Repeated intramuscular injections of nerve growth factor induced progressive muscle hyperalgesia, facilitated temporal summation, and expanded pain areas

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PAIN 154 (2013) 2344–2352

www.elsevier.com/locate/pain

Repeated intramuscular injections of nerve growth factor induced progressive muscle hyperalgesia, facilitated temporal summation, and expanded pain areas Koei Hayashi a,b,c, Shinichiro Shiozawa a, Noriyuki Ozaki b, Kazue Mizumura d, Thomas Graven-Nielsen a,⇑ a Laboratory for Musculoskeletal Pain and Motor Control, Center for Sensory-Motor Interaction (SMI), Department of Health Science and Technology, Faculty of Medicine, Aalborg University, Aalborg, Denmark b Department of Functional Anatomy, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan c Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan d Department of Physical Therapy, College of Life and Health Sciences, Chubu University, Kasugai, Japan

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

a r t i c l e

i n f o

Article history: Received 30 December 2012 Received in revised form 6 July 2013 Accepted 9 July 2013

Keywords: Nerve growth factor (NGF) Soreness Muscle hyperalgesia Temporal summation Referred pain

a b s t r a c t Intramuscular injection of nerve growth factor (NGF) is known to induce deep-tissue mechanical hyperalgesia. In this study it was hypothesised that daily intramuscular injections of NGF produce a progressive manifestation of soreness, mechanical hyperalgesia, and temporal summation of pain. In a double-blind placebo-controlled design, 12 healthy subjects were injected on 3 days with NGF into the tibialis anterior muscle and with isotonic saline on the contralateral side. Assessments were performed before and after the injections on days 0, 1, and 2, and repeated on days 3, 6, and 10. The self-perceived muscle soreness was assessed on a Likert scale. Computer-controlled pressure algometry was used to assess the pressure pain thresholds (PPTs). Temporal summation of pain after repeated pressure stimulations was assessed by computer-controlled pressure algometry. The pain distribution following painful pressure stimulation was also recorded. Compared with baseline and isotonic saline, the NGF injections caused (P < 0.05): (1) progressively increasing soreness scores from 3 hours after the first injection until day 2, after which it remained increased; (2) decreased PPTs at days 1 to 3; (3) facilitated temporal summation of pressure pain at days 1 to 10; and (4) enlarged pressure-induced pain area after the injection on day 1 to day 6. The daily injections of NGF produced a progressive manifestation of muscle soreness, mechanical hyperalgesia, temporal summation of pressure pain, and pressure-induced pain distribution. These data illustrate that the prolonged NGF application affects peripheral and central mechanisms and may reflect process in musculoskeletal pain conditions. Ó 2013 Published by Elsevier B.V. on behalf of International Association for the Study of Pain.

1. Introduction The myofascial pain syndrome is an important chronic muscle pain condition characterised by myofascial trigger points located within taut bands of skeletal muscle, mechanical hyperalgesia, and referred pain induced by pressure on the trigger point [7]. The pathophysiological mechanisms involved in myofascial trigger points are, however, still unclear. Localised hyperalgesia to mechanical stimulation accompanied by referred pain is the main characteristic of myofascial trigger

⇑ Corresponding author. Address: Laboratory for Musculoskeletal Pain and Motor Control, Center for Sensory-Motor Interaction (SMI), Department of Health Science and Technology, Faculty of Medicine, Aalborg University, Fredrik Bajers Vej 7D-3, Aalborg E 9220, Denmark. Tel.: +45 9940 9832; fax: +45 9815 4008. E-mail address: [email protected] (T. Graven-Nielsen).

points. Similar focal mechanical hyperalgesia in the deep tissue has been reported experimentally in exercise-induced delayedonset muscle soreness [2]. Interestingly, facilitated central manifestations have also been reported after exercise-induced delayed-onset muscle soreness as facilitated temporal summation of pressure-induced muscle pain [20]. Computer-controlled sequential pressure stimulation on muscle evoked facilitated temporal summation of pain in chronic musculoskeletal pain patients, and central sensitisation has been proposed as an explanation for the findings [4,26–28]. It is likely that myofascial trigger points involve both facilitated peripheral and central mechanisms. However, it is not known if focal and prolonged muscle hyperalgesia has similar facilitating effects on central mechanisms such as referred pain and temporal summation of pain. Although no direct evidence has been presented, nerve growth factor (NGF) has been proposed as an important substance

0304-3959/$36.00 Ó 2013 Published by Elsevier B.V. on behalf of International Association for the Study of Pain. http://dx.doi.org/10.1016/j.pain.2013.07.007

