Systemic paracetamol-induced analgesic and antihyperalgesic effects through activation of descending serotonergic pathways involving spinal 5-HT7 receptors

European Journal of Pharmacology 677 (2012) 93–101

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Neuropharmacology and Analgesia

Systemic paracetamol-induced analgesic and antihyperalgesic effects through activation of descending serotonergic pathways involving spinal 5-HT7 receptors Ahmet Dogrul a,⁎, Melik Seyrek a, Emin Ozgur Akgul b, Tuncer Cayci b, Serdar Kahraman c, Hayrunnisa Bolay d a

Department of Pharmacology, Gulhane Academy of Medicine, 06010, Ankara, Turkey Department of Biochemistry, Gulhane Academy of Medicine, 06010, Ankara, Turkey Department of Neurosurgery, Gulhane Academy of Medicine, 06010, Ankara, Turkey d Department of Neurology, Gazi Medical Faculty, Ankara, Turkey b c

a r t i c l e

i n f o

Article history: Received 27 August 2011 Received in revised form 1 December 2011 Accepted 9 December 2011 Available online 21 December 2011 Keywords: Acetaminophen Paracetamol Analgesia Serotonin 5-HT7 Spinal Hyperalgesia Postoperative pain Descending Incision

a b s t r a c t Although some studies have shown the essential role of descending serotonergic pathways and spinal 5HT1A, 5-HT2A, or 5-HT3 receptors in the antinociceptive effects of paracetamol, other studies have presented conflicting results, and the particular subtype of spinal 5-HT receptors involved in paracetamol-induced analgesia remains to be clarified. Recent studies have demonstrated the importance of spinal 5-HT7 receptors in descending serotonergic pain inhibitory pathways. In this study, we investigated the role of descending serotonergic pathways and spinal 5-HT7 receptors compared with 5-HT3 and 5-HT2A receptors in the antinociceptive and antihyperalgesic effects of paracetamol. Tail-flick, hot plate and plantar incision tests were used to determine nociception in male BALB/c mice. Lesion of serotonergic bulbospinal pathways was performed by intrathecal (i.th.) injection of 5,7-dihydroxytryptamine (5,7-DHT), and spinal 5-HT levels were measured by HPLC. To evaluate the particular subtypes of the spinal 5-HT receptors, the selective 5-HT7, 5-HT3 and 5HT2A receptor antagonists SB 269970, ondansetron and ketanserin, respectively, were given i.th. after oral administration of paracetamol. Oral paracetamol (200, 400 and 600 mg/kg) elicits dose-dependent antinociceptive and antihyperalgesic effects. I.th. pretreatment with 5,7-DHT (50 μg) sharply reduced 5-HT levels in the spinal cord. Depletion of spinal 5-HT totally abolished the antinociceptive and antihyperalgesic effects of paracetamol. I.th. injection of SB 2669970 (10 μg) blocked the antinociceptive and antihyperalgesic effects of paracetamol, but ondansetron and ketanserin (10 μg) did not. Our findings suggest that systemic administration of paracetamol may activate descending serotonergic pathways and spinal 5-HT7 receptors to produce a central antinociceptive and antihyperalgesic effects. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Oral paracetamol is one of the most commonly used pain relievers, and the recent introduction of an intravenous paracetamol formulation has increased its use in postoperative and perioperative settings (Smith, 2009; Toussaint et al., 2010). Although one of the primary mechanisms of paracetamol-induced analgesic and antihyperalgesic effects relies on cyclooxygenase inhibition, experimental studies have shown that other mechanisms contribute to the effects of paracetamol on nociception as well (Anderson, 2008; Bonnefont et al., 2007; Hamza and Dionne, 2009; Smith, 2009). Preclinical and clinical studies have indicated that there exists a central serotonergic mechanism for paracetamol-induced analgesia (Anderson, 2008; Smith, 2009). Descending serotonergic pathways make up one of the main ⁎ Corresponding author at: Department of Pharmacology, Gulhane Academy of Medicine, 06018-Etlik, Ankara, Turkey. Tel.: +90 312 3044767; fax: +90 312 3042150. E-mail address: [email protected] (A. Dogrul). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.12.016

endogenous analgesic systems and various analgesic drug actions are associated with the activation of these pathways (Dogrul and Seyrek, 2006, 2009; Rahman et al., 2011; Roca-Vinardell et al., 2003; Seyrek et al., 2010;Toussaint et al., 2010). The marked attenuation of the antinociceptive effects of systemic paracetamol in animals depleted of spinal 5-HT provides evidence that paracetamol induces antinociceptive effects by reinforcing descending serotonergic pathways that inhibit nociceptive transmission in the spinal cord (Mallet et al., 2008; Tjolsen et al., 1991). Descending serotonergic (5-hydroxytryptamine, 5-HT) pathways are known to modulate spinal nociceptive processes via seven 5-HT receptor families (5-HT1–7) (Millan, 2002). Although some studies have reported on the contribution of spinal 5-HT1A, 5-HT2A, and 5-HT3 receptors to the antinociceptive effects of paracetamol (Bonnefont et al., 2005; Courade et al., 2001b; Mallet et al., 2008; Roca-Vinardell et al., 2003), other studies have provided conflicting results. For example, while i.th. administration of tropisetron, a selective 5-HT3 receptor antagonist blocked antinociceptive effects of paracetamol in paw pressure

