Fluorescent Labeling and 2-Photon Imaging of Mouse Tooth Pulp Nociceptors.

Retrograde fluorescent labeling of dental primary afferent neurons (DPANs) has been described in rats through crystalline fluorescent DiI, while in the mouse, this technique was achieved with only Fluoro-Gold, a neurotoxic fluorescent dye with membrane penetration characteristics superior to the carbocyanine dyes. We reevaluated this technique in the rat with the aim to transfer it to the mouse because comprehensive physiologic studies require access to the mouse as a model organism. Using conventional immunohistochemistry, we assessed in rats and mice the speed of axonal dye transport from the application site to the trigeminal ganglion, the numbers of stained DPANs, and the fluorescence intensity via 1) conventional crystalline DiI and 2) a novel DiI formulation with improved penetration properties and staining efficiency. A 3-dimensional reconstruction of an entire trigeminal ganglion with 2-photon laser scanning fluorescence microscopy permitted visualization of DPANs in all 3 divisions of the trigeminal nerve. We quantified DPANs in mice expressing the farnesylated enhanced green fluorescent protein (EGFPf) from the transient receptor potential cation channel subfamily M member 8 (TRPM8EGFPf/+) locus in the 3 branches. We also evaluated the viability of the labeled DPANs in dissociated trigeminal ganglion cultures using calcium microfluorometry, and we assessed the sensitivity to capsaicin, an agonist of the TRPV1 receptor. Reproducible DiI labeling of DPANs in the mouse is an important tool 1) to investigate the molecular and functional specialization of DPANs within the trigeminal nociceptive system and 2) to recognize exclusive molecular characteristics that differentiate nociception in the trigeminal system from that in the somatic system. A versatile tool to enhance our understanding of the molecular composition and characteristics of DPANs will be essential for the development of mechanism-based therapeutic approaches for dentine hypersensitivity and inflammatory tooth pain.

Role of transient receptor potential ankyrin 1 receptors in rodent models of meningeal nociception - Experiments in vitro.

BACKGROUND: The TRP channel ankyrin type 1 (TRPA1) is a nonselective cation channel known to be activated by environmental irritants, cold and endogenous mediators of inflammation. Activation of TRPA1 in trigeminal afferents innervating meningeal structures has recently been suggested to be involved in the generation of headaches. METHODS: Two in vitro models of meningeal nociception were employed using the hemisected rodent head preparation, (1) recording of single meningeal afferents and (2) release of calcitonin gene-related peptide (CGRP) from the cranial dura mater. The role of TRPA1 was examined using the TRPA1 agonists acrolein and mustard oil (MO). BCTC, an inhibitor of TRP vanilloid type 1 receptor channels (TRPV1), and the TRPA1 inhibitor HC030031 as well as mice with genetically deleted TRPA1 and TRPV1 proteins, were used to differentiate between effects. RESULTS: Acrolein did not cause discharge activity in meningeal Aδ- or C-fibres but increased the electrical activation threshold. Acrolein was also effective in releasing CGRP from the dura of TRPV1-/- but not of TRPA1-/- mice. MO increased the discharge activity of afferent fibres from rat as well as C57 wild-type and TRPA1-/- but not TRPV1-/- mice. The effect was higher in C57 compared to TRPA1-/- mice. CONCLUSION: Sole TRPA1 receptor channel activation releases CGRP and increases the activation threshold of meningeal afferents but does not generate propagated activity, and so would be capable of causing local effects like vasodilatation but not pain generation. In contrast, combined TRPA1 and TRPV1 activation may be rather pronociceptive supporting headache generation. SIGNIFICANCE: Sole activation of TRPA1 receptor channels increases the activation threshold but does not cause propagated action potentials in meningeal afferents. TRPA1 agonists cause CGRP release from rodent dura mater. Peripheral TRPA1 receptors may have a pronociceptive function in trigeminal nociception only in combination with TRPV1.

[Physiology of pain]. / Physiologie des Schmerzes.

