Trigeminovascular Calcitonin Gene-Related Peptide Release and Peripheral Vascular Responses in a Mouse Model of Accelerated Aging: Implications for Migraine

Trigeminovascular Calcitonin Gene-Related Peptide Release and Peripheral Vascular Responses in a Mouse Model of Accelerated Aging: Implications for Migraine | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Trigeminovascular Calcitonin Gene-Related Peptide Release and Peripheral Vascular Responses in a Mouse Model of Accelerated Aging: Implications for Migraine Linda Al-Hassany, Eloisa Rubio-Beltrán, Alejandro Labastida-Ramirez, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9187264/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Objective Understanding age-related changes in migraine is pivotal, considering the increasing global life expectancy. In addition, both aging and migraine are prominent cardiovascular risk factors. It remains unclear whether calcitonin gene-related peptide (CGRP) release changes with age across trigeminovascular components, and how this relates to peripheral responses to migraine-related vasodilatory molecules. The primary aim was to investigate age-related effects on CGRP release from the trigeminovascular system by studying a mouse model of combined accelerated neuronal and vascular aging, the DNA repair-deficient Ercc1 Δ/− mice. Second, we assessed the effects of aging on isolated coronary vasodilatory responses to CGRP and forskolin. Methods Experiments were conducted using DNA repair-deficient Ercc1 Δ/− mice and their wild type controls. After sacrifice, the trigeminal nucleus caudalis (TNC), trigeminal ganglion (TG), and dura mater (DM) were isolated. Ex vivo KCl-induced CGRP release was measured, and CGRP release was compared between Ercc1 Δ/− and wild type mice. In a subset of mice, concentration-response curves to CGRP and forskolin were generated in isolated coronary arteries. The pEC 50 (negative log of the molar concentration of an agonist needed to reach half of its maximal effect) and E max (maximum relaxation response) values were compared between both groups. Results CGRP release (expressed as ratio compared to baseline release) of the DM was significantly lower in Ercc1 Δ/− (2.01±0.24) versus wild type mice (3.22±0.48) ( P =0.040). No differences were observed in CGRP release between Ercc1 Δ/− and wild type mice in the TNC (8.07±1.15 versus 6.10±0.57, P =0.364) or the TG (4.84±0.92 versus 4.41±0.60, P =0.838). In addition, there were no differences in pEC 50 and E max values in response to CGRP and forskolin. Conclusion Our findings suggest that aging is linked to reduced CGRP release of the DM, potentially partly explaining the reduction in migraine attacks in elderly. This decreased CGRP release is not accompanied by altered peripheral vascular reactivity to CGRP. Aging CGRP Mice Migraine Peripheral vasculature Trigeminovascular system Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Migraine is a highly disabling and stigmatizing primary headache disorder that is characterized by unilateral moderate to severe headache attacks and associated symptoms ( i.e. , nausea, vomiting, photo- and phonophobia), which may be preceded or accompanied by aura symptoms in approximately one-third of patients ( 1 – 3 ). The overall migraine prevalence is (at least) two-fold higher in women compared to men ( 4 ), while the migraine peak prevalence occurs at similar ages in both sexes – namely during the reproductive years (approximately 20s and 30s) with a second peak in women around fifty years of age ( 5 ). Both the incidence and prevalence of migraine tend to decrease with advancing age ( 6 , 7 ). Understanding the neurovascular basis of these age-related changes is pivotal, especially considering the increase in global life expectancy ( 8 ). The exact pathophysiological age-dependent mechanisms are not completely understood, but neuronal and vascular changes, besides hormonal senescence, have been hypothesized to be involved in migraine remission at older ages ( 6 ). Activation of the trigeminovascular system and concomitant release of the vasodilatory neuropeptide calcitonin gene-related peptide (CGRP), expressed in both the central and peripheral components of the trigeminovascular system, play a causal role in the headache phase of migraine ( 9 – 12 ). Interestingly, in rats, transport of CGRP in nerve fibers has been shown to decline significantly with advancing age ( 13 ). In addition, in vivo studies have shown age-dependent changes in both pre- and post-synaptic peripheral fibers of these animals. These data indicate a reduction in the number of sensory fibers and/or a decrease in their peptide content or release, impacting the activation of the trigeminovascular system ( 14 , 15 ). Such a decline in CGRP content was also observed in specific brain structures, i.e. , substantia nigra and striatum, as well as in cardiovascular tissue of aging rats ( 16 ) and in guinea pigs, whose maximum density of CGRP-containing perivascular nerve plexuses declined to approximately half in older age ( 17 ). Being a neurovascular condition and an important cardiovascular risk factor ( 18 ), these alterations in trigeminal pain pathways should not be considered in isolation from the vascular age-related changes associated with migraine. Accordingly, previous research suggests that aging is associated with reduced cerebrovascular vasodilatory capacitance, probably accompanied by decreased activation of sensory neurons ( 6 , 19 ) and disrupted neurovascular coupling ( 20 ). It is yet unclear whether age-dependent differences in CGRP release exist across the central and peripheral structures of the trigeminovascular system. Therefore, the primary aim of this study was to investigate the effects of aging on CGRP release from the trigeminal nucleus caudalis (central trigeminovascular component), trigeminal ganglion, and dura mater (both peripheral trigeminovascular components) by comparing CGRP release in a mouse model of accelerated aging, the DNA repair-deficient Ercc1 Δ/− mice, versus their wild type littermates. The secondary objective was to deepen our understanding of the connection between neuronal and vascular age-related changes. We therefore studied vasodilatory responses of coronary arteries to CGRP and forskolin in Ercc1 Δ/− and wild type mice, as both lead to the accumulation of intracellular levels of cyclic adenosine monophosphate (cAMP) ( 21 , 22 ), which, in turn, has been described to play a role in migraine induction ( 23 , 24 ). Further explorative analyses were conducted to evaluate the differences in trigeminovascular CGRP release between i) male and female mice and ii) the three components of the trigeminovascular system. MATERIALS AND METHODS Experimental mice Experiments were performed using both male and female DNA repair-deficient Ercc1 Δ/− mice and their controls, i.e. , wild type littermates ( Ercc1 +/+ mice). The Ercc1 Δ/− mice have one exon 7-truncated and one completely inactivated allele of the endonuclease Ercc1 , thus partially inactivating this endonuclease that is involved in nucleotide excision and interstrand DNA crosslink repair. Ercc1 Δ/− mice with an F1 C57BL6J/FVB hybrid background were obtained, preventing strain-specific phenotypes, as previously described ( 25 , 26 ). Ercc1 Δ/− mice represent a well-established model of accelerated neurovascular aging due to genomic instability, characterized by age-related diseases and a reduced lifespan (24–28 weeks compared to at least 100 weeks, and up to 146 weeks, in wild type mice). Overall, the accelerated aging phenotype includes neurodegeneration, osteoporosis, early-onset hypertension, vascular stiffness, accelerated endothelial and vasodilatory dysfunction, and cardiomyopathy ( 27 – 30 ), associated with an increase in phosphodiesterase 1 expression ( 31 ). In addition, aortas from Ercc1 Δ/− mice exhibit phenotypic switching of vascular smooth muscle cells and an increased stress response ( 32 ). Breeding took place at the animal facility of the Erasmus Medical Center (Erasmus MC). Mice were kept in individually ventilated cages under controlled conditions (20–22°C, 12-hour light/dark cycle) with free access to standard chow and water. All mice were weighed and visually inspected daily to monitor their well-being. The study consisted of three mice batches with identical genotypes, and all mice were sacrificed between 88 and 117 days of age. The study was planned and carried out in accordance with the Principles of Laboratory Animal Care and with guidelines approved by an independent Animal Ethics Committee, consulted at the Erasmus MC, as well as the ARRIVE guidelines. Calcitonin gene-related peptide release experiments in the trigeminal nucleus caudalis, trigeminal ganglion, and dura mater The technique used to measure ex vivo CGRP release in the central (trigeminal nucleus caudalis) and peripheral (dura mater and trigeminal ganglion) components of the trigeminovascular system has been described previously ( 33 , 34 ). In short, mice were anesthetized using intraperitoneal sodium pentobarbital (80 mg/kg) and decapitated at the atlantooccipital joint. The skin and galea aponeurotica were both pulled back from the cranium. The trigeminal nucleus caudalis, located caudally between 9–13 mm from bregma, was first carefully isolated from the brainstem and divided into two parts. The skull was subsequently divided into two halves along the sagittal suture, and both cerebral hemispheres were removed, leaving the cranial dura mater intact and attached to the skull. The trigeminal ganglia of both sides were then obtained by dissecting 1 mm proximal and distal to the point where the mandibular nerve branches off. The final step consisted of carefully hemisecting the cranium and removing the remaining (brain) tissue from the skull, except for the dura mater. Each (half) of the isolated trigeminal nucleus caudalis, trigeminal ganglion, and skull with dura mater were immersed and washed in carbogenated synthetic interstitial fluid, containing NaCl (108 mM), KCl (3.48 mM), MgSO 4 (3.5 mM), NaHCO 3 (26 mM), NaH 2 PO 4 (11.7 mM), CaCl 2 (1.5 mM), sodium gluconate (9.6 mM), glucose (5.55 mM), and sucrose (7.6 mM) for three times 30 minutes at 37°C to reduce false positive results. Both halves of the isolated tissues were then placed in a 24-well plate containing 500 µL of synthetic interstitial fluid per well. The plate was fixed in a water bath, creating a closed, humid and carbogenated chamber of 37°C. Tissues were washed three additional times over a period of up to 15 minutes. Basal CGRP levels were measured in each tissue, after which CGRP release was induced by adding 60 mM KCl. Both the baseline and KCl-infused samples were collected after 10 minutes of incubation and mixed with aprotinin (500 KIU/mL). Samples were stored at − 80°C until CGRP content was assessed using a commercial CGRP RIA kit according to the manual (Phoenix Pharmaceuticals, Burlingame, CA, USA). The assay has a detection level of 1.0 pg/mL; samples with CGRP concentrations below this limit were assigned this value of 1.0 