Neuropeptides 47 (2013) 199–206

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Neuropeptides

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Peripheral substance P and neurokinin-1 receptors have a role in inflammatoryand neuropathic orofacial pain models

Fernanda C. Teodoro a, Marcos F. Tronco Júnior a, Aleksander R. Zampronio a, Alessandra C. Martini b,Giles A. Rae b, Juliana G. Chichorro a,⇑a Department of Pharmacology, Federal University of Parana, Curitiba, PR, Brazilb Departament of Pharmacology, Federal University of Santa Catarina, SC, Brazil

a r t i c l e i n f o

Article history:Received 20 August 2012Accepted 12 October 2012Available online 22 November 2012

Keywords:Substance PNK1 receptorHyperalgesiaInflammatory painNeuropathic painTrigeminal nerve

0143-4179/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.npep.2012.10.005

⇑ Corresponding author. Address: Federal Universences Sector, Department of Pharmacology, Curitiba,1720; fax: +55 41 3266 2042.

E-mail address: juliana.chichorro@ufpr.br (J.G. Chi

a b s t r a c t

There is accumulating evidence that substance P released from peripheral sensory neurons participates ininflammatory and neuropathic pain. In this study it was investigated the ability of substance P to induceorofacial nociception and thermal and mechanical hyperalgesia, as well as the role of NK1 receptors onmodels of orofacial inflammatory and neuropathic pain. Substance P injected into the upper lip at 1,10 and 100 lg/50 lL failed to induce nociceptive behavior. Also, substance P (0.1–10 lg/50 lL) injectedinto the upper lip did not evoke orofacial cold hyperalgesia and when injected at 1 lg/50 lL did notinduce mechanical hyperalgesia. However, substance P at this latter dose induced orofacial heat hyper-algesia, which was reduced by the pre-treatment of rats with a non-peptide NK1 receptor antagonist(SR140333B, 3 mg/kg). Systemic treatment with SR140333B (3 mg/kg) also reduced carrageenan-inducedheat hyperalgesia, but did not exert any influence on carrageenan-induced cold hyperalgesia. Blockade ofNK1 receptors with SR140333B also reduced by about 50% both phases of the formalin response evaluatedin the orofacial region. Moreover, heat, but not cold or mechanical, hyperalgesia induced by constrictionof the infraorbital nerve, a model of trigeminal neuropathic pain, was abolished by pretreatment withSR140333B. Considering that substance P was peripherally injected (i.e. upper lip) and the NK1 antagonistused lacks the ability to cross the blood–brain-barrier, our results demonstrate that the peripheral SP/NK1

system participates in the heat hyperalgesia associated with inflammation or nerve injury and in the per-sistent pain evoked by formalin in the orofacial region.

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1. Introduction

Orofacial pain represents a major medical and social problem. Itis estimate that, in the United States, about 22% of the Americansolder than 18 years report pain in the orofacial region (Liptonet al., 1993). Orofacial pain encompasses various disorders, includ-ing those that directly affect the trigeminal nerve, such as trigeminalneuralgia, invasion by tumors that compromise joint or another oro-facial structures, temporomandibular joint dysfunction, headache,migraine, as well as idiopathic pain. In addition, orofacial painmay be classified in three categories, acute pain after injury, chronicinflammatory pain (e.g., temporomandibular disorders) and neuro-pathic pain (e.g., trigeminal neuralgia) (Le Resche, 1997; Eide andRabben, 1998).

There are several models to study orofacial pain, both of inflam-matory and neuropathic nature. The orofacial formalin test, first

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chorro).

proposed by Clavelou et al. (1989), is extensively used to assessnociceptive process in the orofacial region. Additionally, injectionof carrageenan into the upper lip, as well as other orofacial struc-tures, is widely employed to study orofacial inflammatory hyperal-gesia (Chichorro et al., 2006a; Rodrigues et al., 2006; Teixeira et al.,2010). Regarding orofacial neuropathic pain, the constriction of theinfraorbital nerve, a model established by Vos and Maciewicz(1991) has been shown to reproduce many sensory alterationsobserved in patients suffering of trigeminal neuropathic pain, suchas spontaneous pain, mechanical allodynia and thermal hyperalge-sia (Vos et al., 1994; Imamura et al., 1997; Chichorro et al.,2006a,b; Chichorro et al., 2009).

