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Magnesium sulfate reduces formalin-induced orofacial pain in rats with normal magnesium serum levels
Accepted Manuscript Title: Magnesium sulfate reduces formalin-induced orofacial pain in rats with normal magnesium serum levels Authors: Drgana P. Srebro, Sonja M. Vuˇckovi´c, Ivan S. Doˇzi´c, Branko S. Doˇzi´c, Katarina R. Savi´c Vujovi´c, Aleksandar P. ˇ Prostran Milovanovi´c, Branislav V. Karadˇzi´c, Milica S. PII: DOI: Reference:
S1734-1140(17)30357-2 http://dx.doi.org/10.1016/j.pharep.2017.08.005 PHAREP 777
To appear in: Received date: Revised date: Accepted date:
21-5-2017 1-8-2017 17-8-2017
Please cite this article as: Drgana P.Srebro, Sonja M.Vuˇckovi´c, Ivan S.Doˇzi´c, Branko S.Doˇzi´c, Katarina R.Savi´c Vujovi´c, Aleksandar P.Milovanovi´c, ˇ Branislav V.Karadˇzi´c, Milica S.Prostran, Magnesium sulfate reduces formalininduced orofacial pain in rats with normal magnesium serum levels (2010), http://dx.doi.org/10.1016/j.pharep.2017.08.005 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 proof before it is published in its final 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.
Magnesium sulfate reduces formalin-induced orofacial pain in rats with normal magnesium serum levels Running title: Mg effectively reducing the orofacial pain
Drgana P. Srebroa,*, Sonja M. Vučkovića, Ivan S. Dožićb, Branko S. Dožićc, Katarina R. Savić Vujovića, Aleksandar P. Milovanovićd, Branislav V. Karadžiće, Milica Š. Prostrana aDepartment
of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of
Belgrade, Belgrade, Serbia b
c
Department of Biochemistry, School of Dental Medicine, University of Belgrade, Belgrade, Serbia
Department of Pathology, School of Dental Medicine, University of Belgrade, Belgrade, Serbia
d
Institute of Occupational Health "Dr Dragomir Karajovic", Faculty of Medicine, University of Belgrade,
Belgrade, Serbia e
Department of conservative dentistry and endodontics, School of Dental Medicine, University of Belgrade,
Belgrade, Serbia *Corresponding author: Dr Dragana P. Srebro, Department of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, Serbia Dr Subotića-starijeg 1, P.O. Box 38, 11129 Belgrade, Serbia Tel: +381 11 3643 390 E mail: [email protected] Acknowledgements This work was supported by the Ministry of Education, Science and Technological Development of Serbia (Grant 175023). The authors declare no potential conflicts of interest related to this study.
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Abstract Background: In humans, orofacial pain has a high prevalence and is often difficult to treat. Magnesium is an essential element in biological a system which controls the activity of many ion channels, neurotransmitters and enzymes. Magnesium produces an antinociceptive effect in neuropathic pain, while in inflammatory pain results are not consistent. We examined the effects of magnesium sulfate using the rat orofacial formalin test, a model of trigeminal pain. Methods: Male Wistar rats were injected with 1.5% formalin into the perinasal area, and the total time spent in pain-related behavior (face rubbing) was quantified. We also spectrophotometrically determined the concentration of magnesium and creatine kinase activity in blood serum. Results: Magnesium sulfate administered subcutaneously (0.005-45 mg/kg) produced significant antinociception in the second phase of the orofacial formalin test in rats at physiological serum concentration of magnesium. The effect was not dose-dependent. The maximum antinociceptive effect of magnesium sulfate was about 50% and was achieved at doses of 15 and 45 mg/kg. Magnesium did not affect increase the levels of serum creatine kinase activity. Conclusions: Preemptive systemic administration of magnesium sulfate as the only drug can be used to prevent inflammatory pain in the orofacial region. Its analgesic effect is not associated with magnesium deficiency.
Keywords: magnesium; preemptive analgesics; orofacial pain; creatine kinase; serum magnesium
Introduction Orofacial pain is one of the most prevalent pain coditions, which is not surprising given that the trigeminal nerve innervates the face, oral and nasal cavities, the anterior scalp, and meninges. About 26% of the population is affected by some form of orofacial pain [1], most of which are dental-related, with over 5% of instances that are 2
chronic [2]. Nondental forms of pain which specifically affect the trigeminal nerve include trigeminal neuralgia, migraine, temporomandibular disorders, burning mouth syndrome [3-5], eye pain [6], and salivary gland pain [7]. Following substantial injury to trigeminal nerve branches, chronic pain develops in about 3-5% of patients [8]. Orofacial pain is often a therapeutic challenge for physicians. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the main pharmaceuticals used to relieve acute orofacial pain, with triptans or ergotamine also available for acute migraine, and anticonvulsants for treating chronic orofacial pain. The use of novel drugs may offer improvements in therapy of orofacial pain since current therapy is often insufficiently effective and can be limited by adverse effects. The formalin model is believed to simulate the essential aspects of clinical pain [9]. The formalin orofacial test assesses orofacial nociceptive, inflammatory and neuropathic processes in the trigeminal area. Subcutaneous (sc) injection of diluted formalin in the vibrissa pad in rats produces a characteristic biphasic response of face rubbing and rapid head flinching. The first phase is the result of direct activation of peripheral nociceptors, while the second phase is associated with an inflammatory response and central sensitization [4, 9]. Up to now, there is no evidence that in this model of nociception muscle damage can contribute to pain. Magnesium is an essential element in biological systems. Magnesium is involved in various biochemical enzymatic and metabolic processes, it modulates ion transport by pumps, carriers and channels, thereby modulating signal transduction and cytosolic concentrations of calcium, potassium and sodium ions [10]. Magnesium has an important role in adenosine triphosphate synthesis and muscle metabolism. Creatine kinase (CK) is an enzyme which is directly activated by magnesium. Also, the activity of creatine kinase is useful in determining the functional status of magnesium in severe magnesium deficiency [11]. There is considerable interest to explore dietary supplements such as magnesium in relation to pain perception and pain control. In different neuropathic models of pain magnesium produces an antinociceptive effect [12, 13]. Studies of the effects of magnesium in inflammatory pain have yielded controversial findings that depend on the type of magnesium salt, the applied dose and route of administration, as well as on the inflammatory pain model [14-19].
