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5-HT acts on nociceptive primary afferents through an indirect mechanism to induce hyperalgesia in the subcutaneous tissue
Neuroscience 145 (2007) 708 –714
5-HT ACTS ON NOCICEPTIVE PRIMARY AFFERENTS THROUGH AN INDIRECT MECHANISM TO INDUCE HYPERALGESIA IN THE SUBCUTANEOUS TISSUE M. C. G. OLIVEIRA,a A. PELEGRINI-DA-SILVA,a C. A. PARADAb AND C. H. TAMBELIa*
becomes effective in inducing nociception. This primary sensory nociceptor sensitization is referred to as hyperalgesia. 5-Hydroxytryptamine (5-HT) is an important inflammatory mediator (Pierce et al., 1995; Xie et al., 2003) released from platelets and mast cells in response to tissue injury. 5-HT induces hyperalgesia and nociception by sensitizing and exciting afferent nerve fibers of different peripheral tissues such as articular (Herbert and Schmidt, 1992), dermal (Taiwo and Levine, 1992), muscular (Ernberg et al., 2000) and s.c. (Sufka et al., 1992; Parada et al., 2001; Tambeli et al., 2006). We have recently demonstrated (Tambeli et al., 2006) that the nociceptive effect of 5-HT in the s.c. tissue is exerted by an indirect action of 5-HT on the primary afferent neurons, because this effect is attenuated by polymorphonuclear leukocyte depletion, cyclooxygenase inhibition, norepinephrine depletion from the sympathetic terminals, and by local blockade of the 1- or 2-adrenergic receptor. Although it has been previously suggested that the mechanisms mediating hyperalgesia can be quite separate and distinct from those mediating nociception (Taiwo and Levine, 1991; Waldron and Sawynok, 2004), this suggestion does not exclude the involvement of common mechanisms. The aim of this study was to test the hypothesis that 5-HT induces mechanical hyperalgesia by mechanisms similar to those mediating nociception, that is, by a combination of mechanisms involved in inflammation such as neutrophil migration (Secco et al., 2003) and the release of local prostaglandin (Claria and Romano, 2005) and/or sympathomimetics amines (Levine et al., 1986; Green et al., 1993). The experimental design of the current study was very similar to that of our previous one (Tambeli et al., 2006). However, the Randall-Selitto nociceptive paw-withdrawal flexion reflex test was used as experimental assay for hyperalgesia in the current study and the spontaneous nociceptive flinching and shaking behavior was used as experimental assays for nociception in our previous study (Tambeli et al., 2006).
a
Department of Physiology, Laboratory of Orofacial Pain, Faculty of Dentistry of Piracicaba, University of Campinas, UNICAMP, Av. Limeira, 901, Zip Code 13414-900, Piracicaba, São Paulo, Brazil
b
Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, USP, Av. Bandeirantes, 3900, Zip Code 14-049-900, São Paulo, Brazil
Abstract—We have recently demonstrated that s.c.-injected 5-hydroxytryptamine (5-HT) induces nociception by an indirect action on the primary afferent nociceptor in addition to its previously described direct action. Although the mechanisms mediating hyperalgesia can be quite separate and distinct from those mediating nociception, the aim of this study was to test the hypothesis that 5-HT induces mechanical hyperalgesia by mechanisms similar to those mediating nociception. s.c. injection of 5-HT induced a dose-dependent mechanical hyperalgesia measured by the mechanical paw withdrawal nociceptive threshold test in the rat. 5-HT-induced hyperalgesia was significantly reduced by local blockade of the 5-HT3 receptor by tropisetron, by the nonspecific selectin inhibitor fucoidan, by the cyclooxygenase inhibitor indomethacin, by guanethidine depletion of norepinephrine in the sympathetic terminals, and by local blockade of the 1- or 2-adrenergic receptor by atenolol or ICI 118,551, respectively. Taken together, these findings indicate that like nociception, hyperalgesia induced by the injection of 5-HT in the s.c. tissue is also mediated by an indirect action of 5-HT on the primary afferent nociceptor. This indirect hyperalgesic action of 5-HT is mediated by a combination of mechanisms involved in inflammation such as neutrophil migration and the local release of prostaglandin and norepinephrine. However, in contrast to nociception, hyperalgesia induced by 5-HT in the s.c. tissue is mediated by a norepinephrine-dependent mechanism that involves the activation of peripheral 2 adrenoceptors. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: serotonin, hyperalgesia, prostaglandins, norepinephrine, neutrophil migration, s.c. tissue.
Nociception results from the activation of primary afferent nociceptors and the transmission of the nociceptive information to the spinal cord from where it is relayed to supra spinal levels (Millan, 1999; Julius and Basbaum, 2001). Following tissue injury and inflammation, primary afferent nociceptors are sensitized by mediators released from diseased or damaged tissue or from the immune system in such a way that previously slight or ineffective stimulation
EXPERIMENTAL PROCEDURES Subjects Male albino Wistar rats weighing 150 –250 g were used, and the experiments were conducted in accordance with the IASP guidelines on using laboratory animals (Zimmermann, 1983). All animal experimental procedures and protocols were approved by the Committee on Animal Research of the University of Campinas. Animal suffering and number of animals per group were kept at a minimum. The animals were housed in plastic cages with soft bedding (five/cage) on a 12-h light/dark cycle (lights on at 6:00
*Corresponding author. Tel: ⫹55-19-3412-5305; fax: ⫹55-19-3412-5212. E-mail address: [email protected] (C. H. Tambeli). Abbreviations: NO, nitric oxide; 5-HT, 5-hydroxytryptamine.
0306-4522/07$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.12.021
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M. C. G. Oliveira et al. / Neuroscience 145 (2007) 708 –714 AM) with food and water available ad libitum. They were maintained on a temperature-controlled room test (⫾23 °C) for a 1-hour habituation period prior to the test.
S.c. injections Drugs or vehicle was s.c. injected in the dorsum of the rat’s hind paw by tenting the skin and puncturing it with a 30-gauge needle prior to injecting the test agent, as previously described (Khasar et al., 1993; Vivancos et al., 2003). The needle was connected to a catheter of polyethylene (PE-50; Intramedic, Clay Adams, BectonDickinson, Franklin Lakes, NJ, USA) and also to a 50-l syringe (Hamilton, Reno, NV, USA). The animals were briefly restrained and the volume of injection was 50 l.
Mechanical paw withdrawal nociceptive threshold test Testing sessions took place during light phase (between 9:00 AM and 5:00 PM) in a quiet room maintained at 23 °C (Rosland, 1991). The Randall-Selitto nociceptive paw-withdrawal flexion reflex test (Randall and Selitto, 1957) was performed using an Ugo-Basile analgesymeter (Stoelting, Chicago, IL, USA), which applies a linearly increasing mechanical force to the dorsum of the rat’s hind paw (Aley et al., 2001). The rats were allowed to acclimatize to the restrainer for 5–10 min, after which the hind paws were exposed to the test stimulus. The nociceptive threshold was defined as the force in grams at which the rat withdrew its paw, and baseline paw-withdrawal threshold was defined as the mean of three tests performed at 5-min intervals before test agents were injected. Mechanical hyperalgesia was quantified as the change in mechanical nociceptive threshold calculated by subtracting the mean of three mechanical nociceptive threshold measurements taken 60 min after injection of the test agent from the mean of the three pre-injection baseline measurements.
Drugs and doses The protocols of drug administration were based on previous studies as follows: 5-HT (0.1, 1.0 and 10 g/paw, Taiwo and Levine, 1992); tropisetron (15, 50 and 150 g/paw, Parada et al., 2001); fucoidan (20 mg/kg, i.v., Zhang et al., 2001); indomethacin (10, 30 and 100 g/paw, Cunha et al., 2004); prostaglandin 100 ng/paw, Vivancos et al., 2003), epinephrine (1 g/paw, Khasar et al., 1999), atenolol (0.5, 2.0 and 6.0 g/paw, Cunha et al., 1991); ICI 118,551 (0.5, 1.0 and 1.5 g/paw, Parada et al., 2003); guanethidine (30 mg/kg s.c., Chang et al., 1965) and were all obtained from Sigma Chemicals, St. Louis, MO, USA. All drugs were dissolved in saline (0.9% NaCl). The doses of 5-HT used in the current study to produce mechanical hyperalgesia measured by the mechanical paw-withdrawal nociceptive threshold in the rat were lower than those used in our previous study to produce nociception measured by flinching and shaking behavior (Tambeli et al., 2006), because lower doses are needed to produce hyperalgesia than for nociception.
