Protein kinase C-inhibition reduces critical weight loss and improves functional outcome after experimental subarachnoid haemorrhage.

OBJECTIVES: Subarachnoid haemorrhage (SAH) carries a high burden of morbidity and mortality. One in three patients develop vasospasm, which is associated with Delayed Cerebral Ischemia. The pathophysiology includes vasoconstrictor receptor upregulation in cerebral arteries. The protein kinase C - inhibitor RO-31-7549 reduces the expression of several vasoconstrictor receptors and normalizes cerebral blood flow in experimental SAH but functional and behavioural effects are unknown. This study was undertaken to analyse functional outcomes up to 14 days after experimental SAH. MATERIALS AND METHODS: 54 male rats were randomised to experimental SAH or sham, using the pre-chiasmatic, single injection model, and subsequent treatment or vehicle. 42 remained for final analysis. The animals were euthanized on day 14 or when reaching a humane endpoint. The primary endpoint was overall survival, defined as either spontaneous mortality or when reaching a predefined humane endpoint. The secondary outcomes were differences in the rotating pole test, weight, open field test, novel object recognition and qPCR of selected inflammatory markers. RESULTS: In the vehicle group 6/15 rats reached the humane endpoint of >20 % weight loss compared to 1/14 in the treatment group. This resulted in a significant reduced risk of early euthanasia due to >20 % weight loss of HR 0.15 (0.03-0.66, p = 0.04). Furthermore, the treatment group did significantly better on the rotating pole test, RR 0.64 (0.47-0.91, p = 0.02). CONCLUSION: RO-31-7549 improved outcomes in terms >20 % weight loss and rotating pole performance after experimental SAH and could be investigated.

Pharmacological Nature of the Purinergic P2Y Receptor Subtypes That Participate in the Blood Pressure Changes Produced by ADPßS in Rats.

Purine nucleosides (adenosine) and nucleotides such as adenosine mono/di/triphosphate (AMP/ADP/ATP) may produce complex cardiovascular responses. For example, adenosine-5'-(ß-thio)-diphosphate (ADPßS; a stable synthetic analogue of ADP) can induce vasodilatation/vasodepressor responses by endothelium-dependent and independent mechanisms involving purinergic P2Y receptors; however, the specific subtypes participating in these responses remain unknown. Therefore, this study investigated the receptor subtypes mediating the blood pressure changes induced by intravenous bolus of ADPßS in male Wistar rats in the absence and presence of central mechanisms with the antagonists MRS2500 (P2Y1), PSB0739 (P2Y12), and MRS2211 (P2Y13). For this purpose, 120 rats were divided into 60 anaesthetised rats and 60 pithed rats, and further subdivided into four groups (n = 30 each), namely: (a) anaesthetised rats, (b) anaesthetised rats with bilateral vagotomy, (c) pithed rats, and (d) pithed rats continuously infused (intravenously) with methoxamine (an α1-adrenergic agonist that restores systemic vascular tone). We observed, in all four groups, that the immediate decreases in diastolic blood pressure produced by ADPßS were exclusively mediated by peripheral activation of P2Y1 receptors. Nevertheless, the subsequent increases in systolic blood pressure elicited by ADPßS in pithed rats infused with methoxamine probably involved peripheral activation of P2Y1, P2Y12, and P2Y13 receptors.

Progesterone distribution in the trigeminal system and its role to modulate sensory neurotransmission: influence of sex.