pg/mL, in line with our previous study ( 33 ). Blanks containing only synthetic interstitial fluid, without CGRP, were used as a control to exclude false-positive measurements. For the CGRP assays, the analyst was blinded to the mice genotype. CGRP release was assessed in duplicate for both individual halves of each trigeminovascular structure, of which averages were used for further analyses. CGRP release was expressed as the ratio of KCl-induced CGRP release to basal CGRP release, which represents the relative stimulated release of CGRP ( i.e. , CGRP release ratio). Tissues that exhibited either no or decreased relative CGRP release in response to 60 mM KCl ( i.e. , CGRP release ratios ≤ 1.05) or an unusually high relative release ( i.e. , CGRP release ratios ≥ 100) were excluded from further analyses, consistent with our previous study on CGRP release in mice ( 34 ). As the ex vivo experiments consisted of three mouse batches, an outlier test – i.e. , the ROUT method (Q = 1%) – was performed on each individual batch and the combined data of all batches. Peripheral vasodilatory experiments in coronary arteries Immediately after sacrificing the mice, coronary arteries were carefully isolated in oxygenated and carbogenated Krebs buffer solution (NaCl 118 mM, KCl 4.7 mM, CaCl 2 2.5 mM, MgSO 4 1.2 mM, KH 2 PO 4 1.2 mM, NaHCO 3 25 mM, and glucose 8.3 mM in distilled water, pH 7.4). Functional experiments on the coronaries were conducted only for 6 wild type and 6 Ercc1 Δ/− mice concurrently with the CGRP release experiments. For these experiments, one vessel segment of 2 mm length per mouse was mounted in small wire Mulvany myograph organ baths (Danish Myograph Technology, Aarhus, Denmark), containing 6 mL of oxygenated and carbogenated Krebs solution at 37°C. The mounted vessel segments were first left to equilibrate before the tension was normalized by stepwise stretching vessels to a tension corresponding to 90% of the estimated diameter at a transmural pressure of 100 mmHg ( 35 ). After four washing steps, each separated by a 5-minute interval, maximum contractile responses were determined using 60 mmol/L KCl as a reference contraction. To evaluate vasodilatory responses, coronary segments were precontracted with 30 nmol/L of U46619 (a thromboxane A 2 analog) to study the effects of forskolin and with 30 mmol/L of KCl (facilitating comparison with human data from our lab, where precontractions are also induced by KCl) to study the effects of CGRP. After reaching a contraction plateau, concentration-response curves were sequentially generated in a single segment for the following vasodilators using cumulative doses: CGRP (10 − 11 –10 − 6 mol/L in half-logarithmic steps) and forskolin (10 − 9 –10 − 4 mol/L in half-logarithmic steps). Data were recorded using LabChart data acquisition software (AD Instruments Ltd, Oxford, UK). Relaxation responses to the vasodilatory substances are expressed as percentages (%) relative to the contraction induced by either 30 nmol/L U46619 or 30 mmol/L KCl, which was set at 100%. Concentration-response curves with a sigmoidal shape were constructed using a computerized curve-fitting technique, in which the bottom was constrained to zero. Non-linear regression analysis was used to determine the pEC 50 (negative log of the molar concentration of an agonist needed to reach half of its maximal effect) and E max (maximum relaxation response) values. Statistical analyses CGRP release ratios and percentages of relaxation are all expressed as mean ± SEM. Age is expressed as median [Q 1 –Q 3 ]. Statistical differences in the relative CGRP release, pEC 50 and E max values between wild type and Ercc1 Δ/− mice were calculated using the two-tailed Mann-Whitney test for unpaired observations. A nonparametric test was applied to compare differences in CGRP release, in accordance with previous studies ( 33 , 34 ), and due to the small sample size of vasodilatory responses in coronary arteries. To additionally explore differences between male and female wild type and Ercc1 Δ/− mice, a Kruskal-Wallis test with a post-hoc test, corrected Dunn's multiple comparisons tests, was applied. Similarly, differences in CGRP release between the trigeminal nucleus caudalis, trigeminal ganglion, and dura mater were analyzed using the Kruskal-Wallis test with Dunn's multiple comparisons test, stratified by wild type and Ercc1 Δ/− mice. The statistical significance level was set at P ≤ 0.05. All analyses were performed using GraphPad Prism version 8.0.1 for Windows (GraphPad Software, Boston, Massachusetts USA). Compounds Forskolin was obtained from Tocris BioScience (Bristol, UK) and dissolved in physiological saline. U46619 was purchased from Sigma Chemical Co. (St. Louis, MO, USA); r-α-CGRP was obtained from PolyPeptide (Strasbourg, France). RESULTS Calcitonin gene-related peptide release in the trigeminal nucleus caudalis, trigeminal ganglion, and dura mater CGRP release experiments were performed on a total of 27 Ercc1 Δ/− mice and 29 wild type mice, of whom tissues were obtained and analyzed from both halves. From 1 Ercc1 Δ/− mouse, no trigeminal nucleus caudalis and trigeminal ganglion were obtained due to damaged tissue. Trigeminal nucleus caudalis Exclusion of individual tissues of the trigeminal nucleus caudalis based on the previously mentioned criteria of the CGRP release ratio and outlier tests yielded averaged ratios from 23 Ercc1 Δ/− mice and 27 wild type mice. The Ercc1 Δ/− mice consisted of 9 males (39%) and 14 females (61%) with a median age of 92 [90–111] days. The wild type mice consisted of 14 males (52%) and 13 females (48%) with a median age of 110 [96–114] days. No significant differences were observed in CGRP release of the trigeminal nucleus caudalis between Ercc1 Δ/− mice (8.07 ± 1.15) and wild type mice (6.10 ± 0.57) ( P = 0.364), Fig. 1 . Trigeminal ganglion Exclusion of individual tissues of the trigeminal ganglion based on the previously mentioned criteria yielded averaged ratios from 20 Ercc1 Δ/− mice and 23 wild type mice. The Ercc1 Δ/− mice consisted of 10 males (50%) and 10 females (50%) with a median [Q 1 –Q 3 ] age of 91 [90–106.8] days. The wild type mice consisted of 11 males (48%) and 12 females (52%) with a median age of 110 [92–114] days. No significant differences were observed in CGRP release of the trigeminal ganglion between Ercc1 Δ/− mice (4.84 ± 0.92) and wild type mice (4.41 ± 0.60) ( P = 0.838), Fig. 2 . Dura mater Exclusion of individual tissues of the dura mater based on the previously mentioned criteria yielded averaged ratios from 15 Ercc1 Δ/− mice and 19 wild type mice. The Ercc1 Δ/− mice consisted of 8 males (53%) and 7 females (47%) with a median [Q 1 –Q 3 ] age of 92 [90–112] days. The wild type mice consisted of 11 males (58%) and 8 females (42%) with a median age of 112 [95–114] days. Significantly lower CGRP release of the dura mater were observed in Ercc1 Δ/− mice (2.01 ± 0.24) compared to wild type mice (3.22 ± 0.48) ( P = 0.040), Fig. 3 . Analyses on male-female differences and different trigeminovascular system components We further explored differences in CGRP release between male and female Ercc1 Δ/− mice and wild type mice and observed no differences ( data not shown ). However, significant differences were observed among components of the trigeminovascular system in both groups of mice. In both, Ercc1 Δ/− mice (overall Kruskal-Wallis test P < 0.0001) and wild type mice (overall Kruskal-Wallis test P = 0.002), CGRP release was the highest in the trigeminal nucleus caudalis, followed by the trigeminal ganglion, and the lowest in the dura mater. In Ercc1 Δ/− mice, post-hoc testing indicated significant differences between the trigeminal nucleus caudalis and trigeminal ganglion (adjusted P = 0.036) and the dura mater (adjusted P < 0.0001), but not between the trigeminal ganglion and dura mater (adjusted P = 0.146), Supplemental Fig. 1 . In wild type mice, post-hoc testing indicated only significant differences between the trigeminal nucleus caudalis and dura mater (adjusted P = 0.002) but not between the trigeminal nucleus caudalis and trigeminal ganglion (adjusted P = 0.093) nor between the trigeminal ganglion and dura mater (adjusted P = 0.575), Supplemental Fig. 2 . Peripheral vasodilatory responses in coronary arteries Coronary arteries were obtained from a total of 6 Ercc1 Δ/− mice (aged 90 [90–90] days, including 2 females) and 6 wild type mice (aged 91.5 [90.5–92.3] days, including 2 females). Responses to 30 mmol/L KCl resulted in a significantly lower mean contraction of 0.72 ± 0.13 mN in Ercc1 Δ/− mice and 1.91 ± 0.21 mN in wild type mice ( P = 0.002). Similarly, responses to 30 nmol/L of U46619 resulted in a significantly lower mean contraction of 1.10 ± 0.21 mN in Ercc1 Δ/− mice and 2.63 ± 0.24 mN in wild type mice ( P = 0.004), Supplemental Figs. 3 and 4 . Concentration-response curves of vasodilatory responses to CGRP and forskolin in coronaries obtained from wild type and Ercc1 Δ/− mice are depicted in Fig. 4 . Corresponding pEC 50 and E max values are presented for both mice groups in Table 1 . The concentration-response curves to CGRP and forskolin showed no differences in the potency, nor in the maximum response, between Ercc1 Δ/− and wild type mice. Table 1 – Vasodilatory responses to calcitonin-gene related peptide (CGRP) and forskolin. The mean ± SEM of the corresponding pEC 50 (i.e., negative log of the molar concentration of an agonist needed to reach half of its maximal effect) and average E max (maximum relaxation response) values are presented and compared between both wild type mice (n = 6) and Ercc1 Δ /− mice (n = 6). An asterisk ( * ) indicates P 0.999 102.68 ± 3.03 99.39 ± 2.79 0.132 DISCUSSION In this study, the effects of aging on trigeminovascular CGRP release and peripheral vascular responses of coronaries were investigated, comparing DNA repair-deficient Ercc1 Δ/− mice to their controls. We observed reduced CGRP release of the dura mater in Ercc1 Δ/− mice compared to wild type mice. No differences were observed in the trigeminal ganglion or in the central part of the trigeminovascular system, i.e. , the trigeminal nucleus caudalis. To the best of our knowledge, no previous studies have focused on the effect of aging on CGRP release in the trigeminovascular system. Admittedly, Bergman and colleagues observed an age-related reduction in CGRP expression, both at the cellular peptide level and mRNA level, in primary sensory neurons of the cervical and lumbar dorsal root ganglia of aged rats compared to young adults ( 36 ). These processes are hypothesized to be caused by aging-related lesions of axons. Our results might suggest that aging selectively affects peripheral nociceptive nerves in the dura mater rather than the entire trigeminovascular system – hinting towards a reduction in the density or functional integrity of nociceptive CGRP-positive nerve fibers in this structure. Indeed, previous preclinical studies in rats have shown a decrease in axoplasmic transport of CGRP in peripheral nerves ( 13 ) and changes in sensory nerve function, including impaired neuropeptide