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involved in muscle hyperalgesia, such as in myofascial trigger points. NGF is an important neurotrophic protein implicated in maintenance and differentiation of sensory and sympathetic neurons and later in adulthood in nociception and pathological pain condition [16]. NGF is secreted from various structures, including skeletal muscle tissue [1], and during inflammation NGF levels increase in the periphery [22]. When NGF is present for a long time, such as during inflammatory conditions, it is transported retrograde from the periphery to the dorsal root ganglion and is thereby possibly involved in central sensitisation [16]. The potency of NGF for deep tissue is illustrated by long-lasting upregulated muscle NGF accompanied with guarding pain behaviours following a muscle incision [34]. In animal studies, NGF has been implicated in the delayed-onset muscle soreness where NGF was upregulated in the muscle after a single bout of eccentric contraction and essential for maintaining muscular mechanical hyperalgesia after eccentric exercise [18]. Furthermore, a recent study showed that repeated bouts of eccentric contractions for several days induced increased NGF expression in regenerating muscle cells, and produced a long-lasting and progressive muscular mechanical hyperalgesia [11]. In humans, intramuscular injection of NGF induced mechanical hyperalgesia 3 hours after administration, which peaked after 24 hours and lasted for 7 days [3,29,31]. Similar effects have been reported after NGF injection into the fascia [5] and after intradermal NGF injection in humans [5]. However, the prolonged effect of NGF on the muscle sensitivity has not been studied. In the current study it is proposed that daily repeated intramuscular injections of NGF mimic the peripheral and central mechanisms potentially effective in some musculoskeletal pain conditions such as myofascial trigger points. Moreover, it is hypothesised that daily intramuscular injections of NGF induce a progressive manifestation of self-perceived muscle soreness, hyperalgesia, temporal summation of pain, and pressure-induced pain areas. 2. Materials and methods 2.1. Subjects A total of 12 (5 female) healthy subjects participated in the study; subjects were aged 24–39 years (mean 30) and had body mass indexes of 18–29 (kg/m2). The number of subjects was based on a previous study with a similar design to assess the NGF effects [29]. The subjects had no pain complaints or history of injuries to the lower leg and were not taking any medication. The women included in the study all took oral contraceptives. Previously, the NGF-induced muscle soreness assessed by pressure pain thresholds and Likert scores was not found to be different between males and females [3], arguing for inclusion of both genders in the present study. The inclusion time for females was not synchronised with the contraceptive break, but the control leg used in the experimental design allowed accounting for general changes in the pain sensitivity. All subjects were given a verbal explanation of the study, and written informed consent was obtained from each subject prior to inclusion in the study. The study was approved by the local ethics committee (N-20100095) and conducted in accordance with the Declaration of Helsinki. 2.2. Experimental design The study was performed as a randomized, partially doubleblind, placebo-controlled design. Both legs received 3 injections of either NGF or isotonic saline (control) into the belly of the tibialis anterior muscle (TA). The sensory sensitivity was assessed on both legs. The right leg was always examined first, but the administration side of NGF and control was randomized (ie, in 6 subjects the right TA

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was injected with NGF, and in the other 6 subjects the left TA was injected with NGF). Pressure pain thresholds (PPTs), cutaneous mechanical sensitivity, temporal summation to sequential pressure stimulation, and pressure-induced referred pain were assessed before and 1 hour after the injection on days 0, 1, and 2, then repeated on days 3, 6, and 10. The area of tenderness was assessed before and 1, 3, and 6 hours after the first injection on day 0, then repeated on days 1, 2, 3, 6, and 10. A modified Likert scale diary was used to assess muscle soreness before and 1, 3, and 6 hours after the first injection on day 0, and thereafter on days 1 to 10, 16, and 22. The same examiner performed all the assessments and experimental procedures, but was blinded to which leg was injected with NGF. Another examiner prepared and randomized the side of NGF or control injections. All subjects were blinded with respect to administration of NGF or control. At least in the beginning of the experiment the subjects were effectively blinded to the experimental condition, but after some days most subjects felt which leg was sorer. 2.3. Assessment sites Three sites (sites 1, 2, and 3) were selected for assessments of PPT, cutaneous mechanical sensitivity, and temporal summation. Sites 1 and 2 were located over the TA muscle belly and site 3 was on the web between the first and second metatarsals. Site 1 was identified as one-third of the distance from the inferior border of the patella to the midpoint of the transverse crease of the ankle, and 2.5 cm lateral to the tibial tuberosity. Site 2 was identified as 8 cm distal to site 1. Site 3 was chosen to test for potential widespread effects because this site is innervated by the deep peroneal nerve, likewise the TA muscle [3]. All sites were marked with a permanent marker, and the subjects were asked to keep the marked sites visible for the duration of the experiment. 2.4. NGF administration and assessment of injection pain The sterile solutions of recombinant human NGF were prepared by the pharmacy at Aalborg hospital. A dose of 5 lg NGF (0.2 mL) was given as a bolus injection, as described previously [3,6,29], into the muscle belly of the TA (assessment site 1), and this was repeated for 3 days. As a control, the same volume of isotonic saline (0.2 mL) was injected into the contralateral TA muscle. During and immediately after each injection, the subjects scored continuously the pain intensity on an electronic visual analogue scale (VAS) from 0 to 10 cm, where 0 indicated ‘‘no pain’’ and 10 cm indicated ‘‘maximum pain.’’ The data were sampled every 1 second and area under the VAS-time curve (VAS area) and maximum scores (VAS peak) were calculated for analysis. 2.5. Assessment of self-perceived muscle soreness To assess muscle soreness during the 22 days of the study, the subjects were asked to fill in a modified 7-point Likert scale [24]: 0 = a complete absence of soreness; 1 = a light soreness in the muscle felt only when touched – a vague ache; 2 = a moderate soreness felt only when touched – a slight persistent pain; 3 = a light muscle soreness when walking up and down stairs; 4 = a light muscle soreness when walking on flat surface; 5 = a moderate muscle soreness, stiffness, or weakness when walking; 6 = a severe muscle soreness, stiffness, or weakness that limits the ability to move. 2.6. Pressure algometry Pressure stimulation was applied perpendicularly to the skin surface by a computer-controlled pressure algometer (Aalborg University, Aalborg, Denmark) to record the PPT [10]. Step motors

