European Journal of Pharmacology 673 (2011) 13–19

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European Journal of Pharmacology

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Neuropharmacology and Analgesia

Acetylsalicylic acid inhibits α,β-meATP-induced facilitation of neck musclenociception in mice — Implications for acute treatment of tension-type headache

Dejan Ristic a, Peter Spangenberg b, Jens Ellrich a,⁎a Medical Physiology and Experimental Pharmacology Group, Department of Health Science and Technology, Medical Faculty, Aalborg University, Aalborg, Denmarkb Department of Neurosurgery, Ruhr University Bochum, Knappschaftskrankenhaus Bochum, Germany

⁎ Corresponding author at: Medical Physiology andGroup, Center for Sensory-Motor Interaction SMI, DepaTechnology, Medical Faculty, Aalborg University, FredAalborg, Denmark. Tel.: +49 151 1616 9842.

E-mail address: jellrich@hst.aau.dk (J. Ellrich).

0014-2999/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.ejphar.2011.10.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 September 2011Accepted 10 October 2011Available online 18 October 2011

Keywords:BrainstemCyclooxygenaseJaw-opening reflexAspirin®PainTension-type headache

Infusion of α,β-methylene ATP (α,β-meATP) into murine neck muscle facilitates brainstem nociception. Thisanimal experimental model is suggested to be appropriate for investigating pathophysiological mechanismsin tension-type headache. It was hypothesized that D-lysine acetylsalicylic acid (ASA, aspirin®) reverses thisα,β-meATP effect. Facilitation of neck muscle nociceptive processing was induced via bilateral infusion ofα,β-meATP into semispinal neck muscles (100 nM, 20 μl each) in 42 anesthetized mice. Brainstem nocicep-tion was monitored by the jaw-opening reflex elicited via electrical tongue stimulation. The hypothesiswas addressed by subsequent (15, 30, 60 mg/kg) and preceding (60 mg/kg) intraperitoneal ASA injection.Saline served as control to ASA solution. Subsequent ASA dose-dependently reversed α,β-meATP-inducedreflex facilitation and was the most prominent with 60 mg/kg. Preceding 60 mg/kg ASA prevented reflexfacilitation. Cyclooxygenases are involved in nociceptive transmission. Former experiments showed thatunspecific inhibition of cyclooxygenases does not alter the α,β-meATP effect. This suggests a specific modeof action of ASA. The concept is accepted that neck muscle nociception is involved in the pathophysiologyof tension-type headache. Thus, objective proof of ASA effects in this experimental model may emphasizeits major role in pharmacological treatment of tension-type headache attacks.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Tension-type headache is themost frequent primary headachewithimportant socioeconomic impact (Stovner et al., 2007). Increased peri-cranial muscle tenderness is a hallmark of tension-type headache(Ashina et al., 1999b; Buchgreitz et al., 2007). Acetylsalicylic acid (as-pirin®, ASA) is one of the most frequently applied over-the-counterdrugs in tension-type headache patients (Diener et al., 2005;Martinez-Martin et al., 2001; Peters et al., 1983; Steiner et al., 2003).ASA was first synthesized in 1897 fromwillow bark extracts and is rec-ognized for its analgesic, antiinflammatory and antipyretic action aswell as for prevention of both ischemic stroke and cardiovascularevents. The main mode of action is irreversible inhibition of cyclooxy-genase (COX) activity by acetylating serine in the active site of the en-zyme. This causes the inhibition of prostaglandin synthesis (Roth andMajerus, 1975; Vane, 1971; Vane and Botting, 2003). ASA-driven acet-ylation of COX-1 and COX-2 isoenzymes is most intensely studied.