Experimental design The mechanical hyperalgesia produced by 5-HT was tested by the injection of increasing doses of 5-HT or of its vehicle (0.9% NaCl) into the s.c. tissue of the dorsum of the rat’s hind paw. Three mechanical paw-withdrawal thresholds measurement were initially made at 0, 30, 60, 120 and 180 min after the injection of 5-HT, and the mean of each of these three readings was defined as the nociceptive threshold for each period. Only the period of 60 min was used in further experiments. To evaluate if the hyperalgesia produced by 5-HT in the s.c. tissue is a 5-HT3-receptormediated response, the selective 5-HT3 receptor antagonist tropisetron was co-injected with 5-HT or with its vehicle (0.9% NaCl)
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in the ipsilateral paw. In order to confirm its local action, tropisetron was also injected in the contralateral paw. To investigate if 5-HT induces mechanical hyperalgesia by an indirect action on the primary afferent nociceptor of the s.c. tissue, fucoidan was i.v. injected 20 min prior to the injection of 5-HT or of its vehicle (0.9% NaCl) to inhibit neutrophil migration, indomethacin or its vehicle (0.9% NaCl) was s.c. injected 30 min prior to the injection of 5-HT or of its vehicle (0.9% NaCl) to induce cyclooxygenase inhibition and guanethidine was s.c. injected 3 days prior to the injection of 5-HT or of its vehicle (0.9% NaCl) to deplete norepinephrine in the sympathetic terminals. Also, the 1-adrenoceptor antagonist atenolol or the 2-adrenoceptor antagonist ICI 118,551 was co-injected with 5-HT or with its vehicle (0.9% NaCl) in the ipsilateral paw to block each of these receptors. In order to confirm their local action, atenolol and ICI 118,551 were also injected in the contralateral paw. To confirm that the effect of fucoidan was not due to an increase in the synthesis of nitric oxide (NO) (Goor et al., 2006; Nakamura et al., 2006; Yang et al., 2006), fucoidan was also i.v. injected 20 min prior to the s.c. injection of prostaglandin (100 ng) or epinephrine (1 g).
Statistical analysis To determine if there were significant differences (P⬍0.05) between treatment groups, one-way ANOVA was performed. If there was a significant between-subjects main effect of treatment group following one-way ANOVA, post hoc contrasts, using the Tukey test, were performed to determine the basis of the significant difference. Data from Figs. 2 and 4A were analyzed by t-test. Data are expressed in figures by the mechanical hyperalgesia and presented as means⫾S.E.M.
RESULTS 5-HT-induced mechanical hyperalgesia The s.c. injection of 5-HT (1.0 and 10 g) in the dorsum of the rat’s hind paw induced a dose-dependent mechanical hyperalgesia (Fig. 1A). Saline vehicle did not affect nociceptive threshold at any time-point during the 180-min testing period. The peak of the hyperalgesic response induced by 5-HT was at 60 min. Therefore, a 60-min interval between the 5-HT injection (1.0 g) and the mechanical stimuli application was used in further experiments. Local application of tropisetron (150 g) in the ipsilateral but not in the contralateral paw significantly reduced (Fig. 1B, P⬍0.05, Tukey test) 5-HT-induced mechanical hyperalgesia, confirming its local action. Tropisetron (150 g) did not affect nociceptive threshold when co-injected with saline vehicle. Fucoidan Pretreatment with fucoidan (20 mg/kg, i.v.) blocked 5-HTinduced mechanical hyperalgesia (Fig. 2, P⬍0.05, Tukey test) suggesting that neutrophil migration contributes to the hyperalgesic action of 5-HT in the s.c. tissue. Pretreatment with fucoidan did not affect nociceptive threshold when administered prior to the s.c. injection of saline vehicle. Prostaglandin (mean⫾S.E.M: 27.33⫾2.38) or epinephrine-induced hyperalgesia (30.00⫾3.37) in naïve rats was similar to that of rats pretreated with fucoidan (30.67⫾4.75; 36.00⫾3.45, respectively), ruling out the possibility that the effect of fucoidan on 5-HT-induced hyperalgesia could be due to an increase in the synthesis of NO.
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Indomethacin Local pretreatment with indomethacin (100 g) but not with its vehicle (0.9% NaCl) significantly reduced 5-HT-induced mechanical hyperalgesia (Fig. 3, P⬍0.05, Tukey test). Indomethacin (100 g) had no effect when applied in the contralateral paw, confirming its local action, and did not affect nociceptive threshold when administered prior to the s.c. injection of saline vehicle. These findings indicate that prostaglandins contribute to 5-HT-induced mechanical hyperalgesia.
Fig. 2. Effect of fucoidan on 5-HT-induced mechanical hyperalgesia. Pretreatment with fucoidan (20 mg/kg, i.v.) significantly blocked 5-HTinduced mechanical hyperalgesia (P⬍0.05, t-test) as indicated by the symbol “#”.
Sympathetic nervous system Pretreatment with guanethidine (30 mg/kg, s.c.) significantly reduced 5-HT-induced mechanical hyperalgesia (Fig. 4A, P⬍0.05, Tukey test) and did not affect nociceptive threshold when administered prior to the s.c. injection of saline vehicle. Local application of either atenolol (6.0 g) or ICI 118,551 (1.5 g) in the ipsilateral but not in the contralateral paw significantly reduced (Fig. 1B, P⬍0.05, Tukey test) 5-HT-induced mechanical hyperalgesia, confirming a local action. Neither atenolol (6.0 g) nor ICI 118,551 (1.5 g) affected nociceptive threshold when co-injected with saline vehicle. Taken together, these findings indicate that 5-HT induces mechanical hyperalgesia by a norepinephrine-dependent mechanism mediated by the activation of 1 and 2 adrenoceptors.
Fig. 1. Hyperalgesia induced by s.c. injection of 5-Hydroxytryptamine. (A) Dose-response curves of the mechanical hyperalgesia induced by s.c. injection of 5-HT (0.1, 1.0 and 10 g/paw). 5-HT (white symbols) induced a significant mechanical hyperalgesia (P⬍0.05, ANOVA). The effect of 5-HT at the doses of 1.0 and 10 g/paw was significantly greater than that of saline (P⬍0.05 in both cases, Tukey test), but not significantly different from each other (P⬎0.05, Tukey test). In this and subsequent figures, group sample sizes are shown in parentheses. (B) Effect of tropisetron on 5-HT-induced mechanical hyperalgesia. Co-application of the selective 5-HT3 receptor antagonist tropisetron completely blocked (P⬍0.05, ANOVA) the mechanical hyperalgesia induced by 5-HT (1.0 g/paw). The effect of tropisetron (150 g) was significantly lower than that of all other groups as indicated by the symbol “#” (P⬍0.05; Tukey test). Application of tropisetron (150 g) into the ct paw did not affect the magnitude of 5-HT-induced mechanical hyperalgesia since the effect of 5-HT and 5-HT⫹tropisetron ct paw was not significantly different from each other (P⬎0.05, Tukey test). In this and subsequent figures, each bar represents the mechanical hyperalgesia tested at 60 min post the s.c. injection of 5-HT. Abbreviation: ct, contralateral.
Fig. 3. Effect of indomethacin on 5-HT-induced mechanical hyperalgesia. Pretreatment with indomethacin (100 g/paw) blocked 5-HTinduced mechanical hyperalgesia (P⬍0.05, ANOVA). The effect of 5-HT⫹indomethacin was significantly lower than that of all other treatments as indicated by the symbol “#” (P⬍0.05 in all cases, Tukey test). Application of indomethacin (100 g) into the ct paw did not affect the magnitude of 5-HT-induced mechanical hyperalgesia since the effect of 5-HT and 5-HT⫹indomethacin ct paw was not significantly different from each other (P⬎0.05, Tukey test). Abbreviation: ct, contralateral.
