Exp Brain Res (2008) 189:1–10

DOI 10.1007/s00221-008-1399-4

RESEARCH ARTICLE

EVects of NGF-induced muscle sensitization on proprioception and nociception

Peter Svensson · Kelun Wang · Lars Arendt-Nielsen · Brian E. Cairns

Received: 26 November 2007 / Accepted: 16 April 2008 / Published online: 14 May 2008© Springer-Verlag 2008

Abstract Temporomandibular disorders (TMDs) areassociated with perturbation of proprioceptive and noci-ceptive function. Recent studies have shown that injec-tion of the neurotrophic protein nerve growth factor(NGF) into the masseter muscle causes sensitization tomechanical pressure stimuli; however, it is not clear ifvibration sense and jaw stretch reXexes as measures ofproprioceptive function as well as glutamate-evokedpain are also altered. We tested the hypothesis that NGF-induced mechanical sensitization would be associatedwith changes in vibration sense and stretch reXex sensi-tivity as well as facilitation of glutamate-evoked painresponses. A double-blind, randomized and placebo-controlled study was conducted on 14 healthy men. Inone session subjects received an injection of NGF (5 �gin 0.2 ml) into the masseter muscle and in a control ses-sion an injection of buVered isotonic saline (0.9%,0.2 ml). Subjects assessed their pain intensity on a0–10 cm visual analogue scale (VAS) for 15 min after

the injections. Pressure pain thresholds (PPT), vibrationsense and jaw stretch reXexes were recorded at baselineand 1, 2, 3 and 24 h post-injection. The sensitivity toinjections of glutamate into the masseter muscle (1 M,0.2 ml) was assessed after 24 h. ANOVAs were used toassess signiWcant diVerences. NGF did not cause morepain than isotonic saline, but signiWcantly reduced PPTs1, 2, 3 and 24 h post-injection (P < 0.001) whereas iso-tonic saline had no eVects on PPTs (P = 0.583). Theinjection of glutamate after 24 h was associated withreduced PPTs in both sessions, but the PPTs remainedlower in the NGF pretreated masseter than in the controlmasseter (P < 0.001). Ratings of vibratory stimuli andthe normalized amplitude of the jaw stretch reXex werenot aVected by the NGF-induced sensitization; however,after glutamate injection a signiWcant increase in thestretch reXex was observed in the injected masseter mus-cle in both sessions (P = 0.002). There were no signiW-cant diVerences in the perceived pain intensity of theglutamate injection between the masseter muscle pre-treated with NGF or control (P > 0.414), although theglutamate-evoked pain drawing areas were larger for theNGF-pretreated masseter muscle (P = 0.009). In conclu-sion, this study conWrms that masseter muscle injectionof NGF is associated with a distinct and prolonged sen-sitization to mechanical stimuli, but without an eVect onlarge-diameter mechanoreceptive and the muscle spin-dle aVerents. Additional challenge of the NGF pretreatedmuscle with glutamate did not indicate a conspicuoussensitization to noxious chemical stimuli. These Wnd-ings are discussed in terms of the concept of “proprio-ceptive allodynia”.

Keywords TMD pain · NGF · Stretch reXex · Proprioceptive allodynia

P. Svensson (&)Department of Clinical Oral Physiology, School of Dentistry, University of Aarhus, Vennelyst Boulevard 9, 8000 Aarhus C, Denmarke-mail: psvensson@odont.au.dk

P. SvenssonDepartment of Oral and Maxillofacial Surgery, Aarhus University Hospital, 8000 Aarhus C, Denmark

P. Svensson · K. Wang · L. Arendt-NielsenOrofacial Pain Laboratory, Center for Sensory–Motor Interaction, Aalborg University, 9220 Aalborg, Denmark

B. E. CairnsFaculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, V6T 1Z3, Vancouver, Canada

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Introduction

It is well established that the neurotrophin nerve growthfactor (NGF) interferes with nociceptive transmission andleads to sensitization of peripheral tissues (Pezet andMcMahon 2006; McMahon et al. 2006). We have shown inhumans that direct administration of small doses of NGF(5 �g) into the masseter muscle of healthy male and femalesubjects does not cause any spontaneous pain, but is associ-ated with a prolonged period of increased sensitivity tomechanical stimuli (Svensson et al. 2003a). Administrationof human NGF into the masseter muscle of rats does notevoke aVerent discharge but is associated with signiWcantdecreases in the mechanical thresholds of slowly-conduct-ing masseter muscle aVerent Wbers, which indicates thatthere is a peripheral component to the sensitization process(Mann et al. 2006). Injections of human NGF into themouse cervical muscles have shown long-lasting facilita-tory eVects on the jaw-opening reXex, which may indicatethat there is also a central component to NGF-inducedmechanical sensitization (Makowska et al. 2005). The Wnd-ings that intramuscular injection of NGF appears to recruitboth peripheral and central sensitization mechanisms sug-gests that injection of NGF into the masticatory musclemight exert a broader inXuence on sensitivity to noxiousand innocuous stimuli than previously recognized.

