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Role of ventrolateral orbital cortex muscarinic and nicotinic receptors in modulation of capsaicin-induced orofacial pain-related behaviors in rats
Author’s Accepted Manuscript Role of ventrolateral orbital cortex muscarinic and nicotinic receptors in modulation of capsaicininduced orofacial pain-related behaviors in rats Esmaeal Tamaddonfard, Amir Erfanparast, Amir Abbas Farshid, Fatmeh Delkhosh-Kasmaie www.elsevier.com/locate/ejphar
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S0014-2999(17)30635-0 https://doi.org/10.1016/j.ejphar.2017.09.048 EJP71434
To appear in: European Journal of Pharmacology Received date: 20 April 2017 Revised date: 12 September 2017 Accepted date: 28 September 2017 Cite this article as: Esmaeal Tamaddonfard, Amir Erfanparast, Amir Abbas Farshid and Fatmeh Delkhosh-Kasmaie, Role of ventrolateral orbital cortex muscarinic and nicotinic receptors in modulation of capsaicin-induced orofacial pain-related behaviors in rats, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2017.09.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Role of ventrolateral orbital cortex muscarinic and nicotinic receptors in modulation of capsaicin-induced orofacial pain-related behaviors in rats
Esmaeal Tamaddonfard1*, Amir Erfanparast1, Amir Abbas Farshid2, Fatmeh DelkhoshKasmaie2 1
Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine,
Urmia University, Urmia 5756151818, Iran. 2
Division of Pathology, Department of Pathobiology, Faculty of Veterinary Medicine,
Urmia University, Urmia 5756151818, Iran.
*Corresponding author: Esmaeal Tamaddonfard, Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia 5756151818, Iran, Phone: +98 44 32770508, Fax: +98 44 32771926 E-mail: E-Mail: [email protected]; [email protected]
ABSTRACT Acetylcholine, as a major neurotransmitter, mediates many brain functions such as pain. This study was aimed to investigate the effects of microinjection of muscarinic and nicotinic acetylcholine receptor antagonists and agonists into the ventrolateral orbital cortex (VLOC) on capsaicin-induced orofacial nociception and subsequent hyperalgesia. The right side of VLOC was surgically implanted with a guide cannula in anaesthetized rats. Orofacial pain-related behaviors were induced by subcutaneous injection of a capsaicin solution (1.5 µg/20 µl) into the left vibrissa pad. The time spent face rubbing with
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ipsilateral forepaw and general behavior were recorded for 10 min, and then mechanical hyperalgesia was determined using von Frey filaments at 15, 30, 45 and 60 min postcapsaicin injection. Alone intra-VLOC microinjection of atropine (a muscarinic acetylcholine receptor antagonist) and mecamylamine (a nicotinic acetylcholine receptor antagonist) at a similar dose of 200 ng/site did not alter nocifensive behavior and hyperalgesia. Microinjection of oxotremorine (a muscarinic acetylcholine receptor agonist) at doses of 50 and 100 ng/site and epibatidine (a nicotinic acetylcholine receptor agonist) at doses of 12.5, 25, 50 and 100 ng/site into the VLOC suppressed pain-related behaviors. Prior microinjections of 200 ng/site atropine and mecamylamine (200 ng/site) prevented oxotremorine (100 ng/site)-, and epibatidine (100 ng/site)-induced antinociception, respectively. None of the above-mentioned chemicals changed general behavior. These results showed that the VLOC muscarinic and nicotinic acetylcholine receptors might be involved in modulation of orofacial nociception and hypersensitivity. Keywords: Capsaicin, Muscarinic acetylcholine receptor, Nicotinic acetylcholine receptor, Orofacial pain, Ventrolateral orbital cortex. Chemical compounds studied in this article oxotremorine (Pubchem CID: 4630); atropine sulfate (Pubchem CID: 5927); (+/-)epibatidine dihydrochloride (PubChem CID: 16219300); mecamylamine hydrochloride (PubChem CID: 13221); capsaicin (Pubchem CID: 1548943); normal saline (PubChem: 5234) 1. Introduction Muscarinic and nicotinic acetylcholine receptors mediate the integrative activity of acetylcholine in many functions of the brain such as cognition, attention, emotion and
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synaptic plasticity (Hahn, 2015; Fuenzalida et al., 2016; Prado et al., 2017). Besides to local peripheral and spinal contributions in mediating pain, recent evidences have also suggested the involvement of these receptors in supraspinal modulation of pain (Bartolini et al., 2011; Fiorino and Garcia-Guzman, 2012; De Angelis and Tata, 2016). For example, microinjection of dihydro-β-erythroidine (DHβE; α4β2 nicotinic receptor antagonist) into the rostral ventromedial medulla (RVM) blocked the epibatidineʼs antihyperalgesia in Complete Freundʼs Adjuant (CFA)-induced inflammatory pain in rats (Jareczek et al., 2017). In addition, prior microinjection of methoctramine, a M2 muscarinic receptor antagonist, into the posterior insular cortex (pIC) reduced the analgesic effects of donepezil, a centrally active acetylcholinestrase inhibitor, in both oxaliplatin and spared nerve injury models of neuropathic pain in rats (Ferrier et al., 2015). Ventrolateral orbital cortex (VLOC) is a subdivision structure of the orbital cortex (OC) (Van De Werd and Uylings, 2008; Hoover and Vertes, 2011) and involves in modulation of many brain functions such as memory and depression (Zhao et al., 2013; Xing et al., 2014). Anatomical, pharmacological and behavioral studies in rats have established that the VLOC is an important cerebral cortex area related to pain modulation (Tang et al., 2009). In this context, electrical stimulation of the VLOC depressed jaw-opening reflex in rats (Zhang et al., 1998). In addition, bilateral electrolytic lesions of the VLOC reduced the inhibitory effects of high intensity electroacupuncture on tail flick reflex (Lu et al., 1996). A variety of neurotransmitters including glutamate, GABA, serotonin, dopamine, noradrenaline and opioids are involved in VLOC modulation of pain (Tang et al., 2009; Zhu et al., 2013). The trigeminal nerve relays the sensory information including pain arising from cornea, oral and nasal mucosa, face skin, tooth pulp and temporomandibular joint to the higher
