Effect of experimental posterior temporalis muscle pain on human brainstem reflexes

Clinical Neurophysiology 116 (2005) 1611–1620 www.elsevier.com/locate/clinph

Effect of experimental posterior temporalis muscle pain on human brainstem reflexes Anitha Peddireddya, Kelun Wanga, Peter Svenssona,b, Lars Arendt-Nielsena,* a

Orofacial Pain Laboratory, Center for Sensory-Motor Interaction, Aalborg University, Fredrik Bajars Vej 7D-3, 9220 Aalborg, Denmark b Department of Clinical Oral Physiology, School of Dentistry, University of Aarhus, Aarhus, Denmark Accepted 23 February 2005 Available online 29 April 2005

Abstract Objective: To study the modulation of jaw-stretch and blink reflexes by experimental posterior temporalis muscle pain. Methods: Thirty healthy volunteers (15 males, 25.5G0.6 years and 15 females, 27.4G1.2 years) were included. Short-latency stretch reflex responses were evoked in the masseter and temporalis muscles by fast stretches (1 mm displacement, 10 ms ramp time) and the blink reflexes were evoked by painful electrical pulses (0.5 ms duration), delivered by a concentric electrode placed on the left lower forehead close to the supraorbital foramen before, during and 15 min after a period with experimentally induced muscle pain. Results: The normalized peak-to-peak amplitude of the stretch reflex in the painful temporalis was significantly higher during pain in both males and females compared with pre- and post-pain conditions (P!0.004). The R2 root mean square (RMS) of the blink reflex decreased significantly during muscle pain as compared to the pre-pain (P!0.03) in both males and females. Conclusions: The present results indicated that experimental posterior temporalis muscle pain facilitates the jaw-stretch reflex, whereas the nociceptive specific blink reflex is inhibited. Significance: Present study suggested that these reflexes are suitable models for probing pontine and medullary pain processing. q 2005 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Human experimental muscle pain; Stretch reflex; Blink reflex; Trigeminal brainstem reflexes

1. Introduction Some of the most common acute and chronic pain conditions occur in the face and mouth (for example, toothaches, migraine headaches, tension-type headaches, temporomandibular disorders (TMD)). The masseter stretch reflexes, and the blink reflexes are useful diagnostic tools for evaluation of brain stem disorders (Hopf, 1994). Monitoring jaw stretch reflex parameters makes it possible to look into the excitability of the trigeminal motoneuron pool. The level of this excitability may be modified by inputs from the orofacial area during function and dysfunction (Murray and Klineberg, 1984). Sudden stretches of the jaw-closing muscle can elicit short-latency excitatory response in * Corresponding author. Tel.: C45 9635 8830; fax: C45 9815 4008. E-mail address: [email protected] (L. Arendt-Nielsen).

the muscles, so called jaw-jerk reflexes or stretch reflexes. The stretch reflex in jaw-closing muscles is the trigeminal equivalent of the monosynaptic, myotatic spinal reflexes of the limb muscles (Lund et al., 1983). One of the functions of the stretch reflex in the jaw-closing muscles is to maintain and restore the postural position of the mandible when it is perturbed during rapid head movements (Miles et al., 2003). It has recently been shown that injection of algogenic substances such as hypertonic saline, capsaicin and glutamate into the human masseter muscle increases the short-latency reflex response to stretch in both the ipsi- and contra-lateral masseter muscles, when measured in the surface electromyogram (EMG) (Cairns et al., 2003; Svensson et al., 2000; Wang et al., 2000, 2001, 2002, 2004). However, no studies have so far examined the influence of posterior temporalis experimental pain on the human jaw-stretch reflex. There is evidence that tension

1388-2457/$30.00 q 2005 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2005.02.022

