Sage Journals: Discover world-class research

Abstract

Aim

The aim of this article is to investigate whether the nitric oxide (NO) donator diethylenetriamine/nitric oxide (DETA/NO) affects trigeminal sensory processing through the trigeminal ganglion in part by activating trigeminal satellite glial cells (SGCs) and whether this effect is attenuated by the anti-inflammatory compound palmitoylethanolamide (PEA).

Methods

DETA/NO was administered to isolated rat trigeminal SGCs in vitro, and injected into the rat trigeminal ganglion in vivo, in the presence or absence of PEA.

Results

Administration of DETA/NO (1000 µM) increased the release of prostaglandin E₂ by SGCs. PEA (1 and 10 µM) significantly attenuated prostaglandin E₂ release. Two intraganglionic injections of DETA/NO (10 mM, 3 µl) or prostaglandin E₂ at a 30-minute interval did not evoke discharge in trigeminal ganglion neurons that innervate the rat jaw-closer muscles, but did reduce the mechanical activation threshold of their peripheral endings by 30%–50%. Intravenous administration of PEA (1 mg/kg) or ketorolac (0.5 mg/kg) prevented DETA/NO-induced afferent mechanical sensitization.

Conclusions

Elevation of NO in the trigeminal ganglion results in the sensitization of the peripheral endings of masticatory muscle nociceptors to mechanical stimulation through a mechanism that involves prostaglandin E₂ release from SGCs. Attenuation of this sensitization by PEA suggests a possible option for acute management of craniofacial pain and headache.

Keywords

Headache satellite glial cells trigeminal ganglion nitric oxide glial modulators palmitoylethanolamide

Introduction

Systemic administration of nitric oxide (NO) donators and nitrates, such as nitroglycerin, to healthy humans results in reports of acute headache and, in migraine headache patients, a delayed migraine-like headache (1,2). The mechanism underlying the effect of NO donators is incompletely understood. It is known that NO donators relax cerebral vascular smooth muscle by increasing intracellular levels of cyclic guanosine monophosphate (cGMP) through an action on soluble guanylate cyclase (sGC) (3,4). NO is also known to increase the release of calcitonin-related gene peptide (CGRP), a potent vasodilatory neuropeptide associated with migraine headache (1). In addition, NO donators are able to directly increase the activity of both isoforms of cyclooxygenase (COX), which results in an increased synthesis of prostaglandins (PGs) (5). It has been proposed that through these and other mechanisms, NO facilitates trigeminal nociceptive transmission, although the sites of action of NO are still being determined (1). As far as a peripheral site of action, a recent study found that systemic infusion or topical administration of an NO donator to the dura did not excite dural afferent fibers, but did, after a delay of several hours, sensitize the majority of these fibers to mechanical stimuli (6). As NO donator-induced headaches in humans are rapid in onset, this finding suggests that NO may act at other levels of the trigeminal system to alter sensory processing.

All trigeminal sensory afferent fibers project to the central nervous system by way of the trigeminal ganglion, where their cell bodies are found (7). The trigeminal ganglion is composed of these cell bodies or neurons and their support cells, called satellite glial cells (SGCs), which completely surround each ganglion neuron (8). It was recently demonstrated that the NO donator diethylenetriamine/nitric oxide (DETA/NO) could act on SGCs to increase the release of prostaglandin (PG) E₂, which is a pro-inflammatory and algesic PG (8). About half of all trigeminal ganglion neurons express two of the four receptors that are activated by PGE₂: the EP2 and EP3 receptors (9). In vitro, PGE₂ has been reported to decrease the activation threshold for trigeminal ganglion neurons through actions on tetrodotoxin (TTX)-resistant sodium channels and hyperpolarization-activated currents (10,11). This suggests the possibility that NO donators could act on cells within the trigeminal ganglion to facilitate nociception through an increase in the release of PGE₂.

Indomethacin, a nonsteroidal anti-inflammatory drug (NSAID) that inhibits COX, is effective in preventing the early headache induced by NO donators (12). Palmitoylethanolamide (PEA) is an endogenous fatty acid amide with analgesic as well as anti-inflammatory effects equivalent to indomethacin, and may act, in part, by altering COX expression (13,14). There is accumulating evidence that many of the anti-inflammatory effects of PEA are mediated through its ability to activate the peroxisome proliferator activated receptor-α (PPARα that has broad effects on metabolism and immune response (15,16). There has been increasing interest in the use of PEA to treat various craniofacial pain conditions, and recently PEA has been reported to be better than the NSAID ibuprofen for pain relief in temporomandibular joint osteoarthritis or arthralgia (17).