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The first aim of the present study was to investigate whether magnesium is effective for treating orofacial pain using an suitable animal model. To that end we used the orofacial formalin test because this experimental model is considered to (i) be more closely related to clinical pain than tests producing phasic pain; (ii) mimic features of inflammatory pain in humans; and (iii) since no animal study has yet examined the activity of magnesium in this model of pain. Our second aim was to determine whether the effect of magnesium depends on the magnesium status in rats. In addition, how creatine kinase is an indicator of muscle damage and how there is a direct relationship between magnesium and creatine kinase, we wanted to determine the activity of creatine kinase and the effect of magnesium on creatine kinase activity in the formalin model of orofacial pain. Materials and methods Animals and experimental conditions Experiments were performed using 54 male Wistar rats weighting 200-250 g. The animals obtained from the Military Medical Academy Breeding Farm. The animals were housed in groups of three in Plexiglas cages (42.5×27×19 cm) under standard laboratory conditions of temperature (22±1°C), relative humidity (60%), and a 12 h light/dark cycle, with lights on at 08:00 h. Food and water were freely available, except during the experimental procedures. The animals were fed with standard rat pellets. Before each experiment, the animals were habituated to handling for at least two consecutive days. The animals were acclimatized to the laboratory environment for at least 2 h before use in the test. Prior to performing the formalin test, the rats were habituated to the observation chambers for 30 min. The rats were individually removed from their cages for each test and were returned to the cages immediately after the test. Biochemical analysis was performed using a separate group of rats. The animals in all groups had the same origin and were fed as the animals used for nociception assessment. The rats were subjected to a similar protocol as for pain assessment. The animals were restrained in plexiglass holders for several minutes during taking of blood. All experimental groups were consisting of 6 rats. The experiments were conducted by the same experimenter on consecutive days, between 08:00 and 14:00 h, in order to avoid diurnal variations in the behavioral tests. Each animal was used only once (except for the biochemical analysis as blood was taken four times from one animal), it received only one dose of the tested drug and was killed at the end of the 4
experiments by an intraperitoneal (ip) injection of sodium thiopental (200 mg/kg). The experiments were approved by the Local Ethical Committee of the Medical University and the Ethical Council of the Ministry of Agriculture, Forestry and Water Management, which are in complied with the European Communities Council Directive of November 24th, 1986 (86/609/EEC). Drugs and their administration Magnesium sulfate (Magnesio Solfato; S.A.L.F. Spa-Cenate Sotto Bergamo, Italy) was dissolved in isotonic saline and sc administered only once with a dose that ranged from 0.005-45 mg/kg (0.001-9 mg/kg of pure magnesium). Control animals received sc injections of saline. Either saline or magnesium sulfate was administered at a final volume of 2 mL/kg 5 min before the formalin injection. The formalin solution was prepared from commercially available aqueous stock solution of 37% formaldehyde (Gram, Serbia) that was diluted in isotonic saline to a final concentration of 1.5% (0.55% formaldehyde). The formalin was administered sc in a final volume of 100 μL using a 1 mL syringe with a 29-gauge needle. Orofacial formalin test and antinociception assessment The animal was sc injected with 100 μL of 1.5% formalin into the right vibrissa pad (the upper lip, right next to the nose). After formalin injection, the rats were immediately and individually returned to the transparent observation chambers for a 45-min observation period. The intensity of nociception in each rat was registered as the total time spent in pain-related behavior (face rubbing) after the injection of formalin or saline. The recording time was divided into 15 blocks of 3 min and a pain score was determined for each block by measuring the number of seconds that the animals spent in rubbing the injected area with the ipsilateral forepaw or hind paw [20]. Using this data, the cumulative nociceptive time was calculated during the first (0-9 min) and second phases (9-45 min) after formalin injection. Analgesic activity (AA%) for each rat was calculated according to the formula [19]: AA% = (control rubbing time formalin − post drug rubbing time) / (control rubbing time formalin) × 100. Determination of magnesium concentration in blood serum Immediately before drug administration and 25 min, 24 and 48 h after the injection of formalin, blood samples (for all biochemical analyses 0.5-1 ml per rat on consecutive time points) were collected from the tail vein. During 5
blood collecting, the rats were kept in plexiglass holders with minimal animal restraint and blood collecting was as quick as possible. Blood samples were collected in plastic syringes and immediately transferred to heparinized metal-free Vacutainer tubes with a gel serum separator (Plymouth, UK). The serum was isolated by centrifugation at 3,000xg for 10 min. The total magnesium concentration in blood serum was determined spectrophotometrically (absorbance was read at 520 nm) (RaytoRT-1904C, USA) according to the calmogite blue method (Linear, Barcelona, Spain). Magnesium concentrations were expressed as mmol/L. The degree of change of magnesium concentration in each rat during the observed times was quantified as the difference in concentration [d (mmol/L)] measured after and before (baseline concentration) the injection of magnesium sulfate or saline. Determination of creatine kinase activity in blood serum Blood samples (see above) were collected immediately before drug administration and 25 min, 24 and 48 h after injection of the formalin solution. During the procedure, the rats were kept in plexiglass holders with minimal restraint, and blood was collected as quickly as possible. Blood was collected in plastic syringes and immediately transferred to heparinized metal-free Vacutainer tubes with a serum separator gel (Plymouth, UK). Serum was prepared by centrifugation at 3,000xg for 10 min. The total creatine kinase activity in blood serum was determined spectrophotometrically (Rayto RT-1904C, USA) according to the modified standard method, as recommended by the European Committee for Clinical Laboratory Standards (ECCLS) and the International Federation of Clinical Chemistry (IFCC) (Human, Wiesbaden, Germany). Creatine kinase activity were expressed as U/L. The degree of change in creatine kinase activity in each rat during the observed times was quantified as the difference in activity [d (U/L)] measured after and before (baseline concentration) the injection of magnesium sulfate or saline. Statistical analysis The results of the formalin pain test are presented as mean total times spent on nociceptive behavior ± standard errors of the mean (SEM) obtained in six rats. For biochemical analysis, the results are presented as the mean differences of the concentrations or activity d (mmol/L or U/L) ± SEM, obtained in six rats. Statistical comparisons were made by a two-way analysis of variance (ANOVA) with repeated measures, followed by Tukey’s HSD post hoc test. A p value lower than 0.05 was considered to represent a statistically significant difference. 6