BACKGROUND: Women are disproportionately affected by migraine, representing up to 75% of all migraine cases. This discrepancy has been proposed to be influenced by differences in hormone levels between the sexes. One such hormone is progesterone. Calcitonin gene-related peptide (CGRP) system is an important factor in migraine pathophysiology and could be influenced by circulating hormones. The purpose of this study was to investigate the distribution of progesterone and its receptor (PR) in the trigeminovascular system, and to examine the role of progesterone to modulate sensory neurotransmission. METHODS: Trigeminal ganglion (TG), hypothalamus, dura mater, and the basilar artery from male and female rats were carefully dissected. Expression of progesterone and PR proteins, and mRNA levels from TG and hypothalamus were analyzed by immunohistochemistry and real-time quantitative PCR. CGRP release from TG and dura mater were measured using an enzyme-linked immunosorbent assay. In addition, the vasomotor effect of progesterone on male and female basilar artery segments was investigated with myography. RESULTS: Progesterone and progesterone receptor -A (PR-A) immunoreactivity were found in TG. Progesterone was located predominantly in cell membranes and in Aδ-fibers, and PR-A was found in neuronal cytoplasm and nucleus, and in satellite glial cells. The number of positive progesterone immunoreactive cells in the TG was higher in female compared to male rats. The PR mRNA was expressed in both hypothalamus and TG; however, the PR expression level was significantly higher in the hypothalamus. Progesterone did not induce a significant change neither in basal level nor upon stimulated release of CGRP from dura mater or TG in male or female rats when compared to the vehicle control. However, pre-treated with 10 µM progesterone weakly enhanced capsaicin induced CGRP release observed in the dura mater of male rats. Similarly, in male basilar arteries, progesterone significantly amplified the dilation in response to capsaicin. CONCLUSIONS: In conclusion, these results highlight the potential for progesterone to modulate sensory neurotransmission and vascular responses in a complex manner, with effects varying by sex, tissue type, and the nature of the stimulus. Further investigations are needed to elucidate the underlying mechanisms and physiological implications of these findings.

Xenobiotic Exposure and Migraine-Associated Signaling: A Multimethod Experimental Study Exploring Cellular Assays in Combination with Ex Vivo and In Vivo Mouse Models.

BACKGROUND: Mechanisms for how environmental chemicals might influence pain has received little attention. Epidemiological studies suggest that environmental factors such as pollutants might play a role in migraine prevalence. Potential targets for pollutants are the transient receptor potential (TRP) channels ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), which on activation release pain-inducing neuropeptide calcitonin gene-related peptide (CGRP). OBJECTIVE: In this study, we aimed to examine the hypothesis that environmental pollutants via TRP channel signaling and subsequent CGRP release trigger migraine signaling and pain. METHODS: A calcium imaging-based screen of environmental chemicals was used to investigate activation of migraine pain-associated TRP channels TRPA1 and TRPV1. Based on this screen, whole-cell patch clamp and in silico docking were performed for the pesticide pentachlorophenol (PCP) as proof of concept. Subsequently, PCP-mediated release of CGRP and vasodilatory responses of cerebral arteries were investigated. Finally, we tested whether PCP could induce a TRPA1-dependent induction of cutaneous hypersensitivity in vivo in mice as a model of migraine-like pain. RESULTS: A total of 16 out of the 52 screened environmental chemicals activated TRPA1 at 10 or 100µM. None of the investigated compounds activated TRPV1. Using PCP as a model of chemical interaction with TRPA1, in silico molecular modeling suggested that PCP is stabilized in a lipid-binding pocket of TRPA1 in comparison with TRPV1. In vitro, ex vivo, and in vivo experiments showed that PCP induced calcium influx in neurons and resulted in a TRPA1-dependent CGRP release from the brainstem and dilation of cerebral arteries. In a mouse model of migraine-like pain, PCP induced a TRPA1-dependent increased pain response (Ntotal=144). DISCUSSION: Here we show that multiple environmental pollutants interact with the TRPA1-CGRP migraine pain pathway. The data provide valuable insights into how environmental chemicals can interact with neurobiology and provide a potential mechanism for putative increases in migraine prevalence over the last decades. https://doi.org/10.1289/EHP12413.

Intraocular Adeno-Associated Virus-Mediated Transgene Endothelin-1 Delivery to the Rat Eye Induces Functional Changes Indicative of Retinal Ischemia-A Potential Chronic Glaucoma Model.

