Inhibition of nitric oxide synthases prevents and reverses α,β-meATP-induced neck muscle nociception in mice

Abstract

Introduction: Tension-type headache (TTH) is associated with noxious input from neck muscles. Intravenous administration of the unspecific nitric oxide synthase inhibitor L-NMMA in chronic TTH patients caused analgesia and reduction of neck muscle tenderness.

Methods: The unspecific nitric oxide synthase inhibitor L-NMMA was applied in an experimental model for neck muscle nociception in anesthetized mice (N = 25).

Results: Local injection of α,β-meATP into semispinal neck muscles induced sustained facilitation of brainstem nociception as monitored by the jaw-opening reflex. Preceding intraperitoneal administration of L-NMMA (0.05, 0.1, 1 mg/kg) prevented reflex facilitation evoked by α,β-meATP in a dose-dependent manner. Intraperitoneal injection of L-NMMA subsequent to intramuscular α,β-meATP application reversed established brainstem reflex facilitation back to baseline values.

Discussion: Both experiments with preceding and subsequent L-NMMA indicate the involvement of nitric oxide synthases in the induction and maintenance of facilitation. However, future experiments will have to address the involvement of various isoenzymes in order to provide for new therapeutic concepts in TTH.

Introduction

Neck muscle nociception probably plays a decisive role in the pathophysiology of tension-type headache (TTH) (1–5). Consequently, a recently developed experimental animal model addresses the impact of nociceptive afferent input from neck muscles on pain processing in the brainstem (6–12). A single intramuscular injection of the adenosine 5′-triphosphate analog α,β-methylene ATP (α,β-meATP) induces sustained increase of neuronal excitability in the brainstem for at least four hours. This long-term excitatory effect on neck muscle nociception is mediated by P2X receptors (10).

Nitric oxide (NO), a free diffusible messenger molecule, is involved in various biological processes, including nociceptive transmission in the central and peripheral nervous system (13–15). The NO synthase (NOS) catalyses the transformation of L-arginine into NO and citrulline (16). Experiments in animals have shown that NO is an important messenger molecule in pain pathways of the spinal cord and that formation of NO may trigger or be associated with central sensitization (17–19). Moreover, NOS inhibitors reduce central sensitization in animal pain models (20–22) and nociceptive responses in these models are augmented by NO donors (23,24).

The involvement of NO in the pathophysiology of TTH has been demonstrated in clinical studies in patients (25–28). Systemic administration of the NO donor glyceryl trinitrate induced an immediate and a delayed headache (26). The characteristics of the delayed headache were similar to the attacks of the primary headache disorder. Intravenous infusion of the unspecific NOS inhibitor L-NMMA in TTH patients reduced pain intensity on the visual analogue scale significantly more than placebo (27). Both studies suggest that NO significantly contributes to mechanisms of TTH.

The present study addresses the impact of the unspecific NOS inhibitor L-NMMA on α,β-meATP-evoked facilitation of neck muscle nociception in anesthetized mice.

Methods

Electrophysiological experiments were performed in 25 adult male C57BL/6 mice (approximately 12 weeks old; 22–27 g; Charles River Laboratories, Wilmington, MA, USA, www.criver.com). All procedures received institutional approval from the local ethics committee. The principles of laboratory animal care and use of laboratory animals (European Council Directive of November 24, 1986(86/609/EEC)) were followed. All efforts were made to minimise animal suffering and to use only the number of animals necessary to produce reliable scientific data.

