The influence of the time course of inflammation and spinalization on the antinociceptive activity of the α2-adrenoceptor agonist medetomidine

European Journal of Pharmacology 532 (2006) 50 – 60 www.elsevier.com/locate/ejphar

The influence of the time course of inflammation and spinalization on the antinociceptive activity of the α2-adrenoceptor agonist medetomidine Carlos Molina, Juan F. Herrero ⁎ Departamento de Fisiología, Facultad de Medicina, Campus Universitario, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain Received 20 October 2005; received in revised form 14 December 2005; accepted 15 December 2005 Available online 10 February 2006

Abstract The purpose of the present study was to investigate the influence of the time course of inflammation and the implication of spinal and supraspinal sites on the antihyperalgesic effects of the α2-adrenoceptor agonist medetomidine. Behavioral experiments showed a more intense antihyperalgesia in the phase of maintenance of inflammation than in the early or resolution stages. Maximum effect, without sedation, was observed with a dose of 40 μg/kg (66 ± 12% and 76 ± 15% reduction of mechanical and thermal hyperalgesia). No change was observed in the paw swelling, indicating that its effects were not secondary to a reduction of inflammation. In electrophysiological experiments, the effect was more pronounced in animals with an intact spinal cord than in spinalized animals (max. effects of 2 ± 0.7% vs. 48 ± 11% of control, noxious mechanical stimulation). We conclude that the antihyperalgesic effect of medetomidine depends on the time course of inflammation and that it is mainly located supraspinally. © 2006 Elsevier B.V. All rights reserved. Keywords: Pain; Hyperalgesia; Carrageenan; Single motor unit; Behavioral

1. Introduction The spinal administration of α2-adrenoceptor agonists results in a dose-dependent analgesia by different mechanisms of action, including the modulation of transmitter release and the hyperpolarization of neurons (Williams et al., 1985; Bean, 1989; Lipscombe et al., 1989; Takano et al., 1993; Eisenach et al., 1996). Numerous studies have indicated that α2-adrenoceptor agonists are very effective drugs in nociceptive modulation, but they are also more active in situations of inflammationinduced sensitization (Hylden et al., 1991; Idänpään-Heikkilä et al., 1994; Stanfa and Dickenson, 1994; Mansikka and Pertovaara, 1995). This is supported by the increase of the density of α2-adrenoceptors in laminae I and II of the spinal cord during inflammation (Brandt and Livingston, 1990) and by the increase of the turnover of noradrenalin on arthritis (WeilFugazza et al., 1986). The modulation of inflammatory pain by the α2-adrenoceptor system is not limited to a direct action on spinal cord ⁎ Corresponding author. Tel.: +34 91 885 45 16; fax: +34 91 885 45 90. E-mail address: [email protected] (J.F. Herrero).

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neurons. Descending inhibitory pain control mechanisms are activated in inflammatory pain (Herrero and Cervero, 1996; Ren and Dubner, 1996) and, among these, the noradrenergic system is considered to be one of the major systems originating from the brainstem (Peng et al., 1996; Cui et al., 1999). However, the antinociceptive and antihyperalgesic actions of α2-adrenoceptor agonists have been evaluated under different experimental protocols and a variable number of inflammatory conditions. This includes, for example, 24 h monoarthritis induced by kaolin and carrageenan (Buerkle et al., 1999), 3 h softtissue inflammation induced by carrageenan s.c. in the paw (Kawamata et al., 1999), and many others (Idänpään-Heikkilä et al., 1994; Mansikka and Pertovaara, 1995; Stanfa and Dickenson, 1994; Xu et al., 2000; Mansikka et al., 2002). Controversial results on the antihyperalgesic activity of α2adrenoceptor agonists might derive from the differences in the protocols used, especially those related to the stage of inflammation, anesthesia and integrity of supraspinal modulatory systems. The purpose of the present study was to investigate, first, the variability of the intensity of hyperalgesia and allodynia on the evolution of carrageenan-induced soft-tissue