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COX-1 is constitutively expressed in the endoplasmic reticulum ofmost cells (Morita et al., 1995) and regulates platelet activation and ag-gregation (Smith, 1992). Its inhibition causes reduced prostaglandinbiosynthesis resulting in antiplatelet actions of ASA. COX-2 is rapidlyinducible by e.g. inflammatory stimuli and contributes to inflammatoryresponses (Kujubu et al., 1991; Xie et al., 1991). Based on its mode ofaction on the COX system, solid evidence was provided for peripheralantinociceptive (Choi et al., 2001; Dogrul et al., 2007; Eschalier et al.,1983) and central analgetic effects of ASA (Ellrich et al., 1999; Tortoriciand Vanegas, 1995; Ulucan et al., 2003) in various animal models andmen (Bromm et al., 1991; Katsarava et al., 2004; Kaube et al., 2002).A translational mouse model on putative pathophysiologicalmechanisms of tension-type headache addresses the impact of noci-ceptive afferent input from neck muscles on central nervous systemnociceptive processing monitored by the jaw-opening reflex (Ellrich,2007; Ellrich and Makowska, 2007; Ellrich et al., 2010; Makowska etal., 2005a,b, 2006b; Reitz et al., 2009; Ristic et al., 2010). This reflex isan accepted model for the investigation of altered excitability in senso-ry brainstem neurons with convergent afferent input from differentcraniofacial tissues such as neck muscles. Intramuscular infusion(i.m.) of α,β-methylene adenosine 5′-triphosphate (α,β-meATP)provides noxious input from neck muscles that causes sustained reflexpotentiation. This indicates heterosynaptic facilitation due to access ofnociceptive afferents from neck muscle to the reflex neuronal networkin the brainstem. The present animal model is suggested to be a

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translational model for the investigation of pathophysiological aspectsof tension-type headache.

The present study addressed the effect of systemically adminis-tered ASA on α,β-meATP-induced facilitation of neck muscle nocicep-tive processing in mice. Particular focus lied on ASA applicationsubsequent to α,β-meATP infusion under stable facilitation. Thiswould resemble daily routine in acute medication of tension-typeheadache attacks.

2. Methods

Electrophysiological experiments were performed in adult maleC57BL/6 mice (approximately 12 weeks old; 22 to 33 g; Taconic,www.taconic.com). All procedures received institutional approvalfrom the local ethics committee. The principles were followed for lab-oratory animal care and use of laboratory animals (European CouncilDirective of November 24, 1986(86/609/EEC)). All efforts were madeto minimize animal suffering and to use only the number of animalsnecessary to produce reliable scientific data.

2.1. Animal preparation

Detailed description of anesthesia, surgery and electrophysiologi-cal recording has been published previously (Ellrich and Wesselak,2003). Briefly, mice were anesthetized by an initial intraperitoneal(i.p.) injection of a 0.5% pentobarbital sodium salt solution(Amgros I/S, Copenhagen, Denmark, www.amgros.dk) with a doseof 70 mg/kg. Depth of anesthesia was checked by ensuring thatnoxious pinch stimulation (blunt forceps) of hindpaw, forepaw, andear did not evoke any sensorimotor reflexes. When the animal wassufficiently deeply anesthetized, the skin of the throat and neckwere carefully shaved and lidocaine hydrochloride gel (Xylocaine®2%, AstraZeneca A/S, Albertslund, Denmark, www.astrazeneca.com)was applied to the skin of the throat in order to induce local anesthe-sia. Dexpanthenol eye ointment (Bepanthen®, Roche, Grenzach-Wyhlen, Germany, www.roche.com) was applied to the cornea andconjunctiva of both eyes in order to protect them from drying. Theright external jugular vein was catheterized for continuous adminis-tration of a 2% methohexital sodium salt solution (Brevimytal®Hikma, Hikma Pharmaceuticals PLC, London, United Kingdom,www.hikma.de) with a dose of 60 mg/kg per hour corresponding toa flow rate of about 0.07 ml/h for a 23 g mouse. In order to recordelectromyographic activity (EMG) and the jaw-opening reflex via adifferential amplifier, a pair of Teflon-coated stainless steel wires(140 μm diameter) was inserted into the right anterior digastric mus-cle. After tracheotomy, animals were placed in a stereotactic frame.They were artificially respired with a stroke volume of about 150 μland about 200 strokes per min (MiniVent Model 845, Harvard Appa-ratus, Hugo Sachs Elektronik, March-Hugstetten, Germany, www.harvardapparatus.com). Body core temperature was maintained at37.3 °C with a heating blanket and a fine rectal thermal probe (FMIGmbH, Seeheim-Ober-Beerbach, Germany, www.fmigmbh.de). Oneplatinum needle electrode each (300 μm diameter) was subcutane-ously inserted into right forepaw and left hind paw to record the elec-trocardiogram (ECG) via a differential amplifier. Two stainless steelneedle electrodes (150 μm diameter) were longitudinally insertedinto the tongue musculature (parallel, 2 mm distance) in order toapply electrical stimuli and to evoke the jaw-opening reflex. Theoral cavity was filled up with white vaseline (Riemser ArzneimittelAG, Greifswald - Insel Riems, Germany, www.riemser.de) in orderto protect oral mucous membrane from drying. Neck skin was locallyanesthetized via Xylocaine®. Semispinal neck muscles on both sideswere carefully exposed. One injection cannula each (0.4 mm diame-ter) was inserted into the muscle belly of both semispinal neckmuscles. Each cannula was connected via thin and short tubing to aliquid switch (CMA/110, CMA Microdialysis AB, Solna, Sweden,