Recent studies have implicated large-diameter mechano-receptive aVerents in the generation of delayed onset mus-cle soreness (DOMS) whereas these aVerent Wbersappeared to have no inXuence on hypertonic saline-evokedmuscle pain (Weerakkody et al. 2001, 2003). It has beenproposed that this could represent a mechanism resemblingsecondary hyperalgesia (i.e., enhanced pain evoked by anormally painful stimulus in an area adjacent to an injury)and the term “proprioceptive allodynia” (i.e., pain due to anormally non-painful stimulus which activates propriocep-tive aVerent nerve Wbers) has been suggested (Treede et al.2006). Vibration is an eVective stimulus of large-diametermechanoreceptive aVerents and muscle spindles and impor-tantly in this context it has been shown that vibration senseis disturbed, although not painful, in myofascial TMD painpatients (Hollins et al. 1996, 2001; Hollins and Sigurdsson1998). Moreover, muscle spindle aVerents in addition toalpha-motoneuron excitability inXuence the human jawstretch reXex, which our previous studies have shown isfacilitated by experimental jaw muscle pain (Wang et al.2000, 2002, 2004; Svensson et al. 2001; Cairns et al.2003a; Peddireddy et al. 2005). This muscle pain-relatedfacilitation of the stretch reXex is not seen in conditionswith experimentally-evoked jaw muscle fatigue (van Selmset al. 2005) or in conditions with high levels of pain fromnon-muscular tissues in the craniofacial region (Biasiottaet al. 2007), which suggests a diVerential eVect of trigemi-

nal aVerent inputs on jaw stretch reXex sensitivity. In addi-tion, a series of animal studies have demonstrated asigniWcant eVect of nociceptive inputs from jaw muscles onthe sensitivity of muscle spindle aVerents recorded in themesencephalic trigeminal (mesV) nucleus (for a review seeCapra et al. 2007). This modulatory eVect appears to becrucially dependent on functional integrity of the caudal tri-geminal nuclei and to involve gamma-motoneurons (Capraet al. 2007). Based on the suggestion of “proprioceptiveallodynia” and our own observations that intramuscularadministration of NGF in humans has a pronounced eVecton jaw motor function (Svensson et al. 2003a, 2008) and inanimals on trigeminal reXex pathways (Makowska et al. 2005),we decided to test the eVects of NGF-evoked sensitizationon sensitivity to vibration and human jaw stretch reXexes.

Finally, recent observations in patients with primaryWbromyalgia have shown signiWcant correlations betweenlevels of NGF and glutamate in the cerebrospinal Xuid (Sar-chielli et al. 2007) and it was suggested that NGF could exertat least part of its action through a glutamatergic mechanism(Jarvis et al. 1997; Michael et al. 1997). Furthermore, this isin line with the Wnding that NGF induces phosphorylation ofthe NMDA receptor subunits NR2A and NR2B in the spinalcord (Di Luca et al. 2001) and a recent observation of NR2Breceptors on masseter muscle aVerent Wbers and their co-localization with the tyrosine kinase A (TrkA) and p75receptors on which NGF acts (Dong et al. 2007; Svenssonet al. 2008). We have in recent studies shown that injectionof glutamate into the masseter muscle is associated with painand a signiWcant decrease in pressure pain thresholds (PPT),i.e., mechanical sensitization, that was mediated, in part,through NMDA receptor activation as well as increased jawstretch reXex responses (Cairns et al. 2001, 2003a, b, 2006;Svensson et al. 2003b); however, how pretreatment of themuscle with NGF might aVect glutamate-evoked pain andsensitization has not been investigated. Therefore, we alsotested the eVects of painful glutamate injections in the pres-ence of NGF-induced mechanical sensitization.

The present study speciWcally tested the hypothesis thatNGF-induced mechanical sensitization of the massetermuscle would be associated with a facilitation of the vibra-tion sense and the human jaw stretch reXex and that a sub-sequent injection of glutamate would evoke greater painresponses in NGF pretreated muscle than in isotonic salinepretreated muscle.

Materials and methods

Subjects

A total of 14 healthy male volunteers with a mean age of25.1 § 0.8 years were recruited from among the students at

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Aalborg University. None of the subjects reported painfulTMD or other orofacial pain complaints or had taken anal-gesics within 48 h of the investigation. The subjects werescreened for TMD in accordance with the research diagnos-tic criteria (RDC) for TMD (Dworkin and LeResche 1992).The local Ethics Committee approved the experiments(20040171) and informed consent was obtained from allparticipants. The study was conducted in accordance withthe Declaration of Helsinki.