release from peripheral terminals, with advancing age ( 14 ). Rodent studies have also confirmed an age-related decline in CGRP in the peripheral vascular system. Further, immunoreactive CGRP levels were observed to be lower in both plasma and mesenteric (resistance) arteries of middle-aged female rats compared to young adult rats ( 37 ), and a decline in aortic CGRP-positive neuronal fibers was observed during aging ( 38 ). An age-dependent decline in CGRP concentrations has also been observed in human middle cerebral arteries ( 39 ). Our results on the dura mater confirm a previous study by De Vries et al. on age- and sex-dependent CGRP-induced vasodilation in human isolated vessels, distinguishing responses in both younger and older men and women ( 40 ). While the latter study did not focus on CGRP release, age-related differences were observed in middle meningeal arteries, with a negative correlation between the maximum response to CGRP with age in men. Whether this is caused by the decreased release in elderly, potentially leading to receptor downregulation, remains to be demonstrated. In our study, the dura mater exhibited the lowest levels of CGRP release compared to the trigeminal ganglion and trigeminal nucleus caudalis in both Ercc1 Δ/− and wild type mice. A previous rodent study showed that in the dura mater, CGRP is expressed in unmyelinated C-fibers, probably acting on CLR and RAMP1-expressing myelinated Aδ-fibers – of which an age-related decline has been previously observed ( 41 ) – mast cells, and vascular smooth muscle cells ( 11 ). Another study found no expression of these CGRP-receptor components on sensory axons in the cranial dura mater ( 42 ). It should be acknowledged that the applied hemiskull model in mice is relatively fragile, potentially resulting in large variability between mice and a relatively greater exclusion of CGRP release ratios of the dura mater; many research groups therefore favor the rat model instead ( 43 , 44 ). However, we were confined to the use of these mice, which allowed us to investigate the effects of neurovascular aging. We did not study the meningeal artery in the myograph because the dura mater had already been used for the aforementioned experiments. Therefore, we studied the coronary artery to assess potential peripheral responses and observed no significant differences in responses to CGRP and forskolin. While this is consistent with human data showing no differences in functional responses to CGRP in young and aged human coronary arteries ( 40 ), we cannot categorically exclude that the small number of coronary experiments limited the statistical power of our study, given the trend we observed for diminished CGRP responses in the Ercc1 Δ/− animals. If this would indeed be the case, as would be in accordance with other mouse studies ( 45 ), this would most likely involve a mechanism independent of downstream alterations in the smooth muscle cAMP signaling pathway, as in our study responses to forskolin were identical between groups, which is in accordance with a previous study showing no age-related changes in adenylate cyclase activity in rat aorta ( 46 ). In contrast, a potentially decreased response to CGRP could involve endothelial and/or cyclic guanosine monophosphate (cGMP)-dependent mechanisms, which we previously showed to be affected in these Ercc1 Δ/− mice ( 47 ). Indeed, while CGRP-induced relaxations in human coronary arteries are endothelium-independent ( 48 ), endothelium-dependent mechanisms seem to be involved in mice ( 49 ) and in rodent arteries precontracted with noradrenaline ( 50 ). Lastly, we compared trigeminovascular CGRP release between male and female mice and observed no differences. As the menstrual cycle is known to influence the CGRP-mediated trigeminovascular responsiveness in human experiments ( 51 , 52 ), it is important to note that both male and female Ercc1 Δ/− mice were previously shown to be infertile ( 53 ), and thus, sex differences may have been obscured. Clinical translation: Implications for migraine and future research The vascular dilatory capacity decreases with age, while the frequency and severity of migraine decline with age as well ( 6 ). However, no conclusive evidence exists on the exact neuronal and vascular age-related changes in migraine. While direct comparisons regarding CGRP release or activity cannot be made, our findings in the current study are in accordance with our previous study in humans, showing that capsaicin-induced trigeminal nerve-mediated vasodilation at the forehead was significantly reduced in postmenopausal women compared to younger women, while peripheral microvascular reactivity was preserved and comparable to that of younger women ( 52 ). Our findings might partly explain the reduction in migraine headache attacks in elderly, considering the important meningeal contribution to migraine pain. Indeed, the dura mater is pain-sensitive in proximity to the meningeal arteries, related to activation of dural perivascular nociceptors ( 54 ). Release of neuropeptides leads to vasodilation and degranulation of mast cells in the dura mater in animal models ( 12 ). In this context, it is tempting to hypothesize that the previously described reduction in migraine headache with advancing age ( 55 ) may relate to diminished dural CGRP release, possibly acting alongside age-related vascular stiffening in patients where migraine declines during lifetime ( 56 , 57 ). In contrast, migraine aura symptoms may persist into older age due to central mechanisms ( 55 ) – potentially reflected by unaltered CGRP release in the central structures of the trigeminovascular system as observed in our study – that are less affected by age. However, while we currently lack evidence to confirm or refute this hypothesis, future studies should address these peripheral and central age-dependent effects, including the influence on cortical spreading depression – a key mechanism underlying migraine aura ( 58 ). In our study, the decreased trigeminovascular CGRP release from the dura mater in Ercc1 Δ/− mice did not seem to be accompanied by an altered peripheral vascular reactivity to CGRP, although no conclusions regarding peripheral CGRP release can be drawn. Notably, we did not investigate the release of other neuropeptides, such as adrenomedullin and amylin, which often colocalize with CGRP, in the trigeminovascular system ( 59 , 60 ). Given that aging is one of the most significant risk factors for cardiovascular disease, that migraine is increasingly recognized as a cardiovascular risk factor, and that CGRP plays a key role in cardiovascular health and vascular aging processes ( 61 – 63 ), our results offer new pathophysiological insights into the involvement of specific vasodilatory mechanisms herein. CONCLUSION Taken together, this study demonstrates a reduction in CGRP release from the dura mater – but not from the trigeminal nucleus caudalis or the trigeminal ganglion – in a mouse model of accelerated aging. No differences in peripheral vasodilatory responses to CGRP and forskolin were found, suggesting that reduced trigeminovascular CGRP release is not associated with altered peripheral vascular responsiveness to CGRP in older mice – a finding which may, however, differ for other peptides and therefore warrants further research. An age-related decline in the density or functional integrity of nociceptive CGRP-positive nerve fibers in the dura mater may, at least partly, explain the reduction in migraine headache attacks in the elderly. Declarations Ethics approval The study was planned and carried out in accordance with the Principles of Laboratory Animal Care and with guidelines approved by an independent Animal Ethics Committee, consulted at the Erasmus MC, as well as the ARRIVE guidelines. Consent to participate or for publication Not applicable. Availability of data and materials Data are available upon reasonable request to the corresponding author. Conflict of Interest Statement Linda Al-Hassany, Eloisa Rubio-Beltrán, Alejandro Labastida-Ramirez, Anton JM Roks, Ingrid M Garrelds, and A.H. Jan Danser declare no conflict of interest with respect to the research, authorship, and/or publication of this article. Antoinette MaassenVanDenBrink received research grants and/or consultation fees from AbbVie, Amgen/Novartis, Eli Lilly, Lundbeck, Manistee, Pfizer, Satsuma, Teva, and Tonix. Funding This work was supported by the Dutch Research Council (ZonMw/NWO, Vici Grant (09150181910040). Authors’ Contributions LA-H performed the measurements, analyzed the data, and took the lead in writing the manuscript and visualizing the data. ER-B conceptualized the study, performed measurements, analyzed the data, and reviewed/edited the manuscript. AL-R conceptualized the study, performed measurements, and reviewed/edited the manuscript. IMG performed the CGRP measurements and reviewed/edited the manuscript. AJMR, AMvdB and AHJD conceptualized, designed and funded the study, and reviewed/edited the manuscript. AMvdB additionally supervised the study. All authors approved the final version of the manuscript. Acknowledgements We would like to thank Keivan Golshiri, Ehsan Ataei Ataabadi, Annika A. 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NeuroImage 85:592–607 Walker CS, Conner AC, Poyner DR, Hay DL (2010) Regulation of signal transduction by calcitonin gene-related peptide receptors. Trends Pharmacol Sci 31(10):476–483 Laurenza A, Sutkowski EM, Seamon KB (1989) Forskolin: a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action? Trends Pharmacol Sci 10(11):442–447 Guo S, Olesen J, Ashina M (2014) Phosphodiesterase 3 inhibitor cilostazol induces migraine-like attacks via cyclic AMP increase. Brain 137(Pt 11):2951–2959 Fried N, Elliott M, Oshinsky M (2017) The role of adenosine signaling in headache: A review. Brain Sci 7(3):30 Weeda G, Donker I, de Wit J, Morreau H, Janssens R, Vissers CJ et al (1997) Disruption of mouse ERCC1 results in a novel repair syndrome with growth failure, nuclear abnormalities and senescence. Curr Biol 7(6):427–439 Vermeij WP, Dollé MET, Reiling E, Jaarsma D, Payan-Gomez C, Bombardieri CR et al (2016) Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice. Nature 537(7620):427–431 Durik M, Kavousi M, van der Pluijm I, Isaacs A, Cheng C, Verdonk K et al (2012) Nucleotide excision DNA repair is associated with age-related vascular dysfunction. Circulation 126(4):468–478 Wu H, van Thiel BS, Bautista-Niño PK, Reiling E, Durik M, Leijten FPJ et al (2017) Dietary restriction but not angiotensin II type 1 receptor blockade improves DNA damage-related vasodilator dysfunction in rapidly aging Ercc1∆/- mice. Clin Sci (Lond) 131(15):1941–1953 Bautista-Niño PK, Portilla-Fernandez E, Rubio-Beltrán E, van der Linden JJ, de Vries R, van Veghel R et al (2020) Local endothelial DNA repair deficiency causes aging-resembling endothelial-specific dysfunction. Clin Sci (Lond) 134(7):727–746 Dollé MET, Kuiper RV, Roodbergen M, Robinson J, de Vlugt S, Wijnhoven SWP et al (2011) Broad segmental progeroid changes in short-lived Ercc1(-/∆7) mice. Pathobiol