Xylazine Induced Central Antinociception Mediated by Endogenous Opioids and Μ-Opioid Receptor, but Not Δ-Or Κ-Opioid Receptor

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

Thiago Roberto Lima Romero1, Daniela da Fonseca Pacheco1, Igor Dimitri Gama Duarten

Department of Pharmacology, Institute of Biological Sciences, UFMG, Av. Antoˆnio Carlos, 6627, CEP 31.270.100, Belo Horizonte, MG, Brazil

article info abstract

Article history: Endogenous opioids have been implicated in compound-induced antinociception, and our Accepted 18 February 2013 group previously suggested that xylazine induces peripheral antinociception by releasing Available online 26 February 2013 endogenous opioids that act on their respective receptors. In this study, we investigated a Keywords: the involvement of endogenous opioids in 2-adrenoceptor agonist xylazine-induced Xylazine central antinociception. The nociceptive threshold for thermal stimulation was measured Endogenous opioid in Swiss mice using the tail-ﬂick test. The drugs were administered via the intracerebro- Central antinociception ventricular route. Probabilities less than 5% (po0.05) were considered to be statistically signiﬁcant (ANOVA/Bonferroni’s test). Our results demonstrated that opioid receptor a2-adrenoceptor 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 short-duration surgery in both wild and domesticated pigs (Ajadi et al., 2008). Synergistic antinociceptive interactions

Xylazine is an a2-adrenoceptor agonist (Hsu, 1981)thatis between epidurally administered xylazine and lidocaine extensively used in veterinary medical practice and animal induced good-quality antinociception in water buffalo calves experimentation for sedation, antinociception, and muscle (Singh et al., 2005). Recently, it was observed that xylazine- relaxation (Willian et al., 2001). Xylazine exhibits direct, local tramadol in combination is useful for equine sedation and anesthetic sensory and motor nerve inhibition in addition to its analgesia (Seo et al., 2011).

spinal cord a2-adrenoceptor-mediated analgesic effects (Knight, Xylazine’s antinociceptive actions were initially investi- 1980). Furthermore, xylazine in combination with other sub- gated using the murine acetic acid-induced writhing test by stances has been useful in many surgical procedures. For Bentley et al. (1977). Others observed that the antinociceptive example, xylazine-ketamine mixtures have been used for effects of intraperitoneal xylazine administration were

nCorresponding author. Fax: þ55 31 409 2695. E-mail address: [email protected] (I.D.G. Duarte). 1Both authors Romero and Pacheco have the same participation.

0006-8993 & 2013 Elsevier B.V. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.brainres.2013.02.030 brain research 1506 (2013) 58–63 59

antagonized by yohimbine, but not by the opioid receptor antagonist naloxone. These data suggest that this effect is dependent on an a2-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 a2-adrenoceptor agonists activate opioid receptors via endogenous opioid release.

In 1988, it was shown that a2-adrenoceptor agonist clonidine- induced 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). Fig. 1 – Antinociception induced by intracerebroventricular Endogenous opioids have been implicated in antinocicep- administration of xylazine in mice. Xylazine (Xyl; 5, 10 and tion induced by several compounds including non-steroidal 20 lg) was administered 1 min prior to measured in the tail- anti-inflammatory drugs (Rezende et al., 2009), cannabinoids flick test. Each line represents the mean7SEM for four mice (Cox and Welch, 2004) and natural coffee-derived diterpenes per group. Ã indicates a significant difference compared to (Guzzo et al., 2012). They act mainly through three types of Sal-injected group (ANOVAþBonferroni test; interaction: opioid receptors m, d and k (Singh et al., 1997). Centrally, the Df¼12, F¼20.96; column factor: Df¼3, F¼82.60; row factor: periaqueductal gray area (PAG) contains high concentrations Df¼4, F¼101.20; po0.05). Sal, saline (0.5% of Evans blue). 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 antinoci- (10 mg). These drugs did not significantly modify the nocicep- ception at supraspinal sites (Terashvili et al., 2008). tive threshold in control mice. To elucidate the mechanisms involved in supraspinal xylazine administration, the aim of the present study was to determine whether endogenous opioids are involved in 3. Discussion xylazine-induced central antinociception and to identify the opioid receptors that are involved. Opioid peptides have been implicated in antinociception induced by several compounds, including non-steroidal anti- inflammatory drugs (Rezende et al., 2009), cannabinoids (Cox 2. Results and Welch, 2004), natural coffee-derived diterpenes (Guzzo et al., 2012)anda2-adrenoceptor agonists (Nakamura and 2.1. Antinociceptive effect of xylazine Ferreira, 1988). Our group suggested previously that xylazine induces peripheral antinociception by releasing endogenous The intracerebroventricular administration of xylazine (5, 10 opioids that act on their receptors (Romero et al., 2009). and 20 mg) produced an antinociceptive response, in a dose- Because there is a lack of information regarding opioid peptide dependent manner (Fig. 1). There was no statistical difference participation in the central analgesic mechanism of xylazine, between the 10 and 20 mg doses, being the dose of 10 mg the present work investigated this hypothesis. chosen for the present study. The antinociceptive effect of xylazine administered systemically (Bentley et al., 1977; Naves and Freire, 1989), intrathecally (Goodchildetal.,1996) or intracerebroventricu- 2.2. Antagonism of xylazine-induced antinociception by larly (Schmitt et al., 1974) has been described by several naloxone or clocinnamox authors. Our results demonstrated that xylazine produced central antinociceptive effects in the tail-flick test in a dose- As shown in Fig. 2a and b, the intracerebroventricular admin- dependent manner, and a 10 mg dose was chosen for the istration of naloxone (2.5 and 5 mg) or clocinnamox (2 and 4 mg) present study. It has been shown that administration of other inhibited the xylazine-induced central antinociception (10 mg), adrenergic agonists intracerebroventricularly induces marked respectively. The highest effective dose of the antagonists antinociception to thermal stimuli (Buerkle and Yaksh, 1998; per se did not modify the withdrawal threshold (Fig. 2a and b). Pertovaara et al., 1991). The opioid receptor antagonist naloxone prevented xylazine- 2.3. Effect of naltrindole and nor-binaltorphimine on induced central antinociception, suggesting participation of xylazine-induced antinociception opioid receptors in this effect. Several reports in the literature suggest interaction of opioid- and a-adrenoceptor agonists at the The intracerebroventricular administration of naltrindole receptor level. In 1974, it was observed that naloxone inhibited (6 and 12 mg; Fig. 3a) and nor-binaltorphimine (10 and 20 mg; phenoxybenzamine and phentolamine binding to brain homo- Fig. 3b) did not modify the central antinociception of xylazine genates (Cicero et al., 1974). Other authors demonstrated that 60 brain research 1506 (2013) 58–63