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headache reported by TMD sufferers is related to temporalis muscle/tendon dysfunction (Chua et al., 1989). High temporal muscle activities were found in myogenous craniomandibular disorder patients compared to healthy controls (Visser et al., 1995). The so-called ‘nociceptive specific’ blink reflex (BR) is an alternative, non-invasive and reliable electrophysiological technique to measure the nociceptive transmission state of the trigeminal system in humans (Ellrich, 2002; Katsarava et al., 2002; Romaniello et al., 2002). According to the investigation of spinal nociception by cutaneomuscular reflexes, the pathophysiological mechanisms of central sensitisation, hyperalgesia and allodynia can be investigated in the trigeminal system with the use of BR (Ellrich, 2000). The BR is a trigeminofacial brainstem reflex that is usually elicited by electrical stimulation of the supraorbital nerve. The BR consists of an ipsilateral, pontine R1 component with an onset latency of 11 ms, and two bilateral, medullary R2 and R3 components at 33 and 84 ms (Ellrich and Hopf, 1996; Kimura, 1989; Rossi et al., 1989). Recently it was shown that the glutamate-evoked masseter muscle afferent fibre activity was significantly greater in female rats than in male rats (Cairns et al., 2001), which was paralleled by higher pain scores in female subjects compared to male subjects (Cairns et al., 2001; Svensson et al., 2003a,b). These sex-related differences in acute experimental muscle pain are particularly interesting given the higher prevalence of many chronic muscle pain conditions in women, e.g. TMD and TTH. It was hypothesized that, as the BR and stretch reflex are mediated by different neuronal circuitrics in the brainstem, these components may be influenced to varying degrees by painful inputs from the craniofacial tissues. The aims of the present study were to investigate the effects of experimentally induced posterior temporalis muscle pain on the human jaw-stretch reflex and nociceptive specific BR and to examine if pain-related changes in brainstem reflex sensitivity are dependent on gender.

2. Materials and methods 2.1. Subjects Thirty subjects (15 males and 15 females) with a mean age of 25.5G0.6 years (males) and 27.4G1.2 years (females) were included. All subjects were healthy and did not take any medication. Their history and clinical examinations revealed no signs or symptoms of TMD (Dworkin and LeResche, 1992) and any type of headache (The International Classification of Headache Disorders, 2004) at the time of testing. The study was conducted in accordance with the Declaration of Helsinki, approved by the local ethics committee, and written informed consent was obtained from all participants prior to inclusion.

2.2. Experimental protocol The experiments were performed to study the influence of tonic pain on the jaw-stretch reflex and nociceptive specific BR. On two sessions separated by 1 week, the subjects received infusions of 5.8 or 0.9% saline given into the posterior part of left temporalis in random sequence. Hypertonic saline in a concentration of 4–6% has by far been the most used chemical substance to induce muscle pain (Graven-Nielsen et al., 1997; Svensson et al., 1998a,b; Wang et al., 2000, 2001). The EMG activity in both masseter and temporalis muscles was recorded. The EMG activity in the left temporalis (painful side) was used for feedback. The subjects were asked to perform three maximal clenches each lasting up to 3 s on the bar with their incisor teeth to obtain the mean EMG value of the maximal voluntary contraction (MVC) in the four muscles. During the experiment, the subjects were guided by visual feedback to keep their EMG level at 15% MVC. For the BR recordings, the EMG activity in orbicularis oculi muscles was recorded bilaterally. During recording of the BR, the subject was sitting comfortably in a chair with eyes closed. Recordings of stretch reflex and BR were obtained during three experimental conditions: before pain, during pain and 15 min after the end of pain induction. The sequence of the stretch and BR recordings was randomised in a balanced way. 2.3. Experimental muscle pain The subjects were randomised to receive intramuscular infusions of 5.8% hypertonic saline and 0.9% of isotonic saline. Each infusion was given over 10 min. Infusions were given in a standardized anatomical point at the centre of the deep mid portion of left posterior temporalis muscle (8–10 cm from the corner of the eye and 1.5–2 cm above the ear lobe). A 27 G needle was connected via a tube (IVAC; G30303, USA) to a computer-controlled infusion pump (IVAC; model 770, USA) (Graven-Nielsen et al., 1997). A standardized infusion paradigm was used with 0.2 ml saline infused over 20 s followed by a steady infusion rate of 6 ml/h for the next 440 s and finally 9 ml/h for the next 440 s (Svensson et al., 1998a). For safety purposes individual changes in infusion rate could be made in steps of 3 ml/h and the infusion system could be halted by a keyboard command. The subjects continuously scored the pain intensity on a 10 cm electronic visual analogue scale (VAS) with the lower extreme marked ‘no pain’ and the upper extreme marked ‘most pain imaginable’. Stretch and BR were recorded in a random order 2 min after the start of infusion, when the VAS pain scores had reached a constant level (Svensson et al., 1998a; Wang et al., 1999, 2000). The mean VAS pain score was calculated starting at the same time as the reflex recordings.