Results Formalin-induced nociceptive behavioral response The injection of 100 μL 1.5% formalin into the perinasal area (vibrissa pad) produced characteristic biphasic nociceptive behavior consisting of a first short-lasting response, followed by a second, prolonged response. The two phases were separated by a period of relative inactivity. The time course of the nociceptive responses is presented in Fig. 1. The first phase (measured during the first 9 min) started immediately after the formalin injection, with the mean duration of rubbing activity being 55.2±9.8 s. The second phase (measured between 9-45 min after the formalin injection) was characterized by intense rubbing activity; its total duration was 579±23.7 s. Compared to the formalin injection, the injection of an equivalent volume of saline did not produce any significant behavioral effect (not shown). Antinociceptive effects of magnesium sulfate Systemic administration of magnesium sulfate (0.005-45 mg/kg) produced a statistically significant reduction of the nociceptive response (F=7.6; p=0.000; for intervals), and significantly changed its time course (F=2.8; p=0.000; for intervals). The dose-response curves of magnesium sulfate are displayed in Fig. 1. There was no dose-response relationship. Although in the first time interval (0-3 min) magnesium sulfate at doses of 5 and 15 mg/kg reduced pain behavior by about 50-60%, the effect was not statistically significant (p=0.179 and p=0.110, respectively). The amplitude of the rubbing response was significantly reduced during the second phase by 31.6±4.9% (p=0.005), 18.5±2.1% (p=0.01), 33.1±4.8% (p=0.003), 50.1±3.8% (p=0.000), and 50.1±3.4% (p=0.000) after administration of 0.005, 0.5, 5, 15, and 45 mg/kg, respectively. The highest antinociceptive effect of magnesium sulfate was detected between 18 and 27 min after pain induction, at the time when the greatest severity of pain is observed. Cumulatively, the maximum antinociceptive effect of magnesium sulfate was 66.7±14.5% during the first and 50.1±3.8% during the second phase of the test, and was achieved at a dose of 15 mg/kg. Serum concentrations of magnesium The basal concentrations of magnesium in rat serum were 1.04±0.07 (0.8-1.2), 1.19±0.03 (1.1-1.4), and 1.16±0.04 (1.1-1.3) mmol/L in saline, 5 mg/kg and 45 mg/kg magnesium administered groups, respectively. There were no 7
significant differences in the basal levels between groups. As presented in Fig. 2, all three treatments significantly changed the concentration of magnesium during the tested time (F=3.2; p=0.021), however, the concentrations remained within the normal range. Only the higher dose of magnesium (45 mg/kg) caused a statistically significant (p=0.05) increase in magnesium concentration at the 25-min time point. However, 24 h after administration of both doses of magnesium sulfate and saline, the serum magnesium concentration decreased and returned to the basal level at 48 h. There were no significant differences in magnesium concentrations between groups at 24 and 48 h. Serum activity of creatine kinase The mean basal creatine kinase specific activities were 727±55 (452-830), 845±110 (426-1300), and 884±52 (6961122) U/L in saline, 5 mg/kg and 45 mg/kg magnesium groups, respectively. In tested rats, we did not observe significant differences in basal creatine kinase activity. In all three groups, creatine kinase activity changed significantly (F=5.9; p=0.013) during the study period. First, all groups showed an increase in concentration creatine kinase activity, with a maximum of about 3000 U/L measured at the 25-min time point. About 24 h later, creatine kinase activity decreased and returned to the basal level at the 48 h time point. Compared to the saline group, the highest dose of administered magnesium (45 mg/kg) caused a statistically significant (p=0.042) decrease in creatine kinase activity at 48 h. We did not detect a significant difference in creatine kinase activity between groups at 25 min and 24 h post treatment. Discussion The major finding of this study is that systemically preemptive administered magnesium sulfate reduced inflammatory pain in the orofacial area in rats with a normal magnesium serum concentration. Its analgesic effect was not dose-dependent and the maximal effect was achieved with dose of 15 mg/kg. Our results are in agreement with the antinociceptive activity of magnesium described in different animal models of pain. Hyperalgesia evoked by neuropathy [12, 13] and inflammation [18, 19] was attenuated by magnesium. However, the effects of magnesium in the paw formalin test are not consistent. It has been reported that magnesium reduced or had no effect on formalin-induced paw pain, depending on the type of pain measured, the phase of the formalin test, the applied magnesium salts, their dosage and route of administration [14-17]. The present study for 8
the first time describes the analgesic effects of magnesium sulfate in the formalin-induced orofacial test in rats. Herein we show that a single sc administration of magnesium sulfate (0.005-45 mg/kg) produces a significant dose-independent reduction of nociceptive behavior induced by injection of formalin into the orofacial area in rats. The doses used range from 0.005 mg/kg (0.001 mg/kg of pure magnesium) which is about 5000 times smaller than the daily dose for supplementation in humans (RDA) [10], to the dose of 45 mg/kg (9 mg/kg of pure magnesium) which is about two-fold higher than the daily dosage for supplementation in humans. Since magnesium sulfate has poor oral bioavailability (about 4%), it is important to note that we gave it by parenteral route. In present the study magnesium reduces pain in the first phase, but the effect is not statistically significant. These results confirm that magnesium at lower doses does not significantly affect acute nociceptive pain caused by a directly acting chemical irritant. However higher doses of magnesium can reduce acute pain, but it is followed by side effects (i.e. motor weakness and paralysis [14]). Up to the present, there is only one investigation examining the analgesic effect of magnesium in orofacial pain [21]. The authors showed that supplementation with magnesium chloride reduced mechanical hyperalgesia during carragennan-induced temporomandibular arthritis. However, in this study magnesium was used in the form of chloride, and was repeatedly administered and at a much higher dose (180 mg/kg per day, po). For pain induction we applied 1.5% formalin in order to increase the sensitivity of the test; we detected both hypo- and hyperalgesic effects and minimized the suffering of the experimental animals [22]. Also, we avoided peripheral fiber desensitization induced by very high local concentrations of formalin [23]. In the formalin test, the early phase is caused by direct chemical stimulation of nociceptors, while the later phase is dependent on the combination of peripheral inflammation and functional changes in central pain processing [24, 25]. Because magnesium sulfate produced statistically significant antinociception in the second phase of the orofacial formalin test, it can be assumed that it can reduce pain in the trigeminal region which involves inflammation and/or central sensitization but does not affect acute nociceptive pain. Additionally, the results of this behavioral test indicate that magnesium possesses the potential to modulate central pain processes in inflammation [18, 21, 26]. Since magnesium deficiency induces a greatly increased response of the NMDA receptor and hypernociceptive state [21, 27], in our study we examined the concentrations 9