There is accumulating evidence that the undecapeptide sub-stance P (SP) is a neurotransmitter of small diameter nociceptiveafferents in the trigeminal system (Hökfelt et al., 1975; Keller andMarfurt, 1991) and in terminals in the spinal dorsal horn (Takemuraet al., 2006; for review see Hargreaves, 2011). The biological actionsof SP can be mediated through the activation of three different neu-rokinin (NK) receptors named NK1, NK2 and NK3, but the NK1 recep-tors demonstrates a preferential affinity for SP (for review see Satakeet al., 2012). Indeed, several studies support the role of substance P,

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acting via NK1 receptors, in the processing of noxious information inthe spinal cord (Abbadie et al., 1997; Mansikka et al., 2000; LaGraizeet al., 2010), as its participation in models of inflammatory and neu-ropathic pain (González et al., 1998; Cahill and Coderre, 2002; Gaoet al., 2003). In addition, studies have provided evidence for the roleof peripheral SP, acting through NK1 receptors, in hyperalgesic re-sponses of rats evaluated in different models (Nakamura-Craigand Smith, 1989; Jang et al., 2004). In this regard, it has been sug-gested that activation of peripheral NK1 receptors resulted in heathyperalgesia by sensitizing TRPV1 receptors (Zhang et al., 2007).

Considering orofacial pain, few studies have shown theinvolvement of the peripheral SP/NK1 system in experimentalinflammatory hyperalgesia and allodynia (Takeda et al., 2005;Denadai-Souza et al., 2009). In addition, it has been demonstratedthat SP and NK1 receptors are expressed by peripheral trigeminalstructures, such as the dental pulp, and elevated levels of SP havebeen reported in pulpitis (Bowles et al., 2003; Caviedes-Bucheliet al., 2006) and correlate with pain experience and caries progres-sion (Rodd and Boissonade, 2000; Awawdeh et al., 2002). Moreover,stimulation of peripheral afferent fibers resulted in release of SPwithin the trigeminal ganglia and this release is greatly increasedafter orofacial inflammation (Neubert et al., 2000). These datastrongly suggest that peripheral SP plays a role in the trigeminalnociceptive process.

In light of the above considerations, the present study aimed toinvestigate whether SP is able to evoke nociceptive behavior andthermal and mechanical hyperalgesia when injected into the upperlip of rats, as well as, to assess if this neuropeptide, acting throughNK1 receptors, participates in inflammatory and neuropathic orofa-cial pain models.

2. Experimental procedures

2.1. Animals

Experiments were conducted on male Wistar rats weighing180–220 g, maintained five to a cage at controlled temperature(22 ± 1 �C) under a 12/12 h light/dark cycle (lights on at 08:00 h)with free access to food and tap water. They were acclimatizedto the laboratory for at least 48 h before use. Each experimentalgroup consisted of 5–10 rats and all procedures were previouslyapproved by the Federal University of Parana Committee on theEthical Use of Animals (authorization number 424), where thestudy was carried out, and conducted in accordance with the eth-ical guidelines of the International Association for the Study of Pain(Zimmermann, 1983) and Brazilian regulations on animal welfare.All efforts were made to minimize the number of animals used andtheir suffering.

2.2. Cold stimulation

This test was performed as previously described by Chichorroet al. (2006a). Before each testing session, animals were placed inindividual plastic observation cages and left to adapt to the envi-ronment for at least 30 min. After this period, they usually dis-played considerable sniffing, but very little locomotor activity.Cold stimulation was applied by the experimenter in the form ofa brief 2 s spray of tetrafluoroethane to the center of the rightvibrissal pads, while gently restraining the animal in its cage byplacing one hand around its trunk. The total duration of bilateralfacial grooming behavior with both forepaws directed to the snoutwas recorded, using a stopwatch, over the first 2 min followingapplication of the cold stimulus, as an index of the intensity ofnocifensive responsiveness.

