Topical dura mater application of CFA induces enhanced expression of c-fos and glutamate in rat trigeminal nucleus caudalis: attenuated by KYNA derivate (SZR72).

BACKGROUND: Migraine is a debilitating neurological disorder where trigeminovascular activation plays a key role. We have previously reported that local application of Complete Freund's Adjuvant (CFA) onto the dura mater caused activation in rat trigeminal ganglion (TG) which was abolished by a systemic administration of kynurenic acid (KYNA) derivate (SZR72). Here, we hypothesize that this activation may extend to the trigeminal complex in the brainstem and is attenuated by treatment with SZR72. METHODS: Activation in the trigeminal nucleus caudalis (TNC) and the trigeminal tract (Sp5) was achieved by application of CFA onto the dural parietal surface. SZR72 was given intraperitoneally (i.p.), one dose prior CFA deposition and repeatedly daily for 7 days. Immunohistochemical studies were performed for mapping glutamate, c-fos, PACAP, substance P, IL-6, IL-1ß and TNFα in the TNC/Sp5 and other regions of the brainstem and at the C1-C2 regions of the spinal cord. RESULTS: We found that CFA increased c-fos and glutamate immunoreactivity in TNC and C1-C2 neurons. This effect was mitigated by SZR72. PACAP positive fibers were detected in the fasciculus cuneatus and gracilis. Substance P, TNFα, IL-6 and IL-1ß immunopositivity were detected in fibers of Sp5 and neither of these molecules showed any change in immunoreactivity following CFA administration. CONCLUSION: This is the first study demonstrating that dural application of CFA increases the expression of c-fos and glutamate in TNC neurons. Treatment with the KYNA analogue prevented this expression.

KYNA analogue SZR72 modifies CFA-induced dural inflammation- regarding expression of pERK1/2 and IL-1ß in the rat trigeminal ganglion.

BACKGROUND: Neurogenic inflammation has for decades been considered an important part of migraine pathophysiology. In the present study, we asked the question if administration of a novel kynurenic acid analogue (SZR72), precursor of an excitotoxin antagonist and anti-inflammatory substance, can modify the neurogenic inflammatory response in the trigeminal ganglion. METHODS: Inflammation in the trigeminal ganglion was induced by local dural application of Complete Freunds Adjuvant (CFA). Levels of phosphorylated MAP kinase pERK1/2 and IL-1ß expression in V1 region of the trigeminal ganglion were investigated using immunohistochemistry and Western blot. FINDINGS: Pretreatment with one dose of SZR72 abolished the CFA-induced pERK1/2 and IL-1ß activation in the trigeminal ganglion. No significant change was noted in case of repeated treatment with SZR72 as compared to a single dose. CONCLUSIONS: This is the first study that demonstrates that one dose of KYNA analog before application of CFA can give anti-inflammatory response in a model of trigeminal activation, opening a new line for further investigations regarding possible effects of KYNA derivates.

Dural administration of inflammatory soup or Complete Freund's Adjuvant induces activation and inflammatory response in the rat trigeminal ganglion.

BACKGROUND: Migraine is a painful disorder with a huge impact on individual and public health. We hypothesize that migraine pain originates from a central mechanism that results secondarily in hypersensitivity in peripheral afferents associated with the cerebral and cranial blood vessels. It has previously been shown that application of inflammatory or algesic substances onto the dura mater or chemical stimulation of the dural receptive fields causes hypersensitivity to mechanical and thermal stimulation together with direct activation of the TG. We asked whether local inflammation of dura mater induces inflammatory activation in the trigeminal ganglion. METHODS: We performed topical administration of inflammatory soup (IS) or Complete Freund's Adjuvant (CFA) onto an exposed area of the rat dura mater in vivo for 20 min. The window was closed and the rats were sacrificed after 4 h and up to 7 days. Myography was performed on middle meningeal arteries. The trigeminal ganglia were removed and processed for immunohistochemistry or Western blot. RESULTS: Both CFA and IS induced enhanced expression of pERK1/2, IL-1ß and CGRP in the trigeminal ganglia. The pERK1/2 immunoreactivity was mainly seen in the satellite glial cells, while IL-1ß reactivity was observed in the neuronal cytoplasm, close to the cell membrane, seemingly as sign of neuro-glial interaction. The CGRP expression in the neurons and nerve fibres was enhanced after the application of either inflammatory agent. Myography resulted in a strong vasoconstrictor response to IS, but not to CFA. CONCLUSIONS: These results suggest that the application of IS or CFA onto the dura mater causes long-term activation of the TG and demonstrate the importance of the neuro-glial interaction in the activation of the trigeminovascular system.