of magnesium in the serum. Although the concentrations of magnesium in the serum and plasma (which contain less than 1% of the total body magnesium) do not adequately reflect the total body magnesium status, this remains the standard procedure for assessing magnesium status. Our results indicate that rats have a normal basal concentration of magnesium [28] and that when magnesium is administered immediately before inflammation onset at a dose of 5 mg/kg, the exogenous magnesium did not produce a significant effect on the blood magnesium concentration during inflammation. However, a dose of 45 mg/kg, leads to an increase in magnesium concentration to the upper reference value by about 30 min after inflammation onset, and to its subsequent normalization about 48 h later. However, the highest used dose of magnesium (45 mg/kg) does not exceed the toxic plasma magnesium concentration of 3 mM [29]. Additionally, we previously showed that this dose of magnesium did not affect motor performance [19], which confirms its antinociceptive effect in the orofacial formalin test. Although in the orofacial formalin test magnesium reduces pain by about the 30th min (when the increase in magnesium concentration is highest), the results of the present study show that the analgesic effect of magnesium is dose-independent. Also, this indicates that magnesium has no strictly receptor related mechanism of action. The mechanisms by which magnesium induces antinociception during inflammation have been widely discussed in our previous studies [18, 19, 30]. Magnesium possesses a number of pharmacologic actions, including effects on serotoninergic, adrenergic, cholinergic, and dopaminergic receptors [10, 31]. It modulates sodium and potassium ion channel activities and consequently has a considerable influence on membrane potentials [32]. Also, as an endogenous blocker of calcium channels, magnesium is an antagonist of N-methyl-D-aspartate (NMDA) receptors [26, 33]. Recently it was confirmed that during temporomandibular inflammation in the spinal cord, the expression of all NMDA receptor subunit mRNAs and phospho-NR1 immunostaining are increased [21]. The analgesic effect of magnesium is achieved by modulating nitric oxide (NO) synthesis. We recently reported a surprising finding that while the NO activation pathway was involved in the analgesic effect of magnesium in somatic inflammatory pain [18], NO was not involved in the analgesic effect of magnesium in visceral pain [19, 29]. Aside from modulating ion channels and receptor signaling, magnesium affects a number of cellular processes and is a cofactor for enzymes linked with energy metabolism. 10
Our study is also the first to show that in the formalin-induced orofacial pain test in rats there is a significant increase in creatine kinase activity at the time (at the 30-min time point) of the most pronounced behavioral response to pain, and probably tissue damage. As creatine kinase activity was similarly increased in all treatment groups, we concluded that magnesium did not increase the levels of creatine kinase activity. A likely explanation is that the increase in creatine kinase activity was the result of increased motor activity in animals and/or muscle damage due to formalin inflammation. Also, a transient and slight decrease in magnesium concentration could be the result of increased magnesium consumption due to elevated creatine kinase activity in inflammation. Orofacial pain reduces the quality of life [34], compromises occupational performance and is responsible for an increased loss of work days and use of health care, negatively impacting the economy [35, 36]. Therefore, the presented findings could have clinical relevance since the formalin orofacial test examines spontaneous pain and is the only animal model of persistent cutaneous nociception in the trigeminal area. In conclusion, our results indicate that the systemic and preemptive administration of magnesium sulfate as the only drug can prevent inflammatory pain in the orofacial region. The analgesic effect is not associated with magnesium deficiency. Magnesium does not affect the activity of creatine kinase in the formalin model of orofacial pain. Magnesium sulfate displays highly effective, safe and potent analgesic action in inflammatory pain in the trigeminal area. Conflicts of Interest The authors declare that they have no conflicts of interest. References 1. Setty S, David J. Classification and Epidemiology of Orofacial Pain. In: Vadivelu N, Vadivelu A and Kaye DA, editors. Orofacial pain. Swtizerland: Springer, 2014:15-24. 2. Zakrzewska JM. Facial pain. In: Stannard E, Kalso C and Ballantyne J, editors. Evidence-based chronic pain management. Oxford: Blackwell Publishing, 2010:134-50. 3. Kitt CA, Gruber K, Davis M, Woolf CJ, Levine JD. Trigeminal neuralgia: opportunities for research and treatment. Pain 2000;85(1-2):3-7. 11
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26. Dubner R, Ren K. Brainstem mechanisms of persistent pain following injury. J Orofac Pain 2004;18(4):299-305. 27. Begon S, Pickering G, Eschalier A, Mazur A, Rayssiguier Y, Dubray C. Role of spinal NMDA receptors, protein kinase C and nitric oxide synthase in the hyperalgesia induced by magnesium deficiency in rats. Br J Pharmacol 2001;134(6):1227-36. 28. Wlaź P, Serefko A, Szopa A, Poleszak E. The effect of an acute and 7-day administration of magnesium chloride on magnesium concentration in the serum, erythrocytes, and brain of rats. Pharmacol Rep. 2016;68(2):289-91. 29. Felsby S, Nielsen J, Arendt-Nielsen L, Jensen TS. NMDA receptor blockade in chronic neuropathic pain: a comparison of ketamine and magnesium chloride. Pain 1996;64(2):283-91. 30. Srebro D, Vučković S, Prostran M. Inhibition of neuronal and inducible nitric synthase does not affect the analgesic effects of NMDA antagonists in visceral inflammatory pain. Acta Neurobiol. Exp. (Wars) 2016; 76(2):110-6. 31. Cardoso CC, Lobato KR, Binfaré RW, Ferreira PK, Rosa AO, Santos AR, Rodrigues AL. Evidence for the involvement of the monoaminergic system in the antidepressant-like effect of magnesium. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:235-42. 32. Vastani N, Seifert B, Spahn DR, Maurer K. Sensitivities of rat primary sensory afferent nerves to magnesium: implications for differential nerve blocks. Eur J Anaesthesiol 2013;30(1):21–8. 33. Fawcett WJ, Haxby EJ, Male DA. Magnesium: Physiology and pharmacology. Br J Anaesth 1999;83:30220. 34. Shueb SS, Nixdorf DR, John MT, Alonso BF, Durham J. What is the impact of acute and chronic orofacial pain on quality of life? J Dent 2015;43(10):1203-10. 35. Bastos JL, Gigante DP, Peres KG. Toothache prevalence and associated factors: a population based study in southern Brazil. Oral Dis 2008;14(4):320-6.
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36. Lacerda JT, Ribeiro JD, Ribeiro DM, Traebert J. [Prevalence of orofacial pain and its impact on the oral health-related quality of life of textile industries workers of Laguna, SC, Brazil]. Cien Saude Colet 2011;16(10):4275-82. Figure captions Fig. 1. The antinociceptive effect of magnesium sulfate (MS) in rats injected with formalin into the orofacial region. Time intervals of nociceptive behavior in 3-minute bins are showed in the graph A, and the total time spent in nociceptive behavior in phase I (0–9 minutes) and phase II (9–45 minutes after formalin injection) is showed in the graph B. MS or saline was administered subcutaneously (sc) 5 min before formalin (F, 1.5%, 0.1 ml, sc). Each point represents the mean time in face rubbing ± SEM of 6 rats per group. The 0 time point represents the time of administration of formalin. Statistical significance was determined by comparing with the control (0.9% NaCl) (*p< 0.05, **p< 0.01). Statistical significance was found between MS 15 and 45 (## p<0.01). Fig. 2. Effect of single treatment with magnesium sulfate (MS) on magnesium concentration in the blood serum of rats with orofacial pain. MS or saline was administered subcutaneously (sc) 5 min before formalin (F, 1.5%, 0.1 ml, sc). Each point represents the difference mean in concentration d (mmol/l) measured in the blood serum after (0.42, 24 and 48 h) and before (0 h) injection of formalin ± SEM of 6 rats per group. Statistical significance was determined by comparing with the control (0.9% NaCl) (*p< 0.05). Fig. 3. Effect of treatment with magnesium sulfate (MS) on creatin kinase activity in the blood serum of rats with orofacial pain. MS or saline was administered subcutaneously (sc) 5 min before formalin (F, 1.5%, 0.1 ml, sc). Each point represents the difference mean in activity d (U/L) measured in the blood serum after (0.42, 24 and 48h) and before (0 h) injection of formalin ± SEM of 6 rats per group. Statistical significance was determined by comparing with the control (0.9% NaCl) (*p< 0.05).