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nuclei and regions of the brain such as trigeminal sensory nucleus complex (TSNC), periaqueductal gray (PAG), thalamus, and cerebral cortex (Sessle, 2011). The TSNC send axons to the submedius (Sm) nucleus of the thalamus, where nerve fibers project to the VLOC (Yoshida et al., 1991, 1992). Although, there are no reports showing the involvement of the VLOC muscarinic and nicotinic acetylcholine receptors in modulation of orofacial pain, Zaborszky et al. (2015) found a cholinergic input from basal forebrain to the OC. Moreover, the distribution of muscarinic and nicotinic acetylcholine receptors in areas such as OC has been reported (Zilles et al., 1989; Mendez et al., 2013). In the present study, we investigated the effects of microinjection of muscarinic and nicotinic acetylcholine receptors antagonist and agonist into the VLOC on the pain-related behaviors induced by subcutaneous (sc) injection of capsaicin into the vibrissa pad in rats. Moreover, the effects of the above-mentioned compounds on general behavior were also investigated. 2. Materials and Methods 2.1. Animals Experiments were carried out on healthy adult male Wistar rats weighing 270–300 g. They were housed in groups of six per cage with free access to food and water. A 12:12-h lightdark cycle (lights on at 07:00 h) at a controlled ambient temperature (22 ± 0.5°C) was maintained. Experiments were performed between 13:00 h and 16:00 h. Veterinary Ethics Committee of the Faculty of Veterinary Medicine of Urmia University approved all protocols used in the present study. 2.2. Drugs
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Oxotremorine (a muscarinic acetylcholine receptor agonist), atropine (a muscarinic acetylcholine receptor antagonist), epibatidine (a nicotinic acetylcholine receptor agonist), mecamylamine (a nicotinic acetylcholine receptor antagonist) and capsaicin were purchased from Sigma-Aldrich Chemical Co. St Louis, MO, USA. Capsaicin was dissolved in ethanol/dimethyl sulfoxide (DMSO)/distilled water (1:1:8 v/v/v) (Pelissier et al., 2002; Holanda Pinto et al., 2008; Tamaddonfard et al., 2015, 2016). Other chemicals (oxotremorine, atropine, epibatidine and mecamylamine) were dissolved in sterile normal saline 30 min before intra-VLOC microinjection. 2.3. Surgery The rats were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (8 mg/kg). Thereafter, using a stereotaxic apparatus (Stoelting, Wood Lane, IL, USA), a stainless-steel guide cannulas (23-gauge, 12-mm) was inserted 1 mm above the right side of the VLOC at the following coordinates: 3.2 mm posterior to the bregma, 2.6 mm left side of the midline, and 4.6 mm below the top of the skull (Paxinos and Watson, 1986). We used of unilateral (contralateral) cannulation of the VLOC, because it has been found that trigeminosubmedius projection is bilateral but with a contralateral dominance. The contralateral projection is about six times larger than the ipsilateral one in rats (Yoshida et al., 1991). On the other hand, reciprocal connections exist between Sm and VLOC in the rat. These connections are largely ipsilateral, with only a small contralateral component (Yoshida et al., 1992). The cannula was fixed to the skull by three screws and dental acrylic. A 29-guage, 12-mm stylet was inserted into the cannula to keep it patent prior to microinjection. At least 10 days were allowed for recovery from the surgery. 2.4. Intra-VLOC microinjection
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Intra-VLOC microinjections of oxotremorine and epibatidine at similar doses of 12.5, 25, 50 and 100 ng/site and atropine and mecamylamine at a same dose of 200 ng/site were performed using a 30-gauge, 13-mm length injection needle attached to a 1 µl Hamilton syringe. Drug solution was microinjected into each VLOC in a volume of the 0.25 µl and the microinjection was slowly made over a period of 45 second. For facilitating diffusion of the drug, the injection needle was left in place for a further 45 second after completion of microinjection. Atropine and mecamylamine were microinjected 6 min, and oxotremorine and epibatidine were administered 3 min before induction of pain. The chemical agent doses and time schedule used here were according to previous studies (Curzon et al., 1998; Gilbert et al., 2001; Ferrier et al., 2015; Yousofizadeh et al., 2015). 2.5. Orofacial pain-related behaviors There are few behavioral models in laboratory animals dedicated to the study of nociception in orofacial region; however, capsaicin-induced orofacial pain test has been frequently used for this purpose (Pelissier et al., 2002; Tamaddonfard et al., 2015, 2016). In this study, capsaicin-induced orofacial pain-related behaviors test was used as shown in Fig. 1. Initially (60 min before intra-VLOC microinjection), the rats were placed on a 50 × 50 cm table for 15 min. At the end of this period (-45 min), mechanical hyperalgesia were measured using an electronic von Frey filament (IITC-Life Science Instruments, Woodland Hill, CA). Thereafter, the animals were placed in Plexiglass observation chambers (30 cm × 30 cm × 30 cm) with a mirror mounted at 45° beneath the floor to allow an unobstructed view of the orofacial region for a period of 30 min. After this adaptation period, a capsaicin solution (1.5 µg/20 µl) was sc injected into the left vibrissa pad using a 27-gauge injection needle (Tamaddonfard et al., 2015, 2016). Face rubbing duration with ipsilateral forepaw
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was recorded for 10 min, and then, the rats were returned to the 50 × 50 cm table and secondary mechanical hyperalgesia was recorded at 15, 30, 45 and 60 min post-capsaicin injection. A von Frey filament with bending force 28 g (No. 14) was applied to the 10-12 mm distal to capsaicin injection site and care was taken to avoid the primary site of capsaicin injection. This method of capsaicin-induced mechanical hyperalgesia was used after injection of capsaicin into the lateral facial skin surface in rats, and head withdrawal frequency (HWF) was considered as a positive response (Honda et al., 2008). The stimulation with filament was repeated 10 times at 10-15 s intervals. The response frequency to von Frey filament application was expressed as percent of response frequency ([number of head withdrawals/number of trails] × 100) (Tamaddonfard et al., 2013). It has been reported that the occurrence of responses in normal rats to a high force indicates mechanical hyperalgesia (Barragan et al., 2014). Mechanical hyperalgesia was considered as secondary as stimulation with von Frey filament was applied to the site far away from the capsaicin injection (Hansen et al., 2012). In addition, during the 10 min recording of nocifensive behavior, general behaviors including rearing numbers and exploring and grooming durations were also recorded. 2.6. Cannulas tip verification At the end of each experiment, animals were overdosed with a mixture of ketamine and xylazine and perfused intracardially with physiological saline followed by 10% of formalin solution. Brains were then removed and placed in a formalin solution (10%), and after seven days, transverse sections (10–20 µm) were provided. Sections were viewed under a light microscope to localize the injection site according to the atlas of Paxinos and Watson (1986).
