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Home , Cholinesterase inhibitor, Echothiophate, Physostigmine

Short Communication

Role of central muscarinic receptors in the formalin-induced pain in rats Ali Mojtahedin, Esmaeal Tamaddonfard, Ali Zanbouri

ABSTRACT

OObjectives:bjectives: In the present study, central effects of and have investigated in the formalin-induced pain in rats. Division of Physiology, Department of Basic Sciences, Faculty of MMaterialsaterials aandnd MMethods:ethods: In conscious rats implanted with an intracerebroventricular (i.c.v.) Veterinary Medicine, Urmia cannula, the effects of i.c.v. injection of physostigmine and atropine were investigated University, Urmia, Iran on the formalin test in the rat. Formalin test was induced by subcutaneous (s.c.) injection of formalin (50 µl, 1%) in ventral surface of left hind paw, and durations of licking and RReceived:eceived: 02.01.2009 biting of the injected paw were measured in 5-min blocks for 1 h. RRevised:evised: 20.02.2009 RResults:esults: Formalin produced a biphasic response (first phase: 0–5 and second phase: 15–40 AAccepted:ccepted: 08.06.2009 min) in durations of licking and biting of the injected paw. Physostigmine at doses of 2.5, 5 and 10 ug significantly (P < 0.05) attenuated both first and second phases of pain DDOI:OI: 110.4103/0253-7613.552050.4103/0253-7613.55205 response. Atropine (5 and 10 ug), used alone, produced no significant effect on pain, CCorrespondenceorrespondence to:to: but pretreatment with atropine (10 ug) significantly (P < 0.05) blocked antinociception Dr. Esmaeal Tamaddonfard, induced by physostigmine (5 ug). E-mail: e_tamaddonfard@yahoo. CConclusion:onclusion: These results indicate that i.c.v. physostigmine can affect both neurogenic com and inflammatory phases of formalin-induced pain through a mechanism in which the muscarinic cholinergic receptors are involved.

KKEYEY WWORDS:ORDS: Atropine, brain, formalin-induced pain, physostigmine, rats

Introduction reported.[9] It has been reported that intraperitoneal injection , through its muscarinic and nicotinic receptors, of physostigmine produced an antinociceptive effect in plays pivotal roles in a diverse array of physiological processes both tail flick and acetic acid-induced writhing tests in the such as learning, memory, anxiety, epilepsy, attention, cognition mouse.[10] Intrathecal injection of physostigmine inhibited and consciousness.[1-4] The activity of acetylcholine is controlled bradykinin-induced bioelectric activity in spinal ventrolateral through enzymatic degradation by acetylcholine esterase tract in rats.[11] The antinociceptive effect of intrathecally in the sites of cholinergic transmission.[5] There are some administered acetylcholine esterase inhibitors such as evidences, which suggest that inhibitors, such , physostigmine and have been as neostigmine and physostigmine, are involved in modulation reported in the formalin test in rats.[12-15] of pain. Intraarticular and intrathecal injections of neostigmine Physostigmine as a centrally acting produced analgesia in inflamed knee-joint model of pain in rats, and atropine as a centrally acting ,[16] and this effect was reversed by intrathecal pretreatment with were used in the study of pain mechanisms by peripheral and atropine.[6] Moreover, intrathecal administration of neostigmine intrathecal routes of administration.[8-15] There are no reports increased withdrawal threshold in tactile allodynia using showing the effect of intracerebroventricular (i.c.v.) injection of physostigmine and atropine on the formalin-induced pain in von Frey filaments in the L5–L6 spinal nerves tight ligature model of neuropathic pain in rats.[7] Subcutaneous injection rats and mice. Therefore, the aim of the present study was to of physostigmine produced antinociceptive effect in the spinal investigate the direct supraspinal effects of physostigmine, a nerve ligature model of neuropathic pain in rats.[8] In the tail cholinesterase inhibitor and atropine, a muscarinic cholinergic immersion test of nociception in the rat, an antinociceptive antagonist applied by i.c.v. route on formalin-induced effect of subcutaneous (s.c.)-injected physostigmine was pain in the rat.