Study design

The study was performed in a randomized, double-blinded,placebo-controlled manner. All subjects participated intwo sessions: one session with injection of NGF and theother session with injection of buVered isotonic saline. Thesequence of the NGF and buVered isotonic saline (control)injections was randomized. The subjects were asked toscore the perceived intensity of pain on a 0–10 cm elec-tronic visual analogue scale (VAS) after each injection.One examiner prepared the NGF or saline injection whileexaminer two performed all assessments at baseline beforeinjection of NGF or saline. The tests performed wereassessment of PPT in the masseter muscles on both sides,numerical rating scale (NRS) scores of vibratory stimuliapplied to the masseter on the injection side and bilateralrecordings of the stretch reXex responses in the masseterand anterior temporalis muscles. All subjects described thesensation of pain after the injections on a Danish versionof the McGill Pain Questionnaire (MPQ) (Drewes et al.1993) and drew the distribution of pain on a anatomicalWgure of the head (Melzack 1975). One, two and threehours after the injection, examiner two repeated the mea-surements. In both sessions, the same measurements wererepeated 1 day after the NGF or isotonic saline injections.Then an injection of 0.2 ml, 1 M glutamate was made intothe NGF or isotonic saline pretreated muscle and subjectsasked to score the perceived pain intensity on the 10-cmVAS, to Wll out the MPQ and draw the distribution of pain.PPTs, NRS scores to vibration and stretch reXexes weremeasured 2 min (during ongoing glutamate-evoked pain)and 1 h after the glutamate injection. All subjects andexaminer two were blinded with respect to administrationof NGF or saline and there were at least three weeksbetween the two sessions. The code was broken after dataentry and analysis.

Injection of NGF, isotonic saline and glutamate

Based on previous studies (Svensson et al. 2003a; Pettyet al. 1994; Dyck et al. 1997) a single dose of 5 �g NGFwas given in 0.2 ml (»0.1 �g/kg) as a bolus injection toall 14 subjects. The manual injection into the right mas-

seter muscle followed published techniques (Svenssonet al. 1995). As a control, 0.2 ml buVered isotonic salinewas injected into the right masseter muscle in the controlsession. Sterile solutions of recombinant human NGF(25 �g/ml) were prepared by the pharmacy at AalborgHospital.

In accordance with our previous studies (e.g. Svenssonet al. 1995, 2003a, b), the subjects continuously used anelectronic 0–10 cm VAS to score their perceived painintensity of the NGF and isotonic saline injection and todraw the perceived area of pain on the MPQ drawing(Fig. 1). VAS recordings were started immediately after thecompletion of the injections. The VAS signal was sampledand stored in a computer every 5 s. The duration of pain(VASduration) from onset of pain to oVset of pain, area underthe VAS curve (VASauc) and the maximum pain (VASpeak)were calculated.

The area of perceived pain on the MPQ drawings wasdigitized and expressed in arbitrary units. In addition, thepain rating indices (PRI) of the sensory, aVective, evalua-tive, miscellaneous and total dimension of pain [PRI (S, A,E, M, T)] were calculated according to Melzack (1975).

A total of 24 hours after the NGF or isotonic salineinjection, a total of 0.2 ml 1 M glutamate was injected intothe right masseter muscle. A plastic template was used toidentify the same spot in the masseter muscle where theNGF or isotonic saline injection had been previously made(Arima et al. 2000). Subjects were asked to rate their per-ceived pain intensity after injection of glutamate on theelectronic 0–10 cm VAS and to draw the perceived area ofpain on the MPQ drawing.

Fig. 1 Subject-based drawings (n = 14) of perceived pain evoked byinjection of isotonic saline (control) or NGF into the right massetermuscle. Subjects scored the pain intensity on a 0–10 cm electronicvisual analogue scale (VAS) for 900 s. Note that NGF did not evokemore pain than isotonic saline

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Mechanical sensitivity and vibration sense

A pressure algometer (Somedic, Hörby, Sweden) was usedto test sensitivity to deep stimuli applied to the massetermuscles. The PPT was deWned as the amount of pressure(kPa) which the subjects Wrst perceived to be painful(Svensson et al. 1995). The subject pushed a button to stopthe pressure stimulation when the threshold was reached.Subjects were instructed to keep their teeth slightly apart toavoid contraction of the jaw-closing muscles during pres-sure stimulation. The PPTs were determined in duplicatewith a constant application rate of 30 kPa/s and a probearea of 1 cm2. The mean value was used for further statisti-cal evaluation.

