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Citronellol, a natural acyclic monoterpene, attenuates mechanical hyperalgesia response in mice: Evidence of the spinal cord lamina I inhibition
Chemico-Biological Interactions 239 (2015) 111–117
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Citronellol, a natural acyclic monoterpene, attenuates mechanical hyperalgesia response in mice: Evidence of the spinal cord lamina I inhibition Renan G. Brito a, Priscila L. dos Santos a, Jullyana S.S. Quintans a, Waldecy de Lucca Júnior b, Adriano A.S. Araújo c, Shanmugam Saravanan c, Irwin R.A. Menezes d, Henrique D.M. Coutinho d, Lucindo J. Quintans-Júnior a,⇑ a
Department of Physiology, Federal University of Sergipe, São Cristóvão, Sergipe, Brazil Department of Morphology, Federal University of Sergipe, São Cristóvão, Sergipe, Brazil c Department of Pharmacy, Federal University of Sergipe, São Cristóvão, Sergipe, Brazil d Department of Biological Chemistry, Regional University of Cariri, Crato, Ceará, Brazil b
a r t i c l e
i n f o
Article history: Received 5 May 2015 Received in revised form 11 June 2015 Accepted 30 June 2015 Available online 2 July 2015 Keywords: Citronellol Monoterpenes Hyperalgesia Spinal Cord Fos Pain
a b s t r a c t We evaluated the anti-hyperalgesic effect of citronellol (CT) and investigated the spinal cord lamina I involvement in this effect. Male mice were pre-treated with CT (25, 50 and 100 mg/kg, i.p.), indomethacin (10 mg/kg, i.p.), dipyrone (60 mg/kg, i.p.) or vehicle (saline + Tween 80 0.2%). Thirty minutes after the treatment, 20 lL of carrageenan (CG; 300 lg/paw), PGE2 (100 ng/paw), dopamine (DA; 30 lg/paw) or TNF-a (100 pg/paw) were injected into the hind paw subplantar region and the mechanical threshold was evaluated with an electronic anesthesiometer. The CT effect on edema formation was evaluated after the right paw subplantar injection of CG (40 lL; 1%) through the plethysmometer apparatus. To evaluate the CT action on the spinal cord, the animals were treated with CT (100 mg/kg; i.p.) or vehicle (Saline + Tween 80 0.2%; i.p.) and, after 30 min, 20 lL of CG (300 lg/paw; i.pl.) was injected. Ninety minutes after the treatment, the animals were perfused, the lumbar spinal cord collected, crioprotected, cut and submitted in an immunofluorescence protocol for Fos protein. CT administration produced a significantly reduction (p < 0.05) in the mechanical hyperalgesia induced by CG, TNF-a, PGE2 and DA when compared with control group. The treatment with CT also significantly (p < 0.05) decreased the paw edema. The immunofluorescence showed that the CT decrease significantly (p < 0.05) the spinal cord lamina I activation. Thus, our results provide that CT attenuates the hyperalgesia, at least in part, through the spinal cord lamina I inhibition. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Pain perception is a defense system that alarms the organism about danger and produces immediate response to stress factors.
Abbreviations: NSAIDs, Nonsteroidal Anti-inflammatory Drugs; CT, citronellol; CG, carrageenan; TNF-a, tumor necrosis factor-alpha; PGE2, prostaglandins-E2; DA, dopamine; BSA, bovine serum albumin; PBS, phosphate buffer (0.01 M) saline isotonic; COX, cyclooxygenase; TNFR1, tumor necrosis factor receptor 1; TNFR2, tumor necrosis factor receptor 2; IL, interleukin; KC, keratinocyte-derived chemokine; EP2, prostaglandin E2 receptor; PAG, periaqueductal gray area; RVM, rostral ventromedial medulla; CNS, central nervous system. ⇑ Corresponding author at: Department of Physiology, Federal University of Sergipe, Av. Marechal Rondom, s/n, São Cristóvão, Sergipe 49.100-000, Brazil. E-mail addresses: [email protected], [email protected] (L.J. Quintans-Júnior). http://dx.doi.org/10.1016/j.cbi.2015.06.039 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
In inflamed tissue, however, primary sensory neurons become hypersensitive, produce pain response to normally innocuous stimulation or enhanced pain response to painful stimulation [1]. This response is established with the participation of several mediators, such as neurotrophic factors, neuropeptides, prostanoids, kinins and some cytokines [2,3]. The carrageenan (CG), a high-value seaweed hydrocolloid, have been used in the inflammatory pain studies, once it induces mechanical hyperalgesia through a cascade of cytokines, like TNF-a, IL-6 and IL-1b, released by resident or migrating cells initiated by production of bradykinin, with consequent synthesis of prostaglandins and release of sympathetic amines [4,5]. The Nonsteroidal Anti-inflammatory Drugs (NSAIDs) is a name for a number of chemically distinct drugs, representing the most widely used drug class. The NSAIDs, such indomethacin and
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dipyrone, inhibitors of prostaglandins synthesis, are used for the pain inhibition at the site of inflammation. However, significant side effects and low efficacy of NSAIDs in quite a number of inflammatory states primed search of other targets for inflammatory pain relief [1,6,7]. For this reason, there are many studies focusing in the search of new therapeutic options for treating painful conditions. In this context is inserted the monoterpenes, a compound group formed from the coupling of two isoprene units (C10), constituting 90% of the essential oils. Some systematic reviews described the analgesic and anti-inflammatory activities of some monoterpenes, demonstrating the therapeutic potential of these compounds, including to development of pharmaceutical products for pain [8–11]. Citronellol (CT) is a monoterpene compound prevalent in essential oils of various aromatic plant species, such as Cymbopogon citrates and Cymbopogon winterianus [12,13], and its hypotensive, vasorelaxant, anticonvulsant, analgesic and anti-inflammatory profiles are described in the literature [14–17]. However, there are few data that have evaluated the effects of CT on inflammatory pain. Hence, the purpose of the present study was to evaluate the anti-hyperalgesic effect of CT in mice and investigate the involvement of the spinal cord lamina I in this possible effect.
2. Materials and methods 2.1. Chemicals Carrageenan (CG), tumor necrosis factor-alpha (TNF-a), prostaglandins-E2 (PGE2), dopamine (DA), Tween 80, ((S)-( )-Bcitronellol, CT, 97% purity), glycerol solution, glycine and bovine serum albumin (BSA) were purchased from Sigma (USA). Indomethacin and dipyrone were obtained from União Química (Brazil). Rabbit anti-Fos k-25 was obtained from Santa Cruz Biotechnology (USA) and the donkey anti-rabbit Alexa Fluor 488 was purchased from Life Technologies (USA).
2.2. Animals Adult (3-month-old) male albino Swiss mice (28–32 g) were randomly housed in appropriate cages at 21 ± 2 °C with a 12-h light: dark cycle (light from 06:00 to 18:00), with free access to food (PurinaÒ, Brazil) and tap water. All experiments were carried out between 09:00 am and 02:00 pm in a quiet room. Experimental protocols were approved by the Animal Care and Use Committee at the Federal University of Sergipe (CEPA/UFS #72/11). All tests were carried out by the same visual observer, double-blinded and all efforts were made to minimize the number of animals used as well as minimize any discomfort.
2.3. Hyperalgesia induced by CG, TNF-a, PGE2 and DA The hyperalgesia protocols was performed as previous described [18,5,19]. The animals were divided into five groups (n = 6, per group) and treated with vehicle (Saline + Tween 80 0.2%, i.p.), CT (25, 50 or 100 mg/kg, i.p.), indomethacin (10 mg/kg, i.p.) or dipyrone (60 mg/kg, i.p.). Thirty minutes after the treatment, 20 lL of CG (300 lg/paw), PGE2 (100 ng/paw), DA (30 lg/paw) or TNF-a (100 pg/paw) were injected subcutaneously into the subplantar region of the hind paw. The mechanical hyperalgesia was evaluated at 0.5, 1, 2 and 3 h after the hyperalgesic agents injections.