www.microdialysis.se). Glass microsyringes (1 ml) were connectedto the liquid switch by thin tubing and were fixed in a microdialysispump (CMA 102, www.microdialysis.se). This procedure allowed bi-lateral induction of noxious input from neck muscles in order tomimic bilateral neck muscle pain in tension-type headache patients.1 μM α,β-meATP (Sigma-Aldrich Chemie Gmbh, Munich, Germany,www.sigmaaldrich.com) was i.m. administered with a volume of20 μl per semispinal neck muscle during a time period of 1 min.

2.2. Electrophysiology, drug administration and experimental setup

After preparation, the anesthetized animal was rested for at least1 h. During this time period, levels of anesthesia and heart ratewere routinely checked and documented. The depth of anesthesiawas maintained. All electrical signals (EMG, ECG) were recorded bybioamplifiers. EMG signals led into a data collection system (CEDMicro1401, CED, Cambridge Electronic Design Limited, Cambridge,United Kingdom, www.ced.co.uk) and a personal computer usingSignal® software program (CED, Cambridge Electronic Design Limited,Cambridge, United Kingdom, www.ced.co.uk).

The jaw-opening reflex was elicited by electrical stimulation of af-ferent nerve fibers in the tongue musculature via two needleelectrodes with rectangular electrical pulses of 500 μs duration anda stimulation frequency of 0.1 Hz (Fig. 1). Reflex responses wererecorded in the anterior digastric muscle by electromyography(EMG). A reflex response elicited by a single stimulus is quantifiedby its onset latency, duration and integral (Fig. 1A). The durationcovers the time window between onset latency and end of the reflexresponse in the digastric muscle EMG. In this time window, the reflexintegral (area under the curve) was calculated for each sweep. Theelectrical threshold of the jaw-opening reflex was determined by ap-plying one series of increasing and decreasing stimulus intensitiesfrom 0 to 2 mA in steps of 100 μA. The reflex threshold was definedas the lowest stimulus intensity that just evoked a reflex response.Test stimulus intensity was adjusted to approximately 150% of thereflex threshold. The jaw-opening reflex was evoked in series ofeight stimuli (Fig. 1B). These series were repeated every 5 min.Drugs were aspirin® i.v. (ASA, d,L-lysinacetylsalicylat, Bayer Vital,Leverkusen, Germany), sodium chloride solution and α,β-meATP.α,β-meATP was resolved in isotonic saline solution. ASA was resolvedin aqua ad iniectibilia. As control served intraperitoneal injection(i.p.) of a saline solution isoosmolar to 15 mg/ml ASA (correspondingto 60 mg/kg). Isotonic saline was diluted with aqua ad iniectibilia to amean osmolarity of 190 mosmol/l.

Two different experiments were conducted (Fig. 1) in 42 mice(n=7 each). In the first experiment, the effect of subsequently ad-ministered ASA onα,β-meATP-induced reflex facilitation was investi-gated (Fig. 1C). After stable baseline recording, α,β-meATP wasinfused i.m. For the following 60 min, purinergic reflex facilitationwas observed. Then, either saline or ASA was administered i.p. andthe reflex was then monitored for further 90 min. Three differentdosages of ASA (15, 30 and 60 mg/kg) were applied (100 μl)according to preceding pilot experiments and recommended maxi-mal daily ASA dosage in humans (60 mg/kg).

