Abstract
The trigeminal innervation of the dura and its vessels has a prominent role in the mechanism of cluster headache. Nociceptin, an opioid neuropeptide, is the endogenous ligand of the OP-4 receptor, with both algesic and analgesic properties depending on the site of action. Nociceptin and its receptor are expressed by trigeminal ganglion cells where they co-localize with calcitonin gene-related peptide, a marker peptide of the trigeminovascular neurones. Nociceptin inhibits neurogenic dural vasodilatation, a phenomenon related to trigeminovascular activation. To explore its possible involvement in cluster headache, we studied circulating levels of nociceptin when attack-free during the cluster period, and also after the termination of the cluster period, using radioimmunoassay. In 14 cluster headache patients nociceptin levels during the cluster period were significantly lower than in age-, and sex-matched controls (4.91 ± 1.96 vs. 9.58 ± 2.57 pg/ml, P < 0.01). After the termination of the cluster period nociceptin levels (8.60 ± 1.47 pg/ml) were not statistically different from controls. Nociceptin levels did not correlate with age, length of disease or episode length. Lower nociceptin levels during the cluster period may result in a defective regulation of trigeminal activity that might not protect sufficiently against the attacks.
Introduction
In recent years, two major discoveries have changed our view of the pathomechanism of cluster headache (CH). Studies in animal models revealed that activation of the first branch of the trigeminal nerve, the substrate of pain in CH, results in neurogenic inflammation (1). During this process, vasoactive neuropeptides [calcitonin gene-related peptide (CGRP), substance P (SP) and neurokinin A] are released from trigeminal sensory terminals, causing vasodilatation, while the increased permeability of vessel walls leads to protein extravasation. The observation of elevated levels of CGRP and vasoactive intestinal polypeptide (VIP) in blood samples from the ipsilateral external jugular vein during the CH attack (2) provided human evidence of the activation of the trigeminal system and, probably via the trigemino-autonomic reflex, of the cranial parasympathetic outflow. That the trigeminal activation is related to a change in the function of the hypothalamic pacemaker was suggested by functional neuroimaging studies confirming the activation of the posterior hypothalamus during the attack, which ceased when the patient became headache free (3).
Nociceptin is an endogenous ligand for the orphan opioid receptor-like (ORL-1) receptor (4, 5), currently classified as OP-4 (6). Activation of OP-4 receptors inhibits high-voltage-activated Ca2+ channel currents and activates an inwardly rectifying K+ conductance. These intracellular effects contribute to the reduction of neuronal excitability and to the inhibition of presynaptic neurotransmitter release (7). The role of nociceptin in pain processing within the central nervous system (CNS) is suggested by both anatomical and functional data. The OP-4 receptor and its mRNA have been detected in many CNS regions that are involved in sensory processing, including the dorsal horn of the spinal cord, the periaqueductal grey, locus ceruleus, brain stem nuclei, the intralaminar nuclei of the thalamus, and the somatosensory cortex (8). OP-4 receptors are localized on both the main ascending (the trigeminal, spinothalamic and spinoreticular) and descending (the periaqueductal grey, the raphe magnus nucleus, the reticular formation) pathways. In the peripheral nervous system the action of nociceptin resembles that of the classical opioids (9). In the CNS nociceptin may have both algesic and analgesic effects, depending on the species, method of administration, dose and examinational paradigm (10, 11).
The majority of trigeminal ganglia neurones exhibit nociceptin immunoreactivity (12). In these cells nociceptin is co-localized with CGRP, SP, nitric oxide synthase (NOS) and pituitary adenylate-cyclase activating peptide (PACAP) (12). Moreover, neurogenic dural vasodilatation (NDV) was dose-dependently suppressed by nociceptin (13). These data suggest that the OP-4 receptor and its ligand may have a role in trigeminal sensory transmission, and, perhaps, in neurovascular headaches such as migraine or CH. To explore its possible involvement in CH we measured nociceptin levels during the cluster period.