2.4. Mechanical hyperalgesia measurement Mechanical hyperalgesia was tested in mice as previous reported [18]. In a quiet room, the mice were placed in acrylic cages (12 10 17 cm) with wire grid floors for 15–30 min. before the test. This method consisted of evoking a hind paw flexion reflex with a hand-held force transducer (electronic anesthesiometer; InsightÒ, São Paulo, Brazil) adapted with a polypropylene tip. The investigator was trained to apply the tip perpendicularly to the central area of the hind paw with a gradual increase in pressure. The end point was characterized by the withdrawal of the paw followed by clear flinching movements. After this response, the pressure intensity was automatically recorded. The intensity of stimulus was obtained by averaging five measurements taken with minimal intervals of 3 min. The animals were tested before and after the treatment. 2.5. Paw edema measurement The CT effect on edema formation caused by the intraplantar injection of CG was analyzed according to the method previously reported [20]. The animals were divided in five groups (n = 6, per group) and treated with vehicle (Saline + Tween 80 0.2%, i.p.), CT (25, 50 or 100 mg/kg, i.p.), or indomethacin (10 mg/kg; i.p.). The right paw volume was measured by the water column dislocation of a plethysmometer (InsightÒ, Brazil) before (time zero) and 1, 2, 3, 4, 5 and 6 h after subplantar injection of 40 lL of CG (1%). The paw edema was expressed (in milliliter) as the difference between the volume of the paw after and before CG injection. The area under the curve (AUC [0–240 min]; in milliliter per minute) was also calculated using the trapezoidal rule. 2.6. Immunofluorescence To evaluate the action of the test drug on the spinal cord, the animals (n = 6, per group) were treated with CT (100 mg/kg; i.p.) or vehicle (Saline + Tween 80 0.2%; i.p.) and, after 30 min, 20 lL of CG (300 lg/paw) was injected subcutaneously into the subplantar region of the hind paw. One group did not receive any kind of treatment (sham group), which was used as a baseline control. Ninety minutes after the treatment, all animals were perfused and the lumbar spinal cord collected and crio-protected for immunofluorescence processing to Fos protein. The time for realization of immunofluorescence protocol was based on the studies of Barr [21] and Bai et al. [22]. Frozen serial transverse sections (20 lm) of all spinal cords were collected on gelatinized glass slides. The tissue sections were stored at 80 °C until use. The sections were washed with phosphate buffer (0.01 M) saline isotonic (PBS) 5 times for 5 min. and incubated with 0.01 M glycine in PBS for 10 min. Non-specific protein binding was blocked by incubation of the sections for 30 min. in a solution containing 2% BSA. Then, the sections were incubated overnight with rabbit anti-Fos as primary antibodies (1:2000). Afterwards, the sections were incubated for two hours with donkey anti-rabbit Alexa Fluor 594 as secondary antibodies (1:2000). The cover slip was mounted with glycerol solution. As an immunofluorescence control for non-specific labeling, sections were incubated without primary antibody. After each stage, slides were washed with PBS 5 times for 5 min. 2.7. Acquisition and analyses of images A blinded investigator took 20 pictures from Fos positive lumbar spinal cords areas for each animal using an Olympus IX2-ICB (Tokyo, Japan). The lumbar spinal cord regions were classified according to Paxinus and Franklin Atlas [23]. After that, the same
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blinded investigator choose the best 10 pictures of each animal and opened, together, all the pictures of all animal in the free software Image J (National Institute of Health). For counting process, the investigator applied a threshold ranging between 91 and 255. It was counted all cells with size between 0.0002 and 0.0015. Finally, it was calculated the average of 10 images selected for each animal. 2.8. Statistical analysis Values are expressed as mean ± SEM. The data obtained were evaluated through the one-way analysis of variance (ANOVA) followed by Tukey’s test. In all cases, differences were considered significant if p < 0.05. All statistical analyses were done using the software GraphPad Prism 5.0 (GraphPad Prism Software Inc., San Diego, CA, USA). 3. Results Administration of CT, in all doses, produced a significantly reduction (p < 0.05) in the mechanical hyperalgesia induced by CG and TNF-a when compared with control (Fig. 1). In the hyperalgesia induced by PGE2 and DA protocols, CT also significantly reduced (p < 0.05) the mechanical threshold. However, the intraperitoneal injection of CT at a dose of 50 mg/kg did not change the mechanical threshold in the times 0.5 and 1 h after the DA injection when compared with control (Fig. 2). As shown in Fig. 3, the treatment with CT significantly (p < 0.05) decreased the paw edema. The doses of 25 and 100 mg/kg were able to maintain the edema reduction during the 6 h evaluation
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period. The dose of 50 mg/kg only reduced the edema in the last three hours of the test. The inhibition percentages, based on the AUC values, were 41.9%, 35.8% (p < 0.01), and 50.6% (p < 0.001) for 25, 50, and 100 mg/kg, respectively. The indomethacin showed an inhibition of 55.3% (p < 0.001). In the spinal cord lamina I of the animals, the average number of neurons showing Fos protein was significantly reduced (p < 0.05) by an intraperitoneal injection of CT (100 mg/kg) when compared with control (Fig. 4). 4. Discussion The present study evaluated the effect of CT in several models of hyperalgesia and its effect in the edema formation. Our data demonstrate that systemic administration of CT significantly decreased the hyperalgesic response in acute models of inflammatory nociception, reducing the edema. Furthermore, we suggested the possible involvement of the spinal cord lamina I inhibition in this effect. The hyperalgesia induced by intraplantar injection of CG is widely used for evaluating new anti-inflammatory and antihyperalgesic compounds in rodents. The CG administration induces the development of the cardinal signs of inflammation, the result of the action of proinflammatory agents, complement and reactive oxygen and nitrogen species. This signalization cascade leads the release of prostanoids and sympathomimetic amines, stimulating peripheral Ad and C fiber sensory nerve terminals. This neurogenic inflammation contributes for the resultant peripheral and central hyperalgesia [5,24,25]. In this protocol, mice pretreated with CT, all doses, showed an increase in the mechanical threshold, similar
Fig. 1. Effect of the acute administration of vehicle, citronellol (CT; 25, 50, or 100 mg/kg; i.p.) or indomethacin (INDO; 10 mg/kg; i.p.) on mechanical hyperalgesia induced by CG (A) and TNF-a (B). Each point represents the mean ± SEM (n = 6, per group) of the paw withdrawal threshold (in grams) to tactile stimulation of the ipsilateral hind paw. * p < 0.05, **p < 0.01 and ***p < 0.001 vs. control group (ANOVA followed by Tukey test).
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Fig. 2. Effect of the acute administration of vehicle, citronellol (CT; 25, 50, or 100 mg/kg; i.p.) or dipyrone (DYP; 60 mg/kg; i.p.) on mechanical hyperalgesia induced by PGE2 (A) and DA (B). Each point represents the mean ± SEM (n = 6, per group) of the paw withdrawal threshold (in grams) to tactile stimulation of the ipsilateral hind paw. *p < 0.05, ** p < 0.01 and ***p < 0.001 vs. control group (ANOVA followed by Tukey test).
with mice who received indomethacin, a COX inhibitor. This effect is probably the result of the CT anti-inflammatory activity, which has been reported in the literature [17,26,27]. The edema formation is one of the fundamental actions of acute inflammation and it is an essential parameter to be considered when evaluating compounds with potential anti-inflammatory activity [28]. The intraplantar CG injection also induces the edema formation in the paw and it has been fully characterized in the past showing acute inflammation by vasodilatation, exudation of protein-rich fluid and cell migration, primarily neutrophils, into the side of injury [24,29,30]. In this study, we have attempted to demonstrate the effects of CT on the CG-induced mouse paw edema and the results showed that all doses of CT presented antiedematogenic activity. This effect is probably due of the ability of CT to inhibit the neutrophils migration and some pro-inflammatory cytokines, as described previously [17,27]. The tumor necrosis factor-alpha (TNF-a) is one of the main cytokines that control the inflammatory process. It is the first to be released after an inflammatory stimulus, such as the carrageenan injection, leading the increase of the COX and PGE2 expression, which is mediated by the TNFR1 and TNFR2 receptors [5,31]. TNF-a also induces the neutrophils migration to the injury site by indirect mechanisms, such as the induction of the chemotactic factors release through the resident macrophages and the leukotriene pathway stimulation [32,33]. The CT was able to increase the threshold sensitivity of mechanical hyperalgesia induced by TNF-a, as observed in the treatment with indomethacin. This effect is probably associated with the inhibition of TNF-a and PGE2 caused by CT [17,27].