Endothelin-1 (ET-1) overactivity has been implicated as a factor contributing to glaucomatous neuropathy, and it has been utilized in animal models of retinal ischemia. The functional effects of long-term ET-1 exposure and possible compensatory mechanisms have, however, not been investigated. This was therefore the purpose of our study. ET-1 was delivered into rat eyes via a single intravitreal injection of 500 µM or via transgene delivery using an adeno-associated viral (AAV) vector. Retinal function was assessed using electroretinography (ERG) and the retinal expression of potentially compensatory genes was evaluated by means of qRT-PCR. Acute ET-1 delivery led to vasoconstriction and a significant reduction in the ERG response. AAV-ET-1 resulted in substantial transgene expression and ERG results similar to the acute ET-1 injections and comparable to other models of retinal ischemia. Compensatory changes were observed, including an increase in calcitonin gene-related peptide (CGRP) gene expression, which may both counterbalance the vasoconstrictive effects of ET-1 and provide neuroprotection. This chronic ET-1 ischemia model might be especially relevant to glaucoma research, mimicking the mild and repeated ischemic events in patients with long-term vascular dysfunction. The compensatory mechanisms, and particularly the role of vasodilatory CGRP in mitigating the retinal damage, warrant further investigation with the aim of evaluating new therapeutic strategies.

Rodent behavior following a dural inflammation model with anti-CGRP migraine medication treatment.

Background: Migraine is a widespread and prevalent disease with a complex pathophysiology, of which neuroinflammation and increased pain sensitivity have been suggested to be involved. Various studies have investigated the presence of different inflammatory markers in migraineurs and investigated the role of inflammation in inflammatory models with complete Freund's adjuvant (CFA) or inflammatory soup added to the dura mater. Objective: The aim of the current study was to examine whether application of CFA to the dura mater would cause behavioral alterations that are migraine relevant. In addition, we investigated the potential mitigating effects of fremanezumab, a CGRP (calcitonin gene-related peptide) specific antibody, following CFA application. Methods: Male Sprague-Dawley rats were randomly divided into six groups: fresh (n = 7), fresh + carprofen (n = 6), fresh + anti-CGRP (n = 6), sham (n = 7), CFA (n = 16), CFA + anti-CGRP (n = 8). CFA was applied for 15 min on a 3 × 3 mm clearing of the skull exposing the dura mater of male Sprague-Dawley rats. We applied the Light/Dark box and Open Field test, combined with the electronic von Frey test to evaluate outcomes. Finally, we observed CGRP immunoreactivity in the trigeminal ganglion. Results: No differences were observed in the Light/Dark box test. The Open Field test detected behavior differences, notably that sham rats spend less time in the central zone, reared less and groomed more than fresh + carprofen rats. The other groups were not significantly different compared to sham rats, indicating that activation of the TGVS is present in sham surgery and cannot be exacerbated by CFA. However, for the allodynia, we observed specific periorbital sensitization, not observed in the sham animals. This could not be mitigated by fremanezumab, although it clearly reduced the amount of CGRP positive fibers. Conclusion: CFA surgically administered to the dura causes periorbital allodynia and increases CGRP positive fibers in the trigeminal ganglion. Fremanezumab does not reduce periorbital allodynia even though it reduces CGRP positive fibers in the TG. Further work is needed to investigate whether CFA administered to the dura could be used as a non-CGRP inflammatory migraine model.

Pharmacological Profile of the Purinergic P2Y Receptors That Modulate, in Response to ADPßS, the Vasodepressor Sensory CGRPergic Outflow in Pithed Rats.