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ensured correct coordinates for stimulation sites. All sites were assessed 3 times with 1-minute intervals, and the average PPT of the 3 stimulations was used for the statistical analysis. During the pressure stimulation the leg was positioned below the probe for pressure stimulation. A kapok-filled vacuum cushion stabilized the shin during the stimulation and the angle of pressure application was similar for the same site in each session. The probe was circular (1 cm2) and covered with firm rubber. The pressure stimulation was force-feedback controlled and increased at a constant rate (0.6 kg/s). The subject was asked to stop the pressure stimulation at PPT via a push button. The PPT was defined as the point at which a sensation of pressure changed to a sensation of pain. The PPT examination was first performed on site 1, followed by sites 2 and 3. The PPTs were normalised by the PPT measures from the baseline session (day 0). 2.7. Temporal summation of pain induced by sequential pressure stimulation Sequential stimulation consisted of 10 pressure stimuli (1-second duration) with 2-second intervals at the PPT level assessed at baseline for the individual site [19]. Skin contact between the individual pressure stimuli was ensured by keeping a constant force of 0.1 kg; that is, during the series of sequential stimulation, the probe had skin contact and was withdrawn after 10 stimuli. The contact force of 0.1 kg between 2 stimuli does not evoke pain [19]. The subjects scored the pain intensity continuously during the sequential stimulation on an electronic VAS from 0 to 10 cm, where 0 indicated ‘‘no pain,’’ and 10 cm indicated ‘‘maximum pain.’’ The mean VAS scores during 1 second after each stimulus were extracted (ie, 10 VAS scores for each series of sequential stimuli). For further analysis, the 10 VAS scores were normalised by subtraction of the score from the first stimulus. Moreover, the sum of normalised VAS scores was extracted as a parameter reflecting the degree of temporal summation. 2.8. Assessment of tenderness area and pressure-induced pain areas The tenderness areas were assessed by manual palpation, which was applied 1 cm apart. The applied pressure to the shin was about 1 kg and lasted for 1 second. After each pressure, the subjects indicated if the palpation point was sore or not, and then the examiner filled in a body chart marking the tenderness area. To evaluate the extension of localised and referred pain areas, a pressure stimulation equal to 120% of the baseline PPT was applied on site 1 in the NGF and control side for 2 seconds by the computer-controlled pressure algometer (Aalborg University) [8]. After the 2-second pressure stimulus, the subjects filled in a body chart marking all areas of local and referred pain. This was later digitised and the pain areas were estimated [8]. Moreover, to assess spread or referral of pain, the lower limb of the body charts was divided into 5 zones and the frequency of pain occurrence within each zone was estimated. Zone 5 (V) was defined as the foot and ankle below a line drawn between the malleoli. Zones 1 (I) to 4 (IV) were calcu-

lated as being the 4 equal subdivisions of the distance between the line joining the malleoli and the tibial plateau. The pressure stimulation site relates to Zone II. Local pain was defined as pain occurring in Zone II. Referred pain (typically Zones IV and V) was defined as being pain isolated and distinct from the local pain caused by the stimulation. Enlarged pain areas were defined as pain that spread out with the Zone II. 2.9. Cutaneous sensitivity The cutaneous mechanical sensitivity was assessed with von Frey nylon monofilament (Somedic production AB, Hörby, Sweden) before the PPT assessment on all sites [3]. The skin was stimulated by standardised application of filament number 17 (133 g/mm2), and the subjects scored the sensation on a scale from 0 to 10 cm, where 0 indicated ‘‘no sensation,’’ 5 cm indicated ‘‘pain threshold,’’ and 10 cm meant ‘‘maximum pain.’’ These von Frey hair scores allowed assessment of both increased and decreased sensations in the painful and nonpainful range. 2.10. Statistical analyses In case a majority of time points within one parameter passed the Shapiro-Wilk normality and Kolmogorov-Smirnov tests, parametric statistical analysis was used. In consequence, the baseline PPT, baseline tactile sensitivity, and normalised pressure-induced pain areas were analysed by 2-way repeated-measures analysis of variance (RM-ANOVA) with the factors: Injection type (NGF,isotonic saline)and assessment sites (1–3) or time (baseline and subsequent assessments). The normalised PPT and temporal summation VAS scores were analysed by a 3-way RM-ANOVA with factors: Injection type (NGF,isotonic saline),time (baseline and 8 subsequent assessments), and stimulation number (1–10) orassessment sites (1–3). The Newman– Keuls (NK) test was used for post hoc comparisons incorporating correction for the multiple comparisons in case of significant factors or interactions. The VAS parameters after injections, Likert scores, and normalised tactile sensitivity data were analysed with the nonparametric Friedman analysis of variance and, if significant, followed by Wilcoxon post hoc comparisons; Bonferroni corrections were used to adjust P-values for multiple comparisons within tests of data from the control side or when comparing with baseline data, respectively. The data are presented as mean and SEM in text and figures or median and interquartile range when appropriate. A P-value of 6 0.05 was considered significant. 3. Result 3.1. Pain intensity after NGF/control injections The NGF injections induced a short-lasting weak pain around the injection site. The VAS area after the second and third NGF injections was significantly higher compared with the control injections [Table 1; Friedman: v2 (5) = 18.6, P < 0.002; Wilcoxon & Bonferroni: P < 0.016]. In line, the VAS peak after the second NGF injection was

Table 1 Median [25th and 75th % quartile] area under the curve VAS-time curves and maximum VAS after the 3 NGF/control injections. VAS area (cm s)

1st injection 2nd injection 3rd injection

VAS peak (cm)

NGF

Saline

NGF

Saline

2.3 [0.0–25.4] 24.2 [10.5–67.5]⁄,# 14.0 [8.3–101.8]⁄

14.0 [0.0–32.9] 0.1 [0.0–28.8] 0.9 [0.9–18.1]

0.1 [0.0–0.9] 0.7 [0.6–1.6]⁄ 0.6 [0.4–2.6]

0.6 [0.0–1.3] 0.1 [0.0–0.9] 0.1 [0.1–1.0]

VAS, visual analogue scale; NGF, nerve growth factor. Significantly higher VAS scores compared with isotonic saline (⁄Wilcoxon & Bonferroni: P < 0.04). Significantly higher VAS scores compared with the first injection of NGF # ( Wilcoxon test: P < 0.04).