Other cytokines, such as IL-1b, act in the release of prostanoids such as prostaglandins, while keratinocyte-derived chemokine (KC) acts in the release of sympathomimetic amines like dopamine, triggering the activation of nociceptors and transmission impulse by primary nociceptive neurons [5]. PGE2 and dopamine act on the EP2 and D1-type metabotropic receptors, respectively, activating the second messenger pathways, which is responsible for lowering the nociceptor threshold and increasing neuronal membrane excitability [34,35]. Because of this, we investigated the possible CT effect on the hyperalgesia induced by PGE2 and dopamine. It was observed that CT inhibited the mechanical hyperalgesia induced by these hyperalgesic mediators, like dipyrone. These findings are probably a result of the CT ability to inhibit the release of TNF and PGE [17,27], in addition with its capacity in suppress the COX-2 expression [26]. After the tissue injury with subsequent installation of the inflammatory process, stimulating peripheral Ad and C fiber sensory nerve terminals, the first synapse in the transmission of this information to the brain is in the superficial dorsal horn of the spinal cord, which is comprised of lamina I and II. So, the Lamina I plays key role in the modulation of pain transmission, which is the major source of ascending output from the superficial dorsal horn to brain regions that have a role in pain mechanisms, including the medullary reticular formation, parabrachial area, periaqueductal gray (PAG) and thalamus [36–38]. The lamina I also receive an inhibitory input of the descending pain pathways, specifically from PAG area in the mesencephalon and from the rostral ventromedial medulla (RVM), which comprises the nucleus raphe magnus, the paragigantocellular and gigantocellular nuclei, areas
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Fig. 3. Effect of the acute administration of vehicle, citronellol (CT; 25, 50, or 100 mg/kg; i.p.) or indomethacin (INDO; 10 mg/kg; i.p.) on edema induced by CG. Each point represents the mean ± SEM (n = 6, per group) of the paw volume (in milliliter, A) or the area under curve (AUC) from 0 to 6 h (B). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control group (ANOVA followed by Tukey test).
Fig. 4. Neurons Fos positive in the lumbar spinal cord lamina I (A). Vehicle (C) or CT (D; 100 mg/kg) were administered intraperitoneally and, after 30 min, CG was injected subcutaneously into the subplantar region of the hind paw. The sham group (B) did not receive any treatment. The animals were perfused 90 min after the treatment. Values represent mean ± SEM (n = 6, per group). **p < 0.01 vs. control group (unpaired t test). 20 lm.
involved in the pain inhibition [39]. In order to study the central pathways of pain, the c-Fos expression has been used to examine these neural circuitries, once Fos is expressed in the nuclei of cells whereas retrograde tracers accumulate in the cytoplasm, thus facilitating their use in combination [40,41].
On the basis of these findings, we evaluated the expression of Fos protein in spinal cord lamina I. It was observed that the treatment with CT was able to inhibit the neurons activation in the spinal cord lamina I. This inhibition can a be caused by the indirect action of CT through its action in the pro-inflammatory cytokines,
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like described previously, or through the activation of the opioid system in the PAG, once CT is able to activate the PAG and its antinociceptive effect was blocked by the administration of naloxone on the hot plate test, suggesting the involvement of the opioid central receptors in this response, like described by [17,42]. On the other hand, CT can be acting directly in the spinal cord lamina I, probably blocking the glutamatergic system, since CT produced an inhibition of nociceptive behavior induced by injection of glutamate into the right upper limb of mouse [42]. In addition, CT can be reducing the excitability of the neurons in the spinal cord lamina I, once De Sousa et al. demonstrated that CT partially blocked voltage-dependent Na+ channels [16]. Studies have suggested that the CNS depression and the non-specific muscle relaxation effects can reduce the response of motor coordination and might invalidate the behavioral tests [43–45]. However, previous studies with CT, using the same doses as the present study, did not show any interference on motor coordination of the animals in the rota-rod test and in the spontaneous locomotor activity [17,42]. So, the action of CT on mechanical hyperalgesia, observed in this study, is not entirely due to an inhibitory effect on the CNS or muscle relaxation. Thus, it can be concluded that CT reduced the mechanical hyperalgesia and the edema in mice, probably by the inhibition of peripheral mediators, such as TNF-a and PGE2, as well as the inhibition of spinal cord lamina I, probably by the activation of CNS regions, especially the PAG, encephalic area involved in pain. Conflict of interest The authors report no conflict of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
Acknowledgements We thank Mr. Osvaldo Andrade Santos for technical support. This work was supported by grants from the National Council of Technological and Scientific Development (CNPq/Brazil), the Research Supporting Foundation of the State of Sergipe (FAPITEC-SE/Brazil) and Coordinating Development of Senior Staff (CAPES/Brazil). References [1] Y.A. Andreev, A.A. Vassilevski, S.A. Kozlov, Molecules to selectively target receptors for treatment of pain and neurogenic inflammation, Recent Pat. Inflammation Allergy Drug Discovery 6 (2012) 35–45. [2] A. Coutaux, F. Adam, J.-C. Willer, D. Le Bars, Hyperalgesia and allodynia: peripheral mechanisms, Joint Bone Spine Rev. Rhum. 72 (2005) 359–371, http://dx.doi.org/10.1016/j.jbspin.2004.01.010. [3] O. Obreja, P.K. Rathee, K.S. Lips, C. Distler, M. Kress, IL-1 beta potentiates heatactivated currents in rat sensory neurons: involvement of IL-1RI, tyrosine kinase, and protein kinase C, FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 16 (2002) 1497–1503, http://dx.doi.org/10.1096/fj.02-0101com. [4] N. Rhein-Knudsen, M.T. Ale, A.S. Meyer, Seaweed hydrocolloid production: an update on enzyme assisted extraction and modification technologies, Mar. Drugs 13 (2015) 3340–3359, http://dx.doi.org/10.3390/md13063340. [5] T.M. Cunha, W.A. Verri Jr., J.S. Silva, S. Poole, F.Q. Cunha, S.H. Ferreira, A cascade of cytokines mediates mechanical inflammatory hypernociception in mice, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 1755–1760, http://dx.doi.org/10.1073/ pnas.0409225102. [6] J.A. Mitchell, T.D. Warner, Cyclo-oxygenase-2: pharmacology, physiology, biochemistry and relevance to NSAID therapy, Br. J. Pharmacol. 128 (1999) 1121–1132, http://dx.doi.org/10.1038/sj.bjp.0702897. [7] R.O. Day, G.G. Graham, Non-steroidal anti-inflammatory drugs (NSAIDs), BMJ 346 (2013) f3195.