Detailed description of anesthesia, surgery and electrophysiological recording has been published (29). Mice were anesthetized by an initial intraperitoneal (IP) injection of a 0.5% pentobarbital sodium salt solution (Sigma-Aldrich, Munich, Germany, www.sigmaaldrich.com) with a dose of 70 mg/kg. Depth of anesthesia was checked by ensuring that noxious pinch stimulation (blunt forceps) of hindpaw, forepaw, and ear did not evoke any sensorimotor reflexes. When the animal was sufficiently deeply anesthetized, the skin of the throat was carefully shaved and lidocaine hydrochloride gel (Xylocaine® 2%, AstraZeneca, Wedel, Germany, www.astrazeneca.com) was applied to induce local anesthesia. Dexpanthenol eye ointment (Bepanthen®, Roche, Berlin, Germany, www.roche.com) was applied to cornea and conjunctiva of both eyes to protect them from drying. The right external jugular vein was catheterized for continuous administration of a 2% methohexital sodium salt solution (Brevimytal®, Hikma, Gräfelfing, Germany, www.hikma.com) with a dose of 60 mg/kg per hour corresponding to a flow rate of about 0.07 ml/h for a 23 g mouse. A pair of Teflon-coated stainless steel wires (140 µm diameter) was inserted into the right anterior digastric muscle (Dig) to record electromyographic activity (EMG) and the jaw-opening reflex (JOR) via a differential amplifier. After tracheotomy, animals were placed in a stereotaxic frame and were artificially respired with a stroke volume of about 150 µl and about 200 strokes per minute during complete experiment (MiniVent Model 845, Harvard Apparatus, Holliston, MA, USA, www.harvardapparatus.com). Body core temperature was maintained at 37.5°C with a heating blanket and a fine rectal thermal probe (FMI, Seeheim-Ober Beerbach, Germany, www.fmigmbh.de). One platinum needle electrode each (300 µm diameter) was subcutaneously inserted into the left forepaw and right hindpaw to record the electrocardiogram (ECG) via a differential amplifier. Two stainless-steel needle electrodes (150 µm diameter) were longitudinally inserted into the tongue musculature (parallel, 2 mm distance) in order to apply electrical stimuli and to evoke JOR. The oral cavity was filled up with white petrolatum jelly (Riemser, Insel Riems, Germany, www.riemser.de) to protect oral mucous membrane from drying. Semispinal neck muscles on both sides were carefully exposed. One injection canula each (0.4 mm diameter) was inserted into the muscle belly of both semispinal neck muscles. Each canula was connected via thin and short tubing to a liquid switch (CMA/110, www.microdialysis.se). Glass microsyringes (1 ml) were connected to the liquid switch by thin tubing and were fixed in a microdialysis pump (CMA 102, CMA Microdialyis, Solna, Sweden, www.microdialysis.se). This procedure allowed bilateral induction of noxious input from neck muscles in mice in order to mimic bilateral neck muscle pain in TTH patients. The ATP analog α,β-meATP 5′-triphosphate lithium salt (α,β-meATP, 100 nmol/l, Sigma-Aldrich, www.sigmaaldrich.com) was intramuscularly (IM) administered with a volume of 20 µl per muscle during a time period of one minute.

After surgery and placement of all electrodes, the anesthetized animal was rested for at least one hour. During this time period, level of anesthesia and heart rate were routinely checked and documented, and depth of anesthesia was maintained. All electrical signals (EMG, ECG) were recorded by bioamplifiers and led into a data collection system (CED Micro1401, Cambridge Electronic Design, Cambridge, UK, www.ced.co.uk) and a personal computer using Signal® and Spike2® software programs (CED, www.ced.co.uk).

The JOR was elicited by rectangular electrical pulses of 500 µs duration with a stimulation frequency of 0.1 Hz (Figure 1A). The electrical threshold of the JOR was determined by applying increasing and decreasing stimulus intensities from 0 to 2 mA in steps of 100 µA. The lowest stimulus intensity that just evoked a reflex response was defined as the JOR threshold (I_JOR). Test stimulus intensity was adjusted to about 125% of the I_JOR. The JOR was evoked in series of eight stimuli each. These series were repeated every five minutes. After three stable baseline JOR series, α,β-meATP was IM administered and the effect was monitored for at least two hours (Figure 1).

Figure 1.