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S1734-1140(17)30357-2 http://dx.doi.org/10.1016/j.pharep.2017.08.005 PHAREP 777
To appear in: Received date: Revised date: Accepted date:
21-5-2017 1-8-2017 17-8-2017
Please cite this article as: Drgana P.Srebro, Sonja M.Vuˇckovi´c, Ivan S.Doˇzi´c, Branko S.Doˇzi´c, Katarina R.Savi´c Vujovi´c, Aleksandar P.Milovanovi´c, ˇ Branislav V.Karadˇzi´c, Milica S.Prostran, Magnesium sulfate reduces formalininduced orofacial pain in rats with normal magnesium serum levels (2010), http://dx.doi.org/10.1016/j.pharep.2017.08.005 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 proof before it is published in its final 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.
Magnesium sulfate reduces formalin-induced orofacial pain in rats with normal magnesium serum levels Running title: Mg effectively reducing the orofacial pain
Drgana P. Srebroa,*, Sonja M. Vučkovića, Ivan S. Dožićb, Branko S. Dožićc, Katarina R. Savić Vujovića, Aleksandar P. Milovanovićd, Branislav V. Karadžiće, Milica Š. Prostrana aDepartment
of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of
Belgrade, Belgrade, Serbia b
c
Department of Biochemistry, School of Dental Medicine, University of Belgrade, Belgrade, Serbia
Department of Pathology, School of Dental Medicine, University of Belgrade, Belgrade, Serbia
d
Institute of Occupational Health "Dr Dragomir Karajovic", Faculty of Medicine, University of Belgrade,
Belgrade, Serbia e
Department of conservative dentistry and endodontics, School of Dental Medicine, University of Belgrade,
Belgrade, Serbia *Corresponding author: Dr Dragana P. Srebro, Department of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, Serbia Dr Subotića-starijeg 1, P.O. Box 38, 11129 Belgrade, Serbia Tel: +381 11 3643 390 E mail: [email protected] Acknowledgements This work was supported by the Ministry of Education, Science and Technological Development of Serbia (Grant 175023). The authors declare no potential conflicts of interest related to this study.
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Abstract Background: In humans, orofacial pain has a high prevalence and is often difficult to treat. Magnesium is an essential element in biological a system which controls the activity of many ion channels, neurotransmitters and enzymes. Magnesium produces an antinociceptive effect in neuropathic pain, while in inflammatory pain results are not consistent. We examined the effects of magnesium sulfate using the rat orofacial formalin test, a model of trigeminal pain. Methods: Male Wistar rats were injected with 1.5% formalin into the perinasal area, and the total time spent in pain-related behavior (face rubbing) was quantified. We also spectrophotometrically determined the concentration of magnesium and creatine kinase activity in blood serum. Results: Magnesium sulfate administered subcutaneously (0.005-45 mg/kg) produced significant antinociception in the second phase of the orofacial formalin test in rats at physiological serum concentration of magnesium. The effect was not dose-dependent. The maximum antinociceptive effect of magnesium sulfate was about 50% and was achieved at doses of 15 and 45 mg/kg. Magnesium did not affect increase the levels of serum creatine kinase activity. Conclusions: Preemptive systemic administration of magnesium sulfate as the only drug can be used to prevent inflammatory pain in the orofacial region. Its analgesic effect is not associated with magnesium deficiency.
Keywords: magnesium; preemptive analgesics; orofacial pain; creatine kinase; serum magnesium
Introduction Orofacial pain is one of the most prevalent pain coditions, which is not surprising given that the trigeminal nerve innervates the face, oral and nasal cavities, the anterior scalp, and meninges. About 26% of the population is affected by some form of orofacial pain [1], most of which are dental-related, with over 5% of instances that are 2
chronic [2]. Nondental forms of pain which specifically affect the trigeminal nerve include trigeminal neuralgia, migraine, temporomandibular disorders, burning mouth syndrome [3-5], eye pain [6], and salivary gland pain [7]. Following substantial injury to trigeminal nerve branches, chronic pain develops in about 3-5% of patients [8]. Orofacial pain is often a therapeutic challenge for physicians. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the main pharmaceuticals used to relieve acute orofacial pain, with triptans or ergotamine also available for acute migraine, and anticonvulsants for treating chronic orofacial pain. The use of novel drugs may offer improvements in therapy of orofacial pain since current therapy is often insufficiently effective and can be limited by adverse effects. The formalin model is believed to simulate the essential aspects of clinical pain [9]. The formalin orofacial test assesses orofacial nociceptive, inflammatory and neuropathic processes in the trigeminal area. Subcutaneous (sc) injection of diluted formalin in the vibrissa pad in rats produces a characteristic biphasic response of face rubbing and rapid head flinching. The first phase is the result of direct activation of peripheral nociceptors, while the second phase is associated with an inflammatory response and central sensitization [4, 9]. Up to now, there is no evidence that in this model of nociception muscle damage can contribute to pain. Magnesium is an essential element in biological systems. Magnesium is involved in various biochemical enzymatic and metabolic processes, it modulates ion transport by pumps, carriers and channels, thereby modulating signal transduction and cytosolic concentrations of calcium, potassium and sodium ions [10]. Magnesium has an important role in adenosine triphosphate synthesis and muscle metabolism. Creatine kinase (CK) is an enzyme which is directly activated by magnesium. Also, the activity of creatine kinase is useful in determining the functional status of magnesium in severe magnesium deficiency [11]. There is considerable interest to explore dietary supplements such as magnesium in relation to pain perception and pain control. In different neuropathic models of pain magnesium produces an antinociceptive effect [12, 13]. Studies of the effects of magnesium in inflammatory pain have yielded controversial findings that depend on the type of magnesium salt, the applied dose and route of administration, as well as on the inflammatory pain model [14-19].
