Activation of PAR‐2 Elicits NO‐Dependent and CGRP‐Independent Dilation of the Dural Artery

(Headache 2010;50:1017‐1030) Objectives.— The goal of this study was to determine the vascular effects of protease‐activated receptor‐2 (PAR‐2) activation in the rat cranial vasculature. Background.— The role of PAR‐2 in pain and inflammatory conditions has been established but the information available on its effects and receptor distribution in the trigeminal vascular axis is limited. We studied the dilatory function and expression of PAR‐2 in the neuro‐vascular circuit, critical in migraine pathogenesis. We also investigated the interaction of PAR‐2 with calcitonin gene‐related peptide (CGRP) and dural mast cells. Methods.— We used an improved model of intravital microscopy on the closed cranial window in rats to study the vascular effects of PAR‐2 activating peptides (PAR‐2 APs; SLIGRL‐NH₂, 2‐Furoyl‐LIGRLO‐NH₂) in the dural vasculature. Measurement of immunoreactive CGRP in skull halves and in trigeminal nucleus caudalis was done by using an enzyme‐linked immunosorbent assay. We also analyzed the presence of PAR‐2 in different migraine relevant tissues by quantitative real‐time PCR and Western blot analysis. Results.— PAR‐2 APs and trypsin induced a dose‐dependent increase in dural artery diameter. The topical application of a nonspecific nitric oxide synthase (NOS) inhibitor, L‐N^G‐Nitroarginine methyl ester, attenuated SLIGRL‐NH₂ responses. Olcegepant, a CGRP receptor antagonist, did not a have significant effect on the SLIGRL‐NH₂ responses, though exogenous CGRP responses were completely blocked. There was no significant release of CGRP from skull halves incubated with SLIGRL‐NH₂ as compared with those incubated with the corresponding negative peptide. Chronic mast cell degranulation did not change the vascular effects of PAR‐2 APs. mRNA and protein expression of PAR‐2 were found throughout trigeminovasuclar axis. Conclusion.— PAR‐2 activation leads to vasodilation of dural arteries and these responses are partially mediated by nitric oxide. As PAR‐2 is present throughout trigeminovasuclar axis, it may have a role in migraine pathogenesis, independent of CGRP and mast cell mediated mechanism.     

Effect of dural inflammatory soup application on activation and sensitization markers in the caudal trigeminal nucleus of the rat and the modulatory effects of sumatriptan and kynurenic acid

BackgroundThe topical inflammatory soup can model the inflammation of the dura mater causing hypersensitivity and activation of the trigeminal system, a phenomenon present in migraineurs. Calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase are important in the sensitization process there.5-HT_1B/1D receptor agonists, triptans are used as a treatment of migraine. Kynurenic acid an NMDA antagonist can act on structures involved in trigeminal activation.AimWe investigated the effect of inflammatory soup induced dural inflammation on the calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase levels in the caudal trigeminal nucleus. We also tested whether pretreatment with a well-known antimigraine drug, such as sumatriptan and kynurenic acid, a compound with a different mechanism of action, can affect these changes and if their modulatory effects are comparable.Material and methodsAfter subcutaneous sumatriptan or intraperitoneal kynurenic acid the dura mater of adult male Sprague-Dawley rats (n = 72) was treated with inflammatory soup or its vehicle (synthetic interstitial fluid). Two and a half or four hours later perfusion was performed and the caudal trigeminal nucleus was removed for immunohistochemistry.Results and conclusionInflammatory soup increased calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase in the caudal trigeminal nucleus compared to placebo, which was attenuated by sumatriptan and kynurenic acid. This suggests the involvement of 5-HT_1B/1D and NMDA receptors in neurogenic inflammation development of the dura and thus in migraine attacks.     

Sex differences in behavior and expression of CGRP-related genes in a rodent model of chronic migraine