Aging Age Relat Dis 1(1):7219 Bautista Niño PK, Durik M, Danser AHJ, de Vries R, Musterd-Bhaggoe UM, Meima ME et al (2015) Phosphodiesterase 1 regulation is a key mechanism in vascular aging. Clin Sci (Lond) 129(12):1061–1075 van der Linden J, Stefens SJM, Heredia-Genestar JM, Ridwan Y, Brandt RMC, van Vliet N et al (2024) Ercc1 DNA repair deficiency results in vascular aging characterized by VSMC phenotype switching, ECM remodeling, and an increased stress response. Aging Cell 23(5):e14126 Chan KY, Labastida-Ramírez A, Ramírez-Rosas MB, Labruijere S, Garrelds IM, Danser AH et al (2019) Trigeminovascular calcitonin gene-related peptide function in Cacna1a R192Q-mutated knock-in mice. J Cereb Blood Flow Metab 39(4):718–729 Labastida-Ramírez A, Rubio-Beltrán E, Haanes KA, Chan KY, Garrelds IM, Johnson KW et al (2020) Lasmiditan inhibits calcitonin gene-related peptide release in the rodent trigeminovascular system. Pain 161(5):1092–1099 Mulvany MJ, Halpern W (1977) Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res 41(1):19–26 Bergman E, Johnson H, Zhang X, Hökfelt T, Ulfhake B (1996) Neuropeptides and neurotrophin receptor mRNAs in primary sensory neurons of aged rats. J Comp Neurol 375(2):303–319 Gangula PRR, Chauhan M, Reed L, Yallampalli C (2009) Age-related changes in dorsal root ganglia, circulating and vascular calcitonin gene-related peptide (CGRP) concentrations in female rats: effect of female sex steroid hormones. Neurosci Lett 454(2):118–123 Connat JL, Busseuil D, Gambert S, Ody M, Tébaldini M, Gamboni S et al (2001) Modification of the rat aortic wall during ageing; possible relation with decrease of peptidergic innervation. Anat Embryol (Berl) 204(6):455–468 Edvinsson L, Ekman R, Jansen I, Ottosson A, Uddman R (1987) Peptide-containing nerve fibers in human cerebral arteries: immunocytochemistry, radioimmunoassay, and in vitro pharmacology. Ann Neurol 21(5):431–437 de Vries T, Boucherie DM, Chan KY, Rubio-Beltrán E, Labastida-Ramírez A, Labruijere S et al (2024) Sex differences in CGRP-induced vasodilation of human middle meningeal arteries but not human coronary arteries: implications for migraine. Cephalalgia 44(7):3331024241254088 Chakour MC, Gibson SJ, Bradbeer M, Helme RD (1996) The effect of age on Aδ- and C-fibre thermal pain perception. Pain 64(1):143–152 Lennerz JK, Rühle V, Ceppa EP, Neuhuber WL, Bunnett NW, Grady EF et al (2008) Calcitonin receptor-like receptor (CLR), receptor activity‐modifying protein 1 (RAMP1), and calcitonin gene‐related peptide (CGRP) immunoreactivity in the rat trigeminovascular system: Differences between peripheral and central CGRP receptor distribution. J Comp Neurol 507(4):spc1–spc1 Jansen-Olesen I, Baun M, Amrutkar DV, Ramachandran R, Christophersen DV, Olesen J (2014) PACAP-38 but not VIP induces release of CGRP from trigeminal nucleus caudalis via a receptor distinct from the PAC1 receptor. Neuropeptides 48(2):53–64 Dux M, Vogler B, Kuhn A, Mackenzie KD, Stratton J, Messlinger K (2022) The anti-CGRP antibody fremanezumab lowers CGRP release from rat Dura mater and meningeal blood flow. Cells 11(11):1768 King R, Smillie SJ, Bodkin JV, Brain SD (2012) Impaired vascular responses to calcitonin gene-related peptide in the ageing α-cgrp knockout mouse. Heart 98(Suppl 5):A91–A9 Schoeffter P, Stocklet JC (1990) Age-related differences in cyclic AMP metabolism and their consequences on relaxation induced by isoproterenol and phosphodiesterase inhibitors in rat isolated aorta. Mech Ageing Dev 54(3):197–205 Golshiri K, Ataei Ataabadi E, Rubio-Beltran E, Dutheil S, Yao W, Snyder GL et al (2021) Selective phosphodiesterase 1 inhibition ameliorates vascular function, reduces inflammatory response, and lowers blood pressure in aging animals. J Pharmacol Exp Ther 378(2):173–183 de Vries T, Labruijere S, Rivera-Mancilla E, Garrelds IM, de Vries R, Schutter D et al (2024) Intracellular pathways of calcitonin gene-related peptide-induced relaxation of human coronary arteries: A key role for Gβγ subunit instead of cAMP. Br J Pharmacol 181(15):2478–2491 Rosenblum WI, Shimizu T, Nelson GH (1993) Endothelium-dependent effects of substance P and calcitonin gene-related peptide on mouse pial arterioles. Stroke 24(7):1043–1047 discussion 1047–8 Wang X, Han C, Fiscus RR (1991) Calcitonin gene-related peptide (CGRP) causes endothelium-dependent cyclic AMP, cyclic GMP and vasorelaxant responses in rat abdominal aorta. Neuropeptides 20(2):115–124 Ibrahimi K, Vermeersch S, Frederiks P, Geldhof V, Draulans C, Buntinx L et al (2017) The influence of migraine and female hormones on capsaicin-induced dermal blood flow. Cephalalgia 37(12):1164–1172 Ibrahimi K, van Oosterhout WPJ, van Dorp W, Danser AHJ, Garrelds IM, Kushner SA et al (2015) Reduced trigeminovascular cyclicity in patients with menstrually related migraine. Neurology 84(2):125–131 Hsia KT, Millar MR, King S, Selfridge J, Redhead NJ, Melton DW et al (2003) DNA repair gene Ercc1 is essential for normal spermatogenesis and oogenesis and for functional integrity of germ cell DNA in the mouse. Development 130(2):369–378 Khan S, Amin FM, Christensen CE, Ghanizada H, Younis S, Olinger ACR et al (2019) Meningeal contribution to migraine pain: a magnetic resonance angiography study. Brain 142(1):93–102 Bigal ME, Liberman JN, Lipton RB (2006) Age-dependent prevalence and clinical features of migraine. Neurology 67(2):246–251 Ibrahimi K, Rist PM, Carpenet C, Lee Rohmann J, Buring JE, van den Maassen A et al (2022) Vascular Risk Score and Associations With Past, Current, or Future Migraine in Women: Cohort Study. Neurology 99(16):e1694–e1701 Al-Hassany L, MaassenVanDenBrink A, Kurth T (2024) Cardiovascular risk scores and migraine status. JAMA Netw Open 7(10):e2440577 Pietrobon D, Brennan KC (2019) Genetic mouse models of migraine. J Headache Pain. ;20(79) Sundrum T, Walker CS (2018) Pituitary adenylate cyclase-activating polypeptide receptors in the trigeminovascular system: implications for migraine. Br J Pharmacol 175(21):4109–4120 Edvinsson L, Warfvinge K (2021) In: Headache (ed) The CGRP family of neuropeptides and their receptors in the trigeminovascular system. Springer International Publishing, Cham, pp 1–12 Favoni V, Giani L, Al-Hassany L, Asioli GM, Butera C, de Boer I et al (2019) CGRP and migraine from a cardiovascular point of view: what do we expect from blocking CGRP? J Headache Pain 20(1):27 Mahmoud AN, Mentias A, Elgendy AY, Qazi A, Barakat AF, Saad M et al (2018) Migraine and the risk of cardiovascular and cerebrovascular events: a meta-analysis of 16 cohort studies including 1 152 407 subjects. BMJ Open 8(3):e020498 Cui XY, Zhan JK (2025) Capsaicin and TRPV1: A novel therapeutic approach to mitigate vascular aging. Aging Dis 17(1):256–274 Additional Declarations Competing interest reported. Linda Al-Hassany, Eloisa Rubio-Beltrán, Alejandro Labastida-Ramirez, Anton JM Roks, Ingrid M Garrelds, and A.H. Jan Danser declare no conflict of interest with respect to the research, authorship, and/or publication of this article. Antoinette MaassenVanDenBrink received research grants and/or consultation fees from AbbVie, Amgen/Novartis, Eli Lilly, Lundbeck, Manistee, Pfizer, Satsuma, Teva, and Tonix. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9187264","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":611341937,"identity":"37250160-0851-49dc-81f7-aa415a6a9301","order_by":0,"name":"Linda Al-Hassany","email":"","orcid":"","institution":"Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"Linda","middleName":"","lastName":"Al-Hassany","suffix":""},{"id":611341938,"identity":"42025cf8-3794-42c4-8ac6-d62df082f262","order_by":1,"name":"Eloisa Rubio-Beltrán","email":"","orcid":"","institution":"Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"Eloisa","middleName":"","lastName":"Rubio-Beltrán","suffix":""},{"id":611341939,"identity":"572a84ff-3ab5-442d-a083-8a5a82208da1","order_by":2,"name":"Alejandro Labastida-Ramirez","email":"","orcid":"","institution":"Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"Alejandro","middleName":"","lastName":"Labastida-Ramirez","suffix":""},{"id":611341940,"identity":"2eea7570-8ca5-4e89-93c7-07fc38c8e7b9","order_by":3,"name":"Ingrid M Garrelds","email":"","orcid":"","institution":"Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"Ingrid","middleName":"M","lastName":"Garrelds","suffix":""},{"id":611341941,"identity":"a0bcdcc6-e098-401f-ba02-22ed1398d769","order_by":4,"name":"A. H. Jan Danser","email":"","orcid":"","institution":"Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"H. Jan","lastName":"Danser","suffix":""},{"id":611341942,"identity":"71da601a-0eb3-4a44-80c6-445237a30d13","order_by":5,"name":"Anton JM Roks","email":"","orcid":"","institution":"Erasmus MC","correspondingAuthor":false,"prefix":"","firstName":"Anton","middleName":"JM","lastName":"Roks","suffix":""},{"id":611341943,"identity":"ebccaca6-b771-4cc4-838d-60fde471402e","order_by":6,"name":"Antoinette MaassenVanDenBrink","email":"data:image/png;base64,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","orcid":"","institution":"Erasmus MC","correspondingAuthor":true,"prefix":"","firstName":"Antoinette","middleName":"","lastName":"MaassenVanDenBrink","suffix":""}],"badges":[],"createdAt":"2026-03-21 17:08:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9187264/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9187264/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105378650,"identity":"85e6b18a-090a-40fc-89dd-d2612d1733c9","added_by":"auto","created_at":"2026-03-25 10:42:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28649,"visible":true,"origin":"","legend":"\u003cp\u003eBar plots with the mean ± SEM of the relative stimulated calcitonin gene-related peptide (CGRP) release ratios of the trigeminal nucleus caudalis (TNC) after potassium chloride (KCl) administration in wild type (n = 27) and \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/−\u003c/em\u003e\u003c/sup\u003e mice (n = 23) (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9187264/v1/15f3489bcdaac09deed6b306.png"},{"id":105378651,"identity":"68d13de4-b288-48e0-88d0-0bcd2efa7b4f","added_by":"auto","created_at":"2026-03-25 10:42:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":26018,"visible":true,"origin":"","legend":"\u003cp\u003eBar plots with the mean ± SEM of the relative stimulated calcitonin gene-related peptide (CGRP) release ratios of the trigeminal ganglion (TG) after potassium chloride (KCl) administration in wild type (n = 23) and \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/−\u003c/em\u003e\u003c/sup\u003e mice (n = 20) (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9187264/v1/82fb4b8d8fe250a78f20aa74.png"},{"id":105378653,"identity":"5e7a20c7-a1d7-4858-a20a-f2c95003bbcb","added_by":"auto","created_at":"2026-03-25 10:42:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24701,"visible":true,"origin":"","legend":"\u003cp\u003eBar plots with the mean ± SEM of the relative stimulated calcitonin gene-related peptide (CGRP) release ratios of the dura mater after potassium chloride (KCl) administration in wild type (n = 19) and \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/−\u003c/em\u003e\u003c/sup\u003e mice (n = 15) (\u003cstrong\u003e*\u003c/strong\u003e indicates \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9187264/v1/9f60f7e3372f1bdf256bfc58.png"},{"id":105378642,"identity":"df66e0a3-56bd-41b7-b817-b3eda5a3c9a2","added_by":"auto","created_at":"2026-03-25 10:42:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68732,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration-response curve showing relaxation in response to calcitonin-gene related peptide (CGRP) (A) and forskolin (B), relative to either potassium chloride (KCl) or U46619, in coronary arteries of wild type mice (n = 6) and\u003cem\u003e Ercc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/−\u003c/em\u003e\u003c/sup\u003e mice (n = 6).