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The first aim of the present study was to investigate whether magnesium is effective for treating orofacial pain using an suitable animal model. To that end we used the orofacial formalin test because this experimental model is considered to (i) be more closely related to clinical pain than tests producing phasic pain; (ii) mimic features of inflammatory pain in humans; and (iii) since no animal study has yet examined the activity of magnesium in this model of pain. Our second aim was to determine whether the effect of magnesium depends on the magnesium status in rats. In addition, how creatine kinase is an indicator of muscle damage and how there is a direct relationship between magnesium and creatine kinase, we wanted to determine the activity of creatine kinase and the effect of magnesium on creatine kinase activity in the formalin model of orofacial pain. Materials and methods Animals and experimental conditions Experiments were performed using 54 male Wistar rats weighting 200-250 g. The animals obtained from the Military Medical Academy Breeding Farm. The animals were housed in groups of three in Plexiglas cages (42.5×27×19 cm) under standard laboratory conditions of temperature (22±1°C), relative humidity (60%), and a 12 h light/dark cycle, with lights on at 08:00 h. Food and water were freely available, except during the experimental procedures. The animals were fed with standard rat pellets. Before each experiment, the animals were habituated to handling for at least two consecutive days. The animals were acclimatized to the laboratory environment for at least 2 h before use in the test. Prior to performing the formalin test, the rats were habituated to the observation chambers for 30 min. The rats were individually removed from their cages for each test and were returned to the cages immediately after the test. Biochemical analysis was performed using a separate group of rats. The animals in all groups had the same origin and were fed as the animals used for nociception assessment. The rats were subjected to a similar protocol as for pain assessment. The animals were restrained in plexiglass holders for several minutes during taking of blood. All experimental groups were consisting of 6 rats. The experiments were conducted by the same experimenter on consecutive days, between 08:00 and 14:00 h, in order to avoid diurnal variations in the behavioral tests. Each animal was used only once (except for the biochemical analysis as blood was taken four times from one animal), it received only one dose of the tested drug and was killed at the end of the 4
experiments by an intraperitoneal (ip) injection of sodium thiopental (200 mg/kg). The experiments were approved by the Local Ethical Committee of the Medical University and the Ethical Council of the Ministry of Agriculture, Forestry and Water Management, which are in complied with the European Communities Council Directive of November 24th, 1986 (86/609/EEC). Drugs and their administration Magnesium sulfate (Magnesio Solfato; S.A.L.F. Spa-Cenate Sotto Bergamo, Italy) was dissolved in isotonic saline and sc administered only once with a dose that ranged from 0.005-45 mg/kg (0.001-9 mg/kg of pure magnesium). Control animals received sc injections of saline. Either saline or magnesium sulfate was administered at a final volume of 2 mL/kg 5 min before the formalin injection. The formalin solution was prepared from commercially available aqueous stock solution of 37% formaldehyde (Gram, Serbia) that was diluted in isotonic saline to a final concentration of 1.5% (0.55% formaldehyde). The formalin was administered sc in a final volume of 100 μL using a 1 mL syringe with a 29-gauge needle. Orofacial formalin test and antinociception assessment The animal was sc injected with 100 μL of 1.5% formalin into the right vibrissa pad (the upper lip, right next to the nose). After formalin injection, the rats were immediately and individually returned to the transparent observation chambers for a 45-min observation period. The intensity of nociception in each rat was registered as the total time spent in pain-related behavior (face rubbing) after the injection of formalin or saline. The recording time was divided into 15 blocks of 3 min and a pain score was determined for each block by measuring the number of seconds that the animals spent in rubbing the injected area with the ipsilateral forepaw or hind paw [20]. Using this data, the cumulative nociceptive time was calculated during the first (0-9 min) and second phases (9-45 min) after formalin injection. Analgesic activity (AA%) for each rat was calculated according to the formula [19]: AA% = (control rubbing time formalin − post drug rubbing time) / (control rubbing time formalin) × 100. Determination of magnesium concentration in blood serum Immediately before drug administration and 25 min, 24 and 48 h after the injection of formalin, blood samples (for all biochemical analyses 0.5-1 ml per rat on consecutive time points) were collected from the tail vein. During 5
blood collecting, the rats were kept in plexiglass holders with minimal animal restraint and blood collecting was as quick as possible. Blood samples were collected in plastic syringes and immediately transferred to heparinized metal-free Vacutainer tubes with a gel serum separator (Plymouth, UK). The serum was isolated by centrifugation at 3,000xg for 10 min. The total magnesium concentration in blood serum was determined spectrophotometrically (absorbance was read at 520 nm) (RaytoRT-1904C, USA) according to the calmogite blue method (Linear, Barcelona, Spain). Magnesium concentrations were expressed as mmol/L. The degree of change of magnesium concentration in each rat during the observed times was quantified as the difference in concentration [d (mmol/L)] measured after and before (baseline concentration) the injection of magnesium sulfate or saline. Determination of creatine kinase activity in blood serum Blood samples (see above) were collected immediately before drug administration and 25 min, 24 and 48 h after injection of the formalin solution. During the procedure, the rats were kept in plexiglass holders with minimal restraint, and blood was collected as quickly as possible. Blood was collected in plastic syringes and immediately transferred to heparinized metal-free Vacutainer tubes with a serum separator gel (Plymouth, UK). Serum was prepared by centrifugation at 3,000xg for 10 min. The total creatine kinase activity in blood serum was determined spectrophotometrically (Rayto RT-1904C, USA) according to the modified standard method, as recommended by the European Committee for Clinical Laboratory Standards (ECCLS) and the International Federation of Clinical Chemistry (IFCC) (Human, Wiesbaden, Germany). Creatine kinase activity were expressed as U/L. The degree of change in creatine kinase activity in each rat during the observed times was quantified as the difference in activity [d (U/L)] measured after and before (baseline concentration) the injection of magnesium sulfate or saline. Statistical analysis The results of the formalin pain test are presented as mean total times spent on nociceptive behavior ± standard errors of the mean (SEM) obtained in six rats. For biochemical analysis, the results are presented as the mean differences of the concentrations or activity d (mmol/L or U/L) ± SEM, obtained in six rats. Statistical comparisons were made by a two-way analysis of variance (ANOVA) with repeated measures, followed by Tukey’s HSD post hoc test. A p value lower than 0.05 was considered to represent a statistically significant difference. 6