2.3. Heat stimulation

Heat stimulation of the orofacial area was performed as previ-ously described (Chichorro et al., 2009). On each occasion, the ani-mal was temporarily removed from its home cage and gently heldby the experimenter and a radiant heat source was positioned 1 cmfrom the surface of the right vibrissal pad. The latency to displayeither head withdrawal or vigorous flicking of the snout was re-corded (in seconds) using a stopwatch, and a 20 s cut-off timewas used to prevent tissue damage.

2.4. Mechanical stimulation

To assess the mechanical threshold, before each testing session,animals were placed in individual plastic cages and left to adapt tothe environment for at least 2 h. The mechanical threshold wasmeasured using a graded series of eight von Frey filaments rangingfrom 0.04 to 8 g (Semmes–Weinstein monofilaments, Stoelting,Wood Dale, IL) as described previously (Chichorro et al. 2006b).Each filament was applied three times near the center of the rightvibrissal pad. Each stimulation series began with the filament pro-ducing the lowest force, and proceeded up to the filament thatevoked one of the following nocifensive behaviors twice: briskhead withdrawal, escape or attack reactions or asymmetric facialgrooming. Only rats that did not react to application of the 8 g fil-ament in the preoperative tests or previous injection of substance Pwere included in this study, to avoid unspecific responses.

2.5. Formalin induced-response

This test was based on the study by Clavelou et al. (1989) withminor modifications. Briefly, each animal was placed in individualplastic cages and left to adapt to the environment for at least15 min. Subsequently, the animals were gently held and receiveda subcutaneous 50 ll injection of formalin (2.5%) or vehicle (saline)into the right upper lip and were returned immediately to theobservation cage. The time each animal spent rubbing the injectedarea with the forepaws was recorded in consecutive 3 min intervalsover a 30 min period, and it was considered as an index of nocicep-tion. The nociceptive responses were measured in both phases afterinjection of formalin into upper lip, the first phase lasting 0–3 minand the second was considered 12–30 min after formalin injection.

2.6. Constriction of the infraorbital nerve

The constriction of the infraorbital nerve (CION) was performedaccording to Chichorro et al. (2006a,b), a modify procedure fromthat originally proposed by Vos and Maciewicz (1991). Briefly, ratswere anesthetized with an intraperitoneal (i.p.) injection of a mix-ture of cetamine and xylazine (50 and 10 mg/kg, respectively) andan incision was made in the skin of the snout, under the right eye,about 3 mm caudal to the mystacial pads. The superior lip elevatorand anterior superficial masseter muscle were bluntly dissected toexpose the rostral end of the infraorbital nerve, as it emerged fromthe infraorbital fissure. Special care was taken not to damage thefacial nerve. Two silk 4–0 ligatures were then tied loosely and2 mm apart around the infraorbital nerve and the wound wasclosed with additional silk sutures (4–0). Sham-operated rats weretreated identically, but no ligatures were applied to the infraorbitalnerve. After surgery, all rats were maintained in a warm room untilthey recovered from anesthesia.

2.7. Drugs

Substance P and carrageenan were obtained from Sigma–Al-drich Co. (St. Louis, USA) and dissolved in 0.9% sterile saline

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solution. SR140333B ((S)1-{3-(3,4-dichlorophenyl)-3-[2(4-phenyl-1-azabicyclo[2.2.2]oct-1-yl]-ethyl]-piperidin-1-yl}-2-(3- isopro-poxyphenyl)-ethanone benzenesulfonate) was donated bySanofi-Aventis (France) and dissolved in Tween 80 (1%) and 0.9%sterile saline solution (99%). Formalin was obtained from VetecQuímica Fina Ltda (Rio de Janeiro, Brazil) and dissolved in 0.9%sterile saline solution. Ketamine was obtained from RhobifarmaInd. Farmacêutica (Hortolândia, SP, Brazil) and xylazine from Lab-oratórios König S.A. (Avellaneda, Argentina).