(Headache 2011;51:674‐692) Objective.— The objectives of this study were to develop a preclinical rodent model that produces migraine‐like behaviors based on International Headache Society diagnostic criteria, to determine whether sex differences are present, and to determine whether expression of calcitonin gene‐related peptide (CGRP) and the genes encoding its receptor in trigeminal ganglion or medulla correlates with those behaviors. Background.— Few animal studies of migraine have tested behaviors associated with migraine diagnostic criteria. In this study, changes in activity and in mechanical sensitivity of facial regions following application of inflammatory soup (IS) or vehicle (phosphate‐buffered saline [PBS]) to the dura were measured to model changes in routine activity and allodynia. CGRP, an important mediator of migraine pathogenesis, and the 3 components of its receptor, calcitonin‐like receptor (CLR), receptor activity‐modifying protein 1 (RAMP1), and receptor component protein (RCP) mRNAs were quantified in the trigeminal ganglion and medulla to identify baseline sex differences and changes associated with application of IS or PBS to the dura. Methods.— Male and female Sprague‐Dawley rats were implanted with a dural cannula. Groups of rats were treated with 10 or 20 µL volumes of IS or PBS. Baseline behavioral testing was conducted prior to surgery and again at 7 days postsurgery, and dural application of IS or PBS was performed repeatedly for a total of 8 applications. Locomotor activity was assessed using force plate actimetry during and following application to provide information on distance traveled, bouts of low mobility, spatial confinement, and focused energy. Periorbital and perimasseter sensory testing was performed 20 minutes post‐application to measure allodynia. The rats were sacrificed 30 minutes following the final dural treatment, tissue was dissected and total RNAs were isolated from ipsilateral trigeminal ganglia and ipsilateral medulla. Quantitative real‐time polymerase chain reactions were used to measure the expression of amplified constructs using gene‐specific primers for CGRP, RAMP1, CLR, and RCP. Results.— Both males and females showed behavioral effects of IS application, but there were pronounced sex differences. Females showed effects at the lower dose, and activity changes were present for a longer duration, but males required fewer applications of IS to exhibit behavioral changes. Females showed increased withdrawal responses for periorbital and perimasseter mechanical testing (10 µL IS groups), and males showed increased perimasseter withdrawal responses (20 µL IS group). In the trigeminal ganglion, there were no baseline sex differences in CGRP‐encoding mRNA, but females had lower baseline expression of RAMP1, CLR, and RCP‐encoding mRNAs. In the medulla, females had higher baseline levels of CGRP‐encoding mRNAs and lower baseline levels of RAMP1, CLR, and RCP‐encoding mRNAs than males. Both IS and PBS increased expression of mRNAs encoding CGRP, RAMP1, RCP, and CLR in the trigeminal ganglion in males, but in females, only CLR and RCP were increased. In the medulla both IS and PBS increased expression of CGRP, CLR in males and CLR and RCP in females. Thus, expression of CGRP‐related genes did not mirror the behavioral differences between IS and PBS groups. Instead, CGRP‐related genes were upregulated by both IS and PBS applications. Conclusions.— This study demonstrates significant changes in locomotor activity and facial allodynia associated with application of IS to the dura as well as significant sex differences, demonstrating that International Headache Society diagnostic criteria can be used to design a rodent behavioral model of migraine. In addition, there were prominent baseline sex differences in expression of CGRP and its receptor in both the trigeminal ganglion and medulla, but the majority of changes in expression of CGRP and its receptor were present in both the IS and PBS treated rats. This suggests that the CGRP pathway responds to changes in intracranial pressure or meningeal stretch, while migraine‐like behaviors occur after meningeal inflammation.     

Oestrogen-modulated increase of calmodulin-dependent protein kinase II (CamKII) in rat spinal trigeminal nucleus after systemic nitroglycerin

Migraine can be triggered by systemic administration of the nitric oxide (NO) donor nitroglycerin (NTG) and by abrupt falls in plasma oestradiol. Calmodulin-dependent protein kinase II (CamKII) present in superficial dorsal horns is thought to play a role in sensitization of central nociceptors, a phenomen present in migraineurs. We therefore examined in rats the expression of CamKII in the caudal trigeminal nucleus (TNC) after subcutaneous NTG (10 mg/kg) and its modulation by oestrogen. In male rats and in ovariectomized females, after 4 h NTG increased significantly CamKII expression in the superficial layers of TNC, but not in the upper thoracic spinal cord. NTG had no effect on CamKII expression in oestradiol-treated ovariectomized animals. Thus NTG, i.e. NO, selectively enhances CamKII in the rat TNC and oestradiol blocks this effect. These data may help to understand the mechanisms by which NO triggers migraine attacks and oestrogens influence migraine severity.     