Results Formalin-induced nociceptive behavioral response The injection of 100 μL 1.5% formalin into the perinasal area (vibrissa pad) produced characteristic biphasic nociceptive behavior consisting of a first short-lasting response, followed by a second, prolonged response. The two phases were separated by a period of relative inactivity. The time course of the nociceptive responses is presented in Fig. 1. The first phase (measured during the first 9 min) started immediately after the formalin injection, with the mean duration of rubbing activity being 55.2±9.8 s. The second phase (measured between 9-45 min after the formalin injection) was characterized by intense rubbing activity; its total duration was 579±23.7 s. Compared to the formalin injection, the injection of an equivalent volume of saline did not produce any significant behavioral effect (not shown). Antinociceptive effects of magnesium sulfate Systemic administration of magnesium sulfate (0.005-45 mg/kg) produced a statistically significant reduction of the nociceptive response (F=7.6; p=0.000; for intervals), and significantly changed its time course (F=2.8; p=0.000; for intervals). The dose-response curves of magnesium sulfate are displayed in Fig. 1. There was no dose-response relationship. Although in the first time interval (0-3 min) magnesium sulfate at doses of 5 and 15 mg/kg reduced pain behavior by about 50-60%, the effect was not statistically significant (p=0.179 and p=0.110, respectively). The amplitude of the rubbing response was significantly reduced during the second phase by 31.6±4.9% (p=0.005), 18.5±2.1% (p=0.01), 33.1±4.8% (p=0.003), 50.1±3.8% (p=0.000), and 50.1±3.4% (p=0.000) after administration of 0.005, 0.5, 5, 15, and 45 mg/kg, respectively. The highest antinociceptive effect of magnesium sulfate was detected between 18 and 27 min after pain induction, at the time when the greatest severity of pain is observed. Cumulatively, the maximum antinociceptive effect of magnesium sulfate was 66.7±14.5% during the first and 50.1±3.8% during the second phase of the test, and was achieved at a dose of 15 mg/kg. Serum concentrations of magnesium The basal concentrations of magnesium in rat serum were 1.04±0.07 (0.8-1.2), 1.19±0.03 (1.1-1.4), and 1.16±0.04 (1.1-1.3) mmol/L in saline, 5 mg/kg and 45 mg/kg magnesium administered groups, respectively. There were no 7
significant differences in the basal levels between groups. As presented in Fig. 2, all three treatments significantly changed the concentration of magnesium during the tested time (F=3.2; p=0.021), however, the concentrations remained within the normal range. Only the higher dose of magnesium (45 mg/kg) caused a statistically significant (p=0.05) increase in magnesium concentration at the 25-min time point. However, 24 h after administration of both doses of magnesium sulfate and saline, the serum magnesium concentration decreased and returned to the basal level at 48 h. There were no significant differences in magnesium concentrations between groups at 24 and 48 h. Serum activity of creatine kinase The mean basal creatine kinase specific activities were 727±55 (452-830), 845±110 (426-1300), and 884±52 (6961122) U/L in saline, 5 mg/kg and 45 mg/kg magnesium groups, respectively. In tested rats, we did not observe significant differences in basal creatine kinase activity. In all three groups, creatine kinase activity changed significantly (F=5.9; p=0.013) during the study period. First, all groups showed an increase in concentration creatine kinase activity, with a maximum of about 3000 U/L measured at the 25-min time point. About 24 h later, creatine kinase activity decreased and returned to the basal level at the 48 h time point. Compared to the saline group, the highest dose of administered magnesium (45 mg/kg) caused a statistically significant (p=0.042) decrease in creatine kinase activity at 48 h. We did not detect a significant difference in creatine kinase activity between groups at 25 min and 24 h post treatment. Discussion The major finding of this study is that systemically preemptive administered magnesium sulfate reduced inflammatory pain in the orofacial area in rats with a normal magnesium serum concentration. Its analgesic effect was not dose-dependent and the maximal effect was achieved with dose of 15 mg/kg. Our results are in agreement with the antinociceptive activity of magnesium described in different animal models of pain. Hyperalgesia evoked by neuropathy [12, 13] and inflammation [18, 19] was attenuated by magnesium. However, the effects of magnesium in the paw formalin test are not consistent. It has been reported that magnesium reduced or had no effect on formalin-induced paw pain, depending on the type of pain measured, the phase of the formalin test, the applied magnesium salts, their dosage and route of administration [14-17]. The present study for 8
the first time describes the analgesic effects of magnesium sulfate in the formalin-induced orofacial test in rats. Herein we show that a single sc administration of magnesium sulfate (0.005-45 mg/kg) produces a significant dose-independent reduction of nociceptive behavior induced by injection of formalin into the orofacial area in rats. The doses used range from 0.005 mg/kg (0.001 mg/kg of pure magnesium) which is about 5000 times smaller than the daily dose for supplementation in humans (RDA) [10], to the dose of 45 mg/kg (9 mg/kg of pure magnesium) which is about two-fold higher than the daily dosage for supplementation in humans. Since magnesium sulfate has poor oral bioavailability (about 4%), it is important to note that we gave it by parenteral route. In present the study magnesium reduces pain in the first phase, but the effect is not statistically significant. These results confirm that magnesium at lower doses does not significantly affect acute nociceptive pain caused by a directly acting chemical irritant. However higher doses of magnesium can reduce acute pain, but it is followed by side effects (i.e. motor weakness and paralysis [14]). Up to the present, there is only one investigation examining the analgesic effect of magnesium in orofacial pain [21]. The authors showed that supplementation with magnesium chloride reduced mechanical hyperalgesia during carragennan-induced temporomandibular arthritis. However, in this study magnesium was used in the form of chloride, and was repeatedly administered and at a much higher dose (180 mg/kg per day, po). For pain induction we applied 1.5% formalin in order to increase the sensitivity of the test; we detected both hypo- and hyperalgesic effects and minimized the suffering of the experimental animals [22]. Also, we avoided peripheral fiber desensitization induced by very high local concentrations of formalin [23]. In the formalin test, the early phase is caused by direct chemical stimulation of nociceptors, while the later phase is dependent on the combination of peripheral inflammation and functional changes in central pain processing [24, 25]. Because magnesium sulfate produced statistically significant antinociception in the second phase of the orofacial formalin test, it can be assumed that it can reduce pain in the trigeminal region which involves inflammation and/or central sensitization but does not affect acute nociceptive pain. Additionally, the results of this behavioral test indicate that magnesium possesses the potential to modulate central pain processes in inflammation [18, 21, 26]. Since magnesium deficiency induces a greatly increased response of the NMDA receptor and hypernociceptive state [21, 27], in our study we examined the concentrations 9