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2.7. Statistical analysis The results were analyzed using Graph Pad Prism version 5 (Graph Pad Software Inc, Son Diego, CA USA). The data obtained from spontaneous pain (face rubbing durations) were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test. Secondary mechanical hyperalgesia results were analyzed using two-way ANOVA followed by Bonferroni post hoc test. In figures, all values are expressed as mean ± S.E.M. Statistical significance was set at P < 0.05. 3. Results 3.1. Cannula tip verification The placement of the tip of the cannula in the VLOC of rats is shown in Fig. 2. The location of the cannula tip placement in the VLOC was confirmed in the VLOC sections (Fig. 2, right side). The rat brain section (Fig. 2, left side) was adopted from the atlas of Paxinos and Watson, (1986). 3.2. Effects of intra-VLOC microinjection of muscarinic acetylcholine receptor antagonist and agonist on nocifensive behavior induced by capsaicin Alone microinjection of atropine (200 ng/site) into the VLOC did not change face rubbing duration. Intra-VLOC microinjection of oxotremorine (12.5 and 25 ng/site) was without effect, whereas at doses of 50 and 100 ng/site, it significantly (P < 0.05) decreased the intensity of capsaicin-induced orofacial pain. Prior microinjection of atropine (200 ng/site) before oxotremorine (100 ng/site) significantly (P < 0.05) prevented the suppressive effect of oxotremorine on nocifensive behavior (Fig. 3). 3.3. Effects of intra-VLOC microinjection of nicotinic acetylcholine receptor antagonist and agonist on nocifensive behavior induced by capsaicin
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Alone microinjection of mecamylamine (200 ng/site) into the VLOC did not alter orofacial pain intensity. Intra-VLOC microinjection of epibatidine at doses of 12.5, 25, 50 and 100 ng/site significantly (P < 0.05) decreased face rubbing duration. The suppressive effect of intra-VLOC-microinjected epibatidine (100 ng/site) on capsaicin-induced orofacial pain was significantly (P < 0.05) inhibited by prior microinjection of mecamylamine (200 ng/site) into the same site (Fig. 4). 3.4. Effects of intra-VLOC microinjection of muscarinic acetylcholine receptor antagonist and agonist on capsaicin-induced secondary hyperalgesia No significant differences were observed among experimental groups at 45 min before sc injection of capsaicin. Atropine (200 ng/site) alone did not change head frequency percentage (HWF %). Oxotremorine at doses of 12.5 and 25 ng/site produced no significant effect, whereas at doses of 50 and 100 ng/site, it significantly (P < 0.05) decreased HWF % at 15, 30, 45 and 60 min post-capsaicin injection. Prior administration of atropine (200 ng/site) significantly (P < 0.05) inhibited the suppressive effects of oxotremorine (100 ng/site) on HWF % at 15, 30, 45 and 60 min post-capsaicin injection (Fig. 5). 3.5. Effects of intra-VLOC microinjection of nicotinic acetylcholine receptor antagonist and agonist on capsaicin-induced secondary hyperalgesia Mecamylamine (200 ng/site) alone did not alter HWF %. Epibatidine (12.5 ng/site) significantly (P < 0.05) decreased HWF % at 15 and 30 min post-capsaicin-injection. At doses of 25, 50 and 100 ng/site, epibatidine significantly (P < 0.05) decreased HWF % at 15, 30, 45 and 60 min post-capsaicin injection. No significant differences were observed between the suppressive effects of 100 ng/site epibatidine at 30, 45 and 60 min postcapsaicin injection in comparison with 45 min pre-capsaicin injection. The suppressive
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effects of epibatidine (100 ng/site) on HWF% at 15, 30, 45 and 60 min post-capsaicin injection were significantly (P < 0.05) prevented by prior administration of 200 ng/site mecamylamine (Fig. 6). 3.6. Effects of intra-VLOC microinjection of muscarinic and nicotinic acetylcholine receptor antagonist and agonist on general behavior The rearing number and exploring and grooming durations in intra-VLOC saline normal treated rats were 10.7 ± 1 (n/10min), 42 ± 4.1 and 136.7 ± 9.6 s, respectively after sc injection of capsaicin into the vibrissa pad. Intra-VLOC microinjection of atropine, oxotremorine, mecamylamine and epibatidine did not alter the number and duration of general behaviors (Fig. 7A and 7B). 4. Discussion This study shows that sc injection of capsaicin into the vibrissa pad produced a distinct face rubbing and subsequent secondary hyperalgesia in rats. Capsaicin, the principal pungent component in the cayenne peppers, has been used to study nociception and hyperalgesia due to activate transient receptor potential vanilloid type 1 (TRPV1), a heat-sensitive cation channel on nociceptor terminal (Szolcsányi, 2014). These receptors are critical to the sensing of a variety of stimuli such as noxious, heat, and subsequent activation of polymodal C and A-δ nociceptive fibers (Evangelista, 2015). Face rubbing due to capsaicin injection into the orofacial region such as upper lip and vibrissa pad, has been mentioned as a specific nociceptive response (Pelissier et al., 2002; Holanda Pinto et al., 2008; Tamaddonfard et al., 2015). Although there are not any reports showing secondary mechanical hyperalgesia after injection of capsaicin into vibrissa pad, our present study results showed that the induced secondary mechanical hyperalgesia began 15 min after
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capsaicin injection and lasted for 60 min. Hansen et al. (2012) reported that secondary mechanical hypersensitivity was produced following intraplantar injection of capsaicin, which lasted for 240 min. In addition, capsaicin injection into the upper lip produced heat hyperalgesia in mice that lasted for 60 min (Nomura et al., 2013). Besides inducing acute nocifensive responses, capsaicin was found to produce sensitization in central pain pathways (Lam et al., 2008; Ro et al., 2009; Hansen et al., 2012; Nomura et al., 2013). Central sensitization manifested as a reduction in threshold (allodynia), an increase in responsiveness and prolonged aftereffect on noxious stimuli (hyperalgesia) and a receptive field expansion input from non-injured tissue to produce pain (Woolf, 2011). The results of the present study showed that intra-VLOC microinjection of oxotremorine attenuated spontaneous pain and secondary hyperalgesia, and prior treatment with atropine inhibited this effect. A non-significant increase of pain intensity due to alone microinjection of atropine observed in the present study may be related to blockade of muscarinic receptors. This indicates the VLOC muscarinic acetylcholine receptor involvement in modulation of orofacial pain and subsequent central hypersensitivity. Five muscarinic acetylcholine receptor subtypes (M1, M2, M3, M4 and M5 receptors) are widely expressed throughout the central nervous system, and are involved in modulation of neuronal functions (Haga, 2013). Oxotremorine and atropine are non-selective muscarinic acetylcholine receptor agonist and antagonist, respectively, and have used to explore the role of these receptors in brain function modulation (Gholami et al., 2012; Zhou et al., 2015). Although there are no reports showing the effects of microinjection of oxotremorine into the VLOC on orofacial pain and hyperalgesia, Villarreal and Prado (2007) reported that alone microinjection of atropine into the anterior pretectal nucleus (APtN) increased
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allodynia, and prior treatment with atropine prevented oxotremorine-induced antiallodynic effect in a rat model of hind paw incision. In addition, oxotremorine increased tail-flick latency when administered into the brain lateral ventricle and microinjected into the medial, central, basolateral and posterior lateral nuclei of amygdala complex (Oliveira and Prado, 1994). However, in one study, microinjection of oxotremorine into the posterior insular cortex (PIC) attenuated cold and mechanical allodynia induced by oxaliplatin in rats (Ferrier et al., 2015). Our results include the first report on the possible involvement of VLOC muscarinic acetylcholine receptor in modulation of capsaicin-induced orofacial pain and hyperalgesia. Our present results showed that prior microinjection of mecamylamine into the VLOC prevented the suppressive effect of epibatidine on capsaicin-induced pain-related behaviors. Alone microinjection of mecamylamine non-significantly increased pain intensity and this effect may be related to blockade of nicotinic receptors. These mean that at the OC level, nicotinic acetylcholine receptor contributes to processing of orofacial region pain. Mammalian nicotinic acetylcholine receptor is composed of five subunits. The neuronal subunits are divided into alpha (α2-α7, α9 and α10) and beta (β2-β4) classification based on the presence of adjacent cysteine groups in the extracellular domain of only α subunits (Zoli et al., 2014). The most abundant brain nicotinic acetylcholine receptor subtypes are heteromeric α4β2 and homomeric α7 nicotinic acetylcholine receptor subtypes (Wu et al., 2016). In the brain areas such as cortex, thalamus, hippocampus, basal ganglia and cerebellum, the distribution of α4β2 nicotinic acetylcholine receptor subtype has been reported (Hurst et al., 2013). Epibatidine is a potent but nonselective nicotinic acetylcholine receptor agonist, and its biological effects appear to be mediated largely by α4β2 nicotinic