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3.3. Pressure pain thresholds The baseline PPTs on the 3 sites were not different between legs (site 1: 4.0 ± 0.2 kg; site 2: 3.8 ± 0.2 kg; site 3: 2.4 ± 0.1 kg), but site 3 had significantly lower PPT compared with sites 1 and 2 [RM-ANOVA: F(2) = 29.1, P < 0.000001; NK test: P < 0.0001]. A significant interaction between sites, time, and injection type were found for the PPTs [Fig. 2A; RM-ANOVA: F(16) = 2.27, P < 0.005]. The PPTs at site 1 were significantly decreased after NGF injections at days 1, 2, 3, 6, and 10 when compared with isotonic saline injections, and at day 1, 2, and 3 after NGF injections compared with baseline and day 10 recordings (NK test: P < 0.02). After isotonic saline injections, the PPTs were significantly increased compared with baseline at day 6 and 10 (NK test: P < 0.0001). No progressive decrease of PPT was observed after repetition of NGF injection. The PPTs at site 2 were significantly decreased after NGF injections at days 2 (before injection), 3, and 6 when compared with isotonic saline injections (NK test: P < 0.04) and significantly increased at day 10 after both isotonic saline and NGF injections compared with all other recordings except day 6 (Fig. 2B; NK test: P < 0.003). The PPT at day 6 in the isotonic saline series was also increased compared with baseline. The PPTs at site 3 were not changed during the observation period (Fig. 2C).

PP PT (% of basseline)

The Likert scores showed higher soreness scores in the NGF side from day 1 after the first NGF injection until day 16 compared with baseline and the control side [Fig. 1; Friedman: v2 (31) = 332.5, P < 0.00001; Wilcoxon & Bonferroni: P 6 0.05].

site 1

50

B 150

$

$

100

PPT (% of baselinee)

3.2. Self-perceived muscle soreness

A 150

*#

*#

BI

AI

*#

*#

BI

AI

*#

*

*

#

Base

1h

3

6

site 2

10 $

$

$

*

100

50

C 150 PPT (% off baseline)

significantly higher compared with the control injection [Friedman: v2 (5) = 13.2, P < 0.02; Wilcoxon & Bonferroni: P < 0.041].

*

Base

1h

BI

AI

site 3

*

BI

AI

3

6

10

NGF side Control side (Isotonic saline)

100

50

Base 0

1h

BI

AI 1

BI

AI

3

6

10

2

Days

3.4. Temporal summation of pressure pain A significant main effect of stimulus number was found for VAS scores [RM-ANOVA: F(9) > 28.8, P < 0.000001], indicating a progressive increase in the VAS scores over the 10 sequential stimulation for all assessment sites (Fig. 3). VAS scores at site 1 showed a significant interaction between stimulus number, time, and injection type [RM-ANOVA: F(72) = 2.62, P < 0.000001]. All VAS scores from the 2nd to 10th stimulus at day 1 to 10 was significantly higher after the NGF injec-

5

Likert scale (0-6)

NGF Control side/iso

4

* * *

3

*

*

2

* * * *

1

0

Base 1h 3h 6h 0

1

2

3

4

5

6

7

8

9

*

*

10 16 22

Days Fig. 1. Median Likert scale score (25th and 75th % quartile, n = 12) of muscle soreness after injection of nerve growth factor (NGF) and control injection of isotonic saline. Arrows indicate the timing of the injections. Significantly higher score compared with the control side and baseline, day 0 (⁄Wilcoxon & Bonferroni: P 6 0.05).

Fig. 2. Mean (± SEM, n = 12) normalised pressure pain threshold (PPT) of the nerve growth factor (NGF) and control side at sites 1 (A), 2 (B), and 3 (C). PPTs were measured on day 0 until day 10. Base: before the first injection, 1 h: 1 hour after the first injection, BI: before the injection, AI: 1 hour after the injection. Arrows indicate the timing of the injections. The PPT values were normalised to baseline value and shown as a percentage of baseline-PPT. Significantly reduced compared with the control side (⁄Newman–Keuls [NK] test: P < 0.04) and baseline, day 0 (#NK test: P < 0.02). Significantly increased compared with the baseline, day 0 ($NK test: P < 0.02).