Calcitonin gene-related peptide (CGRP), an endogenous neuropeptide released from perivascular sensory nerves, exerts a powerful vasodilatation. Interestingly, adenosine triphosphate (ATP) stimulates the release of CGRP by activation of prejunctional P2X2/3 receptors, and adenosine 5'-O-2-thiodiphosphate (ADPßS), a stable adenosine diphosphate (ADP) analogue, produces vasodilator/vasodepressor responses by endothelial P2Y1 receptors. Since the role of ADP in the prejunctional modulation of the vasodepressor sensory CGRPergic drive and the receptors involved remain unknown, this study investigated whether ADPßS inhibits this CGRPergic drive. Accordingly, 132 male Wistar rats were pithed and subsequently divided into two sets. In set 1, ADPßS (5.6 and 10 µg/kg·min) inhibited the vasodepressor CGRPergic responses by electrical stimulation of the spinal T9-T12 segment. This inhibition by ADPßS (5.6 µg/kg·min) was reverted after i.v. administration of the purinergic antagonists MRS2500 (300 µg/kg; P2Y1) or MRS2211 (3000 µg/kg; P2Y13), but not by PSB0739 (300 µg/kg; P2Y12), MRS2211 (1000 µg/kg; P2Y13) or the KATP blocker glibenclamide (20 mg/kg). In set 2, ADPßS (5.6 µg/kg·min) failed to modify the vasodepressor responses to exogenous α-CGRP. These results suggest that ADPßS inhibits CGRP release in perivascular sensory nerves. This inhibition, apparently unrelated to activation of ATP-sensitive K+ channels, involves P2Y1 and probably P2Y13, but not P2Y12 receptors.

Lasmiditan and 5-Hydroxytryptamine in the rat trigeminal system; expression, release and interactions with 5-HT1 receptors.

BACKGROUND: 5-Hydroxytryptamine (5-HT) receptors 1B, 1D and 1F have key roles in migraine pharmacotherapy. Selective agonists targeting these receptors, such as triptans and ditans, are effective in aborting acute migraine attacks and inhibit the in vivo release of calcitonin gene-related peptide (CGRP) in human and animal models. The study aimed to examine the localization, genetic expression and functional aspects of 5- HT1B/1D/1F receptors in the trigeminal system in order to further understand the molecular sites of action of triptans (5-HT1B/1D) and ditans (5-HT1F). METHODS: Utilizing immunohistochemistry, the localization of 5-HT and of 5-HT1B/1D/1F receptors was examined in rat trigeminal ganglion (TG) and combined with quantitative polymerase chain reaction to quantify the level of expression for 5-HT1B/1D/1F receptors in the TG. The functional role of these receptors was examined ex vivo with a capsaicin/potassium induced 5-HT and CGRP release. RESULTS: 5-HT immunoreactivity (ir) was observed in a minority of CGRP negative C-fibres, most neuron somas and faintly in A-fibres and Schwann cell neurolemma. 5-HT1B/1D receptors were expressed in the TG, while the 5-HT1F receptor displayed a weak ir. The 5-HT1D receptor co-localized with receptor activity-modifying protein 1 (RAMP1) in Aδ-fibres in the TG, while 5-HT1B-ir was weakly expressed and 5-HT1F-ir was not detected in these fibres. None of the 5-HT1 receptors co-localized with CGRP-ir in C-fibres. 5-HT1D receptor mRNA was the most prominently expressed, followed by the 5-HT1B receptor and lastly the 5-HT1F receptor. The 5-HT1B and 5-HT1D receptor antagonist, GR127935, could reverse the inhibitory effect of Lasmiditan (a selective 5-HT1F receptor agonist) on CGRP release in the soma-rich TG but not in soma-poor TG or dura mater. 5-HT release in the soma-rich TG, and 5-HT content in the baseline samples, negatively correlated with CGRP levels, showing for the first time a physiological role for 5-HT induced inhibition. CONCLUSION: This study reveals the presence of a subgroup of C-fibres that store 5-HT. The data shows high expression of 5-HT1B/1D receptors and suggests that the 5-HT1F receptor is a relatively unlikely target in the rat TG. Furthermore, Lasmiditan works as a partial agonist on 5-HT1B/1D receptors in clinically relevant dose regiments.

Biological and small molecule strategies in migraine therapy with relation to the calcitonin gene-related peptide family of peptides.