DHE Repression of ATP‐Mediated Sensitization of Trigeminal Ganglion Neurons

Objective.— To investigate the mechanism by which adenosine triphosphate (ATP) causes sensitization of trigeminal neurons and how dihydroergotamine (DHE) represses this modulatory effect. Background.— Dihydroergotamine is an effective treatment of migraine. The cellular mechanisms of action of DHE in treating migraine attacks remain unclear. Methods.— In this study, neonatal rat trigeminal ganglia cultures were used to investigate effects of ATP, alpha, beta‐methyl ATP (α,β‐meATP), and DHE on intracellular calcium levels and calcitonin gene‐related peptide (CGRP) secretion. Results.— Pretreatment with ATP or α,β‐meATP caused sensitization of neurons, via P2X ₃ receptors, such that a subthreshold amount of potassium chloride (KCl) significantly increased intracellular calcium levels and CGRP secretion. Pretreatment with DHE repressed increases in calcium and CGRP secretion in response to ATP‐KCl or α,β‐meATP‐KCl treatment. Importantly, these inhibitory effects of DHE were blocked with an α ₂ ‐adrenoceptor antagonist and unaffected by a 5HT _1B/D receptor antagonist. DHE also decreased neuronal membrane expression of the P2X ₃ receptor. Conclusions.— Our findings provide evidence for a novel mechanism of action for DHE that involves blocking ATP‐mediated sensitization of trigeminal neurons, repressing stimulated CGRP release, and decreasing P2X ₃ membrane expression via activation of α ₂ ‐adrenoceptors.     

Selective Inhibition of 5‐HT7 Receptor Reduces CGRP Release in an Experimental Model for Migraine

Objective.— To investigate the role of 5‐HT₇ receptors on the release of calcitonin gene‐related peptide (CGRP) in an animal model of migraine. Background.— Calcitonin gene‐related peptide has been identified as a key neuropeptide in the pathophysiology of migraine. It is elevated in the external jugular vein during migraine attacks in humans and after stimulation of the trigeminal ganglion in animal models of migraine. This can be treated with the 5‐HT_1B/1D receptor agonist sumatriptan concomitant with headache relief. Nevertheless, triptans, the most effective agents for the treatment of acute migraine attacks, are not effective in more than 1/3 of migraineurs and less than 50% of migraineurs achieve complete pain freedom. This indicates other serotonin receptors may be involved in the pathophysiology of migraine. Increasing evidence has shown that 5‐HT₇ receptors may be involved in migraine pathogenesis. However, direct evidence for the role of 5‐HT₇ receptors in migraine is still lacking. Methods.— Unilateral electrical stimulation of the trigeminal ganglion (TGES) was performed in anesthetized male Sprague‐Dawley rats. Animals were pretreated with sumatriptan (300 µg/kg, i.v.), selective 5‐HT₇ receptor antagonist SB269970 (5, 10 mg/kg, s.c.), potential 5‐HT₇ receptor agonist AS19 (5, 10 mg/kg, s.c.) or co‐administration of SB269970 and AS19 (10 mg/kg, s.c.). Serum CGRP concentrations in the ipsilateral jugular vein were determined before and at 2 and 5 minutes after the start of TGES. Results.— Our results showed that sumatriptan almost completely inhibited the release of CGRP evoked by TGES. Pre‐administration of SB269970 (5, 10 mg/kg) caused a significant decrease in serum CGRP concentrations at 2 and 5 minutes following the onset of TGES, with a less inhibitory effect compared with sumatriptan. AS19 had no significant effect on CGRP release, while the SB269970‐induced inhibitory effect was reversed by AS19. Conclusions.— Selective inhibition of 5‐HT₇ receptors partly reduced CGRP release evoked by TGES. These findings suggest that 5‐HT₇ receptors may play a role in the pathophysiology of migraine. (Headache 2010;50:579‐587)     

Involvement of Pro‐Nociceptive 5‐HT2A Receptor in the Pathogenesis of Medication‐Overuse Headache

(Headache 2010;50:185‐197) Objectives.— To determine the involvement of 5‐HT_2A (5‐HT_2A) receptor in the process of trigeminal plasticity induced by chronic analgesic exposure and in the process of inflammatory‐induced thermal hyperalgesia. Background.— Derangement in 5‐HT_2A serotonin receptor has been reported to implicate in pathogenesis of medication‐overuse headache. No clear explanation concerning the precise roles of these receptors in the process. Methods.— Wistar rats were daily administered with paracetamol (200 mg/kg) for 30 days. On the next day, ketanserin, a 5‐HT_2A antagonist, or saline was given prior to cortical spreading depression (CSD) induction. Electrocorticogram, cortical blood flow, Fos and 5‐HT_2A‐immunoreactivity in cortex and trigeminal pathway were studied. In the other experiment, complete Freund's adjuvant was injected into the rat hind paw to induce tissue inflammation. Three days later, ketanserin was given and noxious heat was applied to both inflamed and noninflamed paws. The response between 2 sides was compared by measuring paw withdrawal latency. Results.— Chronic paracetamol exposure led to an increase in CSD frequency and CSD‐evoked Fos expression in cerebral cortex indicating the increase in neuronal excitability. Prolonged medication exposure also facilitated trigeminal nociception as evident by an increase in CSD‐evoked Fos expression in trigeminal nucleus caudalis. The expression of 5‐HT_2A receptor in cerebral cortex and trigeminal ganglia was enhanced by chronic paracetamol administration. Pretreatment with ketanserin significantly attenuated these effects. The second experiment showed that ketanserin was able to lengthen the paw withdrawal latency in the inflamed side but did not alter nociceptive response in the noninflamed side. Conclusion.— These findings suggest that up‐regulation of pro‐nociceptive 5‐HT_2A receptor is an important step in the process of cortical hyper‐excitation and nociceptive facilitation induced by chronic analgesic exposure.     