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2.4. Stretch reflex Stretch reflexes were evoked in the jaw-closing muscles with a muscle-stretcher (Miles et al., 1993). Briefly, subjects bit onto metal bars with their incisor teeth. The vertical position of the lower bar was controlled precisely with a powerful electromagnetic vibrator whose moving core was under servo control (1 mm displacement, 10 ms ramp time) (see Wang et al., 2000). Stretch reflex responses were recorded with the use of bipolar disposable surface electrodes (4!7 mm recording area, 720-01-k, Neuroline, Medicotest, Denmark) placed 10 mm apart along the central part of the masseter and the anterior temporalis muscles on both sides. The masseter muscle was felt by placing the fingers on the jaw angle and the anterior temporalis was felt by placing the fingers on the subjects temples (on the sides of their heads, just behind the eyebrows), while clenching and unclenching their teeth. The skin over the recording positions was cleaned with alcohol. A ground electrode soaked with saline was attached to the right wrist. The EMG signals were amplified 2000–5000 times (Counterpoint MK2, Denmark), filtered with a band pass of 20 Hz–1 kHz, sampled at 4 kHz and stored for off-line analysis. Subjects were instructed to contract their muscle at a steady EMG level corresponding to 15% MVC. To help them achieve this they were shown a screen display of the root-mean-square (RMS) value in 200 ms intervals of their EMG. The screen also showed the levels of EMG corresponding with 13.5 and 16.5% of the EMG recorded during the MVC. The display of their EMG level changed from green to red when it crossed the upper and lower limits of the window (Svensson et al., 1998b). The program automatically triggered the jawmuscle stretcher when the EMG activity remained within the pre-set window for more than 400 ms. A total of 300 ms EMG activity was recorded with 100 ms prestimulus and 200 ms post-stimulus. Twenty trials with an inter-stimulus interval about 10 s were recorded in each condition, and the data were stored in a computer for later analysis. 2.5. Blink reflex Recording electrodes were placed infraorbitally (active) and at the outer canthus of the eye (indifferent), bilaterally (Counterpoint MK2), band width 1 Hz–1 kHz; sampling rate 2.5 kHz, sweep length 150 ms. A concentric electrode (CE) (stimulation area 22 mm2) was placed on the left lower forehead close to the supraorbital foramen. Monopolar electrical square wave pulses of 0.5 ms duration and 0.6–1.5 mA intensity were produced at an inter stimulus interval of 10–15 s by a constant current stimulator (Counterpoint MK2) controlled by a computerized system. Twelve blink reflex sweeps were evoked. The stimulus intensities to evoke individual

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sensory threshold (Is) and pin-prick pain sensation (Ip) were assessed. The current intensity was set at zero and increased at 0.1 mA increments and the subjects were asked to identify the first electric sensation, which was marked as individual sensory threshold and later to identify a sharper pin-prick like pain sensation with the increased current intensity, which was marked as pinprick pain sensation. A fixed stimulation intensity of 1.5 times Ip was used to evoke the BR. The perceived pain intensity of the electrical stimulus (1.5!Ip) was assessed by the subjects on a 0–10 cm VAS scale. This stimulus intensity preferentially activates Ad fibres but not Ab and C-fibres (Kaube et al., 2000). 2.6. Analysis For the stretch reflex, a special-purpose computer program processed the reflex responses evoked in the EMG. First, the mean EMG in the pre-stimulus interval (K100 to 0 ms) of the averaged and rectified signal was calculated (Lobbezoo et al., 1996). The onset and peak-topeak amplitude of the early reflex component, which appeared as a biphasic potential in the average of the nonrectified recordings, was measured in the different experimental conditions (Cruccu et al., 1997). The peakto-peak amplitudes were expressed as a percentage of prestimulus EMG activity (Wang et al., 2000). For the BR, four different parameters were determined, the pre-stimulus EMG activity (K100 to 0 ms), RMS, area under the curve (AUC) and onset latency of the R2. Each block of stimuli consisted of twelve recordings. The first recording of each block was discarded in an attempt to reduce the influence of the startle reaction. The eleven trials of each stimulus series were rectified and averaged off-line. RMS (mV) and AUC (mVms) values of the R2 were analysed by a computer program in a time window from 27 to 87 ms (Ellrich and Treede, 1998). The RMS values were expressed as a percentage of pre-stimulus EMG activity like in stretch reflex (Wang et al., 2000). Onset latencies for R2 responses were measured for each sweep after rectification of the curves. Mean values for each block were calculated. (Kaube et al., 2002). 2.7. Statistics One-way and two-way analyses of variance (ANOVA) with repeated measures were performed and followed by pair-wise multiple comparison procedures (Tukey test). The factors in the ANOVA were experimental condition (three levels: pre-, during, and post-pain) and muscle (four levels: left/right masseter/temporalis for stretch and two levels: left/right orbicularis oculi for the blink reflex). The significance level was set at P!0.05. Mean valuesGSEM are presented in the text and figures.

