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Spinal 5-HT receptors that contribute to the pain-relieving effects of spinal cord stimulation in a rat model of neuropathy
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PAIN 152 (2011) 1666–1673
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Spinal 5-HT receptors that contribute to the pain-relieving effects of spinal cord stimulation in a rat model of neuropathy Zhiyang Song ⇑, Björn A. Meyerson, Bengt Linderoth Department of Clinical Neuroscience, Section of Neurosurgery, Karolinska Institutet, Stockholm, Sweden
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
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Article history: Received 14 September 2010 Received in revised form 3 February 2011 Accepted 8 March 2011
Keywords: Neuropathic pain Spinal cord stimulation 5-HT receptor Rat
a b s t r a c t Spinal cord stimulation (SCS) is extensively employed in the management of neuropathic pain, but the underlying mechanisms are only partially understood. Recently, we demonstrated that the pain-relieving effect of SCS appears to involve the spinal serotonin system, and the present study aimed at identifying the types of the spinal serotonin receptors involved. Experiments were performed on rats with neuropathy produced by partial ligation of the sciatic nerve. Tactile sensitivity was assessed using von Frey filaments, and cold and heat sensitivity with cold spray and radiant heat, respectively. Selective 5-HT receptor antagonists, methiothepin (5-HT1,6,7), ketanserin tartrate (5-HT2A), TICM (5-HT3), SDZ-205,557 (5-HT4), as well as receptor agonists, a-m-5-HT (5-HT2), m-CPBG (5-HT3) in per se ineffective doses, or vehicle, were administrated intrathecally 5 minutes prior to the application of SCS. Ketanserin and SDZ-205,557 significantly attenuated the suppressive effect of SCS on tactile hypersensitivity, while methiothepin and TICM were ineffective. The suppressive effect on cold hypersensitivity of SCS was counteracted by ketanserin only. None of the 5-HT receptor antagonists attenuated the suppressive effect on heat hyperalgesia of SCS. Subeffective doses of a-m-5-HT and m-CPBG enhanced the suppressive effect of SCS on tactile hypersensitivity. The enhancing effect of m-CPBG was abolished by a c-aminobutyric acid (GABA)A or GABAB antagonist intrathecally. These results suggest that the activation of 5-HT2A, 5-HT3, and 5-HT4 receptors plays an important role in SCS-induced relief of neuropathic pain. The activation of 5-HT3 receptors appears to operate via spinal GABAergic interneurons. Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
1. Introduction Spinal cord stimulation (SCS) provides useful, long-lasting pain relief in 60–70% of patients suffering from certain forms of neuropathic pain [33,50], which are often resistant to conventional pharmacotherapy [15]. However, knowledge concerning its mode of action is still fragmentary. Therefore, a better understanding of the neurobiological mechanisms behind the pain-suppressing effect of SCS may help to develop new treatment strategies. In our previous studies, c-aminobutyric acid (GABA)ergic and cholinergic mechanisms in the pain relief produced by SCS have been explored. It was shown that SCS could augment spinal dorsal horn GABA and acetylcholine (ACh) release, with associated decreased release of glutamate and aspartate in animal models of neuropathic pain [10,47,54]. It was also demonstrated that the corresponding receptor agonists (GABAB-baclofen; cholinergic musca⇑ Corresponding author. Address: Department of Clinical Neuroscience, Section of Neurosurgery, Karolinska University Hospital, S-171 76 Stockholm, Sweden. Tel.: +46 8 51775806; fax: +46 8 51771778. E-mail address: [email protected] (Z. Song).
rinic–oxotremorine) could significantly enhance the pain-relieving effect of SCS both in animal models and in patients [9,26,27,52]. Antinociceptive effects of serotonin are well documented in normal animals and in pain models [1,3,7]. In several early studies it was shown that electrical stimulation applied in the brain stem, to the dorsolateral funiculus and peripheral nerves, can activate serotonergic descending pathways with an increased release of 5-HT and an elevation of pain thresholds [29,51,58]. In a series of experimental studies, Saade et al. [13,45] have demonstrated an attenuating effect of electrical stimulation at the level of the dorsal column nuclei on the flexor reflex as well as on tactile and thermal hypersensitivity following peripheral nerve injury. They have thus provided evidence for the involvement of a supraspinal loop in the mode of action of SCS, implying that the effect is exerted, at least partially, via the activation of descending serotonergic pain inhibitory pathways. It was earlier demonstrated in cats that SCS induced serotonin release in the spinal dorsal horn [28]. More recently, we reported that SCS may induce an increase of the endogenous serotonin content in the rat dorsal horn, indicating an activation of the descending serotonergic pathways. This effect was also shown to partially
0304-3959/$36.00 Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2011.03.012
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involve spinal GABAergic circuitries [53]. Inconsistent outcomes of pain modulation by exogenous 5-HT may be attributed to nonselective actions of 5-HT at multiple receptor types and subtypes [1–4,17,60]. At least 5 types of serotonergic receptors (5-HT1, 5HT2, 5-HT3, 5-HT4, and 5-HT7) are present in the spinal cord and are claimed to be involved in the modulation of nociceptive transmission [12,18,19,23,30,59]. The specific 5-HT receptors contributing to SCS-induced pain relief have not yet been explored. The aim of the current study was to gain insight into the functional significance of different types of 5-HT receptors involved in the SCS effect. Several selective 5-HT receptor antagonists and agonists were intrathecally (i.t.) administered to investigate their ability to counteract or enhance the pain-relieving effect induced by SCS in a neuropathic pain model. 2. Materials and methods 2.1. Animals and anesthesia The experiments were performed on male Sprague-Dawley rats (B&K Universal AB, Sollentuna, Sweden), weighing 250–350 g, in accordance with the recommendations of the Committee for Research and Ethical Issues of the International Association for the Study of Pain (1983) and with a protocol approved by the local ethical committee for animal research. The surgical procedures were performed under general anesthesia delivered through an open mask system. Anesthesia was induced by 4% Isoflurane (Forene, Abbott, Solna, Sweden) and maintained with 1–2% in a 1:1 mixture of air and oxygen at a flow rate of 2 L/min. During surgery, the body temperature was maintained at 37 ± 0.5°C by an automatic heating pad (CMA/150, CMA Microdialysis AB, Stockholm, Sweden). Postoperative analgesia was provided by subcutaneous injection of 5 mg/kg carprofen (Rimadyl, Pfizer, New york, NY, USA). 2.2. Sciatic nerve ligation A sciatic nerve lesion was created according to the method of Seltzer et al. [49] to produce mononeuropathy. In short, the left sciatic nerve was exposed at the proximal thigh level. An 8/0 suture with a 3/8 curved and reverse cutting mini-needle (Ethicon/Johnson & Johnson, St-Stevens-Wolume, Belgium) was inserted into the dorsal part of the nerve and firmly tied, severing approximately 1/3 to 1/2 of the nerve. The wound was then sutured in layers.
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set of regularly calibrated von Frey filaments with stiffness corresponding to 0.5, 0.8, 1.5, 2.7, 4.0, 4.5, 5.5, 7, 8.5, 10, 12.5, 15, 18.5, 20, 22, 26, and 30 g bending force to quantify the degree of hypersensitivity. The test always started with the least stiff filament and continued in ascending order of stiffness to avoid sensitization. The softest filament that produced a brisk withdrawal to at least 3 of 5 applications was defined as the WT. Cold sensitivity was assessed using ethyl chloride spray (HistoLab, Göteborg, Sweden). The responses were classified according to an arbitrary inversed ranking scale: vocalization and/or biting (0), paw licking (1), paw shaking (2), paw withdrawal (3), and no response (4). Responses from a brisk paw withdrawal (3) to vocalization and other generalized aversive reactions (0) were considered as indicative of cold hypersensitivity. Prior to testing, a few bursts of ethyl chloride were sprayed next to the cage to habituate the animal to the noise of the discharge. Cold hypersensitivity was determined by the median response score of 3 quick-burst stimuli applied with 3-minute intervals [52]. Heat sensitivity was assessed using the rat plantar test (Ugo Basile, Comerio, Italy), a modified Hargreaves method. A mobile radiant heat source was located under the glass table and focused onto the hind paw. Paw withdrawal latencies (PWLs) were recorded in seconds. An automatic cut-off time of 30 seconds was set to avoid tissue damage. The mean of 3 recordings, obtained from both hind paws, with 3-minute recovery periods was taken as the final PWL. The apparatus was calibrated to give a mean value of PWL of approximately 22 seconds in control rats. 2.4. Implantation of a spinal cord stimulation system Under general anesthesia, a monopolar electrode system for SCS was implanted at the level of T11. The cathode, a solid rectangular platina-iridium plate, 3 1.5 mm, thickness 0.25 mm, was placed in the dorsal epidural space via a small laminectomy at the T11 level. The anode, a platina-iridium disc Ø = 6 mm, was implanted in subcutaneous tissue on the left chest wall. This technique has been described in detail earlier and has been subsequently used in many studies [34,48]. The two poles were connected via insulated stainless steel wires (Medtronic Inc., Minneapolis, MN, USA) tunneled subcutaneously to microcontacts fixed to the neck skin. After electrode implantation, rats were allowed to recover for at least 48 hours before starting further experiments. They were each kept in separate cages to avoid damage to the microcontacts by other rats. Rats with signs of neurological sequelae after the surgery were excluded from subsequent experiments.
2.3. Assessment of pain-related behavior
2.5. Spinal cord stimulation
The behavioral studies were carried out under standardized conditions in a separate quiet room 2 weeks after the induction of nerve injury. For the tactile and cold hypersensitivity tests, the rat was placed in a circular observation Plexiglas cage equipped with a metal mesh floor and allowed to acclimatize to the environment for at least 15 minutes before starting the experiments. For the heat sensitivity test, rats were habituated for the same period of time to the apparatus, which consisted of Plexiglas boxes on an elevated glass table with a movable light source below it. Tactile sensitivity was assessed using 2 regularly calibrated von Frey filaments (Marstock Nervtest, Marburg, Germany) corresponding to 2.7 g (26 mN) and 12.5 g (147 mN), respectively. Each hair was applied to the lateral aspect of the plantar surface of the paw with an upward force to bend the filament. The number of times the rat withdrew its leg, per 10 trials, was recorded. The filaments were applied with a minimum interval of 10 seconds. To determine the maximum possible effect (see later) of some selected drugs, withdrawal threshold (WT) was determined using a
For the spinal cord stimulation, the rat was again placed in the circular observation cage. The microcontacts of the SCS electrodes were connected to a Grass S44 stimulator via a constant current unit, CCU1 (Grass Instruments, Quinsey, MA, USA). The rat was able to move freely in the cage during the experiments. The stimulation parameters were chosen to mimic clinical practice and were the same as used in our previous studies [34,48]. Monopolar electrical stimulation was applied for 30 minutes with a frequency of 50 Hz and a pulse width of 0.2 ms. The amplitude was individually determined to 80% of the intensity required to produce a slight twitching in the lower trunk muscles or leg stretching (ie, motor threshold [MT]). The MT was always assessed before starting each experiment with SCS. During 30 minutes of SCS, the withdrawal responses to mechanical stimuli were assessed every 10 minutes and testing continued until the pretreatment threshold values were restored. Cold hypersensitivity and heat responses were checked immediately after the end of the SCS period.