Serotonin Depletion Increases Nociception‐Evoked Trigeminal NMDA Receptor Phosphorylation

Objective.— To investigate the effect of serotonin depletion on phosphorylation and expression of NR1 subunit of N‐methyl‐D‐aspartate (NMDA) receptor in trigeminal nucleus caudalis (TNC), and on trigeminal nociception evoked by dural inflammation. Background.— Migraine is associated with low serotonin condition and an increased neuronal excitability. NMDA receptor is implicated in central plasticity change that leads to neural sensitization. Alteration in NMDA receptor phosphorylation or expression in TNC may be responsible for increased trigeminal nociception in serotonin‐depleted state. Methods.— Adult male Wistar rats were separated into normal and low serotonin groups. Serotonin was depleted by intraperitoneal injection with para‐chlorophenylalanine 3 days before the experiment. Trigeminal nociception was induced by applying inflammatory soup on exposed dura. Two hours after induction, phosphorylated NR1, NR1, and Fos expressions were studied in TNC by immunohistochemistry. Results.— Dural application of inflammatory soup led to the activation of trigeminal nociceptive system as well as the phosphorylation of NR1, which were further enhanced in the low serotonin condition. There was a strong relationship between NR1 phosphorylation and trigeminal nociception. However, neither meningeal inflammation nor serotonin depletion altered NR1 expression. Conclusions.— Low serotonin condition facilitates dural inflammation‐induced NR1 phosphorylation and trigeminal nociception. It is suggested that the mechanism of nociceptive facilitation in serotonin‐depleted state may involve the increase in NR1 phosphorylation rather than the upregulation of NR1 subunit of NMDA receptor.     

Involvement of AMPA Receptors in CSD‐Induced Impairment of LTP in the Hippocampus

Objective.— To investigate the alteration of hippocampal long‐term plasticity and basal synaptic transmission induced by repetitive cortical spreading depressions (CSDs). Background.— There is a relationship between migraine aura and amnesia attack. CSD, a state underlying migraine attacks, may be responsible for hippocampus‐related symptoms. However, the precise role of CSD on hippocampal activity has not been investigated. Methods.— Male Wistar rats were divided into CSD and control groups. Repetitive CSDs were induced in vivo by topical application of solid KCl. Forty‐five minutes later, the ipsilateral hippocampus was removed, and hippocampal slices were prepared for a series of electrophysiological studies. Results.— Repetitive CSDs led to a decrease in the magnitude of long‐term potentiation in the hippocampus. CSD also reduced hippocampal synaptic efficacy, as shown by a reduction in post‐synaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor responses. In contrast, the post‐synaptic N‐methyl‐d ‐aspartate receptor responses remained unchanged. In addition, there were no changes in paired‐pulse profiles between the groups, indicating that CSD did not induce any presynaptic alterations. Conclusion.— These findings suggest that a reduction of post‐synaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor responses is the mechanism responsible for impaired hippocampal long‐term potentiation induced by CSD.     

Vasodilator and antihypertensive effects of a novel N‐acylhydrazone derivative mediated by the inhibition of L‐type Ca2+ channels