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3. Results

and females both for mean VAS pain scores and VAS peak values (One-way ANOVA: PO0.4).

3.1. Experimental muscle pain 3.2. Stretch reflex Of the 30 subjects, 15 received the hypertonic saline first and 15 received the isotonic saline first. The sequence was randomised in a balanced way. An average of 0.47G 0.04 ml hypertonic saline was infused into the left posterior temporalis muscle over 10 min. The mean VAS pain score during EMG recordings was 5.3G0.4 cm with a peak value of 5.9G0.4 cm in males and 5.9 G 0.5 cm with a peak value of 6.3G0.5 cm in females. There were no significant gender differences in mean VAS pain scores (One-way ANOVA: PZ0.3) or VAS peak values (One-way ANOVA: PZ0.5). All subjects reported no pain (VASZ0) 5–10 min after stopping the infusion of hypertonic saline. However, a slight soreness usually persisted during the post-infusion recordings. Infusion of a similar amount of isotonic saline into the left posterior temporalis caused no or very low levels of pain. There were no significant differences between males

Averaged reflex responses (20 sweeps for stretch) in the left temporalis evoked by fast jaw-stretches (1 mm displacement, 10 ms ramp time) and nociception specific blink reflex (11 sweeps rectified and averaged, 10 s inter-stimulus interval) in a single subject in the pre-, during and post-pain conditions was illustrated in Fig. 1. Two-way ANOVAs indicated significant effects of experimental condition on pre-stimulus EMG activity for infusion of hypertonic saline (two-way ANOVA: F(2,14)Z 3.9, PZ0.03 in males and F(2,14)Z3.7, PZ0.03 in females) (Fig. 2A and E), but not for infusion of isotonic saline (Fig. 2B and F). Post hoc analysis showed that the EMG activity in the left temporalis (painful), which served as the feedback muscle during the recordings, was constant during all the conditions (Tukey: PO0.5), whereas significant decreases were observed in the pre-stimulus activity in the

Fig. 1. Averaged reflex responses (20 sweeps for stretch) in the left temporalis evoked by fast jaw-stretches (1 mm displacement, 10 ms ramp time) and nociception specific blink reflex (11 sweeps rectified and averaged, 10 s inter-stimulus interval) in a single subject in the pre-, during and post-pain conditions. Arrow shows the onset of the stimulus.

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Hypertonic saline

Isotonic saline

100

*

*

Pre-Stimulus EMG, Male

B

*

80 Base Pain Post

60 40 20 0 MAL

MAR

TAL

Average EMG (µV)

Average EMG (µV)

Pre-stimulus EMG , Male

A

100 80 60 40 20 0

TAR

MAL

*

*

*

D

40 30 20 10 0 MAL

MAR

TAL

TAR

Change from Pre-Stimulus Level (%)

Change from Pre-Stimulus Level (%)

* 50

40 30 20 10 0 MAL

F

* *

60 *

40 20 0

G

Change from Pre-Stimulus Level (%)

MAL

MAR

TAL

*

*

*

*

*

40 30 20 10 0 MAL

MAR

TAL

TAL

TAR

80 60 40 20 0 MAL

TAR

Normalized Peak-to Peak, Female 50

MAR

Pre-stimulus EMG, Female Average EMG (µV)

80

TAR

100

TAR

H

Change from Pre-stimulus Level (%)

Average EMG (µV)