The aim of the current study was to investigate the hypothesis that NO donators affect trigeminal sensory processing at the level of the trigeminal ganglion, in part, by activating trigeminal SGCs. This hypothesis was investigated by determining whether i) DETA/NO could stimulate the release of PGE₂ from isolated trigeminal SGCs, ii) intraganglionic injection of DETA/NO altered the excitability of trigeminal sensory fibers and iii) systemic administration of PEA and the NSAID ketorolac could reverse the effects of DETA/NO.

Materials and methods

For all experiments, adult male Sprague-Dawley rats were used. In vivo experimental protocols were approved by the University of British Columbia Animal Care Committee (No. #A11-0279) and the studies were performed in accordance with the guidelines established by the Canadian Council on Animal Care and the International Association for the Study of Pain.

In vitro PGE₂ release experiments

Trigeminal satellite glial cell isolation

SGCs were isolated from the trigeminal ganglion of adult male Sprague-Dawley rats (Taconic, DK; n = 8) based on a previous study (8). In brief, rats were anaesthetized with a combination of Hypnorm (Vetapharma, UK), midazolam (Hameln Pharma, Germany) and sterile water (25/25/50% v/v; 0.3 ml/100 g subcutaneously (s.c.)) and both ganglia were removed and transferred to ice-cold phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺ (Sigma Aldrich, USA) supplemented with 1% penicillin and streptomycin (Invitrogen, USA). The tissue was then digested in collagenase (5 mg/ml; Sigma Aldrich, Denmark) for 15 minutes at 37 ℃. Subsequently the tissue was digested in 0.125% trypsin (Invitrogen, USA) for 10 minutes and then suspended in Ham’s F12 medium (Invitrogen, Denmark) supplemented with 10% fetal calf serum (Invitrogen, USA) and 1% penicillin and streptomycin and mechanically dissociated with a pipette to obtain a single-cell suspension. This solution was added to a T25 culture flask and incubated for three hours to separate SGCs from neurons. Afterwards, the medium was replaced with fresh growth medium, which was changed after 24 hours and subsequently every second day until the cells were used in experiments.

PGE₂ release assays

In preliminary experiments, the time- and concentration-relationship for PGE₂ release from isolated SGCs was assessed (n = 3 rats). SGCs were seeded at a density of 40,000 cells/well in 96-well-plates and treated with 0 (control), 10, 100, or 1000 µM of the NO donator DETA/NO (Sigma Aldrich, DK) for four, eight, and 24 hours.

Based on these preliminary experiments, stimulation with 1000 µM DETA/NO for eight hours was chosen as the activating stimulus for the subsequent PEA experiments (n = 5 rats). In these experiments, SGCs were seeded at a density of 40,000 cells/well in 96-well-plates and pretreated for 24 hours with 0.1, 1, and 10 µM PEA (Tocris, UK). The concentrations of PEA were based on previous studies (18,19). The following day pretreatment media were replaced with control medium or treatment medium containing 1000 µM DETA/NO together with 0, 0.1, 1, and 10 µM PEA. After eight hours of incubation, duplicate samples of 50 µl culture supernatant were collected and stored at −20 ℃ until analyzed.

The PGE₂ concentration in the medium was determined by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Cayman Chemicals, USA). Duplicate readings were performed at 405 nm on a plate reader.

In vivo electrophysiology

Anesthesia and surgical preparation

Prior to use, rats were housed, two per cage, in an animal facility and were given free access to food and water. Rats were anaesthetized with isoflurane (2%–2.5%) and oxygen (97%–98%). A catheter was inserted into the carotid artery to monitor the blood pressure and a tracheal tube was inserted into the trachea to permit artificial ventilation. A second catheter was inserted into the femoral vein to permit delivery of drugs systemically. Body temperature was measured with a rectal thermometer and maintained at 37.0 ± 0.2 ℃ with an electric heating pad. The heart rate, blood pressure, and core body temperature were monitored throughout the experiments.

The animals were positioned prone in a stereotaxic frame, the skin over the dorsal surface of the skull was reflected, and a small trephination made to allow lowering of a microelectrode through the brain into the trigeminal ganglion for recording. The skin and muscle overlying the neck were gently dissected to expose the brain stem and the dura overlying the brain stem was removed to permit a stimulating electrode to contact the caudal brain stem. A 27-gauge needle attached to a 10 µl Hamilton syringe by polyethylene tubing was inserted into the trigeminal ganglion with a Kopf electrode manipulator at an angle of 30 degrees until the needle tip was felt to hit the base of the skull. This catheter was used to make microinjections (3 µl per injection) into the trigeminal ganglion.