2.8. Experimental procedures

2.8.1. Evaluation of the ability of SP to induce orofacial nociceptiveresponse, thermal and mechanical hyperalgesia

To assess whether SP could evoke nociceptive behavior in theorofacial area, animals received a subcutaneous injection of SP(1, 10 and 100 lg/50 lL, s.c.) into the upper lip and the time theyspent rubbing the injected area with the forepaws was recordedover 15 min. Control animals were treated identically with the cor-responding vehicles. To evaluate the influence of SP on orofacialthermal stimulation, SP at 0.1, 1 and 10 lg/50 lL or vehicle (saline,50 lL) were injected subcutaneously into the upper lip and theradiant heat source was applied before the injection and at 1 h-intervals up to 4 h. An additional group of rats received SP (0.1,1 and 10 lg/50 lL) or vehicle (saline, 50 lL) into the upper lipand the cold stimulus was applied prior to its administration andat 1 h-intervals up to 4 h. To assess the influence of SP on the oro-facial mechanical threshold, rats received SP (1 lg/50 lL, s.c.) orvehicle and the stimulation was performed before and at 1 h-inter-vals up to 4 h after the injection. In another set of experiments,rats were treated systemically with the NK1 receptor antagonist(SR140333B, 1 or 3 mg/kg, i.p.) or vehicle (1 mL/kg) 30 min beforeSP (1 lg/50 lL) injection into the upper lip and the heat stimuluswas applied before and at 1 h-intervals up to 4 h after the localinjection.

2.8.2. Influence of NK1 receptors blockade on carrageenan-inducedthermal hyperalgesia and formalin-evoked grooming behavior

Rats received SR140333B (3 mg/kg, i.p.) or vehicle (1 mL/kg, i.p.)30 min before carrageenan (50 lg/50 lL) injection into the upperlip and heat or cold stimulation was performed before and at1 h-intervals up to 4 h after the local injection, respectively. Othergroup of animals were injected with formalin (2.5%, 50 lL) or vehi-cle (saline, 50 lL) into the upper lip and then the time each animalspent rubbing the injected area with the forepaws (facial groom-ing) was recorded in consecutive 3 min-intervals over a 30 minperiod. An additional group of rats received SR140333B (3 mg/kg,i.p.) 30 min before the injection of formalin (2.5%, 50 lL) or vehicle(saline, 50 lL) into upper lip and the facial grooming was recordedover 30 min.

2.8.3. Influence of NK1 receptors blockade on thermal and mechanicalhyperalgesia induced by infraorbital nerve constriction

Rats were submitted to CION or sham surgery, and on day 4postoperative, they were treated systemically with SR140333B(3 mg/kg) or vehicle (1 mL/kg) and the thermal hyperalgesia (toheat and cold) was assessed, in different group of rats, beforeand at 1 h-intervals up to 6 h after the treatments. In an additionalgroup of rats, the mechanical threshold was evaluated before thesham or CION surgery and on postoperative days 8, 10, 12 and16, when they received SR140333B (3 mg/kg, i.p.) or vehicle(1 mL/kg) and the mechanical stimulation was performed at 1 h-intervals up to 6 h after the treatments. The dose of SP was basedon the study of Sahbaie et al. (2009) while the dose of SR140333Bwas based on the studies of Bradesi et al. (2006), Denadai-Souzaet al. (2009) and Reis et al. (2011).

2.9. Statistical analysis

Two-way repeated measures ANOVA was used to analyze thedata from both, thermal and mechanical stimulation, with drugtreatment as the independent factor and the different evaluationtime points of nociceptive behavior as the repeated measure. Incase of significant differences with the independent factor or withthe interaction between the independent and repeated factors,one-way ANOVA, followed by the Duncan’s post hoc test, was per-formed. The results obtained from experiments assessing sub-stance P- or formalin-induced nociception were submitted toone-way ANOVA followed by the Duncan’s post hoc test. In all sta-tistical analysis P 6 0.05 was considered statistically significant.