Reflex specimen and stimulation protocol. (A). The jaw-opening reflex was evoked by electrical stimulation of afferent nerve fibres in the tongue musculature via two needle electrodes. Reflex responses were recorded by electromyography (EMG) in the anterior digastric muscle (a.u.: arbitrary units). The graph shows a typical reflex response elicited by a single electrical stimulus. Dotted lines mark onset and end latencies. The area under the curve is highlighted in grey. The reflex was elicited in series (vertical grey bars) of eight stimuli each. Reflex series were repeated every 5 minutes. In two different experimental groups, substances were injected intraperitoneally (IP) or intramuscularly (IM) into both semispinal neck muscles (as marked by arrows). (B) L-NMMA (100 µl, 0.05 mg/kg, 0.1 mg/kg, 1 mg/kg, N = 5 each) or isotonic saline (100 µl, N = 5) were IP injected. After three baseline reflex series and one hour after IP injection of L-NMMA or saline, α,β-meATP (20 µl, 100 nmol/l) was IM administered and the effect on the reflex was monitored for at least two hours. (C) After three baseline series, α,β-meATP (20 µl, 100 nmol/l) was IM administered and the effect on the reflex was monitored. After two hours during established reflex facilitation L-NMMA (1 mg/kg, n = 5) was IP injected and the reflex was recorded for another two hours. ms = millisecond.

Two different experimental procedures were performed in order to address effects of preceding and subsequent NOS inhibition on α,β-meATP-evoked reflex facilitation (Figure 1). In both experiments, α,β-meATP was IM administered at time point 0. In one experiment, the unspecific NOS inhibitor N^G-monomethyl-L-arginine acetate (L-NMMA; dissolved in 100 µl saline; 0.05, 0.1, 1 mg/kg; Tocris Bioscience, Bristol, UK, www.tocris.com) or isotonic saline as a control (100 µl, DeltaSelect GmbH, Pfullingen, Germany, www.deltaselect.de) was IP injected one hour before IM administration of α,β-meATP. JOR series continued for two hours after local application of α,β-meATP (Figure 1B). In another experiment, L-NMMA (1 mg/kg) was IP administered two hours after IM injection of α,β-meATP during established reflex facilitation. JOR series continued for two hours after systemic application of L-NMMA (Figure 1C). Former studies in patients applied 6 mg/kg L-NMMA (27,28). Another study in mice systemically applied 10 mg/kg L-NMMA (30). The maximum dose in the present manuscript in mice was 1 mg/kg L-NMMA. In preliminary experiments different doses ranging from 10 to 0.01 mg/kg L-NMMA were exemplarily tested. These dose-finding experiments indicated that 1 mg/kg L-NMMA and lower doses did neither affect the baseline reflex nor the heart rate.

Onset latency, duration and integral of JOR were analysed in each single sweep. In recent studies, reflex integral turned out to be the key parameter of reflex alteration in the present animal model (6,8,10,11). Reflex integral, indeed, partly depends on duration of reflex. For the sake of clarity and readability, the present manuscript focuses on statistical analysis of reflex integral. Arithmetic mean and standard error were calculated (mean ± standard error of the mean [SEM]). One-way repeated measures analysis of variance (ANOVA) or Friedman-repeated measures ANOVA on ranks (Friedman ANOVA) were applied in order to analyse differences within groups. Multiple comparison procedures within groups were performed by Student-Newman-Keuls (SNK) post hoc test. Differences between groups were analysed by one-way ANOVA. Multiple comparison procedures between groups were performed by Student-Newman-Keuls (SNK) post hoc test.

Results

In 25 mice, electrical tongue stimulation elicited the JOR with a threshold intensity of I_JOR= 685 ± 30 µA (mean ± SEM). Test stimulus intensity was adjusted to 865 ± 38 µA corresponding to 126 ± 1% of I_JOR.

Combined administration of saline or L-NMMA and subsequent α,β-meATP

In 5 mice, saline was IP injected one hour before local administration of α,β-meATP solution into semispinal neck muscles (Figures 1B, 2, 3). Reflex integrals significantly increased within two hours after α,β-meATP injection (Figure 2). At time point 120 minutes, the percentage increase of the integral was 130 ± 36%. With combined administration of preceding IP L-NMMA 0.05 mg/kg or 0.1 mg/kg and subsequent IM α,β-meATP significant reflex facilitaton established and achieved integral changes of 143 ± 49% or 44 ± 22%, respectively, at time point 120 minutes (Figure 2). With 1 mg/kg L-NMMA IP and subsequent IM α,β-meATP the reflex integral remained unchanged for at least two hours after local injection (Figure 2). Statistical comparison of average integral changes after local administration of α,β-meATP revealed a dose-dependent prevention of reflex facilitation by the NOS inhibitor L-NMMA (Figure 3).