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inflammation on rat behavioral experiments. Secondly, the influence of the time course of inflammation on the intensity and duration of the antihyperalgesic and antiallodynic effect of the selective α2-adrenoceptor agonist medetomidine (Virtanen et al., 1988; Pertovaara, 1993). Following this series of experiments, we evaluated the influence of the integrity of supraspinal modulation on the antinociceptive actions of medetomidine using an electrophysiological preparation in anesthetized normal and spinalized animals. 2. Materials and methods 2.1. Behavioral experiments 2.1.1. Animals and preparations The experiments were performed on male Wistar rats weighing 235–355 g which were maintained on a 12 h light dark cycle and provided with food and water ad libitum. In all cases the animals were allowed to habituate for 5 days to the testing environment. Soft-tissue inflammation was induced under brief halothane anesthesia (5% in oxygen for induction, 2% for maintenance) by the intraplantar administration of 100 μl of carrageenan λ (10 mg/ml in distilled water, Sigma) in the right hind paw. Another 100 μl of saline was injected in the left hind paw as a control for inflammation. The level of inflammation was evaluated by measuring the volume of the paw by plethysmometry (Letica plethysmometer) before the administration of carrageenan or saline, and after each of the tests performed. The development of hyperalgesia induced by carrageenan inflammation and the antinociceptive effect of medetomidine were studied in withdrawal reflex responses evoked by mechanical and thermal stimulation. The methods were adapted from those described by Gilchrist et al. (1996) and have been described in detail elsewhere (Mazario et al., 2001; Lahdesmaki et al., 2003). The rats were placed on a raised wire mesh grid in plastic chambers. Seven von Frey filaments (50, 60, 80, 100, 200, 300 and 500 mN) were applied ten times for approximately 1 s to the plantar surface of each hind paw in an ascending series. The frequency of paw withdrawal was calculated for each monofilament. Thermal hyperalgesia was assessed by measuring paw withdrawal latencies to 55 °C radiant heat generated by an algesimeter (Ugo Basile plantar test, Hargreaves et al., 1988). Animals were placed in a clear plastic chamber and were allowed to accommodate to the testing apparatus for 5 min. Two consecutive thermal stimuli were applied to each of the paws with an interval of 2–3 min between tests and the maximum cut-off time was set to 17 s to avoid tissue damage. Control tests were established previous to the injection of carrageenan. Three further tests were studied 4, 20 and 44 h of inflammation to check the displacement of the baseline responses and to follow the evolution of allodynia and hyperalgesia on different stages of inflammation. In the animals treated with medetomidine, a control test was made immediately before the administration of the drug and was followed by tests at 10, 30 and 60 min. In all

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cases the tests were carried out in the paw treated with carrageenan and in the paw treated with saline. The effect of 40 μg/kg medetomidine was challenged in four animals with 20-h inflammation with the selective α2-adrenoceptor antagonist atipamezole (100 μg/kg, i.p., Virtanen et al., 1989). In an independent group of experiments (n = 4), a dose of 100 μg/kg, i.p. of atipamezole was tested on its own to discard an effect of the antagonist on the nociceptive responses. 2.1.2. Drugs and analysis of data Medetomidine (Domtor, Pfizer) was dissolved in saline and administered i.p. in a total volume of 0.5 ml at doses of 20, 40 or 80 μg/kg. Atipamezole (Antisedan, SB) was also dissolved in saline and injected i.p. in a total volume of 0.5 ml at a dose of 100 μg/kg. The experimenter was blind to the treatment of animals. The results were compared as raw data using the one-way analysis of variance (ANOVA) for repeated measures with the Dunnett's post-test (GraphPad Prism and GraphPad Instat for Windows). Data obtained with 50 and 60 mN force hairs were analyzed separately, since the responses obtained with them were below the threshold intensity in tests performed before the administration of carrageenan. An increase in the responses obtained with these stimuli due to the carrageenaninduced inflammation was considered as allodynia, and, therefore, a reduction of these responses after the administration of a drug was considered as a reduction or attenuation of allodynia. Responses obtained with 80 to 200 mN force hairs were situated between stimuli near threshold intensity and stimuli that produce, or are close to producing, saturation of responses in tests performed before the administration of carrageenan. The increase in responses observed with these filaments, due to the administration of carrageenan, was considered as hyperalgesia, and the reduction of responses in this range was therefore considered as inhibition or attenuation of hyperalgesia (Mazario et al., 2001). Withdrawal responses were represented as raw data and, for comparison between groups, were converted to percentage of maximum possible effect (%MPE; Harris and Pierson, 1964; Romero-Sandoval et al., 2005) according to the following formula: 100 × [(post-drug value − pre-drug value) / (max. possible response − pre-drug value)], where the post-drug value represents the latency (in the case of thermal stimulation) or the number of positive responses (in the case of von Frey filaments stimuli) after the administration of medetomidine; pre-drug value is the latency or number of positive responses before drug was given; maximum possible response represents a cut-off time of 17 s for thermal stimulation or the maximal number of responses that can possibly be observed with mechanical stimulation (10 withdrawal responses with each of the filaments used). Side-effects including excessive staring, grooming and exploring, chewing and sedation were scored in a semi-quantitative manner according to their intensity (none; mild; moderate; severe) and nature (absent; intermittent; continuous) immediately following testing at each assessment time (Nielsen et al., 2005). Data are expressed as mean ± S.E.M.