[8] F. Bakkali, S. Averbeck, D. Averbeck, M. Idaomar, Biological effects of essential oils – a review, Food Chem. Toxicol. Int. J. Publ. Br. Ind. Biol. Res. Assoc. 46 (2008) 446–475, http://dx.doi.org/10.1016/j.fct.2007.09.106. [9] A.G. Guimarães, J.S.S. Quintans, L.J. Quintans Jr., Monoterpenes with analgesic activity – a systematic review, Phytother. Res. PTR 27 (2013) 1–15, http:// dx.doi.org/10.1002/ptr.4686. [10] A.G. Guimarães, M.R. Serafini, L.J. Quintans-Júnior, Terpenes and derivatives as a new perspective for pain treatment: a patent review, Expert Opin. Ther. Pat. 24 (2014) 243–265, http://dx.doi.org/10.1517/13543776.2014.870154. [11] R. De Cássia da Silveira e Sá, L.N. Andrade, D.P. de Sousa, A review on antiinflammatory activity of monoterpenes, Mol. Basel, Switz. 18 (2013) 1227– 1254, http://dx.doi.org/10.3390/molecules18011227. [12] B. Abegaz, P.G. Yohannes, R.K. Dieter, Constituents of the essential oil of Ethiopian Cymbopogon citratus stapf, J. Nat. Prod. 46 (1983) 424–426, http:// dx.doi.org/10.1021/np50027a022. [13] L.J. Quintans-Júnior, T.T. Souza, B.S. Leite, N.M.N. Lessa, L.R. Bonjardim, M.R.V. Santos, et al., Phythochemical screening and anticonvulsant activity of Cymbopogon winterianus Jowitt (Poaceae) leaf essential oil in rodents, Phytomed. Int. J. Phytother. Phytopharm. 15 (2008) 619–624, http:// dx.doi.org/10.1016/j.phymed.2007.09.018. [14] M.R.V. Santos, F.V. Moreira, B.P. Fraga, D.P. de Souza, L.R. Bonjardim, L.J. Quintans-Junior, Cardiovascular effects of monoterpenes: a review, Rev. Bras. Farmacogn. 21 (2011) 764–771, http://dx.doi.org/10.1590/S0102695X2011005000119. [15] J.F.A. Bastos, I.J.A. Moreira, T.P. Ribeiro, I.A. Medeiros, A.R. Antoniolli, D.P. De Sousa, et al., Hypotensive and vasorelaxant effects of citronellol, a monoterpene alcohol, in rats, Basic Clin. Pharmacol. Toxicol. 106 (2010) 331–337, http://dx.doi.org/10.1111/j.1742-7843.2009.00492.x. [16] D.P. de Sousa, J.C.R. Gonçalves, L. Quintans-Júnior, J.S. Cruz, D.A.M. Araújo, R.N. de Almeida, Study of anticonvulsant effect of citronellol, a monoterpene alcohol, in rodents, Neurosci. Lett. 401 (2006) 231–235, http://dx.doi.org/ 10.1016/j.neulet.2006.03.030. [17] R.G. Brito, A.G. Guimarães, J.S.S. Quintans, M.R.V. Santos, D.P. De Sousa, D. Badaue-Passos Jr., et al., Citronellol, a monoterpene alcohol, reduces nociceptive and inflammatory activities in rodents, J. Nat. Med. 66 (2012) 637–644, http://dx.doi.org/10.1007/s11418-012-0632-4. [18] T.M. Cunha, W.A. Verri Jr., G.G. Vivancos, I.F. Moreira, S. Reis, C.A. Parada, et al., An electronic pressure-meter nociception paw test for mice, Braz. J. Med. Biol. Res. Rev. (Bras. Pesqui. Médicas E Biológicas Soc. Bras. Biofísica Al.) 37 (2004) 401–407. [19] C.F. Villarreal, M.I. Funez, F. Figueiredo, F.Q. Cunha, C.A. Parada, S.H. Ferreira, Acute and persistent nociceptive paw sensitisation in mice: the involvement of distinct signalling pathways, Life Sci. 85 (2009) 822–829, http://dx.doi.org/ 10.1016/j.lfs.2009.10.018. [20] L. Levy, Carrageenan paw edema in the mouse, Life Sci. 8 (1969) 601–606. [21] G.A. Barr, Formalin-induced c-fos expression in the brain of infant rats, J. Pain Off. J. Am. Pain Soc. 12 (2011) 263–271, http://dx.doi.org/10.1016/ j.jpain.2010.09.005. [22] Z.-T. Bai, T. Liu, X.-Y. Pang, Z.-F. Chai, Y.-H. Ji, Suppression by intrathecal BmK IT2 on rat spontaneous pain behaviors and spinal c-Fos expression induced by formalin, Brain Res. Bull. 73 (2007) 248–253, http://dx.doi.org/10.1016/ j.brainresbull.2007.03.007. [23] G. Paxinos, K. Franklin, The Mouse Brain in Stereotaxic Coordinates, second ed., Elsevier, California, USA, 2001. [24] C.J. Morris, Carrageenan-induced paw edema in the rat and mouse, Methods Mol. Biol. Clifton, NJ 225 (2003) 115–121, http://dx.doi.org/10.1385/1-59259374-7:115. [25] S.H. Ferreira, B.B. Lorenzetti, S. Poole, Bradykinin initiates cytokine-mediated inflammatory hyperalgesia, Br. J. Pharmacol. 110 (1993) 1227–1231. [26] M. Katsukawa, R. Nakata, S. Koeji, K. Hori, S. Takahashi, H. Inoue, Citronellol and geraniol, components of rose oil, activate peroxisome proliferatoractivated receptor a and c and suppress cyclooxygenase-2 expression, Biosci. Biotechnol. Biochem. 75 (2011) 1010–1012. [27] Y.-W. Su, S.-H. Chao, M.-H. Lee, T.-Y. Ou, Y.-C. Tsai, Inhibitory effects of citronellol and geraniol on nitric oxide and prostaglandin E2 production in macrophages, Planta Med. 76 (2010) 1666–1671, http://dx.doi.org/10.1055/s0030-1249947. [28] A. Russo, C. Costagliola, L. Delcassi, F. Parmeggiani, M.R. Romano, R. Dell’Omo, et al., Topical nonsteroidal anti-inflammatory drugs for macular edema, Mediators Inflamm. 2013 (2013) 476525, http://dx.doi.org/10.1155/2013/ 476525. [29] S.Z. Hussein, K. Mohd Yusoff, S. Makpol, Y.A. Mohd Yusof, Gelam honey inhibits the production of proinflammatory, mediators NO, PGE2, TNF-a, and IL-6 in carrageenan-induced acute paw edema in rats, Evidence Based Complement Altern. Med. ECAM 2012 (2012), http://dx.doi.org/10.1155/2012/109636. [30] E.F.C. Reis, S.B.R. Castro, C.C.S. Alves, E.E. Oliveira, T.A. Corrêa, M.V. Almeida, et al., Lipophilic amino alcohols reduces carrageenan-induced paw edema and anti-OVA DTH in BALB/c mice, Int. Immunopharmacol. 17 (2013) 727–732, http://dx.doi.org/10.1016/j.intimp.2013.08.017. [31] C. Sommer, C. Schmidt, A. George, Hyperalgesia in experimental neuropathy is dependent on the TNF receptor 1, Exp. Neurol. 151 (1998) 138–142, http:// dx.doi.org/10.1006/exnr.1998.6797. [32] L.H. Faccioli, G.E.P. Souza, F.Q. Cunha, S. Poole, D.S.H. Ferreira, Recombinant interleukin-1 and tumor necrosis factor induce neutrophil migration ‘‘in vivo’’ by indirect mechanisms, Agents Actions 30 (1990) 344–349, http://dx.doi.org/ 10.1007/BF01966298.
R.G. Brito et al. / Chemico-Biological Interactions 239 (2015) 111–117 [33] C. Canetti, J.S. Silva, S.H. Ferreira, F.Q. Cunha, Tumour necrosis factor-alpha and leukotriene B(4) mediate the neutrophil migration in immune inflammation, Br. J. Pharmacol. 134 (2001) 1619–1628, http://dx.doi.org/10.1038/ sj.bjp.0704403. [34] S.H. Ferreira, M. Nakamura, I – Prostaglandin hyperalgesia, a cAMP/Ca2+ dependent process, Prostaglandins 18 (1979) 179–190. [35] D. Sachs, C. Villarreal, F. Cunha, C. Parada, S. Ferreira, The role of PKA and PKCe pathways in prostaglandin E2-mediated hypernociception, Br. J. Pharmacol. 156 (2009) 826–834, http://dx.doi.org/10.1111/j.1476-5381.2008.00093.x. [36] R. Suzuki, A. Dickenson, Spinal and supraspinal contributions to central sensitization in peripheral neuropathy, Neurosignals 14 (2005) 175–181, http://dx.doi.org/10.1159/000087656. [37] A.D. Craig, J.O. Dostrovsky, Differential projections of thermoreceptive and nociceptive lamina I trigeminothalamic and spinothalamic neurons in the cat, J. Neurophysiol. 86 (2001) 856–870. [38] A.J. Todd, Z. Puskar, R.C. Spike, C. Hughes, C. Watt, L. Forrest, Projection neurons in lamina I of rat spinal cord with the neurokinin 1 receptor are selectively innervated by substance p-containing afferents and respond to noxious stimulation, J. Neurosci. Off. J. Soc. Neurosci. 22 (2002) 4103–4113. doi:20026261.
117
[39] B. Calvino, R.M. Grilo, Central pain control, Joint Bone Spine 73 (2006) 10–16, http://dx.doi.org/10.1016/j.jbspin.2004.11.006. [40] J.A. Harris, Using c-fos as a neural marker of pain, Brain Res. Bull. 45 (1998) 1–8. [41] R.K. Snowball, F.M. Semenenko, B.M. Lumb, Visceral inputs to neurons in the anterior hypothalamus including those that project to the periaqueductal gray: a functional anatomical and electrophysiological study, Neuroscience 99 (2000) 351–361. [42] R.G. Brito, P.L. Santos, D.S. Prado, M.T. Santana, A.A.S. Araújo, L.R. Bonjardim, et al., Citronellol reduces orofacial nociceptive behaviour in mice – evidence of involvement of retrosplenial cortex and periaqueductal grey areas, Basic Clin. Pharmacol. Toxicol. 112 (2013) 215–221, http://dx.doi.org/10.1111/ bcpt.12018. [43] D. Le Bars, M. Gozariu, S.W. Cadden, Animal models of nociception, Pharmacol. Rev. 53 (2001) 597–652. [44] P. Raboisson, R. Dallel, The orofacial formalin test, Neurosci. Biobehav. Rev. 28 (2004) 219–226, http://dx.doi.org/10.1016/j.neubiorev.2003.12.003. [45] C.S. Passos, M.D. Arbo, S.M.K. Rates, G.L. von Poser, Terpenoids with activity in the Central Nervous System (CNS), Rev. Bras. Farmacogn. 19 (2009) 140–149, http://dx.doi.org/10.1590/S0102-695X2009000100024.