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9187264/v1/ddbf1e3df2dd1ea48fe39bfe.png"},{"id":105565893,"identity":"9ffa61cb-5f80-4c9f-8964-5883a466fa63","added_by":"auto","created_at":"2026-03-27 12:54:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1076959,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9187264/v1/bfeb8dc3-93ee-4511-8f72-efce14e9e12a.pdf"},{"id":105378629,"identity":"25849e94-9e8c-4fe0-ae8c-e2d2f0d99984","added_by":"auto","created_at":"2026-03-25 10:42:35","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1757782,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9187264/v1/f5fbb6758147da96e6a507b2.docx"}],"financialInterests":"Competing interest reported. Linda Al-Hassany, Eloisa Rubio-Beltrán, Alejandro Labastida-Ramirez, Anton JM Roks, Ingrid M Garrelds, and A.H. Jan Danser declare no conflict of interest with respect to the research, authorship, and/or publication of this article.\n\nAntoinette MaassenVanDenBrink received research grants and/or consultation fees from AbbVie, Amgen/Novartis, Eli Lilly, Lundbeck, Manistee, Pfizer, Satsuma, Teva, and Tonix.","formattedTitle":"Trigeminovascular Calcitonin Gene-Related Peptide Release and Peripheral Vascular Responses in a Mouse Model of Accelerated Aging: Implications for Migraine","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMigraine is a highly disabling and stigmatizing primary headache disorder that is characterized by unilateral moderate to severe headache attacks and associated symptoms (\u003cem\u003ei.e.\u003c/em\u003e, nausea, vomiting, photo- and phonophobia), which may be preceded or accompanied by aura symptoms in approximately one-third of patients (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The overall migraine prevalence is (at least) two-fold higher in women compared to men (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), while the migraine peak prevalence occurs at similar ages in both sexes \u0026ndash; namely during the reproductive years (approximately 20s and 30s) with a second peak in women around fifty years of age (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Both the incidence and prevalence of migraine tend to decrease with advancing age (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Understanding the neurovascular basis of these age-related changes is pivotal, especially considering the increase in global life expectancy (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe exact pathophysiological age-dependent mechanisms are not completely understood, but neuronal and vascular changes, besides hormonal senescence, have been hypothesized to be involved in migraine remission at older ages (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Activation of the trigeminovascular system and concomitant release of the vasodilatory neuropeptide calcitonin gene-related peptide (CGRP), expressed in both the central and peripheral components of the trigeminovascular system, play a causal role in the headache phase of migraine (\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Interestingly, in rats, transport of CGRP in nerve fibers has been shown to decline significantly with advancing age (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In addition, \u003cem\u003ein vivo\u003c/em\u003e studies have shown age-dependent changes in both pre- and post-synaptic peripheral fibers of these animals. These data indicate a reduction in the number of sensory fibers and/or a decrease in their peptide content or release, impacting the activation of the trigeminovascular system (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Such a decline in CGRP content was also observed in specific brain structures, \u003cem\u003ei.e.\u003c/em\u003e, substantia nigra and striatum, as well as in cardiovascular tissue of aging rats (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) and in guinea pigs, whose maximum density of CGRP-containing perivascular nerve plexuses declined to approximately half in older age (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Being a neurovascular condition and an important cardiovascular risk factor (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), these alterations in trigeminal pain pathways should not be considered in isolation from the vascular age-related changes associated with migraine. Accordingly, previous research suggests that aging is associated with reduced cerebrovascular vasodilatory capacitance, probably accompanied by decreased activation of sensory neurons (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) and disrupted neurovascular coupling (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is yet unclear whether age-dependent differences in CGRP release exist across the central and peripheral structures of the trigeminovascular system. Therefore, the primary aim of this study was to investigate the effects of aging on CGRP release from the trigeminal nucleus caudalis (central trigeminovascular component), trigeminal ganglion, and dura mater (both peripheral trigeminovascular components) by comparing CGRP release in a mouse model of accelerated aging, the DNA repair-deficient \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, \u003cem\u003eversus\u003c/em\u003e their wild type littermates. The secondary objective was to deepen our understanding of the connection between neuronal and vascular age-related changes. We therefore studied vasodilatory responses of coronary arteries to CGRP and forskolin in \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and wild type mice, as both lead to the accumulation of intracellular levels of cyclic adenosine monophosphate (cAMP) (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), which, in turn, has been described to play a role in migraine induction (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Further explorative analyses were conducted to evaluate the differences in trigeminovascular CGRP release between i) male and female mice and ii) the three components of the trigeminovascular system.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental mice\u003c/h2\u003e \u003cp\u003eExperiments were performed using both male and female DNA repair-deficient \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and their controls, \u003cem\u003ei.e.\u003c/em\u003e, wild type littermates (\u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice). The \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice have one exon 7-truncated and one completely inactivated allele of the endonuclease \u003cem\u003eErcc1\u003c/em\u003e, thus partially inactivating this endonuclease that is involved in nucleotide excision and interstrand DNA crosslink repair. \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice with an F1 C57BL6J/FVB hybrid background were obtained, preventing strain-specific phenotypes, as previously described (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice represent a well-established model of accelerated neurovascular aging due to genomic instability, characterized by age-related diseases and a reduced lifespan (24\u0026ndash;28 weeks compared to at least 100 weeks, and up to 146 weeks, in wild type mice). Overall, the accelerated aging phenotype includes neurodegeneration, osteoporosis, early-onset hypertension, vascular stiffness, accelerated endothelial and vasodilatory dysfunction, and cardiomyopathy (\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), associated with an increase in phosphodiesterase 1 expression (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In addition, aortas from \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibit phenotypic switching of vascular smooth muscle cells and an increased stress response (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBreeding took place at the animal facility of the Erasmus Medical Center (Erasmus MC). Mice were kept in individually ventilated cages under controlled conditions (20\u0026ndash;22\u0026deg;C, 12-hour light/dark cycle) with free access to standard chow and water. All mice were weighed and visually inspected daily to monitor their well-being. The study consisted of three mice batches with identical genotypes, and all mice were sacrificed between 88 and 117 days of age. The study was planned and carried out in accordance with the Principles of Laboratory Animal Care and with guidelines approved by an independent Animal Ethics Committee, consulted at the Erasmus MC, as well as the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCalcitonin gene-related peptide release experiments in the trigeminal nucleus caudalis, trigeminal ganglion, and dura mater\u003c/h3\u003e\n\u003cp\u003eThe technique used to measure \u003cem\u003eex vivo\u003c/em\u003e CGRP release in the central (trigeminal nucleus caudalis) and peripheral (dura mater and trigeminal ganglion) components of the trigeminovascular system has been described previously (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In short, mice were anesthetized using intraperitoneal sodium pentobarbital (80 mg/kg) and decapitated at the atlantooccipital joint. The skin and galea aponeurotica were both pulled back from the cranium. The trigeminal nucleus caudalis, located caudally between 9\u0026ndash;13 mm from bregma, was first carefully isolated from the brainstem and divided into two parts. The skull was subsequently divided into two halves along the sagittal suture, and both cerebral hemispheres were removed, leaving the cranial dura mater intact and attached to the skull. The trigeminal ganglia of both sides were then obtained by dissecting 1 mm proximal and distal to the point where the mandibular nerve branches off. The final step consisted of carefully hemisecting the cranium and removing the remaining (brain) tissue from the skull, except for the dura mater. Each (half) of the isolated trigeminal nucleus caudalis, trigeminal ganglion, and skull with dura mater were immersed and washed in carbogenated synthetic interstitial fluid, containing NaCl (108 mM), KCl (3.48 mM), MgSO\u003csub\u003e4\u003c/sub\u003e (3.5 mM), NaHCO\u003csub\u003e3\u003c/sub\u003e (26 mM), NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (11.7 mM), CaCl\u003csub\u003e2\u003c/sub\u003e (1.5 mM), sodium gluconate (9.6 mM), glucose (5.55 mM), and sucrose (7.6 mM) for three times 30 minutes at 37\u0026deg;C to reduce false positive results.