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DISCUSSION In this study we confirmed previous reports of a hyperalgesic effect of 5-HT (Taiwo and Levine, 1992; Chen et al., 2006) and showed that 5-HT3 receptors contribute to this effect in the s.c. tissue. This latter finding is consistent with the involvement of 5-HT3 receptors in the mechanical hyperalgesia induced by the injection of complete Freund’s adjuvant in the s.c. tissue (Giordano and Sacks, 1997). We also showed for the first time that 5-HT induces hyperalgesia in the s.c. tissue by a combination of mechanisms involved in inflammation such as neutrophil migration, the release of local prostaglandins and norepinephrine. Therefore, in addition to its well-described direct action on the nociceptive primary afferent neurons (Holz and Anderson, 1984; Holz et al., 1985; Malone et al., 1991; Taiwo and Levine, 1992; Moalem et al., 2005), 5-HT acts indirectly on these neurons to induce hyperalgesia as it does to induce nociception (Tambeli et al., 2006) in the s.c. tissue. Although it was already known that 5-HT induces neutrophil migration in the s.c. tissue to contribute to 5-HTinduced nociception (Tambeli et al., 2006), it was not known whether the inhibition of neutrophil migration by fucoidan was able to reduce 5-HT-induced hyperalgesia. Our findings that fucoidan blocked 5-HT-induced hyperalgesia suggest that neutrophil migration contributes to the hyperalgesic action of 5-HT in the s.c. tissue. This suggestion was further supported by the lack of effect of fucoidan pretreatment on prostaglandin or epinephrine-induced hyperalgesia, ruling out the possibility that the effect of fucoidan on 5-HT-induced hyperalgesia could be due to an increase in the synthesis of NO (Goor et al., 2006; Nakamura et al., 2006; Yang et al., 2006). Neutrophil migration to the injured tissue may contribute to 5-HT-induced hyperalgesia by stimulating the release of local prostaglandins (Weissmann, 1982; Fasano et al., 1998). Consistent with that, we showed that cyclooxygenase inhibition by local administration of indomethacin prevented 5-HT-induced mechanical hyperalgesia indicating that the release of prostaglandins contributes to 5-HT-induced sensitization of the primary afferent nociceptor of the s.c. tissue. Based on these findings, we suggest that prostaglandin is locally released, at least in part, by the neutrophil migration induced by 5-HT to contribute to 5-HT-induced hyperalgesia. Further evidence that 5-HT induces hyperalgesia by an indirect action on the primary afferent nociceptor of the s.c. tissue was provided by the ability of both the 1 adrenoceptor antagonist atenolol and the 2 adrenoceptor antagFig. 4. Effect of sympathetic nervous system on 5-HT-induced mechanical hyperalgesia. (A) Effect of guanethidine (Gua) on 5-HTinduced mechanical hyperalgesia. Pretreatment with Gua (30 mg/kg s.c.) blocked 5-HT-induced mechanical hyperalgesia (P⬍0.05, t-test) as indicated by the symbol “#”. (B) Effect of the 1 adrenoceptor antagonist atenolol on 5-HT-induced mechanical hyperalgesia. Co-application of atenolol blocked 5-HT-induced hyperalgesia (P⬍0.05, ANOVA). The effect of 5-HT⫹atenolol 6 g was significantly lower than that of all other treatments as indicated by the symbol “#” (P⬍0.05 in all cases, Tukey test). Application of atenolol (6 g) in the ct paw did not affect the magnitude of 5-HT-induced mechanical hyperalgesia since the effect of 5-HT and 5-HT⫹atenolol ct paw was not
significantly different from each other (P⬎0.05, Tukey test). Abbreviation: ct, contralateral. (C) Effect of the 2 adrenoceptor antagonist ICI 118,551 on 5-HT-induced mechanical hyperalgesia. Co-application of ICI 118,551 blocked 5-HT-induced mechanical hyperalgesia. The effect of 5-HT⫹ICI 118,551 1.5 g was significantly lower than that of all other treatments as indicated by the symbol “#” (P⬍0.05 in all cases, Tukey test). Application of ICI 118,551 (1.5 g) in the ct paw did not affect the magnitude of 5-HT-induced mechanical hyperalgesia since the effect of 5-HT and 5-HT⫹118,551 ct paw was not significantly different from each other (P⬎0.05, Tukey test).
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onist ICI 118,551 to significantly reduce 5-HT-induced hyperalgesia. These findings indicate that 5-HT-induced hyperalgesia is also mediated by the endogenous release of local norepinephrine, which was further confirmed by the lack of effect of atenolol and ICI 118,551 when injected in the contralateral paw and by the significant reduction of 5-HT-induced hyperalgesia induced by the depletion of norepinephrine in the sympathetic terminals by guanethidine (Maxwell et al., 1960; Johnson et al., 1975). 5-HT may induce the release of norepinephrine by activating 5-HT receptors expressed on peripheral sympathetic postganglionic terminals. This suggestion is supported by the existence of 5-HT receptors, specifically the 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A and 5-HT3 receptors subtypes in the lumbar sympathetic ganglia (Pierce et al., 1995), and by the findings that 5-HT depolarizes sympathetic postganglionic axons (Nash and Wallis, 1981) and excites certain axon terminals, resulting in the release of norepinephrine (Fozard and Mwaluko, 1976; Gothert et al., 1979). We believe that 5-HT may activate 5-HT3 receptors expressed on peripheral sympathetic postganglionic terminals to induce the release of norepinephrine, which in turn, would activate 1 and 2 receptors to contribute to 5-HT-induced hyperalgesia in the s.c. tissue. However, this suggestion has not as yet been tested and does not exclude the involvement of other 5-HT receptors. Although the inflammatory reaction triggered by exogenous 5-HT may be different and larger from what happens after physiological 5-HT release, the exogenous administration of 5-HT can give important insights to understand the mechanism by which endogenous 5-HT sensitizes the nociceptor. In this context, it is important to point out that the concentration of exogenous 5-HT necessary to induce hyperalgesia in the current study (1 g) is similar to that used in a previous study (Taiwo and Levine, 1992), although larger than the concentration of 5-HT released in peripheral tissues after injury (34.55 pg; Sasaki et al., 2006). However, the dose-dependent effect of exogenous 5-HT and the blockade of 5-HT-induced hyperalgesia by the 5-HT3 receptor antagonist tropisetron showed in the current study, minimize the limitation of the conclusions for the in vivo situation with the use of exogenous 5-HT. It is well known that 5-HT induces nociception (Sufka et al., 1992; Giordano et al., 1998; Parada et al., 2001; Tambeli et al., 2006). Like hyperalgesia, nociception quantified by the nociceptive behavior (e.g. number of flinches) induced by the injection of 5-HT in the s.c. tissue is also mediated by an indirect action of 5-HT on the primary afferent nociceptor mediated by neutrophil migration and local release of prostaglandin, as recently demonstrated by our group (Tambeli et al., 2006). However, in contrast to hyperalgesia, nociception induced by s.c.-injected 5-HT is not affected by either the selective 2 adrenoceptor antagonist ICI 118,551 or by the depletion of norepinephrine in the sympathetic terminals by guanethidine suggesting that 5-HT induces nociception by a norepinephrine-independent mechanism that involves a local release of dopamine. Taken together, these findings add support to the sugges-
tion that the mechanisms mediating nociception can be separate and distinct, at least in part, from those mediating hyperalgesia (Taiwo and Levine, 1991; Waldron and Sawynok, 2004). In addition to the distinct mechanisms involved in 5-HT-induced nociception and hyperalgesia, the mechanisms underlying 5-HT-induced hyperalgesia differ on different tissues, such as dermal and s.c. In contrast to our findings that 5-HT induces hyperalgesia by an indirect action of 5-HT on the primary afferent neurons of the s.c. tissue, it has been previously suggested that the hyperalgesic action of 5-HT in the dermal tissue is due to a direct action of 5-HT on these neurons (Taiwo and Levine, 1992). This suggestion was based on the observations that procedures that eliminate some of the known indirect pathways that contribute to hyperalgesia such as sympathectomy, polymorphonuclear leukocyte depletion or cyclooxygenase inhibition do not attenuate 5-HT-induced hyperalgesia in the dermal tissue (Taiwo and Levine, 1992). Like 5-HT, PGE2 also induces hyperalgesia in both dermal and s.c. tissue, but the mechanism underlying the hyperalgesic action of PGE2 also differs in these tissues because the peak and the duration of its action are significantly lower and shorter in the dermal than in the s.c. tissue (Vivancos et al., 2003). Importantly, inflammatory mediators may also induce opposing effects in different depths of skin of rats. For example, NO as well as the arginine/NO/cGMP pathway are antinociceptive in the s.c. but pronociceptive in the dermal tissue (Vivancos et al., 2003). Given that an inflammatory mediator can induce the same effect through distinct mechanisms and also opposing effects in different tissues, the site of application is crucial in the study of the role of the inflammatory mediators in hyperalgesia. We believe that the effect of a given inflammatory mediator and the mechanism underlying its effect depend on the existence of different subsets of nociceptive primary sensory neurons, and of different population of cells expressing different numbers and types of receptors in different tissues. Therefore, the difference in the mechanism underlying 5-HT-induced hyperalgesia in dermal and in s.c. tissue may result from activation of distinct 5-HT receptors subtypes. Consistent with this idea, the hyperalgesia induced by 5-HT is mediated by 5-HT3 receptor activation in the s.c. tissue (current study), but not in the dermal tissue (Taiwo and Levine, 1992).
CONCLUSION In summary, we have demonstrated that like nociception, hyperalgesia induced by the injection of 5-HT in the s.c. tissue is also mediated by an indirect action of 5-HT on the primary afferent nociceptor. This indirect hyperalgesic action of 5-HT is mediated by a combination of mechanisms involved in inflammation such as neutrophil migration and the local release of prostaglandin and norepinephrine. We believe that the neutrophil migration induced by 5-HT contributes to the release of prostaglandins at the injured tissue. We also believe that 5-HT may activate peripheral sympathetic terminals to locally release norepinephrine that, taken
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together with prostaglandins, sensitizes the primary afferent nociceptor of the s.c. tissue to contribute to 5HTinduced hyperalgesia. Our findings demonstrate that in contrast to nociception, hyperalgesia induced by 5-HT in the s.c. tissue is mediated by a norepinephrine-dependent mechanism that involves the activation of peripheral 2 adrenoceptors.