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acetylcholine receptors (Nirogi et al., 2013). In this context, prior microinjection of mecamylamine into the dorsal raphe nucleus (DRN) and the locus coeruleus (LC), brain important pain processing centers, inhibited the same sites microinjected epibatidineinduced antinociceptive effects in the paw formalin test of pain (Cucchiaro et al., 2005, 2006). Curzon et al. (1998) reported that epibatidine acts largely via descending inhibitory pathway arising from nucleus raphe magnus (NRM). It is possible that some of the antinociceptive effects of VLOC microinjected epibatidine may be related to the diffusion of the drug solution to the adjacent structures such as ventral orbital cortex (VOC). In this context, it has been reported that microinjection of epibatidine into the ventral orbital cortex (VOC), a subdivision of OC, attenuated formalin-induced orofacial pain, and prior microinjection of mecamylamine into the same site inhibited this effect (Yousofizadeh et al., 2015). It seems that at the VLOC, the nicotinic acetylcholine receptor involves in processing of capsaicin-induced orofacial pain and related hyperalgesia. In the present study we did not show any significant changes in general behavior after intraVLOC microinjection of oxotremorine, atropine, epibatidine and mecamylamine. There are no reports showing the effects of microinjection of these chemicals into the VLOC on general behavior. The decrease in nocifensive responses induced by most, but not all analgesics may be related to locomotor activity reduction resulting from sedative-like activity (hypoactivity) production, rather than a strict analgesic effect (Cortright et al., 2008). For example, in formalin-induced orofacial pain, a higher dose of memantine inhibited both phases of pain but also produced other motor effects (increased exploratory and decreased freezing behavior, hind-paw weakness and gait ataxia) which were not observed at the lower doses (Eisenberg et al., 1993). Therefore, a special care is needed to
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assure that the analgesic effect is due to a reduction in pain perception and not caused by motor impairment blocking the withdrawal response. Ophthalmic, mandible and maxillary branches of trigeminal nerve relay nociceptive information from orofacial structures into the various parts of TSNC (Sessle, 2011). In addition, thalamic Sm nucleus receives major projections from the trigeminal TSNC and projects primarily to the VLOC (Yoshida et al., 1991, 1992). The VLOC contains neurons that project to the periaqueductal gray (PAG) (Zhang et al., 1997). Considerable neurons of PAG send projection fibers to the TSNC (Li et al., 1993). The TSNC-Sm-VLOC-PAGTNSC may constitute a trigeminal pain processing pathway (Tang et al., 2009). A variety of neurotransmitters and their corresponding receptors are involved in this pathway processing functions (Tang et al., 2009; Zhu et al., 2013; Erfanparast et al., 2015). Because some of the most prevalent and debilitating pain conditions arise from the structures innervated by the trigeminal system (head, face and associated structures), understanding of the neural pathways that mediate trigeminal pain should enhance treatment for the clinical conditions (Romeo-Reyes and Uyanik, 2014). 5. Conclusion In conclusion, the results of the present study showed that at the VLOC level of the brain, activation of muscarinic and nicotinic acetylcholine receptors by oxotrmorine and epibatidine, respectively, suppressed the orofacial pain and subsequent secondary hyperalgesia induced by capsaicin. Prior microinjections of atropine and mecamylamine before oxotremorine and epibatidine, respectively, inhibited oxotremorine-, and epibatidine-induced antinociception. Therefore, muscarinic and nicotinic acetylcholine
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receptors of acetylcholine can involve in orofacial region pain modulation at the VLOC level of the brain. Acknowledgement This article was financially supported by Faculty of Veterinary Medicine of Urmia University. Conflict of interest The authors declare that there are no conflicts of interest. References Barragan, P., Rocha-Gonzalez, H.I., Pineda-Farias, J.B., Murabartian, J., GodinezChaparro, B., Reinach, P.S., Cunha, T.M., Cunha, F.Q., Granodos-Soto, V., 2014. Inhibition of peripheral anion exchanger 3 decreses formalin-induced pain. Eur. J. Pharmacol. 738, 91–100. Bartolini, A., Di Cesare Mannelli, L., Ghelardini, C., 2011. Analgesic and antineuropathic drugs acting through central cholinergic mechanisms. Recent Pat. CNS Drug Discov. 6(2), 119–140. Cortright, D.N., Matson, D.J., Broom, D.C., 2008. New frontiers in assessing pain and analgesia in laboratory animals. Expert Opin. Drug Discov. 3(9), 1099–1108. Cucchiaro, G., Chaijale, N., Commons, K.G., 2005. The dorsal raphe nucleus as a site of action of the antinociceptive and behavioral effects of the α4 nicotinic receptor agonist epibatidine. J. Pharmacol. Exp. Ther. 313 (1), 389–394. Cucchiaro, G., Chaijale, N., Commons, K.G., 2006. The locus coeruleus nucleus as a site of action of the antinociceptive and behavioral effects of nicotinic receptor antagonist, epibatidine. Neuropharmacology 50 (7), 769–776.
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Fig. 1. Chemicals microinjection and pain recording time line. At 60 and 30 min before sc injection of capsaicin, adaptation of animals to experimental conditions was performed. At 45 min before and 15, 30, 45 and 60 min after sc injection of capsaicin, mechanical hyperalgesia were recorded. Immediately after sc injection of capsaicin, nocifensive and general behaviors were recorded for 10 min. Alone and prior intra-VPL microinjection of antagonists and post-antagonist administration of agonists were performed 6 and 3 min before sc injection of capsaicin, respectively.
Fig. 2. Schematic illustration of the rat brain showing the approximate location of the VLOC microinjection site in this study adapted from Paxinos and Watson, (1986) (left). Transverse section of the rat brain shows the location of the permanent cannula tip (black arrow) in the VLOC of rats included in the present study (right). Fr: Frontal cortex, AI: agranular insular cortex, LO: lateral orbital cortex, VLO: ventrolateral orbital cortex, Pir: piriform cortex, AOP: anterior olfactory nucleus, posterior part, IL: infralimbic cortex; Cg:cingulate cortex.
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Fig. 3. Effects of intra-VPL microinjection of atropine and oxotremorine alone and atropine plus oxotremorine on injected area rubbing (A) and face grooming (B) induced by capsaicin. Capsaicin was sc injected into the left vibrissa pad of rats. All values are expressed as mean ± S.E.M. (n = 6). * P < 0.05 significantly different compared with normal saline treated groups. † P < 0.05 significantly different compared with oxotremorine (100 ng/site) treated group.
Fig. 4. Effects of intra-VPL microinjection of mecamylamine and epibatidine alone and mecamylamine plus epibatidine on injected area rubbing (A) and face grooming (B) induced by capsaicin. Capsaicin was sc injected into the left vibrissa pad of rats. All values are expressed as mean ± S.E.M. (n = 6). * P < 0.05 significantly different compared with normal saline treated groups. † P < 0.05 significantly different compared with epibatidine (100 ng/site) treated group.