Animals were euthanized at the end of experiments (Nembutal, 100 mg/kg; Abbott Laboratories, USA).

Stimulation and recording

Extracellular action potentials were recorded from the trigeminal ganglion with a parylene-coated tungsten microelectrode (0.10 inches, 2 M, A-M Systems Inc, USA) routed through a 1401 A-D board to a computer with Spike 2 software (Cambridge Electronics, UK). A fine-tipped cotton swab was applied to the orofacial region as a mechanical search stimulus while the electrode was slowly lowered to identify afferent discharge in response to mechanical stimulation of the face. Trigeminal ganglion neurons, which responded to mechanical stimulation of either the masseter or temporalis muscle, but not to mechanical stimulation of the skin overlying the muscle, were considered to innervate the muscle.

It has been shown that the caudal brain stem is a key projection site for putative nociceptors that innervate craniofacial muscles (20). Antidromic collision was used to confirm the projection of trigeminal ganglion neurons to the caudal brain stem and thus identify them as putative nociceptors. To estimate the afferent conduction velocity (CV), the distance between the stimulating and recording electrodes was divided by the latency of the antidromic action potential of each nociceptor (20). To assess the mechanical activation threshold (minimum force required to evoke afferent discharge), an electronic von Frey hair (model 1601C, Life Science, USA) was applied to the mechano-receptive field of the masticatory muscle afferent.

Experimental design

Experiments were first conducted to assess the effect of microinjection of DETA/NO into the ipsilateral (n = 6) and contralateral (n = 3) trigeminal ganglion on neuronal discharges and afferent mechanical activation threshold. A baseline afferent mechanical activation threshold was first assessed by applying the electronic von Frey hair at an interval of one minute between each mechanical stimulus for a total of 10 minutes. Then, baseline discharge was recorded for 10 minutes, after which an initial injection of DETA/NO (10 mM, 3 µl) was made into the trigeminal ganglion and discharge was monitored. The concentration of DETA/NO was chosen, in part, based on the results obtained in the in vitro SGC assays. Thirty minutes later, a second injection of DETA/NO (10 mM, 3 µl) was performed and the resulting discharge was again monitored. Twenty minutes after the second injection, the mechanical activation threshold was again measured with an electronic von Frey hair for a total of 10 minutes. The same experimental design was later used to assess the effect of repeated injection of PGE₂ (0.1 mg/ml, 3 µl; n = 5). The concentration of PGE₂ used was chosen because it had been previously shown to induce mechanical sensitization of masticatory muscle afferent fibers when injected intramuscularly (21).

In subsequent experiments, PEA (0.1, 1.0 mg/kg; n = 12), ketorolac (0.5 mg/kg, positive control; n = 6) or the PEA vehicle (10% ethanol in PBS; n = 6) were administered by intravenous (i.v.) injection to determine their effects on DETA/NO-induced mechanical sensitization. In these experiments, a baseline mechanical activation threshold was first assessed as described in the previous paragraph, after which the drug being tested was administered intravenously. Ten minutes after i.v. administration, the mechanical activation threshold was re-assessed and then the experiment proceeded with the same design and timeline as described above, i.e. two intraganglionic injections followed by a post-injection assessment of mechanical activation threshold.

The doses of PEA were based on previously published studies (13,14). As a pilot experiment with 2 mg/kg ketorolac alone resulted in a substantial increase in baseline mechanical activation threshold, 0.5 mg/kg ketorolac, which had a less pronounced effect on baseline mechanical activation threshold, was used for this study.

Data and statistical analysis

Cumulative discharge was calculated by subtracting the sum of action potentials over the 10 minutes prior to each injection from the sum of action potentials that occurred over a 10-minute period after the injection. The baseline(s) and post-injection mechanical activation threshold for each trigeminal ganglion neuron was calculated as the mean of the 10 mechanical activation values measured before and after the trigeminal ganglion injections, respectively.

Paired data were assessed with a paired t test. Data from more than two populations were analyzed with one-way analysis of variance (ANOVA) followed by the Holm-Sidak post hoc test. All data were analyzed with SigmaStat for Windows (Systat, USA). P < 0.05 was considered statistical significant. Data are expressed as mean ± standard error of the mean (SEM).