3. Results

3.1. Evaluation of the ability of SP to induce orofacial nociceptiveresponse, thermal and mechanical hyperalgesia

Substance P (1, 10 and 100 lg/50 lL) injected into the upper lipfailed to increase the time animals spent executing facial groomingbehavior compared to animals treated with saline (Fig. 1A). In con-trast, when injected at 0.1, 1 and 10 lg/50 lL, SP was able to in-duce heat hyperalgesia, starting 3 h and persisting up to 4 h afterits injection (Fig. 1B). It is also noteworthy that the thermal hyper-algesia induced by SP at 1 lg/50 lL persisted up to 6 h after itsinjection (data not shown). On the other hand, SP (0.1–10 lg/50 lL) injected into the upper lip did not induce hyperalgesia toa cold stimulus applied to the orofacial region or altered themechanical threshold assessed by the application of Von Frey fila-ments, when injected at 1 lg/50 lL (Fig. 1C and D). The SP-inducedheat hyperalgesia was reduced by the prior treatment with the NK1

receptor antagonist SR140333B systemically injected at 3 mg/kg,but not at 1 mg/kg, and only at the third hour after the treatment(Fig. 2).

3.2. Influence of NK1 receptors blockade on carrageenan-inducedthermal hyperalgesia and formalin-evoked grooming behavior

As illustrated in Fig. 3, carrageenan (50 lg/50 lL) injection intothe upper lip induced heat and cold hyperalgesia, which were sta-tistically significant at the third and fourth hours after injection.Pre-treatment with SR140333B (3 mg/kg, i.p.) completely pre-vented carrageenan-induced heat hyperalgesia (Fig. 3A), but failedto modify carrageenan-induced cold hyperalgesia (Fig. 3B).

Formalin (2.5%, 50 lL) injection into upper lip of rats induced abiphasic nociceptive response, which was measured as the time ani-mals spent executing facial grooming on the first 3 min (phase I) andfrom 12 to 30 min after the injection (phase II). The systemic pre-treatment of rats with SR140333B (3 mg/kg) reduced by about 50%both the first and the second phases of nociception induced by for-malin, but it did not influence the grooming behavior time of ani-mals that received a local (i.e. upper lip) injection of vehicle (Fig. 4).

3.3. Influence of NK1 receptors blockade on thermal and mechanicalhyperalgesia induced by infraorbital nerve constriction

On day 4 after CION surgery (peak of thermal hyperalgesia,Chichorro et al., 2006a, 2009), treatment of animals withSR140333B (3 mg/kg, i.p.) reversed the heat hyperalgesia from thefirst up to the sixth hour after the treatment (Fig. 5A). It is importantto point out that sham animals treated with SR140333B (3 mg/kg,i.p.) showed the same profile of sham animals treated with vehicle(data not shown). On the other hand, CION-induced cold hyperalge-sia, also assessed on day 4 after surgery, was not affected by prior

Fig. 1. Influence of substance P on orofacial nociceptive behavior and on thermal and mechanical threshold in rats. Animals received an injection of SP (1, 10 or 100 lg/50 lL,s.c.) or the corresponding vehicle into the upper lip and the the facial grooming time was registered over the next 15 min (panel A). SP (0.1, 1 and 10 lg/50 lL, s.c.) or vehiclewas injected into the upper lip and reaction time to a radiant heat stimulus applied to the face or the grooming behavior after the application of a cold stimulus wereevaluated at 1 h-intervals up to 4 h (panels B and C, respectively). On panel D, SP (1 lg/50 lL) or vehicle was injected into the upper lip and the mechanical threshold wasassessed at 1 h-intervals up to 4 h. Each point represents mean ± S.E.M. of 5–10 animals. P < 0.05 compared with the vehicle group (one-way ANOVA followed by the Duncan’spost hoc test, panel A, and two-way repeated measures ANOVA followed by the Duncan’s post hoc test on panels B, C and D).