Kynurenic acid modulates experimentally induced inflammation in the trigeminal ganglion.

BACKGROUND: The trigeminal ganglion (TG) plays a central role in cranial pain. Administration of complete Freund's adjuvant (CFA) into the temporomandibular joint (TMJ) elicits activation of TG. Kynurenic acid (KYNA) is an endogenous excitatory amino acid receptor blocker, which may have an anti-inflammatory effect. We hypothesize that KYNA may reduce CFA-induced activation within the TG. METHODS: A local inflammation was induced by administration of CFA into the TMJ in rats. KYNA and kynurenic acid amide 2 (KYNAA2) were intraperitoneally administered. We investigated changes of mitogen-activated protein kinases (MAPKs as ERK1/2, p38 and SAPK/JNK), NF-κB, CaMKII and DREAM, in addition to calcitonin gene-related peptide (CGRP) and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in the TG, with immunohistochemistry and Western blot at 2 and 10 days post-CFA injection. RESULTS: We showed CFA-induces increases in pERK1/2, pp38, CaMKII, NF-κB and DREAM immunohistochemistry after 2 and 10 days. KYNAA2 displayed stronger effects on MAPKs than KYNA. Increased expression of CaMKII, NF-κB and DREAM were found in the neurons. Western blot showed significantly increase in pERK expression at 10 days post-CFA, which decreased after 10 days of KYNA treatment. Two days post-CFA, a significantly increase in pp38 expression was found, which decreased after 2 days of KYNA and KYNAA2 treatment. CONCLUSIONS: The CFA-induced inflammatory model for the TG activation provided a time-related expression of MAPK (pERK1/2, pp38) and NF-κB. It involves both the neuronal and glial activation, which points to possible neuron-glia interactions during this process. The administration of the endogenous NMDA-receptor antagonists, KYNA and its derivative KYNAA2, resulted in the inhibition of the induced signaling system of the TG, which further points the importance of the glutamate receptors in this mechanism.

Where does a migraine attack originate? In the brainstem.

Migraine is a common, paroxysmal, highly disabling primary headache disorder. The origin of migraine attacks is enigmatic. Numerous clinical and experimental results suggest that the activation of distinct brainstem nuclei is crucial in its pathogenesis, but the primary cause of this activation is not fully understood. We conclude that the initialization of a migraine attack can be explained as an altered function of the neuronal elements of the brainstem nuclei. In light of our findings and the literature data, we can assume that migraine is a subcortical disorder of a specific brainstem area.

Migraine is a neuronal disease.

Migraine is a common, paroxysmal, highly disabling primary headache disorder with a genetic background. The primary cause and the origin of migraine attacks are enigmatic. Numerous clinical and experimental results suggest that activation of the trigeminal system (TS) is crucial in its pathogenesis, but the primary cause of this activation is not fully understood. Since activation of the peripheral and central arms of the TS might be related to cortical spreading depression and to the activity of distinct brainstem nuclei (e.g. the periaqueductal grey), we conclude that migraine can be explained as an altered function of the neuronal elements of the TS, the brainstem, and the cortex, the centre of this process comprising activation of the TS. In light of our findings and the literature data, therefore, we can assume that migraine is mainly a neuronal disease.

Pattern of changes in salicylic acid-induced protein kinase (SIPK) gene expression and salicylic acid accumulation in wheat under cadmium exposure.