Results

In vitro, DETA/NO-evoked PGE₂ release is attenuated by PEA

Figure 1(a) shows a representative phase-contrast image of the SGCs seven days after isolation. In preliminary tests it was found that eight and 24 hours after administration of DETA/NO (1000 µM), there was a significantly increased release of PGE₂ from the SGCs (p < 0.05; Figure 1). As a result, subsequent experiments to assess the effect of PEA on PGE₂ release by SGCs utilized this concentration of DETA/NO.

Figure 1.

The time- and concentration-dependent effects of DETA/NO on release of PGE₂ from SGCs in vitro. (a) A representative phase-contrast image of SGCs after seven days in culture. (b) Stimulation of SGCs with DETA/NO produced a concentration- and time-dependent effect on PGE₂ release, which was most pronounced for 1000 µM DETA/NO. Data are presented as mean ± SEM (n = 3). *, p < 0.05 one-way repeated measures ANOVA and Holm-Sidak post-hoc test. DETA/NO: diethylenetriamine/nitric oxide; PGE₂: prostaglandin E₂; SGCs: satellite glial cells; ANOVA: analysis of variance.

Application of PEA brought about a ∼30% decrease in the DETA/NO-evoked PGE₂ release in a concentration-dependent manner (Figure 2). Accordingly, the PGE₂ concentrations were significantly lowered from 106.2 ± 9.6 pg/ml (DETA/NO alone) to 77.3 ± 4.6 and 75.4 ± 3.1 pg/ml for pretreatment with 1 and 10 µM PEA, respectively (p < 0.01; Figure 2).

Figure 2.

Effect of PEA on DETA/NO-evoked PGE₂ release from SGCs in vitro. When SGCs were stimulated with 1000 µM DETA/NO for eight hours, there was a three-fold increased PGE₂ accumulation in the culture medium. Pretreatment with PEA (1 and 10 µM) significantly attenuated this DETA/NO-evoked PGE₂ accumulation. Data are presented as mean ± SEM (n = 5). ***, p < 0.001 vs. control; ##, p < 0.05 vs. DETA/NO alone one-way repeated measures ANOVA and Holm-Sidak post-hoc test. PEA: palmitoylethanolamide; DETA/NO: diethylenetriamine/nitric oxide; PGE₂: prostaglandin E₂; SGCs: satellite glial cells; ANOVA: analysis of variance.

In vivo, intraganglionic injection of DETA/NO sensitizes masticatory muscle afferent fibers

Two injections of DETA/NO into the trigeminal ganglion were made to determine whether two injections at a 30-minute interval would evoke reproducible neuronal discharge in six ganglion neurons with afferent projections to either the temporalis or masseter muscles. Ten minutes after the second injection of DETA/NO, the mechanical activation threshold of the ganglion neurons was reassessed. Injection of DETA/NO evoked afferent discharge after both injections in only one of the six neurons examined (Figure 3(a)). Overall, the median cumulative discharge evoked by the first and second injections was 0. However, assessment of the mechanical activation threshold after the second injection of DETA/NO indicated that it was reduced by ∼30% (Figure 3(b) and (c)). This finding suggested that repeated injection of DETA/NO into the ganglion was sensitizing the terminal endings of masticatory muscle afferent fibers to mechanical stimulation. To rule out the possibility that this was due to a systemic effect, DETA/NO was injected into the contralateral ganglion (n = 3) to determine whether this would alter the mechanical activation threshold of the afferent fibers. There was no effect of contralateral injections of DETA/NO on the mechanical activation threshold of the afferent fibers (Figure 3(c)).

Figure 3.

(a) The peri-stimulus histogram shows the response of a masseter muscle trigeminal ganglion neuron (CV: 13.5 m/s) to two injections of DETA/NO (10 mM, 3 µl, arrows). This neuron was not spontaneously active, and injection of DETA/NO did evoke discharges after a delay of several minutes. This was the only ganglion neuron out of the six examined that exhibited any discharge in response to injection of DETA/NO. (b) An example of extracellular discharge of the same ganglion neuron to mechanical stimulation of the masseter muscle is illustrated. Injection of DETA/NO reduced the mechanical activation threshold of the afferent fiber from 6.9 g at baseline to 3.4 g. (c) The vertical bar graphs show the mean (±SEM) mechanical activation threshold of masticatory muscle afferent fibers before and after two injections of DETA/NO. Injection of DETA/NO into the ipsilateral trigeminal ganglion decreased the mechanical activation threshold of six masticatory muscle afferent fibers by 31%. No effect of injection of DETA/NO into the contralateral trigeminal ganglion was observed on the mechanical activation threshold of three masticatory muscle afferent fibers in a subsequent series of experiments. *p < 0.05 paired t test. CV: conduction velocity; DETA/NO: diethylenetriamine/nitric oxide; BMT: baseline mechanical threshold.