A vibrometer (Somedic Hörby, Sweden) was appliedwith constant pressure (30 kPa) only to the injection side(right masseter) in order to save time but otherwise fol-lowed the same experimental protocol as the assessment ofPPTs. The perception threshold, i.e., the smallest amplitude(�m) of vibration at 100 Hz was determined in triplicate atbaseline. A constant amplitude vibratory stimulus (100 Hz,24£ perception threshold) was applied to the masseter for3 min. The subjects were asked to rate the intensity on a 0–10 NRS where 0 was denoted no sense of vibration at alland 10 the most intense sensation of vibration.

Stretch reXex

Stretch reXexes were evoked in the jaw-closing muscleswith a muscle-stretcher (Miles et al. 1993). BrieXy, subjectsbit onto metal bars with their incisor teeth. The verticalposition of the lower bar was controlled precisely with apowerful electromagnetic vibrator whose moving core wasunder servo control (1 mm displacement, 10 ms ramp time)(Wang et al. 2000). Stretch reXex responses were recordedwith the use of bipolar disposable surface electrodes(4 £ 7 mm recording area, 720-01-k, Neuroline, Medico-test, Denmark) placed 10 mm apart along the central part ofthe masseter and the anterior temporalis muscles on bothsides. The skin over the recording positions was cleanedwith alcohol. An isolated common electrode soaked withsaline was attached to the right wrist. The EMG signalswere ampliWed 2,000–50,00 times (Counterpoint MK2,Denmark), Wltered with a band pass of 20 Hz–1 kHz, sam-pled at 4 kHz and stored for oV-line analysis. Subjects wereinstructed to contract their muscle at a steady EMG levelcorresponding to 15% of the maximal voluntary contractionlevel (MVC). The MVC was established as the mean ofthree repeated contractions at about 2–3 min intervals inaccordance with our previous studies (e.g. Svensson andArendt-Nielsen 1996). To help subjects achieve the targetEMG level they were shown a screen display of the root-mean-square (RMS) value in 200-ms intervals of their

EMG from the left masseter muscle (feedback muscle). Thescreen also showed the levels of EMG corresponding with13.5 and 16.5% of the EMG recorded during the MVC. Thedisplay of their EMG level changed from green to red whenit crossed the upper and lower limits of the window (Wanget al. 2000). The program automatically triggered the jaw-muscle stretcher when the EMG activity remained withinthe pre-set window for more than 400 ms. A total of 300 msEMG activity was recorded with 100 ms pre-stimulus and200 ms post-stimulus. Sixteen stretch stimuli were appliedwith an inter-stimulus interval of about 10 s (Wang et al.2000; Svensson et al. 2001).

A special-purpose computer program processed thereXex responses evoked in the EMG. First, the mean EMGin the pre-stimulus interval (¡100 to 0 ms) of the rectiWedand averaged (n = 16) signal was calculated. The onset andpeak-to-peak amplitude of the early reXex component,which appeared as a biphasic potential in the average of thenon-rectiWed recordings, was measured. The normalizedpeak-to-peak amplitudes were expressed as a percentage ofpre-stimulus EMG activity (Wang et al. 2000).

Statistics

According to the Kolomogorov–Smirnov test, the data setwas normally distributed and was therefore analyzed withparametric statistics. The only exceptions were VAS painresponses (VASpeak and VASauc) and pain drawing areaswhich therefore were reported and analyzed with non-para-metric statistics. Other results are presented asmean § standard errors of the mean (SEM). The PPTs werenormalized to the baseline values to calculate the relativechanges. Two-factor repeated measures analysis of vari-ance (ANOVA) was used to test the normalized PPT, NRSscores of vibratory stimuli, pre-stimulus EMG, peak-to-peak amplitude and normalized peak-to-peak amplitudewith session (2 levels: NGF and isotonic saline) as one fac-tor and time as the other factor (7 levels: baseline, 1, 2, 3,24 h; and 2 min and 1 h after glutamate injection). Post hoccomparisons were done with the use of Tukey tests. Thelevel of signiWcance was set at P < 0.05.

Results

EVects on spontaneous pain reports

Intramuscular injections of NGF and buVered isotonicsaline were associated with very low VAS pain scores anddid not diVer signiWcantly [Median NGF VASpeak = 0.5(interquartile range: 0.3–0.7 cm), median saline VASpeak =0.1 (0–1.6) cm; P = 1.000; NGF VASduration = 59 § 12 s,saline VASduration = 36 § 9 s, P = 0.124; and median NGF

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VASauc = 28.7 (5.8–46.8) cm £ s, median salineVASauc = 4.4 (0–49.7) cm £ s, P = 0.455] (Fig. 1). Thepain drawing areas were small and did not diVer betweenthe NGF [Median 0.0 (0.0–0.0) a.u.] and the isotonic saline[0.0 (0.0–0.1) a.u.; P = 0.188] injections (Fig. 1).