In the second experiment, the effect of precedingly administeredASA or saline on α,β-meATP-induced reflex facilitation was investi-gated (Fig. 1D, E). After stable baseline recordings, the maximal ASAdosage (60 mg/kg) was i.p. injected and the reflex was monitoredfor the following 60 min. α,β-meATP was i.m. infused. The reflexwas monitored for at least 60 min. In a further series, saline was i.p.administered after stable baseline recordings and the reflex wasrecorded for the following 30 min. Then, α,β-meATP was i.m. infusedand the reflex was recorded for at least 60 min. I.m. infusion of isoton-ic saline as a vehicle for α,β-meATP did not affect the jaw-opening re-flex (Reitz et al., 2009). These published experiments were not

Fig. 1. Stimulation protocol. (A) The jaw-opening reflex was evoked by electrical stim-ulation of afferent nerve fibers in the tongue via two needle electrodes. Reflex re-sponses were recorded in the anterior digastric muscle by electromyography (EMG)(a.u.: arbitrary units). The graph shows a typical reflex response elicited by a singlestimulus. Dotted lines mark onset and end latency which determine the reflex dura-tion. In this time window, the reflex integral (area under the curve in gray) was ana-lyzed as the most prominent reflex parameter. (B) The jaw-opening reflex wasevoked in a series of eight stimuli each (black bars). Reflex series were repeatedevery 5 min (gray bars). (C, D, E) In two different experimental groups, drugs were ap-plied intraperitoneally (i.p.) or intramuscularly (i.m.) into both semispinal neck mus-cles as marked by arrows. (C) After three baseline series, α,β-meATP was i.m.administered and the reflex was recorded for the following 60 min. Subsequently, sa-line or ASA (15, 30, 60 mg/kg) was intraperitoneally (i.p.) injected. The reflex wasrecorded for further 90 min. (D) After three baseline series, 60 mg/kg ASA was i.p.injected and the reflex recorded for further 60 min. Then, α,β-meATP was i.m. infusedand the jaw-opening reflex was recorded for the following 60 min. (E) After threebaseline series, saline was i.p. injected and the jaw-opening reflex recorded for further30 min. Then, α,β-meATP was i.m. infused and the reflex was recorded for 60 min.

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repeated in order to minimize the amount of animals used in thisstudy.

2.3. Data analysis

Onset latency, duration, and integral as the area under the curve ofthe jaw-opening reflex were analyzed in each single sweep. In recentstudies, reflex integral turned out to be the key parameter of reflex al-teration in the present animal model. Indeed, the reflex integral part-ly depends on the duration of reflex. For the sake of clarity andreadability, the present manuscript focuses on statistical analysis ofreflex integral expressed as percentage changes from baseline.Arithmetic mean and standard error of mean (mean±S.E.M.) werecalculated. For the effect of subsequent ASA or saline on the facilitatedjaw-opening reflex, percentage changes from baseline based upon av-erage reflex integrals in the time window from 50 min to 60 min(Fig. 1C). For the effect of preceding ASA or saline on ATP-induced fa-cilitation, percentage changes from baseline based upon average

reflexes in the time window from −15 min to −5 min (Fig. 1D).For the effect of preceding ASA on basal reflex, percentage changesbased upon average basal reflexes (Fig. 1D). Depending on data distri-bution, comparison within groups was performed via one way re-peated measures ANOVA (one-way RM ANOVA) or Friedman one-way repeated measures ANOVA. Differences between and withingroups were analyzed via two-way repeated measures analysis ofvariance (two-way RM ANOVA, F and P values). Factors were sub-stance and time. Data was also analyzed for interaction betweenthese factors. Depending on data distribution, data was ranktransformed for two-way RM ANOVA. Post-hoc comparisons wereperformed with the Student–Newman–Keuls test (SNK, P values).Level of significance was set to Pb0.05.

3. Results

In 42 mice, electrical tongue stimulation elicited the jaw-openingreflex with a threshold intensity of 0.7±0.2 μA (mean±S.E.M). Teststimulus intensity was adjusted to 1.0±0.3 μA corresponding to145±4% of the reflex threshold.