Salicylic acid-induced protein kinase (SIPK) is known as a 'master switch' for stress responses in plants. It can be induced by salicylic acid (SA) and several stress factors. The main aim of the present study was to reveal the relationship between SA accumulation and the gene expression level of SIPK during 50 and 250 µm Cd stress in wheat plants. Quantitative real-time PCR was used for determination of the gene expression level of SIPK. Salicylic acid content measurement was performed with an HPLC system equipped with a fluorescence detector. Cadmium treatment increased the endogenous SA level and expression level of SIPK in a concentration-dependent manner. Induction of SIPK expression preceded the accumulation of endogenous SA. Although SA treatment induced dramatic endogenous SA accumulation, its SIPK-inducing effect was moderate. In roots, higher induction of SIPK was observed than in leaves. The same tendency of SIPK expression was observed in both Cd- and SA-treated plants, as decisively the highest transcript level was detected after 30 min of treatment, but thereafter the expression decreased rapidly to control level or even below. The induction of SIPK was transient in all cases, and even a very high SA level in either the leaves or roots was not able to maintain the elevated expression level of this gene. The results suggest that SIPK has a role in initiating Cd stress response and the exogenous SA-induced signalling process.

Lymphocytic infiltrates in the spinal cord in amyotrophic lateral sclerosis.

OBJECTIVE: Immunohistochemical examination was undertaken to assess the presence of lymphocytes and lymphocyte subsets in the spinal cord in amyotrophic lateral sclerosis (ALS). DESIGN: Twenty-seven consecutive ALS autopsy cases and 11 consecutive disease--control autopsy cases were examined. Tissue sections were reacted with mouse monoclonal antibody to human leukocyte common antigen, or monoclonal antibody to human B-cell L26 antigen, and detected with immunoperoxidase techniques. Unfixed sections were reacted with antihuman Leu-3a and Leu-3b/CD4 or antihuman Leu-2a/CD8 mouse monoclonal antibody and then detected with peroxidase techniques. SETTING: Tertiary care hospital. CASES: Amyotrophic lateral sclerosis and non-ALS control autopsy specimens. MAIN OUTCOME MEASURE: Detection of lymphocytes by histological and immunohistochemical reactivity. RESULTS: Perivascular and intraparenchymal lymphocytic infiltrates were found in the spinal cord of 18 of 27 consecutive ALS autopsy cases. The lymphocytes possessed only T-cell markers; no B-cell markers could be demonstrated. T-helper cells were found in proximity to degenerating corticospinal tracts, while T-helper and T-suppressor/cytotoxic cells were demonstrated in ventral horns. Lymphocytes were present in the spinal cord of only one control specimen (multiple sclerosis) and in none of the remaining 10 control specimens. In ALS tissue, the lymphocytic infiltrates did not correlate with the rate of progression or stage of the disease or with the presence or absence of terminal infections. CONCLUSIONS: T-cell lymphocytes are present in the spinal cord of patients with ALS. T-helper cells are found in proximity to corticospinal tract degeneration, while T-helper and T-suppressor/cytotoxic cells are present in ventral horns. The role of these lymphocytes remains to be elucidated.

Cyclophosphamide alters the clinical and pathological expression of experimental autoimmune gray matter disease.

Guinea pigs inoculated with bovine spinal cord ventral horn homogenate develop a syndrome termed experimental autoimmune gray matter disease (EAGMD) characterized by extremity weakness, bulbar signs, and a loss of lower and upper motoneurons. To provide evidence for the role of autoimmune mechanisms, we have administered the immunosuppressant cyclophosphamide prior to and after gray matter immunization. Pretreatment with cyclophosphamide prevented the appearance of clinical signs of disease and decreased the loss of spinal cord motoneurons, the appearance of damaged motoneurons, and the antibody titer to motoneurons. Treatment 7 days after immunization attenuated the expression of disease. Treatment immediately after signs also improved the clinical and pathological findings. In all cyclophosphamide-treated animals there was less IgG within motoneurons and less inflammation. These results support the role for autoimmune mechanisms in motoneuron loss and degeneration in EAGMD.