Fig. 3 – Effect of intracerebroventricular administration of Fig. 2 – Antagonism induced by intracerebroventricular naltrindole (a) and nor-binaltorphimine (b) on the central administration of naloxone (a) and clocinnamox (b) on the antinociception produced by xylazine. Naltrindole (NTD; 6 central antinociception produced by xylazine. Naloxone and 12 lg) or nor-binaltorphimine (NorBni; 10 and 20 lg) (Nal; 2.5 and 5 lg) or clocinnamox (Clo; 2 and 4 lg) were were administered 1 min prior to the xylazile (Xyl; 10 lg). administered 1 min prior to xylazine (Xyl; 10 lg). These Each line represents the mean7SEM for 4 mice per group. antagonists did not significantly modify the nociceptive Ã indicates a significant difference compared to Sal-injected threshold in control mice. Each line represents the group (ANOVAþBonferroni test; interaction: Df¼16, F¼7.52; mean7SEM for four mice per group. Ã and # indicate a column factor: Df¼4, F¼52.82; row factor: Df¼4, F¼56.49; significant difference compared to (SalþSal) and (SalþXyl p 0.05 (a) and interaction: Df¼16, F¼6.37; column factor: 10)-injected controls, respectively (ANOVAþBonferroni test; o Df¼4, F¼31.83; row factor: Df¼4, F¼52.36; po0.05 (b)) Sal, interaction: Df¼16, F¼10.96; column factor: Df¼4, F¼42.35; saline (0.5% of Evans blue). 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). for m-andk-opioid receptors (Portoghese et al., 1990), and nor- binaltorphimine shows 27- to 29-fold less potency, respec- phenoxybenzamine caused a dose-dependent displacement of tively, for m and d binding sites compared with k receptors stereospecifically bound naloxone from mouse brain homoge- binding sites (Rothman et al., 1988). The results demonstrated nates (Spiehler et al., 1978). Thus, it has been hypothesized that that clocinnamox, but not naltrindole or nor-binaltorphimine, a2-adrenoceptor and opioid receptors may be linked, either via antagonized xylazine-induced central antinociception, reinfor- secondary messenger systems or physical membrane interation cing the participation of m-opioid receptors in this effect. (Bentley et al., 1983). Aley and Levine (1997) proposed that the The periaqueductal gray area (PAG) contains high concen- 9 existence of a m a2 receptor complex mediating peripheral trations of endogenous opioid peptides b-endorphin and antinociception explained the naloxone-mediated inhibition of enkephalin and receptors m and d (Yaksh et al., 1988). Activa- peripheral xylazine-induced antinociception. tion of any of these opioid receptors by PAG administration of Naloxone has been useful for receptor affinity determina- b-endorphin, morphine or other opioids produces potent tion of type-selective opioids in mammals; however, nalox- antinociception at supraspinal sites and also activates the one preferentially interacts with m binding sites (KD ¼3.9 nM), descending pain control pathways, which are mediated by but also has significant affinity for k-opioid receptors (KD ¼16 the brainstem rostral ventromedial medulla (RVM) (Basbaum nM) and less affinity for d-opioid receptors (KD ¼95 nM) (Satoh and Fields, 1984; Pavlovic and Bodnar, 1998; Smith et al., and Minami, 1995). To clarify which receptor subtype would 1988). The RVM contains m-, d-, and k-opioid receptors and it be involved in the central antinociceptive effect of xylazine, is a major locus for the descending control of nociception and highly selective antagonists clocinnamox, naltrindole and nor- opioid analgesia (Bowker and Dilts, 1988; Gutstein et al., 1998; binaltorphimine were used. Clocinnamox is an irreversible m- Kalyuzhny and Wessendorf, 1998). The analgesia produced by opioid receptor antagonist with Ki values of 0.7, 1.9 and 5.7 nM microinjection of morphine into the PAG is dependent upon for mouse m, d and k receptors, respectively (Burke et al., 1994). the connection to the RVM and the direct stimulation of the Naltrindole has 223- and 346-fold greater activity for d-than RVM also can produce potent analgesia (Pan and Fields, 1996; brain research 1506 (2013) 58–63 61