New bioactive N‐acylhydrazone derivatives synthesized from safrole previously have been found to promote intense vasodilation and antihypertensive activity. In this study, we describe the synthesis and the cardiovascular effects of the new N‐acylhydrazone derivative (E)‐N‐methyl‐N′‐(thiophen‐3‐ylmethylene)benzo[d][1,3]dioxole‐5‐carbohydrazide (LASSBio‐1289). Thoracic aorta and left papillary muscles from Wistar–Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were prepared for isometric tension recording. LASSBio‐1289 promoted relaxation of endothelium‐intact and denuded aortic rings with respective pIC₅₀ (?log IC₅₀) values of 5.07 ± 0.09 and 4.26 ± 0.09 (P < 0.001) for WKY rats and 5.43 ± 0.05 and 5.58 ± 0.07 (P > 0.05) for SHR. The vasodilator activity of LASSBio‐1289 was increased in the KCl‐contracted aorta. LASSBio‐1289 attenuated the contracture elicited by Ca²⁺ in depolarized aorta from both WKY rats and SHR. In endothelium‐intact aorta from WKY rats, LASSBio‐1289‐induced relaxation was unchanged after incubation with propranolol, ZM 241385, atropine, diphenhydramine, and HOE140, but was significantly reduced by L‐NAME and ODQ. LASSBio‐1289 decreased papillary muscles contractility only at concentrations above 200 μm . Acute intravenous injection of LASSBio‐1289 (3 mg/kg) produced a significant hypotensive response in SHR but not in WKY rats, suggesting its antihypertensive profile. The antihypertensive effect was also observed in SHR during 14 days of intraperitoneal and oral administration. In conclusion, our data demonstrated that LASSBio‐1289 induces both endothelium‐independent vasorelaxation involving the inhibition of Ca²⁺ influx through L‐type Ca²⁺ channels in aorta from WKY rats and SHR, and endothelium‐dependent relaxation mediated by the NO/cyclic GMP pathway in WKY rats.     

Two Mechanisms Involved in Trigeminal CGRP Release: Implications for Migraine Treatment

Objective/Background.— The goal of this study was to better understand the cellular mechanisms involved in proton stimulation of calcitonin gene‐related peptide (CGRP) secretion from cultured trigeminal neurons by investigating the effects of 2 antimigraine therapies, onabotulinumtoxinA and rizatriptan. Stimulated CGRP release from peripheral and central terminating processes of trigeminal ganglia neurons is implicated in migraine pathology by promoting inflammation and nociception. Based on models of migraine pathology, several inflammatory molecules including protons are thought to facilitate sensitization and activation of trigeminal nociceptive neurons and stimulate CGRP secretion. Despite the reported efficacy of triptans and onabotulinumtoxinA to treat acute and chronic migraine, respectively, a substantial number of migraineurs do not get adequate relief with these therapies. A possible explanation is that triptans and onabotulinumtoxinA are not able to block proton‐mediated CGRP secretion. Methods.— CGRP secretion from cultured primary trigeminal ganglia neurons was quantitated by radioimmunoassay while intracellular calcium and sodium levels were measured in neurons via live cell imaging using Fura‐2 AM and SBFI AM, respectively. The expression of acid‐sensing ion channel 3 (ASIC3) was determined by immunocytochemistry and Western blot analysis. In addition, the involvement of ASICs in mediating proton stimulation of CGRP was investigated using the potent and selective ASIC3 inhibitor APETx2. Results.— While KCl caused a significant increase in CGRP secretion that was significantly repressed by treatment with ethylene glycol tetraacetic acid (EGTA), onabotulinumtoxinA, and rizatriptan, the stimulatory effect of protons (pH 5.5) was not suppressed by EGTA, onabotulinumtoxinA, or rizatriptan. In addition, while KCl caused a transient increase in intracellular calcium levels that was blocked by EGTA, no appreciable change in calcium levels was observed with proton treatment. However, protons did significantly increase the intracellular level of sodium ions. Under our culture conditions, ASIC3 was shown to be expressed in most trigeminal ganglion neurons. Importantly, proton stimulation of CGRP secretion was repressed by pretreatment with the ASIC3 inhibitor APETx2, but not the transient receptor potential vanilloid‐1 antagonist capsazepine. Conclusions.— Our findings provide evidence that proton regulated release of CGRP from trigeminal neurons utilizes a different mechanism than the calcium and synaptosomal‐associated protein 25‐dependent pathways that are inhibited by the antimigraine therapies, rizatriptan and onabotulinumtoxinA.     