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Materials and Methods after injection of formalin represented phase two (late phase). Cannula verification Animals During the surgery and before i.c.v. injections, a rise in Healthy adult male Wistar rats, weighing 220–250 g were the CSF through the cannula was observed. For additional used in this study. Rats were maintained in polyethylene cages confirmation of the placement of the cannula in the lateral with food and water available ad libitum, in a laboratory with ventricle of the brain, at the end of experiments, the rats were controlled ambient temperature (23 ± 0.5°C) and under a 12-h i.c.v. injected with 10 µl , and they were then light–dark cycle (lights on from 07:00 h). Six rats were used deeply anesthetized with a high dose of ether and decapitated. in each experiment. Experiments were carried out between The brains were removed and placed in a formaldehyde (10%) 12:00 and 16:00 h. The experimental protocol was approved solution. After 24 h, the brains were sliced into 1 mm slices by the Laboratory Animal Care and Use Center of the Faculty and the placement of the tip of the cannula and distribution of of Veterinary Medicine of Urmia University. the dye in the lateral ventricle were confirmed using a loop. Data from rats with an incorrect placement of the cannula were Drugs used in the present study included physostigmine excluded from the analysis. (Eserine) and atropine sulfate (Sigma – Aldrich, Steinheim, Statistical analysis Germany). All drugs were dissolved in normal saline. To evaluate significant differences among treated groups, Surgery one-way analysis of variance (ANOVA) and Duncan’s test were After a 15-day adaptation period, each rat was anesthetized applied. All values are expressed as the mean ± SEM. Statistical with a mixture of ketamine hydrochloride 10% (80 mg/kg) and significance was set at p < 0.05. xylazine hydrochloride 2% (10 mg/kg) injected intraplantar (i.p.), and then placed in a stereotaxic apparatus (Stoelting, Results Wood Lane, IL, USA). The bone was exposed after incision of The durations of licking and biting of the injected paw in the skin. A hole was drilled in the bone and a 22 gauge, 12 mm first and second phases after intraplantar injection of normal stainless-steel guide cannula was inserted in the right lateral saline were 3.0 ± 0.9 and 1.2 ± 0.5 s, respectively. The results ventricle of the brain. The tip of the cannula was aimed at the obtained from i.p. injection of normal saline are not presented following coordinates: 0.8 mm posterior to the bregma, 2 mm in the figures. Intraplantar injection of formalin 30 min after lateral to the midline and 4 mm below the top of the skull.[17] intracerebroventricular injection of normal saline produced The cannula was then fixed to the skull using three screw and a biphasic (54.7 ± 4.8 s in 0–5 min and 162.5 ± 14.0 s in dental acrylic. A 12.5 mm stylet was inserted in the cannula to 15–40 min) response in duration of licking and biting of the keep it patent prior to injection. Animals were allowed a 10-day injected paw [Figures 1 and 2]. recovery period before initiation of the experiments. Intracerebroventricular injection of physostigmine at doses injection of 2.5, 5 and 10 µg significantly (p < 0.05) decreased duration For i.c.v. injections of normal saline (control), physostigmine of licking and biting of the injected paw in both first and second and atropine, a 28 gauge, 12.5 mm injection needle was phases of formalin-induced pain. No significant difference was attached to a 30 cm polyethylene tube fitted to a 5 µl Hamilton observed among the different doses of physostigmine in first syringe. Then, the rat was restrained by hand, the stylet was phase of pain, but in the second phase of pain, suppressive withdrawn and the injection needle was inserted into the guide effect of physostigmine at doses of 5 and 10 µg was significantly cannula. The volume of the solutions to be injected into the (p < 0.05) higher than that of 2.5 µg. There was no significant lateral ventricle was 1 µl and the injection was slowly made over a period of 30 s. The injection needle was left in place for an additional 15 s to aid the diffusion of drug into cerebrospinal Figure 1: Effect of i.c.v. injection of physostigmine on the formalin- fluid (CSF). Atropine and physostigmine were adminisrered i.c.v. induced pain in rats. Each column represents mean ± SEM (n = 6). *P < 0.05 vs. control (one-way ANOVA followed by Duncan’s test). i.p.l.: 10 and 5 min before induction of formalin pain, respectively. intraplantar, i.c.v.: intracerebroventricular Formalin test Formalin test was used for induction of pain. Before rats were pain tested, they were placed in a Plexiglass observation 200 Control (for malin, 1%, ipl) chamber (30 × 30 × 25 cm) for 30 min on three successive days 180 Phy sos tigmine (2.5 ug, icv ) [18] to minimize stress-activated pain suppressive mechanisms. 160 Phy sos tigmine (5 ug, icv) Phy sos tigmine (10 ug, icv ) The formalin test was applied as follows. Fifty microliters of 1% 140 formalin was injected subcutaneously into the ventral surface of 120 [19] * right hind paw using a 29-gauge injection needle. The rat was 100 then placed in the observation chamber with a mirror mounted 80 ** at 45û beneath the floor to allow an unobstructed view of the 60 *** paw. The time spent licking and biting the injected paw was 40 taken as a measure of nociceptive response and was recorded 20 Licking and biting of the injected paw (s ) in 5-min intervals for 1 h. In the present study, data collected 0 between 0- and 5-min post-formalin injection represented phase First phase Second phas e (0-5 min) (15-40min) one (early phase) and data collected between 15 and 45 min