Migraine is one of the most common of neurological disorders with a global prevalence of up to 15%. One in five migraineurs have frequent episodic or chronic migraine requiring prophylactic treatment. In recent years, specific pharmacological treatments targeting calcitonin gene-related peptide (CGRP) signalling molecules have provided safe and effective treatments, monoclonal antibodies for prophylaxis and gepants for acute therapy. Albeit beneficial, it is important to understand the molecular mechanisms of these new drugs to better understand migraine pathophysiology and improve therapy. Here, we describe current views on the role of the CGRP family of peptides - CGRP, calcitonin, adrenomedullin, amylin - and their receptors in the trigeminovascular system. All these molecules are present within the trigeminovascular system but differ in expression and localization. It is likely that they have different roles, which can be utilized in providing additional drug targets. LINKED ARTICLES: This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc.

Neurokinins and their receptors in the rat trigeminal system: Differential localization and release with implications for migraine pain.

Substance P (SP) and calcitonin gene-related peptide (CGRP) have both been considered potential drug candidates in migraine therapy. In recent years, CGRP receptor inhibition has been established as an effective treatment, in particular as a prophylactic for chronic migraine. Curiously, inhibition of neurokinin receptor 1 (NK1R) failed to alleviate acute migraine attacks in clinical trials, and the neurokinins were consequently abandoned as potential antimigraine candidates. The reason behind this has remained enigmatic.Utilizing immunohistochemistry and semi-quantitative cell counts the expression of neurokinins and their associated receptors was examined in the rat trigeminal ganglion.Immunohistochemistry results revealed SP co-localization in CGRP positive neurons and C-fibres, where it mainly concentrated at boutons. Neurokinin A (NKA) was observed in a population of C-fibres and small neurons where it could co-localize with SP. In contrast, neurokinin B (NKB) did not co-localize with SP and was observed in large/medium sized neurons and Aδ-fibres. All neurokinin receptors (NK1-3R) were found to be expressed in a majority of trigeminal ganglion neurons and A-fibres.The functional release of SP and CGRP in the trigeminovascular system was stimulated with either 60 mM K+ or 100 nM capsaicin and measured with an enzyme-linked immunosorbent assay (ELISA). ELISA results established that SP can be released locally from trigeminovascular system. The released SP was comparatively minor compared to the CGRP release from stimulated dura mater, trigeminal ganglion neurons and fibres. We hypothesize that SP and CGRP signalling pathways may work in tandem to exacerbate painful stimuli in the TGV system.

CGRP-dependent signalling pathways involved in mouse models of GTN- cilostazol- and levcromakalim-induced migraine.

BACKGROUND: Knowledge of exact signalling events during migraine attacks is lacking. Various substances are known to trigger migraine attacks in patients and calcitonin gene-related peptide antagonising drugs are effective against migraine pain. Here, we investigated the signalling pathways involved in three different mouse models of provoked migraine and relate them to calcitonin gene-related peptide and other migraine-relevant targets. METHODS: In vivo mouse models of glyceryl trinitrate-, cilostazol- and levcromakalim-induced migraine were applied utilising tactile sensitivity to von Frey filaments as measuring readout. Signalling pathways involved in the three models were dissected by use of specific knockout mice and chemical inhibitors. In vivo results were supported by ex vivo wire myograph experiments measuring arterial dilatory responses and ex vivo calcitonin gene-related peptide release from trigeminal ganglion and trigeminal nucleus caudalis from mice. RESULTS: Glyceryl trinitrate-induced hypersensitivity was dependent on both prostaglandins and transient receptor potential cation channel, subfamily A, member 1, whereas cilostazol- and levcromakalim-induced hypersensitivity were independent of both. All three migraine triggers activated calcitonin gene-related peptide signalling, as both receptor antagonism and antibody neutralisation of calcitonin gene-related peptide were effective inhibitors of hypersensitivity in all three models. Stimulation of trigeminal ganglia and brain stem tissue samples with cilostazol and levcromakalim did not result in release of calcitonin gene-related peptide, and vasodilation following levcromakalim stimulation was independent of CGRP receptor antagonism. CONCLUSION: The mouse models of glyceryl trinitrate-, cilostazol- and levcromakalim- induced migraine all involve calcitonin gene-related peptide signalling in a complex interplay between different cell/tissue types. These models are useful in the study of migraine mechanisms.