\u003c/p\u003e \u003cp\u003eBoth halves of the isolated tissues were then placed in a 24-well plate containing 500 \u0026micro;L of synthetic interstitial fluid per well. The plate was fixed in a water bath, creating a closed, humid and carbogenated chamber of 37\u0026deg;C. Tissues were washed three additional times over a period of up to 15 minutes. Basal CGRP levels were measured in each tissue, after which CGRP release was induced by adding 60 mM KCl. Both the baseline and KCl-infused samples were collected after 10 minutes of incubation and mixed with aprotinin (500 KIU/mL).\u003c/p\u003e \u003cp\u003eSamples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until CGRP content was assessed using a commercial CGRP RIA kit according to the manual (Phoenix Pharmaceuticals, Burlingame, CA, USA). The assay has a detection level of 1.0 pg/mL; samples with CGRP concentrations below this limit were assigned this value of 1.0 pg/mL, in line with our previous study (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Blanks containing only synthetic interstitial fluid, without CGRP, were used as a control to exclude false-positive measurements. For the CGRP assays, the analyst was blinded to the mice genotype. CGRP release was assessed in duplicate for both individual halves of each trigeminovascular structure, of which averages were used for further analyses. CGRP release was expressed as the ratio of KCl-induced CGRP release to basal CGRP release, which represents the relative stimulated release of CGRP (\u003cem\u003ei.e.\u003c/em\u003e, CGRP release ratio). Tissues that exhibited either no or decreased relative CGRP release in response to 60 mM KCl (\u003cem\u003ei.e.\u003c/em\u003e, CGRP release ratios\u0026thinsp;\u0026le;\u0026thinsp;1.05) or an unusually high relative release (\u003cem\u003ei.e.\u003c/em\u003e, CGRP release ratios\u0026thinsp;\u0026ge;\u0026thinsp;100) were excluded from further analyses, consistent with our previous study on CGRP release in mice (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). As the \u003cem\u003eex vivo\u003c/em\u003e experiments consisted of three mouse batches, an outlier test \u0026ndash; \u003cem\u003ei.e.\u003c/em\u003e, the ROUT method (Q\u0026thinsp;=\u0026thinsp;1%) \u0026ndash; was performed on each individual batch and the combined data of all batches.\u003c/p\u003e\n\u003ch3\u003ePeripheral vasodilatory experiments in coronary arteries\u003c/h3\u003e\n\u003cp\u003eImmediately after sacrificing the mice, coronary arteries were carefully isolated in oxygenated and carbogenated Krebs buffer solution (NaCl 118 mM, KCl 4.7 mM, CaCl\u003csub\u003e2\u003c/sub\u003e 2.5 mM, MgSO\u003csub\u003e4\u003c/sub\u003e 1.2 mM, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1.2 mM, NaHCO\u003csub\u003e3\u003c/sub\u003e 25 mM, and glucose 8.3 mM in distilled water, pH 7.4).\u003c/p\u003e \u003cp\u003eFunctional experiments on the coronaries were conducted only for 6 wild type and 6 \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice concurrently with the CGRP release experiments. For these experiments, one vessel segment of 2 mm length per mouse was mounted in small wire Mulvany myograph organ baths (Danish Myograph Technology, Aarhus, Denmark), containing 6 mL of oxygenated and carbogenated Krebs solution at 37\u0026deg;C. The mounted vessel segments were first left to equilibrate before the tension was normalized by stepwise stretching vessels to a tension corresponding to 90% of the estimated diameter at a transmural pressure of 100 mmHg (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). After four washing steps, each separated by a 5-minute interval, maximum contractile responses were determined using 60 mmol/L KCl as a reference contraction. To evaluate vasodilatory responses, coronary segments were precontracted with 30 nmol/L of U46619 (a thromboxane A\u003csub\u003e2\u003c/sub\u003e analog) to study the effects of forskolin and with 30 mmol/L of KCl (facilitating comparison with human data from our lab, where precontractions are also induced by KCl) to study the effects of CGRP. After reaching a contraction plateau, concentration-response curves were sequentially generated in a single segment for the following vasodilators using cumulative doses: CGRP (10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e\u0026ndash;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e mol/L in half-logarithmic steps) and forskolin (10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u0026ndash;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e mol/L in half-logarithmic steps).\u003c/p\u003e \u003cp\u003eData were recorded using LabChart data acquisition software (AD Instruments Ltd, Oxford, UK). Relaxation responses to the vasodilatory substances are expressed as percentages (%) relative to the contraction induced by either 30 nmol/L U46619 or 30 mmol/L KCl, which was set at 100%. Concentration-response curves with a sigmoidal shape were constructed using a computerized curve-fitting technique, in which the bottom was constrained to zero. Non-linear regression analysis was used to determine the pEC\u003csub\u003e50\u003c/sub\u003e (negative log of the molar concentration of an agonist needed to reach half of its maximal effect) and E\u003csub\u003emax\u003c/sub\u003e (maximum relaxation response) values.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eCGRP release ratios and percentages of relaxation are all expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Age is expressed as median [Q\u003csub\u003e1\u003c/sub\u003e\u0026ndash;Q\u003csub\u003e3\u003c/sub\u003e]. Statistical differences in the relative CGRP release, pEC\u003csub\u003e50\u003c/sub\u003e and E\u003csub\u003emax\u003c/sub\u003e values between wild type and \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were calculated using the two-tailed Mann-Whitney test for unpaired observations. A nonparametric test was applied to compare differences in CGRP release, in accordance with previous studies (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), and due to the small sample size of vasodilatory responses in coronary arteries. To additionally explore differences between male and female wild type and \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, a Kruskal-Wallis test with a post-hoc test, corrected Dunn's multiple comparisons tests, was applied. Similarly, differences in CGRP release between the trigeminal nucleus caudalis, trigeminal ganglion, and dura mater were analyzed using the Kruskal-Wallis test with Dunn's multiple comparisons test, stratified by wild type and \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003eThe statistical significance level was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05. All analyses were performed using GraphPad Prism version 8.0.1 for Windows (GraphPad Software, Boston, Massachusetts USA).\u003c/p\u003e\n\u003ch3\u003eCompounds\u003c/h3\u003e\n\u003cp\u003eForskolin was obtained from Tocris BioScience (Bristol, UK) and dissolved in physiological saline. U46619 was purchased from Sigma Chemical Co. (St. Louis, MO, USA); r-α-CGRP was obtained from PolyPeptide (Strasbourg, France).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCalcitonin gene-related peptide release in the trigeminal nucleus caudalis, trigeminal ganglion, and dura mater\u003c/h2\u003e \u003cp\u003eCGRP release experiments were performed on a total of 27 \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and 29 wild type mice, of whom tissues were obtained and analyzed from both halves. From 1 \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mouse, no trigeminal nucleus caudalis and trigeminal ganglion were obtained due to damaged tissue.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTrigeminal nucleus caudalis\u003c/h3\u003e\n\u003cp\u003eExclusion of individual tissues of the trigeminal nucleus caudalis based on the previously mentioned criteria of the CGRP release ratio and outlier tests yielded averaged ratios from 23 \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and 27 wild type mice. The \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice consisted of 9 males (39%) and 14 females (61%) with a median age of 92 [90\u0026ndash;111] days. The wild type mice consisted of 14 males (52%) and 13 females (48%) with a median age of 110 [96\u0026ndash;114] days. No significant differences were observed in CGRP release of the trigeminal nucleus caudalis between \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (8.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15) and wild type mice (6.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.364), Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTrigeminal ganglion\u003c/h2\u003e \u003cp\u003eExclusion of individual tissues of the trigeminal ganglion based on the previously mentioned criteria yielded averaged ratios from 20 \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and 23 wild type mice. The \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice consisted of 10 males (50%) and 10 females (50%) with a median [Q\u003csub\u003e1\u003c/sub\u003e\u0026ndash;Q\u003csub\u003e3\u003c/sub\u003e] age of 91 [90\u0026ndash;106.8] days. The wild type mice consisted of 11 males (48%) and 12 females (52%) with a median age of 110 [92\u0026ndash;114] days. No significant differences were observed in CGRP release of the trigeminal ganglion between \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (4.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92) and wild type mice (4.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.838), Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDura mater\u003c/h2\u003e \u003cp\u003eExclusion of individual tissues of the dura mater based on the previously mentioned criteria yielded averaged ratios from 15 \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and 19 wild type mice. The \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice consisted of 8 males (53%) and 7 females (47%) with a median [Q\u003csub\u003e1\u003c/sub\u003e\u0026ndash;Q\u003csub\u003e3\u003c/sub\u003e] age of 92 [90\u0026ndash;112] days. The wild type mice consisted of 11 males (58%) and 8 females (42%) with a median age of 112 [95\u0026ndash;114] days. Significantly lower CGRP release of the dura mater were observed in \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (2.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24) compared to wild type mice (3.