, K. Brune, Suppressed injury-induced rise in spinal prostaglandin E2 production and reduced early thermal hyperalgesia in iNOS-deficient mice, J. Neurosci. 20 (2000) 6714 – 6720. [23] J.Z. Guo, K. Yoshioka, F.Y. Zhao, R. Hosoki, T. Maehara, M. Yanagisawa, R.M. Hagan, M. Otsuka, Pharmacological characterization of GR82334, a tachykinin NK1 receptor antagonist, in the isolated spinal cord of the neonatal rat, Eur. J. Pharmacol. 281 (1995) 49 – 54. [24] J.E. Haley, A.F. Sullivan, A.H. Dickenson, Evidence for spinal Nmethyl-d-aspartate receptor involvement in prolonged chemical nociception in the rat, Brain Res. 518 (1990) 218 – 226. [25] R.L. Handy, P.K. Moore, Effects of selective inhibitors of neuronal nitric oxide synthase on carrageenan-induced mechanical and thermal hyperalgesia, Neuropharmacology 37 (1998) 37 – 43. [26] C. Hay, J. de Belleroche, Carrageenan-induced hyperalgesia is associated with increased cyclo-oxygenase-2 expression in spinal cord, NeuroReport 8 (1997) 1249 – 1251. [27] M.T. Heneka, D.L. Feinstein, Expression and function of inducible nitric oxide synthase in neurons, J. Neuroimmunol. 114 (2001) 8 – 18. [28] A. Holguim, J. Biedenkapp, J. Campisi, J. Wieseler-Frank, K.A. O´Connor, E.D. Milligan, E. Maksimova, C. Bravmann, M.K. Hansen, D. Martin, M. Fleshner, S.F. Maier, L.R. Watkins, HIV-1 gp 120 stimulates proinflammatory cytokine-mediated pathological pain via activation of nitric oxide synthase-I (nNOS), Pain (2004).