In contrast to the administration of NGF and isotonicsaline, the subsequent injection of glutamate was associatedwith moderate to strong levels of spontaneous pain in boththe NGF pretreated masseter [Median VASpeak = 4.9 (4.2–6.3) cm; VASduration = 500 § 61 s; Median VASauc =1246.6 (751.7–2492.8) cm £ s] and the control masseter[Median VASpeak = 5.2 (3.2–6.3) cm; VASduration = 498 §46 s; Median VASauc = 1230.4 (747.9–2014.0) cm £ s], butwith no signiWcant diVerences between sessions (P = 0.455;P = 0.414; P = 0.787, respectively) (Fig. 2). There was aslight, but signiWcantly larger pain drawing area in theNGF pretreated masseter muscle [1.48 (0.70–1.93) a.u]compared with the control masseter [0.84 (0.29–1.48) a.u;P = 0.009].

The PRI (S, A, E, M, T) of the glutamate-evoked painwere not diVerent between the two sessions (P > 0.082).The words most often chosen by the subjects to describe theglutamate-evoked pain in both sessions were “pressing”(8/14), “sharp” (5/14) and “intense” (5/14).

There were no reports of systemic adverse eVects afterthe NGF or glutamate injections.

EVects on mechanical sensitivity and vibration sense

The absolute PPT values at baseline were 252 § 17 kPawith no signiWcant diVerences between sides (ANOVA:P = 0.685) or between sessions (ANOVA: P = 0.352).

A two-way ANOVA test of the normalized PPTs on theinjected side showed that there was a strong main eVect oftime (ANOVA: P < 0.001), as well as session (ANOVA:P < 0.001) with a signiWcant interaction between factors(ANOVA: P < 0.001). For the masseter muscle injectedwith NGF, the normalized PPTs were signiWcantly lowerthan baseline values after 1, 2, 3 and 24 h (i.e., mechanicalallodynia) (Tukey: P < 0.05) (Fig. 3a) and signiWcantlylower than the corresponding values in the isotonic salinesession (Tukey: P < 0.05) (Fig. 3a). There were no signiW-cant changes in the normalized PPTs on the contralateralside (ANOVA: P = 0.583) (Fig. 3b).

Compared with baseline values the injection of gluta-mate was associated with a signiWcant decrease in normal-ized PPTs both in the NGF pretreated and isotonic saline-injected masseter muscles (Tukey: P < 0.009), which stillwas present 1 h after the glutamate injection (Tukey:P < 0.001) (Fig. 3a). In the isotonic saline session, the nor-malized PPTs after the glutamate injection were also sig-niWcantly lower than the PPT values at 24 h (Tukey:P = 0.047). In the NGF pre-treated muscle there were nosigniWcant diVerences between the PPTs at 24 h and afterthe glutamate injection (Tukey: P = 0.999); however, thechanges in normalized PPTs were still greater than in thecontrol masseter muscle (Tukey: P < 0.001) (Fig. 3a). Inthe buVered isotonic saline pre-treated muscle there was asigniWcant diVerence between the PPTs at 24 h and after theglutamate injection.

The absolute perception threshold to vibratory stimuliapplied to the right masseter muscle (8.1 § 7.9 �m) was notsigniWcantly diVerent at baseline or 24 h after the injectionsof NGF or isotonic saline (ANOVA: P = 0.208). The NRSscores of the 24 h perception threshold vibratory stimuliwere not diVerent between sessions (ANOVA: P = 0.330)and there was no eVect of time or of glutamate injection(ANOVA: P = 0.386) (Fig. 4). None of the subjects everreported a painful sensation of the vibration in the massetermuscle.

EVects on stretch reXex responses

Analysis of the pre-stimulus EMG activity revealed a sig-niWcant eVect of time (ANOVA: P = 0.039) but not session(ANOVA: P = 0.080). Post hoc analysis did not indicateany signiWcant Xuctuations during the Wrst 24 h in the NGFor isotonic saline session for any of the four jaw muscles(Tukey: P = 0.311) (Fig. 5a–d). In the left masseter muscle,there was a signiWcant increase in pre-stimulus EMG activity

Fig. 2 Subject-based pain drawings and scores (n = 14) on a 0–10 cmelectronic visual analogue scale (VAS) of injections of 0.2 ml gluta-mate into the isotonic saline pretreated or NGF pretreated massetermuscle. Glutamate-evoked pain was perceived to be mainly around theinjection side in the masseter with some spreading towards the temple.Although quantitative analysis of the pain drawing areas revealed sig-niWcantly larger areas in the NGF session, no other major diVerencesin pain responses were apparent

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2 min after the injection of glutamate into the right masse-ter muscle compared with baseline (Tukey: P = 0.016)but post hoc tests could not diVerentiate between the twosessions.

The latency of the stretch reXex in the jaw closing mus-cles was 8.1 § 0.1 ms in the isotonic saline session and8.4 § 0.1 ms in the NGF session. There were no diVerencesbetween sessions (ANOVAs: P = 0.071) or time eVects(ANOVAs: P = 0.382) (data not shown).