Pain research is based broadly on physiological disciplines and its development follows the methodological progress of the era, from classical psychophysiology to electrophysiological investigations at peripheral and central nociceptive systems, single cells and ion channels to modern imaging of nociceptive processing. Physiological pain research in Germany has long been part of an interdisciplinary research network extending beyond all political boundaries, and this situation has continued since molecular techniques started to dominate all biomedical research. Current scientific questions, such as intracellular nociceptive signal mechanisms, interactions with other physiological systems including the immune system, or the genetic basis of epidemic and chronic pain diseases can only be solved interdisciplinary and with international collaboration.

Mice and rats differ with respect to activity-dependent slowing of conduction velocity in the saphenous peripheral nerve.

We assess in mice, the electrophysiological criteria developed in humans and rats in vivo for unmyelinated (C) fibre differentiation into sub-classes, derived from the activity-induced latency increase ("slowing") in response to electrical stimulation during 6 min at 0.25 Hz followed by 3 min at 2 Hz. Fibres are considered nociceptors if they show more than 10% slowing at 2 Hz; nociceptors are further divided into mechanosensitive ("polymodal") and mechanoinsensitive ("silent") ones according to a latency shift of less and more than 1% during the first minute at 0.25 Hz, respectively. Sympathetic postganglionics are recognised by 2-10% slowing at 2 Hz; units slowing less than 2% at 2 Hz remain uncategorised. For assessment of these criteria, we also developed a novel in vivo technique for recording of peripheral single-fibres in the mouse. We compared the theoretical slowing-rate discriminator criteria with experimental data obtained from mice in vivo/in vitro and rats in vitro. Out of 69 cutaneous mouse C-fibres in vitro and 19 in vivo, only 38 (67%) and 9 (47%) met the above 1% criterion, respectively; sympathetics were not identified. In contrast, out of 20 rats nerve fibres in vitro, 19 (95%) met this criterion. We conclude that (A) our novel electrophysiological technique is a practical method for examining mouse cutaneous single-fibres in vivo and (B) the published criterion for identifying silent nociceptors in rats and humans is not applicable in mice.

Localization of receptors for calcitonin-gene-related peptide to intraganglionic laminar endings of the mouse esophagus: peripheral interaction between vagal and spinal afferents?

The calcitonin-gene-related peptide (CGRP) receptor is a heterodimer of calcitonin-receptor-like receptor (CLR) and receptor-activity-modifying protein 1 (RAMP1). Despite the importance of CGRP in regulating gastrointestinal functions, nothing is known about the distribution and function of CLR/RAMP1 in the esophagus, where up to 90 % of spinal afferent neurons contain CGRP. We detected CLR/RAMP1 in the mouse esophagus using immunofluorescence and confocal laser scanning microscopy and examined their relationship with neuronal elements of the myenteric plexus. Immunoreactivity for CLR and RAMP1 colocalized with VGLUT2-positive intraganglionic laminar endings (IGLEs), which were contacted by CGRP-positive varicose axons presumably of spinal afferent origin, typically at sites of CRL/RAMP1 immunoreactivity. This provides an anatomical basis for interaction between spinal afferent fibers and IGLEs. Immunoreactive CLR and RAMP1 also colocalized in myenteric neurons. Thus, CGRP-containing spinal afferents may interact with both vagal IGLEs and myenteric neurons in the mouse esophagus, possibly modulating motility reflexes and inflammatory hypersensitivity.

[Neuropeptide effects on the trigeminal system: pathophysiology and clinical significance for migraine]. / Neuropeptidwirkungen im trigeminalen System: Pathophysiologie und klinische Bedeutung bei der Migräne.

Neuropeptides, such as calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal polypeptide (VIP) are considered important mediators in primary headaches. Increased concentrations of CGRP have been found in jugular venous plasma during attacks of migraine and, concomitant with VIP elevation, during cluster headache. Substance P and CGRP are produced from subsets of trigeminal afferents whereas VIP derives from parasympathetic efferents. Release of these neuropeptides in the meninges causes arterial vasodilatation, mast cell degranulation and plasma extravasation in animal experiments. Particularly CGRP seems to be important, as receptor antagonists have recently been shown to have a therapeutic effect on migraine. Animal models have confirmed the role of CGRP in meningeal nociception. The activity of spinal trigeminal neurons is a sensitive integrative measure of trigeminal activity and CGRP released from central terminals of trigeminal afferents in the spinal trigeminal nucleus has been shown to facilitate nociceptive transmission, most likely by a presynaptic action. The proposed CGRP functions are supported by the distribution of CGRP receptor components localized in the rat cranial dura mater, the trigeminal ganglion and the spinal trigeminal nucleus. The currently available data indicate multiple sites of CGRP action in trigeminal nociception and the pathogenesis of migraine but central CGRP receptors are probably the essential targets in the treatment of migraine using CGRP receptor antagonists.