Systemic administration of PEA dose dependently attenuates mechanical sensitization

Experiments were conducted on 24 ganglion neurons that projected to either the masseter or temporalis muscle to determine whether systemic injection PEA (1, 0.1 mg/kg) or ketorolac (0.5 mg/kg) could affect baseline mechanical activation threshold or alter DETA/NO-induced afferent mechanical sensitization. Systemic administration of the vehicle (PBS/10% ethanol) and ketorolac were found to increase mechanical activation threshold by 18% and 16%, respectively (Figure 4(a)). Injection of PEA did not significantly affect the baseline mechanical activation threshold (PEA 1 mg/kg: 3%, 0.1 mg/kg: –4%). After systemic administration of the vehicle, repeat injections of DETA/NO reduced the mechanical activation threshold by 27%. Systemic administration of ketorolac and PEA (1 mg/kg) prevented DETA/NO-induced mechanical sensitization, whereas injection of PEA (0.1 mg/kg) did not affect DETA/NO-induced mechanical sensitization.

Figure 4.

(a) The vertical bar graph shows the mean (±SEM) relative mechanical activation threshold 10 minutes after systemic injection of vehicle (PBS/ethanol 10%, n = 6), ketorolac (Ket; 0.5 mg/kg, n = 6) and PEA (0.1, 1.0 mg/kg, n = 6). Systemic injections of the vehicle or ketorolac increased the mechanical activation threshold slightly, whereas injections of PEA had less effect on the mechanical activation threshold. (b) Repeated injection of DETA/NO reduced mechanical activation threshold by 27% after administration of vehicle. Compared to vehicle, both PEA (1 mg/kg) and ketorolac (0.5 mg/kg) significantly inhibited DETA/NO-induced mechanical sensitization. PEA (0.1 mg/kg) was not effective in altering DETA/NO-induced mechanical sensitization. *p < 0.05 compared with vehicle control, one-way ANOVA and Holm-Sidak post hoc test. PBS: phosphate-buffered saline; PEA: palmitoylethanolamide; DETA/NO: diethylenetriamine/nitric oxide; Ket: ketorolac; ANOVA: analysis of variance.

Intraganglionic injection of PGE₂ induces mechanical sensitization

To determine whether PGE₂ release could be responsible for DETA/NO-induced mechanical sensitization, additional experiments were conducted to investigate whether injection of PGE₂ into the trigeminal ganglion could produce a similar change in the mechanical activation threshold. Repeated injections of PGE₂ resulted in a significant decrease in the mechanical activation threshold by 47% (Figure 5). After systemic administration of PEA (1 mg/kg) prior to intraganglionic injection of PGE₂, the mechanical activation threshold was lowered by 44%.

Figure 5.

The bar graphs show the average mechanical activation threshold before and after repeated administration of PGE₂ to the trigeminal ganglion. PGE₂ significantly decreased the mechanical activation threshold of five muscle nociceptors (*: p < 0.05, paired t test). Systemic administration of PEA (1 mg/kg) nonsignificantly elevated the mechanical activation threshold, but did not prevent PGE₂-induced mechanical sensitization (* p < 0.05 repeated-measures ANOVA, Holm-Sidak test). BMT: baseline mechanical threshold; PGE₂: prostaglandin E₂; PEA: palmitoylethanolamide; ANOVA: analysis of variance.

Discussion

The findings of the current study indicate that elevation of NO in the trigeminal ganglion results in the sensitization of the peripheral endings of masticatory muscle nociceptors to mechanical stimulation through a mechanism that involves PGE₂ release. NO-induced mechanical sensitization of masticatory muscle nociceptors was inhibited by a low systemic dose of the COX inhibitor ketorolac, in support of the view that it is mediated through the release of PGs. Further, intraganglionic injection of PGE₂ produced a robust mechanical sensitization of masticatory muscle nociceptors, which is consistent with the idea that intraganlionic injection of the NO-donator DETA/NO induces its effects through a localized release of PGE₂. Elevation of NO concentrations resulted in a significant release of PGE₂ by SGCs in culture, which indicates that one important source of the PGE₂ in the trigeminal ganglion after elevation of NO is likely to be SGCs. PEA dose-dependently inhibited NO-induced mechanical sensitization of masticatory muscle nociceptors and PGE₂ release by SGCs. However, PEA administration had no effect on mechanical sensitization induced by intraganglionic injection of PGE₂, which suggests that the effect of PEA was likely mediated through inhibition of COX. We propose that PEA may exert its analgesic and anti-inflammatory activity through a mechanism that involves inhibition of PG synthesis by SGCs.