3.1. Effects of saline and subsequent ASA on establishedα,β-meATP-induced reflex facilitation (Figs. 2 and 3)

I.m. α,β-meATP reliably induced reflex facilitation. The averageoverall (n=28) reflex values before subsequent saline or ASA admin-istration (50 to 60 min) were 155±12% from baseline values (−15to −5 min). Basal reflex duration showed a low variability with 6±0.1 ms (−15 to −5 min) before i.m. α,β-meATP infusion amonganimals.

In both subsequent ASA and saline groups, α,β-meATP reliably in-duced an increase of the jaw-opening reflex in 28 mice. Time courseof reflex integrals revealed purinergic facilitation and its reversalwith 60 mg/kg ASA to basal levels (Fig. 2A). Presented control datawith subsequent i.p. saline demonstrates persistent purinergic facili-tation of brainstem nociceptive processing for at least 155 min(Figs. 2B, 3). Average reflex sweeps illustrated different levels ofbrainstem nociceptive processing between groups with subsequentsaline and 60 mg/kg ASA (Fig. 2B). Recorded sweeps demonstrateclearly divergent levels of brainstem nociceptive processing with sub-sequent ASA and saline whereas reflexes seemed similar within base-line and facilitation time segments. This α,β-meATP-induced reflexfacilitation was reversed with higher ASA dosages in a dose depen-dent manner (Figs. 2, 3). Comparison within groups revealed reversalof the facilitated jaw-opening reflex with subsequently administeredASA dosages of 30 and 60 mg/kg (Fig. 3A). In contrast, subsequentsaline and low dose ASA (15 mg/kg) showed no significant impact.

Reflex time course analysis was performed on reflex integralswithin 90 min after vehicle and ASA administration (Fig. 3B). For fur-ther analysis, data was averaged in 10 min intervals for each group.Data was rank transformed in order to perform a two-way repeatedmeasure ANOVA. There were significant effects of factors time, sub-stance and interaction (Fig. 3B). For the sake of comprehension,untransformed data is shown. Differences between groups occurredfrom 80 min on and persisted until the end of the experiment. At90 min, the facilitated reflex was stably reverted to basal levels with60 mg/kg ASA. At 110 min, there were no more differences between30 and 60 mg/kg ASA. At 150 min, reflex integrals differed between15 mg/kg ASA and control group.

3.2. Effects of preceding saline and ASA on α,β-meATP-induced reflexfacilitation (Fig. 4)

In 14 mice (n=7 each), the impact of precedingly applied salineor 60 mg/kg ASA on the development of α,β-meATP-induced reflexfacilitation was assessed (Fig. 4). Comparison within groups revealed

Fig. 2. Reversal of α,β-meATP-induced reflex facilitation with subsequent 60 mg/kgASA. Exemplary data sets demonstrate the complete time course of recorded jaw-opening reflex integrals (JOR) and original reflex recordings at distinct time points(n=1 each). (A) The time course shows JOR integrals covering baseline (gray circles),facilitation (black upward triangles) and subsequent 60 mg/kg ASA (dark gray down-ward triangles) administration periods in 5 min intervals (n=1). Reflex integral datais shown in arbitrary units (a.u.). One value represents the average of eight reflex inte-grals. Arrows indicate time point of intramuscular (i.m.) α,β-meATP and intraperitone-al (i.p.) ASA administration. (B) Eight rectified sweeps are averaged under baselineconditions (−5 min, gray), 1 h after i.m. α,β-meATP infusion (60 min, black) and90 min after subsequent i.p. administration (155 min, dark gray) of either saline(left) or 60 mg/kg ASA (right, n=1 each). Electromyographic activity is given in arbi-trary units (a.u.). Facilitation of jaw-opening reflex was reversed to basal levels withsubsequent ASA (60 mg/kg) whereas facilitated digastric muscle EMG remainedunchanged or facilitation even continued with subsequent saline injection (control).