The duration of the stretch reXex in the jaw closing mus-cles was 10.9 § 0.2 ms in the isotonic saline session and11.1 § 0.2 ms in the NGF session. There were no signiW-cant diVerences between the two sessions (ANOVAs:P = 0.604) or time eVects (ANOVAs: P = 0.395) (data notshown).

Analysis of the normalized peak-to-peak amplitude didnot indicate any signiWcant changes during the Wrst 24 h ordiVerences between sessions (ANOVAs: P = 0.054)

(Fig. 5e–h). Only for the right masseter was there anincrease in the normalized peak-to-peak amplitude 2 minafter the glutamate injection compared with baseline val-ues, which occurred in both the NGF and isotonic salinesessions (Tukey: P = 0.002) (Fig. 5f).

Discussion

The main Wndings in this study were (1) the lack of sponta-neous pain evoked by intramuscular injection of NGF, (2)the consistent and long-lasting increase in mechanical sen-sitivity to pressure stimuli, but not to vibratory stimuli inthe NGF-injected masseter muscle, (3) the lack of NGF-induced changes on stretch reXex sensitivity and 4) the lackof signiWcant interactions between NGF and glutamate.

EVects of NGF on spontaneous pain and mechanical sensitization

The present study has shown that NGF injections into themasseter muscle of men do not cause more pain than injec-tions of isotonic saline consistent with our previous reportin men (Svensson et al. 2003a). This Wnding suggests thatNGF either does not excite nociceptive aVerent Wbers ordoes not activate a suYcient number of nociceptive aVerentWbers necessary for a conscious sensation of pain inhumans. Recent Wndings suggest that injection of humanNGF into the masseter muscle does not evoke signiWcantdischarge in slowly-conducting, principally A� (group III)aVerent Wbers, but can induce a prolonged period ofmechanical sensitization in these aVerent Wbers (Mann et al.2006; Svensson et al. 2008). In contrast, Hoheisel et al.(2005) reported NGF excited 10 of 28 C (group IV) Wberstested in the gastrocnemius-soleus muscle of rats withoutsigniWcant changes in C-Wber discharges in response toWxed mechanical stimuli, which suggested that NGF injec-tions did not alter the mechanical sensitivity of these Wbers.

Fig. 4 NRS scores for the constant vibratory stimulus applied to theinjection side (right masseter) at baseline (base) and in response toinjection with NGF and isotonic saline (mean values § SEM; n = 14).After the assessment at 24 h, an injection of 0.2 ml glutamate was doneand the PPTs assessed immediately after (24 h + G) and 1 h after(G + 1 h)

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PPTs assessed immediately after (24 h + G) and 1 h after (G + 1 h).* indicates signiWcant diVerent from baseline values (Tukey: P < 0.05)and # indicates signiWcant diVerence between NGF and isotonic saline(Tukey: P < 0.05)

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This discrepancy is diYcult to explain although it has beenproposed that a summation of multiple C-Wber inputs wouldbe necessary to evoke a subjective sensation of muscle pain(Hoheisel et al. 2005) and there could be diVerences inNGF-mediated response characteristics between A�- andC-Wbers, expression of TrkA receptors and other functionaldiVerences between the masseter (trigeminal) and gastroc-nemeus-soleus (spinal) muscles.

A consistent Wnding in this study and our previous studyin healthy men (Svensson et al. 2003a) was the pro-nounced sensitization of the masseter in a localized areaaround the injection site. The lack of PPT changes on thecontralateral side (Fig. 3b) and the rapid time eVect (after1 h) strongly suggest that there is a peripheral componentto this mechanical sensitization eVect. In our previousstudy there was a pronounced sensitization to mechanicalstimuli after 1 and 7 days, but not after 1 h (Svensson et al.2003a). The reason for this minor discrepancy in timecourse is not clear but the rapid (within hours) sensitiza-tion eVects in humans are consistent with the time courseof NGF-induced mechanical sensitization of slowly-con-ducting masseter aVerent Wbers in rats (Mann et al. 2006).In summary, injection of NGF into human muscles appearsto be a very reliable and consistent model in terms of arapid (hours) and long-lasting (days) mechanical sensitiza-tion without spontaneous pain.