Prevalence and association of headaches, temporomandibular joint disorders, and occlusal interferences.

STATEMENT OF PROBLEM: Although an interaction of malocclusion, parafunction, and temporomandibular joint disorders (TMD) can be inferred from the experience of daily practice, scientific evidence to corroborate this hypothesis does not exist. However, there are indications that TMD and headaches may be intertwined. PURPOSE: The purpose of this study was to identify the presence or absence of an association of occlusal interferences, parafunction, TMD, or physiologic, muscular, or prosthodontic factors with the occurrence of headache. MATERIAL AND METHODS: In a private practice population of 1031 subjects (436 men and 595 women, mean age 49.6 years) the demographic parameters, headache and general pain history, habits and general personal information were recorded. Clinical examination for dental, muscular, and temporomandibular joint pathology was accomplished. Data were statistically analyzed using the Mann-Whitney U, Kruskal-Wallis, and Chi-Square tests (α = .05). A multinomial logistic regression analysis was performed with respect to confounding variables. RESULTS: Headache affliction was found to affect women more frequently than men (1.7:1). Students and non academics were more prone to suffer from headache. Parafunction (P=.001), TMD (P=.001) and gross differences between centric occlusion and maximum intercuspation of more than a 3 mm visible track marked with 8 µm articulation foil (P=.001) significantly influenced the presence of headache. Headache intensity and frequency decreased with age. While tension-type headache was most frequently diagnosed, the parameters studied were not significantly associated with one certain headache diagnosis more frequently than others. CONCLUSIONS: Stomatognathic factors of TMD, parafunction, and gross differences between centric occlusion and maximum intercuspation of more than 3 mm are associated with headache. These findings should be interpreted with caution due to the cross-sectional nature of this study.

Increase in NADPH-diaphorase-positive and neuronal NO synthase immunoreactive neurons in the rat spinal trigeminal nucleus following infusion of a NO donor--evidence for a feed-forward process in NO production involved in trigeminal nociception.

Nitric oxide (NO) donors, which cause delayed headaches in migraineurs, have been shown to activate central trigeminal neurons with meningeal afferent input in animal experiments. Previous reports indicate that this response may be due to up-regulation of NO-producing cells in the trigeminal brainstem. To investigate this phenomenon further, we determined nitric oxide synthase (NOS)-containing neurons in the rat spinal trigeminal nucleus (STN), the projection site of nociceptive trigeminal afferents, following infusion of the NO donor sodium nitroprusside (SNP). Barbiturate anaesthetized rats were infused intravenously with SNP (50 microg/kg) or vehicle for 20 min or 2 h, and after periods of 3-8 h fixed by perfusion. Cryostat sections of the medulla oblongata containing the caudal STN were histochemically processed for detection of nicotineamide adenine dinucleotide phosphate (NADPH)-diaphorase or immunohistochemically stained for NOS isoforms and examined by light and fluorescence microscopy. The number of neurons positive for these markers was determined. Various forms of neurons positive for NADPH-diaphorase or immunoreactive to neuronal NOS (nNOS) were found in superficial and deep laminae of the STN caudalis and around the central canal. Neurons were not immunopositive for endothelial (eNOS) or inducible (iNOS) NOS isoforms. The number of NADPH-diaphorase-positive neurons increased time dependently after SNP infusion by a factor of more than two. Likewise, the number of nNOS-immunopositive neurons was increased after SNP compared with vehicle infusion. Around the central canal the number of NADPH-diaphorase-positive neurons was slightly increased and the number of nNOS+ neurons not changed after SNP treatment. NO donors increase the number of neurons that produce NO in the STN, possibly by induction of nNOS expression. Increased NO production may facilitate neurotransmitter release and promote nociceptive transmission in the STN. This mechanism may explain the delayed increase in neuronal activity and headache after infusion of NO donors.

