Central XYLAZINE-Induced Antinociception, but not Peripheral, is Mediated by Ca2+-Activated Cl- Channels (Caccs) in Rats

N-(2,6-dimethylphenyl)-5,6-dihydro-4H-1,3-thiazin-2-amine, commonly known as xylazine, is a drug first synthesized by Bayer in 1962, which initially promoted its use as an antihypertensive agent [1]. Later, its anesthetic properties were identified, leading to its widespread use in veterinary medicine as a sedative, antinociceptive, and muscle relaxant [2]. However, due to its significant hypotensive effects and central nervous system depression, it was not approved for human use [3]. It was first described that the effects of xylazine were mainly mediated through α2-adrenoceptor agonism [4]. Since then, several additional mechanisms of action have been reported. Specifically, in the field of analgesia, xylazine may induce antinociception through multiple pathways, including activation of postsynaptic α2-adrenoceptors [5], modulation of the endogenous opioid system [6], involvement of the endocannabinoid system [7], and inhibition of hyperpolarization-activated cyclic nucleotide-gated ion channel (HCN channel) currents [8].

Similar to HCN channels, Ca2+-activated Cl- channels (CaCCs) are present in excitable cells like smooth muscle and various neurons, including those in the dorsal root ganglion (DRG), spinal cord, and autonomic system [9]. When these channels open, they cause membrane hyperpolarization, which depends on the chloride equilibrium potential and the resting membrane potential. These processes help produce an antinociceptive effect in afferent neurons through a hyperpolarization mechanism [10]. Based on the data presented, this study aims to determine whether peripheral and intrathecal injections of xylazine produce antinociception in an acute pain model induced by PGE2 and if CaCCs are involved in this process.

Animals

Male Wistar rats, weighing between 170 and 200 g and obtained from the Central Animal Facility of the Federal University of Minas Gerais in Minas Gerais, Brazil, were used in the experiments. The animals were housed in plastic cages with bedding made from forage shavings, with free access to water and food, and kept in the testing room for 2 days prior to the experiments for habituation. They were maintained in a temperature-controlled environment (24°C ± 2°C) with a 12-hour light/dark cycle (7:00 AM to 7:00 PM). All tests were conducted during the light phase (8:00 AM to 5:00 PM). All animal procedures and protocols were approved by the Ethics Committee on Animal Experimentation of the Federal University of Minas Gerais (protocol number 191/2023) and complied with the guidelines for assessing experimental pain in animals [11].

Algesimetric method

Hyperalgesia was induced by injecting prostaglandin E2 (PGE2; 2 μg) subcutaneously into the plantar surface of the hind paw. The mechanical nociceptive threshold was measured by assessing the response to a paw pressure test, as described by Randall and Selitto (1957) [12]. An analgesimeter (Ugo-Basile, Varese, Italy) was used to apply a steadily increasing force to the rat’s paw. The weight in grams (g) needed to trigger a paw withdrawal response was recorded as the nociceptive threshold. A cut-off value of 300 g was set to reduce the risk of paw injury. The nociceptive threshold, expressed in grams, was calculated as the average of three consecutive trials. The peak effect after PGE2 injection was considered to occur at 180 minutes (the 3rd hour), at which point Xylazine was administered. For time-response experiments, measurements were taken at zero minutes and at 185, 190, 200, and 210 minutes. In delta (Δ) experiments, measurements were recorded at zero minutes and during the 3rd hour, with Δ calculated as the difference between these values. An n=4 was used for all experimental groups tested.

Experimental design

In all experiments, PGE2 was injected into the subcutaneous plantar surface of the right hind paw in a final volume of 100 μl. For intrathecal administration, rats were shaved in the dorsal lumbar region. After sedation with isoflurane (3.5 %) (CRISTÁLIA®, Brazil), xylazine and niflumic acid were injected in a volume of 20 μl using a 13 x 0,3 mm needle attached to a hypodermic syringe (BD®, Brazil) directly into the subarachnoid space between the sixth and seventh lumbar vertebrae [13]. This volume remains constant for the drugs and their respective vehicles. An animal group treated with 4 % lidocaine (20 μl) was used to confirm injection efficacy, showing temporary paralysis of the posterior limbs (data not shown). Prostaglandin E2 (PGE2, Sigma, USA) was dissolved in 2% ethanol. Xylazine (10% Syntec, BRA) was dissolved in physiological saline, and Niflumic acid (Sigma, USA) was dissolved in 10% DMSO in saline.

Statistical analyses

All results were analyzed using GraphPad Prism 10.1 and are shown as mean ± SEM. Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. A difference was considered significant when P < 0.01.

When Xylazine was administered intrathecally (10 μg), it fully reversed PGE2-induced hyperalgesia, bringing the nociceptive threshold back to baseline (Figure 1A). When injected into the hind paw, xylazine produced antinociception at a dose of 100 μg (Figure 1B). The maximum antinociceptive effect was observed 5 minutes after injection. Intrathecal administration of niflumic acid (2, 4, and 8 μg), a CaCC blocker, dose-dependently reduced xylazine-mediated antinociception (10 μg), with the 8-μg dose completely reversing the effect (Figure 1A). However, when injected into the hind paw, niflumic acid (32 μg/paw) did not reverse xylazine-induced analgesia (100 μg). Niflumic acid alone did not alter the nociceptive threshold in animals injected with PGE₂ or vehicle in both routes of administration (Figure 1).