Patients and methods
Patients
Fourteen episodic CH patients (three females and 11 males, mean age 49.1 years, SD 15.4) with a mean disease duration of 13.2 years (SD 10.6, range 3 months to 34 years) and a group of 22 age- and sex-matched healthy controls were enrolled. CH patients were examined during the CH period and at least 1 month after its termination. Patients receiving prophylactic treatment or any medical treatment (related to other conditions) were not involved. Informed consent was obtained. The study was approved by the local Ethics Committee.
Blood samples and nociceptin measurements
Blood was sampled in the headache-free state, at an interval of at least 3 h from the CH attack. Blood drawn from the antecubital vein of fasting subjects between 08.00 and 09.00 h was collected in 6-ml vacutainers containing K-EDTA as anticoagulant. Aprotinin (0.6 trypsin inhibitory units (TIU)/ml of blood; Calbiochem, San Diego, CA, USA) was added immediately as a protease inhibitor. The plasma was collected in mini-sorb tubes (Omker, Budapest, Hungary) and kept frozen at − 80 °C until analysis. The extraction and radioimmunoassay of nociceptin were carried out as described previously (14). In short, 1.0-ml aliquots of plasma samples were mixed with equal volume of trifluoroacetic acid (TFA; 1% v/v) and centrifuged. Acidified plasma samples were loaded onto C18 Sep-Pack cartridges, washed twice with 1.0% TFA, eluted with 60% acetonitrile in 0.1% TFA, then freeze-dried. Radioimmunoassay of the reconstituted eluate was performed using a commercially available 125I-Nociceptin kit (Phoenix Pharmaceuticals Inc., Belmont, CA, USA) with minimum sensitivity of 1 pg/ml. The assay was performed blind to the subject group.
Statistical analysis
The distribution of data was tested using the Kolmogorov–Smirnov test with Lilliefors’ method. As the distribution was Gaussian, comparison of the plasma nociceptin concentration in groups of subjects was made using an ANOVA with Bonferroni multiple comparisons test. The level of significance was set at P < 0.05. Correlation between nociceptin level and various patient characteristics was evaluated by Pearson's tests.
Results
In CH patients headache-free nociceptin levels were 4.91 ± 1.96 pg/ml (range 2.88–9.58), markedly and significantly lower (P < 0.01) than in healthy controls (9.58 ± 2.57 pg/ml, range 7.11–15.86) (Fig. 1). After the termination of the cluster period, nociceptin levels were 8.60 ± 1.47 (range 4.20–10.26), not statistically different from controls. Nociceptin levels did not correlate with age, length of disease, length of the present CH episode or the average number of attacks per day (Table 1).
Nociceptin levels in headache-free cluster headache (CH) patients during and after the cluster period and in controls. Nociceptin levels are expressed in pg/ml. During the cluster period nociceptin levels are significantly lower than in other groups. ∗Significant differences (P < 0.01, Bonferroni multiple comparisons test). NS, Not significant (P > 0.05).
Correlation of nociceptin levels during the cluster headache episode with patient characteristics
Values are Pearson's correlation coefficients followed by P-values. There was no significant correlation between nociceptin levels and these variables.
Discussion
We have demonstrated that CH patients have lower circulating nociceptin levels than controls during the CH period and that nociceptin levels normalize after its termination. To our knowledge, this is the first report of nociceptin levels in a primary headache syndrome.
Although intense pain per se does not necessarily lead to a change in blood or CSF nociceptin levels (15), we studied blood samples taken at least 3 h after or before an attack. We have not found human data about the half-life of nociceptin. In pharmacological studies conducted in Chinese hamster ovary (CHO) cell lines and rodents, the effects of a single dose of nociceptin lasted no longer than 60 min (16, 17). Therefore it is very unlikely that the lower nociceptin levels we observed were the consequence of the attack itself.