Fig. 5. Effects of intra-VLOC microinjection of atropine and oxotremorine alone and atropine plus oxotremorine on head withdrawal frequency induced by capsaicin. Capsaicin was sc injected into the left vibrissa pad of rats. All values are expressed as mean ± S.E.M. (n = 6). * P < 0.05 significantly different compared with normal saline treated group and 45 min. † P < 0.05 significantly different compared with oxotremorine (100 ng/site) treated group.
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Fig. 6. Effects of intra-VLOC microinjection of mecamylamine and epibatidine alone and mecamylamine plus epibatidine on head withdrawal frequency induced by capsaicin. Capsaicin was sc injected into the left vibrissa pad of rats. All values are expressed as mean ± S.E.M. (n = 6). * P < 0.05 significantly different compared with normal saline treated group and -45 min. † P < 0.05 significantly different compared with epibatidine (100 ng/site) treated group.
Fig. 7. Effects of intra-VLOC microinjection of mAChR and nAChR antagonists and agonists on general behavior during capsaicin-induced orofacial pain. Capsaicin was sc injected into the left vibrissa pad of rats. All values are expressed as mean ± S.E.M. (n = 6).
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PII: DOI: Reference:
S0014-2999(17)30635-0 https://doi.org/10.1016/j.ejphar.2017.09.048 EJP71434
To appear in: European Journal of Pharmacology Received date: 20 April 2017 Revised date: 12 September 2017 Accepted date: 28 September 2017 Cite this article as: Esmaeal Tamaddonfard, Amir Erfanparast, Amir Abbas Farshid and Fatmeh Delkhosh-Kasmaie, Role of ventrolateral orbital cortex muscarinic and nicotinic receptors in modulation of capsaicin-induced orofacial pain-related behaviors in rats, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2017.09.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Role of ventrolateral orbital cortex muscarinic and nicotinic receptors in modulation of capsaicin-induced orofacial pain-related behaviors in rats
Esmaeal Tamaddonfard1*, Amir Erfanparast1, Amir Abbas Farshid2, Fatmeh DelkhoshKasmaie2 1
Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine,
Urmia University, Urmia 5756151818, Iran. 2
Division of Pathology, Department of Pathobiology, Faculty of Veterinary Medicine,
Urmia University, Urmia 5756151818, Iran.
*Corresponding author: Esmaeal Tamaddonfard, Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia 5756151818, Iran, Phone: +98 44 32770508, Fax: +98 44 32771926 E-mail: E-Mail: [email protected]; [email protected]
ABSTRACT Acetylcholine, as a major neurotransmitter, mediates many brain functions such as pain. This study was aimed to investigate the effects of microinjection of muscarinic and nicotinic acetylcholine receptor antagonists and agonists into the ventrolateral orbital cortex (VLOC) on capsaicin-induced orofacial nociception and subsequent hyperalgesia. The right side of VLOC was surgically implanted with a guide cannula in anaesthetized rats. Orofacial pain-related behaviors were induced by subcutaneous injection of a capsaicin solution (1.5 µg/20 µl) into the left vibrissa pad. The time spent face rubbing with
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ipsilateral forepaw and general behavior were recorded for 10 min, and then mechanical hyperalgesia was determined using von Frey filaments at 15, 30, 45 and 60 min postcapsaicin injection. Alone intra-VLOC microinjection of atropine (a muscarinic acetylcholine receptor antagonist) and mecamylamine (a nicotinic acetylcholine receptor antagonist) at a similar dose of 200 ng/site did not alter nocifensive behavior and hyperalgesia. Microinjection of oxotremorine (a muscarinic acetylcholine receptor agonist) at doses of 50 and 100 ng/site and epibatidine (a nicotinic acetylcholine receptor agonist) at doses of 12.5, 25, 50 and 100 ng/site into the VLOC suppressed pain-related behaviors. Prior microinjections of 200 ng/site atropine and mecamylamine (200 ng/site) prevented oxotremorine (100 ng/site)-, and epibatidine (100 ng/site)-induced antinociception, respectively. None of the above-mentioned chemicals changed general behavior. These results showed that the VLOC muscarinic and nicotinic acetylcholine receptors might be involved in modulation of orofacial nociception and hypersensitivity. Keywords: Capsaicin, Muscarinic acetylcholine receptor, Nicotinic acetylcholine receptor, Orofacial pain, Ventrolateral orbital cortex. Chemical compounds studied in this article oxotremorine (Pubchem CID: 4630); atropine sulfate (Pubchem CID: 5927); (+/-)epibatidine dihydrochloride (PubChem CID: 16219300); mecamylamine hydrochloride (PubChem CID: 13221); capsaicin (Pubchem CID: 1548943); normal saline (PubChem: 5234) 1. Introduction Muscarinic and nicotinic acetylcholine receptors mediate the integrative activity of acetylcholine in many functions of the brain such as cognition, attention, emotion and
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synaptic plasticity (Hahn, 2015; Fuenzalida et al., 2016; Prado et al., 2017). Besides to local peripheral and spinal contributions in mediating pain, recent evidences have also suggested the involvement of these receptors in supraspinal modulation of pain (Bartolini et al., 2011; Fiorino and Garcia-Guzman, 2012; De Angelis and Tata, 2016). For example, microinjection of dihydro-β-erythroidine (DHβE; α4β2 nicotinic receptor antagonist) into the rostral ventromedial medulla (RVM) blocked the epibatidineʼs antihyperalgesia in Complete Freundʼs Adjuant (CFA)-induced inflammatory pain in rats (Jareczek et al., 2017). In addition, prior microinjection of methoctramine, a M2 muscarinic receptor antagonist, into the posterior insular cortex (pIC) reduced the analgesic effects of donepezil, a centrally active acetylcholinestrase inhibitor, in both oxaliplatin and spared nerve injury models of neuropathic pain in rats (Ferrier et al., 2015). Ventrolateral orbital cortex (VLOC) is a subdivision structure of the orbital cortex (OC) (Van De Werd and Uylings, 2008; Hoover and Vertes, 2011) and involves in modulation of many brain functions such as memory and depression (Zhao et al., 2013; Xing et al., 2014). Anatomical, pharmacological and behavioral studies in rats have established that the VLOC is an important cerebral cortex area related to pain modulation (Tang et al., 2009). In this context, electrical stimulation of the VLOC depressed jaw-opening reflex in rats (Zhang et al., 1998). In addition, bilateral electrolytic lesions of the VLOC reduced the inhibitory effects of high intensity electroacupuncture on tail flick reflex (Lu et al., 1996). A variety of neurotransmitters including glutamate, GABA, serotonin, dopamine, noradrenaline and opioids are involved in VLOC modulation of pain (Tang et al., 2009; Zhu et al., 2013). The trigeminal nerve relays the sensory information including pain arising from cornea, oral and nasal mucosa, face skin, tooth pulp and temporomandibular joint to the higher