EVects of NGF on vibration sense and stretch reXexes

Vibration is an eVective stimulus for large-diameter mecha-noreceptive aVerents and muscle spindles and is innocuousin healthy subjects, but several lines of evidence have dem-onstrated an intriguing link between vibratory sensorychannels and nociceptive pathways. For example, recentstudies have implicated large-diameter mechanoreceptiveaVerents in the generation of DOMS whereas these aVerentWbers appear to have no inXuence on hypertonic saline-evoked muscle pain (Weerakkody et al. 2001, 2003). Thiscould represent a mechanism resembling secondary hyper-algesia and the term “proprioceptive allodynia” has beensuggested despite the diVerences between “hyperalgesia”and “allodynia” (Treede et al. 2006). Psychophysical stud-ies have also revealed that vibration sense is disturbed, butnot directly painful, in most myofascial TMD pain patientswhen compared to matched control subjects (Hollins et al.1996, 2001; Hollins and Sigurdsson 1998). TMD patientsappear to be impaired in terms of frequency discriminationbut not amplitude discrimination (Hollins and Sigurdsson1998) but also to have a diminished capability to detectlow-frequency vibratory stimuli (Hollins et al. 1996). Inone case-report of a TMD patient, a generalized vibrotactile(or proprioceptive) allodynia was described which wasalleviated by the systemic administration of a NMDA

Fig. 5 Analysis of the stretch reXex responses and the eVects of NGF and isotonic saline on the pre-stimulus RMS EMG activity (�V) (a–d) and the nor-malized peak-to-peak amplitude (e–h) in four diVerent jaw-clos-ing muscles: masseter left (MAL), masseter right (MAR = injected muscle), ante-rior temporalis left (TAL) and anterior temporalis right (TAR). Stretch reXexes were recorded at baseline (base), 1, 2, 3 and 24 h after the NGF and isotonic saline injections. After the recordings at 24 h, an injection of 0.2 ml glutamate was done and the stretch reXexes recorded imme-diately after (24 h + G) and 1 h after (G + 1 h). * indicates sig-niWcant diVerent from baseline values (Tukey: P < 0.05)

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8 Exp Brain Res (2008) 189:1–10

receptor antagonist (dextromethorphan) (Fillingim et al.1998).

In the present study we found no evidence of a “proprio-ceptive allodynia” or “proprioceptive hyperesthesia” in themasseter muscle despite a pronounced mechanical sensitiza-tion. The vibratory stimulus was applied at a constant pres-sure, which is necessary to ensure a correct stimulation;however, the constant pressure was considerably lower(30 kPa) than the PPTs at baseline (»250 kPa) and duringthe lowest PPTs (»50% of baseline values at 3 h). This sug-gests that the method of vibration application did not inter-fere with the subjects’ ability to rate the vibratory stimulus.Consistent with the constant NRS scores of the vibratorystimulus, there were no signiWcant diVerences in perceptionthresholds between the sessions strongly indicating thatvibration sense remained unaVected by the NGF-inducedmechanical sensitization. This Wnding in humans providesnew insight into the aVerent nerve Wber population aVectedby NGF and suggests that large-diameter and low-thresholdmechanoreceptive units do not contribute to NGF-inducedmechanical allodynia. Hoheisel et al. (2005) also noted thatlow-threshold C-Wbers were unaVected by intramuscularadministration of NGF in their experimental set-up. It seemslikely that high-threshold A�-Wbers could mediate this typeof mechanical sensitization in humans and would be consis-tent with our Wndings in animal studies of primary aVerentrecordings from the masseter muscle (Mann et al. 2006).

In addition, we assessed the jaw stretch reXex which ismediated by muscle spindle aVerents from the masseter mus-cle. The present study did not Wnd any evidence of a NGF-related change in this monosynaptic reXex. Experimental jawmuscle pain evoked by painful injections of glutamate,hypertonic saline, and capsaicin have consistently beenshown to facilitate the amplitude of the jaw stretch reXex(Wang et al. 2000, 2002, 2004; Svensson et al. 2001; Cairnset al. 2003a; Peddireddy et al. 2005). Interestingly, experi-mentally-evoked fatigue in jaw muscles does not cause anychanges in the jaw stretch reXex sensitivity (van Selms et al.2005) and we have recently shown that very intense painfrom non-muscular tissues in the craniofacial region does notmodulate the jaw stretch reXex (Biasiotta et al. 2007). Com-bined these Wndings suggest a diVerential inXuence of aVer-ent Wber inputs on the jaw stretch reXex circuitries. Animalstudies have demonstrated a modulatory eVect of algogenicsubstances (hypertonic saline, mustard oil) injected into thejaw muscles on the response properties of muscle spindleaVerents recorded in the mesV nucleus (Capra et al. 2007) aswell as jaw-closer reXexes evoked by electrical stimulationof the mesV (Kurose et al. 2005). This modulatory eVect isdependent on the functional integrity of the caudal trigeminalsensory nuclear complex and appears to involve the gamma-motoneurons (Capra et al. 2007). These Wndings suggest themuscle spindle aVerents are not directly sensitized but that

central pathways may be involved in the muscle-pain relatedincrease in stretch reXex sensitivity. Although NGF receptorsare expressed by mesV ganglion neurons (Koh et al. 1989),the present study did not indicate that NGF-induced mechan-ical sensitization in any way inXuenced the circuitries relatedto the stretch reXex.