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2.6. Implantation of intrathecal catheter For the implantation of intrathecal catheters, a small dorsal midline incision was made at the level of the iliac crest under isoflurane anaesthesia. The spinal canal was punctured at the L5–6 level with a 23-gauge needle. A 32-gauge polyethylene catheter (ReCathoco, Allison park, PA, USA) was introduced in the subarachnoidal space and advanced rostrally 3.5–4 cm to reach the lumbar enlargement. The catheter was fixed to the fascia, and its proximal end was tunneled subcutaneously and then fixed to the skin between the scapulae. To physiologically ascertain the proper position of the catheter tip, 10 lL of 2% lidocaine (Xylocain, AstraZeneca, Södertälje, Sweden) was injected to induce a transient flaccid paralysis of the hind limbs. Before the animals were killed, 10 lL methylene blue dye solution was injected by the same route to enable spread of the dye into the spinal cord. Data from animals in which no dye could be traced at the L4–L6 levels of spinal cord were not included in the study.
tivity produced by SCS. The same doses of methiothepin i.t. alone did not influence the basal tactile thresholds (Fig. 1A and B). Ketanserin (30 lg, i.t.), a 5-HT2A receptor antagonist, significantly counteracted the suppressive effect on tactile hypersensitivity during and 10 minutes after SCS (P < .05). The same dose of ketanserin or DMSO i.t. did not affect the basal tactile thresholds (Fig. 1C and D). Likewise, SDZ (30 lg, i.t.), a 5-HT4 receptor antagonist, significantly counteracted the suppressive effect on tactile hypersensitivity during and 10 minutes after SCS (P < .05). The same dose of SDZ did not affect the basal tactile thresholds (Fig. 1E and F). Ketanserin markedly attenuated the cold hypersensitivity suppressive effect during SCS (P < .05), whereas the other 5-HT receptor antagonists (methiothepin, SDZ) produced no attenuation. None of the antagonists, at the doses used, or DMSO i.t. affected basal cold scores (Fig. 2A through C). None of the 5-HT receptor antagonists (ketanserin, methiothepin, SDZ) had an effect on the SCS-attenuated heat hyperalgesia, as assessed by PWL. Also the same doses of the antagonists per se or DMSO did not affect the basal PWL (Fig. 3A through C).
2.7. Drugs The following drugs and doses (based on literature data) were used: methiothepin (5-HT1, 5-HT6, 5-HT7 receptor antagonist, 0.3, 3 lg [30]); ketanserin tartrate (5-HT2A receptor antagonist, 30 lg [23]); a-methylserotonin maleate salt, a-m-5-HT (5-HT2 receptor agonist, 5 lg [39]); 1-(3-chloropheny) biguanide hydrochloride, m-CPBG (5-HT3 receptor agonist, 10 lg [18]); 3-tropanylindole-3carboxylate methiodide, TICM (5-HT3 receptor antagonist, 1 lg [19]); SDZ-205,557,SDZ (5-HT4 receptor antagonist, 30 lg [18]); bicuculline, BIC (GABAA receptor antagonist, 15 lg [40]), and CGP 35348, CGP (GABAB receptor antagonist, 50 lg [53]). The substances were freshly dissolved in volumes of 10 lL saline except for ketanserin tartrate, which was dissolved in a volume of 10 lL 20% dimethylsulfoxide (DMSO). All drugs were prewarmed to 37.5°C, and administered i.t. using a Hamilton syringe, followed by 10 lL prewarmed saline to rinse the catheter. All tested drugs were injected i.t. 5 minutes before starting SCS expect for bicuculline and CGP, which were administered i.t. 10 minutes prior to SCS. To further determine the subeffective doses of the 5-HT2 agonist, 5-HT3 agonist, and antagonist we performed pilot experiments to identify a dose that produced less than 25% of the maximum possible effect (% MPE). % MPE was calculated by using the formula % MPE = [(postdrug withdrawal threshold predrug withdrawal threshold)/(cut-off gram predrug withdrawal threshold)] 100. 2.8. Data presentation and statistics The changes of behavior with respective treatments (control, SCS, and drug treatments) were analyzed using Kruskal–Wallis nonparametric analysis of variance followed by Dunn’s post-hoc test. Data are presented as means ± standard error of the mean (SEM) or medians with 5th to 95th percentiles, when appropriate. P < .05 was considered significant in all tests. Graphics and calculations were performed using GraphPad PRISM version 5.0 (GraphPad, San Dieg, CA, Chicago, IL, USA), and SPSS Version 15.0 (SPSS Inc, USA).
3. Results 3.1. Possible involvement of different 5-HT receptors in the SCSinduced effects on tactile and cold hypersensitivity and heat hyperalgesia Methiothepin (0.3, 3 lg, i.t.), a 5-HT1, 5-HT6, 5-HT7 receptor antagonist, had no effect on the attenuation of tactile hypersensi-
3.2. Possible enhancing effects of a 5-HT2 receptor agonist, a 5-HT3 receptor agonist, and an antagonist on the SCS-induced attenuation of tactile hypersensitivity Our pilot experiments confirmed that individual i.t. administration of a-m-5-HT (5-HT2 receptor agonist), m-CPBG (5-HT3 receptor agonist), or TICM (5-HT3 receptor antagonist) exhibited dosedependent antinociceptive effects in the present model of mononeuropathy [18,19,37,39]. The doses of a-m-5-HT (5 lg), m-CPBG (10 lg), and TICM (10 lg) that produced 6.7 ± 0.9%, 5.8 ± 1.2%, and 7.1 ± 2.1% MPE, respectively, in tactile hypersensitivity were chosen as test doses in the subsequent experiments. Injection of a-m-5-HT or m-CPBG 5 minutes prior to the SCS application had an enhancing effect as shown by significant reductions of the response percentage evoked by mechanical stimulation with a 12.5 g filament at time points 20 minutes and 30 minutes, but not with a 2.7 g filament (Fig. 4A through D). On the other hand, TICM failed to influence the SCS effects on tactile and cold hypersensitivity and on heat hyperalgesia (Fig. 5A through C). 3.3. Effect of GABAA, B receptor antagonists on the enhanced SCS effect produced by a 5-HT3 receptor agonist Consistent with our previous study [9], i.t. injection of CGP (GABAB receptor antagonist) abolished the entire effect of SCS alone or when enhanced by m-CPBG. Conversely, BIC (GABAA receptor antagonist) selectively eliminated the 5-HT3-induced enhancement while the original SCS effect remained intact (Fig. 6A and B).
4. Discussion The current study was performed as a result of a previous investigation in which we, in a neuropathic pain model, demonstrated that the pain-relieving effect of SCS is produced partially by activation of 5-HT descending pathways [53]. The findings in the present study show that spinal serotonin receptors, at least in part, mediate the SCS-induced pain-relieving effect, because ketanserin [23] and SDZ [18] significantly attenuated the suppressive effect of SCS on tactile hypersensitivity, whereas methiothepin [30] and TICM [19] were ineffective. Further, the suppressive effect on cold hypersensitivity of SCS was counteracted only by ketanserin. In addition, subeffective doses of am-5-HT [39] and m-CPBG [18] enhanced the suppressive effect of SCS on tactile hypersensitivity. The enhancing effect of mCPBG was abolished by a preceding administration of bicuculline
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Fig. 1. Effects of different types of 5-HT receptor antagonists (i.t.) on SCS-induced attenuation of tactile hypersensitivity. Percentages of paw withdrawal responses to 2 von Frey filaments (2.7 and 12.5 g) were recorded from the intact (Contra) hind paw and the nerve injured paw without (basal WT) and with SCS, SCS with pretreatment with a receptor antagonist, and with the antagonist alone; for ketanserin, a control test with DMSO was included. (A and B) Methiothepin (0.3, 3 lg), a 5-HT1, 5-HT6, 5-HT7 receptor antagonist, influenced neither the basal thresholds nor the SCS effect. (C and D) Ketanserin (30 lg), a 5-HT2A receptor antagonist, significantly counteracted the hypersensitivity suppressive effect during and 10 minutes after SCS. The same dose of ketanserin or DMSO did not affect the basal thresholds. (E and F) SDZ (30 lg), a 5-HT4 receptor antagonist, significantly counteracted the hypersensitivity suppressive effect during and 10 minutes after SCS. The same dose of SDZ did not affect the basal thresholds. Data are presented as means ± SEM percentage of paw withdrawals to 10 filament applications. n = 4 to 7. ⁄P < .05. DMSO = dimethylsulfoxide; i.t. = intrathecally; SCS = spinal cord stimulation; SDZ = SDZ-205, 557; SEM = standard error of the mean; WT = withdrawal threshold.
Fig. 2. Effects of different types of 5-HT receptor antagonists (i.t.) on SCS-induced attenuation of cold hypersensitivity. Comparison of scored responses to cold spray applied to the nerve-injured hind paw without (Basal) and with SCS, with the antagonist alone, and SCS pretreated with a receptor antagonist. Ketanserin counteracted the cold hypersensitivity suppression produced by SCS (B), whereas the other 5-HT receptor antagonists, methiothepin (A) and SDZ (C), had no effect. The same doses of respective antagonists or DMSO did not affect the basal PWL. Data are presented as medians with 5th to 95th percentiles. n = 7; ⁄P < .05. DMSO = dimethylsulfoxide; i.t. = intrathecally; PWL = paw withdrawal latency; SCS = spinal cord stimulation; SDZ = SDZ-205, 557.
Fig. 3. Effects of different types of 5-HT receptor antagonists (i.t.) on SCS-induced attenuation of heat hyperalgesia. Comparison of PWL to radiant heat applied to the nerveinjured hind paw without (Basal) and with SCS, with the antagonist alone, and SCS pretreated with a receptor antagonist. None of the 5-HT subtype receptor antagonists (ketanserin (A), methiothepin (B), SDZ-205,557(C)) had an effect on PWLs augmented by SCS. The same doses of respective antagonists or DMSO did not affect the basal PWL. Data are presented as medians with 5th to 95th percentiles. n = 7. ⁄P < .05. DMSO = dimethylsulfoxide; i.t. = intrathecally; PWL = paw withdrawal latency; SCS = spinal cord stimulation; SDZ = SDZ-205, 557.
[40] or CGP [53]. These findings suggest that the 5-HT2A, 5-HT3, and 5-HT4 receptors, but not the 5-HT1, 5-HT6, and 5-HT7 receptors, are involved in the SCS-induced pain-relieving effect at the spinal cord level.