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test (Bonnefont et al., 2005), another study reported that i.th. injection of other selective 5-HT3 receptor antagonists did not alter paracetamol action in the same nociceptive test (Courade et al., 2001a, 2001b, 2001c). Similar contradiction also exists with regard to the role of 5HT1A and 5-HT2A receptors in paracetamol analgesia. Thus, the detailed mechanism by which paracetamol activates descending serotonergic pathways, together with the particular subtype of spinal 5-HT receptors responsible for paracetamol-induced analgesia remains to be clarified. The most recently discovered subtypes of 5-HT receptor are the 5HT7 receptors (Leopoldo et al., 2011). Several studies have indicated that spinal 5-HT7 receptors mediate descending serotonergic inhibition of pain (Andrews and O'Neill, 2011; Dogrul et al., 2009; Leopoldo et al., 2011; Yanarates et al., 2010). It has been observed that nerve injury increased the expression of 5-HT7 receptors in the dorsal horn of the spinal cord and that acute systemic administration of the 5-HT7 receptor agonists exerted antihyperalgesic effects in nerve injury or capsaicin challenge models, highlighting the role of spinal 5-HT7 receptors in the control of nociceptive hypersensitivity (Andrews and O'Neill, 2011; Brenchat et al. 2009, 2010; Leopoldo et al., 2011). To the best of our knowledge, there has been no study to evaluate the role of spinal 5-HT7 receptors in paracetamol-induced analgesic and antihyperalgesic effect. Therefore, the purpose of the current study was to examine the role of the descending serotonergic pathways and spinal 5-HT7 receptors compared to 5-HT3 and 5-HT2A receptors in the antinociceptive and antihyperalgesic effects of paracetamol on acute thermal and postoperative pain models. 2. Materials and methods 2.1. Animals Male BALB/c mice (25–30 g) were housed under controlled temperature and humidity conditions and a 12-h light/dark cycle (lights on at 8:00 AM) with available laboratory chow and tap water ad libitum. All experimental procedures were performed between 10:00 and 14:00 h and were in accordance with recommendations of the International Association for the Study of Pain and ethical guidelines of Gulhane Animal Care and Use Committee (2008-38). 2.2. Drugs Paracetamol was obtained from Sigma (USA), dissolved in a vehicle solution containing 20:1:1:78 (v/v/v/v) mixture of DMSO:ethanol: Tween 80:0.9% saline, and given orally at a volume of 0.3 mL/10 g via a stainless steel tube to the hand-restrained animals. The selective serotonergic neurotoxin 5,7-dihydroxytryptamine creatinine sulfate salt (5,7-DHT) and highly selective 5-HT7 antagonist SB-269970 ((R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl) pyrrolidine-1-sulfonyl) phenol) hydrochloride (Lovell et al., 2000) were purchased from Sigma-RBI (USA). Selective 5-HT3 receptor antagonist ondansetron (Freeman et al., 1992) was obtained from Glaxo SmithKline (Zofran, Turkey). Selective 5-HT2A antagonist, ketanserin (Herndon et al., 1992) tartrate was obtained from RBI (USA). 5,7-DHT, SB 269970, ondansetron and ketanserin were dissolved in 0.9% saline and given intrathecally (i.th.) in a volume of 10 μL. The i.th. injections were administered by the method of Hylden and Wilcox (1980), in which a 30-gauge needle is inserted into the subarachnoid space through the lumbar space between the L5 and L6 vertebrae of unanesthetized mice. 2.3. Assessment of antinociception Antinociception was determined by the tail-flick (Columbus, OH, USA; Type 812) and hot plate (plate temperature maintained at 52 °C) tests. For the radiant heat tail-flick test, a heat source was focused on the dorsal surface of the animal's tail 1–2 cm from its proximal end,