tions compared with the comparable scores after isotonic saline injections (Fig. 3A; NK test: P < 0.02). At site 2, an interaction between injection type and stimulus number showed that NGF injections caused significantly higher VAS scores for the 2nd to 10th stimuli after NGF injections, compared with isotonic saline [RM-ANOVA: F(9) = 4.82, P < 0.0004; NK test: P < 0.0006]. This was further qualified with a significant interaction between time and stimulus number, illustrating that VAS scores after stimuli 3 to 10 were significantly higher at days 1 and 2 compared with assessments at day 0 and day 10 [RM-ANOVA: F(72) = 1.71, P < 0.0004; NK: P < 0.04]. Combining these measures at site 2 illustrates that NGF gives progressively higher VAS scores, especially at day 1 and 2 compared with isotonic saline (Fig. 3B). At site 3, an interaction between injection type and stimulus number showed that NGF injections caused significantly higher VAS scores for the 2nd to 10th stimuli after NGF injections compared with isotonic saline [RM-ANOVA: F(9) = 4.94, P < 0.00002; NK test: P < 0.005]. In addition, there was a significant interaction between time and stimulus number, illustrating that VAS scores after stimulus 7 to 10 were significantly higher at day 2 before the NGF injection compared with assessments at day 0 and day 10 [RM-ANOVA: F(72) = 1.34, P < 0.04; NK test: P < 0.04]. Thus, compared with

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NGF side

Normalised VAS (cm)

A 3

Normalised VAS (cm m)

B

** ** * * *

2 1 0 3

3.5. Tenderness area and pressure-induced pain areas

Site 1 Baseline Day 2 AI

Day 0 AI Day 3

One hour after the first NGF injection (day 0), 25% of subjects felt tenderness at the injection site. Three hours after the first NGF injection, 83% of subjects felt the tenderness still localised around the injection site. On day 1 all subjects felt the tenderness in the NGF side, and it continued until day 10. The tenderness area in the NGF side gradually expanded both proximally and distally with time (Fig. 4). In contrast, the tenderness was observed in only 2 (17%) subjects in the control side. The pain induced by the pressure stimulation at 120% of the pain threshold on site 1 was localised at day 0; at day 2, and especially day 3, the pain was referred from site 1 downward over the shin to the ankle and dorsal foot on the NGF-injected side (Fig. 5A). The pressure-induced pain area in the NGF side was significantly enlarged after the second injection on day 1 until day 6, compared with baseline and with the control injection of isotonic saline [Fig. 5B; RM-ANOVA: F(8) = 6.04, P < 0.000004; NK test: P < 0.003]. The distribution of pressure-induced pain illustrated that the baseline pressure-induced pain was only localised to Zone 2, and in the course of NGF injections the number of zones with pain occurrence increased progressively with both enlarged pain and pain referral (Table 3). In the control leg (contralateral to the NGF leg), the pain was localised throughout the experiment, except for one subject who interestingly developed referred pain at days 2 and 3.

Site 1

1 2 3 4 5 6 7 8 9 10

2

1 2 3 4 5 6 7 8 9 10

Day 1 BI Day 6

Day 1 AI Day 10

Day 2 BI

** * *

1 0

C 3 Normalised VAS (cm)

RM-ANOVA: F(8) = 3.66, P < 0.001], illustrating that the VAS sum at days 1 to 6 were significantly increased after the NGF injections compared with isotonic saline and baseline (NK test: P < 0.04). At sites 2 and 3, a main effect of injection type was found with higher VAS sum for NGF injections compared with isotonic saline [RMANOVA: F(1) > 5.91, P < 0.03; NK: P < 0.03].

Control side

Site 2

Site 2

1 2 3 4 5 6 7 8 9 10

2

1 2 3 4 5 6 7 8 9 10

*

1 0

Site 3

Site 3

1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

Stimulation number

Stimulation number

3.6. Cutaneous sensitivity

Fig. 3. Average visual analogue scale (VAS) scores reflecting temporal summation of sequential pressure pain (n = 12) of nerve growth factor (NGF) and control side to stimuli with 2-second interstimulus interval (ISI) at sites 1 (A), 2 (B), and 3 (C). Each average VAS score reflects the pain sensation to one pressure stimulus (1 second duration) delivered with 2-second interval at the pressure pain threshold level assessed at baseline for the individual site. VAS scores were offset corrected by subtracting the VAS score from the first stimulation. Temporal summation was measured on day 0 until day 10. Baseline: before the first injection (day 0); BI: before the injection; AI: 1 hour after the injection. Last scores significantly higher than the comparable recording after isotonic saline (⁄see text for details).

isotonic saline, NGF gives higher VAS scores at site 3 for the last pressure stimulations, in particular at day 2 before the injection (Fig. 3C). In addition to the above detailed analysis of individual VAS scores during repeated pressure stimulations, the sum of VAS scores was also evaluated. At site 1 a significant interaction was found between time and injection type for the VAS sum [Table 2;

The cutaneous mechanical sensitivity assessed at baseline by scores after von Frey hair stimulations was not different between legs (site 1: 0.70 ± 0.09 cm; site 2: 0.77 ± 0.2 cm; site 3: 1.03 ± 0.11 cm), but site 3 had significantly higher von Frey hair scores compared with sites 1 and 2 [RM-ANOVA: F(2) = 9.18, P < 0.001; NK test: P < 0.0001]. The subsequent time points did not change due to the injections (data not reported). 4. Discussion The novel findings in the present study were that repeated intramuscular administration of NGF (1) evoked muscle soreness, prolonged decrease of PPT, facilitated temporal summation of pressure pain, and enlargement of the pressure-induced pain distribution, and (2) produced sensitised spots similar to myofascial trigger

Table 2 Mean (± SEM, n = 12) sum of VAS scores following the 10 repeated pressure stimulations to assess temporal summation on the days before (BI) and 1 hour after injection (AI) of NGF and isotonic saline. VAS sum (cm) Site 1 (inj. site) Site 2 Site 3