Serotonin may bind to several different types of receptors, of which 7 have been identified so far (5-HT1-7), and a total of 14 subtypes. All types, except 5-HT3, are G-protein coupled. At least 5 types of 5-HT receptors (5-HT1, 5-HT2, 5-HT3, 5-HT4, and 5-HT7)
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Fig. 4. Enhancing effects on SCS-induced attenuation of tactile hypersensitivity by a-m-5-HT (5-HT2 receptor agonist, 5 lg) (A and B) or m-CPBG (5-HT3 receptor agonist, 10 lg) (C and D). The drugs were given 5 minutes before 30 minutes of SCS. Data were presented as means ± SEM percentage of paw withdrawals to 10 filaments applications. n = 7. ⁄P < .05. SCS = spinal cord stimulation; SEM = standard error of the mean.
are believed to be involved in the modulation of nociceptive processing in the spinal dorsal horn [12,18,19,23,30,59]. In the current study, ketanserin, a 5-HT2A receptor antagonist, attenuated the SCS
suppressive effect on tactile and cold hypersensitivity, but had no influence on heat hyperalgesia, indicating that the SCS-induced pain-relieving effect is only partially mediated by the 5-HT2A
Fig. 5. Absent effect of TICM (5-HT3 receptor antagonist, 10 lg i.t) on SCS-induced attenuation of tactile and cold hypersensitivity, as well as of heat hyperalgesia. (A and B) Percents of paw withdrawal responses to 2 von Frey filaments (2.7 and 12.5 g) were recorded from the intact (Contra) hind paw and the nerve-injured paw without (Basal WT) and with SCS, SCS with pretreatment with a receptor antagonist, and with the antagonist alone. (C) Comparison of scored responses to cold spray and (D) PWL to radiant heat, applied to the nerve-injured hind paw without (Basal) and with SCS, with the antagonist alone and SCS pretreated with a receptor antagonist. Data are presented as means ± SEM percentage of paw withdrawals to 10 filament applications (A and B) and as medians with 5th to 95th percentiles (C and D). n = 7. DMSO = dimethylsulfoxide; i.t. = intrathecally; PWL = paw withdrawal latency; SCS = spinal cord stimulation; SDZ = SDZ-205, 557; SEM = standard error of the mean; TICM = 3-tropanylindole-3carboxylate methiodide; WT = withdrawal threshold.
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Fig. 6. Involvement of GABAA,B receptors in m-CPBG (5-HT3 receptor agonist)-induced enhancing effect of SCS on tactile hypersensitivity. BIC (GABAA receptor antagonist, 15 lg i.t.) or CGP (GABAB receptor antagonist, 50 lg i.t.) were given 5 minutes before m-CPBG (10 lg) followed by SCS. n = 7. ⁄P < .05 refers to effect by BIC; +P < .05, ++P < .01 refer to effect by CGP. BIC = bicuculline; CGP = CGP 35348; GABA = c-aminobutyric acid; i.t. = intrathecally; SCS = spinal cord stimulation.
receptor. Nevertheless, a 5-HT2 receptor agonist could enhance the SCS effect, as demonstrated with the use of a 12.5 g von Frey filament. There was no significant further attenuation of the paw withdrawals to a thin filament (2.7 g), conceivably due to the fact that they were almost normalized by SCS alone. Several studies indicate that the 5-HT2 receptor is involved in antinociception, as demonstrated in the formalin test [46] and in a nerve injury model [38]. Radhakrishnan et al. in 2003 [43] reported that the 5-HT2A receptor appears to mediate transcutaneous electrical nerve stimulation–induced antihyperalgesia in an inflammatory pain model. In the rat spinal cord, 5-HT2 receptors are present in both superficial and deep laminae of the dorsal horn, although their density is low [31,56]. The 5-HT2 receptor mRNA is expressed in both large and small DRG neurons in the rat, suggesting that these receptors are localized at central as well as peripheral terminals of both large and small afferent fibers [57]. Mechanical hypersensitivity is mediated via myelinated fibers (mainly Ab fibers, but also Ad fibers), whereas heat hyperalgesia involves unmyelinated C-fibers, and both Ad- and C-fibers mediate cold hypersensitivity [21]. However, in the present study, intrathecal administration of ketanserin (30 lg) merely eliminated the SCS-induced attenuation of the hypersensitivity (tactile and cold) and had no influence on the SCS-induced changes in heat hyperalgesia. The absence of effect of ketanserin on heat hyperalgesia may be explained by its high affinity to certain subtypes of 5-HT2 receptors. The binding affinity (1/Ki value; Ki = inhibition constant) of ketanserin for the 5-HT2A receptor site has been reported to be 42% [25]. It also might be that the SCS effect on hypersensitivity (tactile and cold) is selectively mediated by activation of the 5-HT2A receptors, while another receptor subtype is involved in heat hyperalgesia. However, considering that none of the other 5-HT receptor antagonists attenuated the suppressive effect of SCS on heat hyperalgesia, it is more likely that the mechanisms involved in the effect of SCS on this form of neuropathology are more or less independent from the spinal serotonergic system as demonstrated previously [53]. The present results confirmed that both the 5-HT3 receptor agonist m-CPBG and antagonist TICM have an antihypersensitivity effect in partial sciatic nerve ligation rats [40,55]. Unlike the other 5-HT receptors referred to previously, the 5-HT3 receptor is an excitatory ligand-gated ion channel [11]. Autoradiography, in situ hybridization, and immunocytochemical studies demonstrate a dense band of 5-HT3 receptors in the superficial laminae of the dorsal horn [22,24,36]. The exact neuronal populations expressing the 5-HT3 receptor remain controversial. 5-HT3 receptor immunoreactivity is not found in glutamic acid decarboxylase-expressing interneurons, but is present in primary afferent fibers. A small proportion of these fibers contains the calcitonin gene-related peptide, with the remaining 5-HT3-positive fibers found in an unidentified subset of myelinated and unmyelinated nociceptors [32,61]. Hence, blockade of the spinal 5-HT3 receptor reduces release of pronociceptive pep-
tides from primary afferents, and this is also supported by the finding that the analgesia after i.t. administration of 5-HT is attenuated by selectively knocking down the spinal 5-HT3 receptor [41]. There is some controversy over the identity of the descending serotonergic system responsible for the facilitatory or inhibitory effect in neuropathic pain [6,35,42,44]. To inhibit neuropathic pain, OFF cells in the rostroventromedial medulla activate 5-HT3 receptors on inhibitory interneurons in the dorsal horn, and hence suppress pain by an increase of inhibitory transmitters [35]. It has been reported that i.t. administration of a selective 5-HT3 receptor agonist can increase spinal GABA concentration without changing the glutamate and glycine levels in the spinal cord, suggesting an additional inhibitory effect on second-order neurons [20,40]. A subeffective dose of the 5-HT3 receptor agonist m-CPBG was found to enhance the SCS effect, whereas the corresponding antagonist neither enhanced nor counteracted SCS. Considering that there is evidence that neuropathic pain is maintained by a spinal excitatory tone of serotonin via the 5-HT3 receptor, we had expected TICM to enhance the SCS effect. The absence of effect of TICM may reflect the predominant importance of the 5-HT3 receptor linkage to local GABAergic interneurons [20,40]. To further explore this issue, GABAA and GABAB receptor antagonists were given shortly before the administration of m-CPBG, which produced an enhancement of the SCS effect. This enhancing effect on SCS was totally abolished by a GABAB antagonist (CGP) and partially blocked by a GABAA antagonist (BIC). These results are consistent with our previous study showing the crucial role of the GABAB receptor in the SCS-induced pain-relieving effect, whereas the GABAA receptor appears to be of less importance [8]. The possible role played by 5-HT4 receptors in pain modulation has up to now not been much investigated. Nevertheless, there are data indicating that activation of 5-HT4 receptors may reduce visceral pain [14], and it has also been shown that this receptor type is involved in serotonin-induced antinociception. There is also evidence that 5-HT4 agonists may elicit antinociception via cholinergic and GABAergic mechanisms [5,16]. The present study shows that a 5-HT4 receptor antagonist markedly attenuated the SCS-induced reduction of tactile hypersensitivity, indicating the likely involvement of this receptor. The role of 5-HT4 in SCS is in agreement with the involvement of the spinal cholinergic and GABAgeric systems, as demonstrated previously [8,47]. In conclusion, the current study demonstrates that the SCS-induced pain-relieving effect involves activation of spinal 5-HT receptors (5-HT2A, 5-HT3, and 5-HT4), partly via GABAergic interneurons. Acknowledgements This study was supported by grants from Medtronic International and the Karolinska Institutet Funds. Special thanks to
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Dr. Linda Sorkin for critical reading of the manuscript. The authors state that there is no conflict of interest. References [1] Bardin L, Bardin M, Lavarenne J, Eschalier A. Effect of intrathecal serotonin on nociception in rats: influence of the pain test used. Exp Brain Res 1997;113:81–7. [2] Bardin L, Lavarenne J, Eschalier A. Serotonin receptor subtypes involved in the spinal antinociceptive effect of 5-HT in rats. Pain 2000;86:11–8. [3] Bardin L, Schmidt J, Alloui A, Eschalier A. Effect of intrathecal administration of serotonin in chronic pain models in rats. Eur J Pharmacol 2000;409:37–43. [4] Barnes NM, Sharp T. A review of central 5-HT receptors and their function. Neuropharmacology 1999;38:1083–152. [5] Bianchi C, Rodi D, Marino S, Beani L, Siniscalchi A. Dual effects of 5-HT4 receptor activation on GABA release from guinea pig hippocampal slices. Neuroreport 2002;13:2177–80. [6] Burgess SE, Gardell LR, Ossipov MH, Malan Jr TP, Vanderah TW, Lai J, Porreca F. Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain. J Neurosci 2002;22:5129–36. [7] Crisp T, Stafinsky JL, Spanos LJ, Uram M, Perni VC, Donepudi HB. Analgesic effects of serotonin and receptor-selective serotonin agonists in the rat spinal cord. Gen Pharmacol 1991;22:247–51. [8] Cui JG, Linderoth B, Meyerson BA. Effects of spinal cord stimulation on touchevoked allodynia involve GABAergic mechanisms. An experimental study in the mononeuropathic rat. Pain 1996;66:287–95. [9] Cui JG, Meyerson BA, Sollevi A, Linderoth B. Effect of spinal cord stimulation on tactile hypersensitivity in mononeuropathic rats is potentiated by simultaneous GABA(B) and adenosine receptor activation. Neurosci Lett 1998;247:183–6. [10] Cui JG, O’Connor WT, Ungerstedt U, Linderoth B, Meyerson BA. Spinal cord stimulation attenuates augmented dorsal horn release of excitatory amino acids in mononeuropathy via a GABAergic mechanism. Pain 1997;73:87–95. [11] Derkach V, Surprenant A, North RA. 5-HT3 receptors are membrane ion channels. Nature 1989;339:706–9. [12] Dogrul A, Ossipov MH, Porreca F. Differential mediation of descending pain facilitation and inhibition by spinal 5HT-3 and 5HT-7 receptors. Brain Res 2009;1280:52–9. [13] El-Khoury C, Hawwa N, Baliki M, Atweh SF, Jabbur SJ, Saade NE. Attenuation of neuropathic pain by segmental and supraspinal activation of the dorsal column system in awake rats. Neuroscience 2002;112:541–53. [14] Espejo EF, Gil E. Antagonism of peripheral 5-HT4 receptors reduces visceral and cutaneous pain in mice, and induces visceral analgesia after simultaneous inactivation of 5-HT3 receptors. Brain Res 1998;788:20–4. [15] Finnerup NB, Otto M, McQuay HJ, Jensen TS, Sindrup SH. Algorithm for neuropathic pain treatment: an evidence based proposal. Pain 2005;118:289–305. [16] Ghelardini C, Galeotti N, Casamenti F, Malmberg-Aiello P, Pepeu G, Gualtieri F, Bartolini A. Central cholinergic antinociception induced by 5HT4 agonists: BIMU 1 and BIMU 8. Life Sci 1996;58:2297–309. [17] Hylden JL, Wilcox GL. Intrathecal serotonin in mice: analgesia and inhibition of a spinal action of substance P. Life Sci 1983;33:789–95. [18] Jeong CY, Choi JI, Yoon MH. Roles of serotonin receptor subtypes for the antinociception of 5-HT in the spinal cord of rats. Eur J Pharmacol 2004;502:205–11. [19] Kawamata T, Omote K, Toriyabe M, Kawamata M, Namiki A. Intracerebroventricular morphine produces antinociception by evoking gamma-aminobutyric acid release through activation of 5hydroxytryptamine 3 receptors in the spinal cord. Anesthesiology 2002;96:1175–82. [20] Kawamata T, Omote K, Toriyabe M, Yamamoto H, Namiki A. The activation of 5-HT(3) receptors evokes GABA release in the spinal cord. Brain Res 2003;978:250–5. [21] Khan GM, Chen SR, Pan HL. Role of primary afferent nerves in allodynia caused by diabetic neuropathy in rats. Neuroscience 2002;114:291–9. [22] Kia HK, Miquel MC, McKernan RM, Laporte AM, Lombard MC, Bourgoin S, Hamon M, Verge D. Localization of 5-HT3 receptors in the rat spinal cord: immunohistochemistry and in situ hybridization. Neuroreport 1995;6:257–61. [23] Kim SK, Park JH, Bae SJ, Kim JH, Hwang BG, Min BI, Park DS, Na HS. Effects of electroacupuncture on cold allodynia in a rat model of neuropathic pain: mediation by spinal adrenergic and serotonergic receptors. Exp Neurol 2005;195:430–6. [24] Laporte AM, Koscielniak T, Ponchant M, Verge D, Hamon M, Gozlan H. Quantitative autoradiographic mapping of 5-HT3 receptors in the rat CNS using [125I]iodo-zacopride and [3H]zacopride as radioligands. Synapse 1992;10:271–81. [25] Leysen JE, Niemegeers CJ, Van Nueten JM, Laduron PM. [3H]Ketanserin (R 41,468), a selective 3H-ligand for serotonin2 receptor binding sites. Binding properties, brain distribution, and functional role. Mol Pharmacol 1982;21:301–14. [26] Lind G, Meyerson BA, Winter J, Linderoth B. Intrathecal baclofen as adjuvant therapy to enhance the effect of spinal cord stimulation in neuropathic pain: a pilot study. Eur J Pain 2004;8:377–83.