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nuclei and regions of the brain such as trigeminal sensory nucleus complex (TSNC), periaqueductal gray (PAG), thalamus, and cerebral cortex (Sessle, 2011). The TSNC send axons to the submedius (Sm) nucleus of the thalamus, where nerve fibers project to the VLOC (Yoshida et al., 1991, 1992). Although, there are no reports showing the involvement of the VLOC muscarinic and nicotinic acetylcholine receptors in modulation of orofacial pain, Zaborszky et al. (2015) found a cholinergic input from basal forebrain to the OC. Moreover, the distribution of muscarinic and nicotinic acetylcholine receptors in areas such as OC has been reported (Zilles et al., 1989; Mendez et al., 2013). In the present study, we investigated the effects of microinjection of muscarinic and nicotinic acetylcholine receptors antagonist and agonist into the VLOC on the pain-related behaviors induced by subcutaneous (sc) injection of capsaicin into the vibrissa pad in rats. Moreover, the effects of the above-mentioned compounds on general behavior were also investigated. 2. Materials and Methods 2.1. Animals Experiments were carried out on healthy adult male Wistar rats weighing 270–300 g. They were housed in groups of six per cage with free access to food and water. A 12:12-h lightdark cycle (lights on at 07:00 h) at a controlled ambient temperature (22 ± 0.5°C) was maintained. Experiments were performed between 13:00 h and 16:00 h. Veterinary Ethics Committee of the Faculty of Veterinary Medicine of Urmia University approved all protocols used in the present study. 2.2. Drugs
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Oxotremorine (a muscarinic acetylcholine receptor agonist), atropine (a muscarinic acetylcholine receptor antagonist), epibatidine (a nicotinic acetylcholine receptor agonist), mecamylamine (a nicotinic acetylcholine receptor antagonist) and capsaicin were purchased from Sigma-Aldrich Chemical Co. St Louis, MO, USA. Capsaicin was dissolved in ethanol/dimethyl sulfoxide (DMSO)/distilled water (1:1:8 v/v/v) (Pelissier et al., 2002; Holanda Pinto et al., 2008; Tamaddonfard et al., 2015, 2016). Other chemicals (oxotremorine, atropine, epibatidine and mecamylamine) were dissolved in sterile normal saline 30 min before intra-VLOC microinjection. 2.3. Surgery The rats were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (8 mg/kg). Thereafter, using a stereotaxic apparatus (Stoelting, Wood Lane, IL, USA), a stainless-steel guide cannulas (23-gauge, 12-mm) was inserted 1 mm above the right side of the VLOC at the following coordinates: 3.2 mm posterior to the bregma, 2.6 mm left side of the midline, and 4.6 mm below the top of the skull (Paxinos and Watson, 1986). We used of unilateral (contralateral) cannulation of the VLOC, because it has been found that trigeminosubmedius projection is bilateral but with a contralateral dominance. The contralateral projection is about six times larger than the ipsilateral one in rats (Yoshida et al., 1991). On the other hand, reciprocal connections exist between Sm and VLOC in the rat. These connections are largely ipsilateral, with only a small contralateral component (Yoshida et al., 1992). The cannula was fixed to the skull by three screws and dental acrylic. A 29-guage, 12-mm stylet was inserted into the cannula to keep it patent prior to microinjection. At least 10 days were allowed for recovery from the surgery. 2.4. Intra-VLOC microinjection
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Intra-VLOC microinjections of oxotremorine and epibatidine at similar doses of 12.5, 25, 50 and 100 ng/site and atropine and mecamylamine at a same dose of 200 ng/site were performed using a 30-gauge, 13-mm length injection needle attached to a 1 µl Hamilton syringe. Drug solution was microinjected into each VLOC in a volume of the 0.25 µl and the microinjection was slowly made over a period of 45 second. For facilitating diffusion of the drug, the injection needle was left in place for a further 45 second after completion of microinjection. Atropine and mecamylamine were microinjected 6 min, and oxotremorine and epibatidine were administered 3 min before induction of pain. The chemical agent doses and time schedule used here were according to previous studies (Curzon et al., 1998; Gilbert et al., 2001; Ferrier et al., 2015; Yousofizadeh et al., 2015). 2.5. Orofacial pain-related behaviors There are few behavioral models in laboratory animals dedicated to the study of nociception in orofacial region; however, capsaicin-induced orofacial pain test has been frequently used for this purpose (Pelissier et al., 2002; Tamaddonfard et al., 2015, 2016). In this study, capsaicin-induced orofacial pain-related behaviors test was used as shown in Fig. 1. Initially (60 min before intra-VLOC microinjection), the rats were placed on a 50 × 50 cm table for 15 min. At the end of this period (-45 min), mechanical hyperalgesia were measured using an electronic von Frey filament (IITC-Life Science Instruments, Woodland Hill, CA). Thereafter, the animals were placed in Plexiglass observation chambers (30 cm × 30 cm × 30 cm) with a mirror mounted at 45° beneath the floor to allow an unobstructed view of the orofacial region for a period of 30 min. After this adaptation period, a capsaicin solution (1.5 µg/20 µl) was sc injected into the left vibrissa pad using a 27-gauge injection needle (Tamaddonfard et al., 2015, 2016). Face rubbing duration with ipsilateral forepaw
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was recorded for 10 min, and then, the rats were returned to the 50 × 50 cm table and secondary mechanical hyperalgesia was recorded at 15, 30, 45 and 60 min post-capsaicin injection. A von Frey filament with bending force 28 g (No. 14) was applied to the 10-12 mm distal to capsaicin injection site and care was taken to avoid the primary site of capsaicin injection. This method of capsaicin-induced mechanical hyperalgesia was used after injection of capsaicin into the lateral facial skin surface in rats, and head withdrawal frequency (HWF) was considered as a positive response (Honda et al., 2008). The stimulation with filament was repeated 10 times at 10-15 s intervals. The response frequency to von Frey filament application was expressed as percent of response frequency ([number of head withdrawals/number of trails] × 100) (Tamaddonfard et al., 2013). It has been reported that the occurrence of responses in normal rats to a high force indicates mechanical hyperalgesia (Barragan et al., 2014). Mechanical hyperalgesia was considered as secondary as stimulation with von Frey filament was applied to the site far away from the capsaicin injection (Hansen et al., 2012). In addition, during the 10 min recording of nocifensive behavior, general behaviors including rearing numbers and exploring and grooming durations were also recorded. 2.6. Cannulas tip verification At the end of each experiment, animals were overdosed with a mixture of ketamine and xylazine and perfused intracardially with physiological saline followed by 10% of formalin solution. Brains were then removed and placed in a formalin solution (10%), and after seven days, transverse sections (10–20 µm) were provided. Sections were viewed under a light microscope to localize the injection site according to the atlas of Paxinos and Watson (1986).