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.040), Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnalyses on male-female differences and different trigeminovascular system components\u003c/h2\u003e \u003cp\u003eWe further explored differences in CGRP release between male and female \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and wild type mice and observed no differences (\u003cem\u003edata not shown\u003c/em\u003e). However, significant differences were observed among components of the trigeminovascular system in both groups of mice. In both, \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (overall Kruskal-Wallis test \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and wild type mice (overall Kruskal-Wallis test \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), CGRP release was the highest in the trigeminal nucleus caudalis, followed by the trigeminal ganglion, and the lowest in the dura mater. In \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, post-hoc testing indicated significant differences between the trigeminal nucleus caudalis and trigeminal ganglion (adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.036) and the dura mater (adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), but not between the trigeminal ganglion and dura mater (adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.146), \u003cb\u003eSupplemental Fig.\u0026nbsp;1\u003c/b\u003e. In wild type mice, post-hoc testing indicated only significant differences between the trigeminal nucleus caudalis and dura mater (adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) but not between the trigeminal nucleus caudalis and trigeminal ganglion (adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.093) nor between the trigeminal ganglion and dura mater (adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.575), \u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePeripheral vasodilatory responses in coronary arteries\u003c/h2\u003e \u003cp\u003eCoronary arteries were obtained from a total of 6 \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (aged 90 [90\u0026ndash;90] days, including 2 females) and 6 wild type mice (aged 91.5 [90.5\u0026ndash;92.3] days, including 2 females). Responses to 30 mmol/L KCl resulted in a significantly lower mean contraction of 0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mN in \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and 1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mN in wild type mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). Similarly, responses to 30 nmol/L of U46619 resulted in a significantly lower mean contraction of 1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mN in \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and 2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 mN in wild type mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004), \u003cb\u003eSupplemental Figs.\u0026nbsp;3 and 4\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eConcentration-response curves of vasodilatory responses to CGRP and forskolin in coronaries obtained from wild type and \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Corresponding pEC\u003csub\u003e50\u003c/sub\u003e and E\u003csub\u003emax\u003c/sub\u003e values are presented for both mice groups in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The concentration-response curves to CGRP and forskolin showed no differences in the potency, nor in the maximum response, between \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and wild type mice.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003e\u0026ndash; Vasodilatory responses to calcitonin-gene related peptide (CGRP) and forskolin. The mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of the corresponding pEC\u003csub\u003e50\u003c/sub\u003e (i.e., negative log of the molar concentration of an agonist needed to reach half of its maximal effect) and average E\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e(maximum relaxation response) values are presented and compared between both wild type mice (n = 6) and\u003cem\u003e\u0026nbsp;Ercc1\u003c/em\u003e\u003cem\u003e\u003csup\u003e\u0026Delta;\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u003csup\u003e/\u0026minus;\u003c/sup\u003e\u003c/em\u003e mice (n = 6). An asterisk (\u003cstrong\u003e*\u003c/strong\u003e) indicates \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"924\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubstance\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 396px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epEC\u003csub\u003e50\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 396px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eE\u003csub\u003emax\u0026nbsp;\u003c/sub\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u003cu\u003eErcc1\u003c/u\u003e\u003c/em\u003e\u003cem\u003e\u003csup\u003e\u0026Delta;/\u0026minus;\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cu\u003eWild type\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cu\u003e\u0026nbsp;\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u003cu\u003eErcc1\u003c/u\u003e\u003c/em\u003e\u003cem\u003e\u003csup\u003e\u0026Delta;/\u0026minus;\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cu\u003eWild type\u003c/u\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCGRP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e8.00 \u0026plusmn; 0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e8.63 \u0026plusmn; 0.08\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e22.86 \u0026plusmn; 1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e43.23 \u0026plusmn; 11.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.132\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eForskolin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e6.07 \u0026plusmn; 0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e6.07 \u0026plusmn; 0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026gt;0.999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e102.68 \u0026plusmn; 3.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e99.39 \u0026plusmn; 2.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.132\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, the effects of aging on trigeminovascular CGRP release and peripheral vascular responses of coronaries were investigated, comparing DNA repair-deficient \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice to their controls. We observed reduced CGRP release of the dura mater in \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice compared to wild type mice. No differences were observed in the trigeminal ganglion or in the central part of the trigeminovascular system, \u003cem\u003ei.e.\u003c/em\u003e, the trigeminal nucleus caudalis.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, no previous studies have focused on the effect of aging on CGRP release in the trigeminovascular system. Admittedly, Bergman and colleagues observed an age-related reduction in CGRP expression, both at the cellular peptide level and mRNA level, in primary sensory neurons of the cervical and lumbar dorsal root ganglia of aged rats compared to young adults (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). These processes are hypothesized to be caused by aging-related lesions of axons. Our results might suggest that aging selectively affects peripheral nociceptive nerves in the dura mater rather than the entire trigeminovascular system \u0026ndash; hinting towards a reduction in the density or functional integrity of nociceptive CGRP-positive nerve fibers in this structure. Indeed, previous preclinical studies in rats have shown a decrease in axoplasmic transport of CGRP in peripheral nerves (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) and changes in sensory nerve function, including impaired neuropeptide release from peripheral terminals, with advancing age (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Rodent studies have also confirmed an age-related decline in CGRP in the peripheral vascular system. Further, immunoreactive CGRP levels were observed to be lower in both plasma and mesenteric (resistance) arteries of middle-aged female rats compared to young adult rats (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), and a decline in aortic CGRP-positive neuronal fibers was observed during aging (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). An age-dependent decline in CGRP concentrations has also been observed in human middle cerebral arteries (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Our results on the dura mater confirm a previous study by De Vries et al. on age- and sex-dependent CGRP-induced vasodilation in human isolated vessels, distinguishing responses in both younger and older men and women (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). While the latter study did not focus on CGRP release, age-related differences were observed in middle meningeal arteries, with a negative correlation between the maximum response to CGRP with age in men. Whether this is caused by the decreased release in elderly, potentially leading to receptor downregulation, remains to be demonstrated.\u003c/p\u003e \u003cp\u003eIn our study, the dura mater exhibited the lowest levels of CGRP release compared to the trigeminal ganglion and trigeminal nucleus caudalis in both \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and wild type mice. A previous rodent study showed that in the dura mater, CGRP is expressed in unmyelinated C-fibers, probably acting on CLR and RAMP1-expressing myelinated Aδ-fibers \u0026ndash; of which an age-related decline has been previously observed (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e) \u0026ndash; mast cells, and vascular smooth muscle cells (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Another study found no expression of these CGRP-receptor components on sensory axons in the cranial dura mater (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). It should be acknowledged that the applied hemiskull model in mice is relatively fragile, potentially resulting in large variability between mice and a relatively greater exclusion of CGRP release ratios of the dura mater; many research groups therefore favor the rat model instead (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). However, we were confined to the use of these mice, which allowed us to investigate the effects of neurovascular aging.