E

TAL

50

Pre-stimulus EMG, Female 100

MAR

Normalized Peak-to-Peak, Male

Normalized Peak-to-Peak, Male

C

1615

MAR

TAL

TAR

Normalized Peak-to-Peak, Female 50 40 30 20 10 0 MAL

MAR

TAL

TAR

Fig. 2. (A–H) Effects of infusion of hypertonic and isotonic saline into the left posterior temporalis muscle on pre-stimulus EMG activity and normalized peakto-peak amplitude of the short-latency reflex response from left and right masseter (MAL, MAR) and temporalis muscles (TAL, TAR). In the pre-stimulus EMG: Y-axis, average EMG in mV; X axis, different muscles MAL, MAR, TAL, TAR plotted on a bar graph. In the normalized peak-to-peak: Y-axis, change from pre-stimulus level (values were expressed as a percentage of pre-stimulus activity); X-axis, different muscles MAL, MAR, TAL, TAR. Mean valuesG SEM (nZ15). * Indicates significant difference between conditions (Tukey: P!0.05).

right and left masseter in males (Tukey: P!0.02) and right and left masseter and right temporalis between pain and post pain conditions in females (P!0.03). There were no significant differences in the pre-stimulus EMG activity between males and females (t test: PO0.5), except in the prepain condition in left temporalis muscle; thus, the mean value of pre-stimulus EMG was larger in males (35.3G4.4 mV) compared to females (22.5G2.6 mV) (t test: PZ0.02). The mean onset latency of the reflex response evoked by the fast stretches (10 ms ramp) in the EMG was 8.3G0.2 ms in males and 8.4G0.2 ms in females. There were no significant differences between the experimental conditions and also between males and females. The mean amplitude of the stretch reflex was: pre-pain 839.2 mv, pain 1120.0 mv, post-pain 953.3 mv in males,

and pre-pain 843.3 mv, pain 1019.6 mV, post-pain 861.9 mV in females. Two-way ANOVAs demonstrated significant differences for the normalized peak-to-peak amplitude of the jaw-stretch reflex between conditions in the experiments with infusion of hypertonic saline (F(2,14)Z7.0, PZ0.003, pre-pain 18.1%, pain 33.4%, post-pain 19.0% in males and F(2,14)Z6.5, PZ0.005, pre-pain 21.4%, pain 34.9%, postpain 21.2% in females) (Fig. 2C and G), but not with infusion of isotonic saline. (Fig. 2D and H). Post hoc analysis showed significantly higher peak-to-peak amplitudes during pain compared with pre-and post-pain conditions and most notably for the right temporalis in both males (Tukey: P! 0.004) and females (Tukey: P!0.002). There were no significant differences in the pre-pain and pain conditions in the left and right masseters in males (Tukey: PO0.09) and in

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Table 1 For the nociception specific stimulation, the mean for sensory thresholds (Is), pin-prick pain thresholds (Ip) and electrical pain thresholds (I) Male, mean (mA)

Female, mean (mA)

P-value

0.5G0.06 1.3G0.1 1.9G0.1

0.3G0.03 1.0G0.07 1.5G0.1

0.01* 0.04* 0.03*

* Significant difference between conditions (Tukey: P!0.05).

the right masseter in females (Tukey: PZ0.2). There were no significant differences between males and females in the relative increases of the stretch reflex (t test: PZ0.6). 3.3. Blink reflex

A

Average EMG (µV)

For the electrical stimulation, the mean values for sensory thresholds (Is), pin-prick pain sensation (Ip) Hypertonic saline

Isotonic saline

Pre-stimulus EMG, Male

Pre-stimulus EMG, Male

B

10 8 Pre-Pain Pain Post-Pain

6 4 2 0 OOL

8 6 4 2 0 OOL

RMS of R2, Male

12 10 8 6 4 2 0

D

*

OOL

12 10 8 6 4 2 0

OOR

OOL

*

*

8

F

6 4 2 0 OOL

10 8 6 4 2 0

OOR

OOL

RMS of R2, Female

*

*

OOL

OOR

RMS of R2, Female

*

*

H RMS (%)

RMS (%)

G

12 10 8 6 4 2 0

OOR

Pre-stimulus EMG, Female Average EMG (µV)

E

Average EMG (µV)

Pre-stimulus EMG, Female 10

OOR RMS of R2, Male

RMS (%)

RMS (%)

10

OOR

* C

Average EMG (µV)