Calcitonin gene-related peptide receptor inhibition reduces neuronal activity induced by prolonged increase in nitric oxide in the rat spinal trigeminal nucleus.

Infusion of nitric oxide (NO) donors is known to induce delayed attacks of migraine and cluster headache or aggravate tension-type headaches in patients suffering from these primary headaches. Previously we have reported that infusion of NO donors in the rat causes delayed neuronal activity in the spinal trigeminal nucleus, which parallels the above clinical observations. Suggesting that endogenous NO production is involved in the generation of primary headaches, we used this animal model of meningeal nociception to determine whether a prolonged increase in NO levels causes an increase in neuronal activity. In anaesthetized rats spinal trigeminal neurons with afferent input from the exposed dura were recorded. Continuous intravenous infusion of the NO donors sodium nitroprusside (25 microg/kg/h) or glycerol trinitrate (250 microg/kg/h) for 2 h induced a persisting increase in neuronal activity but no change in systemic blood pressure. In this activated trigeminal system the calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS (900 microg/kg) was infused. Spinal trigeminal activity was significantly reduced within minutes and to a similar extent as previously reported in animals not treated with NO. Slow continuous NO infusion may be a model of the active headache phase, and inhibition of CGRP receptors can reverse the induced neuronal activity.

Ongoing activity in trigeminal wide-dynamic range neurons is driven from the periphery.

Ongoing activity of spinal trigeminal neurons is observed under various conditions and suggested to be responsible for ongoing headache. It can be spontaneous, i.e. arising intrinsically from the neuron, or the product of descending influences from other central neurons, or maintained by ongoing afferent input. The aim of the present study was to examine if ongoing activity of neurons in different subnuclei of the spinal trigeminal nucleus is driven from peripheral afferent input. Experiments were performed in Wistar rats anesthetized with isoflurane or Nembutal/urethane. Ongoing activity of single wide-dynamic range (WDR) neurons was recorded with carbon fiber glass microelectrodes in two subnuclei of the spinal trigeminal nucleus: oral (Sp5O) and caudal (Sp5C). Peripheral receptive fields were evaluated using von Frey filaments. Sp5O neurons received peripheral input from facial areas innervated by the mandibular branch of the trigeminal nerve. Units in Sp5C had receptive fields in the surgically exposed dura mater and in facial areas innervated by the ophthalmic and maxillary branch of the trigeminal nerve. Saline or the local anesthetic lidocaine was locally applied onto the exposed dura mater or microinjected into V3 (for Sp5O units) or V1/V2 (for Sp5C units) divisions of the trigeminal ganglion via the infraorbital channel. Local application of lidocaine onto the exposed dura caused mechanical insensitivity of dural receptive fields but not significant decrease in ongoing activity. Microinjection of lidocaine but not saline into the trigeminal ganglion was followed by a substantial decrease in both the receptive field size and the activity of the recorded WDR units. Mechanical insensitivity of receptive fields after trigeminal ganglion blockade was accompanied by the disappearance of ongoing activity. We conclude that the ongoing activity of WDR neurons in the spinal trigeminal nucleus, which may be indicative for processes of sensitization, is driven remotely by ongoing afferent input.

Cannabinoid and vanilloid effects of R(+)-methanandamide in the hemisected meningeal preparation.

The endogenous cannabinoid R(+)-methanandamide (mAEA) exerts differential anti- and pronociceptive effects by activating both cannabinoid (CB1) and vanilloid (TRPV1) receptors of nociceptive primary afferents. The significance of these effects in meningeal nociception was evaluated by modulation of calcitonin gene-related peptide (CGRP) release from meningeal afferents measured in an in vitro preparation of the hemisected rat skull. Temperature steps to 39 degrees C and 45 degrees C caused heat-dependent increases in CGRP release. One micromolar mAEA inhibited CGRP release at 32 degrees C but facilitated it at 45 degrees C. This effect was abolished in the presence of the TRPV1 receptor antagonist capsazepine. Lower doses of mAEA had no effect on basal or heat-evoked release. In the presence of the CB1 receptor antagonist SR141716 (0.2 microm) heat-stimulated increase in CGRP release was facilitated. CGRP release in the presence of SR141716 (0.2 microm) was further increased by adding mAEA at a concentration which had no effect on its own. These results confirm an opposing functional role for anandamide at CB1 and TRPV1 receptors on meningeal afferents.