REFERENCES Aley KO, Martin A, McMahon T, Mok J, Levine JD, Messing RO (2001) Nociceptor sensitization by extracellular signal-regulated kinases. J Neurosci 21:6933– 6939. Chang CC, Costa E, Brodie BB (1965) Interaction of guanethidine with adrenergic neurons. J Pharmacol Exp Ther 147:303–312. Chen X, Bing F, Dai P, Hong Y (2006) Involvement of protein kinase C in 5-HT-evoked thermal hyperalgesia and spinal fos protein expression in the rat. Pharmacol Biochem Behav 84:8 –16. Claria J, Romano M (2005) Pharmacological intervention of cyclooxygenase-2 and 5-lipoxygenase pathways. Impact on inflammation and cancer. Curr Pharm Des 11:3431–3447. Cunha FQ, Lorenzetti BB, Poole S, Ferreira SH (1991) Interleukin-8 as a mediator of sympathetic pain. Br J Pharmacol 104:765–767. Cunha TM, Verri WA Jr, Vivancos GG, Moreira IF, Reis S, Parada CA, Cunha FQ, Ferreira SH (2004) An electronic pressure-meter nociception paw test for mice. Braz J Med Biol Res 37:401– 407. Ernberg M, Lundeberg T, Kopp S (2000) Pain and allodynia/hyperalgesia induced by intramuscular injection of serotonin in patients with fibromyalgia and healthy individuals. Pain 85:31–39. Fasano MB, Wells JD, McCall CE (1998) Human neutrophils express the prostaglandin G/H synthase 2 gene when stimulated with bacterial lipopolysaccharide. Clin Immunol Immunopathol 87:304 –308. Fozard JR, Mwaluko GM (1976) Mechanism of the indirect sympathomimetic effect of 5-hydroxytrypt-amine on the isolated heart of the rabbit. Br J Pharmacol 57:115–125. Giordano J, Daleo C, Sacks SM (1998) Topical ondansetron attenuates nociceptive and inflammatory effects of intradermal capsaicin in humans. Eur J Pharmacol 354:R13–R14. Giordano J, Sacks SM (1997) Sub-anesthetic doses of bupivacaine or lidocaine increase peripheral ICS-205 930-induced analgesia against inflammatory pain in rats. Eur J Pharmacol 334:39 – 41. Goor Y, Goor O, Wollman Y, Chernichovski T, Schwartz D, Cabili S, Iaina A (2006) Fucoidin, an inhibitor of leukocyte adhesion, exacerbates acute ischemic renal failure and stimulates nitric oxide synthesis. Scand J Urol Nephrol 40:57– 62. Gothert M, Duhrsen U, Rieckesmann JM (1979) Ethanol, anaesthetics and other lipophilic drugs preferentially inhibit 5-hydroxytryptamine- and acetylcholine-induced noradrenaline release from sympathetic nerves. Arch Int Pharmacodyn Ther 242:196 –209. Green PG, Luo J, Heller PH, Levine JD (1993) Further substantiation of a significant role for the sympathetic nervous system in inflammation. Neuroscience 55:1037–1043. Herbert MK, Schmidt RF (1992) Activation of normal and inflamed fine articular afferent units by serotonin. Pain 50:79 – 88. Holz GG 4th, Anderson EG (1984) The actions of serotonin on frog primary afferent terminals and cell bodies. Comp Biochem Physiol C 77:13–21. Holz GG 4th, Shefner SA, Anderson EG (1985) Serotonin depolarizes type A and C primary afferents: an intracellular study in bullfrog dorsal root ganglion. Brain Res 327:71–79. Johnson EM Jr, Cantor E, Douglas JR Jr (1975) Biochemical and functional evaluation of the sympathectomy produced by the administration of guanethidine to newborn rats. J Pharmacol Exp Ther 193:503–512. Julius D, Basbaum AI (2001) Molecular mechanisms of nociception. Nature 413:203–210.
713
Khasar SG, Green PG, Levine JD (1993) Comparison of intradermal and subcutaneous hyperalgesic effects of inflammatory mediators in the rat. Neurosci Lett 153:215–218. Khasar SG, McCarter G, Levine JD (1999) Epinephrine produces a beta-adrenergic receptor-mediated mechanical hyperalgesia and in vitro sensitization of rat nociceptors. J Neurophysiol 81:1104 –1112. Levine JD, Taiwo YO, Collins SD, Tam JK (1986) Noradrenaline hyperalgesia is mediated through interaction with sympathetic postganglionic neurone terminals rather than activation of primary afferent nociceptors. Nature 323:158 –160. Malone HM, Peters JA, Lambert JJ (1991) Physiological and pharmacological properties of 5-HT3 receptors-a patch clamp-study. Neuropeptides 19 (Suppl):25–30. Maxwell RA, Plummer AJ, Schneider F, Povalski H, Daniel AI (1960) Pharmacology of [2-(octahydro-1-azocinyl)-ethyl]-guanidine sulfate (Su-5864). J Pharmacol Exp Ther 128:22–29. Millan MJ (1999) The induction of pain: an integrative review. Prog Neurobiol 57:1–164. Moalem G, Grafe P, Tracey DJ (2005) Chemical mediators enhance the excitability of unmyelinated sensory axons in normal and injured peripheral nerve of the rat. Neuroscience 134:1399 –1411. Nakamura T, Suzuki H, Wada Y, Kodama T, Doi T (2006) Fucoidan induces nitric oxide production via p38 mitogen-activated protein kinase and NF-kappaB-dependent signaling pathways through macrophage scavenger receptors. Biochem Biophys Res Commun 343:286 –294. Nash HL, Wallis DI (1981) Effects of divalent cations on responses of a sympathetic ganglion to 5-hydroxytryptamine and 1,1-dimethyl4-phenyl piperazinium. Br J Pharmacol 73:759 –772. Parada CA, Tambeli CH, Cunha FQ, Ferreira SH (2001) The major role of peripheral release of histamine and 5-hydroxytryptamine in formalin-induced nociception. Neuroscience 102:937–944. Parada CA, Yeh JJ, Joseph EK, Levine JD (2003) Tumor necrosis factor receptor type-1 in sensory neurons contributes to induction of chronic enhancement of inflammatory hyperalgesia in rat. Eur J Neurosci 17:1847–1852. Pierce PA, Xie GX, Peroutka SJ, Green PG, Levine JD (1995) 5-Hydroxytryptamine-induced synovial plasma extravasation is mediated via 5-hydroxytryptamine2A receptors on sympathetic efferent terminals. J Pharmacol Exp Ther 275:502–508. Randall LO, Selitto JJ (1957) A method for measurement of analgesic activity on inflamed tissue. Arch Int Pharmacodyn Ther 111: 409 – 419. Rosland JH (1991) The formalin test in mice: the influence of ambient temperature. Pain 45:211–216. Sasaki M, Obata H, Kawahara K, Saito S, Goto F (2006) Peripheral 5-HT2A receptor antagonism attenuates primary thermal hyperalgesia and secondary mechanical allodynia after thermal injury in rats. Pain 122:130 –136. Secco DD, Paron JA, de Oliveira SH, Ferreira SH, Silva JS, de Cunha FQ (2003) Neutrophil migration in inflammation: nitric oxide inhibits rolling, adhesion and induces apoptosis. Nitric Oxide 9:153–164. Sufka KJ, Schomburg FM, Giordano J (1992) Receptor mediation of 5-HT-induced inflammation and nociception in rats. Pharmacol Biochem Behav 41:53–56. Taiwo YO, Levine JD (1991) Further confirmation of the role of adenyl cyclase and of cAMP-dependent protein kinase in primary afferent hyperalgesia. Neuroscience 44:131–135. Taiwo YO, Levine JD (1992) Serotonin is a directly-acting hyperalgesic agent in the rat. Neuroscience 48:485– 490. Tambeli CH, Oliveira MC, Clemente JT, Pelegrini-da-Silva A, Parada CA (2006) A novel mechanism involved in 5-hydroxytryptamineinduced nociception: the indirect activation of primary afferents. Neuroscience 141:1517–1524. Vivancos GG, Parada CA, Ferreira SH (2003) Opposite nociceptive effects of the arginine/NO/cGMP pathway stimulation in dermal and subcutaneous tissues. Br J Pharmacol 138:1351–1357.
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Waldron JB, Sawynok J (2004) Peripheral P2X receptors and nociception: interactions with biogenic amine systems. Pain 110:79–89. Weissmann G (1982) The biochemistry of inflammation: rheumatoid arthritis and anti-inflammatory drugs. J Miss State Med Assoc 23:66–73. Xie G, Wang Y, Sharma M, Gabriel A, Mitchell J, Xing Y, Meuser T, Palmer PP (2003) 5-Hydroxytryptamine-induced plasma extravasation in the rat knee joint is mediated by multiple prostaglandins. Inflamm Res 52:32–38.
Yang JW, Yoon SY, Oh SJ, Kim SK, Kang KW (2006) Bifunctional effects of fucoidan on the expression of inducible nitric oxide synthase. Biochem Biophys Res Commun 346:345–350. Zhang XW, Liu Q, Thorlacius H (2001) Inhibition of selectin function and leukocyte rolling protects against dextran sodium sulfate-induced murine colitis. Scand J Gastroenterol 36:270 –275. Zimmermann M (1983) Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16:109 –110.