and the intensity of the beam was adjusted to elicit a mean control baseline latency of 2.5–3.5 s. Subjects were tested with a single baseline tailflick and hot plate latencies (BL) for each mouse prior to (30 min) and then test latencies (TL) were recorded at several time points after drug or vehicle administration. Cut-off latencies of 6 s and 60 s were imposed to prevent tissue damage during the tail-flick and hot plate tests, respectively. To obtain a dose–response curve, tail-flick and hot plate latencies were converted to % antinociception, according to the formula: ((TL− BL)/ (Cutoff time− BL)) × 100. 2.4. Plantar incision and assessment of thermal hyperalgesia Paw incision-induced thermal hyperalgesia was induced according to methods previously described (Pogatzki and Raja, 2003). The mice were anesthetized by ketamine and xylazine (80 and 4 mg/ kg i.p., respectively). After cleaning the plantar surface of the right hindpaw with 10% povidone–iodine, an approximately 5-mm longitudinal incision was made with a no. 11 blade, through the skin and plantaris muscle, starting 2 mm from the proximal edge of the heel in midplantar line. After hemostasis with sterile gauze, the skin was closed with two simple sutures of 5-0 silk, and the mice were allowed to recover in their home cage. Incision-induced thermal hypersensitivity was assessed by radiant heat paw flick test (Hargreaves et al., 1988) between 1 and 7 days after plantar incision or sham operation. Each mouse was individually placed on a glass platform within a plexiglass chamber to allow approximately 15 min to acclimate to the testing environment, and a heat source was focused on the incised midplantar area from a 50-W light bulb mounted in a custom-built movable case (Commat Ltd., Ankara, Turkey). The intensity of the light beam was adjusted to produce mean baseline paw withdrawal latencies of 8–10 s in the right hindpaw in unincised control mice. The time required to withdraw the hindpaw from the thermal stimulus was recorded following drug administrations in the plantarincised animals at several time-points. To generate a dose response curve, paw withdrawal latencies were converted to % Thermal Antihyperalgesia using the formula ((Postdrug latency − Baseline latency) / (Presurgery latency− Baseline latency)) ×100. 2.5. Lesion of descending serotonergic pathways with spinal 5,7-DHT administration The depletion of spinal serotonergic neurons and measurement of lumbar 5-HT levels following i.th. 5,7-DHT injections were performed according to a modified method of Hung et al. (2003). The mice were pretreated with desipramine hydrochloride (25 mg/kg, i.p) to prevent the uptake of 5,7-DHT into noradrenergic terminals. Then, following a 45-min desipramine administration, the mice received an i.th. injection with either 0.9% saline or 50 μg of 5,7-DHT. On the testing day (4 days after 5,7-DHT injections), in order to evaluate the efficacy of 5,7-DHT on spinal 5-HT depletion, a group of mice was sacrificed by high-dose ether anesthesia, followed by a surgical cut made at the S1/S2 level. Inserting a 22-gauge needle with a syringe containing saline into the sacral vertebral canal at the cutting site, the spinal cord was ejected through the cervical opening. The spinal cord was rapidly placed on ice, and the dorsal lumbar sections were dissected and then immediately frozen in liquid nitrogen for storage at −70 °C until the 5-HT measurement procedure. The levels of 5-HT in the lumbar dorsal horn were measured by high-performance liquid chromatography with a fluorescence detector (Agilent 1100, Santa Clara, CA) using a commercially available kit (Eureka, Chiaravalle, Italy) described in our recent study (Yanarates et al., 2010). In brief, the spinal cord containing lumbar regions were weighed and homogenized with 0.2 M perchloric acid solution including 100 M EDTA (1 mL/30 mg of spinal tissue). The precipitated protein was removed by centrifugation at 20,000 g for 15 min at 0 °C. The clear supernatant was filtered using 0.2-μm membrane filters. According to

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kit procedure, 200 μL of deproteinization solution with internal standard was added to 400 μL of sample, vortexed at least 10 s, centrifuged at 5000 g for 5 min, and 200 μL was pipetted in a tube of clear supernatant. After the addition of a 200 μL stabilization solution, we injected 50 μL into the chromatographic system. Separation of 5-HT and internal standard was achieved with a reverse-phase column (VertiSep GES C18; 4.6 × 150 mm; particle size, 4 μm) with a guard precolumn (Vertical Chromatography, Bangkok, Thailand) at a flow rate of 1.2 mL/min. The areas of peaks detected by fluorescent detector (Ex: 285 nm; Em: 344 nm) were used for quantification. 2.6. Experimental protocol First, we evaluated the antinociceptive effects of paracetamol in the tail-flick and hot plate tests. Next, we measured the time course of thermal hyperalgesic effects between 1 and 7 days after plantar incision. Paracetamol-induced antihyperalgesic effects were determined on the day that the maximal thermal hyperalgesia was obtained after plantar incision. Thermal thresholds for tail-flick, hot plate, and plantar tests were determined before and 30, 60, 90, 120 and 180 min after paracetamol or vehicle administration. In the second stage, we evaluated the antinociceptive and antihyperalgesic effects of paracetamol in the spinal 5-HT-depleted mice. Finally, we examined the contribution of spinal 5-HT7 or 5-HT3 receptors by i.th. injection of the selective 5-HT receptor antagonists at 60 min after oral paracetamol administration. In our previous studies, we showed that i.th. injection of SB 269970 at a dose of 10 μg blocks the antinociceptive effects of systemically administered morphine and tramadol in mice (Dogrul and Seyrek, 2006; Yanarates et al., 2010); therefore, a 10-μg dose for selective 5-HT receptor antagonists was chosen for spinal administration. 2.7. Statistical analysis The data were presented as mean ± S.E.M. (n = 8 in each group). Differences among the groups were analyzed by two-way repeated measures ANOVA followed by the Bonferroni post hoc test for individual comparisons, using GraphPad Prism version 4 software (GraphPad, San Diego, CA). Statistical significance between the two groups was evaluated by unpaired Student's t-test, and results of the statistical tests were considered significant at P b 0.05. The A50 values (dose of paracetamol that resulted in a 50% thermal antinociceptive or antihyperalgesic effect) and the 95% confidence intervals (CIs) of paracetamol alone and paracetamol in combination with selective 5-HT receptor antagonists were calculated by the linear regression analysis of the dose–response curves, according to the method described by Tallarida (2001), with the aid of the FlashCalc pharmacological statistics package. If the 95% confidence limits of paracetamol alone did not overlap with the 95% confidence limit of the combination treatment with selective 5-HT antagonists, statistical significance was considered to be present between the groups. The significance of the confidence intervals was determined by applying the t-test at P b 0.05. 3. Results 3.1. The influence of spinal 5-HT depletion and i.th. administration of selective 5-HT7, 5-HT3 and 5-HT2A receptor antagonists on the antinociceptive effects of systemic paracetamol The mean cumulative baseline latencies of the tail-flick and hot plate tests before treatments were found to be 2.82 ± 0.03 s and 25.6 ± 2.1 s, respectively. While vehicle administration alone did not change the baseline tail-flick and hot plate latencies, orally administered paracetamol (200, 400, and 600 mg/kg) generated a dose-dependent antinociceptive action by increasing the tail-flick (F(3,140) = 49.07, P b 0.001) and hot plate (F(3,140) = 41.04, P b 0.001) latencies (Fig. 1A and B).