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Citronellol, a natural acyclic monoterpene, attenuates mechanical hyperalgesia response in mice: Evidence of the spinal cord lamina I inhibition Renan G. Brito a, Priscila L. dos Santos a, Jullyana S.S. Quintans a, Waldecy de Lucca Júnior b, Adriano A.S. Araújo c, Shanmugam Saravanan c, Irwin R.A. Menezes d, Henrique D.M. Coutinho d, Lucindo J. Quintans-Júnior a,⇑ a
Department of Physiology, Federal University of Sergipe, São Cristóvão, Sergipe, Brazil Department of Morphology, Federal University of Sergipe, São Cristóvão, Sergipe, Brazil c Department of Pharmacy, Federal University of Sergipe, São Cristóvão, Sergipe, Brazil d Department of Biological Chemistry, Regional University of Cariri, Crato, Ceará, Brazil b
a r t i c l e
i n f o
Article history: Received 5 May 2015 Received in revised form 11 June 2015 Accepted 30 June 2015 Available online 2 July 2015 Keywords: Citronellol Monoterpenes Hyperalgesia Spinal Cord Fos Pain
a b s t r a c t We evaluated the anti-hyperalgesic effect of citronellol (CT) and investigated the spinal cord lamina I involvement in this effect. Male mice were pre-treated with CT (25, 50 and 100 mg/kg, i.p.), indomethacin (10 mg/kg, i.p.), dipyrone (60 mg/kg, i.p.) or vehicle (saline + Tween 80 0.2%). Thirty minutes after the treatment, 20 lL of carrageenan (CG; 300 lg/paw), PGE2 (100 ng/paw), dopamine (DA; 30 lg/paw) or TNF-a (100 pg/paw) were injected into the hind paw subplantar region and the mechanical threshold was evaluated with an electronic anesthesiometer. The CT effect on edema formation was evaluated after the right paw subplantar injection of CG (40 lL; 1%) through the plethysmometer apparatus. To evaluate the CT action on the spinal cord, the animals were treated with CT (100 mg/kg; i.p.) or vehicle (Saline + Tween 80 0.2%; i.p.) and, after 30 min, 20 lL of CG (300 lg/paw; i.pl.) was injected. Ninety minutes after the treatment, the animals were perfused, the lumbar spinal cord collected, crioprotected, cut and submitted in an immunofluorescence protocol for Fos protein. CT administration produced a significantly reduction (p < 0.05) in the mechanical hyperalgesia induced by CG, TNF-a, PGE2 and DA when compared with control group. The treatment with CT also significantly (p < 0.05) decreased the paw edema. The immunofluorescence showed that the CT decrease significantly (p < 0.05) the spinal cord lamina I activation. Thus, our results provide that CT attenuates the hyperalgesia, at least in part, through the spinal cord lamina I inhibition. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Pain perception is a defense system that alarms the organism about danger and produces immediate response to stress factors.
Abbreviations: NSAIDs, Nonsteroidal Anti-inflammatory Drugs; CT, citronellol; CG, carrageenan; TNF-a, tumor necrosis factor-alpha; PGE2, prostaglandins-E2; DA, dopamine; BSA, bovine serum albumin; PBS, phosphate buffer (0.01 M) saline isotonic; COX, cyclooxygenase; TNFR1, tumor necrosis factor receptor 1; TNFR2, tumor necrosis factor receptor 2; IL, interleukin; KC, keratinocyte-derived chemokine; EP2, prostaglandin E2 receptor; PAG, periaqueductal gray area; RVM, rostral ventromedial medulla; CNS, central nervous system. ⇑ Corresponding author at: Department of Physiology, Federal University of Sergipe, Av. Marechal Rondom, s/n, São Cristóvão, Sergipe 49.100-000, Brazil. E-mail addresses: [email protected], [email protected] (L.J. Quintans-Júnior). http://dx.doi.org/10.1016/j.cbi.2015.06.039 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
In inflamed tissue, however, primary sensory neurons become hypersensitive, produce pain response to normally innocuous stimulation or enhanced pain response to painful stimulation [1]. This response is established with the participation of several mediators, such as neurotrophic factors, neuropeptides, prostanoids, kinins and some cytokines [2,3]. The carrageenan (CG), a high-value seaweed hydrocolloid, have been used in the inflammatory pain studies, once it induces mechanical hyperalgesia through a cascade of cytokines, like TNF-a, IL-6 and IL-1b, released by resident or migrating cells initiated by production of bradykinin, with consequent synthesis of prostaglandins and release of sympathetic amines [4,5]. The Nonsteroidal Anti-inflammatory Drugs (NSAIDs) is a name for a number of chemically distinct drugs, representing the most widely used drug class. The NSAIDs, such indomethacin and
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dipyrone, inhibitors of prostaglandins synthesis, are used for the pain inhibition at the site of inflammation. However, significant side effects and low efficacy of NSAIDs in quite a number of inflammatory states primed search of other targets for inflammatory pain relief [1,6,7]. For this reason, there are many studies focusing in the search of new therapeutic options for treating painful conditions. In this context is inserted the monoterpenes, a compound group formed from the coupling of two isoprene units (C10), constituting 90% of the essential oils. Some systematic reviews described the analgesic and anti-inflammatory activities of some monoterpenes, demonstrating the therapeutic potential of these compounds, including to development of pharmaceutical products for pain [8–11]. Citronellol (CT) is a monoterpene compound prevalent in essential oils of various aromatic plant species, such as Cymbopogon citrates and Cymbopogon winterianus [12,13], and its hypotensive, vasorelaxant, anticonvulsant, analgesic and anti-inflammatory profiles are described in the literature [14–17]. However, there are few data that have evaluated the effects of CT on inflammatory pain. Hence, the purpose of the present study was to evaluate the anti-hyperalgesic effect of CT in mice and investigate the involvement of the spinal cord lamina I in this possible effect.
2. Materials and methods 2.1. Chemicals Carrageenan (CG), tumor necrosis factor-alpha (TNF-a), prostaglandins-E2 (PGE2), dopamine (DA), Tween 80, ((S)-( )-Bcitronellol, CT, 97% purity), glycerol solution, glycine and bovine serum albumin (BSA) were purchased from Sigma (USA). Indomethacin and dipyrone were obtained from União Química (Brazil). Rabbit anti-Fos k-25 was obtained from Santa Cruz Biotechnology (USA) and the donkey anti-rabbit Alexa Fluor 488 was purchased from Life Technologies (USA).
2.2. Animals Adult (3-month-old) male albino Swiss mice (28–32 g) were randomly housed in appropriate cages at 21 ± 2 °C with a 12-h light: dark cycle (light from 06:00 to 18:00), with free access to food (PurinaÒ, Brazil) and tap water. All experiments were carried out between 09:00 am and 02:00 pm in a quiet room. Experimental protocols were approved by the Animal Care and Use Committee at the Federal University of Sergipe (CEPA/UFS #72/11). All tests were carried out by the same visual observer, double-blinded and all efforts were made to minimize the number of animals used as well as minimize any discomfort.
2.3. Hyperalgesia induced by CG, TNF-a, PGE2 and DA The hyperalgesia protocols was performed as previous described [18,5,19]. The animals were divided into five groups (n = 6, per group) and treated with vehicle (Saline + Tween 80 0.2%, i.p.), CT (25, 50 or 100 mg/kg, i.p.), indomethacin (10 mg/kg, i.p.) or dipyrone (60 mg/kg, i.p.). Thirty minutes after the treatment, 20 lL of CG (300 lg/paw), PGE2 (100 ng/paw), DA (30 lg/paw) or TNF-a (100 pg/paw) were injected subcutaneously into the subplantar region of the hind paw. The mechanical hyperalgesia was evaluated at 0.5, 1, 2 and 3 h after the hyperalgesic agents injections.