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2.7. Statistical analysis The results were analyzed using Graph Pad Prism version 5 (Graph Pad Software Inc, Son Diego, CA USA). The data obtained from spontaneous pain (face rubbing durations) were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test. Secondary mechanical hyperalgesia results were analyzed using two-way ANOVA followed by Bonferroni post hoc test. In figures, all values are expressed as mean ± S.E.M. Statistical significance was set at P < 0.05. 3. Results 3.1. Cannula tip verification The placement of the tip of the cannula in the VLOC of rats is shown in Fig. 2. The location of the cannula tip placement in the VLOC was confirmed in the VLOC sections (Fig. 2, right side). The rat brain section (Fig. 2, left side) was adopted from the atlas of Paxinos and Watson, (1986). 3.2. Effects of intra-VLOC microinjection of muscarinic acetylcholine receptor antagonist and agonist on nocifensive behavior induced by capsaicin Alone microinjection of atropine (200 ng/site) into the VLOC did not change face rubbing duration. Intra-VLOC microinjection of oxotremorine (12.5 and 25 ng/site) was without effect, whereas at doses of 50 and 100 ng/site, it significantly (P < 0.05) decreased the intensity of capsaicin-induced orofacial pain. Prior microinjection of atropine (200 ng/site) before oxotremorine (100 ng/site) significantly (P < 0.05) prevented the suppressive effect of oxotremorine on nocifensive behavior (Fig. 3). 3.3. Effects of intra-VLOC microinjection of nicotinic acetylcholine receptor antagonist and agonist on nocifensive behavior induced by capsaicin
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Alone microinjection of mecamylamine (200 ng/site) into the VLOC did not alter orofacial pain intensity. Intra-VLOC microinjection of epibatidine at doses of 12.5, 25, 50 and 100 ng/site significantly (P < 0.05) decreased face rubbing duration. The suppressive effect of intra-VLOC-microinjected epibatidine (100 ng/site) on capsaicin-induced orofacial pain was significantly (P < 0.05) inhibited by prior microinjection of mecamylamine (200 ng/site) into the same site (Fig. 4). 3.4. Effects of intra-VLOC microinjection of muscarinic acetylcholine receptor antagonist and agonist on capsaicin-induced secondary hyperalgesia No significant differences were observed among experimental groups at 45 min before sc injection of capsaicin. Atropine (200 ng/site) alone did not change head frequency percentage (HWF %). Oxotremorine at doses of 12.5 and 25 ng/site produced no significant effect, whereas at doses of 50 and 100 ng/site, it significantly (P < 0.05) decreased HWF % at 15, 30, 45 and 60 min post-capsaicin injection. Prior administration of atropine (200 ng/site) significantly (P < 0.05) inhibited the suppressive effects of oxotremorine (100 ng/site) on HWF % at 15, 30, 45 and 60 min post-capsaicin injection (Fig. 5). 3.5. Effects of intra-VLOC microinjection of nicotinic acetylcholine receptor antagonist and agonist on capsaicin-induced secondary hyperalgesia Mecamylamine (200 ng/site) alone did not alter HWF %. Epibatidine (12.5 ng/site) significantly (P < 0.05) decreased HWF % at 15 and 30 min post-capsaicin-injection. At doses of 25, 50 and 100 ng/site, epibatidine significantly (P < 0.05) decreased HWF % at 15, 30, 45 and 60 min post-capsaicin injection. No significant differences were observed between the suppressive effects of 100 ng/site epibatidine at 30, 45 and 60 min postcapsaicin injection in comparison with 45 min pre-capsaicin injection. The suppressive
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effects of epibatidine (100 ng/site) on HWF% at 15, 30, 45 and 60 min post-capsaicin injection were significantly (P < 0.05) prevented by prior administration of 200 ng/site mecamylamine (Fig. 6). 3.6. Effects of intra-VLOC microinjection of muscarinic and nicotinic acetylcholine receptor antagonist and agonist on general behavior The rearing number and exploring and grooming durations in intra-VLOC saline normal treated rats were 10.7 ± 1 (n/10min), 42 ± 4.1 and 136.7 ± 9.6 s, respectively after sc injection of capsaicin into the vibrissa pad. Intra-VLOC microinjection of atropine, oxotremorine, mecamylamine and epibatidine did not alter the number and duration of general behaviors (Fig. 7A and 7B). 4. Discussion This study shows that sc injection of capsaicin into the vibrissa pad produced a distinct face rubbing and subsequent secondary hyperalgesia in rats. Capsaicin, the principal pungent component in the cayenne peppers, has been used to study nociception and hyperalgesia due to activate transient receptor potential vanilloid type 1 (TRPV1), a heat-sensitive cation channel on nociceptor terminal (Szolcsányi, 2014). These receptors are critical to the sensing of a variety of stimuli such as noxious, heat, and subsequent activation of polymodal C and A-δ nociceptive fibers (Evangelista, 2015). Face rubbing due to capsaicin injection into the orofacial region such as upper lip and vibrissa pad, has been mentioned as a specific nociceptive response (Pelissier et al., 2002; Holanda Pinto et al., 2008; Tamaddonfard et al., 2015). Although there are not any reports showing secondary mechanical hyperalgesia after injection of capsaicin into vibrissa pad, our present study results showed that the induced secondary mechanical hyperalgesia began 15 min after
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capsaicin injection and lasted for 60 min. Hansen et al. (2012) reported that secondary mechanical hypersensitivity was produced following intraplantar injection of capsaicin, which lasted for 240 min. In addition, capsaicin injection into the upper lip produced heat hyperalgesia in mice that lasted for 60 min (Nomura et al., 2013). Besides inducing acute nocifensive responses, capsaicin was found to produce sensitization in central pain pathways (Lam et al., 2008; Ro et al., 2009; Hansen et al., 2012; Nomura et al., 2013). Central sensitization manifested as a reduction in threshold (allodynia), an increase in responsiveness and prolonged aftereffect on noxious stimuli (hyperalgesia) and a receptive field expansion input from non-injured tissue to produce pain (Woolf, 2011). The results of the present study showed that intra-VLOC microinjection of oxotremorine attenuated spontaneous pain and secondary hyperalgesia, and prior treatment with atropine inhibited this effect. A non-significant increase of pain intensity due to alone microinjection of atropine observed in the present study may be related to blockade of muscarinic receptors. This indicates the VLOC muscarinic acetylcholine receptor involvement in modulation of orofacial pain and subsequent central hypersensitivity. Five muscarinic acetylcholine receptor subtypes (M1, M2, M3, M4 and M5 receptors) are widely expressed throughout the central nervous system, and are involved in modulation of neuronal functions (Haga, 2013). Oxotremorine and atropine are non-selective muscarinic acetylcholine receptor agonist and antagonist, respectively, and have used to explore the role of these receptors in brain function modulation (Gholami et al., 2012; Zhou et al., 2015). Although there are no reports showing the effects of microinjection of oxotremorine into the VLOC on orofacial pain and hyperalgesia, Villarreal and Prado (2007) reported that alone microinjection of atropine into the anterior pretectal nucleus (APtN) increased