Interaction of calcitonin gene-related peptide, nitric oxide and histamine release in neurogenic blood flow and afferent activation in the rat cranial dura mater.

Calcitonin gene-related peptide (CGRP), nitric oxide (NO) and histamine are implicated in primary headaches but their role in vascular and nociceptive events in the dura mater is not well described. In an in vitro preparation of the hemisected rat skull, CGRP and histamine release from the cranial dura was measured using enzyme-linked immunoassays. While the NO donator NONO(ate) (10(-4) M) was without effect, CGRP (10(-5) M) induced considerable histamine release from the rat cranial dura, which was blocked by the CGRP receptor antagonist CGRP(8-37) (10(-5) M). Conversely, histamine (10(-4) M) did not stimulate CGRP release. In vitro recordings from single rat meningeal afferents showed that only one of 12 mechanically identified units but several mechanically insensitive units responded to histamine (up to 10(-5) M). Increases in meningeal blood flow after histamine application (10(-4) M) to the rat cranial dura remained unchanged during CGRP receptor blockade with CGRP(8-37), inhibition of NO synthesis with L-NAME (20 mg/kg i.v.) and H(3) receptor blockade with thioperamide (10(-4) M). We conclude that histamine produces direct vasodilatation and activates a subset of largely non-mechanically sensitive, non-CGRP containing afferents in the rat meninges. Histamine is released from meningeal mast cells which are stimulated by CGRP. Similar mechanisms may be involved in the pathogenesis of headaches.

The TRPV1/2/3 activator 2-aminoethoxydiphenyl borate sensitizes native nociceptive neurons to heat in wildtype but not TRPV1 deficient mice.

TRPV1 gene disruption results in a loss of capsaicin and proton responsiveness, but has minimal effects on heat-induced nocifensive behavior, suggesting that sensory transduction of heat is independent of TRPV1. TRPV3, another heat-activated ion channel but insensitive to capsaicin, was shown to be expressed in keratinocytes as well as in sensory neurons projecting to the skin. Recently, 2-aminoethoxydiphenyl borate was introduced as a TRPV3 agonist, but its selectivity was questioned by showing that it activated recombinant TRPV1 and TRPV2 as well. We used the isolated mouse skin-saphenous nerve preparation and whole-cell patch-clamping of cultured dorsal root ganglia neurons from TRPV1-/- and wildtype mice. We found no phenotypic differences between the heat responses of polymodal C-fibers, whereas cultured dorsal root ganglia neurons of TRPV1-/- hardly showed any heat-activated currents. Only C-fibers of wildtype but not TRPV1-/- mice were clearly sensitized to heat by 2-aminoethoxydiphenyl borate 10 and 100 microM; heat-activated current in wildtype neurons was only facilitated at 100 microM. Noxious heat-induced calcitonin gene-related peptide release showed clear deficits (<50%) in TRPV1 deficient skin, but the stimulated calcitonin gene-related peptide release from the isolated skull dura was unaffected. In both models, 2-aminoethoxydiphenyl borate was able to potentiate the heat response (46 degrees C, 5 min) in a concentration-dependent manner, again, only in wildtype but not TRPV1-/- mice, suggesting that TRPV2/3 are not involved in this sensitization to heat. The results further suggest that TRPV1 is not responsible for the normal heat response of native nociceptors but plays the essential role in thermal sensitization and a prominent one in controlling dermal calcitonin gene-related peptide release, i.e. neurogenic inflammation.

Noxious chemical stimulation of rat facial mucosa increases intracranial blood flow through a trigemino-parasympathetic reflex--an experimental model for vascular dysfunctions in cluster headache.