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Paracetamol at doses of 400 and 600 mg/kg elicited significantly increased tail-flick and hot plate latencies at 30, 60, 90, 120, and 180 min compared with the vehicle group (Pb 0.05 for each timepoint) (Fig. 1A and B). Consistent with our recent study, we found that i.th. treatment with 5,7-DHT (Yanarates et al., 2010) significantly reduced the 5-HT levels in the dorsal lumbar part of spinal tissues by 85% (1282 ± 143.6 ng/g vs 196.72 ± 74.7 ng/g of spinal cord tissue for control and 5,7-DHT treated animals, respectively; t = 6.7, P b 0.01, n = 8). We examined the antinociceptive effects of systemically administered paracetamol (200, 400, and 600 mg/kg) in spinal 5,7-DHT-pretreated animals (Fig. 1A and B). The mean baseline tail-flick and hot plate latencies in the mice treated with i.th. 5,7-DHT (50 μg) were found to be 2.87 ± 0.19 s and 27.63 ± 3.6 s, which were not significantly different from the 0.9% salinetreated controls (t= 0.51, P = 0.95 and t = 0.47, P = 0.63, respectively). However, paracetamol (200, 400, and 600 mg/kg)-induced antinociception was completely abolished in the spinal 5-HT-depleted animals when compared to the 0.9% saline-treated group in the tail-flick (F(5,210) = 58.92, P b 0.001) (Fig. 1A) and hot plate (F(5,210) = 39.59, P b 0.001) tests (Fig. 1B). The dose response curves from the data generated 60 min after paracetamol administration in the tail-flick and hot plate tests are presented in Fig. 1C and D, respectively. To evaluate the contribution of spinal 5-HT7 receptors in paracetamolinduced antinociception, we injected SB-269970 (10 μg) i.th. 60 min after paracetamol (200, 400, and 600 mg/kg) administration. While i.th. injection of SB-269970 (10 μg) alone did not alter baseline latencies in the tailflick and hot plate tests (Fig. 2A and B), i.th. SB-269970 (10 μg) significantly blocked paracetamol-induced (200, 400, and 600 mg/kg) antinociception in the tail-flick (F(3,140) F=55.08, Pb 0.001) (Fig. 2A) and hot plate (F(3,140) F=61.51, Pb 0.001) (Fig. 2B) tests. Post hoc analysis indicated that the i.th. injection of SB-269970 elicited a significant reduction in the paracetamol (400 and 600 mg/kg)-induced increase on tail-flick (Fig. 2A) and hot plate (Fig. 2B) latencies at 30, 60, and 120 min after injection. Fig. 2C and D present the dose response curves of paracetamolinduced antinociception generated from the data 30 min after i.th. administration of SB 269970 (90 min after paracetamol) in the tail-flick and hot plate tests, respectively. In contrast to SB 269970, post hoc analysis showed that i.th. administration of ondansetron (10 μg) (Fig. 3A and B) and ketanserin (10 μg) (Fig. 4A and B) did not alter the antinociceptive effects of paracetamol (200, 400, and 600 mg/kg) in the tail-flick and hot plate tests, at any time with our observation period when compared with the corresponding dose of paracetamol, while their administration alone was inactive in both tests. Figs. 3C–D and 4C–D demonstrated the paracetamoldose response curves in the tail-flick and hot plate tests generated from the data 30 min after i.th. injection of ondansetron and ketanserin, respectively. The ED50 values and 95% CL for systemic paracetamol with SB269970, ondansetron or ketanserin (10 μg for each antagonist) in tail-flick test and hot plate tests are shown in Table 1. While the ED 50 values for paracetamol with ondansetron or ketanserin resulted in an experimental ED50 values which were not significantly different (P > 0.05) from paracetamol alone, the ED50 values for paracetamol with SB-269970 (10 μg) was found to be significantly greater than those for paracetamol alone (Table 1). 3.2. The influence of spinal 5-HT depletion and i.th. injected 5-HT7, 5-HT3 and 5-HT2A receptor antagonists on the antihyperalgesic effects of per orally administered paracetamol in plantar incision test Consistent with previous studies (Pogatzki and Raja, 2003; Yanarates et al., 2010), the mice demonstrated time-dependent thermal hyperalgesia after plantar incision (Fig. 5). Thermal hyperalgesia, as indicated by a significant reduction in paw withdrawal latency from baseline values, reached a maximum on day 1 from 8.5 ± 0.44 to 2.5 ± 0.13 (t = 12.16, P b 0.01). The thermal hyperalgesic effects

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Fig. 1. The involvement of serotonergic descending pathways in the antinociceptive effects of paracetamol in the tail-flick (A) and hot plate (B) tests. The antinociceptive effects of paracetamol were determined in control and spinal serotonin-depleted mice by intrathecal (i.th.) injection of 5,7-dihydroxytryptamine (5,7-DHT) in tail-flick (A) and hot plate (B) tests. Data are expressed as mean ± S.E.M. N = 8 each group. *Differences from vehicle treated groups, P b 0.05 (Bonferroni post hoc test). +Differences from corresponding dose of paracetamol alone, P b 0.05 (Bonferroni post hoc test). Tail-flick (C) and hot plate (D) latencies were converted to % antinociception to generate the dose response curve at 90 min after paracetamol.

remained over 3 days and returned time dependently to baseline levels within 7 days after incision (Fig. 5). Plantar incision did not change significantly the paw withdrawal latency of the contralateral hind paws, which was 9.01 ± 0.35 s (t = 1.22, P b 0.23).