Fig. 2. Effect of a selective NK1 receptor antagonist on SP-induced heat hyperal-gesia. Animals were treated systemically with the NK1 receptor antagonistSR140333 (1–3 mg/kg, i.p.) 30 min prior the injection of SP (1 lg/50 lL) or vehicleinto the upper lip. Each point represents mean ± S.E.M. of 6–8 animals. Asterisksdenote P < 0.05 when compared with animals treated with vehicle and fencesdenote P < 0.05 when compared with animals that received vehicle systemicallyand SP into the upper lip (two-way repeated measures ANOVA followed by theDuncan’s post hoc test).

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treatment with SR140333B injected systemically at the same dose(Fig. 5B). As reported before (Chichorro et al., 2006b), the mechanicalhyperalgesia has a different time course compared to thermalhyperalgesia, with a late start and a longer duration. In the presentstudy, we verified a significant decrease in the mechanical thresholdof CION- compared to sham-operated animals on day 8 after surgery(data not shown). On day 16 after CION, when the mechanical hyper-algesia was well established, the systemic treatment withSR140333B (3 mg/kg, i.p.) also failed to modify the mechanicalthreshold of nerve-injured rats (Fig. 5C).

4. Discussion

SP is widely considered a neuromodulator that alters the excit-ability of nociceptive neurons in the spinal dorsal horn. In the

periphery, SP released from the terminals of activated sensoryneurons, including trigeminal neurons, play a fundamental role inthe generation of the process named neurogenic inflammation(Moussaoui et al., 1993; Holzer, 1998; Richardson and Vasko,2002). However, the role of SP and NK1 receptors in the nociceptionat the level of primary sensory neurons are not clearly characterized.

In the present study, administration of SP did not evoke noci-ceptive behavior on the orofacial region. In agreement with ourdata, intraplantar injection of SP (2 lg/paw) in rats did not evokepaw lifting or licking, different from that observed with the injec-tion of prostaglandin E2, bradykinin and 5-hydroxytryptamine(Hong and Abbott, 1994). Several possibilities could explain thelack of nociceptive responses after peripheral SP injection, one isthat SP is neither necessary nor sufficient to elicit pain at theperipheral nociceptive fiber terminal.

On the other hand, there are a number of reports showing thatperipheral application of SP sensitize primary afferents in both nor-mal and inflamed tissues (Nakamura-Craig and Gill, 1991; Kessleret al., 1992; Carlton et al., 1996; Heppelmann and Pawlak, 1997).In this regard, Chen et al. (2006) showed that peripheral injectionof an NK1 receptor agonist induced heat hyperalgesia evaluated atthe rats’ hind paw, which was blocked by co-injection of a NK1

receptor antagonist. Corresponding this data, herein we demon-strated that injected into the upper lip SP was able to induce orofa-cial heat hyperalgesia, which was attenuated by pharmacologicalNK1 receptors blockade with SR140333B, a highly selective andfunctional antagonist for the rat NK1 receptor (Emonds-Alt et al.,1993; Rupniak et al., 2003). Additionally, our data show that thedevelopment of heat hyperalgesia after carrageenan injection intothe upper lip or infraorbital nerve injury was fully prevented by sys-temic SR140333B previous treatment. Some studies have alreadyreported heat hyperalgesia induced by carrageenan in the rat hindpaw was markedly attenuated by different NK1 receptor antago-nists, but in these studies the antagonists were intrathecallyinjected (Traub, 1996; Gao et al., 2003). Considering our data, inwhich SP was peripherally injected (i.e. upper lip) and the