[27] Lind G, Schechtmann G, Winter J, Meyerson BA, Linderoth B. Baclofenenhanced spinal cord stimulation and intrathecal baclofen alone for neuropathic pain: long-term outcome of a pilot study. Eur J Pain 2008;12:132–6. [28] Linderoth B, Gazelius B, Franck J, Brodin E. Dorsal column stimulation induces release of serotonin and substance P in the cat dorsal horn. Neurosurgery 1992;31:289–96. discussion 287–96. [29] Liu MY, Su CF, Lin MT. The antinociceptive role of a bulbospinal serotonergic pathway in the rat brain. Pain 1988;33:123–9. [30] Lozano-Cuenca J, Castaneda-Hernandez G, Granados-Soto V. Peripheral and spinal mechanisms of antinociceptive action of lumiracoxib. Eur J Pharmacol 2005;513:81–91. [31] Marlier L, Teilhac JR, Cerruti C, Privat A. Autoradiographic mapping of 5-HT1, 5-HT1A, 5-HT1B and 5-HT2 receptors in the rat spinal cord. Brain Res 1991;550:15–23. [32] Maxwell DJ, Kerr R, Rashid S, Anderson E. Characterisation of axon terminals in the rat dorsal horn that are immunoreactive for serotonin 5-HT3A receptor subunits. Exp Brain Res 2003;149:114–24. [33] Meyerson BA, Linderoth B. Mechanisms of spinal cord stimulation in neuropathic pain. Neurol Res 2000;22:285–92. [34] Meyerson BA, Ren B, Herregodts P, Linderoth B. Spinal cord stimulation in animal models of mononeuropathy: effects on the withdrawal response and the flexor reflex. Pain 1995;61:229–43. [35] Millan MJ. Descending control of pain. Prog Neurobiol 2002;66:355–474. [36] Morales M, Battenberg E, de Lecea L, Sanna PP, Bloom FE. Cellular and subcellular immunolocalization of the type 3 serotonin receptor in the rat central nervous system. Brain Res Mol Brain Res 1996;36:251–60. [37] Oatway MA, Chen Y, Weaver LC. The 5-HT3 receptor facilitates at-level mechanical allodynia following spinal cord injury. Pain 2004;110:259–68. [38] Obata H, Saito S, Sakurazawa S, Sasaki M, Usui T, Goto F. Antiallodynic effects of intrathecally administered 5-HT(2C) receptor agonists in rats with nerve injury. Pain 2004;108:163–9. [39] Obata H, Saito S, Sasaki M, Ishizaki K, Goto F. Antiallodynic effect of intrathecally administered 5-HT(2) agonists in rats with nerve ligation. Pain 2001;90:173–9. [40] Okazaki R, Namba H, Yoshida H, Okai H, Miura T, Kawamura M. The antiallodynic effect of neurotropin is mediated via activation of descending pain inhibitory systems in rats with spinal nerve ligation. Anesth Analg 2008;107:1064–9. [41] Paul D, Yao D, Zhu P, Minor LD, Garcia MM. 5-Hydroxytryptamine3 (5-HT3) receptors mediate spinal 5-HT antinociception: an antisense approach. J Pharmacol Exp Ther 2001;298:674–8. [42] Pertovaara A, Almeida A. Descending inhibitory systems. Handb Clin Neurol 2006;81:179–92. [43] Radhakrishnan R, King EW, Dickman JK, Herold CA, Johnston NF, Spurgin ML, Sluka KA. Spinal 5-HT(2) and 5-HT(3) receptors mediate low, but not high, frequency TENS-induced antihyperalgesia in rats. Pain 2003;105:205–13. [44] Rahman W, Suzuki R, Webber M, Hunt SP, Dickenson AH. Depletion of endogenous spinal 5-HT attenuates the behavioural hypersensitivity to mechanical and cooling stimuli induced by spinal nerve ligation. Pain 2006;123:264–74. [45] Saade NE, Tabet MS, Soueidan SA, Bitar M, Atweh SF, Jabbur SJ. Supraspinal modulation of nociception in awake rats by stimulation of the dorsal column nuclei. Brain Res 1986;369:307–10. [46] Sasaki M, Obata H, Saito S, Goto F. Antinociception with intrathecal alphamethyl-5-hydroxytryptamine, a 5-hydroxytryptamine 2A/2C receptor agonist, in two rat models of sustained pain. Anesth Analg 2003;96:1072–8. [47] Schechtmann G, Song Z, Ultenius C, Meyerson BA, Linderoth B. Cholinergic mechanisms involved in the pain relieving effect of spinal cord stimulation in a model of neuropathy. Pain 2008;139:136–45. [48] Schechtmann G, Wallin J, Meyerson BA, Linderoth B. Intrathecal clonidine potentiates suppression of tactile hypersensitivity by spinal cord stimulation in a model of neuropathy. Anesth Analg 2004;99:135–9. [49] Seltzer Z, Dubner R, Shir Y. A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 1990;43: 205–8. [50] Simpson BA, Meyerson BA, Linderoth B. Spinal cord and brain stimulation. In: McMahon SB, Koltzenburg M, editors. Textbook of pain. Amsterdam: Elsevier; 2006. p. 563–82. [51] Sluka KA, Lisi TL, Westlund KN. Increased release of serotonin in the spinal cord during low, but not high, frequency transcutaneous electric nerve stimulation in rats with joint inflammation. Arch Phys Med Rehabil 2006;87:1137–40. [52] Song Z, Meyerson BA, Linderoth B. Muscarinic receptor activation potentiates the effect of spinal cord stimulation on pain-related behavior in rats with mononeuropathy. Neurosci Lett 2008;436:7–12. [53] Song Z, Ultenius C, Meyerson BA, Linderoth B. Pain relief by spinal cord stimulation involves serotonergic mechanisms: an experimental study in a rat model of mononeuropathy. Pain 2009;147:241–8. [54] Stiller CO, Cui JG, O’Connor WT, Brodin E, Meyerson BA, Linderoth B. Release of gamma-aminobutyric acid in the dorsal horn and suppression of tactile allodynia by spinal cord stimulation in mononeuropathic rats. Neurosurgery 1996;39:367–74. discussion 365–74. [55] Suzuki R, Rahman W, Hunt SP, Dickenson AH. Descending facilitatory control of mechanically evoked responses is enhanced in deep dorsal horn neurones following peripheral nerve injury. Brain Res 2004;1019:68–76.
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Z. Song et al. / PAIN 152 (2011) 1666–1673 [56] Thor KB, Nickolaus S, Helke CJ. Autoradiographic localization of 5hydroxytryptamine1A, 5-hydroxytryptamine1B and 5-hydroxytryptamine1C/2 binding sites in the rat spinal cord. Neuroscience 1993;55: 235–52. [57] Tokunaga A, Saika M, Senba E. 5-HT2A receptor subtype is involved in the thermal hyperalgesic mechanism of serotonin in the periphery. Pain 1998;76:349–55. [58] Tyce GM, Yaksh TL. Monoamine release from cat spinal cord by somatic stimuli: an intrinsic modulatory system. J Physiol 1981;314:513–29.
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[59] Wu S, Zhu M, Wang W, Wang Y, Li Y, Yew DT. Changes of the expression of 5HT receptor subtype mRNAs in rat dorsal root ganglion by complete Freund’s adjuvant-induced inflammation. Neurosci Lett 2001;307:183–6. [60] Yaksh TL, Wilson PR. Spinal serotonin terminal system mediates antinociception. J Pharmacol Exp Ther 1979;208:446–53. [61] Zeitz KP, Guy N, Malmberg AB, Dirajlal S, Martin WJ, Sun L, Bonhaus DW, Stucky CL, Julius D, Basbaum AI. The 5-HT3 subtype of serotonin receptor contributes to nociceptive processing via a novel subset of myelinated and unmyelinated nociceptors. J Neurosci 2002;22:1010–9.