(Accepted 1 December 2006) (Available online 25 January 2007)
5-HT ACTS ON NOCICEPTIVE PRIMARY AFFERENTS THROUGH AN INDIRECT MECHANISM TO INDUCE HYPERALGESIA IN THE SUBCUTANEOUS TISSUE M. C. G. OLIVEIRA,a A. PELEGRINI-DA-SILVA,a C. A. PARADAb AND C. H. TAMBELIa*
becomes effective in inducing nociception. This primary sensory nociceptor sensitization is referred to as hyperalgesia. 5-Hydroxytryptamine (5-HT) is an important inflammatory mediator (Pierce et al., 1995; Xie et al., 2003) released from platelets and mast cells in response to tissue injury. 5-HT induces hyperalgesia and nociception by sensitizing and exciting afferent nerve fibers of different peripheral tissues such as articular (Herbert and Schmidt, 1992), dermal (Taiwo and Levine, 1992), muscular (Ernberg et al., 2000) and s.c. (Sufka et al., 1992; Parada et al., 2001; Tambeli et al., 2006). We have recently demonstrated (Tambeli et al., 2006) that the nociceptive effect of 5-HT in the s.c. tissue is exerted by an indirect action of 5-HT on the primary afferent neurons, because this effect is attenuated by polymorphonuclear leukocyte depletion, cyclooxygenase inhibition, norepinephrine depletion from the sympathetic terminals, and by local blockade of the 1- or 2-adrenergic receptor. Although it has been previously suggested that the mechanisms mediating hyperalgesia can be quite separate and distinct from those mediating nociception (Taiwo and Levine, 1991; Waldron and Sawynok, 2004), this suggestion does not exclude the involvement of common mechanisms. The aim of this study was to test the hypothesis that 5-HT induces mechanical hyperalgesia by mechanisms similar to those mediating nociception, that is, by a combination of mechanisms involved in inflammation such as neutrophil migration (Secco et al., 2003) and the release of local prostaglandin (Claria and Romano, 2005) and/or sympathomimetics amines (Levine et al., 1986; Green et al., 1993). The experimental design of the current study was very similar to that of our previous one (Tambeli et al., 2006). However, the Randall-Selitto nociceptive paw-withdrawal flexion reflex test was used as experimental assay for hyperalgesia in the current study and the spontaneous nociceptive flinching and shaking behavior was used as experimental assays for nociception in our previous study (Tambeli et al., 2006).
a
Department of Physiology, Laboratory of Orofacial Pain, Faculty of Dentistry of Piracicaba, University of Campinas, UNICAMP, Av. Limeira, 901, Zip Code 13414-900, Piracicaba, São Paulo, Brazil
b
Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, USP, Av. Bandeirantes, 3900, Zip Code 14-049-900, São Paulo, Brazil
Abstract—We have recently demonstrated that s.c.-injected 5-hydroxytryptamine (5-HT) induces nociception by an indirect action on the primary afferent nociceptor in addition to its previously described direct action. Although the mechanisms mediating hyperalgesia can be quite separate and distinct from those mediating nociception, the aim of this study was to test the hypothesis that 5-HT induces mechanical hyperalgesia by mechanisms similar to those mediating nociception. s.c. injection of 5-HT induced a dose-dependent mechanical hyperalgesia measured by the mechanical paw withdrawal nociceptive threshold test in the rat. 5-HT-induced hyperalgesia was significantly reduced by local blockade of the 5-HT3 receptor by tropisetron, by the nonspecific selectin inhibitor fucoidan, by the cyclooxygenase inhibitor indomethacin, by guanethidine depletion of norepinephrine in the sympathetic terminals, and by local blockade of the 1- or 2-adrenergic receptor by atenolol or ICI 118,551, respectively. Taken together, these findings indicate that like nociception, hyperalgesia induced by the injection of 5-HT in the s.c. tissue is also mediated by an indirect action of 5-HT on the primary afferent nociceptor. This indirect hyperalgesic action of 5-HT is mediated by a combination of mechanisms involved in inflammation such as neutrophil migration and the local release of prostaglandin and norepinephrine. However, in contrast to nociception, hyperalgesia induced by 5-HT in the s.c. tissue is mediated by a norepinephrine-dependent mechanism that involves the activation of peripheral 2 adrenoceptors. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: serotonin, hyperalgesia, prostaglandins, norepinephrine, neutrophil migration, s.c. tissue.
Nociception results from the activation of primary afferent nociceptors and the transmission of the nociceptive information to the spinal cord from where it is relayed to supra spinal levels (Millan, 1999; Julius and Basbaum, 2001). Following tissue injury and inflammation, primary afferent nociceptors are sensitized by mediators released from diseased or damaged tissue or from the immune system in such a way that previously slight or ineffective stimulation
EXPERIMENTAL PROCEDURES Subjects Male albino Wistar rats weighing 150 –250 g were used, and the experiments were conducted in accordance with the IASP guidelines on using laboratory animals (Zimmermann, 1983). All animal experimental procedures and protocols were approved by the Committee on Animal Research of the University of Campinas. Animal suffering and number of animals per group were kept at a minimum. The animals were housed in plastic cages with soft bedding (five/cage) on a 12-h light/dark cycle (lights on at 6:00
*Corresponding author. Tel: ⫹55-19-3412-5305; fax: ⫹55-19-3412-5212. E-mail address: [email protected] (C. H. Tambeli). Abbreviations: NO, nitric oxide; 5-HT, 5-hydroxytryptamine.
0306-4522/07$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.12.021
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M. C. G. Oliveira et al. / Neuroscience 145 (2007) 708 –714 AM) with food and water available ad libitum. They were maintained on a temperature-controlled room test (⫾23 °C) for a 1-hour habituation period prior to the test.
S.c. injections Drugs or vehicle was s.c. injected in the dorsum of the rat’s hind paw by tenting the skin and puncturing it with a 30-gauge needle prior to injecting the test agent, as previously described (Khasar et al., 1993; Vivancos et al., 2003). The needle was connected to a catheter of polyethylene (PE-50; Intramedic, Clay Adams, BectonDickinson, Franklin Lakes, NJ, USA) and also to a 50-l syringe (Hamilton, Reno, NV, USA). The animals were briefly restrained and the volume of injection was 50 l.
Mechanical paw withdrawal nociceptive threshold test Testing sessions took place during light phase (between 9:00 AM and 5:00 PM) in a quiet room maintained at 23 °C (Rosland, 1991). The Randall-Selitto nociceptive paw-withdrawal flexion reflex test (Randall and Selitto, 1957) was performed using an Ugo-Basile analgesymeter (Stoelting, Chicago, IL, USA), which applies a linearly increasing mechanical force to the dorsum of the rat’s hind paw (Aley et al., 2001). The rats were allowed to acclimatize to the restrainer for 5–10 min, after which the hind paws were exposed to the test stimulus. The nociceptive threshold was defined as the force in grams at which the rat withdrew its paw, and baseline paw-withdrawal threshold was defined as the mean of three tests performed at 5-min intervals before test agents were injected. Mechanical hyperalgesia was quantified as the change in mechanical nociceptive threshold calculated by subtracting the mean of three mechanical nociceptive threshold measurements taken 60 min after injection of the test agent from the mean of the three pre-injection baseline measurements.
Drugs and doses The protocols of drug administration were based on previous studies as follows: 5-HT (0.1, 1.0 and 10 g/paw, Taiwo and Levine, 1992); tropisetron (15, 50 and 150 g/paw, Parada et al., 2001); fucoidan (20 mg/kg, i.v., Zhang et al., 2001); indomethacin (10, 30 and 100 g/paw, Cunha et al., 2004); prostaglandin 100 ng/paw, Vivancos et al., 2003), epinephrine (1 g/paw, Khasar et al., 1999), atenolol (0.5, 2.0 and 6.0 g/paw, Cunha et al., 1991); ICI 118,551 (0.5, 1.0 and 1.5 g/paw, Parada et al., 2003); guanethidine (30 mg/kg s.c., Chang et al., 1965) and were all obtained from Sigma Chemicals, St. Louis, MO, USA. All drugs were dissolved in saline (0.9% NaCl). The doses of 5-HT used in the current study to produce mechanical hyperalgesia measured by the mechanical paw-withdrawal nociceptive threshold in the rat were lower than those used in our previous study to produce nociception measured by flinching and shaking behavior (Tambeli et al., 2006), because lower doses are needed to produce hyperalgesia than for nociception.