Fig. 3. Dose-dependent reversal of reflex facilitation with subsequent ASA. Duringestablished reflex facilitation 60 min after intramuscular α,β-meATP, ASA (15, 30,60 mg/kg) or saline (n=7 each) was intraperitoneally applied (i.p.). (A) Reflex facili-tation remained unchanged with subsequent saline and 15 mg/kg ASA injectionwhereas integrals significantly decreased after application of 30 and 60 mg/kg ASA(one-way RM ANOVA, Friedman ANOVA). Data is presented as mean±S.E.M. (B) Aver-age reflex integral changes within 10 min intervals after i.p. injection of saline or ASA(15, 30, 60 mg/kg) are presented as box plots (gray line: arithmetic mean) of pre-rank transformed data. F and P values correspond to two-way repeated measuresANOVA on rank transformed data. Student–Newman–Keuls post-hoc tests indicatedsignificant differences. (*: Pb0.05).

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prevention of purinergic facilitation with precedingly administeredASA (60 mg/kg). In contrast, facilitation of the jaw-opening reflex de-veloped with preceding saline within 60 min (Fig. 4A). Reflex timecourse analysis was performed on reflex integrals within 60 minafter saline and 60 mg/kg ASA injection (Fig. 4B). For further analysis,data was accordingly averaged in 10 min intervals for each group.Two-way RM-ANOVA revealed significant effects of time and interac-tion. Differences between groups occurred at 50 and 60 min. Reflexintegrals with preceding 60 mg/kg ASA were lower than with preced-ing saline. The basal jaw-opening reflex was unaffected by both pre-ceding 60 mg/kg ASA administration (Χ2=22.643, n.s.) and bypreceding saline administration (F=1.3, n.s.).

4. Discussion

The results demonstrate inhibitory effects of ASA on α,β-meATP-induced facilitation of neck muscle nociception in anesthetizedmice. Both subsequent and preceding administration of ASA reversedand prevented purinergic facilitation, respectively.

Electrical stimulation of afferent nerve fibers in the tongue muscu-lature reliably evokes the jaw-opening reflex via a brainstem reflex

network (Ellrich and Wesselak, 2003). Sensory neurons in the spinaltrigeminal nucleus receive convergent input from primary afferents(neck muscle, tongue) and project onto digastric motoneurons(Kidokoro et al., 1968; Tsai et al., 1999). Noxious input from neckmuscles leads to sustained reflex potentiation evoked by α,β-meATP(Ellrich and Makowska, 2007; Makowska et al., 2006b; Reitz et al.,2009; Ristic et al., 2010). α,β-meATP interacts with purinergic P2X3

and P2X2/3 receptors (Khakh, 2001) which in turn mediate nociception(Cockayne et al., 2005; North, 2004; Souslova et al., 2001), probably viagroup III muscle afferents (Ellrich andMakowska, 2007). This addition-al excitatory input from neckmuscle nociceptors facilitates the tongue-evoked reflex by heterosynaptic access. The sustained purinergic reflexfacilitation persists for at least 4 h (Reitz et al., 2009). In this study, thefacilitation was unaffected by saline and was documented for 155 min.Purinergic facilitation develops within 30 min and reaches a plateauwithin around 60 to 90 min (Makowska et al., 2006b; Reitz et al.,2009; Ristic et al., 2010). Marginal increases and slight variations inthe facilitated reflex may occur in that time window after α,β-meATPinfusion (Reitz et al., 2009). Presented control data (Fig. 3) mightimply a facilitative effect of systemic saline on the jaw-opening reflex.

Fig. 4. Prevention of α,β-meATP-induced reflex facilitation with preceding 60 mg/kgASA. Preceding to intramuscular (i.m.) α,β-meATP infusion, saline or 60 mg/kg ASAwere injected intraperitoneally (i.p.). (A) Only with preceding i.p. injection of salinebut not with 60 mg/kg ASA, a significant reflex facilitation established after i.m. infu-sion of α,β-meATP into semispinal neck muscles (one-way repeated measuresANOVA and Friedman ANOVA). Data is presented as mean±S.E.M. (B) Average reflexintegral changes within 10 min time frames after i.p. injection of saline or 60 mg/kgASA and subsequent i.m. α,β-meATP are presented as box plots (gray line: arithmeticmean). F and P values correspond to two-way repeated measures ANOVA. Student–Newman–Keuls post-hoc tests indicated significant differences (*: Pb0.05).