Cluster headache is characterized by typical autonomic dysfunctions including facial and intracranial vascular disturbances. Both the trigeminal and the cranial parasympathetic systems may be involved in mediating these dysfunctions. An experimental model was developed in the rat to measure changes in lacrimation and intracranial blood flow following noxious chemical stimulation of facial mucosa. Blood flow was monitored in arteries of the exposed cranial dura mater and the parietal cortex using laser Doppler flowmetry. Capsaicin (0.01-1 mm) applied to oral or nasal mucosa induced increases in dural and cortical blood flow and provoked lacrimation. These responses were blocked by systemic pre-administration of hexamethonium chloride (20 mg/kg). The evoked increases in dural blood flow were also abolished by topical pre-administration of atropine (1 mm) and [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP (0.1 mm), a vasoactive intestinal polypeptide (VIP) antagonist, onto the exposed dura mater. We conclude that noxious stimulation of facial mucosa increases intracranial blood flow and lacrimation via a trigemino-parasympathetic reflex. The blood flow responses seem to be mediated by the release of acetylcholine and VIP within the meninges. Similar mechanisms may be involved in the pathogenesis of cluster headache.

[Neuropeptide release in the dura mater encephali in response to nitric oxide--relevance for the development of vascular headaches?]. / Neuropeptidfreisetzung in den Hirnhäuten durch Stickstoffmonoxid. Bedeutung für die Entstehung vaskulärer Kopfschmerzen?

Nitric oxide (NO) and calcitonin gene-related peptide (CGRP), potent vasodilators in the meninges,may be involved in the pathophysiology of vascular headaches such as migraine pain. NO donators can provoke headache attacks in migraineurs and increased levels of CGRP have been found in the venous outflow from the head during migraine attacks. We therefore examined the effect of both NO and CGRP on dural blood, a process which may parallel nociceptive processes in the meninges. 1. Arterial blood flow was measured in the exposed dura mater encephali of the rat using laser Doppler flowmetry. Local application of different NO donors (SNAP,NONOate, and NOC-12) caused dose-dependent increases in meningeal blood flow. CGRP(8-37) at 10(-4) M did not significantly change the basal flow but attenuated increases in blood flow caused by the NO donors at concentrations of 10(-5)-10(-3) M.2. In another series of experiments, the hemisected skulls of adult Wistar rats, complete with intact dura mater, were filled with oxygenated synthetic interstitial fluid (SIF) and the CGRP content of this fluid was assessed every 5 min. When the NO donator NONOate, at concentrations of 10(-5)-10(-3) M, was added to the SIF, or when the SIF was bubbled with NO gas (1000 ppm in N(2) atmosphere) instead of carbogen, CGRP release increased in a concentration-dependent manner. We conclude that the vasodilatory effect of NO that causes increased meningeal blood flow is in part the result of both stimulating the release of CGRP and promoting the vasodilatory action of CGRP. Since NO donors such as nitroglycerin are known to provoke headache and CGRP is released during migraine pain, the NO-stimulated CGRP release may be relevant for the development of vascular headaches that are accompanied by meningeal hyperaemia.

Increase in meningeal blood flow by nitric oxide--interaction with calcitonin gene-related peptide receptor and prostaglandin synthesis inhibition.

This study addresses possible interactions of the vasodilators nitric oxide (NO), calcitonin gene-related peptide (CGRP) and prostaglandins, which may be implicated in the generation of vascular headaches. Local application of the NO donator diethylamine-NONOate (NONOate) to the exposed dura mater encephali of the rat caused dose-dependent increases in meningeal blood flow recorded by laser Doppler flowmetry. Pre-application of the CGRP receptor antagonist CGRP8-37 significantly attenuated the evoked blood flow increases, while the cyclooxygenase inhibitors acetylsalicylic acid and metamizol were only marginally effective. Stimulation of rat dura mater with NONOate in vitro caused increases in CGRP release. NADPH-diaphorase activity indicating NO production was restricted to the endothelium of dural arterial vessels. We conclude that increases in meningeal blood flow caused by NO depend partly on the release and vasodilatory action of CGRP from dural afferents, while prostaglandins are not significantly involved.

Meningeal nociception: electrophysiological studies related to headache and referred pain.