Functional immunohistochemistry of neuropeptides and nitric oxide synthase in the nerve fibers of the supratentorial dura mater in an experimental migraine model.

The supratentorial cerebral dura of the albino rat is equipped with a rich sensory innervation both in the connective tissue and around blood vessels, which includes nociceptive axons and their terminals; these display intense calcitonin gene-related peptide (CGRP) immunoreactivity. Stereotactic electrical stimulation of the trigeminal (Gasserian) ganglion, regarded as an experimental migraine model, caused marked increase and disintegration of club-like perivascular CGRP-immunopositive nerve endings in the dura mater and induced an apparent increase in the lengths of CGRP-immunoreactive axons. Intravenous administration of sumatriptan or eletriptan, prior to electrical stimulation, prevented disintegration of perivascular terminals and induced accumulation of CGRP in terminal and preterminal portions of peripheral sensory axons. Consequently, immunopositive terminals and varicosities increased in size; accumulation of axoplasmic organelles resulted in the "hollow" appearence of numerous varicosities. Since triptans exert their anti-migraine effect by virtue of agonist action on 5-HT(1D/B) receptors, we suggest that these drugs prevent the release of CGRP from perivascular nerve terminals in the dura mater by an action at 5-HT(1D/B) receptors. Nitroglycerine (NitroPOHL), given subcutaneously to rats, induces increased beading of nitric oxide synthase (NOS)-immunoreactive nerve fibers in the supratentorial cerebral dura mater, and an apparent increase in the number of NOS-immunoreactive nerve fibers in the dural areas supplied by the anterior and middle meningeal arteries, and the sinus sagittalis superior. Structural alterations of nitroxidergic axons innervating blood vessels of the dura mater support the idea that nitric oxide (NO) is involved in the induction of headache, a well-known side effect of coronary dilator agents.

Neuronal messengers and peptide receptors in the human sphenopalatine and otic ganglia.

A majority of the parasympathetic nerve fibers to cranial structures derive from the sphenopalatine and otic ganglia. In particular, blood vessels are invested with a rich supply of dilator fibers of parasympathetic origin. In the present study, we have examined the occurrence of noncholinergic neuromessengers and neuropeptide receptors in the human sphenopalatine and otic ganglia. Vasoactive intestinal peptide (VIP)-immunoreactive (ir) nerve cell bodies occurred in high numbers in the sphenopalatine and otic ganglia. Likewise, high numbers of NOS- and PACAP-containing nerve cell bodies were seen in both ganglia. Autofluorescent lipofuscin, characteristic of adult human nervous tissue, was present within many nerve cell bodies in both ganglia. Receptor mRNA was studied with reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA from the sphenopalatine and otic ganglia was successfully extracted. By using appropriate sense and antisense primers, oligonucleotides were designed from the human sequences derived from GenBank, corresponding to human NPY Y1, CGRP1 and VIP1 receptors. In the sphenopalatine ganglion, we revealed the presence of mRNA for the human NPY Y1 and VIP1 receptors but not the CGRP1 receptor. The otic ganglion was found to react positively only for primers to mRNA for VIP1 but not for CGRP1 or NPY Y1 receptors.

5-HT(1B) and 5-HT(1D) receptors in the human trigeminal ganglion: co-localization with calcitonin gene-related peptide, substance P and nitric oxide synthase.