PAIN 152 (2011) 1666–1673
www.elsevier.com/locate/pain
Spinal 5-HT receptors that contribute to the pain-relieving effects of spinal cord stimulation in a rat model of neuropathy Zhiyang Song ⇑, Björn A. Meyerson, Bengt Linderoth Department of Clinical Neuroscience, Section of Neurosurgery, Karolinska Institutet, Stockholm, Sweden
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
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Article history: Received 14 September 2010 Received in revised form 3 February 2011 Accepted 8 March 2011
Keywords: Neuropathic pain Spinal cord stimulation 5-HT receptor Rat
a b s t r a c t Spinal cord stimulation (SCS) is extensively employed in the management of neuropathic pain, but the underlying mechanisms are only partially understood. Recently, we demonstrated that the pain-relieving effect of SCS appears to involve the spinal serotonin system, and the present study aimed at identifying the types of the spinal serotonin receptors involved. Experiments were performed on rats with neuropathy produced by partial ligation of the sciatic nerve. Tactile sensitivity was assessed using von Frey filaments, and cold and heat sensitivity with cold spray and radiant heat, respectively. Selective 5-HT receptor antagonists, methiothepin (5-HT1,6,7), ketanserin tartrate (5-HT2A), TICM (5-HT3), SDZ-205,557 (5-HT4), as well as receptor agonists, a-m-5-HT (5-HT2), m-CPBG (5-HT3) in per se ineffective doses, or vehicle, were administrated intrathecally 5 minutes prior to the application of SCS. Ketanserin and SDZ-205,557 significantly attenuated the suppressive effect of SCS on tactile hypersensitivity, while methiothepin and TICM were ineffective. The suppressive effect on cold hypersensitivity of SCS was counteracted by ketanserin only. None of the 5-HT receptor antagonists attenuated the suppressive effect on heat hyperalgesia of SCS. Subeffective doses of a-m-5-HT and m-CPBG enhanced the suppressive effect of SCS on tactile hypersensitivity. The enhancing effect of m-CPBG was abolished by a c-aminobutyric acid (GABA)A or GABAB antagonist intrathecally. These results suggest that the activation of 5-HT2A, 5-HT3, and 5-HT4 receptors plays an important role in SCS-induced relief of neuropathic pain. The activation of 5-HT3 receptors appears to operate via spinal GABAergic interneurons. Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
1. Introduction Spinal cord stimulation (SCS) provides useful, long-lasting pain relief in 60–70% of patients suffering from certain forms of neuropathic pain [33,50], which are often resistant to conventional pharmacotherapy [15]. However, knowledge concerning its mode of action is still fragmentary. Therefore, a better understanding of the neurobiological mechanisms behind the pain-suppressing effect of SCS may help to develop new treatment strategies. In our previous studies, c-aminobutyric acid (GABA)ergic and cholinergic mechanisms in the pain relief produced by SCS have been explored. It was shown that SCS could augment spinal dorsal horn GABA and acetylcholine (ACh) release, with associated decreased release of glutamate and aspartate in animal models of neuropathic pain [10,47,54]. It was also demonstrated that the corresponding receptor agonists (GABAB-baclofen; cholinergic musca⇑ Corresponding author. Address: Department of Clinical Neuroscience, Section of Neurosurgery, Karolinska University Hospital, S-171 76 Stockholm, Sweden. Tel.: +46 8 51775806; fax: +46 8 51771778. E-mail address: [email protected] (Z. Song).
rinic–oxotremorine) could significantly enhance the pain-relieving effect of SCS both in animal models and in patients [9,26,27,52]. Antinociceptive effects of serotonin are well documented in normal animals and in pain models [1,3,7]. In several early studies it was shown that electrical stimulation applied in the brain stem, to the dorsolateral funiculus and peripheral nerves, can activate serotonergic descending pathways with an increased release of 5-HT and an elevation of pain thresholds [29,51,58]. In a series of experimental studies, Saade et al. [13,45] have demonstrated an attenuating effect of electrical stimulation at the level of the dorsal column nuclei on the flexor reflex as well as on tactile and thermal hypersensitivity following peripheral nerve injury. They have thus provided evidence for the involvement of a supraspinal loop in the mode of action of SCS, implying that the effect is exerted, at least partially, via the activation of descending serotonergic pain inhibitory pathways. It was earlier demonstrated in cats that SCS induced serotonin release in the spinal dorsal horn [28]. More recently, we reported that SCS may induce an increase of the endogenous serotonin content in the rat dorsal horn, indicating an activation of the descending serotonergic pathways. This effect was also shown to partially
0304-3959/$36.00 Ó 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2011.03.012
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involve spinal GABAergic circuitries [53]. Inconsistent outcomes of pain modulation by exogenous 5-HT may be attributed to nonselective actions of 5-HT at multiple receptor types and subtypes [1–4,17,60]. At least 5 types of serotonergic receptors (5-HT1, 5HT2, 5-HT3, 5-HT4, and 5-HT7) are present in the spinal cord and are claimed to be involved in the modulation of nociceptive transmission [12,18,19,23,30,59]. The specific 5-HT receptors contributing to SCS-induced pain relief have not yet been explored. The aim of the current study was to gain insight into the functional significance of different types of 5-HT receptors involved in the SCS effect. Several selective 5-HT receptor antagonists and agonists were intrathecally (i.t.) administered to investigate their ability to counteract or enhance the pain-relieving effect induced by SCS in a neuropathic pain model. 2. Materials and methods 2.1. Animals and anesthesia The experiments were performed on male Sprague-Dawley rats (B&K Universal AB, Sollentuna, Sweden), weighing 250–350 g, in accordance with the recommendations of the Committee for Research and Ethical Issues of the International Association for the Study of Pain (1983) and with a protocol approved by the local ethical committee for animal research. The surgical procedures were performed under general anesthesia delivered through an open mask system. Anesthesia was induced by 4% Isoflurane (Forene, Abbott, Solna, Sweden) and maintained with 1–2% in a 1:1 mixture of air and oxygen at a flow rate of 2 L/min. During surgery, the body temperature was maintained at 37 ± 0.5°C by an automatic heating pad (CMA/150, CMA Microdialysis AB, Stockholm, Sweden). Postoperative analgesia was provided by subcutaneous injection of 5 mg/kg carprofen (Rimadyl, Pfizer, New york, NY, USA). 2.2. Sciatic nerve ligation A sciatic nerve lesion was created according to the method of Seltzer et al. [49] to produce mononeuropathy. In short, the left sciatic nerve was exposed at the proximal thigh level. An 8/0 suture with a 3/8 curved and reverse cutting mini-needle (Ethicon/Johnson & Johnson, St-Stevens-Wolume, Belgium) was inserted into the dorsal part of the nerve and firmly tied, severing approximately 1/3 to 1/2 of the nerve. The wound was then sutured in layers.
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set of regularly calibrated von Frey filaments with stiffness corresponding to 0.5, 0.8, 1.5, 2.7, 4.0, 4.5, 5.5, 7, 8.5, 10, 12.5, 15, 18.5, 20, 22, 26, and 30 g bending force to quantify the degree of hypersensitivity. The test always started with the least stiff filament and continued in ascending order of stiffness to avoid sensitization. The softest filament that produced a brisk withdrawal to at least 3 of 5 applications was defined as the WT. Cold sensitivity was assessed using ethyl chloride spray (HistoLab, Göteborg, Sweden). The responses were classified according to an arbitrary inversed ranking scale: vocalization and/or biting (0), paw licking (1), paw shaking (2), paw withdrawal (3), and no response (4). Responses from a brisk paw withdrawal (3) to vocalization and other generalized aversive reactions (0) were considered as indicative of cold hypersensitivity. Prior to testing, a few bursts of ethyl chloride were sprayed next to the cage to habituate the animal to the noise of the discharge. Cold hypersensitivity was determined by the median response score of 3 quick-burst stimuli applied with 3-minute intervals [52]. Heat sensitivity was assessed using the rat plantar test (Ugo Basile, Comerio, Italy), a modified Hargreaves method. A mobile radiant heat source was located under the glass table and focused onto the hind paw. Paw withdrawal latencies (PWLs) were recorded in seconds. An automatic cut-off time of 30 seconds was set to avoid tissue damage. The mean of 3 recordings, obtained from both hind paws, with 3-minute recovery periods was taken as the final PWL. The apparatus was calibrated to give a mean value of PWL of approximately 22 seconds in control rats. 2.4. Implantation of a spinal cord stimulation system Under general anesthesia, a monopolar electrode system for SCS was implanted at the level of T11. The cathode, a solid rectangular platina-iridium plate, 3 1.5 mm, thickness 0.25 mm, was placed in the dorsal epidural space via a small laminectomy at the T11 level. The anode, a platina-iridium disc Ø = 6 mm, was implanted in subcutaneous tissue on the left chest wall. This technique has been described in detail earlier and has been subsequently used in many studies [34,48]. The two poles were connected via insulated stainless steel wires (Medtronic Inc., Minneapolis, MN, USA) tunneled subcutaneously to microcontacts fixed to the neck skin. After electrode implantation, rats were allowed to recover for at least 48 hours before starting further experiments. They were each kept in separate cages to avoid damage to the microcontacts by other rats. Rats with signs of neurological sequelae after the surgery were excluded from subsequent experiments.
2.3. Assessment of pain-related behavior
2.5. Spinal cord stimulation
The behavioral studies were carried out under standardized conditions in a separate quiet room 2 weeks after the induction of nerve injury. For the tactile and cold hypersensitivity tests, the rat was placed in a circular observation Plexiglas cage equipped with a metal mesh floor and allowed to acclimatize to the environment for at least 15 minutes before starting the experiments. For the heat sensitivity test, rats were habituated for the same period of time to the apparatus, which consisted of Plexiglas boxes on an elevated glass table with a movable light source below it. Tactile sensitivity was assessed using 2 regularly calibrated von Frey filaments (Marstock Nervtest, Marburg, Germany) corresponding to 2.7 g (26 mN) and 12.5 g (147 mN), respectively. Each hair was applied to the lateral aspect of the plantar surface of the paw with an upward force to bend the filament. The number of times the rat withdrew its leg, per 10 trials, was recorded. The filaments were applied with a minimum interval of 10 seconds. To determine the maximum possible effect (see later) of some selected drugs, withdrawal threshold (WT) was determined using a
For the spinal cord stimulation, the rat was again placed in the circular observation cage. The microcontacts of the SCS electrodes were connected to a Grass S44 stimulator via a constant current unit, CCU1 (Grass Instruments, Quinsey, MA, USA). The rat was able to move freely in the cage during the experiments. The stimulation parameters were chosen to mimic clinical practice and were the same as used in our previous studies [34,48]. Monopolar electrical stimulation was applied for 30 minutes with a frequency of 50 Hz and a pulse width of 0.2 ms. The amplitude was individually determined to 80% of the intensity required to produce a slight twitching in the lower trunk muscles or leg stretching (ie, motor threshold [MT]). The MT was always assessed before starting each experiment with SCS. During 30 minutes of SCS, the withdrawal responses to mechanical stimuli were assessed every 10 minutes and testing continued until the pretreatment threshold values were restored. Cold hypersensitivity and heat responses were checked immediately after the end of the SCS period.