Interactions between NGF and glutamate

NGF exerts its biological eVects by acting on the TrkAreceptor, which is a high aYnity receptor, and the p75receptor, which is a low aYnity receptor (Chao and Hemp-stead 1995). NGF binding to the TrkA receptors causesreceptor activation and dimerization, which results in trans-autophosphorylation and activation of intracellular signal-ing cascades like extracellular signal-regulated kinase(ERK), phosphatidylinositol 3-kinase (PI3 K) and phospho-lipase C� (PLC-�) pathways to rapidly aVect changes indownstream receptor function as well as initiate sloweralterations in receptor expression (Chao 2003). Studieshave shown elevated levels of NGF in the cerebrospinalXuid of patients with headaches and migraines (Sarchielliet al. 2001, 2006). A recent clinical observation in patientswith primary Wbromyalgia also showed signiWcant correla-tions between levels of NGF and glutamate in the cerebro-spinal Xuid (Sarchielli et al. 2007) and it was suggested thatNGF could exert at least part of its action through a gluta-matergic mechanism (Jarvis et al. 1997; Michael et al.1997). Furthermore, the possible interaction between NGFand glutamate is supported by Wndings that NGF inducesphosphorylation of NMDA receptor subunits NR2A andNR2B in the spinal cord (Di Luca et al. 2001) and prelimi-nary observations of co-localization of TrkA, p75 andNR2B receptors on masseter muscle aVerent Wbers (Svens-son et al. 2008). We have in several studies shown thatinjection of glutamate into the masseter muscle is associ-ated with pain and a signiWcant decrease in PPTs as well asincreased jaw stretch reXex responses and that these eVectsare mediated, in part, through activation of peripheralNMDA receptors (Cairns et al. 2001, 2003a, b, 2006; Castr-illon et al. 2007; Svensson et al. 2003b). Consistent withthese Wndings we observed a decrease in PPTs followingthe glutamate injection in the session with isotonic saline.Although the PPTs were already decreased in the NGF ses-sion, there was no further decrease in PPTs after injectionof glutamate. One explanation for the lack of glutamate-induced mechanical sensitization is that a maximaldecrease in PPTs had already been evoked by the NGFinjection so that no additional mechanical sensitization wasdetectable in the present study. Another possibility may bethat the mechanisms by which NGF and glutamate inducemechanical sensitization converge on the same downstreampathways, which prevents additive or synergistic eVects

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Exp Brain Res (2008) 189:1–10 9

when these two compounds are used together. The lack of adetectable interaction between NGF and glutamate was alsoapparent for the stretch reXexes, which as previously shownincreased immediately following the glutamate injectionbut with no signiWcant diVerences between sessions.Despite the feedback control of muscle activity (from theleft masseter), there was a signiWcant increase in pre-stimu-lus EMG activity after the glutamate injection when com-pared to baseline recordings (Fig. 5). Ideally, there shouldhave been no time eVects but subtle diVerences in bitingstrategy during pain (e.g. Wang et al. 2000; Svensson et al.2001) as well as variations in placement of EMG electrodescould be explanations. This Wnding further supports thenotion that normalized reXex responses should be used tocompare diVerent sessions and conditions (Wang et al.2000). Finally, there were no signiWcant diVerences in theperceived pain intensity of the glutamate injection betweenthe masseter muscle pretreated with NGF or control,although the glutamate-evoked pain drawing areas weremarginally, but signiWcantly larger for the NGF-pretreatedmasseter muscle. Although it could be argued that a furthercontrol condition with injections of isotonic saline wouldhave been optimal, we opted not to do this because it wouldhave increased the number of experimental sessions fromtwo to four and pain responses and mechanical sensitizationalready have been shown to be signiWcantly greater with1.0 M glutamate compared to isotonic saline (Castrillonet al. 2007). In summary, the present Wndings suggest inaccordance with preliminary results from aVerent Wbers inrats (Svensson et al. 2008) that there is no or only a minorinteraction (potentiation) between NGF-induced mechani-cal sensitization and glutamate-evoked pain or glutamate-induced mechanical sensitization.

Overall, this study conWrms that masseter muscle injec-tion of NGF is associated with a distinct and prolonged(days) sensitization to mechanical stimuli, but without aneVect on muscle spindle aVerents and stretch reXex sensi-tivity. Additional challenge of the NGF pretreated musclewith glutamate did not indicate a conspicuous sensitizationto a noxious chemical stimulus. Intramuscular injection ofNGF can be a useful model to further explore mechanicalsensitization of muscle tissue in humans.

Acknowledgments The International Association for the Study ofPain kindly awarded a travel grant to BEC and PS. The support fromthe Danish National Research Foundation and Danish Dental Associa-tion is greatly appreciated.

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