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2.2. Electrophysiological experiments 2.2.1. Animals and preparation Electrophysiological experiments were performed on adult male Wistar rats weighing 250–380 g divided in six groups: intact animals without inflammation (n = 7), intact animals with 4-h inflammation (n = 8), intact animals with 20-h inflammation (n = 8), spinalized animals without inflammation (n = 7), spinalized animals with 4-h inflammation (n = 8) and spinalized animals with 20-h inflammation (n = 7). Because it has been shown that surgery to spinal column can enhance the direct spinal potency of some compounds like opioids and adenosine on nociceptive reflexes (Herrero and Headley, 1991; Ramos-Zepeda et al., 2004) it was important to include a sham-operated group in order to compare intact versus spinalized animals. In this case, medetomidine was studied in sham-spinalized animals (n = 6) with 20-h inflammation. Preparatory surgery was performed under halothane anesthesia (5% in oxygen for induction and 2% for maintenance) following the technique described in detail elsewhere (Herrero and Headley, 1991, 1996; Herrero and Cervero, 1996; Solano and Herrero, 1997), and consisted in the cannulation of the trachea, two superficial branches of the jugular veins (for the administration of anesthesia and drugs) and one carotid artery. In the groups of sham-and full-spinalized animals a small laminectomy, with infiltration of lidocaine (1%) with adrenaline (10 μg/ml), was made from thoracic 10 to 8 vertebrae and the dura mater opened. In the group of sham-spinalized animals, no further surgery was performed and the incision was closed. In the group of spinalized animals, the spinal cord was sectioned at thoracic segment 8 or 9, depending on the level of vascularization, using cautery to minimize bleeding. After the surgery the animal was transferred to an appropriate frame, halothane was discontinued and anesthesia maintained with αChloralose (Sigma; 50 mg/kg for induction and 30 mg/kg/h by a perfusion pump for maintenance). The right hind limb was fixed into a Perspex block in inframaximal extension with plaster. Core temperature was maintained at 37 ± 0.5 °C by means of a homeothermic blanket and blood pressure was constantly monitored. The preparation was left to rest for at least 1 h before the experiment started. Animals with a systolic blood pressure under 100 mm Hg before the administration of the drugs were considered unhealthy and discarded. Inflammation was induced following the same protocol as in behavioral experiments. The recording of withdrawal reflexes as single motor units has been used to test the analgesic activity of different drugs and has been described in detail several times (Herrero and Headley, 1991; Solano and Herrero, 1997; RomeroSandoval et al., 2002; Gaitan et al., 2003; Ramos-Zepeda et al., 2004). Briefly, extracellular recordings of single motoneuron reflex activity were made from single motor units activated by mechanical and electrical stimulation and recorded by means of a bipolar tungsten electrode inserted percutaneously into muscles of the right hind limb. Isolation of motor units was performed by moving the electrode with a micromanipulator while a mild pressure was applied to the

paw. The activity of the unit was evoked in cycles of 3 min duration, each cycle consisting of 10 s of noxious mechanical stimulation (0.2 N above the threshold over an area of 14 mm2) and 16 electrical stimuli (2 ms width, 1 Hz, twice the threshold intensity for the recruitment of C-fiber afferences; Herrero and Cervero, 1996) (Fig. 1). Examples of recordings and protocol of stimulation are shown in Figs. 1 and 4. As described previously, electrical stimulation was used to study the phenomenon of wind-up (see Herrero et al., 2000 for review) and was made using two 0.2 mm needles inserted percutaneously in the most sensitive area of the cutaneous receptive field. Mechanical stimulation was performed by a computer-controlled pinch device (Cibertec) that was also used to determine the threshold force required to trigger the withdrawal response using a constantly increasing pressure ramp stimulation. The mean forces used for mechanical stimulation were 0.9 ± 0.1 N in intact animals, 0.83 ± 0.1 N in intact animals with inflammation, 1.1 ± 0.1 N in spinalized animals, 0.96 ± 0.04 N in spinalized animals with inflammation and 1.3 ± 0.6 N in the group of sham-spinalized animals. No significant differences were observed between any of the experimental groups nor in different situations of inflammation. The mean intensities of electrical stimulation were 2.05 ± 0.7 mA in intact animals, 1.85 ± 0.7 mA in intact animals with inflammation, 2 ± 0.6 mA in spinalized animals, 2.17 ± 0.04 N in spinalized animals with inflammation and 2 ± 0.8 mA in the group of sham-spinalized animals. As in the case of mechanical stimulation, no significant differences were observed in the three experimental groups. 2.2.2. Drugs and analysis of data Data are expressed as mean ± S.E.M. of percentage of control, control being the mean of the three responses previous to the administration of the drug. Responses to mechanical and electrical stimulation were counted and analyzed separately. The quantitative analysis was based on the mean number of spikes evoked during each of the two cycles of stimulation between each dose. The data from the electrical stimulation were analyzed by counting the number of spikes evoked between 150 and 650 ms after each stimulus (C-fiber responses; Herrero and Cervero, 1996). Medetomidine was studied at doses of 0.6 to 9.6 μg/kg, dissolved in saline and injected in cumulative log2 regime every two cycles of stimulation (6 min) and injected intravenously at a constant volume of 0.3 ml. As in behavioral experiments, the effect of medetomidine was challenged in all series of experiments with 100 μg/kg of atipamezole in at least three animals. The collection of data and the stimulation protocol were performed using commercial software (CED, U.K.; Spike 2 for Windows). Responses were compared as raw data using the one-way ANOVA for repeated measures with the Dunnett's post-test (GraphPad Prism and GraphPad Instat for Windows). Either in behavioral or electrophysiological experiments, the animals were used for one procedure only and were humanely killed on completion of testing by an overdose of pentobarbitone. All the experimental procedures conformed to the institutional, national and European guidelines