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2.6. Implantation of intrathecal catheter For the implantation of intrathecal catheters, a small dorsal midline incision was made at the level of the iliac crest under isoflurane anaesthesia. The spinal canal was punctured at the L5–6 level with a 23-gauge needle. A 32-gauge polyethylene catheter (ReCathoco, Allison park, PA, USA) was introduced in the subarachnoidal space and advanced rostrally 3.5–4 cm to reach the lumbar enlargement. The catheter was fixed to the fascia, and its proximal end was tunneled subcutaneously and then fixed to the skin between the scapulae. To physiologically ascertain the proper position of the catheter tip, 10 lL of 2% lidocaine (Xylocain, AstraZeneca, Södertälje, Sweden) was injected to induce a transient flaccid paralysis of the hind limbs. Before the animals were killed, 10 lL methylene blue dye solution was injected by the same route to enable spread of the dye into the spinal cord. Data from animals in which no dye could be traced at the L4–L6 levels of spinal cord were not included in the study.
tivity produced by SCS. The same doses of methiothepin i.t. alone did not influence the basal tactile thresholds (Fig. 1A and B). Ketanserin (30 lg, i.t.), a 5-HT2A receptor antagonist, significantly counteracted the suppressive effect on tactile hypersensitivity during and 10 minutes after SCS (P < .05). The same dose of ketanserin or DMSO i.t. did not affect the basal tactile thresholds (Fig. 1C and D). Likewise, SDZ (30 lg, i.t.), a 5-HT4 receptor antagonist, significantly counteracted the suppressive effect on tactile hypersensitivity during and 10 minutes after SCS (P < .05). The same dose of SDZ did not affect the basal tactile thresholds (Fig. 1E and F). Ketanserin markedly attenuated the cold hypersensitivity suppressive effect during SCS (P < .05), whereas the other 5-HT receptor antagonists (methiothepin, SDZ) produced no attenuation. None of the antagonists, at the doses used, or DMSO i.t. affected basal cold scores (Fig. 2A through C). None of the 5-HT receptor antagonists (ketanserin, methiothepin, SDZ) had an effect on the SCS-attenuated heat hyperalgesia, as assessed by PWL. Also the same doses of the antagonists per se or DMSO did not affect the basal PWL (Fig. 3A through C).
2.7. Drugs The following drugs and doses (based on literature data) were used: methiothepin (5-HT1, 5-HT6, 5-HT7 receptor antagonist, 0.3, 3 lg [30]); ketanserin tartrate (5-HT2A receptor antagonist, 30 lg [23]); a-methylserotonin maleate salt, a-m-5-HT (5-HT2 receptor agonist, 5 lg [39]); 1-(3-chloropheny) biguanide hydrochloride, m-CPBG (5-HT3 receptor agonist, 10 lg [18]); 3-tropanylindole-3carboxylate methiodide, TICM (5-HT3 receptor antagonist, 1 lg [19]); SDZ-205,557,SDZ (5-HT4 receptor antagonist, 30 lg [18]); bicuculline, BIC (GABAA receptor antagonist, 15 lg [40]), and CGP 35348, CGP (GABAB receptor antagonist, 50 lg [53]). The substances were freshly dissolved in volumes of 10 lL saline except for ketanserin tartrate, which was dissolved in a volume of 10 lL 20% dimethylsulfoxide (DMSO). All drugs were prewarmed to 37.5°C, and administered i.t. using a Hamilton syringe, followed by 10 lL prewarmed saline to rinse the catheter. All tested drugs were injected i.t. 5 minutes before starting SCS expect for bicuculline and CGP, which were administered i.t. 10 minutes prior to SCS. To further determine the subeffective doses of the 5-HT2 agonist, 5-HT3 agonist, and antagonist we performed pilot experiments to identify a dose that produced less than 25% of the maximum possible effect (% MPE). % MPE was calculated by using the formula % MPE = [(postdrug withdrawal threshold predrug withdrawal threshold)/(cut-off gram predrug withdrawal threshold)] 100. 2.8. Data presentation and statistics The changes of behavior with respective treatments (control, SCS, and drug treatments) were analyzed using Kruskal–Wallis nonparametric analysis of variance followed by Dunn’s post-hoc test. Data are presented as means ± standard error of the mean (SEM) or medians with 5th to 95th percentiles, when appropriate. P < .05 was considered significant in all tests. Graphics and calculations were performed using GraphPad PRISM version 5.0 (GraphPad, San Dieg, CA, Chicago, IL, USA), and SPSS Version 15.0 (SPSS Inc, USA).
3. Results 3.1. Possible involvement of different 5-HT receptors in the SCSinduced effects on tactile and cold hypersensitivity and heat hyperalgesia Methiothepin (0.3, 3 lg, i.t.), a 5-HT1, 5-HT6, 5-HT7 receptor antagonist, had no effect on the attenuation of tactile hypersensi-
3.2. Possible enhancing effects of a 5-HT2 receptor agonist, a 5-HT3 receptor agonist, and an antagonist on the SCS-induced attenuation of tactile hypersensitivity Our pilot experiments confirmed that individual i.t. administration of a-m-5-HT (5-HT2 receptor agonist), m-CPBG (5-HT3 receptor agonist), or TICM (5-HT3 receptor antagonist) exhibited dosedependent antinociceptive effects in the present model of mononeuropathy [18,19,37,39]. The doses of a-m-5-HT (5 lg), m-CPBG (10 lg), and TICM (10 lg) that produced 6.7 ± 0.9%, 5.8 ± 1.2%, and 7.1 ± 2.1% MPE, respectively, in tactile hypersensitivity were chosen as test doses in the subsequent experiments. Injection of a-m-5-HT or m-CPBG 5 minutes prior to the SCS application had an enhancing effect as shown by significant reductions of the response percentage evoked by mechanical stimulation with a 12.5 g filament at time points 20 minutes and 30 minutes, but not with a 2.7 g filament (Fig. 4A through D). On the other hand, TICM failed to influence the SCS effects on tactile and cold hypersensitivity and on heat hyperalgesia (Fig. 5A through C). 3.3. Effect of GABAA, B receptor antagonists on the enhanced SCS effect produced by a 5-HT3 receptor agonist Consistent with our previous study [9], i.t. injection of CGP (GABAB receptor antagonist) abolished the entire effect of SCS alone or when enhanced by m-CPBG. Conversely, BIC (GABAA receptor antagonist) selectively eliminated the 5-HT3-induced enhancement while the original SCS effect remained intact (Fig. 6A and B).
4. Discussion The current study was performed as a result of a previous investigation in which we, in a neuropathic pain model, demonstrated that the pain-relieving effect of SCS is produced partially by activation of 5-HT descending pathways [53]. The findings in the present study show that spinal serotonin receptors, at least in part, mediate the SCS-induced pain-relieving effect, because ketanserin [23] and SDZ [18] significantly attenuated the suppressive effect of SCS on tactile hypersensitivity, whereas methiothepin [30] and TICM [19] were ineffective. Further, the suppressive effect on cold hypersensitivity of SCS was counteracted only by ketanserin. In addition, subeffective doses of am-5-HT [39] and m-CPBG [18] enhanced the suppressive effect of SCS on tactile hypersensitivity. The enhancing effect of mCPBG was abolished by a preceding administration of bicuculline
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Fig. 1. Effects of different types of 5-HT receptor antagonists (i.t.) on SCS-induced attenuation of tactile hypersensitivity. Percentages of paw withdrawal responses to 2 von Frey filaments (2.7 and 12.5 g) were recorded from the intact (Contra) hind paw and the nerve injured paw without (basal WT) and with SCS, SCS with pretreatment with a receptor antagonist, and with the antagonist alone; for ketanserin, a control test with DMSO was included. (A and B) Methiothepin (0.3, 3 lg), a 5-HT1, 5-HT6, 5-HT7 receptor antagonist, influenced neither the basal thresholds nor the SCS effect. (C and D) Ketanserin (30 lg), a 5-HT2A receptor antagonist, significantly counteracted the hypersensitivity suppressive effect during and 10 minutes after SCS. The same dose of ketanserin or DMSO did not affect the basal thresholds. (E and F) SDZ (30 lg), a 5-HT4 receptor antagonist, significantly counteracted the hypersensitivity suppressive effect during and 10 minutes after SCS. The same dose of SDZ did not affect the basal thresholds. Data are presented as means ± SEM percentage of paw withdrawals to 10 filament applications. n = 4 to 7. ⁄P < .05. DMSO = dimethylsulfoxide; i.t. = intrathecally; SCS = spinal cord stimulation; SDZ = SDZ-205, 557; SEM = standard error of the mean; WT = withdrawal threshold.
Fig. 2. Effects of different types of 5-HT receptor antagonists (i.t.) on SCS-induced attenuation of cold hypersensitivity. Comparison of scored responses to cold spray applied to the nerve-injured hind paw without (Basal) and with SCS, with the antagonist alone, and SCS pretreated with a receptor antagonist. Ketanserin counteracted the cold hypersensitivity suppression produced by SCS (B), whereas the other 5-HT receptor antagonists, methiothepin (A) and SDZ (C), had no effect. The same doses of respective antagonists or DMSO did not affect the basal PWL. Data are presented as medians with 5th to 95th percentiles. n = 7; ⁄P < .05. DMSO = dimethylsulfoxide; i.t. = intrathecally; PWL = paw withdrawal latency; SCS = spinal cord stimulation; SDZ = SDZ-205, 557.
Fig. 3. Effects of different types of 5-HT receptor antagonists (i.t.) on SCS-induced attenuation of heat hyperalgesia. Comparison of PWL to radiant heat applied to the nerveinjured hind paw without (Basal) and with SCS, with the antagonist alone, and SCS pretreated with a receptor antagonist. None of the 5-HT subtype receptor antagonists (ketanserin (A), methiothepin (B), SDZ-205,557(C)) had an effect on PWLs augmented by SCS. The same doses of respective antagonists or DMSO did not affect the basal PWL. Data are presented as medians with 5th to 95th percentiles. n = 7. ⁄P < .05. DMSO = dimethylsulfoxide; i.t. = intrathecally; PWL = paw withdrawal latency; SCS = spinal cord stimulation; SDZ = SDZ-205, 557.
[40] or CGP [53]. These findings suggest that the 5-HT2A, 5-HT3, and 5-HT4 receptors, but not the 5-HT1, 5-HT6, and 5-HT7 receptors, are involved in the SCS-induced pain-relieving effect at the spinal cord level.