Experimental design The mechanical hyperalgesia produced by 5-HT was tested by the injection of increasing doses of 5-HT or of its vehicle (0.9% NaCl) into the s.c. tissue of the dorsum of the rat’s hind paw. Three mechanical paw-withdrawal thresholds measurement were initially made at 0, 30, 60, 120 and 180 min after the injection of 5-HT, and the mean of each of these three readings was defined as the nociceptive threshold for each period. Only the period of 60 min was used in further experiments. To evaluate if the hyperalgesia produced by 5-HT in the s.c. tissue is a 5-HT3-receptormediated response, the selective 5-HT3 receptor antagonist tropisetron was co-injected with 5-HT or with its vehicle (0.9% NaCl)
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in the ipsilateral paw. In order to confirm its local action, tropisetron was also injected in the contralateral paw. To investigate if 5-HT induces mechanical hyperalgesia by an indirect action on the primary afferent nociceptor of the s.c. tissue, fucoidan was i.v. injected 20 min prior to the injection of 5-HT or of its vehicle (0.9% NaCl) to inhibit neutrophil migration, indomethacin or its vehicle (0.9% NaCl) was s.c. injected 30 min prior to the injection of 5-HT or of its vehicle (0.9% NaCl) to induce cyclooxygenase inhibition and guanethidine was s.c. injected 3 days prior to the injection of 5-HT or of its vehicle (0.9% NaCl) to deplete norepinephrine in the sympathetic terminals. Also, the 1-adrenoceptor antagonist atenolol or the 2-adrenoceptor antagonist ICI 118,551 was co-injected with 5-HT or with its vehicle (0.9% NaCl) in the ipsilateral paw to block each of these receptors. In order to confirm their local action, atenolol and ICI 118,551 were also injected in the contralateral paw. To confirm that the effect of fucoidan was not due to an increase in the synthesis of nitric oxide (NO) (Goor et al., 2006; Nakamura et al., 2006; Yang et al., 2006), fucoidan was also i.v. injected 20 min prior to the s.c. injection of prostaglandin (100 ng) or epinephrine (1 g).
Statistical analysis To determine if there were significant differences (P⬍0.05) between treatment groups, one-way ANOVA was performed. If there was a significant between-subjects main effect of treatment group following one-way ANOVA, post hoc contrasts, using the Tukey test, were performed to determine the basis of the significant difference. Data from Figs. 2 and 4A were analyzed by t-test. Data are expressed in figures by the mechanical hyperalgesia and presented as means⫾S.E.M.
RESULTS 5-HT-induced mechanical hyperalgesia The s.c. injection of 5-HT (1.0 and 10 g) in the dorsum of the rat’s hind paw induced a dose-dependent mechanical hyperalgesia (Fig. 1A). Saline vehicle did not affect nociceptive threshold at any time-point during the 180-min testing period. The peak of the hyperalgesic response induced by 5-HT was at 60 min. Therefore, a 60-min interval between the 5-HT injection (1.0 g) and the mechanical stimuli application was used in further experiments. Local application of tropisetron (150 g) in the ipsilateral but not in the contralateral paw significantly reduced (Fig. 1B, P⬍0.05, Tukey test) 5-HT-induced mechanical hyperalgesia, confirming its local action. Tropisetron (150 g) did not affect nociceptive threshold when co-injected with saline vehicle. Fucoidan Pretreatment with fucoidan (20 mg/kg, i.v.) blocked 5-HTinduced mechanical hyperalgesia (Fig. 2, P⬍0.05, Tukey test) suggesting that neutrophil migration contributes to the hyperalgesic action of 5-HT in the s.c. tissue. Pretreatment with fucoidan did not affect nociceptive threshold when administered prior to the s.c. injection of saline vehicle. Prostaglandin (mean⫾S.E.M: 27.33⫾2.38) or epinephrine-induced hyperalgesia (30.00⫾3.37) in naïve rats was similar to that of rats pretreated with fucoidan (30.67⫾4.75; 36.00⫾3.45, respectively), ruling out the possibility that the effect of fucoidan on 5-HT-induced hyperalgesia could be due to an increase in the synthesis of NO.
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Indomethacin Local pretreatment with indomethacin (100 g) but not with its vehicle (0.9% NaCl) significantly reduced 5-HT-induced mechanical hyperalgesia (Fig. 3, P⬍0.05, Tukey test). Indomethacin (100 g) had no effect when applied in the contralateral paw, confirming its local action, and did not affect nociceptive threshold when administered prior to the s.c. injection of saline vehicle. These findings indicate that prostaglandins contribute to 5-HT-induced mechanical hyperalgesia.
Fig. 2. Effect of fucoidan on 5-HT-induced mechanical hyperalgesia. Pretreatment with fucoidan (20 mg/kg, i.v.) significantly blocked 5-HTinduced mechanical hyperalgesia (P⬍0.05, t-test) as indicated by the symbol “#”.
Sympathetic nervous system Pretreatment with guanethidine (30 mg/kg, s.c.) significantly reduced 5-HT-induced mechanical hyperalgesia (Fig. 4A, P⬍0.05, Tukey test) and did not affect nociceptive threshold when administered prior to the s.c. injection of saline vehicle. Local application of either atenolol (6.0 g) or ICI 118,551 (1.5 g) in the ipsilateral but not in the contralateral paw significantly reduced (Fig. 1B, P⬍0.05, Tukey test) 5-HT-induced mechanical hyperalgesia, confirming a local action. Neither atenolol (6.0 g) nor ICI 118,551 (1.5 g) affected nociceptive threshold when co-injected with saline vehicle. Taken together, these findings indicate that 5-HT induces mechanical hyperalgesia by a norepinephrine-dependent mechanism mediated by the activation of 1 and 2 adrenoceptors.
Fig. 1. Hyperalgesia induced by s.c. injection of 5-Hydroxytryptamine. (A) Dose-response curves of the mechanical hyperalgesia induced by s.c. injection of 5-HT (0.1, 1.0 and 10 g/paw). 5-HT (white symbols) induced a significant mechanical hyperalgesia (P⬍0.05, ANOVA). The effect of 5-HT at the doses of 1.0 and 10 g/paw was significantly greater than that of saline (P⬍0.05 in both cases, Tukey test), but not significantly different from each other (P⬎0.05, Tukey test). In this and subsequent figures, group sample sizes are shown in parentheses. (B) Effect of tropisetron on 5-HT-induced mechanical hyperalgesia. Co-application of the selective 5-HT3 receptor antagonist tropisetron completely blocked (P⬍0.05, ANOVA) the mechanical hyperalgesia induced by 5-HT (1.0 g/paw). The effect of tropisetron (150 g) was significantly lower than that of all other groups as indicated by the symbol “#” (P⬍0.05; Tukey test). Application of tropisetron (150 g) into the ct paw did not affect the magnitude of 5-HT-induced mechanical hyperalgesia since the effect of 5-HT and 5-HT⫹tropisetron ct paw was not significantly different from each other (P⬎0.05, Tukey test). In this and subsequent figures, each bar represents the mechanical hyperalgesia tested at 60 min post the s.c. injection of 5-HT. Abbreviation: ct, contralateral.
Fig. 3. Effect of indomethacin on 5-HT-induced mechanical hyperalgesia. Pretreatment with indomethacin (100 g/paw) blocked 5-HTinduced mechanical hyperalgesia (P⬍0.05, ANOVA). The effect of 5-HT⫹indomethacin was significantly lower than that of all other treatments as indicated by the symbol “#” (P⬍0.05 in all cases, Tukey test). Application of indomethacin (100 g) into the ct paw did not affect the magnitude of 5-HT-induced mechanical hyperalgesia since the effect of 5-HT and 5-HT⫹indomethacin ct paw was not significantly different from each other (P⬎0.05, Tukey test). Abbreviation: ct, contralateral.