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Fig. 1. Original single motor unit spikes. The figure shows the spikes recorded during three consecutive cycles of stimulation. Each cycle was of 3 min duration and consisted of 10 s of noxious mechanical stimulation and 16 electrical stimuli. Top panel shows the number of spikes/s (sp/s) recorded as bar histograms and is followed by the stimuli applied (mechanical stimulation in N and electrical pulses as TTL pulses) and actual spikes. Lower panel is examples of spikes at different time points of the recording.

for the use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. 3. Results 3.1. Behavioral experiments 3.1.1. Time course of inflammation Withdrawal frequencies to mechanical stimuli were very similar in all control tests, and also very similar to those previously published following similar experimental protocols (Romero-Sandoval et al., 2004; Mazario et al., 2001). A clear shift to the left of the intensity–response curve was observed in the three stages of inflammation studied. The amplitude of the displacement was inversely proportional to the time of inflammation, being higher 4 h after the induction of inflammation and lower 44 h after inflammation. The level of allodynia, mechanical hyperalgesia and paw swelling (Fig. 2) remained significantly high in the three stages of inflammation. Thermal hyperalgesia, however, was observed at 4- and 20-h but not at 44-h inflammation (Fig. 2). Medetomidine was, therefore, not tested at the latter state for thermal hyperalgesia. 3.1.2. Effect of medetomidine on behavioral tests The administration of medetomidine did not induce any change in the paw swelling at any of the doses tested on any of the stages of inflammation studied (Tables 1 and 2, Fig. 3), indicating that, in the present experimental conditions, medetomidine is devoid of an anti-inflammatory action and that any effect on nociceptive responses is not secondary to a reduction of inflammation. In addition, medetomi-

dine did not reduce any of the responses in the paw treated with saline (data not shown) at doses of 20 and 40 μg/kg, but a clear reduction of baseline was observed with the dose of 80 μg/kg (data not shown). This was interpreted as a sign of sedation (see below). Medetomidine induced a dose-dependent reduction of nociceptive responses in animals with inflammation. The effects observed by the administration of 20 μg/kg are shown in Table 1. The main effect was seen in allodynia (67 ± 7%, P b 0.05) followed by the effect on mechanical hyperalgesia (60 ± 9%, P b 0.01) and thermal hyperalgesia (45 ± 18%, P b 0.05). In all cases, the effect of medetomidine 30 min after the injection was higher than that observed 10 and 60 min of injection. In addition, the effect was similar when tested at 20-h and 44-h inflammation and, in either case, higher than that observed at 4-h inflammation (Table 1). The most effective dose of medetomidine without causing sedation (see below) was 40 μg/kg. In responses to mechanical stimulation, the effect observed with this dose was maximum 30 min after injection (Fig. 3). The antinociception was also more intense when tested at 20-h inflammation (allodynia: 85.5 ± 8%, P b 0.01; hyperalgesia: 66 ± 12%, P b 0.01), although the effect was also significant at 4-h (allodynia: 61 ± 11%, P b 0.05; hyperalgesia: 47 ± 14%, P b 0.01) and 44-h inflammation (allodynia: 44 ± 15%, P b 0.05; hyperalgesia: 56 ± 13%, P b 0.05). Thermal hyperalgesia was also mainly reduced 30 min after the administration of 40 μg/kg (Fig. 3) with a maximum of 76 ± 15% (P b 0.01) of effect when studied at 20-h inflammation. Signs of sedation were not observed with either 20 or 40 μg/kg, but they were evident with the dose of 80 μg/kg. In this case (Table 2), full inhibition of nociceptive responses was observed in most tests carried out at 20- and 44-

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Fig. 2. Evolution of allodynia, hyperalgesia and paw swelling in carrageenan-induced inflammation. Nociceptive responses were tested on the paw injected with carrageenan (Inflammation) and compared to the responses observed on the paw treated with saline (Control, **P b 0.01, ***P b 0.001, comparison vs. control response using the one-way ANOVA, with the post hoc Dunnett test).

h inflammation. Sedation was also evident in 4-h inflammation tests, although in this series of experiments full antinociception was not observed (Table 2). The effect of medetomidine was challenged with a dose of 100 μg/kg of the selective α2-adrenoceptor antagonist atipamezole in four animals with 20-h inflammation. In all cases, a recovery above 75% of control was immediately observed. The administration of the same dose of the antagonist on its own did not significantly modify any of the responses tested (data not shown).