5-Hydroxytryptamine (5-HT) is implicated in migraine and agonist directed against 5-HT(1B) and 5-HT(1D) receptors are commonly used as effective therapies. The antimigraine mechanisms involve the inhibition of intracranial sensory neuropeptide release. In order to determine which 5-HT(1) receptor subtypes are involved we have by immunocytochemistry examined the distribution of 5-HT(1B) and 5-HT(1D) receptors in the human trigeminal ganglia, and addressed which of them colocalize with calcitonin gene-related peptide (CGRP), substance P (SP) or nitric oxide synthase (NOS). We detected that 5-HT(1D) receptor immunoreactivity (i.r.) was predominantly expressed in medium-sized cells (86% of positive cells, 30-60 microm). About 9% of the 5-HT(1D) receptor i.r. cells were large in size (> 60 microm) and 5% were small in size (< 30 microm). In a similar pattern, 5-HT(1B) receptor i.r. was mainly expressed in medium-sized cells (81% in 30-60 microm, 15% in > 60 microm and 4% in < 30 microm). Double immunostaining was used to determine whether the 5-HT(1B) or 5-HT(1D) receptor immunoreactive cells co-localized with either CGRP, SP or NOS. Thus, 89% of the CGRP i.r. cells expressed 5-HT(1D) receptor i.r. and 65% of the CGRP positive cells were 5-HT(1B) receptor positive. Most of the 5-HT(1D) (95%) and the 5-HT(1B) (94%) receptor i.r. cells showed SP immunostaining and 83% of 5-HT(1D) receptor and 86% of 5-HT(1B) receptor i.r. cells contained NOS. In conclusion, both 5-HT(1B) and 5-HT(1D) receptors are expressed in the human trigeminal ganglion and they are mainly localized in medium-sized cells and they seem to colocalize with CGRP, SP and NOS.

Characterisation of the effects of a non-peptide CGRP receptor antagonist in SK-N-MC cells and isolated human cerebral arteries.

The cerebral circulation is innervated by calcitonin gene-related peptide (CGRP) containing fibers originating in the trigeminal ganglion. During a migraine attack, there is a release of CGRP in conjunction with the head pain, and triptan administration abolishes both the CGRP release and the pain at the same time. In the search for a novel treatment of migraine, a non-peptide CGRP antagonist has long been sought. Here, we present data on a human cell line and human and guinea-pig isolated cranial arteries for such an antagonist, Compound 1 (4-(2-Oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidine-1-carboxylic acid [1-(3,5-dibromo-4-hydroxy-benzyl)-2-oxo-2-(4-phenyl-piperazin-1-yl)-ethyl]-amide). On SK-N-MC cell membranes, radiolabelled CGRP binding was displaced by both CGRP-(8-37) and Compound 1, yielding pK(i) values of 8.9 and 7.8, respectively. Functional studies with SK-N-MC cells showed that CGRP-induced cAMP production was antagonised by both CGRP-(8-37) and Compound 1 with pA(2) values of 7.8 and 7.7, respectively. Isolated human and guinea pig cerebral arteries were studied with a sensitive myograph technique. CGRP induced a concentration-dependent relaxation in human cerebral arteries which was antagonized by both CGRP-(8-37) and Compound 1 in a competitive manner. In guinea pig basilar arteries, CGRP-(8-37) antagonised the CGRP-induced relaxation while Compound 1 had a weak blocking effect. The clinical studies of non-peptide CGRP antagonists are awaited with great interest.

CGRP and adrenomedullin receptor populations in human cerebral arteries: in vitro pharmacological and molecular investigations in different artery sizes.

The aim of the present study was to determine functional and molecular characteristics of receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin in three different diameter groups of lenticulostriate arteries. Furthermore, the presence of perivascular neuronal sources of CGRP was evaluated in these arteries. In the functional studies, in vitro pharmacological experiments demonstrated that both CGRP and adrenomedullin induce alpha-CGRP-(8-37) sensitive vasodilation in artery segments of various diameters. The maximal amounts of vasodilation induced by CGRP and adrenomedullin were not different, whereas the potency of CGRP exceeded that of adrenomedullin by 2 orders of magnitude. Significant negative correlations between artery diameters and maximal responses were demonstrated for CGRP and adrenomedullin. In addition, the potency of both peptides tended to increase in decreasing artery diameter. In the molecular experiments, levels of mRNAs encoding CGRP receptors and receptor subunits were compared using reverse transcriptase polymerase chain reactions (RT-PCR). The larger the artery, the more mRNA encoding receptor activity-modifying proteins 1 and 2 (RAMP1 and RAMP2) was detected relative to the amount of mRNA encoding the calcitonin receptor-like receptor. By immunohistochemistry, perivascular CGRP containing nerve fibres were demonstrated in all the investigated artery sizes. In conclusion, both CGRP and adrenomedullin induced vasodilation via CGRP receptors in human lenticulostriate artery of various diameter. The artery responsiveness to the CGRP receptor agonists increased with smaller artery diameter, whereas the receptor-phenotype determining mRNA ratios tended to decrease. No evidence for CGRP and adrenomedullin receptor heterogeneity was present in lenticulostriate arteries of different diameters.