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DISCUSSION In this study we confirmed previous reports of a hyperalgesic effect of 5-HT (Taiwo and Levine, 1992; Chen et al., 2006) and showed that 5-HT3 receptors contribute to this effect in the s.c. tissue. This latter finding is consistent with the involvement of 5-HT3 receptors in the mechanical hyperalgesia induced by the injection of complete Freund’s adjuvant in the s.c. tissue (Giordano and Sacks, 1997). We also showed for the first time that 5-HT induces hyperalgesia in the s.c. tissue by a combination of mechanisms involved in inflammation such as neutrophil migration, the release of local prostaglandins and norepinephrine. Therefore, in addition to its well-described direct action on the nociceptive primary afferent neurons (Holz and Anderson, 1984; Holz et al., 1985; Malone et al., 1991; Taiwo and Levine, 1992; Moalem et al., 2005), 5-HT acts indirectly on these neurons to induce hyperalgesia as it does to induce nociception (Tambeli et al., 2006) in the s.c. tissue. Although it was already known that 5-HT induces neutrophil migration in the s.c. tissue to contribute to 5-HTinduced nociception (Tambeli et al., 2006), it was not known whether the inhibition of neutrophil migration by fucoidan was able to reduce 5-HT-induced hyperalgesia. Our findings that fucoidan blocked 5-HT-induced hyperalgesia suggest that neutrophil migration contributes to the hyperalgesic action of 5-HT in the s.c. tissue. This suggestion was further supported by the lack of effect of fucoidan pretreatment on prostaglandin or epinephrine-induced hyperalgesia, ruling out the possibility that the effect of fucoidan on 5-HT-induced hyperalgesia could be due to an increase in the synthesis of NO (Goor et al., 2006; Nakamura et al., 2006; Yang et al., 2006). Neutrophil migration to the injured tissue may contribute to 5-HT-induced hyperalgesia by stimulating the release of local prostaglandins (Weissmann, 1982; Fasano et al., 1998). Consistent with that, we showed that cyclooxygenase inhibition by local administration of indomethacin prevented 5-HT-induced mechanical hyperalgesia indicating that the release of prostaglandins contributes to 5-HT-induced sensitization of the primary afferent nociceptor of the s.c. tissue. Based on these findings, we suggest that prostaglandin is locally released, at least in part, by the neutrophil migration induced by 5-HT to contribute to 5-HT-induced hyperalgesia. Further evidence that 5-HT induces hyperalgesia by an indirect action on the primary afferent nociceptor of the s.c. tissue was provided by the ability of both the 1 adrenoceptor antagonist atenolol and the 2 adrenoceptor antagFig. 4. Effect of sympathetic nervous system on 5-HT-induced mechanical hyperalgesia. (A) Effect of guanethidine (Gua) on 5-HTinduced mechanical hyperalgesia. Pretreatment with Gua (30 mg/kg s.c.) blocked 5-HT-induced mechanical hyperalgesia (P⬍0.05, t-test) as indicated by the symbol “#”. (B) Effect of the 1 adrenoceptor antagonist atenolol on 5-HT-induced mechanical hyperalgesia. Co-application of atenolol blocked 5-HT-induced hyperalgesia (P⬍0.05, ANOVA). The effect of 5-HT⫹atenolol 6 g was significantly lower than that of all other treatments as indicated by the symbol “#” (P⬍0.05 in all cases, Tukey test). Application of atenolol (6 g) in the ct paw did not affect the magnitude of 5-HT-induced mechanical hyperalgesia since the effect of 5-HT and 5-HT⫹atenolol ct paw was not
significantly different from each other (P⬎0.05, Tukey test). Abbreviation: ct, contralateral. (C) Effect of the 2 adrenoceptor antagonist ICI 118,551 on 5-HT-induced mechanical hyperalgesia. Co-application of ICI 118,551 blocked 5-HT-induced mechanical hyperalgesia. The effect of 5-HT⫹ICI 118,551 1.5 g was significantly lower than that of all other treatments as indicated by the symbol “#” (P⬍0.05 in all cases, Tukey test). Application of ICI 118,551 (1.5 g) in the ct paw did not affect the magnitude of 5-HT-induced mechanical hyperalgesia since the effect of 5-HT and 5-HT⫹118,551 ct paw was not significantly different from each other (P⬎0.05, Tukey test).
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onist ICI 118,551 to significantly reduce 5-HT-induced hyperalgesia. These findings indicate that 5-HT-induced hyperalgesia is also mediated by the endogenous release of local norepinephrine, which was further confirmed by the lack of effect of atenolol and ICI 118,551 when injected in the contralateral paw and by the significant reduction of 5-HT-induced hyperalgesia induced by the depletion of norepinephrine in the sympathetic terminals by guanethidine (Maxwell et al., 1960; Johnson et al., 1975). 5-HT may induce the release of norepinephrine by activating 5-HT receptors expressed on peripheral sympathetic postganglionic terminals. This suggestion is supported by the existence of 5-HT receptors, specifically the 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A and 5-HT3 receptors subtypes in the lumbar sympathetic ganglia (Pierce et al., 1995), and by the findings that 5-HT depolarizes sympathetic postganglionic axons (Nash and Wallis, 1981) and excites certain axon terminals, resulting in the release of norepinephrine (Fozard and Mwaluko, 1976; Gothert et al., 1979). We believe that 5-HT may activate 5-HT3 receptors expressed on peripheral sympathetic postganglionic terminals to induce the release of norepinephrine, which in turn, would activate 1 and 2 receptors to contribute to 5-HT-induced hyperalgesia in the s.c. tissue. However, this suggestion has not as yet been tested and does not exclude the involvement of other 5-HT receptors. Although the inflammatory reaction triggered by exogenous 5-HT may be different and larger from what happens after physiological 5-HT release, the exogenous administration of 5-HT can give important insights to understand the mechanism by which endogenous 5-HT sensitizes the nociceptor. In this context, it is important to point out that the concentration of exogenous 5-HT necessary to induce hyperalgesia in the current study (1 g) is similar to that used in a previous study (Taiwo and Levine, 1992), although larger than the concentration of 5-HT released in peripheral tissues after injury (34.55 pg; Sasaki et al., 2006). However, the dose-dependent effect of exogenous 5-HT and the blockade of 5-HT-induced hyperalgesia by the 5-HT3 receptor antagonist tropisetron showed in the current study, minimize the limitation of the conclusions for the in vivo situation with the use of exogenous 5-HT. It is well known that 5-HT induces nociception (Sufka et al., 1992; Giordano et al., 1998; Parada et al., 2001; Tambeli et al., 2006). Like hyperalgesia, nociception quantified by the nociceptive behavior (e.g. number of flinches) induced by the injection of 5-HT in the s.c. tissue is also mediated by an indirect action of 5-HT on the primary afferent nociceptor mediated by neutrophil migration and local release of prostaglandin, as recently demonstrated by our group (Tambeli et al., 2006). However, in contrast to hyperalgesia, nociception induced by s.c.-injected 5-HT is not affected by either the selective 2 adrenoceptor antagonist ICI 118,551 or by the depletion of norepinephrine in the sympathetic terminals by guanethidine suggesting that 5-HT induces nociception by a norepinephrine-independent mechanism that involves a local release of dopamine. Taken together, these findings add support to the sugges-
tion that the mechanisms mediating nociception can be separate and distinct, at least in part, from those mediating hyperalgesia (Taiwo and Levine, 1991; Waldron and Sawynok, 2004). In addition to the distinct mechanisms involved in 5-HT-induced nociception and hyperalgesia, the mechanisms underlying 5-HT-induced hyperalgesia differ on different tissues, such as dermal and s.c. In contrast to our findings that 5-HT induces hyperalgesia by an indirect action of 5-HT on the primary afferent neurons of the s.c. tissue, it has been previously suggested that the hyperalgesic action of 5-HT in the dermal tissue is due to a direct action of 5-HT on these neurons (Taiwo and Levine, 1992). This suggestion was based on the observations that procedures that eliminate some of the known indirect pathways that contribute to hyperalgesia such as sympathectomy, polymorphonuclear leukocyte depletion or cyclooxygenase inhibition do not attenuate 5-HT-induced hyperalgesia in the dermal tissue (Taiwo and Levine, 1992). Like 5-HT, PGE2 also induces hyperalgesia in both dermal and s.c. tissue, but the mechanism underlying the hyperalgesic action of PGE2 also differs in these tissues because the peak and the duration of its action are significantly lower and shorter in the dermal than in the s.c. tissue (Vivancos et al., 2003). Importantly, inflammatory mediators may also induce opposing effects in different depths of skin of rats. For example, NO as well as the arginine/NO/cGMP pathway are antinociceptive in the s.c. but pronociceptive in the dermal tissue (Vivancos et al., 2003). Given that an inflammatory mediator can induce the same effect through distinct mechanisms and also opposing effects in different tissues, the site of application is crucial in the study of the role of the inflammatory mediators in hyperalgesia. We believe that the effect of a given inflammatory mediator and the mechanism underlying its effect depend on the existence of different subsets of nociceptive primary sensory neurons, and of different population of cells expressing different numbers and types of receptors in different tissues. Therefore, the difference in the mechanism underlying 5-HT-induced hyperalgesia in dermal and in s.c. tissue may result from activation of distinct 5-HT receptors subtypes. Consistent with this idea, the hyperalgesia induced by 5-HT is mediated by 5-HT3 receptor activation in the s.c. tissue (current study), but not in the dermal tissue (Taiwo and Levine, 1992).
CONCLUSION In summary, we have demonstrated that like nociception, hyperalgesia induced by the injection of 5-HT in the s.c. tissue is also mediated by an indirect action of 5-HT on the primary afferent nociceptor. This indirect hyperalgesic action of 5-HT is mediated by a combination of mechanisms involved in inflammation such as neutrophil migration and the local release of prostaglandin and norepinephrine. We believe that the neutrophil migration induced by 5-HT contributes to the release of prostaglandins at the injured tissue. We also believe that 5-HT may activate peripheral sympathetic terminals to locally release norepinephrine that, taken
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together with prostaglandins, sensitizes the primary afferent nociceptor of the s.c. tissue to contribute to 5HTinduced hyperalgesia. Our findings demonstrate that in contrast to nociception, hyperalgesia induced by 5-HT in the s.c. tissue is mediated by a norepinephrine-dependent mechanism that involves the activation of peripheral 2 adrenoceptors.