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Firstly, these changes were not significant. Furthermore, both basal re-flex and sustained reflex facilitation remain unaffected by both i.m. andi.p. saline administration (Ellrich and Makowska, 2007; Makowska etal., 2005a; Reitz et al., 2009; Ristic et al., 2010). This α,β-meATP effectseems independent from mechanically induced reflex alteration (vol-ume injection). Instead, it implies pharmacological actions of α,β-meATP. The present animal model is suggested to be a translationalmodel for the investigation of pathophysiological aspects of tension-type headache (Bendtsen and Jensen, 2006).

The analgesic properties of ASA for the treatment of tension-typeheadache are well documented in terms of pain relief (Diener et al.,2005; Langemark and Olesen, 1987; Martinez-Martin et al., 2001;Steiner et al., 2003). Typically, dosages of 1000 mg ranging from650 mg to 1200 mg are orally administered. In case of self-medication with ASA, the German Migraine and Headache Society(DMKG, http://www.dmkg.de) recommends a single oral dosage of1000 mg per day. Antinociceptive and analgetic dosages of ASA in ani-mal experiments range from 10 to 400 mg/kg depending on the modelused (Carlsson et al., 1988; Dogrul et al., 2007; Ellrich et al., 1999;Kaube et al., 1993; Pardutz et al., 2004; Paul-Clark et al., 2004; Tortoriciand Vanegas, 1995). ASA ED50 values for humans and animals – andthus ASA efficacy – may not be directly comparable. Previously, a

correlation was established between human and murine ED50 valuesenabling dosage estimation from mice to humans (Pong et al., 1985).Accordingly, in the presented study applied dosages of 15, 30 and60mg/kg in mice would correspond to single oral dosages of 660,780 and 1030 mg in humans. Thus, applied ASA dosages in this studycover a clinically relevant range.

A peripheral antinociceptive and analgetic mode of action for ASAis usually associated with its antiinflammatory action as documentedin numerous models on inflammatory pain. However, a sole peripher-al mechanism cannot elucidate demonstrated ASA antinociceptivemodes of action anchored presumably in the central nervous system.The exteroceptive suppression periods of activity in the temporalmuscle, an inhibitory antinociceptive brainstem reflex, were in-creased with effervescent ASA (1200 mg) in tension-type headachepatients (Göbel et al., 1992). Oral administration of ASA (1000 mg)reduced pain ratings and somatosensory evoked potentials in ahuman model of phasic pain with intracutaneous electrical stimula-tion (Bromm et al., 1991; Scharein and Bromm, 1998). Data fromvarious animal models on non-inflammatory pain support a possiblecentral mode of action for ASA. I.v. administration of ASA(ED50:74 mg/kg) reduced activity in the dorsomedial part of theventral nucleus in rat thalamus after electrical stimulation of nocicep-tive afferents in the sural nerve whereas the nociceptive activity wasnot affected in ascending axons of the spinal cord (Carlsson et al.,1988). In cats, i.v. ASA (30 mg/kg) induced inhibition of activity inbrainstem nuclei following electrical stimulation of the superiorsagittal sinus which was also inducible with the COX inhibitorketorolac (Kaube et al., 1993).

Consequently, ASA could exert inhibitory effects on central tri-geminal neurons via involvement of COX-related mechanisms.Further evidence on possible central action derived from studies onthe effect of ASA microinjection into the periaqueductal gray (PAG),specifically on on- and off-cells in the rostral ventromedial medulla(RVM) in animal models of heat-elicited tail flick (Vanegas andTortorici, 2002). PAG and RVM are structures of the endogenouspain control system capable of inhibiting nociceptive processing(Mason, 1999). The PAG is rich in opioid receptors and is a majortarget of analgesic action in the central nervous system. Analgesiainduced via μ-opioid related mechanisms can be potentiated by COXinhibitors like ASA (Vaughan et al., 1997). Thus, a central antinocicep-tive action of ASA seems probable in the present study reducing theeffect of increased nociceptive input from the neck muscles evokedby α,β-meATP. This central effect alone could be accountable forobserved ASA effects in this study (Figs. 2–4).