Is Ip I

and electrical pain threshold (I) are shown in Table 1. There were significant differences between males and females for all the three parameters; the stimulus intensities to evoke the three sensations were significantly lower in females than in males (P!0.05) and the VAS scores of the electrically evoked sensation were significantly higher in females compared to males in the pain and post-pain conditions, but not in pre-pain condition (One-way ANOVA: pre-pain mean VAS 2.5G0.3 in males and 3.4G0.3 in females, PZ0.1; pain mean VAS 2.0G0.1 in males and 3.3G0.4 in females, PZ0.02; and in post-pain mean 2.1G0.2 in males and 3.2G0.3 in females (PZ0.007). The re-test reliability of individual sensory and pin-prick pain thresholds demonstrated no significant differences between sessions in both males (One-way ANOVA: sensory, pZ0.6; pin-prick, PZ0.2) and females (One-way ANOVA: sensory, PZ0.7, pin-prick PZ0.8).

OOR

12 10 8 6 4 2 0 OOL

OOR

Fig. 3. (A–H) Effects of infusion of hypertonic and isotonic saline into the left posterior temporalis muscle on pre-stimulus EMG activity and RMS values of the nociceptive specific blink reflex response from left and right orbicularis oculi muscles (OOL, OOR). In the pre-stimulus EMG: Y-axis, average EMG in mV; X-axis, different muscles OOL, OOR plotted on a bar graph. In the RMS: Y-axis, RMS % (values were expressed as a percentage of pre-stimulus activity); X-axis, different muscles OOL, OOR. Mean valuesGSEM (nZ15). * Indicates significant difference between conditions (Tukey: P!0.05).

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There were no significant effects of experimental condition on pre-stimulus EMG activity of the BR in males (two-way ANOVA: F(2,14)Z0.3, PZ0.6) (Fig. 3A), whereas in females significant differences were seen (twoway ANOVA: F(2,14)Z4.6, PZ0.01) (Fig. 3E). Post hoc analysis in females showed significantly higher pre-stimulus EMG values during pain compared with pre-pain and postpain conditions in right orbicularis oculi muscle (PZ0.02), but importantly not in the left orbicularis oculi muscle (PZ0.1). No significant effects of experimental condition on pre-stimulus EMG activity were seen with the infusion of isotonic saline in both males and females (two-way ANOVA: PZ0.3) (Fig. 3B and F). The mean amplitude of the RMS of R2 was: pre-pain 28.3 mV, pain 22.6 mV, post-pain 24.8 mV in males, and prepain 21.1 mV, pain 16.5 mV, post-pain 19.9 mV in females. The RMS of the R2 decreased significantly during hypertonic saline infusion into the posterior temporalis muscle compared with pre-pain and post-pain conditions (two-way ANOVA: F(2,14)Z3.9, PZ0.03, pre-pain 9.0%, pain 6.4%, post-pain 6.8% in males and F(2,14) Z8.2, PZ0.001, pre-pain 6.3%, pain 4.1%, post-pain 5.7% in females) (Fig. 3C and G) but not with infusion of isotonic saline (Fig. 3D and H). Post hoc analysis showed a significantly lower RMS values for right and left orbicularis oculi muscle during pain compared with pre-pain condition in males (Tukey: P!0.03). In females, there was a substantial suppression of RMS value in left orbicularis oculi muscle during pain compared with pre and post-pain conditions (Tukey: P!0.001). There were no significant differences in the R2 RMS activity between males and females (t test: PO0.5). The mean onset latency of the R2 was 33.2G0.1 ms in males and 33.8G0.1 ms in females. The onset latencies were not significantly different between conditions for both hypertonic and isotonic saline infusions. There were no significant differences in the onset latencies between left and right orbicularis oculi and also between males and females. The mean amplitude of the area under the curve (AUC) of R2 was: pre-pain 1.3 mVms, pain 1.2 mVms, post-pain 1.2 mVms in males, and pre-pain 0.9 mVms, pain 0.8 mVms, post-pain 0.8 mVms in females. The AUC of R2 was not statistically significant between conditions for the hypertonic and isotonic saline infusions.