Neuropeptides and the Nodes of Ranvier in Cranial Headaches.

The trigeminovascular system (TGV) comprise of the trigeminal ganglion with neurons and satellite glial cells, with sensory unmyelinated C-fibers and myelinated Aδ-fibers picking up information from different parts of the head and sending signals to the brainstem and the central nervous system. In this review we discuss aspects of signaling at the distal parts of the sensory fibers, the extrasynaptic signaling between C-fibers and Aδ-fibers, and the contact between the trigeminal fibers at the nerve root entry zone where they transit into the CNS. We also address the possible role of the neuropeptides calcitonin gene-related peptide (CGRP), the neurokinin family and pituitary adenylyl cyclase-activating polypeptide 38 (PACAP-38), all found in the TGV system together with their respective receptors. Elucidation of the expression and localization of neuropeptides and their receptors in the TGV system may provide novel ways to understand their roles in migraine pathophysiology and suggest novel ways for treatment of migraine patients.

Estrogen receptors α, ß and GPER in the CNS and trigeminal system - molecular and functional aspects.

BACKGROUND: Migraine occurs 2-3 times more often in females than in males and is in many females associated with the onset of menstruation. The steroid hormone, 17ß-estradiol (estrogen, E2), exerts its effects by binding and activating several estrogen receptors (ERs). Calcitonin gene-related peptide (CGRP) has a strong position in migraine pathophysiology, and interaction with CGRP has resulted in several successful drugs for acute and prophylactic treatment of migraine, effective in all age groups and in both sexes. METHODS: Immunohistochemistry was used for detection and localization of proteins, release of CGRP and PACAP investigated by ELISA and myography/perfusion arteriography was performed on rat and human arterial segments. RESULTS: ERα was found throughout the whole brain, and in several migraine related structures. ERß was mainly found in the hippocampus and the cerebellum. In trigeminal ganglion (TG), ERα was found in the nuclei of neurons; these neurons expressed CGRP or the CGRP receptor in the cytoplasm. G-protein ER (GPER) was observed in the cell membrane and cytoplasm in most TG neurons. We compared TG from males and females, and females expressed more ER receptors. For neuropeptide release, the only observable difference was a baseline CGRP release being higher in the pro-estrous state as compared to estrous state. In the middle cerebral artery (MCA), we observed similar dilatory ER-responses between males and females, except for vasodilatory ERß which we observed only in female arteries. CONCLUSION: These data reveal significant differences in ER receptor expression between male and female rats. This contrasts to CGRP and PACAP release where we did not observe discernable difference between the sexes. Together, this points to a hypothesis where estrogen could have a modulatory role on the trigeminal neuron function in general rather than on the acute CGRP release mechanisms and vasomotor responses.

Oxytocin as a regulatory neuropeptide in the trigeminovascular system: Localization, expression and function of oxytocin and oxytocin receptors.

BACKGROUND: Recent clinical findings suggest that oxytocin could be a novel treatment for migraine. However, little is known about the role of this neuropeptide/hormone and its receptor in the trigeminovascular pathway. Here we determine expression, localization, and function of oxytocin and oxytocin receptors in rat trigeminal ganglia and targets of peripheral (dura mater and cranial arteries) and central (trigeminal nucleus caudalis) afferents. METHODS: The methods include immunohistochemistry, messenger RNA measurements, quantitative PCR, release of calcitonin gene-related peptide and myography of arterial segments. RESULTS: Oxytocin receptor mRNA was expressed in rat trigeminal ganglia and the receptor protein was localized in numerous small to medium-sized neurons and thick axons characteristic of A∂ sensory fibers. Double immunohistochemistry revealed only a small number of neurons expressing both oxytocin receptors and calcitonin gene-related peptide. In contrast, double immunostaining showed expression of the calcitonin gene-related peptide receptor component receptor activity-modifying protein 1 and oxytocin receptors in 23% of the small cells and in 47% of the medium-sized cells. Oxytocin immunofluorescence was observed only in trigeminal ganglia satellite glial cells. Oxytocin mRNA was below detection limit in the trigeminal ganglia. The trigeminal nucleus caudalis expressed mRNA for both oxytocin and its receptor. K+-evoked calcitonin gene-related peptide release from either isolated trigeminal ganglia or dura mater and it was not significantly affected by oxytocin (10 µM). Oxytocin directly constricted cranial arteries ex vivo (pEC50 ∼ 7); however, these effects were inhibited by the vasopressin V1A antagonist SR49059. CONCLUSION: Oxytocin receptors are extensively expressed throughout the rat trigeminovascular system and in particular in trigeminal ganglia A∂ neurons and fibers, but no functional oxytocin receptors were demonstrated in the dura and cranial arteries. Thus, circulating oxytocin may act on oxytocin receptors in the trigeminal ganglia to affect nociception transmission. These effects may help explain hormonal influences in migraine and offer a novel way for treatment.