The human superior cervical ganglion: neuropeptides and peptide receptors.

Noradrenaline (NA)- and neuropeptide Y (NPY)-containing cell bodies were found to occur in high numbers (>75% of all cells were positive) in the human superior cervical ganglion and distributed homogeneously throughout the ganglion and showed colocalisation. A few cell bodies were VIP-immunoreactive (-ir) (less than 5%) but none of them showed NOS-, CGRP- or SP-ir. Receptor mRNA expression was studied with RT-PCR. Total RNA from the superior cervical ganglion was successfully extracted. By using appropriate sense and antisense oligonucleotides designed from the published human sequences, we could show the presence of mRNA for the human NPY Y1, NPY Y2 and VPAC1 receptors but not CGRP1 receptor mRNA.

Electrical stimulation of the Gasserian ganglion induces structural alterations of calcitonin gene-related peptide-immunoreactive perivascular sensory nerve terminals in the rat cerebral dura mater: a possible model of migraine headache.

Calcitonin gene-related peptide (CGRP)-positive sensory nerve fibers in the rat supratentorial dura mater are equipped with varicosities and club-like nerve terminals, often attached to the walls of blood vessels. Brief electrical stimulation of the Gasserian ganglion results in significant swelling and increased immunohistochemical staining of ipsilateral perivascular club-like terminals, while long-lasting electrical stimulation induces their disintegration or bursting, resulting in irregular, corroded outlines of terminals and en passant beads. Stimulation-induced morphological alterations of perivascular terminals may represent a structural basis of increased CGRP content in jugular blood which follows electrical stimulation of the Gasserian ganglion and accompanies migraine attacks.

Experimental immune-mediated motor neuron diseases: models for human ALS.

Amyotrophic lateral sclerosis is an idiopathic, ultimately fatal disease, clinically manifest as progressive weakness and spasticity, associated with the loss of motoneurons. Circumstantial evidence supports a role for autoimmune processes in the progression of this human disorder. Two immune-mediated animal models have been developed in our laboratory for motor neuron loss. Experimental autoimmune motor neuron disease is a lower motor syndrome induced in guinea pigs by the repeated injection of a purified bovine spinal motor neuron antigen. Affected animals demonstrate extremity weakness, associated with electromyographic and morphologic evidence of denervation, a loss of spinal cord motor neurons, high antibody titers against motor neurons, and localization of IgG immunoreactivity to the neuromuscular junction and motor neuron cytoplasm. Experimental autoimmune grey matter disease is a more acute and severe disorder involving both upper and lower motor neurons, induced in guinea pigs by inoculation of a bovine ventral spinal cord homogenate, in which scattered foci of denervation are observed in the motor cortex and ventral spinal cord. Similarities between these diseases and human ALS are reviewed.

Determination of binding capacity and adsorption enthalpy between Human Glutamate Receptor (GluR1) peptide fragments and kynurenic acid by surface plasmon resonance experiments.