Indian J Pharmacol | Jun 2009 | Vol 41 | Issue 3 | 144-147 145 Mojtahedin, et al.: Physostigmine and formalin test

Figure 2: Effect of i.c.v. injection of atropine on physostigmine-induced cholinergic , , into the intralaminar nucleus changes in formalin-induced pain in rats. Each column represents mean parafascicularis of thalamus produced antinociception in rats. ± SEM (n = 6). *P < 0.05 vs. other groups (one-way ANOVA followed Atropine reversed antinociception induced by carbachol[25] by Duncan’s test). i.p.l.: intraplantar, i.c.v.: intracerebroventricular Cholinergic agonist carbachol and cholinesterase inhibitor physostigmine reduced the amplitude of evoked inhibitory and excitatory currents in the periaqueductal gray, and this effect Contr ol ( formalin, 1%, ipl) 200 was abolished by atropine.[26] Atropine (5 ug, icv) 180 In the present study, physostigmine through muscarinic A tropine (10 ug, icv ) 160 cholinergic receptors attenuates both phases of formalin- 140 Phys ostigmine (5 ug, icv ) induced pain. The advantage of formalin test model is that it 120 A tropine (10 ug, icv ) + Phys ostigmine (5 ug, icv ) may provide a tool for studying the effects of pain and analgesia 100 modulating agents for two types (phasic and tonic) of pain at 80 [27,28] * a time. Intrathecal injection of neostigmine reduced the 60 * time spent licking plus elevating the injected paw in the first 40 phase (phasic pain) of formalin (5%, 50 µl)-induced pain in 20 Lick ing and biting of the injected paw (s) rats. The second phase (tonic pain) was slightly reduced only 0 [12] First phase Second phas e by higher dose of neostigmine. Moreover, intrathecal injection (0-5 min) ( 15-40 min) of physostigmine, neostigmine and edrophonium produced a dose-dependent suppression of flinching in both phases of formalin-induced pain in rats. Intrathecal pretreatment with difference observed between effects of 5 and 10 µg of atropine prevented the suppressive effects of cholinesterase physostigmine in second phase of pain [Figure 1]. inhibitors on both phases of pain.[13-15] However, the absence of Intracerebroventricular injection of atropine at doses of 5 a descending cholinergic projection to the spinal cord in rats and 10 µg, used alone produced no significant effect in both was reported.[29] Therefore, it seems that atropine-dependent first and second phases of formalin-induced pain response, but antinociceptive effect of physostigmine observed in present i.c.v. pretreatment with atropine (10 µg) significantly prevented study may be related to action of physostigmine on brain suppressive effect of i.c.v. injected physostigmine (5 µ) in both structures involved in pain perception. first and second phases of pain induced by formalin [Figure 2]. In conclusion, the present data showed that central Discussion physostigmine suppressed both neurogenic (acute) and inflammatory (tonic) phases of formalin-induced pain. Atropine, In the present study, it was found that i.c.v. injection a muscarinic , prevented physostigmine- of physostigmine produced antinociception. In addition, induced antinociception. Hence, brain cholinergic system physostigmine-induced antinociception was blocked by through muscarinic receptors may be involved in modulation atropine pretreatment. These indicate that acetylcholine of pain. esterase inhibitor, physostigmine, through muscarinic cholinergic receptors, may have a role in central modulation References of pain. Physostigmine is a major found in the seeds 1. Hasselmo ME. The role of acetylcholine in learning and memory. Curr Opin of the fabaceous plant , and is a Neurobiol 2006; 16:710–5. powerful and reversible acetylcholine esterase inhibitor that 2. File SE, Kenny PJ, Cheeta S. 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