Xylazine induced central antinociception mediated by endogenous opioids and l-opioid receptor, but not d-or j-opioid receptors

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1 brain research 1506 (2013) Available online at Research Report Xylazine induced central antinociception mediated by endogenous opioids and l-opioid receptor, but not d-or j-opioid receptors Thiago Roberto Lima Romero 1, Daniela da Fonseca Pacheco 1, Igor Dimitri Gama Duarte n Department of Pharmacology, Institute of Biological Sciences, UFMG, Av. Antônio Carlos, 6627, CEP , Belo Horizonte, MG, Brazil article info abstract Article history: Accepted 18 February 2013 Available online 26 February 2013 Keywords: Xylazine Endogenous opioid Central antinociception a 2 -adrenoceptor Endogenous opioids have been implicated in compound-induced antinociception, and our group previously suggested that xylazine induces peripheral antinociception by releasing endogenous opioids that act on their respective receptors. In this study, we investigated the involvement of endogenous opioids in a 2 -adrenoceptor agonist xylazine-induced central antinociception. The nociceptive threshold for thermal stimulation was measured in Swiss mice using the tail-flick test. The drugs were administered via the intracerebroventricular route. Probabilities less than 5% (po0.05) were considered to be statistically significant (ANOVA/Bonferroni s test). Our results demonstrated that opioid receptor antagonist naloxone and m-opioid receptor antagonist clocinnamox, but not d-opioid receptor antagonist naltrindole and k-opioid receptor antagonist nor-binaltorphimine, antagonized xylazine-induced central antinociception. These data provide evidence for the involvement of endogenous opioids and m-opioid receptors in xylazine-induced central antinociception. In contrast, d- and k-opioid receptors do not appear to be involved in this effect. The results contribute to a greater understanding of the central antinociceptive mechanisms of a drug widely used in veterinary therapy. & 2013 Elsevier B.V. Open access under the Elsevier OA license. 1. Introduction Xylazine is an a 2 -adrenoceptor agonist (Hsu, 1981) that is extensively used in veterinary medical practice and animal experimentation for sedation, antinociception, and muscle relaxation (Willian et al., 2001). Xylazine exhibits direct, local anesthetic sensory and motor nerve inhibition in addition to its spinal cord a 2 -adrenoceptor-mediated analgesic effects (Knight, 1980). Furthermore, xylazine in combination with other substances has been useful in many surgical procedures. For example, xylazine-ketamine mixtures have been used for short-duration surgery in both wild and domesticated pigs (Ajadi et al., 2008). Synergistic antinociceptive interactions between epidurally administered xylazine and lidocaine induced good-quality antinociception in water buffalo calves (Singh et al., 2005). Recently, it was observed that xylazinetramadol in combination is useful for equine sedation and analgesia (Seo et al., 2011). Xylazine s antinociceptive actions were initially investigated using the murine acetic acid-induced writhing test by Bentley et al. (1977). Others observed that the antinociceptive effects of intraperitoneal xylazine administration were n Corresponding author. Fax: þ address: dimitri@icb.ufmg.br (I.D.G. Duarte). 1 Both authors Romero and Pacheco have the same participation & 2013 Elsevier B.V. Open access under the Elsevier OA license.