Paracetamol-induced antihyperalgesia was evaluated 24 h (on day 1) after plantar incision. While vehicle administration alone did not change paw withdrawal latencies in incised mice, orally administered paracetamol (200, 400, and 600 mg/kg) produced a dose-dependent

Fig. 2. The effects of an intrathecal (i.th.) injection of selective 5-HT7 receptor antagonist SB-269970 (10 μg) on orally administered paracetamol-induced antinociception in tail-flick (A) and hot plate (B) tests. SB 269970 was given i.th. 60 min after systemic paracetamol administration. Data are expressed as mean ± S.E.M. N = 8 each group. *Differences from corresponding dose of paracetamol alone, P b 0.05 (Bonferroni post hoc test). Tail-flick (C) and hot plate (D) latencies were converted to % antinociception to generate the dose response curve at 30 min after i.th administration of SB 269970 (90 min after paracetamol).

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Fig.3. The effects of an intrathecal (i.th.) injection of selective 5-HT3 receptor antagonist ondansetron (10 μg) on orally administered paracetamol-induced antinociception in tailflick (A) and hot plate (B) tests. Ondansetron was given i.th. 60 min after systemic paracetamol administration. Data are expressed as mean ± S.E.M. N = 8 each group. Tail-flick (C) and hot plate (D) latencies were converted to % antinociception to generate the dose response curve at 30 min after i.th administration of ondansetron (90 min after paracetamol).

A

Paracetamol 600 mg/kg

Tail-flick latencies (sec)

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+ ketanserin Paracetamol 200 mg/kg

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3

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mean paw withdrawal latency of the spinal 5-HT-depleted mice was 8.22 ± 0.58 s, which was not significantly different from the salinetreated naive animals (t = 0.28, P = 0.77). Paw incision significantly decreased paw withdrawal latency to 2.58 ± 0.17 s 24 h after incision in the spinal 5-HT-depleted animals. Similar to the paracetamol-induced antinociceptive effects in the tail-flick and hot plate tests, the thermal

B Hot plate latencies (sec)

thermal antihyperalgesic effect in the paw flick test (Fig. 6A). Paracetamol at doses of 400 and 600 mg/kg increased paw withdrawal latencies to 6.1 ± 0.51 and 9.2 ± 0.67 s, respectively, 60 min following its administration, compared with 2.2 ± 0.11 and 2.52 ± 0.13 s for baseline values. We next evaluated the paracetamol-induced thermal antihyperalgesic effect in the spinal 5-HT-depleted animals (Fig. 6A). The

Paracetamol 600 mg/kg 60

+ ketanserin Paracetamol 400 mg/kg

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60

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120

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D 100 Paracetamol 75

+ ketanserin (10 µg, i.th.)

50 25

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% Antinociception

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+ ketanserin

50

25

0

0 200

400

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Fig.4. The effects of an intrathecal (i.th) injection of selective 5-HT2A receptor antagonist ketanserin (10 μg) on orally administered paracetamol-induced antinociception in tail-flick (A) and hot plate (B) tests. Ketanserin was given i.th. 60 min after systemic paracetamol administration. Data are expressed as mean ± SEM. N = 8 each group. Tail-flick (C) and hot plate (D) latencies were converted to % antinociception to generate the dose response curve at 30 min after i.th administration of ondansetron (90 min after paracetamol).

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Table 1 The influence of intrathecal (i.th.) administration of 5-HT7 (SB 269970), 5-HT3 (ondansetron) and 5-HT2A (ketanserin) receptor antagonists on orally administered (p.o.) paracetamol-induced antinociception in tail-flick and hot plate tests. Drug (mg/kg, p.o.) Paracetamol (200, Paracetamol (200, Paracetamol (200, Paracetamol (200,

400, 400, 400, 400,

and and and and

600 mg/kg) 600 mg/kg) 600 mg/kg) 600 mg/kg)

5-HT receptor antagonists (μg/10 μL, i.th.)

ED 50 and 95% confidence intervals for tail-flick tests

ED 50 and 95% confidence intervals for hot plate tests

0.9% saline SB 269970 Ondansetron Ketanserin

468.1 (392.4–560.5) 2353.1 (1744.4–3174.1) 505.7 (431.6–592.52) 592.7(454.4–773.21)

343.2 (296.7–396.74) 1483.3 (1157.4–1901.1) 384.7 (287.23–515.35) 381.3(301.5–482.2)