Platelet apoptosis by cold‐induced glycoprotein Ibα clustering

Summary. Background: Cold‐storage of platelets followed by rewarming induces changes in Glycoprotein (GP) Ibα‐distribution indicative of receptor clustering and initiates thromboxane A₂‐formation. GPIbα is associated with 14‐3‐3 proteins, which contribute to GPIbα‐signaling and in nucleated cells take part in apoptosis regulation. Objectives and methods: We investigated whether GPIbα‐clustering induces platelet apoptosis through 14‐3‐3 proteins during cold (4 h 0 °C)‐rewarming (1 h 37 °C). Results: During cold‐rewarming, 14‐3‐3 proteins associate with GPIbα and dissociate from Bad inducing Bad‐dephosphorylation and activation. This initiates pro‐apoptosis changes in Bax/Bcl‐x_L and Bax‐translocation to the mitochondria, inducing cytochrome c release. The result is activation of caspase‐9, which triggers phosphatidylserine exposure and platelet phagocytosis by macrophages. Responses are prevented by N‐acetyl‐d ‐glucosamine (GN), which blocks GPIbα‐clustering, and by O‐sialoglycoprotein endopeptidase, which removes extracellular GPIbα. Conclusions: Cold‐rewarming triggers apoptosis through a GN‐sensitive GPIbα‐change indicative of receptor clustering. Attempts to improve platelet transfusion by cold‐storage should focus on prevention of the GPIbα‐change.     

Calcitonin gene-related peptide does not cause migraine attacks in patients with familial hemiplegic migraine

(Headache 2011;51:544‐553) Background.— Calcitonin gene‐related peptide (CGRP) is a key molecule in migraine pathogenesis. Intravenous CGRP triggers migraine‐like attacks in patients with migraine with aura and without aura. In contrast, patients with familial hemiplegic migraine (FHM) with known mutations did not report more migraine‐like attacks compared to controls. Whether CGRP triggers migraine‐like attacks in FHM patients without known mutations is unknown. Objective.— In the present study we therefore examined the migraine‐inducing effect of CGRP in FHM patients without known mutations and healthy controls. Methods and design.— Eleven patients suffering from FHM without known mutations and 11 controls received an intravenous infusion of 1.5 µg/minute CGRP over 20 minutes. The study design was a balanced and controlled provocation study. Headache and other migraine symptoms were scored for 1 hour and self‐recorded hourly thereafter until 13‐hour postinfusion. Results.— We found no difference in the incidence of migraine‐like attacks between the 2 groups, with 9% (1 of 11) of patients and 0% (0 of 10) of controls reporting migraine‐like headache (P = 1.00). CGRP infusion did not induce aura symptoms in any of the participants. There was no difference in the incidence of CGRP‐induced delayed headaches between the groups (P = .18). Conclusion.— In contrast to patients suffering from migraine with aura and without aura, CGRP infusion did not induce more migraine‐like attacks in FHM patients without known mutations compared to controls. It seems that the majority of FHM patients with and without known mutation display no sensitivity to CGRP signaling compared to common types of migraine.     

Increase of capsaicin-induced trigeminal Fos-like immunoreactivity by 5-HT(7) receptors

Objective.— To explore whether pharmacological stimulation of the 5‐hydroxytryptamine₇ (5‐HT₇) receptor modulates Fos‐like immunoreactivity in the trigeminal nucleus caudalis of rats. Background.— The serotonin 5‐HT₇ receptor was proposed to be involved in migraine pathogenesis and evidence suggests it plays a role in peripheral nociception and hyperalgesia through an action on sensory afferent neurons. Methods.— The potential activating or sensitizing role of 5‐HT₇ receptors on trigeminal sensory neurons, as visualized by Fos‐like immunoreactivity in the superficial layers of the trigeminal nucleus caudalis in rats, was investigated using the 5‐HT₇ receptor agonist, LP‐211, in the absence and the presence of intracisternal capsaicin, respectively. The agonist effect was characterized with the 5‐HT₇ receptor antagonist, SB‐656104. Male Wistar rats received a subcutaneous injection of LP‐211, SB‐656104, and SB‐656104 + LP‐211. They were then anesthetized and prepared to receive an intracisternal injection of capsaicin or its vehicle. Animals were perfused and brains removed; sections of the brain stem from the area postrema to the CI level were obtained and processed for Fos immunohistochemistry. Results.— Capsaicin but not its vehicle induced Fos‐like immunoreactivity within laminae I and II of trigeminal nucleus caudalis. Pretreatment with LP‐211 had no effect on Fos‐like immunoreactivity but strongly increased the response produced by capsaicin; this effect was abolished by SB‐656104. Interestingly, capsaicin‐induced Fos‐like immunoreactivity was abolished by SB‐656104 pretreatment thus suggesting involvement of endogenous 5‐HT. Conclusions.— Data suggest that 5‐HT₇ receptors increase activation of meningeal trigeminovascular afferents and/or transmission of nociceptive information in the brain stem. This mechanism could be relevant in migraine and its prophylactic treatment.     