NGF Iso saline NGF# Iso saline NGF# Iso saline

Day 0 Base

Day 0 AI

Day 1 BI

Day 1 AI

Day 2 BI

Day 2 AI

Day 3

Day 6

Day 10

5.8 ± 1.1 6.3 ± 1.6 7.5 ± 1.7 7.1 ± 1.5 8.8 ± 2.6 7.5 ± 1.6

9.5 ± 1.5 7.5 ± 1.3 8.8 ± 2.1 6.3 ± 1.0 8.9 ± 1.8 8.3 ± 1.2

17.0⁄ ± 2.7 7.5 ± 1.3 15.4 ± 2.4 8.8 ± 1.9 9.8 ± 1.5 8.5 ± 1.1

14.4⁄ ± 2.5 5.9 ± 1.4 12.1 ± 2.3 9.9 ± 2.4 9.6 ± 1.5 7.1 ± 1.3

17.7⁄ ± 4.1 9.0 ± 2.1 15.3 ± 3.2 9.1 ± 1.4 12.6 ± 1.9 10.2 ± 1.6

21.0⁄ ± 3.5 7.8 ± 1.6 14.0 ± 3.6 7.9 ± 1.4 13.9 ± 2.4 6.3 ± 1.3

19.2⁄ ± 4.1 4.8 ± 1.1 9.8 ± 3.0 6.7 ± 1.9 10.8 ± 2.3 8.2 ± 3.0

16.4⁄ ± 4.2 6.0 ± 1.9 10.4 ± 3.6 7.3 ± 2.0 10.3 ± 3.1 9.0 ± 1.7

10.9 ± 3.2 3.2 ± 1.1 8.4 ± 2.6 5.0 ± 1.5 5.3 ± 1.4 4.7 ± 0.9

VAS, visual analogue scale; NGF, nerve growth factor. Significantly higher compared with isotonic saline and baseline (⁄Newman–Keuls [NK]: P < 0.04). Significantly main effect in the repeated-measures analysis of variance illustrating generally higher sum of VAS scores after NGF injections compared with isotonic saline (#NK: P < 0.03).

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NGF Control

™

™

™

™

Baseline

1 h after

3 h after

6 h after

Day 0

Day 2

Day 3

Day 6

Day 1

Day 10

Fig. 4. Body chart drawings of tenderness area assessed by manual palpation. The tenderness area was assessed before the first injection, 1 hour, 3 hours, and 6 hours after the first injection on day 0, then, on days 1, 2, 3, 6, and 10. Arrows indicate the timing of the injections. The crosses indicate the assessment sites. Injection sites are indicated by solid circles. NGF, nerve growth factor.

points, which induced pain referral or enlarged pain when pressure was applied. 4.1. NGF-induced muscle pain Consistent with previous studies using single NGF injection, administration of NGF induced short-lasting weak pain [3,29,30], but the present study also demonstrated significantly higher NGF pain intensity after the second and third NGF injection compared with the pain induced by isotonic saline, which is in contrast with previous findings. Animal data reflecting the excitation of muscle nociceptors by NGF injections are conflicting, as excitation of thin muscle-fibre afferents with high mechanical threshold are reported [13], whereas others find that intramuscular injections of NGF do not evoke significant nociceptor discharge [32]. 4.2. Self-perceived soreness following NGF In a study using the same dose of NGF in healthy subjects as in the present study and evaluating the soreness on the Likert scale, it was reported that a single injection induced soreness beginning 3 hours after the injection, peaked at 24 hours (Likert scale: 2.0 ± 0.2), and lasted for 7 days [3]. In the present study, 3 repeated injections of NGF evoked the muscle soreness assessed on the Likert scale, with peak soreness 72 hours after the first injection (ie, 24 hours after the third injection; Likert scale: 3.3 ± 0.3), and lasted for 16 days from the first injection (ie, for 14 days from the third injection). Comparing peak soreness intensities, the present model of repeated NGF injections was superior compared with the single injections, but it is not known if this is due to the prolonged effects of NGF or due to the higher accumulated dose of NGF in this current experimental design. Other injection-based musculoskeletal pain models (eg, hypertonic saline, capsaicin) do not induce long-term and spreading hyperalgesia [9].

4.3. NGF-induced mechanical hyperalgesia NGF-induced tenderness to manual palpation was observed on the NGF injection site 3 hours after the first NGF injection, then gradually the area expanded both proximally and distally with time, and then on day 3 it spread over the shin. The expanded areas of tenderness were maintained until day 10. Repeated NGF injections caused a significant decrease of PPT at the injection site observed at 24 hours after the first injection, and it lasted until 10 days after the first injection (ie, until 8 days after the third injection) when comparing with the control leg, or lasted 3 days when compared with the baseline measures. Assessment of the cutaneous mechanical sensitivity in the days following intramuscular NGF injections revealed no sensitivity changes, excluding the skin as a major contributor to the hyperalgesia detected by pressure algometry. Although this finding is in line with previous reports [3,6], other cutaneous effects cannot be excluded following assessments with a complete battery of cutaneous quantitative sensory tests. The duration of deep tissue hyperalgesia is longer-lasting compared with the previous single-injection study where the hyperalgesia was observed 3 hours after the NGF injection into the TA muscle, and lasted until 24 hours after the injection [3]. Since there was no progressive decrease in the PPT after either second or third injection in the present study, the prolonged duration of the deep tissue hyperalgesia is most likely due to replication of the NGF injections. The PPT values assessed 8 cm away (site 2) from the injection site also demonstrated hyperalgesia to pressure after the NGF injections compared with the control leg, which is in line with the expanded areas of palpable tenderness and previous findings [3]. This indicates that the mechanism involved in the NGF-induced hyperalgesia not only is based on localised peripheral sensitisation, but also may involve sensitisation of central mechanisms. Previously it was found that the spreading hyperalgesia was not affected when anaesthetising the site originally injected with NGF [6], suggesting involvement