antihyperalgesic effects of paracetamol (200, 400, and 600 mg/kg) were totally diminished in the spinal 5,7-DHT-pretreated animals (Fig. 6A). The dose response curves from the data generated 60 min after paracetamol administration in the paw flick test is presented in Fig. 6B In order to explore the contribution of spinal 5-HT7, 5-HT3 or 5HT2A receptors in the antihyperalgesic effects of paracetamol in plantar incision model, we injected SB 269970, ondansetron or ketanserin (10 μg for each antagonist) i.th. 60 min following systemic paracetamol administration. While i.th. administration of SB 269970 alone altered paw withdrawal thermal latency in the paw-incised animals, the antihyperalgesic effects of paracetamol were blocked by SB 269970 (Fig. 7A). In contrast to SB 269970, i.th. injection of ondansetron (Fig. 8A) or ketanserin (Fig. 9A) did not significantly affect the antihyperalgesic effects of paracetamol at any of the time-points tested when compared with the corresponding dose of paracetamol, while their administration alone were inactive. Figs. 7B, 8B and 9B present the dose response curves of paracetamol-induced thermal antihyperalgesic effects generated from the data 30 min after i.th. administration of SB 269970, ondansetron and ketanserin, respectively. The ED50 values and 95% CL for systemic paracetamol-induced antihyperalgesia with i.th. SB-269970, ondansetron or ketanserin (10 μg for each antagonist) are shown in Table 2. While the ED 50 values for systemic paracetamol-induced antihyperalgesic effects with i.th.administered ondansetron or ketanserin were not significantly different (P > 0.05) from paracetamol alone, the ED50 values for paracetamol with SB-269970 (10 μg) were found to be significantly greater than those for paracetamol alone (Table 2).

al., 2003; Srikiatkhachorn et al., 1999). Thus, present data confirm that paracetamol modulates nociception at supraspinal and spinal levels when given systemically (Raffa et al., 2000). The antinociceptive and antihyperalgesic effects of paracetamol were evident at doses of 400 and 600 mg/kg per oral, while lower doses were ineffective in tail-flick, hot plate, and plantar incision tests. These findings were in agreement with previous studies (Furedi et al., 2009; Girard et al., 2011; Pinardi et al., 2002; Pini et al., 1996, 1997;Sandrini et al., 2003). Several studies have reported that paracetamol prevents hyperalgesia in a variety of models of nociception (Bianchi and Panerai 1996; Crawley et al., 2008; Toussaint et al., 2010) and, consistent with present data, a recent study showed that systemically administered paracetamol blocked thermal hyperalgesia in a plantar incision model in rats (Girard et al., 2011). Surgical pain is an important type of acute pain, and the plantar incision model mimics painful conditions after surgery or peripheral nerve lesion (Brennan et al., 1996; Kim et al., 2010). Thermal antihyperalgesic effects of paracetamol in the plantar

4. Discussion Consistent with previous studies, systemic oral administration of paracetamol at doses of 400 and 600 mg/kg, which has been shown to cross the blood brain barrier and distribute throughout the brain and spinal cord (Bannwarth et al., 1992; Courade et al., 2001b), produced a modest effect on the tail-flick test (a spinally mediated nociceptive reflex) and a marked effect on the hot plate and paw flick tests (complex responses that are supraspinally integrated) (Pinardi et al., 2002; Pini et al., 1997; Roca-Vinardell et al., 2003; Sandrini et

Fig. 5. Time-course of thermal hyperalgesia induced by plantar incision surgery in mice. Mean paw withdrawal latencies were significantly reduced after incision on day 1 until post-incision day 5. Data are expressed as mean ± S.E.M. N = 8 each group. *Differences from sham surgery animals, P b 0.05 (Bonferroni post hoc test).

Fig. 6. The effects of orally administered paracetamol on postoperative thermal hyperalgesia in plantar incision model in normal mice and in mice with lesioned serotonergic bulbospinal pathways by intrathecal injection of 5,7-dihydroxytryptamine (5,7-DHT) (A). Paracetamol was given 24 h after plantar incision in healthy and spinal serotonin-lesioned mice. Data are expressed as mean ± S.E.M. N = 8 each group. *Differences from corresponding dose of paracetamol alone, P b 0.05 (Bonferroni post hoc test). Paw flick latencies were converted to % Thermal Antihyperalgesia to generate the dose response curve at 90 min after paracetamol (B).

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Fig. 7. The effects of an intrathecal (i.th) injection of selective 5-HT7 receptor antagonist SB-269970 (10 μg) on orally administered paracetamol-induced thermal antihyperalgesia on postoperative pain model in mice (A). Paracetamol was given 24 h after plantar incision. SB 269970 was given i.th. 60 min after paracetamol administration. Data are expressed as mean± S.E.M. N = 8 each group. *Differences from corresponding dose of paracetamol alone, P b 0.05 (Bonferroni post hoc test). Paw flick latencies were converted to % Thermal Antihyperalgesia to generate the dose response curve at 30 min after i.th. administration of ondansetron (90 min after paracetamol) (B).

Fig. 8. The effects of an intrathecal (i.th) injection of selective 5-HT3 receptor antagonist ondansetron (10 μg) on orally administered paracetamol-induced antihyperalgesia on postoperative pain model in mice (A). Paracetamol was given 24 h after plantar incision. Ondansetron was given i.th. 60 min after paracetamol administration. Data are expressed as mean ± S.E.M. N = 8 each group. *Differences from the corresponding dose of paracetamol alone, P b 0.05 (Bonferroni post hoc test). Paw flick latencies were converted to % Thermal Antihyperalgesia to generate the dose response curve at 30 min after i.th. administration of ondansetron (90 min after paracetamol) (B).