2 brain research 1506 (2013) antagonized by yohimbine, but not by the opioid receptor antagonist naloxone. These data suggest that this effect is dependent on an a 2 -adrenoceptor-mediated, and not on an opioid-mediated, mechanism (Browing et al., 1982; Zemlan et al., 1980). It was additionally shown that the spinal and systemic antinociceptive effects of xylazine are independent of the opioid system (Browing et al., 1982; Goodchildetal., 1996). Conversely, evidence exists that a 2 -adrenoceptor agonists activate opioid receptors via endogenous opioid release. In 1988, it was shown that a 2 -adrenoceptor agonist clonidineinduced peripheral antinociception was prevented by the opioid antagonist naloxone (Nakamura and Ferreira, 1988). Similarly, our group observed that naloxone antagonized xylazine-induced peripheral antinociception in rats (Romero et al., 2009). Endogenous opioids have been implicated in antinociception induced by several compounds including non-steroidal anti-inflammatory drugs (Rezende et al., 2009), cannabinoids (Cox and Welch, 2004) and natural coffee-derived diterpenes (Guzzo et al., 2012). They act mainly through three types of opioid receptors m, d and k (Singh et al., 1997). Centrally, the periaqueductal gray area (PAG) contains high concentrations of endogenous opioid peptides b-endorphin and Met-enkephalin, and opioid receptors m and d. Activation of any of these opioid receptors by b-endorphin, morphine or other opioids administered into the PAG produces potent antinociception at supraspinal sites (Terashvili et al., 2008). To elucidate the mechanisms involved in supraspinal xylazine administration, the aim of the present study was to determine whether endogenous opioids are involved in xylazine-induced central antinociception and to identify the opioid receptors that are involved. 2. Results 2.1. Antinociceptive effect of xylazine The intracerebroventricular administration of xylazine (5, 10 and 20 mg) produced an antinociceptive response, in a dosedependent manner (Fig. 1). There was no statistical difference between the 10 and 20 mg doses, being the dose of 10 mg chosen for the present study Antagonism of xylazine-induced antinociception by naloxone or clocinnamox As shown in Fig. 2a and b, the intracerebroventricular administration of naloxone (2.5 and 5 mg) or clocinnamox (2 and 4 mg) inhibited the xylazine-induced central antinociception (10 mg), respectively. The highest effective dose of the antagonists per se did not modify the withdrawal threshold (Fig. 2a and b) Effect of naltrindole and nor-binaltorphimine on xylazine-induced antinociception The intracerebroventricular administration of naltrindole (6 and 12 mg; Fig. 3a) and nor-binaltorphimine (10 and 20 mg; Fig. 3b) did not modify the central antinociception of xylazine Fig. 1 Antinociception induced by intracerebroventricular administration of xylazine in mice. Xylazine (Xyl; 5, 10 and 20 lg) was administered 1 min prior to measured in the tailflick test. Each line represents the mean7sem for four mice per group. indicates a significant difference compared to Sal-injected group (ANOVAþBonferroni test; interaction: Df¼12, F¼20.96; column factor: Df¼3, F¼82.60; row factor: Df¼4, F¼101.20; po0.05). Sal, saline (0.5% of Evans blue). (10 mg). These drugs did not significantly modify the nociceptive threshold in control mice. 3. Discussion Opioid peptides have been implicated in antinociception induced by several compounds, including non-steroidal antiinflammatory drugs (Rezende et al., 2009), cannabinoids (Cox and Welch, 2004), natural coffee-derived diterpenes (Guzzo et al., 2012) and a 2 -adrenoceptor agonists (Nakamura and Ferreira, 1988). Our group suggested previously that xylazine induces peripheral antinociception by releasing endogenous opioids that act on their receptors (Romero et al., 2009). Because there is a lack of information regarding opioid peptide participation in the central analgesic mechanism of xylazine, the present work investigated this hypothesis. The antinociceptive effect of xylazine administered systemically (Bentley et al., 1977; Naves and Freire, 1989), intrathecally (Goodchild et al., 1996) or intracerebroventricularly (Schmitt et al., 1974) has been described by several authors. Our results demonstrated that xylazine produced central antinociceptive effects in the tail-flick test in a dosedependent manner, and a 10 mg dose was chosen for the present study. It has been shown that administration of other adrenergic agonists intracerebroventricularly induces marked antinociception to thermal stimuli (Buerkle and Yaksh, 1998; Pertovaara et al., 1991). The opioid receptor antagonist naloxone prevented xylazineinduced central antinociception, suggesting participation of opioid receptors in this effect. Several reports in the literature suggest interaction of opioid- and a-adrenoceptor agonists at the receptor level. In 1974, it was observed that naloxone inhibited phenoxybenzamine and phentolamine binding to brain homogenates (Cicero et al., 1974). Other authors demonstrated that