Associations of IgG N‐linked oligosaccharide chains and proteases in sera of prostate cancer patients with and without α2‐macroglobulin deficiency

We previously reported on a number of cases of metastatic prostate cancer (PCa) in which serum α2‐macroglobulin (α2M) levels were markedly decreased to less than 20 mg/dl (α2M deficiency). All PCa patients with α2M deficiency had multiple bone metastases. Proteases in ten PCa patients with and without α2M deficiency were studied and compared against ten healthy controls in order to elucidate the relationships between changes in sugar chain structure and neoplasia. We assessed the relationship between ratios of Fr4 to Fr1 and Fr2 (Fr4/Fr1+Fr2 ratios) of oligosaccharide chains, and ratios of free prostate‐specific antigen (PSA) to total PSA (F/T ratios), and serum levels of matrix‐metalloproteinase‐2 (MMP‐2) in PCa progression. Measurement of serum α2M concentration was performed by laser nephelometry. Serum PSA and MMP‐2 levels were determined by enzyme immunoassay and free PSA by radioimmunoassay. N‐linked oligosaccharides of human serum immunoglobulin G were analyzed using fluorophore‐associated carbohydrate electrophoresis. In those PCa patients with α2M deficiency: (a) serum α2M and F/T ratios were lower (P<0.05) and (b) Fr4/Fr1+Fr2 ratios and serum MMP‐2 levels were higher when compared with those PCa patients without α2M deficiency. There was a significant correlation between Fr4/Fr1+Fr2 ratios and F/T ratios or serum MMP‐2 levels in PCa with α2M deficiency (P<0.05). Therefore, these markers may serve as an auxiliary serum tumor marker for monitoring of the bone metastases or progression of disease in PCa. J. Clin. Lab. Anal. 23:125–131, 2009. © 2009 Wiley‐Liss, Inc.     

Role of K ATP channels in cephalic vasodilatation induced by calcitonin gene-related peptide, nitric oxide, and transcranial electrical stimulation in the rat

Objective.— The objective of this study was to explore the role of K_ATP channels in vasodilatation induced by calcitonin gene‐related peptide (CGRP), nitric oxide (NO), and transcranial electrical stimulation (TES) in intracranial arteries of rat. Background.— Dilatation of cerebral and dural arteries causes a throbbing, migraine‐like pain. Both CGRP and NO are potent vasodilators that can induce migraine. Their antagonists are effective in the treatment of migraine attacks. K_ATP channel openers cause headache in the majority of healthy subjects suggesting a role for K_ATP channels in migraine pathogenesis. We hypothesized that vasodilatation induced by CGRP and the NO donor glyceryltrinitrate (GTN) is mediated via K_ATP channels. Methods.— We examined the effects of the K_ATP channel inhibitor glibenclamide on dural and pial vasodilatation induced by CGRP, NO, and endogenously released CGRP by TES. A rat genuine closed cranial window model was used for in vivo studies and myograph baths for studying the effect in vitro. In the closed cranial window model the diameter of dural vessels was measured directly in anesthetized animals to investigate the vascular effects of infused CGRP, NO, and endogenous CGRP after electrical stimulation. Also diameter changes of pial arteries, mean arterial blood pressure and local cerebral blood flow by Laser Doppler flowmetry (LCBF_Flux) were measured. Results.— CGRP, NO, and TES caused dilatation of the 2 arteries in vivo and in vitro. In anesthetized rats glibenclamide significantly attenuated CGRP induced dural and TES induced dural/pial artery dilatation (P = .001; P = .001; P = .005), but had no effect on dural/pial vasodilatation induced by GTN. In vitro glibenclamide failed to significantly inhibit CGRP‐ and GTN‐induced vasodilatation. Conclusions.— These results show that a K_ATP channel blocker in vivo but not in vitro inhibits CGRP, but not GTN‐induced dilatation of dural and pial arteries, a mechanism thought to be important in migraine.     

Calcitonin gene-related peptide and size of the atrial septal defect in new-onset migraine after transcatheter closure: results of a preliminary study