2.4. Mechanical hyperalgesia measurement Mechanical hyperalgesia was tested in mice as previous reported [18]. In a quiet room, the mice were placed in acrylic cages (12 10 17 cm) with wire grid floors for 15–30 min. before the test. This method consisted of evoking a hind paw flexion reflex with a hand-held force transducer (electronic anesthesiometer; InsightÒ, São Paulo, Brazil) adapted with a polypropylene tip. The investigator was trained to apply the tip perpendicularly to the central area of the hind paw with a gradual increase in pressure. The end point was characterized by the withdrawal of the paw followed by clear flinching movements. After this response, the pressure intensity was automatically recorded. The intensity of stimulus was obtained by averaging five measurements taken with minimal intervals of 3 min. The animals were tested before and after the treatment. 2.5. Paw edema measurement The CT effect on edema formation caused by the intraplantar injection of CG was analyzed according to the method previously reported [20]. The animals were divided in five groups (n = 6, per group) and treated with vehicle (Saline + Tween 80 0.2%, i.p.), CT (25, 50 or 100 mg/kg, i.p.), or indomethacin (10 mg/kg; i.p.). The right paw volume was measured by the water column dislocation of a plethysmometer (InsightÒ, Brazil) before (time zero) and 1, 2, 3, 4, 5 and 6 h after subplantar injection of 40 lL of CG (1%). The paw edema was expressed (in milliliter) as the difference between the volume of the paw after and before CG injection. The area under the curve (AUC [0–240 min]; in milliliter per minute) was also calculated using the trapezoidal rule. 2.6. Immunofluorescence To evaluate the action of the test drug on the spinal cord, the animals (n = 6, per group) were treated with CT (100 mg/kg; i.p.) or vehicle (Saline + Tween 80 0.2%; i.p.) and, after 30 min, 20 lL of CG (300 lg/paw) was injected subcutaneously into the subplantar region of the hind paw. One group did not receive any kind of treatment (sham group), which was used as a baseline control. Ninety minutes after the treatment, all animals were perfused and the lumbar spinal cord collected and crio-protected for immunofluorescence processing to Fos protein. The time for realization of immunofluorescence protocol was based on the studies of Barr [21] and Bai et al. [22]. Frozen serial transverse sections (20 lm) of all spinal cords were collected on gelatinized glass slides. The tissue sections were stored at 80 °C until use. The sections were washed with phosphate buffer (0.01 M) saline isotonic (PBS) 5 times for 5 min. and incubated with 0.01 M glycine in PBS for 10 min. Non-specific protein binding was blocked by incubation of the sections for 30 min. in a solution containing 2% BSA. Then, the sections were incubated overnight with rabbit anti-Fos as primary antibodies (1:2000). Afterwards, the sections were incubated for two hours with donkey anti-rabbit Alexa Fluor 594 as secondary antibodies (1:2000). The cover slip was mounted with glycerol solution. As an immunofluorescence control for non-specific labeling, sections were incubated without primary antibody. After each stage, slides were washed with PBS 5 times for 5 min. 2.7. Acquisition and analyses of images A blinded investigator took 20 pictures from Fos positive lumbar spinal cords areas for each animal using an Olympus IX2-ICB (Tokyo, Japan). The lumbar spinal cord regions were classified according to Paxinus and Franklin Atlas [23]. After that, the same
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blinded investigator choose the best 10 pictures of each animal and opened, together, all the pictures of all animal in the free software Image J (National Institute of Health). For counting process, the investigator applied a threshold ranging between 91 and 255. It was counted all cells with size between 0.0002 and 0.0015. Finally, it was calculated the average of 10 images selected for each animal. 2.8. Statistical analysis Values are expressed as mean ± SEM. The data obtained were evaluated through the one-way analysis of variance (ANOVA) followed by Tukey’s test. In all cases, differences were considered significant if p < 0.05. All statistical analyses were done using the software GraphPad Prism 5.0 (GraphPad Prism Software Inc., San Diego, CA, USA). 3. Results Administration of CT, in all doses, produced a significantly reduction (p < 0.05) in the mechanical hyperalgesia induced by CG and TNF-a when compared with control (Fig. 1). In the hyperalgesia induced by PGE2 and DA protocols, CT also significantly reduced (p < 0.05) the mechanical threshold. However, the intraperitoneal injection of CT at a dose of 50 mg/kg did not change the mechanical threshold in the times 0.5 and 1 h after the DA injection when compared with control (Fig. 2). As shown in Fig. 3, the treatment with CT significantly (p < 0.05) decreased the paw edema. The doses of 25 and 100 mg/kg were able to maintain the edema reduction during the 6 h evaluation
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period. The dose of 50 mg/kg only reduced the edema in the last three hours of the test. The inhibition percentages, based on the AUC values, were 41.9%, 35.8% (p < 0.01), and 50.6% (p < 0.001) for 25, 50, and 100 mg/kg, respectively. The indomethacin showed an inhibition of 55.3% (p < 0.001). In the spinal cord lamina I of the animals, the average number of neurons showing Fos protein was significantly reduced (p < 0.05) by an intraperitoneal injection of CT (100 mg/kg) when compared with control (Fig. 4). 4. Discussion The present study evaluated the effect of CT in several models of hyperalgesia and its effect in the edema formation. Our data demonstrate that systemic administration of CT significantly decreased the hyperalgesic response in acute models of inflammatory nociception, reducing the edema. Furthermore, we suggested the possible involvement of the spinal cord lamina I inhibition in this effect. The hyperalgesia induced by intraplantar injection of CG is widely used for evaluating new anti-inflammatory and antihyperalgesic compounds in rodents. The CG administration induces the development of the cardinal signs of inflammation, the result of the action of proinflammatory agents, complement and reactive oxygen and nitrogen species. This signalization cascade leads the release of prostanoids and sympathomimetic amines, stimulating peripheral Ad and C fiber sensory nerve terminals. This neurogenic inflammation contributes for the resultant peripheral and central hyperalgesia [5,24,25]. In this protocol, mice pretreated with CT, all doses, showed an increase in the mechanical threshold, similar
Fig. 1. Effect of the acute administration of vehicle, citronellol (CT; 25, 50, or 100 mg/kg; i.p.) or indomethacin (INDO; 10 mg/kg; i.p.) on mechanical hyperalgesia induced by CG (A) and TNF-a (B). Each point represents the mean ± SEM (n = 6, per group) of the paw withdrawal threshold (in grams) to tactile stimulation of the ipsilateral hind paw. * p < 0.05, **p < 0.01 and ***p < 0.001 vs. control group (ANOVA followed by Tukey test).
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Fig. 2. Effect of the acute administration of vehicle, citronellol (CT; 25, 50, or 100 mg/kg; i.p.) or dipyrone (DYP; 60 mg/kg; i.p.) on mechanical hyperalgesia induced by PGE2 (A) and DA (B). Each point represents the mean ± SEM (n = 6, per group) of the paw withdrawal threshold (in grams) to tactile stimulation of the ipsilateral hind paw. *p < 0.05, ** p < 0.01 and ***p < 0.001 vs. control group (ANOVA followed by Tukey test).
with mice who received indomethacin, a COX inhibitor. This effect is probably the result of the CT anti-inflammatory activity, which has been reported in the literature [17,26,27]. The edema formation is one of the fundamental actions of acute inflammation and it is an essential parameter to be considered when evaluating compounds with potential anti-inflammatory activity [28]. The intraplantar CG injection also induces the edema formation in the paw and it has been fully characterized in the past showing acute inflammation by vasodilatation, exudation of protein-rich fluid and cell migration, primarily neutrophils, into the side of injury [24,29,30]. In this study, we have attempted to demonstrate the effects of CT on the CG-induced mouse paw edema and the results showed that all doses of CT presented antiedematogenic activity. This effect is probably due of the ability of CT to inhibit the neutrophils migration and some pro-inflammatory cytokines, as described previously [17,27]. The tumor necrosis factor-alpha (TNF-a) is one of the main cytokines that control the inflammatory process. It is the first to be released after an inflammatory stimulus, such as the carrageenan injection, leading the increase of the COX and PGE2 expression, which is mediated by the TNFR1 and TNFR2 receptors [5,31]. TNF-a also induces the neutrophils migration to the injury site by indirect mechanisms, such as the induction of the chemotactic factors release through the resident macrophages and the leukotriene pathway stimulation [32,33]. The CT was able to increase the threshold sensitivity of mechanical hyperalgesia induced by TNF-a, as observed in the treatment with indomethacin. This effect is probably associated with the inhibition of TNF-a and PGE2 caused by CT [17,27].