Serotonin may bind to several different types of receptors, of which 7 have been identified so far (5-HT1-7), and a total of 14 subtypes. All types, except 5-HT3, are G-protein coupled. At least 5 types of 5-HT receptors (5-HT1, 5-HT2, 5-HT3, 5-HT4, and 5-HT7)
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Fig. 4. Enhancing effects on SCS-induced attenuation of tactile hypersensitivity by a-m-5-HT (5-HT2 receptor agonist, 5 lg) (A and B) or m-CPBG (5-HT3 receptor agonist, 10 lg) (C and D). The drugs were given 5 minutes before 30 minutes of SCS. Data were presented as means ± SEM percentage of paw withdrawals to 10 filaments applications. n = 7. ⁄P < .05. SCS = spinal cord stimulation; SEM = standard error of the mean.
are believed to be involved in the modulation of nociceptive processing in the spinal dorsal horn [12,18,19,23,30,59]. In the current study, ketanserin, a 5-HT2A receptor antagonist, attenuated the SCS
suppressive effect on tactile and cold hypersensitivity, but had no influence on heat hyperalgesia, indicating that the SCS-induced pain-relieving effect is only partially mediated by the 5-HT2A
Fig. 5. Absent effect of TICM (5-HT3 receptor antagonist, 10 lg i.t) on SCS-induced attenuation of tactile and cold hypersensitivity, as well as of heat hyperalgesia. (A and B) Percents of paw withdrawal responses to 2 von Frey filaments (2.7 and 12.5 g) were recorded from the intact (Contra) hind paw and the nerve-injured paw without (Basal WT) and with SCS, SCS with pretreatment with a receptor antagonist, and with the antagonist alone. (C) Comparison of scored responses to cold spray and (D) PWL to radiant heat, applied to the nerve-injured hind paw without (Basal) and with SCS, with the antagonist alone and SCS pretreated with a receptor antagonist. Data are presented as means ± SEM percentage of paw withdrawals to 10 filament applications (A and B) and as medians with 5th to 95th percentiles (C and D). n = 7. DMSO = dimethylsulfoxide; i.t. = intrathecally; PWL = paw withdrawal latency; SCS = spinal cord stimulation; SDZ = SDZ-205, 557; SEM = standard error of the mean; TICM = 3-tropanylindole-3carboxylate methiodide; WT = withdrawal threshold.
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Fig. 6. Involvement of GABAA,B receptors in m-CPBG (5-HT3 receptor agonist)-induced enhancing effect of SCS on tactile hypersensitivity. BIC (GABAA receptor antagonist, 15 lg i.t.) or CGP (GABAB receptor antagonist, 50 lg i.t.) were given 5 minutes before m-CPBG (10 lg) followed by SCS. n = 7. ⁄P < .05 refers to effect by BIC; +P < .05, ++P < .01 refer to effect by CGP. BIC = bicuculline; CGP = CGP 35348; GABA = c-aminobutyric acid; i.t. = intrathecally; SCS = spinal cord stimulation.
receptor. Nevertheless, a 5-HT2 receptor agonist could enhance the SCS effect, as demonstrated with the use of a 12.5 g von Frey filament. There was no significant further attenuation of the paw withdrawals to a thin filament (2.7 g), conceivably due to the fact that they were almost normalized by SCS alone. Several studies indicate that the 5-HT2 receptor is involved in antinociception, as demonstrated in the formalin test [46] and in a nerve injury model [38]. Radhakrishnan et al. in 2003 [43] reported that the 5-HT2A receptor appears to mediate transcutaneous electrical nerve stimulation–induced antihyperalgesia in an inflammatory pain model. In the rat spinal cord, 5-HT2 receptors are present in both superficial and deep laminae of the dorsal horn, although their density is low [31,56]. The 5-HT2 receptor mRNA is expressed in both large and small DRG neurons in the rat, suggesting that these receptors are localized at central as well as peripheral terminals of both large and small afferent fibers [57]. Mechanical hypersensitivity is mediated via myelinated fibers (mainly Ab fibers, but also Ad fibers), whereas heat hyperalgesia involves unmyelinated C-fibers, and both Ad- and C-fibers mediate cold hypersensitivity [21]. However, in the present study, intrathecal administration of ketanserin (30 lg) merely eliminated the SCS-induced attenuation of the hypersensitivity (tactile and cold) and had no influence on the SCS-induced changes in heat hyperalgesia. The absence of effect of ketanserin on heat hyperalgesia may be explained by its high affinity to certain subtypes of 5-HT2 receptors. The binding affinity (1/Ki value; Ki = inhibition constant) of ketanserin for the 5-HT2A receptor site has been reported to be 42% [25]. It also might be that the SCS effect on hypersensitivity (tactile and cold) is selectively mediated by activation of the 5-HT2A receptors, while another receptor subtype is involved in heat hyperalgesia. However, considering that none of the other 5-HT receptor antagonists attenuated the suppressive effect of SCS on heat hyperalgesia, it is more likely that the mechanisms involved in the effect of SCS on this form of neuropathology are more or less independent from the spinal serotonergic system as demonstrated previously [53]. The present results confirmed that both the 5-HT3 receptor agonist m-CPBG and antagonist TICM have an antihypersensitivity effect in partial sciatic nerve ligation rats [40,55]. Unlike the other 5-HT receptors referred to previously, the 5-HT3 receptor is an excitatory ligand-gated ion channel [11]. Autoradiography, in situ hybridization, and immunocytochemical studies demonstrate a dense band of 5-HT3 receptors in the superficial laminae of the dorsal horn [22,24,36]. The exact neuronal populations expressing the 5-HT3 receptor remain controversial. 5-HT3 receptor immunoreactivity is not found in glutamic acid decarboxylase-expressing interneurons, but is present in primary afferent fibers. A small proportion of these fibers contains the calcitonin gene-related peptide, with the remaining 5-HT3-positive fibers found in an unidentified subset of myelinated and unmyelinated nociceptors [32,61]. Hence, blockade of the spinal 5-HT3 receptor reduces release of pronociceptive pep-
tides from primary afferents, and this is also supported by the finding that the analgesia after i.t. administration of 5-HT is attenuated by selectively knocking down the spinal 5-HT3 receptor [41]. There is some controversy over the identity of the descending serotonergic system responsible for the facilitatory or inhibitory effect in neuropathic pain [6,35,42,44]. To inhibit neuropathic pain, OFF cells in the rostroventromedial medulla activate 5-HT3 receptors on inhibitory interneurons in the dorsal horn, and hence suppress pain by an increase of inhibitory transmitters [35]. It has been reported that i.t. administration of a selective 5-HT3 receptor agonist can increase spinal GABA concentration without changing the glutamate and glycine levels in the spinal cord, suggesting an additional inhibitory effect on second-order neurons [20,40]. A subeffective dose of the 5-HT3 receptor agonist m-CPBG was found to enhance the SCS effect, whereas the corresponding antagonist neither enhanced nor counteracted SCS. Considering that there is evidence that neuropathic pain is maintained by a spinal excitatory tone of serotonin via the 5-HT3 receptor, we had expected TICM to enhance the SCS effect. The absence of effect of TICM may reflect the predominant importance of the 5-HT3 receptor linkage to local GABAergic interneurons [20,40]. To further explore this issue, GABAA and GABAB receptor antagonists were given shortly before the administration of m-CPBG, which produced an enhancement of the SCS effect. This enhancing effect on SCS was totally abolished by a GABAB antagonist (CGP) and partially blocked by a GABAA antagonist (BIC). These results are consistent with our previous study showing the crucial role of the GABAB receptor in the SCS-induced pain-relieving effect, whereas the GABAA receptor appears to be of less importance [8]. The possible role played by 5-HT4 receptors in pain modulation has up to now not been much investigated. Nevertheless, there are data indicating that activation of 5-HT4 receptors may reduce visceral pain [14], and it has also been shown that this receptor type is involved in serotonin-induced antinociception. There is also evidence that 5-HT4 agonists may elicit antinociception via cholinergic and GABAergic mechanisms [5,16]. The present study shows that a 5-HT4 receptor antagonist markedly attenuated the SCS-induced reduction of tactile hypersensitivity, indicating the likely involvement of this receptor. The role of 5-HT4 in SCS is in agreement with the involvement of the spinal cholinergic and GABAgeric systems, as demonstrated previously [8,47]. In conclusion, the current study demonstrates that the SCS-induced pain-relieving effect involves activation of spinal 5-HT receptors (5-HT2A, 5-HT3, and 5-HT4), partly via GABAergic interneurons. Acknowledgements This study was supported by grants from Medtronic International and the Karolinska Institutet Funds. Special thanks to
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Dr. Linda Sorkin for critical reading of the manuscript. The authors state that there is no conflict of interest. References [1] Bardin L, Bardin M, Lavarenne J, Eschalier A. Effect of intrathecal serotonin on nociception in rats: influence of the pain test used. Exp Brain Res 1997;113:81–7. [2] Bardin L, Lavarenne J, Eschalier A. Serotonin receptor subtypes involved in the spinal antinociceptive effect of 5-HT in rats. Pain 2000;86:11–8. [3] Bardin L, Schmidt J, Alloui A, Eschalier A. Effect of intrathecal administration of serotonin in chronic pain models in rats. Eur J Pharmacol 2000;409:37–43. [4] Barnes NM, Sharp T. A review of central 5-HT receptors and their function. Neuropharmacology 1999;38:1083–152. [5] Bianchi C, Rodi D, Marino S, Beani L, Siniscalchi A. Dual effects of 5-HT4 receptor activation on GABA release from guinea pig hippocampal slices. Neuroreport 2002;13:2177–80. [6] Burgess SE, Gardell LR, Ossipov MH, Malan Jr TP, Vanderah TW, Lai J, Porreca F. Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain. J Neurosci 2002;22:5129–36. [7] Crisp T, Stafinsky JL, Spanos LJ, Uram M, Perni VC, Donepudi HB. Analgesic effects of serotonin and receptor-selective serotonin agonists in the rat spinal cord. Gen Pharmacol 1991;22:247–51. [8] Cui JG, Linderoth B, Meyerson BA. Effects of spinal cord stimulation on touchevoked allodynia involve GABAergic mechanisms. An experimental study in the mononeuropathic rat. Pain 1996;66:287–95. [9] Cui JG, Meyerson BA, Sollevi A, Linderoth B. Effect of spinal cord stimulation on tactile hypersensitivity in mononeuropathic rats is potentiated by simultaneous GABA(B) and adenosine receptor activation. Neurosci Lett 1998;247:183–6. [10] Cui JG, O’Connor WT, Ungerstedt U, Linderoth B, Meyerson BA. Spinal cord stimulation attenuates augmented dorsal horn release of excitatory amino acids in mononeuropathy via a GABAergic mechanism. Pain 1997;73:87–95. [11] Derkach V, Surprenant A, North RA. 5-HT3 receptors are membrane ion channels. Nature 1989;339:706–9. [12] Dogrul A, Ossipov MH, Porreca F. Differential mediation of descending pain facilitation and inhibition by spinal 5HT-3 and 5HT-7 receptors. Brain Res 2009;1280:52–9. [13] El-Khoury C, Hawwa N, Baliki M, Atweh SF, Jabbur SJ, Saade NE. Attenuation of neuropathic pain by segmental and supraspinal activation of the dorsal column system in awake rats. Neuroscience 2002;112:541–53. [14] Espejo EF, Gil E. Antagonism of peripheral 5-HT4 receptors reduces visceral and cutaneous pain in mice, and induces visceral analgesia after simultaneous inactivation of 5-HT3 receptors. Brain Res 1998;788:20–4. [15] Finnerup NB, Otto M, McQuay HJ, Jensen TS, Sindrup SH. Algorithm for neuropathic pain treatment: an evidence based proposal. Pain 2005;118:289–305. [16] Ghelardini C, Galeotti N, Casamenti F, Malmberg-Aiello P, Pepeu G, Gualtieri F, Bartolini A. Central cholinergic antinociception induced by 5HT4 agonists: BIMU 1 and BIMU 8. Life Sci 1996;58:2297–309. [17] Hylden JL, Wilcox GL. Intrathecal serotonin in mice: analgesia and inhibition of a spinal action of substance P. Life Sci 1983;33:789–95. [18] Jeong CY, Choi JI, Yoon MH. Roles of serotonin receptor subtypes for the antinociception of 5-HT in the spinal cord of rats. Eur J Pharmacol 2004;502:205–11. [19] Kawamata T, Omote K, Toriyabe M, Kawamata M, Namiki A. Intracerebroventricular morphine produces antinociception by evoking gamma-aminobutyric acid release through activation of 5hydroxytryptamine 3 receptors in the spinal cord. Anesthesiology 2002;96:1175–82. [20] Kawamata T, Omote K, Toriyabe M, Yamamoto H, Namiki A. The activation of 5-HT(3) receptors evokes GABA release in the spinal cord. Brain Res 2003;978:250–5. [21] Khan GM, Chen SR, Pan HL. Role of primary afferent nerves in allodynia caused by diabetic neuropathy in rats. Neuroscience 2002;114:291–9. [22] Kia HK, Miquel MC, McKernan RM, Laporte AM, Lombard MC, Bourgoin S, Hamon M, Verge D. Localization of 5-HT3 receptors in the rat spinal cord: immunohistochemistry and in situ hybridization. Neuroreport 1995;6:257–61. [23] Kim SK, Park JH, Bae SJ, Kim JH, Hwang BG, Min BI, Park DS, Na HS. Effects of electroacupuncture on cold allodynia in a rat model of neuropathic pain: mediation by spinal adrenergic and serotonergic receptors. Exp Neurol 2005;195:430–6. [24] Laporte AM, Koscielniak T, Ponchant M, Verge D, Hamon M, Gozlan H. Quantitative autoradiographic mapping of 5-HT3 receptors in the rat CNS using [125I]iodo-zacopride and [3H]zacopride as radioligands. Synapse 1992;10:271–81. [25] Leysen JE, Niemegeers CJ, Van Nueten JM, Laduron PM. [3H]Ketanserin (R 41,468), a selective 3H-ligand for serotonin2 receptor binding sites. Binding properties, brain distribution, and functional role. Mol Pharmacol 1982;21:301–14. [26] Lind G, Meyerson BA, Winter J, Linderoth B. Intrathecal baclofen as adjuvant therapy to enhance the effect of spinal cord stimulation in neuropathic pain: a pilot study. Eur J Pain 2004;8:377–83.