4. Discussion For the first time, the effect of experimental posterior temporalis muscle pain was studied on the jaw-stretch reflex and the R2 component of the BR elicited by nociceptive electrical stimuli. The results demonstrated that the jaw-stretch reflex was facilitated, where as the R2 part of the BR component was inhibited during tonic

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jaw-muscle pain. No gender differences in the painmodulation of these two brainstem reflexes could be detected. 4.1. Stretch reflex In the present study, visual feedback and computercontrolled triggering of the jaw-stretch stimulus controlled the level of muscle excitation. In previous studies, the prestimulus EMG level was not controlled to the same extent, which might have contributed to the asymmetry of the jawstretch reflexes in patients with TMD pain (Cruccu et al., 1992, 1997; Murray and Klineberg, 1984). Normalization of the peak-to-peak amplitude with respect to the pre-stimulus EMG level is a sensitive measure of the jaw-stretch reflex (Wang et al., 2000). In the present study the normalized peak-to-peak amplitude demonstrated facilitation in the presence of a painful input. Recently, human studies have provided evidence of facilitation of the early component of the stretch reflex during experimental muscle pain (Matre et al., 1998; Wang et al., 2000). The fusimotor system was suggested to be involved in the increased muscle-spindle sensitivity as a result of muscle pain. In the present study, experimental pain in the posterior temporalis muscle was associated with significant increases in the normalized stretch reflex response in the temporalis muscle, but not in the masseter muscle (Fig. 2C and G). A series of studies in animals has shown facilitatory effects of algesic substances like bradykinin, serotonin, and arachidonic acid on the fusimotor system (Djupsjobacka et al., 1994; Johansson et al., 1993; Pedersen et al., 1997). It was recently reported that intramuscular injections of hypertonic saline, most likely via the g-motor system, increased static sensitivity of the muscle spindle in cats (Hellstro¨m et al., 1999). The observed facilitation of the jaw-stretch reflex by hypertonic saline supports the notion that nociceptive inputs in some conditions may increase the muscle spindle sensitivity (Matre et al., 1998). Injury or inflammation of deep tissues can lead to an increased excitability of Ad and C-fiber primary afferent endings in the tissues and subsequently produce a facilitated nociceptive afferent input into the dorsal horn/or brainstem. This process may lead to an increased excitability of the nociceptive neurons that are involved in the processing of pain (Sessle, 2002). Convergence of cutaneous, muscle and visceral afferents has been described and implicated in the central mechanisms of referred pain as well as superficial and deep pain. Stimulation of high-threshold afferents from jaw and tongue muscles as well as neck muscles not only excites wide-dynamic-range (WDR) neurons, but also may activate low-threshold mechanoreceptive (LTM) and nociceptive-specific (NS) caudalis neurons, which indicates that the muscle afferent input may be predominantly of a nociceptive character (Sessle et al., 1986).

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4.2. Blink reflex In the present study, a special concentric planar stimulating electrode was used (Kaube et al., 2000). By virtue of its concentric design and small anode–cathode distance, a high current density is achieved that allows low current intensities to be used such that depolarisation is limited to the superficial layer of the dermis containing mainly nociceptive fibers but does not reach the deeper, predominantly non-nociceptive fiber containing layers. The R2 response of this modified BR has been shown to be nociception specific (Kaube et al., 2000), although it can never be ruled out that non-nociceptive afferents are activated when an un-specific stimulus modality, such as electrical stimulation is used. The re-test reliability of nociceptive specific BR was studied by Katsarava et al. (2002) and demonstrated that it can be used as a reliable technique in longitudinal studies to detect differences in individuals and patients over time or to assess the long-term effects of drugs. Furthermore, nociceptive specific BR could be able to detect selective impairment of the trigeminal Ad fibres, suggesting that it is a useful tool for neurological clinical assessment. Females showed significantly higher pre-stimulus EMG values during pain compared with pre-pain and post-pain conditions in the right orbicularis oculi muscle, whereas there were no significant effects of experimental condition on pre-stimulus EMG activity of the BR in males. The reasons for this subtle difference (1–2 mV) are not clear but could suggest a slight tonic contraction of the contralateral eye muscles in response to pain. Previous studies have shown that tightening of the orbicularis oculi muscles is one of facial expressions to pain (e.g. LeResche and Dworkin, 1988). Pain ratings and R2 to noxious stimulation of the supraorbital nerve decreased during hypertonic saline infusion into the posterior temporalis muscle, and these effects persisted to some extent afterwards. In previous studies, it has been demonstrated that the electrically evoked BR could be suppressed by remote painful heat stimuli (Ellrich and Treede, 1998) and low frequency peripheral stimulation (electro-acupuncture) (Boureau et al., 1979). The diffuse noxious inhibitory control system (DNIC), probably located in the medullary subnucleus reticularis dorsalis, was activated by remote painful heat, and suppressed the activity of WDR neurons in the medullary dorsal horn mediating the R2 blink reflex (Ellrich and Treede, 1998; Le Bars et al., 1992; Villanueva and Le Bars, 1995). General inhibitory mechanisms such as DNIC appear to suppress R2 during painful stimulation of the limbs (Ellrich and Treede, 1998; Pantaleo et al., 1988). In a previous study, it was reported that the R2 component of the blink reflex was strongly suppressed during and after painful conditioning stimulation (Drummond, 2003). The attenuation of R2 during and after painful conditioning stimulation of the ipsilateral temporalis muscle suggests that DNIC may also