Headaches, which are usually referred to characteristic sites of the skull, are believed to involve meningeal nociceptors located in the dura mater encephali. Animal experiments show that these meningeal nociceptors are polymodal and usually highly sensitive to mechanical stimulation. These properties are also characteristic for the second order neurons in the spinal trigeminal nucleus, most of which receive convergent input from facial receptive sites. Sensitization of primary and secondary neurons by chemical irritants to mechanical stimuli may be an important mechanism in the generation of headaches. The convergent input from extracranial structures, which seems to be differentially organized in rodents and man, may explain the typical features of referred headache. Targets for analgesics used in the therapy of headaches (non-steroidal antiinflammatory drugs, 5-HT(1) receptor agonists) are probably meningeal nociceptors and different sites of the central trigeminal nociceptive and antinociceptive pathways.

Histological demonstration of increased vascular permeability in the dura mater of the rat.

Neurogenic inflammation of the dura mater encephali has been suggested to play an important role in the pathophysiology of headaches. Although functional studies using extravasation techniques indicate an enhanced permeability of blood vessels after chemical or electrical stimulation of C-fibres supplying the dura mater, histological demonstration of leaky blood vessels is still a problem. We used the vascular labelling method combined with i.v. injection of colloidal silver solution to test the permeability increasing effect of intravenous administration of substance P, topical application of mustard oil or acidic phosphate buffer and local electrical stimulation of the exposed dura mater. Histological characteristics of increased vascular permeability were observed exclusively after mustard oil and acidic phosphate buffer. This observation may indicate different mechanisms of increased vascular permeability involving pinocytosis and formation of interendothelial gaps selectively visualized by the vascular labelling method.

Involvement of nitric oxide in the modulation of dural arterial blood flow in the rat.

1. Nitric oxide (NO) has been proposed to be a key molecule in the pathogenesis of migraine pain and other headaches that are linked to vascular disorders. Several lines of evidence indicate that the meningeal vascularization is crucially involved in the generation of these headaches. In an experimental model in the rat a dominating role of calcitonin gene-related peptide (CGRP) in causing neurogenic vasodilatation and increased blood flow has been shown. The aim of the present study was to clarify the role of NO in this model with regard to the meningeal blood flow. 2. The blood flow in and around the medial meningeal artery (dural arterial flow) was recorded in the exposed parietal dura mater encephali of barbiturate anaesthetized rats using laser Doppler flowmetry. Local electrical stimulation of the dura mater (pulses of 0.5 ms delivered at 7.5 - 17.5 V and 5 or 10 Hz for 30 s) caused temporary increases in dural arterial flow for about 1 min that reached peaks of 1.6 - 2.6 times the basal flow. The effects of NO synthase (NOS) inhibitors on the basal flow and the electrically evoked increases in flow were examined. 3. Systemic (i. v.) administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) at cumulative doses of 10 and 50 mg kg(-1) lowered the basal flow to 87 and 72%, respectively, of the control and reduced the evoked increases in blood flow to 82 and 44% on an average. Both these effects could partly be reversed by 300 mg kg(-1) L-arginine. The systemic arterial pressure was increased by L-NAME at both doses. Injection of the stereoisomer D-NAME at same doses did not change basal flow and evoked increases in flow. 4. 4. Topical application of L-NAME (10(-4) - 10(-2) M) was effective only at the highest concentration, which caused lowering of the basal blood flow to 78% of the control; the evoked increases in flow were not changed. Topical application of 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a specific inhibitor of the inducible NOS, at concentrations of 10(-4) - 10(-2) M lowered the basal flow to 89, 87.5 and 85%, respectively, but did not significantly change the evoked flow increases. Same concentrations of 7-nitroindazole monosodium salt (7-NINA), a specific inhibitor of the neuronal NOS, had no significant effects on basal flow and evoked increases in flow. 5. It is concluded that NO is involved in the maintenance of the basal level of dural arterial blood flow as well as in the electrically evoked flow increases, which have been shown to be mainly mediated by CGRP released from dural afferent fibres. The most important source of NO is probably the endothelium of dural arterial vessels. The synergistic effect of NO and CGRP on the stimulated blood flow may be in part due to a NO mediated facilitation of the CGRP release.