REFERENCES Aley KO, Martin A, McMahon T, Mok J, Levine JD, Messing RO (2001) Nociceptor sensitization by extracellular signal-regulated kinases. J Neurosci 21:6933– 6939. Chang CC, Costa E, Brodie BB (1965) Interaction of guanethidine with adrenergic neurons. J Pharmacol Exp Ther 147:303–312. Chen X, Bing F, Dai P, Hong Y (2006) Involvement of protein kinase C in 5-HT-evoked thermal hyperalgesia and spinal fos protein expression in the rat. Pharmacol Biochem Behav 84:8 –16. Claria J, Romano M (2005) Pharmacological intervention of cyclooxygenase-2 and 5-lipoxygenase pathways. Impact on inflammation and cancer. Curr Pharm Des 11:3431–3447. Cunha FQ, Lorenzetti BB, Poole S, Ferreira SH (1991) Interleukin-8 as a mediator of sympathetic pain. Br J Pharmacol 104:765–767. Cunha TM, Verri WA Jr, Vivancos GG, Moreira IF, Reis S, Parada CA, Cunha FQ, Ferreira SH (2004) An electronic pressure-meter nociception paw test for mice. Braz J Med Biol Res 37:401– 407. Ernberg M, Lundeberg T, Kopp S (2000) Pain and allodynia/hyperalgesia induced by intramuscular injection of serotonin in patients with fibromyalgia and healthy individuals. Pain 85:31–39. Fasano MB, Wells JD, McCall CE (1998) Human neutrophils express the prostaglandin G/H synthase 2 gene when stimulated with bacterial lipopolysaccharide. Clin Immunol Immunopathol 87:304 –308. Fozard JR, Mwaluko GM (1976) Mechanism of the indirect sympathomimetic effect of 5-hydroxytrypt-amine on the isolated heart of the rabbit. Br J Pharmacol 57:115–125. Giordano J, Daleo C, Sacks SM (1998) Topical ondansetron attenuates nociceptive and inflammatory effects of intradermal capsaicin in humans. Eur J Pharmacol 354:R13–R14. Giordano J, Sacks SM (1997) Sub-anesthetic doses of bupivacaine or lidocaine increase peripheral ICS-205 930-induced analgesia against inflammatory pain in rats. Eur J Pharmacol 334:39 – 41. Goor Y, Goor O, Wollman Y, Chernichovski T, Schwartz D, Cabili S, Iaina A (2006) Fucoidin, an inhibitor of leukocyte adhesion, exacerbates acute ischemic renal failure and stimulates nitric oxide synthesis. Scand J Urol Nephrol 40:57– 62. Gothert M, Duhrsen U, Rieckesmann JM (1979) Ethanol, anaesthetics and other lipophilic drugs preferentially inhibit 5-hydroxytryptamine- and acetylcholine-induced noradrenaline release from sympathetic nerves. Arch Int Pharmacodyn Ther 242:196 –209. Green PG, Luo J, Heller PH, Levine JD (1993) Further substantiation of a significant role for the sympathetic nervous system in inflammation. Neuroscience 55:1037–1043. Herbert MK, Schmidt RF (1992) Activation of normal and inflamed fine articular afferent units by serotonin. Pain 50:79 – 88. Holz GG 4th, Anderson EG (1984) The actions of serotonin on frog primary afferent terminals and cell bodies. Comp Biochem Physiol C 77:13–21. Holz GG 4th, Shefner SA, Anderson EG (1985) Serotonin depolarizes type A and C primary afferents: an intracellular study in bullfrog dorsal root ganglion. Brain Res 327:71–79. Johnson EM Jr, Cantor E, Douglas JR Jr (1975) Biochemical and functional evaluation of the sympathectomy produced by the administration of guanethidine to newborn rats. J Pharmacol Exp Ther 193:503–512. Julius D, Basbaum AI (2001) Molecular mechanisms of nociception. Nature 413:203–210.
713
Khasar SG, Green PG, Levine JD (1993) Comparison of intradermal and subcutaneous hyperalgesic effects of inflammatory mediators in the rat. Neurosci Lett 153:215–218. Khasar SG, McCarter G, Levine JD (1999) Epinephrine produces a beta-adrenergic receptor-mediated mechanical hyperalgesia and in vitro sensitization of rat nociceptors. J Neurophysiol 81:1104 –1112. Levine JD, Taiwo YO, Collins SD, Tam JK (1986) Noradrenaline hyperalgesia is mediated through interaction with sympathetic postganglionic neurone terminals rather than activation of primary afferent nociceptors. Nature 323:158 –160. Malone HM, Peters JA, Lambert JJ (1991) Physiological and pharmacological properties of 5-HT3 receptors-a patch clamp-study. Neuropeptides 19 (Suppl):25–30. Maxwell RA, Plummer AJ, Schneider F, Povalski H, Daniel AI (1960) Pharmacology of [2-(octahydro-1-azocinyl)-ethyl]-guanidine sulfate (Su-5864). J Pharmacol Exp Ther 128:22–29. Millan MJ (1999) The induction of pain: an integrative review. Prog Neurobiol 57:1–164. Moalem G, Grafe P, Tracey DJ (2005) Chemical mediators enhance the excitability of unmyelinated sensory axons in normal and injured peripheral nerve of the rat. Neuroscience 134:1399 –1411. Nakamura T, Suzuki H, Wada Y, Kodama T, Doi T (2006) Fucoidan induces nitric oxide production via p38 mitogen-activated protein kinase and NF-kappaB-dependent signaling pathways through macrophage scavenger receptors. Biochem Biophys Res Commun 343:286 –294. Nash HL, Wallis DI (1981) Effects of divalent cations on responses of a sympathetic ganglion to 5-hydroxytryptamine and 1,1-dimethyl4-phenyl piperazinium. Br J Pharmacol 73:759 –772. Parada CA, Tambeli CH, Cunha FQ, Ferreira SH (2001) The major role of peripheral release of histamine and 5-hydroxytryptamine in formalin-induced nociception. Neuroscience 102:937–944. Parada CA, Yeh JJ, Joseph EK, Levine JD (2003) Tumor necrosis factor receptor type-1 in sensory neurons contributes to induction of chronic enhancement of inflammatory hyperalgesia in rat. Eur J Neurosci 17:1847–1852. Pierce PA, Xie GX, Peroutka SJ, Green PG, Levine JD (1995) 5-Hydroxytryptamine-induced synovial plasma extravasation is mediated via 5-hydroxytryptamine2A receptors on sympathetic efferent terminals. J Pharmacol Exp Ther 275:502–508. Randall LO, Selitto JJ (1957) A method for measurement of analgesic activity on inflamed tissue. Arch Int Pharmacodyn Ther 111: 409 – 419. Rosland JH (1991) The formalin test in mice: the influence of ambient temperature. Pain 45:211–216. Sasaki M, Obata H, Kawahara K, Saito S, Goto F (2006) Peripheral 5-HT2A receptor antagonism attenuates primary thermal hyperalgesia and secondary mechanical allodynia after thermal injury in rats. Pain 122:130 –136. Secco DD, Paron JA, de Oliveira SH, Ferreira SH, Silva JS, de Cunha FQ (2003) Neutrophil migration in inflammation: nitric oxide inhibits rolling, adhesion and induces apoptosis. Nitric Oxide 9:153–164. Sufka KJ, Schomburg FM, Giordano J (1992) Receptor mediation of 5-HT-induced inflammation and nociception in rats. Pharmacol Biochem Behav 41:53–56. Taiwo YO, Levine JD (1991) Further confirmation of the role of adenyl cyclase and of cAMP-dependent protein kinase in primary afferent hyperalgesia. Neuroscience 44:131–135. Taiwo YO, Levine JD (1992) Serotonin is a directly-acting hyperalgesic agent in the rat. Neuroscience 48:485– 490. Tambeli CH, Oliveira MC, Clemente JT, Pelegrini-da-Silva A, Parada CA (2006) A novel mechanism involved in 5-hydroxytryptamineinduced nociception: the indirect activation of primary afferents. Neuroscience 141:1517–1524. Vivancos GG, Parada CA, Ferreira SH (2003) Opposite nociceptive effects of the arginine/NO/cGMP pathway stimulation in dermal and subcutaneous tissues. Br J Pharmacol 138:1351–1357.
714
M. C. G. Oliveira et al. / Neuroscience 145 (2007) 708 –714
Waldron JB, Sawynok J (2004) Peripheral P2X receptors and nociception: interactions with biogenic amine systems. Pain 110:79–89. Weissmann G (1982) The biochemistry of inflammation: rheumatoid arthritis and anti-inflammatory drugs. J Miss State Med Assoc 23:66–73. Xie G, Wang Y, Sharma M, Gabriel A, Mitchell J, Xing Y, Meuser T, Palmer PP (2003) 5-Hydroxytryptamine-induced plasma extravasation in the rat knee joint is mediated by multiple prostaglandins. Inflamm Res 52:32–38.
Yang JW, Yoon SY, Oh SJ, Kim SK, Kang KW (2006) Bifunctional effects of fucoidan on the expression of inducible nitric oxide synthase. Biochem Biophys Res Commun 346:345–350. Zhang XW, Liu Q, Thorlacius H (2001) Inhibition of selectin function and leukocyte rolling protects against dextran sodium sulfate-induced murine colitis. Scand J Gastroenterol 36:270 –275. Zimmermann M (1983) Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16:109 –110.
(Accepted 1 December 2006) (Available online 25 January 2007)