operate across different divisions of the trigeminal nerve. R2 suppression was greater for low- than high-intensity supraorbital nerve stimulation (Drummond, 2003, 2004). This finding is consistent with the view that the major role of DNIC is to suppress background activity elicited by innocuous stimuli in WDR neurons (Villanueva and Le Bars, 1995). Painful infrared laser stimulation that selectively activates nociceptive Ad fibres in the forehead evokes a bilateral blink reflex at a latency corresponding to R2, but does not evoke an early ipsilateral R1 component (Ellrich et al., 1997). Thus, nociceptive Ad fibres that project to the trigeminal nucleus caudalis apparently contribute to R2. The R2 of the BR can also be reliably elicited by electrical and laser stimulation of the infraorbital and mental nerves (Ellrich and Hopf, 1996; Gandiglio and Fra, 1967; Jaaskelainen, 1995). Ellrich and Treede, (1998) reported that painful thermal stimulation of the forearm inhibited R2 to innocuous stimulation of the supra-orbital nerve consistent with involvement of WDR neurons in R2. In the present study the R2 was significantly decreased which is consistent with the previous findings. There were no differences in onset latencies of BR responses between left and right sides in our population of 30 healthy subjects. This result was consistent with the previous study (Katsarava et al., 2002), in which measurement of lateral differences revealed that the difference of onset latencies more than 4.6 ms could be assumed as abnormal. These findings are in parallel with the observations in patients with acute migraine attacks (Kaube et al., 2002) with reversible but abnormal decreases of onset latencies on the headache site. However, the R2 RMS and AUC values of the left (stimulation side) orbicularis oculi were significantly greater than the right orbicularis oculi muscles in both males and females. The estimation of right vs. left differences regarding onset latencies and response areas could be the main aspect in the detection of BR asymmetries in unilateral involvement of the trigeminal migraine headache, trigeminal neuralgia, acute peripheral nociceptive pain and atypical facial pain (Katsarava et al., 2002). In the present study, the stimulus intensities to evoke the three sensations; sensory thresholds (Is), pin-prick pain sensation (Ip) and electrical pain sensation, were lower in females than in males and the VAS scores for the electrical pain sensation were significantly higher in females compared to males in pain and post-pain conditions. This is in agreement with the previous concept that women seem to show lower pain thresholds, a greater ability to discriminate painful sensations, higher pain ratings, and a lower tolerance for pain (Berkley, 1997; Dao and LeResche, 2000; Svensson et al., 2003a; Vallerand and Polomano, 2000). Gender is an important variable and should be taken into account in both research and the clinical practice of pain management. In conclusion, the present results indicated that experimental posterior temporalis muscle pain facilitates the jawstretch reflex. As the pre-stimulus EMG activity was

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carefully controlled and standardized, this effect cannot be the result of the level of motoneuronal excitation; instead a muscle pain-related increase in sensitivity of the fusimotor system appears to be the net effect of acute deep nociceptive input, whereas, the nociceptive specific BR is inhibited, which could be related to suppressed activity of WDR neurons in the medullary dorsal horn mediating the R2 blink reflex. The present study did not suggest gender-related differences in the effect of experimental muscle pain on these two types of brainstem reflexes. Present study suggested that the BR and stretch reflexes are suitable models for probing pontine and medullary pain processing.

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