It has been proposed that NO is produced by trigeminal ganglion neurons that express neuronal NO synthase (nNOS), even though only about 5%–10% of neurons in the trigeminal ganglion express this enzyme (22). Any NO produced by trigeminal ganglion neurons could readily diffuse across the 20 nm space between the neuron and SGCs (23). NO signaling is mediated through the activation of sGC, resulting in an increase in intracellular cyclic guanosin monophosphate (3,24). One of the downstream consequences of this signaling pathway is activation of COX, which results in an increase in PG synthesis (5,25). Functional guanylate cyclase is a heterodimer composed of one α and one β subunit, e.g. α₁β₁ (26,27). Most of the available evidence indicates that both the α₁ and β₁ subunits are expressed in the trigeminal ganglion and that the α₁ subunit is expressed only in SGCs (and Schwann cells) but is not found in trigeminal ganglion neurons (26,27). It has been suggested that ganglion neurons may not contain functional sGC and thus cannot respond to NO (28,29). A recent study that reported finding the β₁ subunit of guanylate cyclase in ∼50% of trigeminal ganglion neurons appears to contradict this suggestion, although, surprisingly, it did not appear that the β₁ subunit was also expressed by SGCs in this study (30). Findings of PGE₂ release by SGCs after treatment with DETA/NO, coupled to a previous finding of elevated cGMP in SGCs treated with nitroglycerin, strongly suggest that functional sGC is present in SGCs (8,27). Although there is no evidence for a direct effect of NO on trigeminal ganglion neurons, it has been reported the DETA/NO depolarized about 40% of mesencephalic ganglion neurons, which are located within the brain stem and innervate masticatory muscle spindles and periodontal tissue (31). However, in the present study we found no consistent evidence that intraganglionic injection of DETA/NO could excite trigeminal ganglion neurons that innervate the masticatory muscles. In the absence of evidence for a direct effect of NO on trigeminal ganglion neurons, we propose that the ability of repeated injections of NO to decrease the mechanical activation threshold of masticatory muscle nociceptors was due to the release PGE₂ by SGCs. As a result we propose that PEA was able to attenuate the sensitizing effects of intraganglionic injection of DETA/NO because of an action on SGCs.

In a study that assessed the effect of PEA on carrageenan-induced paw inflammation, it was found that daily oral administration of PEA (10 mg/kg) for four days decreased COX activity in the inflamed tissue (14). Single doses of indomethacin or PEA were found to begin decreasing edema volume in the inflamed paw within three hours of administration, which suggests that PEA may have the ability to inhibit COX; however, tissue homogenates were not assayed for either of the isoforms of COX in this study (14), so it is possible that the effect of PEA after four days of administration reflected decreased expression of COX. Indeed, subsequent research has demonstrated that intracerebroventricular administration of 1 µg PEA significantly reduces COX-2 expression in the spinal cord six hours after induction of paw inflammation with carrageenan (32). In contrast, in the present study, PEA administration (1 mg/kg i.v.) was found to block NO-induced mechanical sensitization of masticatory muscle nociceptors within 90 minutes of administration, which would appear too rapid to be explained by an effect on COX expression. Taken together with findings reported in the Costa et al. study, these data suggest that PEA may also exert a direct inhibitory effect on the activation of COX by NO (14).

Evidence in support of a role for NO-soluble guanylate cyclase pathway in the pathogenesis of craniofacial pain includes the effectiveness of an NO synthase inhibitor (L-NMMA) for the treatment of acute migraine headache and the ability to induce pulsing headaches in healthy subjects and full blown migraine headaches in migraineurs by systemic administration of NO donators and nitroglycerin (33,34). Large doses of nitroglycerin are reported to cause headaches indistinguishable from migraine in about 50% of healthy individuals (34). Nitroglycerin-induced headaches have been reported to decrease by about 25% the pressure pain threshold and tolerance over the temporalis muscle in healthy individuals (35). Our finding that an intraganglionic injection of DETA/NO reduces muscle nociceptor mechanical thresholds by about 30% is consistent with the idea that nitroglycerin-induced temporalis muscle sensitivity in humans subjects may be mediated through activation of trigeminal SGCs. The ability of PEA to inhibit DETA/NO-induced nociceptor mechanical sensitization through an apparent action on SGCs suggests that PEA and other glial modulators may prove useful for the treatment of headaches.