Local endothelial DNA repair deficiency causes aging-resembling endothelial-specific dysfunction.

We previously identified genomic instability as a causative factor for vascular aging. In the present study, we determined which vascular aging outcomes are due to local endothelial DNA damage, which was accomplished by genetic removal of ERCC1 (excision repair cross-complementation group 1) DNA repair in mice (EC-knockout (EC-KO) mice). EC-KO showed a progressive decrease in microvascular dilation of the skin, increased microvascular leakage in the kidney, decreased lung perfusion, and increased aortic stiffness compared with wild-type (WT). EC-KO showed expression of DNA damage and potential senescence marker p21 exclusively in the endothelium, as demonstrated in aorta. Also the kidney showed p21-positive cells. Vasodilator responses measured in organ baths were decreased in aorta, iliac and coronary artery EC-KO compared with WT, of which coronary artery was the earliest to be affected. Nitric oxide-mediated endothelium-dependent vasodilation was abolished in aorta and coronary artery, whereas endothelium-derived hyperpolarization and responses to exogenous nitric oxide (NO) were intact. EC-KO showed increased superoxide production compared with WT, as measured in lung tissue, rich in endothelial cells (ECs). Arterial systolic blood pressure (BP) was increased at 3 months, but normal at 5 months, at which age cardiac output (CO) was decreased. Since no further signs of cardiac dysfunction were detected, this decrease might be an adaptation to prevent an increase in BP. In summary, a selective DNA repair defect in the endothelium produces features of age-related endothelial dysfunction, largely attributed to loss of endothelium-derived NO. Increased superoxide generation might contribute to the observed changes affecting end organ perfusion, as demonstrated in kidney and lung.

Lasmiditan inhibits calcitonin gene-related peptide release in the rodent trigeminovascular system.

Migraine headache pathophysiology involves trigeminovascular system activation, calcitonin gene-related peptide (CGRP) release, and dysfunctional nociceptive transmission. Triptans are 5-HT1B/1D/(1F) receptor agonists that prejunctionally inhibit trigeminal CGRP release, but their vasoconstrictor properties limit their use in migraine patients with cardiovascular disease. By contrast, lasmiditan is a novel antimigraine and selective 5-HT1F receptor agonist devoid of vasoconstrictor properties. On this basis, this study has investigated the modulation of trigeminal CGRP release by lasmiditan. For this purpose, we have comparatively analysed the inhibition of several components of the trigeminovascular system induced by lasmiditan and sumatriptan through: ex vivo KCl-induced CGRP release from isolated dura mater, trigeminal ganglion, and trigeminal nucleus caudalis of mice; and in vivo dural vasodilation in the rat closed-cranial window model induced by endogenous (electrical stimulation and capsaicin) and exogenous CGRP. The ex vivo release of CGRP was similarly inhibited by sumatriptan and lasmiditan in all trigeminovascular system components. In vivo, intravenous (i.v.) lasmiditan or higher doses of sumatriptan significantly attenuated the vasodilatory responses to endogenous CGRP release, but not exogenous CGRP effects. These data suggest that lasmiditan prejunctionally inhibits CGRP release in peripheral and central trigeminal nerve terminals. Because lasmiditan is a lipophilic drug that crosses the blood-brain barrier, additional central sites of action remain to be determined.