Urban and Smith, 1994). Importantly, the tail-flick response is 4.3. Intracerebroventricular injection (i.c.v.) modulated in the RVM (Gilbert and Franklin, 2001). Addition- ally, opioid antagonists administered into the PAG signifi- Animals were constrained by a special device, which immobi- cantly reduce both morphine and beta-endorphin analgesia lizes their body, except for their head. With one hand the elicited from the amygdala. This shows the relationship experimenter restrained the animal head and then injected the between the amygdala and PAG in analgesic responsiveness. drugs into its right lateral ventricle, by intracerebroventricular However, the co-administration of subthreshold doses of route using a Hamilton syringe of 5 ml. The site of injection was morphine into both the amygdala and PAG results in a 1 mm from either side of the midline on a line drawn through profound synergistic interaction on the jump test, but not the anterior base of the ears (modified from Haley and the tail-flick test (Pavlovic and Bodnar, 1998). Mccormick, 1957). The syringe was inserted perpendicularly Neurons, astrocytes and microglia express adrenergic through the skull into the brain in the profundity of 2 mm and receptors a1, a2 and b (Laureys et al., 2010; Mori et al., 2002; 2 ml of solution was injected. For ascertaining the areas in the Peng et al., 2010; Porter and McCarthy, 1997), and astrocytes brain ventricular system into which the drugs penetrated, are a major central nervous system target for a2-adrenergic dilution of the drugs in Evans blue 0.5% was realized and the agonists (Hertz et al., 2004). Our data did not directly demon- brain were sectioned for confirmation after experiments under strate xylazine activation of a2-adrenoceptor in these loca- anesthesia. tions, 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 4.4. Experimental protocol the release of endogenous opioids acting on m-opioid receptors. The results of this work contribute to a greater under- All drugs were i.c.v. administered into the lateral ventricle. standing of the central antinociceptive mechanisms of a drug Naloxone, clocinnamox, naltrindole and nor-binaltorphimine widely used in veterinary therapy; however, more work needs were administered 1 min prior to the xylazine. to be done to elucidate the relationship between xylazine and The nociceptive threshold was always measured in the endogenous opioids. 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. 4. Experimental procedure

4.1. Animals 4.5. Statistical analysis

The experiments were performed on 25–30 g male Swiss mice Data were analyzed statistically by two-way analysis of (N¼4 per group) from the CEBIO (The Animal Centre) of the variance (ANOVA) with post hoc Bonferroni’s test for multiple University of Minas Gerais (UFMG). The mice were housed in comparisons. Probabilities less than 5% (po0.05) were con- a temperature-controlled room (2371 1C) on an automatic sidered to be statistically signiﬁcant. 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 experi- 4.6. Chemicals ments. The algesimetric protocol was approved by the Ethics

Committee on Animal Experimentation (CETEA) of UFMG. The following drugs and chemicals were used: a2-adrenoceptor agonist xylazine (Sigma, USA), opioid receptor antagonist naloxone (Sigma), m-opioid receptor antagonist clocinnamox (Tocris, USA), d-opioid receptor antagonist naltrindole (Tocris) 4.2. Algesimetric method and k-opioid receptor antagonist nor-binaltorphimine (Sigma). All drugs were dissolved in saline and injected in a volume of The tail-flick test used in the present study was conducted in 2 ml into the lateral ventricle. The saline used for dilution of all accordance with the procedure described by D’amour and drugs contained 0.5% of Evans blue. 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 Conflicts of interest heat source was recorded as the tail-flick latency. The intensity of the heat was adjusted so that the baseline The authors state no conflict of interest. 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 Acknowledgments trials. To reduce stress, mice were habituated to the appara- tus 1 day prior to conducting the experiments. Fellowship by CNPq (Conselho Nacional de Pesquisa). 62 brain research 1506 (2013) 58–63

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