3.2. Electrophysiological experiments Medetomidine was very effective in intact animals without inflammation. The responses to noxious mechanical stimulation were fully inhibited with a dose of 4.8 μg/kg (Fig. 5, 2 ± 0.7%, P b 0.01) and an ID50 of 1.1 ± 1 μg/kg. In spinalized animals however, medetomidine was less effective and the maximal effect observed was of 48 ± 11% (P b 0.01) of control response with a dose of 9.6 μg/kg. The differences observed in the two experimental situations were significant

Table 1 Effect of 20 μg/kg medetomidine on behavioral experiments Drug MED 20 μg/kg

Time inf (h) 4

20

44

Time test (min)

Vol 1a (ml)

Vol 2a (ml)

THa (% MPE)

Aa (% MPE)

MHa (% MPE)

10 30 60 10 30 60 10 30 60

3.2 ± 0.1

3.3 ± 0.1 3.2 ± 0.1 3.2 ± 0.1 2.7 ± 0.1 2.7 ± 0.1 2.7 ± 0.1 2.4 ± 0.1 2.3 ± 0.04 2.4 ± 0.04

10 ± 5 15 ± 6 b 9±2 34 ± 19 45 ± 18 b 18 ± 8 – – –

12 ± 6 22 ± 7 b 7±5 37 ± 10 57 ± 8 c 43 ± 11b 40 ± 14 67 ± 7b 28 ± 9

9±6 13 ± 6 2±1 30 ± 8b 35 ± 7c 29 ± 7b 23 ± 11 60 ± 9c 28 ± 14

2.7 ± 0.1

2.4 ± 0.1

The effect of medetomidine was studied at 4-, 20- and 44-h inflammation (Time inf) and 10, 30 and 60 min (Time test) after i.p. administration. Tests included the measurement of the paw volume previous to (Vol 1) and after the injection of medetomidine (Vol 2), thermal hyperalgesia (TH), allodynia (A) and mechanical hyperalgesia (MH). Antihyperalgesic and antiallodynic effect of medetomidine are expressed as % of maximum possible effect (% MPE) where 100 % would represent full inhibition of responses by the administration of the drug. Comparisons were made with raw data for each value vs. control response (previous to the administration of the drug) using the one-way ANOVA, with the post hoc Dunnett test. a Data are expressed as mean ± S.E.M. b P b 0.05. c P b 0.01.

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Table 2 Effect of 80 μg/kg medetomidine on behavioral experiments Drug MED 80 μg/kg

Time inf (h) 4

20

44

Time test (min)

Vol 1a (ml)

Vol 2a (ml)

THa (% MPE)

Aa (% MPE)

MHa (% MPE)

10 30 60 10 30 60 10 30 60

3.3 ± 0.04

3.3 ± 0.04 3.3 ± 0.04 3.3 ± 0.05 2.9 ± 0.1 2.9 ± 0.1 2.9 ± 0.1 2.4 ± 0.04 2.4 ± 0.04 2.4 ± 0.05

46 ± 10 67 ± 12 46 ± 13 79 ± 20 86 ± 14 77 ± 19 – – –

69 ± 16 71 ± 16 48 ± 17 89 ± 7 96 ± 4 75 ± 25 100 ± 0 100 ± 0 100 ± 0

52 ± 10 59 ± 17 33 ± 15 97 ± 3 100 ± 0 67 ± 19 90 ± 6 100 ± 0 52 ± 26

3 ± 0.1

2.4 ± 0.03

The effect of medetomidine was studied at 4-, 20- and 44-h inflammation (Time inf) and 10, 30 and 60 min (Time test) after i.p. administration. Tests included the measurement of the paw volume previous to (Vol 1) and after the injection of medetomidine (Vol 2), thermal hyperalgesia (TH), allodynia (A) and mechanical hyperalgesia (MH). Statistical comparison and layout as for Table 1. The effect of medetomidine was always significant though clear signs of sedation were also observed in all cases. a Data are expressed as mean ± S.E.M.