[27] Lind G, Schechtmann G, Winter J, Meyerson BA, Linderoth B. Baclofenenhanced spinal cord stimulation and intrathecal baclofen alone for neuropathic pain: long-term outcome of a pilot study. Eur J Pain 2008;12:132–6. [28] Linderoth B, Gazelius B, Franck J, Brodin E. Dorsal column stimulation induces release of serotonin and substance P in the cat dorsal horn. Neurosurgery 1992;31:289–96. discussion 287–96. [29] Liu MY, Su CF, Lin MT. The antinociceptive role of a bulbospinal serotonergic pathway in the rat brain. Pain 1988;33:123–9. [30] Lozano-Cuenca J, Castaneda-Hernandez G, Granados-Soto V. Peripheral and spinal mechanisms of antinociceptive action of lumiracoxib. Eur J Pharmacol 2005;513:81–91. [31] Marlier L, Teilhac JR, Cerruti C, Privat A. Autoradiographic mapping of 5-HT1, 5-HT1A, 5-HT1B and 5-HT2 receptors in the rat spinal cord. Brain Res 1991;550:15–23. [32] Maxwell DJ, Kerr R, Rashid S, Anderson E. Characterisation of axon terminals in the rat dorsal horn that are immunoreactive for serotonin 5-HT3A receptor subunits. Exp Brain Res 2003;149:114–24. [33] Meyerson BA, Linderoth B. Mechanisms of spinal cord stimulation in neuropathic pain. Neurol Res 2000;22:285–92. [34] Meyerson BA, Ren B, Herregodts P, Linderoth B. Spinal cord stimulation in animal models of mononeuropathy: effects on the withdrawal response and the flexor reflex. Pain 1995;61:229–43. [35] Millan MJ. Descending control of pain. Prog Neurobiol 2002;66:355–474. [36] Morales M, Battenberg E, de Lecea L, Sanna PP, Bloom FE. Cellular and subcellular immunolocalization of the type 3 serotonin receptor in the rat central nervous system. Brain Res Mol Brain Res 1996;36:251–60. [37] Oatway MA, Chen Y, Weaver LC. The 5-HT3 receptor facilitates at-level mechanical allodynia following spinal cord injury. Pain 2004;110:259–68. [38] Obata H, Saito S, Sakurazawa S, Sasaki M, Usui T, Goto F. Antiallodynic effects of intrathecally administered 5-HT(2C) receptor agonists in rats with nerve injury. Pain 2004;108:163–9. [39] Obata H, Saito S, Sasaki M, Ishizaki K, Goto F. Antiallodynic effect of intrathecally administered 5-HT(2) agonists in rats with nerve ligation. Pain 2001;90:173–9. [40] Okazaki R, Namba H, Yoshida H, Okai H, Miura T, Kawamura M. The antiallodynic effect of neurotropin is mediated via activation of descending pain inhibitory systems in rats with spinal nerve ligation. Anesth Analg 2008;107:1064–9. [41] Paul D, Yao D, Zhu P, Minor LD, Garcia MM. 5-Hydroxytryptamine3 (5-HT3) receptors mediate spinal 5-HT antinociception: an antisense approach. J Pharmacol Exp Ther 2001;298:674–8. [42] Pertovaara A, Almeida A. Descending inhibitory systems. Handb Clin Neurol 2006;81:179–92. [43] Radhakrishnan R, King EW, Dickman JK, Herold CA, Johnston NF, Spurgin ML, Sluka KA. Spinal 5-HT(2) and 5-HT(3) receptors mediate low, but not high, frequency TENS-induced antihyperalgesia in rats. Pain 2003;105:205–13. [44] Rahman W, Suzuki R, Webber M, Hunt SP, Dickenson AH. Depletion of endogenous spinal 5-HT attenuates the behavioural hypersensitivity to mechanical and cooling stimuli induced by spinal nerve ligation. Pain 2006;123:264–74. [45] Saade NE, Tabet MS, Soueidan SA, Bitar M, Atweh SF, Jabbur SJ. Supraspinal modulation of nociception in awake rats by stimulation of the dorsal column nuclei. Brain Res 1986;369:307–10. [46] Sasaki M, Obata H, Saito S, Goto F. Antinociception with intrathecal alphamethyl-5-hydroxytryptamine, a 5-hydroxytryptamine 2A/2C receptor agonist, in two rat models of sustained pain. Anesth Analg 2003;96:1072–8. [47] Schechtmann G, Song Z, Ultenius C, Meyerson BA, Linderoth B. Cholinergic mechanisms involved in the pain relieving effect of spinal cord stimulation in a model of neuropathy. Pain 2008;139:136–45. [48] Schechtmann G, Wallin J, Meyerson BA, Linderoth B. Intrathecal clonidine potentiates suppression of tactile hypersensitivity by spinal cord stimulation in a model of neuropathy. Anesth Analg 2004;99:135–9. [49] Seltzer Z, Dubner R, Shir Y. A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 1990;43: 205–8. [50] Simpson BA, Meyerson BA, Linderoth B. Spinal cord and brain stimulation. In: McMahon SB, Koltzenburg M, editors. Textbook of pain. Amsterdam: Elsevier; 2006. p. 563–82. [51] Sluka KA, Lisi TL, Westlund KN. Increased release of serotonin in the spinal cord during low, but not high, frequency transcutaneous electric nerve stimulation in rats with joint inflammation. Arch Phys Med Rehabil 2006;87:1137–40. [52] Song Z, Meyerson BA, Linderoth B. Muscarinic receptor activation potentiates the effect of spinal cord stimulation on pain-related behavior in rats with mononeuropathy. Neurosci Lett 2008;436:7–12. [53] Song Z, Ultenius C, Meyerson BA, Linderoth B. Pain relief by spinal cord stimulation involves serotonergic mechanisms: an experimental study in a rat model of mononeuropathy. Pain 2009;147:241–8. [54] Stiller CO, Cui JG, O’Connor WT, Brodin E, Meyerson BA, Linderoth B. Release of gamma-aminobutyric acid in the dorsal horn and suppression of tactile allodynia by spinal cord stimulation in mononeuropathic rats. Neurosurgery 1996;39:367–74. discussion 365–74. [55] Suzuki R, Rahman W, Hunt SP, Dickenson AH. Descending facilitatory control of mechanically evoked responses is enhanced in deep dorsal horn neurones following peripheral nerve injury. Brain Res 2004;1019:68–76.
Ò
Z. Song et al. / PAIN 152 (2011) 1666–1673 [56] Thor KB, Nickolaus S, Helke CJ. Autoradiographic localization of 5hydroxytryptamine1A, 5-hydroxytryptamine1B and 5-hydroxytryptamine1C/2 binding sites in the rat spinal cord. Neuroscience 1993;55: 235–52. [57] Tokunaga A, Saika M, Senba E. 5-HT2A receptor subtype is involved in the thermal hyperalgesic mechanism of serotonin in the periphery. Pain 1998;76:349–55. [58] Tyce GM, Yaksh TL. Monoamine release from cat spinal cord by somatic stimuli: an intrinsic modulatory system. J Physiol 1981;314:513–29.
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[59] Wu S, Zhu M, Wang W, Wang Y, Li Y, Yew DT. Changes of the expression of 5HT receptor subtype mRNAs in rat dorsal root ganglion by complete Freund’s adjuvant-induced inflammation. Neurosci Lett 2001;307:183–6. [60] Yaksh TL, Wilson PR. Spinal serotonin terminal system mediates antinociception. J Pharmacol Exp Ther 1979;208:446–53. [61] Zeitz KP, Guy N, Malmberg AB, Dirajlal S, Martin WJ, Sun L, Bonhaus DW, Stucky CL, Julius D, Basbaum AI. The 5-HT3 subtype of serotonin receptor contributes to nociceptive processing via a novel subset of myelinated and unmyelinated nociceptors. J Neurosci 2002;22:1010–9.