Figure 2.

The effects of preceding administration of saline or L-NMMA on α,β-meATP-evoked reflex facilitation. With preceding intraperoneal (IP) injection of saline, 0.05 mg/kg L-NMMA or 0.1 mg/kg L-NMMA, a significant reflex facilitation established after additional IM application of α,β-meATP into semispinal neck muscles. Preceding IP administration of 1 mg/kg L-NMMA prevented a reflex facilitation by intramuscular (IM) α,β-meATP. Statistical results of one-way repeated measures analysis of variation (ANOVA) and Friedman ANOVA are given. Percentage changes from baseline reflex integral are presented as mean ± SEM (standard error of the mean). n.s. = not significant.

Figure 3.

Dose-dependent reduction of reflex facilitation with preceding L-NMMA. Average reflex integral changes within two hours after intramuscular (IM) administration of α,β-meATP are presented as box plots (dotted line: arithmetic mean). The effects of preceding intraperoneal (IP) injection of saline or different dosages of L-NMMA on α,β-meATP-evoked reflex facilitation were statistically different (one-way ANOVA). The percentage changes from integral baseline with preceding 1 mg/kg L-NMMA was significantly lower than in all other experimental conditions. Reflex facilitation with preceding 0.1 mg/kg L-NMMA significantly differed from saline experiments. Asterisks mark significant differences such as *: p < .05 and ***: p < .001 as calculated by Student-Newman-Keuls post hoc test.

Combined administration of α,β-meATP and subsequent L-NMMA

In 5 mice, IM application of α,β-meATP induced a significant reflex facilitation within two hours (F = 7.7, p < .001). Within the first hour after local injection, the reflex integral continuously increased and reached a stable level within the second hour of 131 ± 5%. After subsequent IP administration of 1 mg/kg L-NMMA the reflex integral significantly decreased (X² = 70.3, p < .001) and recovered back to baseline (before α,β-meATP) within 60 to 90 minutes (Figure 4).

Figure 4.

Subsequent administration of L-NMMA reverses reflex facilitation. After three baseline reflex series, α,β-meATP (100 nmol/l) was IM injected in both semispinal neck muscles (N = 5). Within the first hour after local injection the reflex integral continuously increased and reached a stable level within the second hour. After subsequent IP administration of 1 mg/kg L-NMMA the reflex integral significantly decreased (X² = 70.3, p < .001) and recovered back to baseline (before α,β-meATP) within 60 to 90 minutes. Reflex integral changes are given as mean ± SEM (standard error of the mean).

Discussion

In the present study, preceding administration of the unspecific NOS inhibitor L-NMMA prevents brainstem reflex facilitation evoked by intramuscular α,β-meATP in a dose-dependent manner. Intraperitoneal injection of L-NMMA subsequent to IM α,β-meATP application reverses established brainstem reflex facilitation back to baseline values.

Recent studies have demonstrated that local administration of α,β-meATP into semispinal neck muscles induces sustained facilitation of the JOR in anesthetized mice for at least four hours (7,10,11,31). This brainstem reflex potentiation is driven by ongoing excitatory input from myofascial nociceptors via interaction of α,β-meATP with ionotropic P2X₃ and P2X_2/3 receptors (10,31). Established reflex facilitation is robust and not affected by subsequent IP or IM administration of saline (7,32). However, established reflex facilitation is interrupted and reversed back to baseline by subsequent local administration of a P2Y₁ receptor agonist (7), P2X₃ receptor antagonists (10,31), or tetrodotoxin (TTX) (11). The inhibitory influence of TTX on reflex facilitation indicates the involvement of thin-myelinated group III nerve fibres in neck muscles excited by local α,β-meATP (11). Brainstem reflex potentiation by additional noxious input from neck muscles is due to heterosynaptic facilitation. Electrical stimulation of afferent nerve fibres in tongue musculature reliably evokes the JOR via a brainstem reflex network (29,33). Nociceptive afferents from neck muscles gain access to the same network as well. Thus, both afferents from tongue musculature and neck muscles converge onto the same central reflex network. Therefore, additional excitatory input from neck muscle nociceptors onto the JOR neuronal network facilitates the tongue-evoked reflex by heterosynaptic access.