incision model deserves attention, because the hyperalgesia evident after tissue incision contributes to postoperative pain (Wilder-Smith and Arendt-Nielsen, 2006), and recent clinical studies have shown intravenous paracetamol to be a very effective postoperative analgesic alternative (Groudine and Fossum, 2011). \Some experimental studies have suggested that the antinociceptive activity of paracetamol depends on the integrity of descending serotonergic pathways in formalin and paw pressure tests (Bonnefont et al., 2007; Tjolsen et al., 1991). In the present investigation, the reduction of antinociceptive potency of paracetamol by spinal 5-HT-depleted mice in tail-flick and hot plate tests extends those previous findings. In addition, our study showed that systemically administered paracetamol needs an intact descending serotonergic pathway to produce thermal antihyperalgesic effects in the early stages of the postoperative pain model. Very few studies have investigated the role of the descending serotonergic pathways in primary hyperalgesia in postoperative pain (Silveira et al., 2010). It has been reported that a surgical incision in the hind paw, which induces a strong long-lasting noxious input, drives the activation of descending serotonergic pathways (Koizuka et al., 2005; Silveira et al., 2010; Villarreal and Prado, 2007). These enhanced descending serotonergic tones after surgery work as an endogenous protective mechanism and contribute to postoperative plasticity (Silveira et al., 2010). Thus, it is possible that there are specific neuronal circuits whereby systemic paracetamol can reinforce the enhanced descending serotonergic inhibitory tones to the spinal cord to block nociception at the spinal level in the acute postoperative period. The lack of effect of spinal administration of ondansetron and ketanserin paracetamol-induced antinociception suggest that spinal 5-HT3 and 5-HT2A receptors do not appear to be involved in the mechanism of paracetamol on acute nociception. Contradiction exists with regard to the role of spinal 5-HT3 and 5-HT2A receptors on

systemic paracetamol-induced analgesia (Courade et al., 2001c; Mattia and Coluzzi, 2009; Rahman et al., 2011; Silveira et al., 2010; Xie et al., 2008). While the blockage of antinociceptive effects of orally administered paracetamol by i.th. administration of tropisetron, a selective 5-HT3 receptor antagonist, suggests that paracetamol action is mediated by spinal 5-HT3 receptors (Mattia and Coluzzi, 2009; Smith, 2009), other studies have reported that selective 5-HT3 antagonists ondansetron and granisetron given i.th. were unable to reverse the antinociceptive effects of paracetamol in the same nociceptive model. Consistent with our data, following studies demonstrate that tropisetron was not a selective 5-HT3 receptor antagonist (Courade et al., 2001a; Libert et al., 2004; Smith, 2009), and using electrophysiological recording and spinal 5-HT3 receptor antisense oligodeoxynucleotide administration approaches, these studies concluded that paracetamol-induced antinociceptive action involves a spinal tropisetron-sensitive receptor that is not the 5-HT3 receptor, which remains to be identified (Libert et al., 2004). Involvement of spinal 5-HT2A receptors in the descending serotonergic modulation of pain is a matter of debate (Rahman et al., 2011). Previous studies on the effects of spinal 5-HT2A receptors have shown both antinociceptive and pronociceptive roles (Seyrek et al., 2010; Silveira et al., 2010; Xie et al., 2008). Courade et al. (2001a, 2001b, 2001c) reported that spinal ketanserin injection reduced an intravenous single dose of propacetamol (400 mg/kg)-induced antinociception (corresponding to 200 mg/kg of paracetamol) in the rat paw pressure test. Although our study shows ineffectiveness of spinal ketanserin in the dose response curve of paracetamol, discrepancies between two studies may be due to differences in the nociceptive test or animal species used (Fu et al., 2010). Similar to paracetamol induced antinociception experiments, our study demonstrate that pharmacological blockade of spinal 5-HT2A and 5-HT3 receptors did not inhibit antihyperalgesic effects of paracetamol on postoperative pain. Supporting

100

Paw flick latencies (sec)

A

A. Dogrul et al. / European Journal of Pharmacology 677 (2012) 93–101

Paracetamol 600 mg/kg 10

+ ketanserin Paracetamol 400 mg/kg

8

+ ketanserin 6

Paracetamol 200 mg/kg + ketanserin

4

Vehicle + ketanserin 2 Before 0 incision

30

60

90

120

150

180

Time (min)

% Thermal Antihyperalgesia

B Paracetamol

100

+ ketanserin (10 µg, i.th.)

75 50 25 0 200

400

600

Paracetamol (mg/kg, p.o.) Fig. 9. The effects of an intrathecal (i.th) injection of selective 5-HT2A receptor antagonist ketanserin (10 μg) on orally administered paracetamol-induced antihyperalgesia on postoperative pain model in mice (A). Paracetamol was given 24 h after plantar incision. Ketanserin was given i.th. 60 min after paracetamol administration. Data are expressed as mean ± S.E.M. N = 8 each group. *Differences from the corresponding dose of paracetamol alone, P b 0.05 (Bonferroni post hoc test). Paw flick latencies were converted to % Thermal Antihyperalgesia to generate the dose response curve at 30 min after i.th. administration of ketanserin (90 min after paracetamol) (B).

our results, a recent study demonstrated that plantar incision induced the increase of c-fos positive neuron in spinal cord which reflects primary and secondary hyperalgesia and facilitation of the response of spinal neurons to noxious stimuli to be mediated by spinal 5-HT2A and 5-HT3 receptors pointing a pronociceptive role of these receptors in postoperative pain (Silveira et al., 2010). In the present study, our observation that spinal administration of SB-269970, a selective 5-HT7 receptor antagonist, blocks the antinociceptive and antihyperalgesic effects of systemic paracetamol, indicates a novel role of the spinal 5-HT7 receptors in the mechanism of paracetamol. It has been hypothesized that descending serotonergic pathways ultimately mediate antinociception through activation of spinal 5-HT7 receptors (Dogrul and Seyrek, 2006; Dogrul et al., 2009). Consistent with this hypothesis, 5-HT7 receptors have been expressed in the dorsolateral funiculus and superficial layers of dorsal

Table 2 The influence of intrathecal (i.th.) administration of 5-HT7 (SB 269970), 5-HT3 (ondansetron) and 5-HT2A (ketanserin) receptor antagonists on thermal antihyperalgesic effects of orally administered (p.o.) paracetamol in plantar incision tests. 5-HT receptor antagonists (μg/10 μL, i.th.)