3 60 brain research 1506 (2013) Fig. 2 Antagonism induced by intracerebroventricular administration of naloxone (a) and clocinnamox (b) on the central antinociception produced by xylazine. Naloxone (Nal; 2.5 and 5 lg) or clocinnamox (Clo; 2 and 4 lg) were administered 1 min prior to xylazine (Xyl; 10 lg). These antagonists did not significantly modify the nociceptive threshold in control mice. Each line represents the mean7sem for four mice per group. and # indicate a significant difference compared to (SalþSal) and (SalþXyl 10)-injected controls, respectively (ANOVAþBonferroni test; interaction: Df¼16, F¼10.96; column factor: Df¼4, F¼42.35; row factor: Df¼4, F¼31.80; po0.05 (a) and interaction: Df¼16, F¼2.21; column factor: Df¼4, F¼18.92; row factor: Df¼4, F¼15.93; po0.05 (b)). Sal, saline (0.5% of Evans blue). phenoxybenzamine caused a dose-dependent displacement of stereospecifically bound naloxone from mouse brain homogenates (Spiehler et al., 1978). Thus, it has been hypothesized that a 2 -adrenoceptor and opioid receptors may be linked, either via secondary messenger systems or physical membrane interation (Bentley et al., 1983). Aley and Levine (1997) proposed that the existence of a m9a 2 receptor complex mediating peripheral antinociception explained the naloxone-mediated inhibition of peripheral xylazine-induced antinociception. Naloxone has been useful for receptor affinity determination of type-selective opioids in mammals; however, naloxone preferentially interacts with m binding sites (K D ¼3.9 nm), but also has significant affinity for k-opioid receptors (K D ¼16 nm) and less affinity for d-opioid receptors (K D ¼95 nm) (Satoh and Minami, 1995). To clarify which receptor subtype would be involved in the central antinociceptive effect of xylazine, highly selective antagonists clocinnamox, naltrindole and norbinaltorphimine were used. Clocinnamox is an irreversible m- opioid receptor antagonist with K i values of 0.7, 1.9 and 5.7 nm for mouse m, d and k receptors, respectively (Burke et al., 1994). Naltrindole has 223- and 346-fold greater activity for d- than Fig. 3 Effect of intracerebroventricular administration of naltrindole (a) and nor-binaltorphimine (b) on the central antinociception produced by xylazine. Naltrindole (NTD; 6 and 12 lg) or nor-binaltorphimine (NorBni; 10 and 20 lg) were administered 1 min prior to the xylazile (Xyl; 10 lg). Each line represents the mean7sem for 4 mice per group. indicates a significant difference compared to Sal-injected group (ANOVAþBonferroni test; interaction: Df¼16, F¼7.52; column factor: Df¼4, F¼52.82; row factor: Df¼4, F¼56.49; po0.05 (a) and interaction: Df¼16, F¼6.37; column factor: Df¼4, F¼31.83; row factor: Df¼4, F¼52.36; po0.05 (b)) Sal, saline (0.5% of Evans blue). for m- and k-opioid receptors (Portoghese et al., 1990), and norbinaltorphimine shows 27- to 29-fold less potency, respectively, for m and d binding sites compared with k receptors binding sites (Rothman et al., 1988). The results demonstrated that clocinnamox, but not naltrindole or nor-binaltorphimine, antagonized xylazine-induced central antinociception, reinforcing the participation of m-opioid receptors in this effect. The periaqueductal gray area (PAG) contains high concentrations of endogenous opioid peptides b-endorphin and enkephalin and receptors m and d (Yaksh et al., 1988). Activation of any of these opioid receptors by PAG administration of b-endorphin, morphine or other opioids produces potent antinociception at supraspinal sites and also activates the descending pain control pathways, which are mediated by the brainstem rostral ventromedial medulla (RVM) (Basbaum and Fields, 1984; Pavlovic and Bodnar, 1998; Smith et al., 1988). The RVM contains m-, d-, and k-opioid receptors and it is a major locus for the descending control of nociception and opioid analgesia (Bowker and Dilts, 1988; Gutstein et al., 1998; Kalyuzhny and Wessendorf, 1998). The analgesia produced by microinjection of morphine into the PAG is dependent upon the connection to the RVM and the direct stimulation of the RVM also can produce potent analgesia (Pan and Fields, 1996;