at doses of 2.4 (P b 0.05) and 4.8 μg/kg (P b 0.01). Wind-up was dose-dependently reduced by medetomidine in intact animals and fully inhibited with the dose of 2.4 μg/kg (8 ± 5%, P b 0.001). In spinalized animals, however, no significant reduction was observed at the doses studied (data not shown). In intact animals with 4-h inflammation, medetomidine was as effective as in the group of animals without inflammation (Fig. 5), observing full inhibition of responses with 4.8 μg/kg (1.3 ± 0.5%, P b 0.01) and an ID50 of 0.7 ± 1.3 μg/kg. In spinalized animals with 4-h inflammation medetomidine was again less effective than in intact animals (Fig. 5) with an effect of

30 ± 3% of control with a dose of 9.6 μg/kg (P b 0.01), and an ID50 of 8.1 ± 1.3 μg/kg. The differences observed in the two experimental situations were significant at doses of 0.6 (P b 0.01) to 4.8 μg/kg (P b 0.001; Fig. 5). As in animals without inflammation, wind-up was totally inhibited in intact animals with a dose of 2.4 μg/kg (3.4 ± 2%, P b 0.001), but only a slight, though significant, reduction was observed in spinalized animals with a dose of 9.6 μg/kg (62 ± 10%, P b 0.05; data not shown in figures). In intact animals with 20-h inflammation, medetomidine was as effective as in previous experimental groups: 8 ± 3% with 4.8 μg/kg, (P b 0.01; Figs. 4 and 5) and an ID50 of

Fig. 3. Effect of 40 μg/kg of medetomidine (MED 40) on nociceptive responses and paw swelling on different stages of carrageenan-induced inflammation. The antinociceptive effect of medetomidine was similar in allodynia, mechanical and thermal hyperalgesia. No reduction of paw inflammation was observed. The effect was more intense 30 min than 10 or 60 min after i.p. administration (statistical comparison and layout as for Fig. 2; #P b 0.05).

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0.81 ± 1.2 μg/kg. The effect of medetomidine in shamoperated animals with 20-h inflammation was very similar to that observed in intact animals: 6.7 ± 2% with 4.8 μg/kg, (P b 0.01; Fig. 4) and an ID50 of 0.9 ± 1.1 μg/kg. In spinalized animals with 20-h inflammation, however, medetomidine was more potent and effective than in the rest of the experiments made on spinalized animals, with an ID50 of 1.3 ± 1 μg/kg. Maximum effect was, in this case, similar to that observed in intact animals (Figs. 4 and 5; 2.8 ± 0.7% with 9.6 μg/kg, P b 0.01), although the effects observed with doses of 2.4 and 4.8 μg/kg were higher in intact animals (P b 0.05). Medetomidine depressed wind-up in intact animals with 20h inflammation at an intensity similar to that seen in previous groups of experiments (Fig. 6), observing full inhibition with a dose of 2.4 μg/kg (12 ± 7%, P b 0.001). The effect was similar in sham-operated animals with a depression of 26 ± 6% (P b 0.01) with the dose of 4.8 μg/kg. In spinalized animals, though the effect was lower than in intact and sham-operated animals, a more pronounced depression of wind-up (Fig. 6) was observed than that in previous groups of experiments, with a maximum effect of 22 ± 7% (P b 0.01) at a dose of 9.6 μg/kg. No significant differences were observed between the effects of medetomidine in the three series of experiments carried out in intact animals with 20-h inflammation. The inhibition of responses to noxious mechanical stimulation by medetomidine in spinalized animals with 20-h inflammation was significantly higher at all doses (P b 0.001, Fig. 5),

when compared to that seen in spinalized animals with 4h inflammation and in spinalized animals without inflammation with doses of 4.8 (P b 0.05) and 9.6 μg/kg (P b 0.01; Fig. 5). As in behavioral experiments, the effect of medetomidine recovered above 80% of control immediately after being challenged with 100 μg/kg of atipamezole (data not shown). 4. Discussion Evident hyperalgesia and allodynia were observed in the three states of inflammation studied in behavioral experiments, although hyperalgesic responses were more intense in the early and middle stages than in the resolution phase of the process. In the latter, the level of inflammation and the responses to mechanical stimulation were still high, but thermal hyperalgesia was not present any more, indicating that the intensity of hyperalgesia in different stages of inflammation depends on the type of stimulus used. This is sustained by the well-established fact that hyperalgesia is mainly dependent on the peripheral afferent barrage in the initial period of inflammation, since a reduction of the ongoing activity at the periphery eliminates hyperalgesia (Andersen et al., 1995). Also, nociceptor sensitization largely accounts for heat hyperalgesia, but not for mechanical allodynia and hyperalgesia (Meyer and Campbell, 1981; Torebjork et al., 1992; Treede et al., 1992).