Other cytokines, such as IL-1b, act in the release of prostanoids such as prostaglandins, while keratinocyte-derived chemokine (KC) acts in the release of sympathomimetic amines like dopamine, triggering the activation of nociceptors and transmission impulse by primary nociceptive neurons [5]. PGE2 and dopamine act on the EP2 and D1-type metabotropic receptors, respectively, activating the second messenger pathways, which is responsible for lowering the nociceptor threshold and increasing neuronal membrane excitability [34,35]. Because of this, we investigated the possible CT effect on the hyperalgesia induced by PGE2 and dopamine. It was observed that CT inhibited the mechanical hyperalgesia induced by these hyperalgesic mediators, like dipyrone. These findings are probably a result of the CT ability to inhibit the release of TNF and PGE [17,27], in addition with its capacity in suppress the COX-2 expression [26]. After the tissue injury with subsequent installation of the inflammatory process, stimulating peripheral Ad and C fiber sensory nerve terminals, the first synapse in the transmission of this information to the brain is in the superficial dorsal horn of the spinal cord, which is comprised of lamina I and II. So, the Lamina I plays key role in the modulation of pain transmission, which is the major source of ascending output from the superficial dorsal horn to brain regions that have a role in pain mechanisms, including the medullary reticular formation, parabrachial area, periaqueductal gray (PAG) and thalamus [36–38]. The lamina I also receive an inhibitory input of the descending pain pathways, specifically from PAG area in the mesencephalon and from the rostral ventromedial medulla (RVM), which comprises the nucleus raphe magnus, the paragigantocellular and gigantocellular nuclei, areas
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Fig. 3. Effect of the acute administration of vehicle, citronellol (CT; 25, 50, or 100 mg/kg; i.p.) or indomethacin (INDO; 10 mg/kg; i.p.) on edema induced by CG. Each point represents the mean ± SEM (n = 6, per group) of the paw volume (in milliliter, A) or the area under curve (AUC) from 0 to 6 h (B). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control group (ANOVA followed by Tukey test).
Fig. 4. Neurons Fos positive in the lumbar spinal cord lamina I (A). Vehicle (C) or CT (D; 100 mg/kg) were administered intraperitoneally and, after 30 min, CG was injected subcutaneously into the subplantar region of the hind paw. The sham group (B) did not receive any treatment. The animals were perfused 90 min after the treatment. Values represent mean ± SEM (n = 6, per group). **p < 0.01 vs. control group (unpaired t test). 20 lm.
involved in the pain inhibition [39]. In order to study the central pathways of pain, the c-Fos expression has been used to examine these neural circuitries, once Fos is expressed in the nuclei of cells whereas retrograde tracers accumulate in the cytoplasm, thus facilitating their use in combination [40,41].
On the basis of these findings, we evaluated the expression of Fos protein in spinal cord lamina I. It was observed that the treatment with CT was able to inhibit the neurons activation in the spinal cord lamina I. This inhibition can a be caused by the indirect action of CT through its action in the pro-inflammatory cytokines,
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like described previously, or through the activation of the opioid system in the PAG, once CT is able to activate the PAG and its antinociceptive effect was blocked by the administration of naloxone on the hot plate test, suggesting the involvement of the opioid central receptors in this response, like described by [17,42]. On the other hand, CT can be acting directly in the spinal cord lamina I, probably blocking the glutamatergic system, since CT produced an inhibition of nociceptive behavior induced by injection of glutamate into the right upper limb of mouse [42]. In addition, CT can be reducing the excitability of the neurons in the spinal cord lamina I, once De Sousa et al. demonstrated that CT partially blocked voltage-dependent Na+ channels [16]. Studies have suggested that the CNS depression and the non-specific muscle relaxation effects can reduce the response of motor coordination and might invalidate the behavioral tests [43–45]. However, previous studies with CT, using the same doses as the present study, did not show any interference on motor coordination of the animals in the rota-rod test and in the spontaneous locomotor activity [17,42]. So, the action of CT on mechanical hyperalgesia, observed in this study, is not entirely due to an inhibitory effect on the CNS or muscle relaxation. Thus, it can be concluded that CT reduced the mechanical hyperalgesia and the edema in mice, probably by the inhibition of peripheral mediators, such as TNF-a and PGE2, as well as the inhibition of spinal cord lamina I, probably by the activation of CNS regions, especially the PAG, encephalic area involved in pain. Conflict of interest The authors report no conflict of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
Acknowledgements We thank Mr. Osvaldo Andrade Santos for technical support. This work was supported by grants from the National Council of Technological and Scientific Development (CNPq/Brazil), the Research Supporting Foundation of the State of Sergipe (FAPITEC-SE/Brazil) and Coordinating Development of Senior Staff (CAPES/Brazil). References [1] Y.A. Andreev, A.A. Vassilevski, S.A. Kozlov, Molecules to selectively target receptors for treatment of pain and neurogenic inflammation, Recent Pat. Inflammation Allergy Drug Discovery 6 (2012) 35–45. [2] A. Coutaux, F. Adam, J.-C. Willer, D. Le Bars, Hyperalgesia and allodynia: peripheral mechanisms, Joint Bone Spine Rev. Rhum. 72 (2005) 359–371, http://dx.doi.org/10.1016/j.jbspin.2004.01.010. [3] O. Obreja, P.K. Rathee, K.S. Lips, C. Distler, M. Kress, IL-1 beta potentiates heatactivated currents in rat sensory neurons: involvement of IL-1RI, tyrosine kinase, and protein kinase C, FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 16 (2002) 1497–1503, http://dx.doi.org/10.1096/fj.02-0101com. [4] N. Rhein-Knudsen, M.T. Ale, A.S. Meyer, Seaweed hydrocolloid production: an update on enzyme assisted extraction and modification technologies, Mar. Drugs 13 (2015) 3340–3359, http://dx.doi.org/10.3390/md13063340. [5] T.M. Cunha, W.A. Verri Jr., J.S. Silva, S. Poole, F.Q. Cunha, S.H. Ferreira, A cascade of cytokines mediates mechanical inflammatory hypernociception in mice, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 1755–1760, http://dx.doi.org/10.1073/ pnas.0409225102. [6] J.A. Mitchell, T.D. Warner, Cyclo-oxygenase-2: pharmacology, physiology, biochemistry and relevance to NSAID therapy, Br. J. Pharmacol. 128 (1999) 1121–1132, http://dx.doi.org/10.1038/sj.bjp.0702897. [7] R.O. Day, G.G. Graham, Non-steroidal anti-inflammatory drugs (NSAIDs), BMJ 346 (2013) f3195.