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allodynia, and prior treatment with atropine prevented oxotremorine-induced antiallodynic effect in a rat model of hind paw incision. In addition, oxotremorine increased tail-flick latency when administered into the brain lateral ventricle and microinjected into the medial, central, basolateral and posterior lateral nuclei of amygdala complex (Oliveira and Prado, 1994). However, in one study, microinjection of oxotremorine into the posterior insular cortex (PIC) attenuated cold and mechanical allodynia induced by oxaliplatin in rats (Ferrier et al., 2015). Our results include the first report on the possible involvement of VLOC muscarinic acetylcholine receptor in modulation of capsaicin-induced orofacial pain and hyperalgesia. Our present results showed that prior microinjection of mecamylamine into the VLOC prevented the suppressive effect of epibatidine on capsaicin-induced pain-related behaviors. Alone microinjection of mecamylamine non-significantly increased pain intensity and this effect may be related to blockade of nicotinic receptors. These mean that at the OC level, nicotinic acetylcholine receptor contributes to processing of orofacial region pain. Mammalian nicotinic acetylcholine receptor is composed of five subunits. The neuronal subunits are divided into alpha (α2-α7, α9 and α10) and beta (β2-β4) classification based on the presence of adjacent cysteine groups in the extracellular domain of only α subunits (Zoli et al., 2014). The most abundant brain nicotinic acetylcholine receptor subtypes are heteromeric α4β2 and homomeric α7 nicotinic acetylcholine receptor subtypes (Wu et al., 2016). In the brain areas such as cortex, thalamus, hippocampus, basal ganglia and cerebellum, the distribution of α4β2 nicotinic acetylcholine receptor subtype has been reported (Hurst et al., 2013). Epibatidine is a potent but nonselective nicotinic acetylcholine receptor agonist, and its biological effects appear to be mediated largely by α4β2 nicotinic
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acetylcholine receptors (Nirogi et al., 2013). In this context, prior microinjection of mecamylamine into the dorsal raphe nucleus (DRN) and the locus coeruleus (LC), brain important pain processing centers, inhibited the same sites microinjected epibatidineinduced antinociceptive effects in the paw formalin test of pain (Cucchiaro et al., 2005, 2006). Curzon et al. (1998) reported that epibatidine acts largely via descending inhibitory pathway arising from nucleus raphe magnus (NRM). It is possible that some of the antinociceptive effects of VLOC microinjected epibatidine may be related to the diffusion of the drug solution to the adjacent structures such as ventral orbital cortex (VOC). In this context, it has been reported that microinjection of epibatidine into the ventral orbital cortex (VOC), a subdivision of OC, attenuated formalin-induced orofacial pain, and prior microinjection of mecamylamine into the same site inhibited this effect (Yousofizadeh et al., 2015). It seems that at the VLOC, the nicotinic acetylcholine receptor involves in processing of capsaicin-induced orofacial pain and related hyperalgesia. In the present study we did not show any significant changes in general behavior after intraVLOC microinjection of oxotremorine, atropine, epibatidine and mecamylamine. There are no reports showing the effects of microinjection of these chemicals into the VLOC on general behavior. The decrease in nocifensive responses induced by most, but not all analgesics may be related to locomotor activity reduction resulting from sedative-like activity (hypoactivity) production, rather than a strict analgesic effect (Cortright et al., 2008). For example, in formalin-induced orofacial pain, a higher dose of memantine inhibited both phases of pain but also produced other motor effects (increased exploratory and decreased freezing behavior, hind-paw weakness and gait ataxia) which were not observed at the lower doses (Eisenberg et al., 1993). Therefore, a special care is needed to
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assure that the analgesic effect is due to a reduction in pain perception and not caused by motor impairment blocking the withdrawal response. Ophthalmic, mandible and maxillary branches of trigeminal nerve relay nociceptive information from orofacial structures into the various parts of TSNC (Sessle, 2011). In addition, thalamic Sm nucleus receives major projections from the trigeminal TSNC and projects primarily to the VLOC (Yoshida et al., 1991, 1992). The VLOC contains neurons that project to the periaqueductal gray (PAG) (Zhang et al., 1997). Considerable neurons of PAG send projection fibers to the TSNC (Li et al., 1993). The TSNC-Sm-VLOC-PAGTNSC may constitute a trigeminal pain processing pathway (Tang et al., 2009). A variety of neurotransmitters and their corresponding receptors are involved in this pathway processing functions (Tang et al., 2009; Zhu et al., 2013; Erfanparast et al., 2015). Because some of the most prevalent and debilitating pain conditions arise from the structures innervated by the trigeminal system (head, face and associated structures), understanding of the neural pathways that mediate trigeminal pain should enhance treatment for the clinical conditions (Romeo-Reyes and Uyanik, 2014). 5. Conclusion In conclusion, the results of the present study showed that at the VLOC level of the brain, activation of muscarinic and nicotinic acetylcholine receptors by oxotrmorine and epibatidine, respectively, suppressed the orofacial pain and subsequent secondary hyperalgesia induced by capsaicin. Prior microinjections of atropine and mecamylamine before oxotremorine and epibatidine, respectively, inhibited oxotremorine-, and epibatidine-induced antinociception. Therefore, muscarinic and nicotinic acetylcholine
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Fig. 1. Chemicals microinjection and pain recording time line. At 60 and 30 min before sc injection of capsaicin, adaptation of animals to experimental conditions was performed. At 45 min before and 15, 30, 45 and 60 min after sc injection of capsaicin, mechanical hyperalgesia were recorded. Immediately after sc injection of capsaicin, nocifensive and general behaviors were recorded for 10 min. Alone and prior intra-VPL microinjection of antagonists and post-antagonist administration of agonists were performed 6 and 3 min before sc injection of capsaicin, respectively.
Fig. 2. Schematic illustration of the rat brain showing the approximate location of the VLOC microinjection site in this study adapted from Paxinos and Watson, (1986) (left). Transverse section of the rat brain shows the location of the permanent cannula tip (black arrow) in the VLOC of rats included in the present study (right). Fr: Frontal cortex, AI: agranular insular cortex, LO: lateral orbital cortex, VLO: ventrolateral orbital cortex, Pir: piriform cortex, AOP: anterior olfactory nucleus, posterior part, IL: infralimbic cortex; Cg:cingulate cortex.
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Fig. 3. Effects of intra-VPL microinjection of atropine and oxotremorine alone and atropine plus oxotremorine on injected area rubbing (A) and face grooming (B) induced by capsaicin. Capsaicin was sc injected into the left vibrissa pad of rats. All values are expressed as mean ± S.E.M. (n = 6). * P < 0.05 significantly different compared with normal saline treated groups. † P < 0.05 significantly different compared with oxotremorine (100 ng/site) treated group.
Fig. 4. Effects of intra-VPL microinjection of mecamylamine and epibatidine alone and mecamylamine plus epibatidine on injected area rubbing (A) and face grooming (B) induced by capsaicin. Capsaicin was sc injected into the left vibrissa pad of rats. All values are expressed as mean ± S.E.M. (n = 6). * P < 0.05 significantly different compared with normal saline treated groups. † P < 0.05 significantly different compared with epibatidine (100 ng/site) treated group.
Fig. 5. Effects of intra-VLOC microinjection of atropine and oxotremorine alone and atropine plus oxotremorine on head withdrawal frequency induced by capsaicin. Capsaicin was sc injected into the left vibrissa pad of rats. All values are expressed as mean ± S.E.M. (n = 6). * P < 0.05 significantly different compared with normal saline treated group and 45 min. † P < 0.05 significantly different compared with oxotremorine (100 ng/site) treated group.
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Fig. 6. Effects of intra-VLOC microinjection of mecamylamine and epibatidine alone and mecamylamine plus epibatidine on head withdrawal frequency induced by capsaicin. Capsaicin was sc injected into the left vibrissa pad of rats. All values are expressed as mean ± S.E.M. (n = 6). * P < 0.05 significantly different compared with normal saline treated group and -45 min. † P < 0.05 significantly different compared with epibatidine (100 ng/site) treated group.
Fig. 7. Effects of intra-VLOC microinjection of mAChR and nAChR antagonists and agonists on general behavior during capsaicin-induced orofacial pain. Capsaicin was sc injected into the left vibrissa pad of rats. All values are expressed as mean ± S.E.M. (n = 6).
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