Background.— New‐onset migraine headache attacks (MHAs) can occur after atrial septal device implantation in patients without previous migraine. Plasma calcitonin gene‐related peptide (CGRP), which plays a crucial role in migraine pathophysiology, has shown to be released from specific cardiac tissues. Methods and Results.— We prospectively collected patients before and after closure and measured plasma CGRP levels using enzyme‐linked immunosorbent assay. Forty atrial septal defect (ASD) patients who had no migraine previously were enrolled. Four (23.5%) of the 17 consecutive patients whose CGRP levels were checked before ASD closure had new‐onset MHAs. The patients with MHAs had bigger ASD size (20 ± 0.9 vs 16 ± 1 mm, P = .009) and lower CGRP levels before closure (21.1 ± 3.9 vs 90.1 ± 27.1 pg/mL, P = .042) than those without. Among the 5 patients with blood samplings both during and between attacks, a paired comparison revealed a significantly increased level during attack (257.2 ± 45.5 vs 45.6 ± 25.5 pg/mL, P = .03). Conclusion.— Bigger ASD size and lower plasma CGRP levels before closure can be a potential predictor of new‐onset MHAs. Furthermore, a significant increase of CGRP levels during migraine attack implies that the occurrences of new‐onset MHAs after ASD closure correlate with the release of CGRP. This suggests CGRP sensitization from a lower baseline may be involved in the occurrence of new‐onset MHAs after ASD closure.     

Distribution of Artemin and GFRα3 Labeled Nerve Fibers in the Dura Mater of Rat

(Headache 2010;50:442‐450) Objective.— We examined the distribution of artemin and its receptor, glial cell line‐derived neurotrophic factor family receptor α3 (GFRα3), in the dura mater of rats. Background.— Artemin, a member of the glial cell line‐derived neurotrophic factor family, is a vasculature‐derived growth factor shown to regulate migration of sympathetic neuroblasts and targeting of sympathetic innervation. The artemin receptor, GFRα3, is present in both sympathetic efferents and a subpopulation of nociceptive afferents. Recent evidence has shown that artemin may contribute to inflammatory hyperalgesia. The extent to which artemin is present in the dural vasculature and its relationship to GFRα3 containing fibers have yet to be investigated. Methods.— We used retrograde labeling, double and triple labeling with immunohistochemistry on the dura mater and trigeminal ganglia of female Sprague‐Dawley rats. Results.— Artemin‐like immunoreactivity (‐LI) was detected in the smooth muscle of dural vasculature. GFRα3‐LI was present in nerve fibers that closely associated with tyrosine hydroxylase or calcitonin gene‐related peptide (CGRP). CGRP‐LI and transient receptor potential ion channel 1 (TRPV1)‐LI were present in all GFRα3‐positive dural afferents, which constituted 22% of the total population of dural afferents. Conclusions.— These anatomical results support the hypothesis that artemin contributes to dural afferent activity, and possibly migraine pain, through modulation of both primary afferent and sympathetic systems.     

Serotonin depletion leads to cortical hyperexcitability and trigeminal nociceptive facilitation via the nitric oxide pathway

(Headache 2011;51:1152‐1160) Objective.— To investigate the role of nitric oxide (NO) in the development of cortical hyperexcitability and trigeminal nociceptive facilitation induced by serotonin (5‐HT) depletion. Background.— Nitric oxide and 5‐HT are important in the pathogenesis of primary headaches. An increase in cortical excitability and trigeminal nociception has been demonstrated in animals with low 5‐HT levels. Although the mechanism underlying this increase is unclear, an alteration of the NO system is one possible explanation. Methods.— Male Wistar rats were divided into control and 5‐HT‐depleted groups. 5‐HT was depleted by i.p. injection of parachlorophenylalanine (100 mg/kg). Three days after injection, a microelectrode was inserted into the cerebral cortex for electrocorticograph recording and waves of cortical spreading depression (CSD) were triggered with KCl application. N‐nitro‐L‐arginine methyl ester (L‐NAME; 10 mg/kg by i.v. injection) or saline was given after the second CSD wave. Following the experiment, the cerebral cortex and brain stem were removed for anti‐neuronal nitric oxide synthase (nNOS) and anti‐Fos immunohistochemistry. Results.— Relative to the control group, the 5‐HT‐depleted group exhibited a higher frequency of CSD waves, more nNOS‐immunoreactive cells in both the cerebral cortex and brainstem and more Fos‐immunoreactive cells in the trigeminal nucleus caudalis (TNC). In the control group, L‐NAME application led to fewer nNOS‐immunoreactive cells in the cerebral cortex and TNC, and fewer Fos‐immunoreactive cells in the TNC; however, L‐NAME was without effect on the CSD pattern. By contrast, in addition to decreased nNOS and Fos expression, L‐NAME significantly reduced the frequency of CSD events in the 5‐HT‐depleted group. Conclusions.— Inhibition of NO production can counter both the cortical hyperexcitability and facilitation of trigeminal nociception that develop in the depleted 5‐HT state. Therefore, NO is likely involved in the increase in both CSD events and CSD‐evoked trigeminal nociception under decreased 5‐HT conditions.