Cerebrovascular effects of endothelin-1 investigated using high-resolution magnetic resonance imaging in healthy volunteers.

Endothelin-1 (ET-1) is a highly potent vasoconstrictor peptide released from vascular endothelium. ET-1 plays a major role in cerebrovascular disorders and likely worsens the outcome of acute ischaemic stroke and aneurismal subarachnoid haemorrhage through vasoconstriction and cerebral blood flow (CBF) reduction. Disorders that increase the risk of stroke, including hypertension, diabetes mellitus, and acute myocardial infarction, are associated with increased plasma levels of ET-1. The in vivo human cerebrovascular effects of systemic ET-1 infusion have not previously been investigated. In a two-way crossover, randomized, double-blind design, we used advanced 3 tesla MRI methods to investigate the effects of high-dose intravenous ET-1 on intra- and extracranial artery circumferences, global and regional CBF, and cerebral metabolic rate of oxygen (CMRO2) in 14 healthy volunteers. Following ET-1 infusion, we observed a 14% increase of mean arterial blood pressure, a 5% decrease of middle cerebral artery (MCA) circumference, but no effects on extracerebral arteries and no effects on CBF or CMRO2. Collectively, the findings indicate MCA constriction secondarily to blood pressure increase and not due to a direct vasoconstrictor effect of ET-1. We suggest that, as opposed to ET-1 in the subarachnoid space, intravascular ET-1 does not exert direct cerebrovascular effects in humans.

C-fibers may modulate adjacent Aδ-fibers through axon-axon CGRP signaling at nodes of Ranvier in the trigeminal system.

BACKGROUND: Monoclonal antibodies (mAbs) towards CGRP or the CGRP receptor show good prophylactic antimigraine efficacy. However, their site of action is still elusive. Due to lack of passage of mAbs across the blood-brain barrier the trigeminal system has been suggested a possible site of action because it lacks blood-brain barrier and hence is available to circulating molecules. The trigeminal ganglion (TG) harbors two types of neurons; half of which store CGRP and the rest that express CGRP receptor elements (CLR/RAMP1). METHODS: With specific immunohistochemistry methods, we demonstrated the localization of CGRP, CLR, RAMP1, and their locations related to expression of the paranodal marker contactin-associated protein 1 (CASPR). Furthermore, we studied functional CGRP release separately from the neuron soma and the part with only nerve fibers of the trigeminal ganglion, using an enzyme-linked immunosorbent assay. RESULTS: Antibodies towards CGRP and CLR/RAMP1 bind to two different populations of neurons in the TG and are found in the C- and the myelinated Aδ-fibers, respectively, within the dura mater and in trigeminal ganglion (TG). CASPR staining revealed paranodal areas of the different myelinated fibers inhabiting the TG and dura mater. Double immunostaining with CASPR and RAMP1 or the functional CGRP receptor antibody (AA58) revealed co-localization of the two peptides in the paranodal region which suggests the presence of the CGRP-receptor. Double immunostaining with CGRP and CASPR revealed that thin C-fibers have CGRP-positive boutons which often localize in close proximity to the nodal areas of the CGRP-receptor positive Aδ-fibers. These boutons are pearl-like synaptic structures, and we show CGRP release from fibers dissociated from their neuronal bodies. In addition, we found that adjacent to the CGRP receptor localization in the node of Ranvier there was PKA immunoreactivity (kinase stimulated by cAMP), providing structural possibility to modify conduction activity within the Aδ-fibers. CONCLUSION: We observed a close relationship between the CGRP containing C-fibers and the Aδ-fibers containing the CGRP-receptor elements, suggesting a point of axon-axon interaction for the released CGRP and a site of action for gepants and the novel mAbs to alleviate migraine.