The interaction between kynurenic acid (KYNA) and two peptide fragments (ca. 30 residues) of Human Glutamate Receptor 201-300 (GluR1) using surface plasmon resonance (SPR) spectroscopy was investigated. Because of the medical interest in the neuroscience, GluR1 is one of the important subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). AMPARs are ionotoropic glutamate receptors, which are mediating fast synaptic transmission and are crucial for plasticity in the brain. On the other hand, KYNA has been suggested to have neuroprotective activity and it has been considered for apply in therapy in certain neurobiological disorders. In this article the adsorption of the GluR1201-230 and GluR1231-259 peptides were studied on gold biosensor chip. The peptides were chemically bonded onto the gold surface via thiol group of L-cysteine resulted in the formation of peptide monolayer on the SPR chip surface. Because the GluR1231-259 peptide does not contain L-cysteine the Val256 was replaced by Cys256. The cross sectional area and the surface orientation of the studied peptides were determined by SPR and theoretical calculations (LOMETS) as well. The binding capability of KYNA on the peptide monolayer was studied in the concentration range of 0.1-5.0 mM using 150 mM NaCl ionic strength at pH 7.4 (±0.02) in phosphate buffer solutions. In order to determine the binding enthalpy the experiments were carried out between +10°C and +40°C. The heat of adsorption was calculated by using adsorption isotherms at different surface loading of KYNA on the SPR chip.

Distribution of vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, nitric oxide synthase, and their receptors in human and rat sphenopalatine ganglion.

Cranial parasympathetic outflow is mediated through the sphenopalatine ganglion (SPG). The present study was performed to examine the expression of the parasympathetic signaling transmitters and their receptors in human and rat SPG. Indirect immunofluorescence technique was used for the demonstration of vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), nitric oxide synthase (NOS), glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), VIP and PACAP common receptors (VPAC1, VPAC2), and PACAP receptor (PAC1). In addition, double labeling was carried out to reveal the co-localization of neurotransmitters. VIP-immunoreactive (-ir) neurons as well as fibers were frequently found in human SPG. Many, homogenously stained NOS-ir cells were found, but no positive fibers. In addition, PACAP-ir was observed in some of the neurons and in fibers. Co-localization was found between VIP and NOS. In rat VIP-, NOS-, and PACAP-ir were found in many neurons and fibers. Co-localization of PACAP and NOS was observed in neurons. PACAP and GS double staining revealed that the PACAP-ir was localized in/close to the cell membrane, but not in the satellite glial cells. PAC1 and VPAC1 immunoreactivity was found in the satellite glial cells of both human and rat. Western blot revealed protein expression of PAC1, VPAC1, and VPAC2 in rat SPG. The trigeminal-autonomic reflex may be active in migraine attacks. We hypothesized that VIP, PACAP, NOS, PAC1, VPAC1, and VPAC2 play a role in the activation of parasympathetic cranial outflow during migraine attacks.

Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion.

Calcitonin gene related peptide (CGRP) has a key role in migraine and recently CGRP receptor antagonists have demonstrated clinical efficacy in the treatment of migraine. However, it remains unclear where the CGRP receptors are located within the CGRP signaling pathway in the human trigeminal system and hence the potential antagonist sites of action remain unknown. Therefore we designed a study to evaluate the localization of CGRP and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein (RAMP) 1 in the human trigeminal ganglion using immunohistochemistry and compare with that of rat. Antibodies against purified CLR and RAMP1 proteins were produced and characterized for this study. Trigeminal ganglia were obtained at autopsy from adult subjects and sections from rat trigeminal ganglia were used to compare the immunostaining pattern. The number of cells expressing CGRP, CLR and RAMP1, respectively, were counted. In addition, the glial cells of trigeminal ganglion, particularly the satellite glial cell, were studied to understand a possible relation. We observed immunoreactivity for CGRP, CLR and RAMP1, in the human trigeminal ganglion: 49% of the neurons expressed CGRP, 37% CLR and 36% RAMP1. Co-localization of CGRP and the receptor components was rarely found. There were no CGRP immunoreactions in the glial cells; however some of the glial cells displayed CLR and RAMP1 immunoreactivity. Similar results were observed in rat trigeminal ganglia. We report that human and rat trigeminal neurons store CGRP, CLR and RAMP1; however, CGRP and CLR/RAMP1 do not co-localize regularly but are found in separate neurons. Glial cells also contain the CGRP receptor components but not CGRP. Our results indicate, for the first time, the possibility of CGRP signaling in the human trigeminal ganglion involving both neurons and satellite glial cells. This suggests a possible site of action for the novel CGRP receptor antagonists in migraine therapy.