L-NMMA is a competitive inhibitor of all three NOS isoforms with IC₅₀ values of 6.6 µM for inducible NOS, 4.9 µM for neuronal NOS, and 3.5 µM for endothelial NOS (16). Similar potencies for all three isoenzymes define L-NMMA as an unspecific NOS inhibitor. Pharmacokinetic properties include an elimination half-life of approximately one hour (34). L-NMMA quickly disappears from plasma but exerts prolonged pharmacodynamic effects after systemic administration. Considering the pharmacokinetics, preceding administration of L-NMMA was performed one hour before local injection of α,β-meATP in one experimental series. Accordingly, after subsequent L-NMMA injection reflex recording continued for two hours in the second experimental series.

NO is suggested to play an important role in the pathophysiology of central sensitization (35). Chronic TTH may be caused by prolonged noxious input from pericranial myofacial tissues, resulting in central sensitization (36,37). Based upon this pathophysiological concept, the analgesic effect of a NOS inhibitor was investigated in TTH patients. In a randomized double-blind, cross-over trial, 16 chronic TTH patients received an intravenous infusion of 6 mg/kg L-NMMA or placebo on two days separated by at least one week. L-NMMA reduced pain intensity on the visual analog scale significantly more than placebo during 120 minutes after start of infusion (27). Muscle hardness in chronic TTH was reduced significantly more following treatment with L-NMMA than with placebo. Compared with baseline, hardness and total tenderness score significantly decreased at 60 and 120 minutes after L-NMMA treatment (28). Most of the side effects in relation to L-NMMA infusion were similar to those after placebo infusion. The difference in the mean arterial blood pressure and pulse rate between L-NMMA 6 mg/kg and placebo disappeared 60 minutes after the start of infusion. In contrast, the analgesic effect on headache intensity and the reduction of muscle hardness and tenderness lasted at least 120 minutes after start of infusion. It therefore seems unlikely that the observed effects of L-NMMA were caused by a hypertensive effect of the agent or influenced by the subjective symptoms reported in relation to the L-NMMA infusion. Thus, the patient studies suggest an analgesic effect of NOS inhibition in chronic TTH. The analgesic effect of NOS in patients with chronic TTH is suggested to be due to a reduction in central sensitization (25).

Neck muscle nociception and pain probably play an important role in the pathophysiology of TTH (1,3,4,38). Consequently, the recently developed experimental animal model addresses the impact of nociceptive afferent input from neck muscles on central nervous system processing (6–12). Besides the potency of the model to address pathophysiological mechanisms, it offers the opportunity for pharmacological studies. The above-mentioned data from patients emphasize the involvement of NOS isoenzymes in TTH pathophysiology (25,27,28). Accordingly, the authors suggest NOS inhibitors as future options for treatment of TTH. However, the unspecific NOS inhibitor L-NMMA inhibits all three isoenzymes, such as neuronal NOS, inducible NOS, and endothelial NOS. Whereas this broad spectrum effect of L-NMMA on NOS seems to induce significant analgesia in patients, it runs the risk of serious side effects under clinical conditions. Future studies will have to address the role of the different NOS isoenzymes in analgesia in TTH as a necessary precondition for appropriate drug development. Selective NOS inhibitors are not available yet for administration in clinical studies. In contrast, a differentiation between the effects of the various isoenzymes on neck muscle nociception is possible by administration of selective NOS inbibitors in an experimental animal model. The present study investigated the effect of the unspecific NOS inhibitor L-NMMA in the animal model of facilitated neck muscle nociception. The drug showed a significant inhibition of established sensitization and prevented induction of sensitization by preceding administration. The results correspond to the clinical effects of the same drug. This study is the prerequisite for further investigation of the roles of different NOS isoenzymes in neck muscle nociception under experimental conditions. Basic experimental studies and clinical investigations will demonstrate whether selective NOS inhibition may become a novel principle in the future treatment of chronic headache.