Nevertheless, it remains not entirely clear what the molecular tar-get of ASA in the central nervous system for antinociception is. Thismode of action is possibly uncoupled from COX inhibition. The COX-unspecific inhibitor indomethacin failed to exert antinociception onthe α,β-meATP effect in the current model (Makowska et al.,2006a). However, indomethacin was only applied subsequently toadministration of α,β-meATP. The facilitative action of α,β-meATPon brainstem nociceptive processing seems to occur in an inductionand a maintenance phase that are distinct from each other (Ristic etal., 2010). Unspecific inhibition of nitric oxide synthase (NOS)prevented and reversed the α,β-meATP effect (Ellrich et al., 2010).Inhibition of neuronal nNOS resulted in the prevention of purinergicfacilitation whereas its reversal was only achievable with inducibleiNOS inhibition, not with nNOS (Ristic et al., 2010). Thus, divergentASA and indomethacin action on the α,β-meATP-induced facilitationof brainstem nociceptive processing is possibly an at least partly COX-independent mechanism. Divergent action of ASA and indomethacinwas also reported in other animal models (Hunskaar et al., 1986;Paul-Clark et al., 2004; Taubert et al., 2004). In the murine formalintest, ASA dose-dependently reduced both early and late responseswhereas indomethacin reduced only the late responses (Hunskaaret al., 1986). Early phase responses supposedly reflect immediate

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nociceptive whereas the late phase reflects inflammatory responsesimplying divergent antinociceptive properties of ASA andindomethacin.

Moreover, a possible impact of ASA on nociceptive processingmight lie in its ability to interact with nitric oxide (NO) synthesizingNOS isoenzymes (Pardutz et al., 2000, 2004). NO and NOS isoenzymesplay an important role not only in nociceptive processing but proba-bly also in the pathophysiology of tension-type headache (Ristic etal., 2010). NO is implicated in pathophysiological mechanisms of cen-tral sensitization (Schmidtko et al., 2009). In chronic tension-typeheadache patients, unspecific NOS inhibition reduced muscle hard-ness and headache intensity (Ashina et al., 1999a,c). Another ASAmode of actions comprises of its dosage dependent effect on mitochon-drial oxidative phosphorylation (Somasundaram et al., 2000). So far, noevidence provides the latter route as antinociceptive mechanisms forASA. Taken together, a central nervous system antinociceptive modeof action for ASA via COX and/or NOSmay be accountable for presentedobservations.

The duration of a tension-type headache attack lasts between30 min and 7 days in both episodic and chronic type in humans(Headache Classification Subcommittee of the International headachesociety, 2004). In the current murine model, facilitation of brainstemnociceptive processing could be observed for at least 4 h (Reitz et al.,2009) resembling fulfilled IHS diagnostic criteria of tension-typeheadache for attack duration. Nociceptive input from neck musclesis regarded as the main trigger for episodic tension-type headachewhereas with prolonged exposition, central sensitization might be in-duced leading to chronification. The setup of the applied murinemodel resembles rather an acute than a chronic model due to single,non-repetitive administration of α,β-meATP. Nevertheless, it mightincorporate transition of pathophysiological mechanisms from epi-sodic to chronic tension-type headache. α,β-meATP-induced facilita-tion of brainstem nociceptive processing establishes in theaforementioned distinct steps, a first induction and a second mainte-nance phase. This two-step mechanism could mirror the transitionfrom an initially peripherally driven induction to a centrallyestablished maintenance phase. Within this scenario, ASA could re-duce periphery-induced increase of central nociceptive processing.Furthermore, ASA could reverse established facilitation of nociceptiveprocessing in the brainstem— independent from the peripheral drive.In analogy to these two distinct phases, a recently applied reflexmodel in tension-type headache patients demonstrated a similar di-vergence (Nardone and Tezzon, 2003). The so-called trigemino-cervical reflex showed abnormal size or latency predominantly pre-sent in chronic but not in episodic tension-type headache patientsimplying established central sensitization with chronification.

5. Conclusion

Demonstrated ASA effects on facilitated brainstem nociceptiveprocessing in this translational murine model supports the treatmentof acute attacks of tension-type headache with ASA.

Acknowledgments

This research project was supported by grants of the GermanHeadache Consortium (01EM0516, project A3), the Lundbeck founda-tion (R17/A1566) and Bayer HealthCare (Leverkusen, Germany). Weappreciate fruitful discussions with Dr. Uwe Gessner and Dr. Mar-ianne Petersen-Braun.

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