[8] F. Bakkali, S. Averbeck, D. Averbeck, M. Idaomar, Biological effects of essential oils – a review, Food Chem. Toxicol. Int. J. Publ. Br. Ind. Biol. Res. Assoc. 46 (2008) 446–475, http://dx.doi.org/10.1016/j.fct.2007.09.106. [9] A.G. Guimarães, J.S.S. Quintans, L.J. Quintans Jr., Monoterpenes with analgesic activity – a systematic review, Phytother. Res. PTR 27 (2013) 1–15, http:// dx.doi.org/10.1002/ptr.4686. [10] A.G. Guimarães, M.R. Serafini, L.J. Quintans-Júnior, Terpenes and derivatives as a new perspective for pain treatment: a patent review, Expert Opin. Ther. Pat. 24 (2014) 243–265, http://dx.doi.org/10.1517/13543776.2014.870154. [11] R. De Cássia da Silveira e Sá, L.N. Andrade, D.P. de Sousa, A review on antiinflammatory activity of monoterpenes, Mol. Basel, Switz. 18 (2013) 1227– 1254, http://dx.doi.org/10.3390/molecules18011227. [12] B. Abegaz, P.G. Yohannes, R.K. Dieter, Constituents of the essential oil of Ethiopian Cymbopogon citratus stapf, J. Nat. Prod. 46 (1983) 424–426, http:// dx.doi.org/10.1021/np50027a022. [13] L.J. Quintans-Júnior, T.T. Souza, B.S. Leite, N.M.N. Lessa, L.R. Bonjardim, M.R.V. Santos, et al., Phythochemical screening and anticonvulsant activity of Cymbopogon winterianus Jowitt (Poaceae) leaf essential oil in rodents, Phytomed. Int. J. Phytother. Phytopharm. 15 (2008) 619–624, http:// dx.doi.org/10.1016/j.phymed.2007.09.018. [14] M.R.V. Santos, F.V. Moreira, B.P. Fraga, D.P. de Souza, L.R. Bonjardim, L.J. Quintans-Junior, Cardiovascular effects of monoterpenes: a review, Rev. Bras. Farmacogn. 21 (2011) 764–771, http://dx.doi.org/10.1590/S0102695X2011005000119. [15] J.F.A. Bastos, I.J.A. Moreira, T.P. Ribeiro, I.A. Medeiros, A.R. Antoniolli, D.P. De Sousa, et al., Hypotensive and vasorelaxant effects of citronellol, a monoterpene alcohol, in rats, Basic Clin. Pharmacol. Toxicol. 106 (2010) 331–337, http://dx.doi.org/10.1111/j.1742-7843.2009.00492.x. [16] D.P. de Sousa, J.C.R. Gonçalves, L. Quintans-Júnior, J.S. Cruz, D.A.M. Araújo, R.N. de Almeida, Study of anticonvulsant effect of citronellol, a monoterpene alcohol, in rodents, Neurosci. Lett. 401 (2006) 231–235, http://dx.doi.org/ 10.1016/j.neulet.2006.03.030. [17] R.G. Brito, A.G. Guimarães, J.S.S. Quintans, M.R.V. Santos, D.P. De Sousa, D. Badaue-Passos Jr., et al., Citronellol, a monoterpene alcohol, reduces nociceptive and inflammatory activities in rodents, J. Nat. Med. 66 (2012) 637–644, http://dx.doi.org/10.1007/s11418-012-0632-4. [18] T.M. Cunha, W.A. Verri Jr., G.G. Vivancos, I.F. Moreira, S. Reis, C.A. Parada, et al., An electronic pressure-meter nociception paw test for mice, Braz. J. Med. Biol. Res. Rev. (Bras. Pesqui. Médicas E Biológicas Soc. Bras. Biofísica Al.) 37 (2004) 401–407. [19] C.F. Villarreal, M.I. Funez, F. Figueiredo, F.Q. Cunha, C.A. Parada, S.H. Ferreira, Acute and persistent nociceptive paw sensitisation in mice: the involvement of distinct signalling pathways, Life Sci. 85 (2009) 822–829, http://dx.doi.org/ 10.1016/j.lfs.2009.10.018. [20] L. Levy, Carrageenan paw edema in the mouse, Life Sci. 8 (1969) 601–606. [21] G.A. Barr, Formalin-induced c-fos expression in the brain of infant rats, J. Pain Off. J. Am. Pain Soc. 12 (2011) 263–271, http://dx.doi.org/10.1016/ j.jpain.2010.09.005. [22] Z.-T. Bai, T. Liu, X.-Y. Pang, Z.-F. Chai, Y.-H. Ji, Suppression by intrathecal BmK IT2 on rat spontaneous pain behaviors and spinal c-Fos expression induced by formalin, Brain Res. Bull. 73 (2007) 248–253, http://dx.doi.org/10.1016/ j.brainresbull.2007.03.007. [23] G. Paxinos, K. Franklin, The Mouse Brain in Stereotaxic Coordinates, second ed., Elsevier, California, USA, 2001. [24] C.J. Morris, Carrageenan-induced paw edema in the rat and mouse, Methods Mol. Biol. Clifton, NJ 225 (2003) 115–121, http://dx.doi.org/10.1385/1-59259374-7:115. [25] S.H. Ferreira, B.B. Lorenzetti, S. Poole, Bradykinin initiates cytokine-mediated inflammatory hyperalgesia, Br. J. Pharmacol. 110 (1993) 1227–1231. [26] M. Katsukawa, R. Nakata, S. Koeji, K. Hori, S. Takahashi, H. Inoue, Citronellol and geraniol, components of rose oil, activate peroxisome proliferatoractivated receptor a and c and suppress cyclooxygenase-2 expression, Biosci. Biotechnol. Biochem. 75 (2011) 1010–1012. [27] Y.-W. Su, S.-H. Chao, M.-H. Lee, T.-Y. Ou, Y.-C. Tsai, Inhibitory effects of citronellol and geraniol on nitric oxide and prostaglandin E2 production in macrophages, Planta Med. 76 (2010) 1666–1671, http://dx.doi.org/10.1055/s0030-1249947. [28] A. Russo, C. Costagliola, L. Delcassi, F. Parmeggiani, M.R. Romano, R. Dell’Omo, et al., Topical nonsteroidal anti-inflammatory drugs for macular edema, Mediators Inflamm. 2013 (2013) 476525, http://dx.doi.org/10.1155/2013/ 476525. [29] S.Z. Hussein, K. Mohd Yusoff, S. Makpol, Y.A. Mohd Yusof, Gelam honey inhibits the production of proinflammatory, mediators NO, PGE2, TNF-a, and IL-6 in carrageenan-induced acute paw edema in rats, Evidence Based Complement Altern. Med. ECAM 2012 (2012), http://dx.doi.org/10.1155/2012/109636. [30] E.F.C. Reis, S.B.R. Castro, C.C.S. Alves, E.E. Oliveira, T.A. Corrêa, M.V. Almeida, et al., Lipophilic amino alcohols reduces carrageenan-induced paw edema and anti-OVA DTH in BALB/c mice, Int. Immunopharmacol. 17 (2013) 727–732, http://dx.doi.org/10.1016/j.intimp.2013.08.017. [31] C. Sommer, C. Schmidt, A. George, Hyperalgesia in experimental neuropathy is dependent on the TNF receptor 1, Exp. Neurol. 151 (1998) 138–142, http:// dx.doi.org/10.1006/exnr.1998.6797. [32] L.H. Faccioli, G.E.P. Souza, F.Q. Cunha, S. Poole, D.S.H. Ferreira, Recombinant interleukin-1 and tumor necrosis factor induce neutrophil migration ‘‘in vivo’’ by indirect mechanisms, Agents Actions 30 (1990) 344–349, http://dx.doi.org/ 10.1007/BF01966298.
R.G. Brito et al. / Chemico-Biological Interactions 239 (2015) 111–117 [33] C. Canetti, J.S. Silva, S.H. Ferreira, F.Q. Cunha, Tumour necrosis factor-alpha and leukotriene B(4) mediate the neutrophil migration in immune inflammation, Br. J. Pharmacol. 134 (2001) 1619–1628, http://dx.doi.org/10.1038/ sj.bjp.0704403. [34] S.H. Ferreira, M. Nakamura, I – Prostaglandin hyperalgesia, a cAMP/Ca2+ dependent process, Prostaglandins 18 (1979) 179–190. [35] D. Sachs, C. Villarreal, F. Cunha, C. Parada, S. Ferreira, The role of PKA and PKCe pathways in prostaglandin E2-mediated hypernociception, Br. J. Pharmacol. 156 (2009) 826–834, http://dx.doi.org/10.1111/j.1476-5381.2008.00093.x. [36] R. Suzuki, A. Dickenson, Spinal and supraspinal contributions to central sensitization in peripheral neuropathy, Neurosignals 14 (2005) 175–181, http://dx.doi.org/10.1159/000087656. [37] A.D. Craig, J.O. Dostrovsky, Differential projections of thermoreceptive and nociceptive lamina I trigeminothalamic and spinothalamic neurons in the cat, J. Neurophysiol. 86 (2001) 856–870. [38] A.J. Todd, Z. Puskar, R.C. Spike, C. Hughes, C. Watt, L. Forrest, Projection neurons in lamina I of rat spinal cord with the neurokinin 1 receptor are selectively innervated by substance p-containing afferents and respond to noxious stimulation, J. Neurosci. Off. J. Soc. Neurosci. 22 (2002) 4103–4113. doi:20026261.
117
[39] B. Calvino, R.M. Grilo, Central pain control, Joint Bone Spine 73 (2006) 10–16, http://dx.doi.org/10.1016/j.jbspin.2004.11.006. [40] J.A. Harris, Using c-fos as a neural marker of pain, Brain Res. Bull. 45 (1998) 1–8. [41] R.K. Snowball, F.M. Semenenko, B.M. Lumb, Visceral inputs to neurons in the anterior hypothalamus including those that project to the periaqueductal gray: a functional anatomical and electrophysiological study, Neuroscience 99 (2000) 351–361. [42] R.G. Brito, P.L. Santos, D.S. Prado, M.T. Santana, A.A.S. Araújo, L.R. Bonjardim, et al., Citronellol reduces orofacial nociceptive behaviour in mice – evidence of involvement of retrosplenial cortex and periaqueductal grey areas, Basic Clin. Pharmacol. Toxicol. 112 (2013) 215–221, http://dx.doi.org/10.1111/ bcpt.12018. [43] D. Le Bars, M. Gozariu, S.W. Cadden, Animal models of nociception, Pharmacol. Rev. 53 (2001) 597–652. [44] P. Raboisson, R. Dallel, The orofacial formalin test, Neurosci. Biobehav. Rev. 28 (2004) 219–226, http://dx.doi.org/10.1016/j.neubiorev.2003.12.003. [45] C.S. Passos, M.D. Arbo, S.M.K. Rates, G.L. von Poser, Terpenoids with activity in the Central Nervous System (CNS), Rev. Bras. Farmacogn. 19 (2009) 140–149, http://dx.doi.org/10.1590/S0102-695X2009000100024.