ED 50 and 95% confidence intervals for thermal antihyperalgesia in plantar incision tests

400,

0.9% saline

257.15 (208.4–317.1)

400,

SB 269970

1145.2 (831.7–1.576.9)

400,

Ondansetron

348.08 (288.1–420.5)

400,

Ketanserin

212.54 (154.4–292.4)

Drug (mg/kg, p.o.)

Paracetamol (200, and 600 mg/kg) Paracetamol (200, and 600 mg/kg) Paracetamol (200, and 600 mg/kg) Paracetamol (200, and 600 mg/kg)

horn (Doly et al., 2005). Thus, it appears that systemic paracetamol should reinforce descending serotonergic pathways and spinal 5HT7 receptors to produce antinociceptive and antihyperalgesic effects. It has been shown that selective 5-HT7 agonists exert thermal antihyperalgesic effects with an increase in spinal 5-HT7 receptors in nerve injury models (Brenchat et al., 2010), suggesting a possible thermal antihyperalgesic mechanism of paracetamol through activation of spinal 5-HT7 receptors in hypersensitivity conditions, such as the plantar incision model in the present study. With regard to the spinal 5-HT7 receptors, the descending serotonergic pathways may inhibit nociceptive stimuli by hyperpolarization of projection neurons or central terminals of primary afferent fibers and excitation of inhibitory interneurons which contain GABA and enkephalin (Bardin, 2011; Pertovaara and Almeida, 2006). 5-HT7 receptors are positively coupled adenylate cyclases, and their stimulation is excitatory on neurons (Millan, 2002). Therefore, paracetamol-induced indirect action through 5-HT7 receptors on localized inhibitory GABAergic or enkephalinergic interneurons which evoke the release of enkephaline or GABA would explain to its antihyperalgesic and antinociceptive effects. Consistent with this hypothesis, 5-HT7 receptors were found to co-localize with GABAergic interneurons in the dorsal horn of the spinal cord (Brenchat et al., 2010). Our study does not indicate how the action of paracetamol is initiated to reinforce descending serotonergic pathways. However, several studies have shown that the endocannabinoid and opioid systems are major components of the antinociceptive activity of paracetamol (Anderson, 2008; Mallet et al., 2008; Mattia and Coluzzi, 2009; Pini et al., 1997; Smith, 2009). In our previous studies, we found that the blockage of spinal 5-HT7 receptors inhibited the antinociceptive effects of both cannabinoids and opioids (Dogrul and Seyrek, 2006; Seyrek et al., 2010). Therefore, similar to the spinal 5HT7-dependent antinociceptive mechanism with cannabinoids and opioids, it is reasonable to suggest that paracetamol may operate by first reinforcing the activity of endogenous opioid and/or cannabinoid systems in the brain and then that of descending serotonergic pathways and spinal 5-HT7 receptors to produce antinociceptive and antihyperalgesic effects. Further studies are needed to clarify this hypothesis. In conclusion, our study provides evidence that activation of descending serotonergic pathways and spinal 5-HT7 receptors following systemic administration of paracetamol produces antinociceptive and antihyperalgesic effects. Acknowledgment This study was supported by Gulhane ARGE Mrk.Bsk.lıgı (201031). References Anderson, B.J., 2008. Paracetamol (Acetaminophen): mechanisms of action. Paediatr. Anaesth. 18, 915–921. Andrews, N., O'Neill, M.F., 2011. It might be a big family but the pain remains—last chance saloon for selective 5-HT receptor ligands? Curr. Opin. Pharmacol. 11, 39–44. Bannwarth, B., Netter, P., Lapicque, F., Gillet, P., Pere, P., Boccard, E., Royer, R.J., Gaucher, A., 1992. Plasma and cerebrospinal fluid concentrations of paracetamol after a single intravenous dose of propacetamol. Br. J. Clin. Pharmacol. 34, 79–81. Bardin, L., 2011. The complex role of serotonin and 5-HT receptors in chronic pain. Behav. Pharmacol. 22, 390–404. Bianchi, M., Panerai, A.E., 1996. The dose-related effects of paracetamol on hyperalgesia and nociception in the rat. Br. J. Pharmacol. 117, 130–132. Bonnefont, J., Chapuy, E., Clottes, E., Alloui, A., Eschalier, A., 2005. Spinal 5-HT1A receptors differentially influence nociceptive processing according to the nature of the noxious stimulus in rats: effect of WAY-100635 on the antinociceptive activities of paracetamol, venlafaxine and 5HT. Pain 114, 482–490. Bonnefont, J., Daulhac, L., Etienne, M., Chapuy, E., Mallet, C., Ouchchane, L., Deval, C., Courade, J.P., Ferrara, M., Eschalier, A., Clottes, E., 2007. Acetaminophen recruits spinal p42/p44 MAPKs and GH/IGF-1 receptors to produce analgesia via the serotonergic system. Mol. Pharmacol. 71, 407–415.

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