Clinical implications

Elevation of nitric oxide (NO) in the trigeminal ganglion results in the mechanical sensitization of the peripheral endings of masticatory muscle nociceptors through a mechanism that involves prostaglandin E₂ release from satellite glial cells (SGCs).

Palmitoylethanolamide (PEA) inhibits prostaglandin synthesis by the trigeminal ganglion SGCs and blocks mechanical sensitization of masticatory muscle nociceptors.

The ability of PEA to inhibit craniofacial muscles’ mechanical sensitization through an apparent action on SGCs suggests that this glial modulator may prove useful for the treatment of craniofacial pain, e.g. headaches.

Footnotes

Funding

This research was supported by the 2010 FSS grant from the Danish Research Council to PG. BEC was the recipient of a Canada Research Chair.

Conflict of interest

The authors declare that they have no conflict of interest.

References

Messlinger

Lennerz

Eberhardt

. CGRP and NO in the trigeminal system: Mechanisms and role in headache generation. Headache 2012; 52: 1411–1427.

Olesen

Thomsen

Lassen

. The nitric oxide hypothesis of migraine and other vascular headaches. Cephalalgia 1995; 15: 94–100.

Derbyshire

Marletta

. Structure and regulation of soluble guanylate cyclase. Annu Rev Biochem 2012; 81: 533–559.

Gao

. The multiple actions of NO. Pflugers Arch 2010; 459: 829–839.

Salvemini

Misko

Masferrer

. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci U S A 1993; 90: 7240–7244.

Zhang

Kainz

Zhao

. Vascular extracellular signal-regulated kinase mediates migraine-related sensitization of meningeal nociceptors. Ann Neurol 2013; 73: 741–750.

Bathla

Hegde

. The trigeminal nerve: An illustrated review of its imaging anatomy and pathology. Clin Radiol 2013; 68: 203–213.

Capuano

De Corato

Lisi

. Proinflammatory-activated trigeminal satellite cells promote neuronal sensitization: Relevance for migraine pathology. Mol Pain 2009; 5: 43–43.

Patwardhan

Vela

Farugia

. Trigeminal nociceptors express prostaglandin receptors. J Dent Res 2008; 87: 262–266.

10.

Gold

Levine

Correa

. Modulation of TTX-R INa by PKC and PKA and their role in PGE₂-induced sensitization of rat sensory neurons in vitro. J Neurosci 1998; 18: 10345–10355.

11.

Ingram

Williams

. Modulation of the hyperpolarization-activated current (Ih) by cyclic nucleotides in guinea-pig primary afferent neurons. J Physiol 1996; 492(Pt 1): 97–106.

12.

Castellano

Micieli

Bellantonio

. Indomethacin increases the effect of isosorbide dinitrate on cerebral hemodynamic in migraine patients: Pathogenetic and therapeutic implications. Cephalalgia 1998; 18: 622–630.

13.

Conti

Costa

Colleoni

. Antiinflammatory action of endocannabinoid palmitoylethanolamide and the synthetic cannabinoid nabilone in a model of acute inflammation in the rat. Br J Pharmacol 2002; 135: 181–187.

14.

Costa

Conti

Giagnoni

. Therapeutic effect of the endogenous fatty acid amide, palmitoylethanolamide, in rat acute inflammation: Inhibition of nitric oxide and cyclo-oxygenase systems. Br J Pharmacol 2002; 137: 413–420.

15.

Costa

Comelli

Bettoni

. The endogenous fatty acid amide, palmitoylethanolamide, has anti-allodynic and anti-hyperalgesic effects in a murine model of neuropathic pain: Involvement of CB(1), TRPV1 and PPARgamma receptors and neurotrophic factors. Pain 2008; 139: 541–550.

16.

Khasabova

Xiong

Coicou

. Peroxisome proliferator-activated receptor alpha mediates acute effects of palmitoylethanolamide on sensory neurons. J Neurosci 2012; 32: 12735–12743.

17.

Marini

Bartolucci

Bortolotti

. Palmitoylethanolamide versus a nonsteroidal anti-inflammatory drug in the treatment of temporomandibular joint inflammatory pain. J Orofac Pain 2012; 26: 99–104.

18.

Benito

Tolón

Castillo

. beta-Amyloid exacerbates inflammation in astrocytes lacking fatty acid amide hydrolase through a mechanism involving PPAR-alpha, PPAR-gamma and TRPV1, but not CB(1) or CB(2) receptors. Br J Pharmacol 2012; 166: 1474–1489.