Green, 1975 [14], described one of the earliest reports on xylazine analgesia, highlighting the challenges in measuring antinociception due to sedative effects following intramuscular injection. The author subjectively noted that a dose of 12 mg/Kg produced “good” analgesia, although it was insufficient and suitable only for superficial procedures in mice used for animal experiments. The subjectivity of Green’s results underscores the importance of accurate and controlled methods of algesimetry in animal research. The development of tests such as the paw pressure test [12], tail flick test [15], and von Frey test has provided researchers with reliable tools to characterize central and peripheral algesimetry [16].

Using the paw pressure test, our group previously demonstrated the effectiveness of xylazine in producing analgesia after hind paw injection, administered 5 minutes before the peak action of PGE2, based on the dose and methodology used in this study [17]. In that research, the 100 μg dose completely eliminated pain responses when injected into the right hind paw of rats. Both the effect and the efficacy of the antinociceptive response against the PGE2 model were confirmed, without systemic effects. Our group has also examined the peripheral mechanisms involved in that xylazine-induced analgesia. We have shown its reliance on the endocannabinoid system [18], endogenous opioid pathways [19], and the l-arginine/nitric oxide/cyclic GMP/KATP pathway [17, 20].

The central administration of xylazine has also been shown in the literature to produce dose-dependent antinociceptive effects at doses of 5, 10, and 20 μg via the intracerebroventricular route [6]. Goodchild et al. (1996) [21] reported that, when injected intrathecally, xylazine depends on the adrenergic system for its antinociceptive effect; however, neither opioid nor Gabaergic propriospinal neurons are involved in mediating this effect. Several studies have shown that opening ATP-sensitive K+ (KATP) channels is the final step in the peripheral antinociceptive mechanism of certain drugs, leading to neuronal membrane hyperpolarization [22-24]. However, hyperpolarization, which may block pain signal transmission, can also be mediated by hyperpolarization-activated cyclic nucleotide- gated (HCN) channels or Ca2+-activated Cl- (CaCC) channels [25].

It was demonstrated that thresholds for both mechanical and thermal nociceptive tests increased in a dose-dependent manner following xylazine administration (30 mg/kg and 40 mg/kg) in both HCN1+/+ and HCN1-/- mice [8]. However, in HCN1+/+ mice, these doses resulted in significantly higher thresholds compared to HCN1-/- mice. Whole-cell patch clamp recordings showed that xylazine inhibited HCN1 and HCN2 ion channel currents, causing a dose-dependent reduction of hyperpolarization-activated currents by xylazine (12.5-100 μmol/L). Similar to HCN channels, Ca2+-activated Cl- channels (CaCCs) may also contribute to membrane hyperpolarization, and their role in the peripheral antinociceptive effects of opioids and cannabinoids has been reported [26, 27]. However, their part in xylazine-induced antinociception has not yet been studied.

In our study, intrathecal injection of niflumic acid (2, 4, and 8 μg), a selective CaCC blocker, dose-dependently reversed the analgesic effect of xylazine when given 5 minutes before its administration. In contrast, injecting niflumic acid (32 μg) into the hind paw did not affect xylazine-induced analgesia. This dose was the same as that which fully antagonized the peripheral antinociceptive effect of δ-opioid receptor activation [26]. These findings indicate that Ca2+-activated Cl- channels contribute to the central, but not peripheral, part of xylazine-induced antinociception through a hyperpolarization- dependent mechanism.

TRLR and IDGD would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil, for a Productivity Fellowship, level 2, numbers 301496/2018-8 and 303876/2023-9. Funding: This work was supported by the CNPq- Brazil (grant number 407943/2018-8).