of magnesium in the serum. Although the concentrations of magnesium in the serum and plasma (which contain less than 1% of the total body magnesium) do not adequately reflect the total body magnesium status, this remains the standard procedure for assessing magnesium status. Our results indicate that rats have a normal basal concentration of magnesium [28] and that when magnesium is administered immediately before inflammation onset at a dose of 5 mg/kg, the exogenous magnesium did not produce a significant effect on the blood magnesium concentration during inflammation. However, a dose of 45 mg/kg, leads to an increase in magnesium concentration to the upper reference value by about 30 min after inflammation onset, and to its subsequent normalization about 48 h later. However, the highest used dose of magnesium (45 mg/kg) does not exceed the toxic plasma magnesium concentration of 3 mM [29]. Additionally, we previously showed that this dose of magnesium did not affect motor performance [19], which confirms its antinociceptive effect in the orofacial formalin test. Although in the orofacial formalin test magnesium reduces pain by about the 30th min (when the increase in magnesium concentration is highest), the results of the present study show that the analgesic effect of magnesium is dose-independent. Also, this indicates that magnesium has no strictly receptor related mechanism of action. The mechanisms by which magnesium induces antinociception during inflammation have been widely discussed in our previous studies [18, 19, 30]. Magnesium possesses a number of pharmacologic actions, including effects on serotoninergic, adrenergic, cholinergic, and dopaminergic receptors [10, 31]. It modulates sodium and potassium ion channel activities and consequently has a considerable influence on membrane potentials [32]. Also, as an endogenous blocker of calcium channels, magnesium is an antagonist of N-methyl-D-aspartate (NMDA) receptors [26, 33]. Recently it was confirmed that during temporomandibular inflammation in the spinal cord, the expression of all NMDA receptor subunit mRNAs and phospho-NR1 immunostaining are increased [21]. The analgesic effect of magnesium is achieved by modulating nitric oxide (NO) synthesis. We recently reported a surprising finding that while the NO activation pathway was involved in the analgesic effect of magnesium in somatic inflammatory pain [18], NO was not involved in the analgesic effect of magnesium in visceral pain [19, 29]. Aside from modulating ion channels and receptor signaling, magnesium affects a number of cellular processes and is a cofactor for enzymes linked with energy metabolism. 10
Our study is also the first to show that in the formalin-induced orofacial pain test in rats there is a significant increase in creatine kinase activity at the time (at the 30-min time point) of the most pronounced behavioral response to pain, and probably tissue damage. As creatine kinase activity was similarly increased in all treatment groups, we concluded that magnesium did not increase the levels of creatine kinase activity. A likely explanation is that the increase in creatine kinase activity was the result of increased motor activity in animals and/or muscle damage due to formalin inflammation. Also, a transient and slight decrease in magnesium concentration could be the result of increased magnesium consumption due to elevated creatine kinase activity in inflammation. Orofacial pain reduces the quality of life [34], compromises occupational performance and is responsible for an increased loss of work days and use of health care, negatively impacting the economy [35, 36]. Therefore, the presented findings could have clinical relevance since the formalin orofacial test examines spontaneous pain and is the only animal model of persistent cutaneous nociception in the trigeminal area. In conclusion, our results indicate that the systemic and preemptive administration of magnesium sulfate as the only drug can prevent inflammatory pain in the orofacial region. The analgesic effect is not associated with magnesium deficiency. Magnesium does not affect the activity of creatine kinase in the formalin model of orofacial pain. Magnesium sulfate displays highly effective, safe and potent analgesic action in inflammatory pain in the trigeminal area. Conflicts of Interest The authors declare that they have no conflicts of interest. References 1. Setty S, David J. Classification and Epidemiology of Orofacial Pain. In: Vadivelu N, Vadivelu A and Kaye DA, editors. Orofacial pain. Swtizerland: Springer, 2014:15-24. 2. Zakrzewska JM. Facial pain. In: Stannard E, Kalso C and Ballantyne J, editors. Evidence-based chronic pain management. Oxford: Blackwell Publishing, 2010:134-50. 3. Kitt CA, Gruber K, Davis M, Woolf CJ, Levine JD. Trigeminal neuralgia: opportunities for research and treatment. Pain 2000;85(1-2):3-7. 11
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26. Dubner R, Ren K. Brainstem mechanisms of persistent pain following injury. J Orofac Pain 2004;18(4):299-305. 27. Begon S, Pickering G, Eschalier A, Mazur A, Rayssiguier Y, Dubray C. Role of spinal NMDA receptors, protein kinase C and nitric oxide synthase in the hyperalgesia induced by magnesium deficiency in rats. Br J Pharmacol 2001;134(6):1227-36. 28. Wlaź P, Serefko A, Szopa A, Poleszak E. The effect of an acute and 7-day administration of magnesium chloride on magnesium concentration in the serum, erythrocytes, and brain of rats. Pharmacol Rep. 2016;68(2):289-91. 29. Felsby S, Nielsen J, Arendt-Nielsen L, Jensen TS. NMDA receptor blockade in chronic neuropathic pain: a comparison of ketamine and magnesium chloride. Pain 1996;64(2):283-91. 30. Srebro D, Vučković S, Prostran M. Inhibition of neuronal and inducible nitric synthase does not affect the analgesic effects of NMDA antagonists in visceral inflammatory pain. Acta Neurobiol. Exp. (Wars) 2016; 76(2):110-6. 31. Cardoso CC, Lobato KR, Binfaré RW, Ferreira PK, Rosa AO, Santos AR, Rodrigues AL. Evidence for the involvement of the monoaminergic system in the antidepressant-like effect of magnesium. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:235-42. 32. Vastani N, Seifert B, Spahn DR, Maurer K. Sensitivities of rat primary sensory afferent nerves to magnesium: implications for differential nerve blocks. Eur J Anaesthesiol 2013;30(1):21–8. 33. Fawcett WJ, Haxby EJ, Male DA. Magnesium: Physiology and pharmacology. Br J Anaesth 1999;83:30220. 34. Shueb SS, Nixdorf DR, John MT, Alonso BF, Durham J. What is the impact of acute and chronic orofacial pain on quality of life? J Dent 2015;43(10):1203-10. 35. Bastos JL, Gigante DP, Peres KG. Toothache prevalence and associated factors: a population based study in southern Brazil. Oral Dis 2008;14(4):320-6.
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36. Lacerda JT, Ribeiro JD, Ribeiro DM, Traebert J. [Prevalence of orofacial pain and its impact on the oral health-related quality of life of textile industries workers of Laguna, SC, Brazil]. Cien Saude Colet 2011;16(10):4275-82. Figure captions Fig. 1. The antinociceptive effect of magnesium sulfate (MS) in rats injected with formalin into the orofacial region. Time intervals of nociceptive behavior in 3-minute bins are showed in the graph A, and the total time spent in nociceptive behavior in phase I (0–9 minutes) and phase II (9–45 minutes after formalin injection) is showed in the graph B. MS or saline was administered subcutaneously (sc) 5 min before formalin (F, 1.5%, 0.1 ml, sc). Each point represents the mean time in face rubbing ± SEM of 6 rats per group. The 0 time point represents the time of administration of formalin. Statistical significance was determined by comparing with the control (0.9% NaCl) (*p< 0.05, **p< 0.01). Statistical significance was found between MS 15 and 45 (## p<0.01). Fig. 2. Effect of single treatment with magnesium sulfate (MS) on magnesium concentration in the blood serum of rats with orofacial pain. MS or saline was administered subcutaneously (sc) 5 min before formalin (F, 1.5%, 0.1 ml, sc). Each point represents the difference mean in concentration d (mmol/l) measured in the blood serum after (0.42, 24 and 48 h) and before (0 h) injection of formalin ± SEM of 6 rats per group. Statistical significance was determined by comparing with the control (0.9% NaCl) (*p< 0.05). Fig. 3. Effect of treatment with magnesium sulfate (MS) on creatin kinase activity in the blood serum of rats with orofacial pain. MS or saline was administered subcutaneously (sc) 5 min before formalin (F, 1.5%, 0.1 ml, sc). Each point represents the difference mean in activity d (U/L) measured in the blood serum after (0.42, 24 and 48h) and before (0 h) injection of formalin ± SEM of 6 rats per group. Statistical significance was determined by comparing with the control (0.9% NaCl) (*p< 0.05).
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