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A NGF Control I II III IV V

Baseline 1h after ft Day 0

BI

AI

Day 3

B

BI Day 2

Day 6

Day 10

1800

*

NGF

s) Pain area a (arbitrary units

AI

Day 1

Control side () (iso) 1200

*

*

*

*

600

0 B Base

1h

BI

0

AI

BI

AI

3

6

10

12 Days

Fig. 5. (A) Body chart drawings of the pain area after 120% pressure stimulation of the baseline-pressure pain threshold (PPT) at site 1. Zones (I–V) for further quantification of the pain spreading is illustrated at the baseline drawings. (B) Mean (± SEM, n = 12) pain areas. Significantly enlarged pressure-induced pain areas compared with baseline and the control side (⁄Newman–Keuls [NK] test: P < 0.003). NGF, nerve growth factor.

Table 3 Number of pain occurrence (max = 12) in 5 zones following pressure stimulation on the injection site on the days before (BI) and after injection (AI) of NGF and isotonic saline. NGF

Control

Zones

I

II

III

IV

V

I

II

III

IV

V

Base 1 hour AI Day 1 BI Day 1 AI Day 2 BI Day 2 AI Day 3 Day 6 Day 10

0 0 0 1 0 0 5 2 1

12 12 12 12 12 12 12 12 12

0 0 1 1 2 4 3 1 0

0 0 0 0 0 3 4 1 0

0 0 1 2 2 2 3 2 1

0 0 0 0 0 0 0 0 0

12 12 12 12 12 12 12 12 12

0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

0 0 0 0 1 1 1 0 0

NGF, nerve growth factor. Zone V includes the foot and ankle. Zones I–IV are 4 equal subdivisions of the leg below the knee and above Zone V. The pressure stimulation site relates to Zone II.

of a central mechanism. Interestingly, contralateral spreading sensitisation has been demonstrated in animal musculoskeletal pain models [25]. In such cases, the current experimental design using the contralateral leg as control may not be ideal. Individual exam-

ples of contralateral effects may be indicated (eg, pressure-induced pain, Fig. 5), but importantly, no systematic effects show sensitisation compared with baseline findings in the control leg. The reduced pressure pain sensitivity detected by pressure pain thresholds in the contralateral leg also has been reported after single injection of NGF [3,21,29] and may be due to peripheral desensitisation or reduced focus on the assessment procedures when approaching the end of the experiment. 4.4. NGF-evoked facilitated temporal summation of pressure pain Temporal summation of pain is the perceptual correlate in humans that is thought to mimic the wind-up process in dorsal horn neurons. Wind-up is a phenomenon where the sensitivity of dorsal horn neurons to C-fibre input can be enhanced by repetitive stimulation at rates above 0.3 Hz [17]. In chronic musculoskeletal pain, temporal summation to repetitive pressure stimulations has been demonstrated to be facilitated compared with healthy controls [4,27]. In the present study, the effects of NGF injections were demonstrated as facilitated temporal summation of pain already evident after 1 day when stimulating the injection site. These data are in

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line with the facilitated temporal summation to pressure stimulation detected 1 day after exercise-induced delayed muscle soreness [20] and detected after 1 day in a model combining the NGF injections with exercise-induced muscle soreness [21]. In addition to the previous findings, the facilitated temporal summation was also detected at day 2 when stimulating the assessment sites away from the injection site. Temporal summation to pressure stimulation seems to be more sensitive in detecting the long-term sensitisation following NGF injections compared with the pressure pain thresholds, which are increased in the NGF leg at days 6 and 10. The degree of temporal summation reflects the excitability of the related central mechanism, whereas the pressure pain thresholds are combined measures that reflect both peripheral and central sensitivity. Thus, the reduced pressure pain sensitivity detected by pressure pain thresholds and not by temporal summation at day 6 is evidence of the robustness of temporal summation in assessing a central mechanism. 4.5. Pressure-induced pain referral The expanded pain areas accompanied with referred pain following intensive pressure stimulation developed in the course of repeated NGF injections. This finding has not been reported before in a series of studies on single-NGF administration. In the present study, the referred pain area induced by the nociceptive pressure stimulation on the NGF injection site was consistent with the pain referral from myofascial trigger points in the TA muscle [23] and experimentally induced by injections of hypertonic saline into the TA muscle [9,15]. The pain referral pattern observed is probably related to the opening of latent excitatory synapses at the spinal cord level expanding the receptive field of nociceptive afferent neurons; the opening of latent excitatory synapses can take place with strong or prolonged nociceptive input [9,12]. The present data illustrating expansion of pain areas and pain referral after repeated NGF injections suggest that the central consequence of the NGF affects the central mechanisms involved in referred pain. 4.6. Peripheral and central sensitisation caused by NGF injections The NGF synthesised in the peripheral tissues binds to highaffinity tyrosine kinase receptor TrkA at the axon terminals of primary afferents, and then it induces a phosphorylation of TrkA, which leads to the activation of multiple intracellular signal pathways. In a recent animal experiment, reduced mechanical thresholds were reported for muscle nociceptors after injecting NGF; although it was not statistically possible to determine the specific time points for sensitisation, the reduced thresholds appeared to level off after 2 hours [32], which also suggests a central mechanism to explain the prolonged sensitising effects. In accordance with this finding, Murase et al. [18] reported that intramuscular NGF injections after 10–20 minutes sensitised group IV muscle afferents compared with vehicle injections, and this sensitisation was maintained until the end of the experiment after 2 hours. In contrast, recordings from single-muscle group IV fibres 30 minutes after an NGF injection did not show a significant effect on the mechano-sensitivity [13]. In addition to peripheral sensitisation, central effects have also been demonstrated. Intracellular recording of the dorsal horn neurons in vivo with input from the muscle tissue demonstrated that intramuscular injection of NGF sensitised the dorsal horn neurons to electrical stimulation of the muscle nerve after 1 day [14]. Similar effects have been reported as increased proportions of dorsal horn neurons responding to electrical stimulation of the muscle nerve 1 day after NGF injections [33]. Such mechanisms may explain the expanded tenderness, pain referral, and facilitated