The few reports concerning nociceptin's possible role in human pain syndromes yield conflicting data. In fibromyalgia patients, decreased levels of nociceptin were reported (18). Acute pain conditions do not necessarily influence nociceptin levels, as both CSF and plasma nociceptin levels were unchanged during labour (15). Ko et al. studied nociceptin levels in aetiologically heterogeneous groups of acute, subacute and chronic pain patients (19), and in all these three groups significantly higher nociceptin levels were found than in healthy volunteers. Nociceptin levels were higher in subjects with chronic pain than in those with acute pain conditions, while there were no significant differences in pain intensity. Unfortunately, nociceptin levels were not compared between the different aetiological groups, which included musculoskeletal diseases, cholecystitis, urinary stone and non-specific chest pain. It was hypothesized that the change in nociceptin concentration represented a response to chronic painful stress, while other authors argued that nociceptin concentrations reflected the existence and duration of pain syndromes (19). The findings of decreased nociceptin levels in fibromyalgia (18) and in CH on the one hand, and increased nociceptin levels in the above-mentioned acute and chronic pain conditions on the other hand, however, seem to argue for a more complex role of this neuropeptide.
M⊘rk et al. examined the effect of intramuscular nociceptin injected into healthy humans (20). In four subjects, nociceptin injection into the temporal muscle did not cause pain. On the other hand, local tenderness increased in 10 subjects who had nociceptin injected into the tender trapezius muscle, although spontaneous pain was not noted. There were no changes of muscle hardness or pressure pain thresholds in the injected muscles. In a separate part of the investigation, the authors could not detect nociceptin-immunoreactive nerve fibres in the temporal artery and dermal tissue. A similar lack of nociceptin or the OP-4 receptor was also reported in synovial tissue and fluid of arthritis patients (21). These findings suggest that nociceptin does not have a major algesic effect on the periphery.
Recently, OP-4 receptor mRNA was demonstrated in human trigeminal ganglia by reverse-transcriptase-polymerase chain reaction and nociceptin immunoreactivity was detected in about 70% of its neurones (12). The majority of nociceptin-positive neurones were medium-sized (30–60 µm). Nociceptin immunoreactivity co-localized with CGRP, SP, NOS and PACAP (12). All the CGRP-, SP-, NOS-, or PACAP-positive neurones of the ganglia were nociceptin-immunoreactive. This distribution suggests that nociceptin might be involved in the regulation of neuropeptide release from trigeminal sensory nerve terminals. A similar effect of systemic nociceptin administration, resulting in the attenuation of neurogenic inflammation, was described in peripheral nerves (9, 17, 22).
Stimulation of the trigeminal nerve results in the dilatation of dural vessels [neurogenic dural vasodilatation (NDV)]. In a rat trigeminovascular model, nociceptin dose-dependently suppressed NDV, while it had no effect on baseline vessel diameter (13). Pretreatment with the selective OP-4 receptor antagonist [Nphe1]NC-(1-13)-NH2 dose-dependently reversed the effect of nociceptin. The human basilar artery does not express nociceptin receptor mRNA, and nociceptin immunoreactivity has not been observed in basilar and middle cerebral arteries (12). In pharmacological tests, nociceptin administration does not modify the contractile properties of human cortical arteries obtained during neurosurgical tumour resections (12). Taken together, these data suggest that the effect of nociceptin on dural vessels is probably mediated by OP-4 receptors located on trigeminal neurones.
Considering these data, it is tempting to suppose that lower circulating levels of nociceptin during the cluster period could result in a defective regulation of the trigeminovascular system, that may not sufficiently protect against CH attacks. However, as the nociceptin doses used in the NDV experiment were much higher than the normal level in humans, it is unclear whether the difference between nociceptin levels of CH patients and controls is relevant to the pathomechanism of CH. Animal studies could help us better understand the effect of nociceptin on trigeminovascular activation and also on pain transmission in the trigeminal nucleus caudalis and in higher-order structures. Further clinical studies are also necessary to clarify whether nociceptin levels change during the attacks and to examine nociceptin levels in other neurovascular headaches such as migraine and rare types of trigemino-autonomic cephalalgias.