110

E.M. Zanchet et al. / Brain Research 1021 (2004) 101–111

[29] S.J. Hopkins, N.J. Rothwell, Cytokines and the nervous system: I. Expression and recognition, Trends Neurosci. 18 (1995) 83 – 88. [30] A. Inoue, K. Ikoma, N. Morioka, K. Kumagai, T. Hashimoto, I. Hide, Y. Nakata, Interleukin-1beta induces substance P release from primary afferent neurons through the cyclooxygenase-2 system, J. Neurochem. 73 (1999) 2206 – 2213. [31] A.P. Jeanjean, S.M. Moussaoui, J.M. Maloteaux, P.M. Laduron, Interleukin-1 beta induces long-term increase of axonally transported opiate receptors and substance P, Neuroscience 68 (1995) 151 – 157. [32] Y.P. Jia, V.S. Seybold, Spinal NK2 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation, Brain Res. 751 (1997) 169 – 174. [33] M.W. Jones, P.M. Headley, Interactions between metabotropic and ionotropic glutamate receptor agonists in the rat spinal cord in vivo, Neuropharmacology 34 (1995) 1025 – 1031. [34] I. Kangrga, M. Randic, Tachykinins and calcitonin gene-related peptide enhance release of endogenous glutamate and aspartate from the rat spinal dorsal horn slice, J. Neurosci. 10 (1990) 2026 – 2038. [35] M. Kawamura, Y. Kuraishi, M. Minami, M. Satoh, Antinociceptive effect of intrathecally administered antiserum against calcitonin generelated peptide on thermal and mechanical noxious stimuli in experimental hyperalgesic rats, Brain Res. 497 (1989) 199 – 203. [36] B.L. Kidd, L.A. Urban, Mechanisms of inflammatory pain, Br. J. Anaesth. 87 (2001) 3 – 11. [37] Y. Long-Chuan, P. Hansson, S. Lundeberg, T. Lundeberg, Effects of calcitonin gene-related peptide-m (8–37) on withdrawal responses in rats with inflammation, Eur. J. Pharmacol. 347 (1998) 275 – 282. [38] T. Maehara, H. Suzuki, K. Yoshioka, M. Otsuka, Characteristics of substance P-evoked release of amino acids from neonatal rat spinal cord, Neuroscience 68 (1995) 577 – 584. [39] A.B. Malmberg, T.L. Yaksh, Spinal nitric oxide synthesis inhibition blocks NMDA-induced thermal hyperalgesia and produces antinociception in the formalin test in rats, Pain 54 (1993) 291 – 300. [40] S.T. Meller, G.F. Gebhart, Nitric oxide (NO) and nociceptive processing in the spinal cord, Pain 52 (1993) 127 – 136. [41] E.D. Milligan, K.K. Mehmert, J.L. Hinde, L.O. Harvey, D. Martin, K.J. Tracey, S.F. Maier, L.R. Watkins, Thermal hyperalgesia and mechanical allodynia produced by intrathecal administration of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein, gp120, Brain Res. 861 (2000) 105 – 116. [42] E.D. Milligan, K.A. O’Connor, K.T. Nguyen, C.B. Armstrong, C. Twining, R.P. Gaykema, A. Holguin, D. Martin, S.F. Maier, L.R. Watkins, Intrathecal HIV-1 envelope glycoprotein gp120 induces enhanced pain states mediated by spinal cord proinflammatory cytokines, J. Neurosci. 21 (2001) 2808 – 2819. [43] P.K. Moore, P. Wallace, Z. Gaffen, S.L. Hart, R.C. Babbedge, Characterization of the novel nitric oxide synthase inhibitor 7-nitro indazole and related indazoles: antinociceptive and cardiovascular effects, Br. J. Pharmacol. 110 (1993) 219 – 224. [44] W.M. Moore, R.K. Webber, G.M. Jerome, F.S. Tjoeng, T.P. Misko, M.G. Currie, L-N6-(1-iminoethyl)lysine: a selective inhibitor of inducible nitric oxide synthase, J. Med. Chem. 37 (1994) 3886 – 3888. [45] F.E. Munro, S.M. Fleetwood-Walker, R.M. Parker, R. Mitchell, The effects of neurokinin receptor antagonists on mustard oil-evoked activation of rat dorsal horn neurons, Neuropeptides 25 (1993) 299 – 305. [46] V. Neugebauer, P. Rumenapp, H.G. Schaible, Calcitonin gene-related peptide is involved in the spinal processing of mechanosensory input from the rat’s knee joint and in the generation and maintenance of hyperexcitability of dorsal horn-neurons during development of acute inflammation, Neuroscience 71 (1996) 1095 – 1109. [47] V. Neugebauer, P. Rumenapp, H.G. Schaible, The role of spinal neurokinin-2 receptors in the processing of nociceptive information from the joint and in the generation and maintenance of inflammationevoked hyperexcitability of dorsal horn neurons in the rat, Eur. J. Neurosci. 8 (1996) 249 – 260.