Fig. 4. Original recordings of single motor unit activity evoked by 3 min cycles of noxious mechanical and electrical stimulation in intact animals (A), shamspinalized animals (B) and spinalized animals (C) with 20-h carrageenan inflammation. Panels show two control responses previous to the administration of cumulative doses of medetomidine (initial dose of 0.6 μg/kg) and the effect observed after the administration of 2.4 and 4.8 μg/kg of medetomidine. The i.v. administration of medetomidine caused a significantly stronger inhibition of nociceptive responses in intact (A) and sham-spinalized animals (B) than in spinalized rats (C).

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The main observation made in the present study, however, was that the antihyperalgesic activity of systemic medetomidine depends on the intensity of hyperalgesia or, in other words, on the time course of inflammation. In electrophysiological experiments, the effect was more pronounced in animals with an intact spinal cord than in spinalized animals, especially in the absence of inflammation and in the early state of inflammation, indicating that supraspinal modulation is crucial for the analgesic effectiveness of medetomidine, either because its main place of action is supraspinal or because the effect is a result of an interaction of medetomidine-mediated antinociceptive activity at spinal and supraspinal sites. Intraperitoneal medetomidine reduced the responses to all tests studied in a dose-dependent manner, and sedation was only observed with the highest dose used of 80 μg/kg. The doses needed to achieve antinociception and sedative effects after intraperitoneal administration were similar (Kalso and Poyhia, 1991) or lower (Pertovaara et al., 1991) to those observed in previous studies. The effect of medetomidine was not accompanied by a reduction of the level of inflammation, indicating that the antinociceptive effect was not secondary to a reduction of nociceptive barrage arriving the spinal cord, as a consequence of the reduction of the paw swelling. Numerous studies have shown that α2-adrenoceptor agonists are more effective analgesic drugs in inflammation than in the normal situation (Kayser et al., 1992; Green et al., 1998; Brandt and Livingston, 1990). Our experiments show that antiallodynic and antihyperalgesic activities of medetomidine are higher in the middle phase of the inflammatory process than in the early or late phases. A variation of the analgesic effect of opioids depending on the time course of inflammation have

Fig. 5. Effect of i.v. medetomidine on pooled single motor unit responses activated by noxious mechanical stimulation. The effect of medetomidine was similar in all the experiments made on intact animals. Its effect was, however, lower in spinalized animals. In the latter series of experiments, the effect of medetomidine was significantly higher in animals with 20-h inflammation than in 4-h inflammation or in the absence of inflammation (statistical comparison with the one-way ANOVA, with the post hoc Dunnett test. ⁎P b 0.05, ⁎⁎P b 0.01, comparison vs. control response. #P b 0.05, ## P b 0.01, ####P b 0.001, comparison vs. intact animals. $P b 0.05, $$P b 0.01 comparison vs. spinalized animals without inflammation. +++P b 0.001, comparison vs. 4-h inflammation spinalized animals).

Fig. 6. Wind-up depression by medetomidine in normal and spinalized animals with 20-h inflammation. The effect of medetomidine on wind-up was more intense in animals with the spinal cord intact than in spinalized animals. Total inhibition of wind-up was observed with a dose of 4.8 μg/kg in intact animals whereas the same dose only reduced wind-up to 63 ± 10% of control in spinalized animals (statistical comparison with the one-way ANOVA, with the post hoc Dunnett test. ⁎P b 0.05, ⁎⁎P b 0.01,⁎⁎⁎P b 0.001, comparison vs. spinalized).