Fig. 3. Effect of the NK1 receptor antagonist on carrageenan-induced thermal hyperalgesia. Animals were treated systemically with the NK1 receptor antagonist SR140333(3 mg/kg, i.p.) 30 min prior a subcutaneous injection of carrageenan (50 lg/50 lL) or vehicle into the upper lip and the reaction time to a radiant heat stimulus applied to theface (panel A) or the grooming behavior after the application of a cold stimulus (panel B), was assessed at 1 h-intervals up to 4 h. Each point represents mean ± S.E.M. of 5–7animals. Asterisks denote P < 0.05 when compared with animals treated with vehicle and fences denote P < 0.05 when compared with animals that received vehiclesystemically and carrageenan into the upper lip (two-way repeated measures ANOVA followed by the Duncan’s post hoc test).

Fig. 4. Effect of the NK1 receptor antagonist on formalin-induced nociception.Animals were treated systemically with the NK1 receptor antagonist SR140333B(3 mg/kg, i.p.) 30 min prior a subcutaneous injection of formalin (2.5%, 50 lL) orvehicle into upper lip and the facial grooming time was registered in 3 min-intervals up to 30 min after formalin injection. Each bar represents mean ± S.E.M. of7–10 animals. Asterisks denote P < 0.05 when compared with animals treated withvehicle and fences denote P < 0.05 when compared with animals that receivedvehicle systemically and formalin into the upper lip (one-way ANOVA followed bythe Duncan’s post hoc test).

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antagonist used do not cross the blood–brain-barrier (Rupniaket al., 2003), we suggest that SP can act via peripheral NK1 receptorsto induce hyperalgesia. In line with these observations, someauthors have proposed recently that activation of peripheral NK1

receptors resulted in heat hyperalgesia by sensitizing TRPV1 recep-tors (Zhang et al., 2007). Thus, it is possible to speculate that heathyperalgesia in the orofacial region involves the activation of NK1receptors which leads to sensitization of TRPV1 receptors in trigem-inal afferents. Although it has not been demonstrated in the trigem-inal nerve yet, a similar mechanism was already described, in whichactivation of NK1 receptors by SP leads to P2X3 receptors sensitiza-tion, which also may contribute to orofacial pain.

In sharp contrast, SP was not able to induce orofacial coldhyperalgesia, as the blockade of NK1 receptors failed to affect

carrageenan- or nerve injury-induced cold hyperalgesia. To ourknowledge, this is the first time that the role of the peripheralSP/NK1 system is investigated in the cold hyperalgesia, and ourdata suggest that this system does not participate of this phenom-enon, at least in the orofacial region. However, is important topoint out that it was recently demonstrated that activation of thecold receptor TRPA1 evokes SP release from the primary sensoryneurons, which participates of TRPA1 induced nociceptive andinflammatory responses (Nakamura et al., 2012).

Also, in the present study we did not observed mechanicalhyperalgesia after SP injection into the upper lip. In addition, withthe method we have used to evaluate changes in the orofacialmechanical threshold, we did not detected mechanical hyperalge-sia after carrageenan injection into the upper lip (data not shown),what unable us to assess the influence of the NK1 receptor antago-nist in this parameter. On the other hand, Denadai-Souza et al.(2009), using the electronic von Frey applied to the face, detectedmechanical hyperalgesia after carrageenan injection into the tem-poromandibular joint, which was not influenced by the systemicpretreatment of rats with SR140333B (3 mg/kg). However, thistreatment substantially inhibited the increase in plasma extravasa-tion, leukocyte influx, as well as the production of the cytokinesTNFa and IL-1b into the joint cavity induced by carrageenan(Denadai-Souza et al., 2009). In line with these observations, wehave also demonstrated that mechanical hyperalgesia after infraor-bital nerve injury was not affected by previous treatment withSR140333B. However, contrasting our observations, a previousstudy showed that mechanical hyperalgesia induced by carra-geenan was attenuated by a non-peptide NK1 receptor antagonist(Birch et al., 1992). In addition, constriction of the sciatic nerve in-duced mechanical hyperalgesia in the rats’ hind paw, which wasattenuated by a non peptide NK1 receptor antagonist (Cahill andCoderre, 2002). Moreover, mechanical hyperalgesia induced by L5