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

1. Greene SA, Thurmon JC (1988) Xylazine-a review of its pharmacology and use in veterinary medicine. J Vet Pharmacol Ther 11(4): 295-313.
2. Willian WM, John H, Roman ST, Richard BM (2001) Fármacos usados na medicação pré-anesté [Drugs used in anesthesia.] W.M. Willian (Ed.), Manual de anestesia veterinária. [Handbook of veterinary anesthesia.], Artmed, Porto Alegre pp: 31-44.
3. Ruiz-Colón K, Chavez-Arias C, Díaz-Alcalá JE, Martínez MA (2014) Xylazine intoxication in humans and its importance as an emerging adulterant in abused drugs: A comprehensive review of the literature. Forensic Sci Int 240:1-8.
4. Hsu WH (1981) Xylazine-induced depression and its antagonism by alpha adrenergic blocking agents. J Pharmacol Exp Ther 218(1): 188-192.
5. Naves LA, Freire AC (1989) Xylazine antinociception in mice: evidence for mediation by postsynaptic adrenoceptors. Braz J Med Biol Res 22(8):1009-1010.
6. Romero TRL, Pacheco DF, Duarte IDG (2013) Xylazine induced central antinociception mediated by endogenous opioids and μ-opioid receptor, but not δ-or κ-opioid receptors, Brain Research 1506: 58-63.
7. Romero TRL, Miranda e Castor MG, Parrella C, Piscitelli F, Di Marzo V, et al. (2020) α₂-Adrenoceptor agonist induces peripheral antinociception via the endocannabinoid system. Pharmacol Reports 72(1): 96-103.
8. Chen FF, Li L, Chen XD, Zhou C, Liao DQ (2019) Xylazine Produced Analgesic Effect via Inhibits Hyperpolarization-activated Cyclic Nucleotide-gated Ion Channels Currents. Sichuan Da Xue Xue Bao Yi Xue Ban 50(1): 20-26.
9. Ferrera L, Zegarra-Moran O, Galietta LJ (2011) Ca²⁺-activated Cl- channels. Compr Physiol 1(4): 2155-2174.
10. Frings S, Reuter D, Kleene SJ (2000) Neuronal Ca²⁺ -activated Cl- channels--homing in on an elusive channel species. Prog Neurobiol 60(3): 247-289.
11. Zimmermann M (1983) Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16(2): 109-110.
12. Randall LO, Selitto JJ (1957) A method for measurement of analgesic activity on inflamed tissue. Arch Int Pharmacodyn Thérapie 111(4): 409-419.
13. Mestre C, Pélissier T, Fialip J, Wilcox G, Eschalier A (1994) A method to perform direct transcutaneous intrathecal injection in rats. J Pharmacol Toxicol Methods 32(4): 197-200.
14. Green Laboratory Animals (1975) Neuroleptanalgesic drug combinations in the anaesthetic management of small laboratory animals 9(3): 161-178.
15. Juszkiewicz-Donsbach J, Levy G (1962) Effect of Small Variations in Heat Stimulus Temperature on the Tail Flick Response of Rats in Analgesimetry. J of Pharmaceut Scien 51(2): 185-186.
16. Levin S, Pearsall G, Ruderman RJ (1978) Von Frey’s method of measuring pressure sensibility in the hand: An engineering analysis of the Weinstein-Semmes pressure aesthesiometer. J of Hand Surgery 3(3): 211-216.
17. Romero TRL, Duarte, IDG (2009a) α₂-Adrenoceptor agonist xylazine induces peripheral antinociceptive effect by activation of the l-arginine/nitric oxide/cyclic GMP pathway in rat. Eur J of Pharmacol 613(1-3): 64-67.
18. Romero TRL, Miranda e Castor MG, Parrella C, Piscitelli F, Di Marzo V, (2020) α₂-Adrenoceptor agonist induces peripheral antinociception via the endocannabinoid system. Pharmacol Reports 72(1): 96-103.
19. Romero TRL, de Castro Perez A, de Francischi JN, Gama Duarte I D (2009b) Probable involvement of α₂C-adrenoceptor subtype and endogenous opioid peptides in the peripheral antinociceptive effect induced by xylazine. Eur J of Pharmacol 608(1-3): 23-27.
20. Romero TRL, Duarte IDG (2009c) Involvement of ATP-Sensitive K+ Channels in the Peripheral Antinociceptive Effect Induced by the α₂-Adrenoceptor Agonist Xylazine. J of Pharmacol Sci 111(4): 323-327.
21. Goodchild C S, Guo Z, Davies A, Gent JP (1996) Antinociceptive actions of intrathecal xylazine: interactions with spinal cord opioid pathways. Bri J of Anaest 76(4): 544-551.
22. Ferreira RCM, Almeida DL, Duarte IDG, Aguiar DC, Moreira, FA, et al. (2022) The antipsychotic aripiprazole induces peripheral antinociceptive effects through PI3Kγ/NO/cGMP/KATP pathway activation. Eur J of Pain 26(4): 825-834.
23. Soares-Santos RR, Machado DP, Romero TRL, Duarte IDG (2024) Nitric oxide and potassium channels but not opioid and cannabinoid receptors mediate tramadol-induced peripheral antinociception in rat model of paw pressure withdrawal. Canadian Journal of Physiology and Pharmacology 102(3): 218-227.
24. Aguiar DD, Petrocchi JA, Silva GC, Lemos VS, Castor MGM, et al. (2024) Participation of the cannabinoid system and the NO/cGMP/KATP pathway in serotonin-induced peripheral antinociception, Neuroscience Letters 818: 137536.
25. Kowdley GC, Ackerman SJ, John JE 3rd, Jones LR, Moorman JR (1994) Hyperpolarization-activated chloride currents in Xenopus oocytes. J Gen Physiol 103(2): 217-230.
26. Pacheco DF, Pacheco CMF, Duarte IDG (2012) δ-Opioid receptor agonist SNC80 induces central antinociception mediated by Ca²⁺-activated Cl-channels. Journal of Pharmacy and Pharmacology 64(8): 1084-1089.
27. Romero TRL, Pacheco DF, Duarte IDG (2013b) Probable involvement of Ca(²⁺)-activated Cl(-) channels (CaCCs) in the activation of CB1 cannabinoid receptors. Brain Research, Life Sci 92(14-16): 815-820.