4 brain research 1506 (2013) Urban and Smith, 1994). Importantly, the tail-flick response is modulated in the RVM (Gilbert and Franklin, 2001). Additionally, opioid antagonists administered into the PAG significantly reduce both morphine and beta-endorphin analgesia elicited from the amygdala. This shows the relationship between the amygdala and PAG in analgesic responsiveness. However, the co-administration of subthreshold doses of morphine into both the amygdala and PAG results in a profound synergistic interaction on the jump test, but not the tail-flick test (Pavlovic and Bodnar, 1998). Neurons, astrocytes and microglia express adrenergic receptors a 1, a 2 and b (Laureys et al., 2010; Mori et al., 2002; Peng et al., 2010; Porter and McCarthy, 1997), and astrocytes are a major central nervous system target for a 2 -adrenergic agonists (Hertz et al., 2004). Our data did not directly demonstrate xylazine activation of a 2 -adrenoceptor in these locations, but they can be implicated as potential cellular sites. In conclusion, our results are the first to suggest that the central antinociceptive effect of xylazine is associated with the release of endogenous opioids acting on m-opioid receptors. The results of this work contribute to a greater understanding of the central antinociceptive mechanisms of a drug widely used in veterinary therapy; however, more work needs to be done to elucidate the relationship between xylazine and endogenous opioids. 4. Experimental procedure 4.1. Animals The experiments were performed on g male Swiss mice (N¼4 per group) from the CEBIO (The Animal Centre) of the University of Minas Gerais (UFMG). The mice were housed in a temperature-controlled room (2371 1C) on an automatic 12-h light/dark cycle (06:00 18:00 h of light phase). All testing was carried out during the light phase (08:00 15:00 h). Food and water were freely available until the onset of the experiments. The algesimetric protocol was approved by the Ethics Committee on Animal Experimentation (CETEA) of UFMG Algesimetric method The tail-flick test used in the present study was conducted in accordance with the procedure described by D amour and Smith (1941) with a slight modification. Briefly, following restraining of the mouse by one of experimenter s hand, a heat source was applied 2 cm from the tip of the tail, and the time(s) required for the animal to withdraw its tail from the heat source was recorded as the tail-flick latency. The intensity of the heat was adjusted so that the baseline latencies were between 3 and 4 s. To avoid tissue damage, the cut-off time was established at 9 s. The baseline latency was obtained for each animal before drug administration (zero time) by calculating an average of three consecutive trials. To reduce stress, mice were habituated to the apparatus 1 day prior to conducting the experiments Intracerebroventricular injection (i.c.v.) Animals were constrained by a special device, which immobilizes their body, except for their head. With one hand the experimenter restrained the animal head and then injected the drugs into its right lateral ventricle, by intracerebroventricular route using a Hamilton syringe of 5 ml. The site of injection was 1 mm from either side of the midline on a line drawn through the anterior base of the ears (modified from Haley and Mccormick, 1957). The syringe was inserted perpendicularly through the skull into the brain in the profundity of 2 mm and 2 ml of solution was injected. For ascertaining the areas in the brain ventricular system into which the drugs penetrated, dilution of the drugs in Evans blue 0.5% was realized and the brain were sectioned for confirmation after experiments under anesthesia Experimental protocol All drugs were i.c.v. administered into the lateral ventricle. Naloxone, clocinnamox, naltrindole and nor-binaltorphimine were administered 1 min prior to the xylazine. The nociceptive threshold was always measured in the mouse tail and the measurements of latency were performed at the following times: 0, 5, 10, 15 and 30 min. The protocol above was assessed in pilot experiments to determine the best moment for the injection of each substance Statistical analysis Data were analyzed statistically by two-way analysis of variance (ANOVA) with post hoc Bonferroni s test for multiple comparisons. Probabilities less than 5% (po0.05) were considered to be statistically significant Chemicals The following drugs and chemicals were used: a 2 -adrenoceptor agonist xylazine (Sigma, USA), opioid receptor antagonist naloxone (Sigma), m-opioid receptor antagonist clocinnamox (Tocris, USA), d-opioid receptor antagonist naltrindole (Tocris) and k-opioid receptor antagonist nor-binaltorphimine (Sigma). All drugs were dissolved in saline and injected in a volume of 2 ml into the lateral ventricle. The saline used for dilution of all drugs contained 0.5% of Evans blue. Conflicts of interest The authors state no conflict of interest. Acknowledgments Fellowship by CNPq (Conselho Nacional de Pesquisa).

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