19.

Scuderi

Esposito

Blasio

. Palmitoylethanolamide counteracts reactive astrogliosis induced by beta-amyloid peptide. J Cell Mol Med 2011; 15: 2664–2674.

20.

Cairns

Gambarota

Svensson

. Glutamate-induced sensitization of rat masseter muscle fibers. Neuroscience 2002; 109: 389–399.

21.

Cairns

Dong

Wong

. Intramuscular ketorolac inhibits activation of rat peripheral NMDA receptors. J Neurophysiol 2012; 107: 3308–3315.

22.

Dieterle

Fischer

Link

. Increase in CGRP- and nNOS-immunoreactive neurons in the rat trigeminal ganglion after infusion of an NO donor. Cephalalgia 2011; 31: 31–42.

23.

Dixon

. Fine structure of nerve-cell bodies and satellite cells in the trigeminal ganglion. J Dent Res 1963; 42: 990–999.

24.

Miclescu

Gordh

. Nitric oxide and pain: 'Something old, something new'. Acta Anaesthesiol Scand 2009; 53: 1107–1120.

25.

Salvemini

Kim

Mollace

. Reciprocal regulation of the nitric oxide and cyclooxygenase pathway in pathophysiology: Relevance and clinical implications. Am J Physiol Regul Integr Comp Physiol 2013; 304: R473–R487.

26.

Kummer

Behrends

Schwarzlmüller

. Subunits of soluble guanylyl cyclase in rat and guinea-pig sensory ganglia. Brain Res 1996; 721: 191–195.

27.

Vit

Jasmin

Bhargava

. Satellite glial cells in the trigeminal ganglion as a determinant of orofacial neuropathic pain. Neuron Glia Biol 2006; 2: 247–257.

28.

Schmidtko

Gao

Konig

. cGMP produced by NO-sensitive guanylyl cyclase essentially contributes to inflammatory and neuropathic pain by using targets different from cGMP-dependent protein kinase I. J Neurosci 2008; 28: 8568–8576.

29.

Schmidtko

Tegeder

Geisslinger

. No NO, no pain? The role of nitric oxide and cGMP in spinal pain processing. Trends Neurosci 2009; 32: 339–346.

30.

Seiler

Nusser

Lennerz

. Changes in calcitonin gene-related peptide (CGRP) receptor component and nitric oxide receptor (sGC) immunoreactivity in rat trigeminal ganglion following glyceroltrinitrate pretreatment. J Headache Pain 2013; 14: 74–74.

31.

Pose

Sampogna

Chase

. Mesencephalic trigeminal neurons are innervated by nitric oxide synthase-containing fibers and respond to nitric oxide. Brain Res 2003; 960: 81–89.

32.

D'Agostino

La Rana

Russo

. Acute intracerebroventricular administration of palmitoylethanolamide, an endogenous peroxisome proliferator-activated receptor-alpha agonist, modulates carrageenan-induced paw edema in mice. J Pharmacol Exp Ther 2007; 322: 1137–1143.

33.

Olesen

. The role of nitric oxide (NO) in migraine, tension-type headache and cluster headache. Pharmacol Ther 2008; 120: 157–171.

34.

Olesen

. Nitric oxide-related drug targets in headache. Neurotherapeutics 2010; 7: 183–190.

35.

Thomsen

Brennum

Iversen

. Effect of a nitric oxide donor (glyceryl trinitrate) on nociceptive thresholds in man. Cephalalgia 1996; 16: 169–174.

Intraganglionic injection of a nitric oxide donator induces afferent mechanical sensitization that is attenuated by palmitoylethanolamide

Abstract

Aim

Methods

Results

Conclusions

Keywords

Introduction

Materials and methods

In vitro PGE2 release experiments

Trigeminal satellite glial cell isolation

PGE2 release assays

In vivo electrophysiology

Anesthesia and surgical preparation

Stimulation and recording

Experimental design

Data and statistical analysis

Results

In vitro, DETA/NO-evoked PGE2 release is attenuated by PEA

In vivo, intraganglionic injection of DETA/NO sensitizes masticatory muscle afferent fibers

Systemic administration of PEA dose dependently attenuates mechanical sensitization

Intraganglionic injection of PGE2 induces mechanical sensitization

Discussion

Clinical implications

Footnotes

Funding

Conflict of interest

References

In vitro PGE₂ release experiments

PGE₂ release assays

In vitro, DETA/NO-evoked PGE₂ release is attenuated by PEA

Intraganglionic injection of PGE₂ induces mechanical sensitization