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temporal summation of pain in the present human experimental model. 4.7. Conclusion In the present study, the repeated intramuscular administration of NGF evoked soreness, increased pressure pain sensitivity, facilitated temporal summation of pressure pain, and enlarged the pressure-induced pain area after the second injection of NGF. The present findings of spot-wise localised hypersensitivity mimic some of the pain manifestations of myofascial trigger points. Conflict of interest statement The authors declare that there are no conflicts of interest related to this study. Acknowledgements This work was supported in part by a collaboration grant between the Japan Science and Technology Agency (JST, Strategic International Cooperative Program) and the Danish Agency for Science, Technology and Innovation. References [1] Amano T, Yamakuni T, Okabe N, Sakimura K, Takahashi Y. Production of nerve growth factor in rat skeletal muscle. Neurosci Lett 1991;132:5–7. [2] Andersen H, Arendt-Nielsen L, Danneskiold-Samsoe B, Graven-Nielsen T. Pressure pain sensitivity and hardness along human normal and sensitized muscle. Somatosens Mot Res 2006;23:97–109. [3] Andersen H, Arendt-Nielsen L, Svensson P, Danneskiold-Samsoe B, GravenNielsen T. Spatial and temporal aspects of muscle hyperalgesia induced by nerve growth factor in humans. Exp Brain Res 2008;191:371–82. [4] Arendt-Nielsen L, Nie H, Laursen MB, Laursen BS, Madeleine P, Simonsen OH, Graven-Nielsen T. Sensitization in patients with painful knee osteoarthritis. PAINÒ 2010;149:573–81. [5] Deising S, Weinkauf B, Blunk J, Obreja O, Schmelz M, Rukwied R. NGF-evoked sensitization of muscle fascia nociceptors in humans. PAINÒ 2012;153:1673–9. [6] Gerber RK, Nie H, Arendt-Nielsen L, Curatolo M, Graven-Nielsen T. Local pain and spreading hyperalgesia induced by intramuscular injection of nerve growth factor are not reduced by local anesthesia of the muscle. Clin J Pain 2011;27:240–7. [7] Gerwin RD. Classification, epidemiology, and natural history of myofascial pain syndrome. Curr Pain Headache Rep 2001;5:412–20. [8] Gibson W, Arendt-Nielsen L, Graven-Nielsen T. Referred pain and hyperalgesia in human tendon and muscle belly tissue. PAINÒ 2006;120:113–23. [9] Graven-Nielsen T. Fundamentals of muscle pain, referred pain, and deep tissue hyperalgesia. Scand J Rheumatol 2006;35:1–43. [10] Graven-Nielsen T, Mense S, Arendt-Nielsen L. Painful and non-painful pressure sensations from human skeletal muscle. Exp Brain Res 2004;159:273–83. [11] Hayashi K, Ozaki N, Kawakita K, Itoh K, Mizumura K, Furukawa K, Yasui M, Hori K, Yi SQ, Yamaguchi T, Sugiura Y. Involvement of NGF in the rat model of persistent muscle pain associated with taut band. J Pain 2011;12:1059–68. [12] Hoheisel U, Mense S, Simons DG, Yu X-M. Appearance of new receptive fields in rat dorsal horn neurons following noxious stimulation of skeletal muscle: a model for referral of muscle pain? Neurosci Lett 1993;153:9–12. [13] Hoheisel U, Unger T, Mense S. Excitatory and modulatory effects of inflammatory cytokines and neurotrophins on mechanosensitive group IV muscle afferents in the rat. PAINÒ 2005;114:168–76. [14] Hoheisel U, Unger T, Mense S. Sensitization of rat dorsal horn neurons by NGFinduced subthreshold potentials and low-frequency activation. A study employing intracellular recordings in vivo. Brain Res 2007;1169:34–43. [15] Kellgren JH. Observations on referred pain arising from muscle. Clin Sci 1938;3:175–90. [16] Lewin GR, Mendell LM. Nerve growth factor and nociception. Trends Neurosci 1993;16:353–9. [17] Mendell LM, Wall PD. Responses of single dorsal cord cells to peripheral cutaneous unmyelinated fibres. Nature 1965;206:97–9. [18] Murase S, Terazawa E, Queme F, Ota H, Matsuda T, Hirate K, Kozaki Y, Katanosaka K, Taguchi T, Urai H, Mizumura K. Bradykinin and nerve growth factor play pivotal roles in muscular mechanical hyperalgesia after exercise (delayed-onset muscle soreness). J Neurosci 2010;30:3752–61. [19] Nie H, Arendt-Nielsen L, Andersen H, Graven-Nielsen T. Temporal summation of pain evoked by mechanical stimulation in deep and superficial tissue. J Pain 2005;6:348–55.

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