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been reported with opioids (Kayser and Guilbaud, 1991; Perrot et al., 1998), but our results seem to be the first evidence showing that this also accounts for α2-adrenergic agonists. In addition, the electrophysiological experiments showed that medetomidine was very effective in non-spinalized animals, independently on the presence or the stage of inflammation. This is in contrast with the observations made on behavioral experiments, and might be due to the presence of anesthesia, though further experiments are needed to find out the actual mechanism underlying this discrepancy. However, the activity of medetomidine was more intense when the spinal cord was intact than after spinalization, and therefore when supraspinal modulation was present. High doses of some less selective α2-adrenoceptor agonists like tizanidine induced an enhancement of flexor reflexes in chronic spinalized rats (1–5 days postoperatively), mediated by an α1-adrenoceptor activation (Kehne et al., 1985; Chen et al., 1987). This might explain why medetomidine was in our experiments less potent in spinalized than in intact animals, however, it does not seem the case since medetomidine is a more selective α2-adrenergic agonist than tizanidine and we did not observe any enhancement of responses to any of the stimulus applied. In addition, the spinalization performed in our experiments was acute, instead of chronic, and the selective α2-adrenergic antagonist atipamezole fully inhibited the effect of medetomidine. The most sensible interpretation of these observations, from our point of view, is that the antinociceptive activity of medetomidine was mainly located at supraspinal sites or, at least, that it required the activation of central areas. The question of whether the antinociceptive effects of α2-adrenoceptor agonists are mediated through spinal or supraspinal mechanisms has been a matter of intense debate. Some experiments, for example, showed that α2-adrenoceptor agonists suppress reflex responses in intact but not in spinal animals (Kehne et al., 1985; Chen et al., 1987). However, Clarke et al. (1988) demonstrated that clonidine was effective in the reduction of spinal cord reflexes in spinalized rabbits (see also Pertovaara, 1993 and references within). Other studies have shown that a high anesthetic dose (300 μg/kg) of systemic medetomidine suppresses nociceptive sensory neuronal and reflex responses by a spinal segmental mechanism of action, but the effect of a sedative dose (100 μg/kg) could only be explained by a supraspinal mechanism of action (Pertovaara et al., 1991). A supraspinal activity of medetomidine is also supported by the well-studied role of the influence of descending noradrenergic systems on spinal cord neurons, mainly throughout α2-adrenoceptors, as well as by the high concentration of these receptors in areas above the spinal cord (see for example Dahlström and Fuxe, 1965; Unnerstall et al., 1984; Westlund et al., 1990; Carlton et al., 1991). Our results provide evidence that the differences observed might be due, not only to the dosage, presence of anesthesia and agonists used, but also to the integrity of the spinal modulation and to the stage of inflammation in which the experiments were performed. It is also possible that, in the present conditions, a peripheral action of medetomidine was involved in the inhibition of hyperalgesia and allodynia. In fact, an attenuation of the excit-

ability of nociceptors by α2-adrenoceptor agonists in inflammation was reported long ago (Nakamura and Ferreira, 1988; Sato and Perl, 1991), and clonidine depresses electrically evoked C-fiber activity (Gozariu et al., 1996). We cannot fully exclude this possibility, however, it does not seem the case since wind-up was more intensely inhibited in intact animals than in spinalized animals. Wind-up is a centrally mediated phenomenon, defined as a progressive and frequency-dependent facilitation of the responses of spinal cord neurons observed on the application of constant and high intensity repetitive electrical stimuli. It is a phenomenon that shares some common mechanisms with central sensitization and is mediated by NMDA and NK1 receptors (Davies and Lodge, 1987; Dickenson and Sullivan, 1987), although other systems are also involved in its generation or maintenance (see Herrero et al., 2000 for further discussion). Wind-up is a spinal cordmediated phenomenon, but it is highly influenced by descending modulatory systems, especially in situations of sensitization (Herrero and Cervero, 1996). The fact that medetomidine inhibited wind-up more intensely in normal than spinalized animals suggests a main centrally mediated activity of the agonist, rather than a peripheral action. In conclusion, the present study seems to show a clear relationship between the antihyperalgesic activity of medetomidine and the time course of carrageenan-induced inflammation. In addition, the antinociceptive activity of the α2adrenoceptor agonist, in inflammation, depends on the integrity of the spinal cord, showing higher activity on intact than on spinalized animals (especially in the absence of inflammation and on the early phase of the inflammatory reaction) and, therefore, having an effect mainly located at supraspinal sites. Acknowledgements This work has been supported by a grant from the Comunidad de Madrid (grant GR/SAL/0815/2004). We are grateful to Lawrence JC Baron for a critical review of the manuscript and for his help with the English revision. References Andersen, O.K., Gracely, R.H., Arendt-Nielsen, L., 1995. Facilitation of the human nociceptive reflex by stimulation of A beta-fibres in a secondary hyperalgesic area sustained by nociceptive input from the primary hyperalgesic area. Acta Physiol. Scand. 155, 87–97. Bean, B.P., 1989. Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence. Nature 340, 153–156. Brandt, S., Livingston, A., 1990. Receptor changes in the spinal cord of sheep associated with exposure to chronic pain. Pain 42, 323–329. Buerkle, H., Schapsmeier, M., Bantel, C., Marcus, M.A., Wusten, R., Van Aken, H., 1999. Thermal and mechanical antinociceptive action of spinal vs. peripherally administered clonidine in the rat inflamed knee joint model. Br. J. Anaesth. 83, 436–441. Carlton, S.M., Honda, C.N., Willcockson, W.S., Lacrampe, M., Zhang, D., Denoroy, L., Chung, J.M., Willis, W.D., 1991. Descending adrenergic input to the primate spinal cord and its possible role in modulation of spinothalamic cells. Brain Res. 543, 77–90. Chen, D.F., Bianchetti, M., Wiesendanger, M., 1987. The adrenergic agonist tizanidine has differential effects on flexor reflexes of intact and spinalized rat. Neuroscience 23, 641–647.

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