Fig. 5. Effect of the NK1 receptor antagonist on the thermal and mechanical threshold of rats submitted to infraorbital nerve constriction. The basal responsiveness of rats tothermal and mechanical stimulation was assessed before sham or CION surgery (�1) and on day 4 after the surgery, but before the treatments (O). On day 4 after the surgery,sham-operated rats received vehicle and nerve-injured rats were treated with vehicle or SR140333 (3 mg/kg, i.p.) and the reaction time to a radiant heat stimulus (panel A) orthe facial grooming time after application of a cold stimulus (panel B) in the orofacial region was assessed at 1 h-intervals up to 6 h. On panel C, on day 16 after the surgery,the groups were treated as describe above and the mechanical threshold was evaluated at 1 h-intervals up to 6 h. Each point represents ± S.E.M. of 5–9 animals. Asterisks andFences denote P < 0.05 when compared to sham animals treated with vehicle and CION animals treated with vehicle, respectively (two-way repeated measures ANOVAfollowed by the Duncan’s post hoc test).

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and/or L6 spinal nerve injury was also significantly reduced bypharmacological NK1 receptors blockade at the peripheral or atthe spinal level, as demonstrated by Jang et al. (2004) and Lee andKim (2007). It is also noteworthy that SP injected into the rats’ hindpaw is able to induce mechanical hyperalgesia, also contrasting ourfindings (Carlton et al., 1996; Sahbaie et al., 2009). Thus, altogetherthese data suggest the peripheral SP/NK1 system does not play arole in the development of the orofacial mechanical hyperalgesia,but participates in the mechanical hyperalgesia related to sensitiza-tion of spinal nerves.

Finally, in the present study we have investigated the influenceof peripheral NK1 receptor blockade in the orofacial formalin test.This test is a commonly used experimental animal model of persis-tent inflammatory pain in the trigeminal system. When formalin isinjected into the rats’ upper lip, it can be seen the typical biphasictime course of the response to formalin, with an early and short-lasting first phase followed, after a quiescent period, by a second,

prolonged (tonic) phase. Several pro-inflammatory and algogenicmediators have been described to participate of formalin-inducednociception (Chichorro et al., 2004), including SP (Lu et al., 2009).Lu et al. (2009) demonstrated that 2 h after orofacial formalininjection there was a significant increase in the expression of SPin the trigeminal spinal nucleus and distal cerebrospinal fluid-con-tacting neurons. In line with this observation, we have shown thatpre-treatment with a NK1 receptor antagonist caused a markedreduction on both phases of formalin-induced orofacial nocicep-tion. This finding is in agreement with studies that employed theformalin test in the rats’ paw, which have shown that pharmaco-logical blockade of NK1 receptors reduced the first or both phasesof nociception evoked by formalin (Birch et al., 1992; Traub,1996). Furthermore, there are several evidences that injection offormalin in the paw leads to an increase of SP levels in the dorsalhorn, probably, as the result of an increased biosynthesis, transportand release of this peptide in primary afferents and spinal neurons

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in response to long-lasting inflow of noxious messages (Kantneret al., 1986; Zhang et al., 1994). Altogether, these results reinforcethe role of SP in persistent pain by acting at both, the peripheral le-vel (present study), as well as at the central level, where most ofthe studies demonstrated increased expression of SP after formalininjection.

In summary, our results demonstrate that the SP/NK1 systemparticipates in the heat hyperalgesia associated with inflammationor nerve injury and in the persistent pain evoked by formalin. Thus,it can be suggested that SP represents an important mediator of thetrigeminal nociceptive process.

Acknowledgments

We gratefully acknowledge to Sanofi-Aventis for the donationof SR140333B. This study was supported by Fundação Araucária.Teodoro, F.C. and Tronco Júnior M.F. were recipients of a CAPES/PROF and UFPR/TN scholarships, respectively.

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