[48] K. Okano, Y. Kuraiashi, M. Satoh, Involvement of spinal substance P and excitatory amino acids in inflammatory hyperalgesia in rats, Jpn. J. Pharmacol. 76 (1998) 15 – 22. [49] R. Oku, M. Satoh, N. Fujii, A. Otaka, H. Yajima, H. Takagi, Calcitonin gene-related peptide promotes mechanical nociception by potentiating release of substance P from the spinal dorsal horn in rats, Brain Res. 403 (1987) 350 – 354. [50] M.G. Osborne, T.J. Coderre, Effects of intrathecal administration of nitric oxide synthase inhibitors on carrageenan-induced thermal hyperalgesia, Br. J. Pharmacol. 126 (1999) 1840 – 1846. [51] D. Papir-Kricheli, J. Frey, R. Laufer, C. Gilon, M. Chorev, Z. Selinger, M. Devor, Behavioral effects of receptor-specific substance P agonists, Pain 31 (1987) 263 – 276. [52] L.O. Randall, J.J. Selitto, A method for measurement of analgesic activity on inflamed tissue, Arch. Int. Pharmacodyn. 111 (1957) 209 – 219. [53] M. Randic, H. Hecimovic, P.D. Ryu, Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurones, Neurosci. Lett. 117 (1990) 74 – 80. [54] D. Regoli, A. Boudon, J.L. Fauchere, Receptors and antagonists for substance P and related peptides, Pharmacol. Rev. 46 (1994) 551 – 599. [55] K. Ren, R. Dubner, NMDA receptor antagonists attenuate mechanical hyperalgesia in rats with unilateral inflammation of the hindpaw, Neurosci. Lett. 163 (1993) 22 – 26. [56] W. Riedel, G. Neeck, Nociception, pain, and antinociception: current concepts, Z. Rheumatol. 60 (2001) 404 – 415. [57] T. Sakurada, Y. Manome, K. Tan-No, S. Sakurada, K. Kisara, M. Ohba, L. Terenius, A selective and extremely potent antagonist of the neurokinin-1 receptor, Brain Res. 593 (1992) 319 – 322. [58] Brazilian Health Ministry, Acidentes por Phoneutria, in: Brazilian Health Ministry (Ed.), Manual de Diagno´stico e Tratamento de Acidentes por Animais Pec¸onhentos, Fundac¸a˜o de Sau´de, Brası´lia, 1998, pp. 54 – 56. [59] V.S. Seybold, Y.P. Jia, L.G. Abrahams, Cyclo-oxygenase-2 contributes to central sensitization in rats with peripheral inflammation, Pain 105 (2003) 47 – 55. [60] S.R. Skilling, D.H. Smullin, A.J. Beitz, A.A. Larson, Extracellular amino acid concentrations in the dorsal spinal cord of freely moving rats following veratridine and nociceptive stimulation, J. Neurochem. 51 (1988) 127 – 132. [61] K.A. Sluka, M.A. Milton, W.D. Willis, K.N. Westlund, Differential roles of neurokinin 1 and neurokinin 2 receptors in the development and maintenance of heat hyperalgesia induced by acute inflammation, Br. J. Pharmacol. 120 (1997) 1263 – 1273. [62] L.C. Stanfa, C. Misra, A.H. Dickenson, Amplification of spinal nociceptive transmission depends on the generation of nitric oxide in normal and carrageenan rats, Brain Res. 737 (1996) 92 – 98. [63] S.M. Sweitzer, R.W. Colburn, M. Rutkowski, J.A. DeLeo, Acute peripheral inflammation induces moderate glial activation and spinal IL-1beta expression that correlates with pain behavior in the rat, Brain Res. 829 (1999) 209 – 221. [64] S. Sweitzer, D. Martin, J.A. DeLeo, Intrathecal interleukin-1 receptor antagonist in combination with soluble tumor necrosis factor receptor exhibits an anti-allodynic action in a rat model of neuropathic pain, Neuroscience 103 (2001) 529 – 539. [65] C.I.Y. Swensson, T. L, The spinal phospholipase–cyclooxygenase– prostanoid cascade in nociceptive processing, Annu. Rev. Pharmacol. Toxicol. 42 (2002) 553 – 583. [66] Y.X. Tao, R.A. Johns, Activation of cGMP-dependent protein kinase I alpha is required for N-methyl-d-aspartate- or nitric oxide-produced spinal thermal hyperalgesia, Eur. J. Pharmacol. 392 (2000) 141 – 145. [67] L. Urban, S.W. Thompson, A. Dray, Modulation of spinal excitability: co-operation between neurokinin and excitatory amino acid neurotransmitters, Trends Neurosci. 17 (1994) 432 – 438. [68] K. Walker, A. Reeve, M. Bowes, J. Winter, G. Wotherspoon, A. Davis, P. Schmid, F. Gasparini, R. Kuhn, L. Urban, mGlu5 receptors and nociceptive function: II. mGlu5 receptors functionally expressed

E.M. Zanchet et al. / Brain Research 1021 (2004) 101–111

[69]

[70] [71]

[72] [73]

[74]

[75]

[76]

[77]

on peripheral sensory neurones mediate inflammatory hyperalgesia, Neuropharmacology 40 (2001) 10 – 19. L.R. Watkins, S.F. Maier, Beyond neurons: evidence that immune and glial cells contribute to pathological pain states, Physiol. Rev. 82 (2002) 981 – 1011. L.R. Watkins, S.F. Maier, GLIA: a novel drug discovery target for clinical pain, Nat. Rev., Drug Discov. 2 (2003) 973 – 985. L.R. Watkins, D. Martin, P. Ulrich, K.J. Tracey, S.F. Maier, Evidence for the involvement of spinal cord glia in subcutaneous formalin induced hyperalgesia in the rat, Pain 71 (1997) 225 – 235. L.R. Watkins, E.D. Milligan, S.F. Maier, Glial activation: a driving force for pathological pain, Trends Neurosci. 24 (2001) 450 – 455. B.A. Winkelstein, M.D. Rutkowski, S.M. Sweitzer, J.L. Pahl, J.A. DeLeo, Nerve injury proximal or distal to the DRG induces similar spinal glial activation and selective cytokine expression but differential behavioral responses to pharmacologic treatment, J. Comp. Neurol. 439 (2001) 127 – 139. C.J. Woolf, M. Costigan, Transcriptional and posttranslational plasticity and the generation of inflammatory pain, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 7723 – 7730. C.J. Woolf, S.W. Thompson, The induction and maintenance of central sensitization is dependent on N-methyl-d-aspartic acid receptor activation; implications for the treatment of post-injury pain hypersensitivity states, Pain 44 (1991) 293 – 299. J. Wu, L. Fang, Q. Lin, W.D. Willis, Nitric oxide synthase in spinal cord central sensitization following intradermal injection of capsaicin, Pain 94 (2001) 47 – 58. T.L. Yaksh, A.B. Malmberg, Spinal actions of NSAIDS in blocking spinally mediated hyperalgesia: the role of cyclooxygenase products, Agents Actions Suppl. 41 (1993) 89 – 100.

111

[78] T.L. Yaksh, X.Y. Hua, I. Kalcheva, N. Nozaki-Taguchi, M. Marsala, The spinal biology in humans and animals of pain states generated by persistent small afferent input, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 7680 – 7686. [79] T. Yamamoto, N. Nozaki-Taguchi, The role of cyclooxygenase-1 and 2 in the rat formalin test, Anesth. Analg. 94 (2002) 962 – 967. [80] P. Yan, Q. Li, G.M. Kim, J. Xu, C.Y. Hsu, X.M. Xu, Cellular localization of tumor necrosis factor-alpha following acute spinal cord injury in adult rats, J. Neurotrauma 18 (2001) 563 – 568. [81] L. Yang, P.C. Blumbergs, N.R. Jones, J. Manavis, G.T. Sarvestani, M.N. Ghabriel, Early expression and cellular localization of proinflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in human traumatic spinal cord injury, Spine 29 (2004) 966 – 971. [82] M.R. Young, S.M. Fleetwood-Walker, T. Dickinson, G. BlackburnMunro, H. Sparrow, P.J. Birch, C. Bountra, Behavioural and electrophysiological evidence supporting a role for group I metabotropic glutamate receptors in the mediation of nociceptive inputs to the rat spinal cord, Brain Res. 777 (1997) 161 – 169. [83] L.C. Yu, P. Hansson, S. Lundeberg, T. Lundeberg, Effects of calcitonin gene-related peptide-(8–37) on withdrawal responses in rats with inflammation, Eur. J. Pharmacol. 347 (1998) 275 – 282. [84] P.K. Zahn, T.J. Brennan, Intrathecal metabotropic glutamate receptor antagonists do not decrease mechanical hyperalgesia in a rat model of postoperative pain, Anesth. Analg. 87 (1998) 1354 – 1359. [85] E.M. Zanchet, Y. Cury, Peripheral tachykinin and excitatory amino acid receptors mediate hyperalgesia induced by Phoneutria nigriventer venom, Eur. J. Pharmacol. 467 (2003) 111 – 118. [86] M. Zimmermann, Ethical guidelines for investigations of experimental pain in conscious animals, Pain 16 (1983) 109 – 110.