\u003c/p\u003e \u003cp\u003eWe did not study the meningeal artery in the myograph because the dura mater had already been used for the aforementioned experiments. Therefore, we studied the coronary artery to assess potential peripheral responses and observed no significant differences in responses to CGRP and forskolin. While this is consistent with human data showing no differences in functional responses to CGRP in young and aged human coronary arteries (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), we cannot categorically exclude that the small number of coronary experiments limited the statistical power of our study, given the trend we observed for diminished CGRP responses in the \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals. If this would indeed be the case, as would be in accordance with other mouse studies (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), this would most likely involve a mechanism independent of downstream alterations in the smooth muscle cAMP signaling pathway, as in our study responses to forskolin were identical between groups, which is in accordance with a previous study showing no age-related changes in adenylate cyclase activity in rat aorta (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). In contrast, a potentially decreased response to CGRP could involve endothelial and/or cyclic guanosine monophosphate (cGMP)-dependent mechanisms, which we previously showed to be affected in these \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Indeed, while CGRP-induced relaxations in human coronary arteries are endothelium-independent (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), endothelium-dependent mechanisms seem to be involved in mice (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) and in rodent arteries precontracted with noradrenaline (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLastly, we compared trigeminovascular CGRP release between male and female mice and observed no differences. As the menstrual cycle is known to influence the CGRP-mediated trigeminovascular responsiveness in human experiments (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), it is important to note that both male and female \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were previously shown to be infertile (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), and thus, sex differences may have been obscured.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eClinical translation: Implications for migraine and future research\u003c/h2\u003e \u003cp\u003eThe vascular dilatory capacity decreases with age, while the frequency and severity of migraine decline with age as well (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, no conclusive evidence exists on the exact neuronal and vascular age-related changes in migraine. While direct comparisons regarding CGRP release or activity cannot be made, our findings in the current study are in accordance with our previous study in humans, showing that capsaicin-induced trigeminal nerve-mediated vasodilation at the forehead was significantly reduced in postmenopausal women compared to younger women, while peripheral microvascular reactivity was preserved and comparable to that of younger women (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur findings might partly explain the reduction in migraine headache attacks in elderly, considering the important meningeal contribution to migraine pain. Indeed, the dura mater is pain-sensitive in proximity to the meningeal arteries, related to activation of dural perivascular nociceptors (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Release of neuropeptides leads to vasodilation and degranulation of mast cells in the dura mater in animal models (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In this context, it is tempting to hypothesize that the previously described reduction in migraine headache with advancing age (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) may relate to diminished dural CGRP release, possibly acting alongside age-related vascular stiffening in patients where migraine declines during lifetime (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). In contrast, migraine aura symptoms may persist into older age due to central mechanisms (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) \u0026ndash; potentially reflected by unaltered CGRP release in the central structures of the trigeminovascular system as observed in our study \u0026ndash; that are less affected by age. However, while we currently lack evidence to confirm or refute this hypothesis, future studies should address these peripheral and central age-dependent effects, including the influence on cortical spreading depression \u0026ndash; a key mechanism underlying migraine aura (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). In our study, the decreased trigeminovascular CGRP release from the dura mater in \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003emice did not seem to be accompanied by an altered peripheral vascular reactivity to CGRP, although no conclusions regarding peripheral CGRP release can be drawn. Notably, we did not investigate the release of other neuropeptides, such as adrenomedullin and amylin, which often colocalize with CGRP, in the trigeminovascular system (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven that aging is one of the most significant risk factors for cardiovascular disease, that migraine is increasingly recognized as a cardiovascular risk factor, and that CGRP plays a key role in cardiovascular health and vascular aging processes (\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e), our results offer new pathophysiological insights into the involvement of specific vasodilatory mechanisms herein.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eTaken together, this study demonstrates a reduction in CGRP release from the dura mater \u0026ndash; but not from the trigeminal nucleus caudalis or the trigeminal ganglion \u0026ndash; in a mouse model of accelerated aging. No differences in peripheral vasodilatory responses to CGRP and forskolin were found, suggesting that reduced trigeminovascular CGRP release is not associated with altered peripheral vascular responsiveness to CGRP in older mice \u0026ndash; a finding which may, however, differ for other peptides and therefore warrants further research. An age-related decline in the density or functional integrity of nociceptive CGRP-positive nerve fibers in the dura mater may, at least partly, explain the reduction in migraine headache attacks in the elderly.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was planned and carried out in accordance with the Principles of Laboratory Animal Care and with guidelines approved by an independent Animal Ethics Committee, consulted at the Erasmus MC, as well as the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate or for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available upon reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLinda Al-Hassany, Eloisa Rubio-Beltr\u0026aacute;n, Alejandro Labastida-Ramirez, Anton JM Roks, Ingrid M Garrelds, and A.H. Jan Danser declare no conflict of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003eAntoinette MaassenVanDenBrink received research grants and/or consultation fees from AbbVie, Amgen/Novartis, Eli Lilly, Lundbeck, Manistee, Pfizer, Satsuma, Teva, and Tonix.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Dutch Research Council (ZonMw/NWO, Vici Grant (09150181910040).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLA-H performed the measurements, analyzed the data, and took the lead in writing the manuscript and visualizing the data. ER-B conceptualized the study, performed measurements, analyzed the data, and reviewed/edited the manuscript. AL-R \u0026nbsp; conceptualized the study, performed measurements, and reviewed/edited the manuscript. IMG performed the CGRP measurements and reviewed/edited the manuscript. AJMR, AMvdB and AHJD conceptualized, designed and funded the study, and reviewed/edited the manuscript. AMvdB additionally supervised the study. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Keivan Golshiri, Ehsan Ataei Ataabadi, Annika A. J\u0026uuml;ttner, and Ren\u0026eacute; de Vries for providing and maintaining the mice and for their assistance with sacrificing the mice.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHeadache Classification Committee of the International Headache Society (IHS) The International Classification of Headache Disorders, 3rd edition. 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BMJ Open 8(3):e020498\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui XY, Zhan JK (2025) Capsaicin and TRPV1: A novel therapeutic approach to mitigate vascular aging. Aging Dis 17(1):256\u0026ndash;274\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-headache-and-pain","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tjhp","sideBox":"Learn more about [The Journal of Headache and Pain](https://thejournalofheadacheandpain.biomedcentral.com/)","snPcode":"10194","submissionUrl":"https://submission.nature.com/new-submission/10194/3","title":"The Journal of Headache and Pain","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Aging, CGRP, Mice, Migraine, Peripheral vasculature, Trigeminovascular system","lastPublishedDoi":"10.21203/rs.3.rs-9187264/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9187264/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnderstanding age-related changes in migraine is pivotal, considering the increasing global life expectancy. In addition, both aging and migraine are prominent cardiovascular risk factors. It remains unclear whether calcitonin gene-related peptide (CGRP) release changes with age across trigeminovascular components, and how this relates to peripheral responses to migraine-related vasodilatory molecules.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The primary aim was to investigate age-related effects on CGRP release from the trigeminovascular system by studying a mouse model of combined accelerated neuronal and vascular aging, the DNA repair-deficient \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/−\u003c/em\u003e\u003c/sup\u003e mice. Second, we assessed the effects of aging on isolated coronary vasodilatory responses to CGRP and forskolin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiments were conducted using DNA repair-deficient \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/−\u003c/em\u003e\u003c/sup\u003e mice and their wild type controls. After sacrifice, the trigeminal nucleus caudalis (TNC), trigeminal ganglion (TG), and dura mater (DM) were isolated. \u003cem\u003eEx vivo\u003c/em\u003e KCl-induced CGRP release was measured, and CGRP release was compared between \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/−\u003c/em\u003e\u003c/sup\u003eand wild type mice.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In a subset of mice, concentration-response curves to CGRP and forskolin were generated in isolated coronary arteries. The pEC\u003csub\u003e50\u003c/sub\u003e (negative log of the molar concentration of an agonist needed to reach half of its maximal effect) and E\u003csub\u003emax\u003c/sub\u003e (maximum relaxation response) values were compared between both groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCGRP release (expressed as ratio compared to baseline release)\u0026nbsp; of the DM was significantly lower in \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/−\u003c/em\u003e\u003c/sup\u003e(2.01±0.24) \u003cem\u003eversus\u003c/em\u003e wild type mice (3.22±0.48) (\u003cem\u003eP\u003c/em\u003e=0.040). No differences were observed in CGRP release between \u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/−\u003c/em\u003e\u003c/sup\u003eand wild type mice in the TNC\u0026nbsp; (8.07±1.15 \u003cem\u003eversus\u003c/em\u003e 6.10±0.57, \u003cem\u003eP\u003c/em\u003e=0.364) or the TG (4.84±0.92 \u003cem\u003eversus\u003c/em\u003e 4.41±0.60, \u003cem\u003eP\u003c/em\u003e=0.838). In addition, there were no differences in\u0026nbsp; pEC\u003csub\u003e50 \u003c/sub\u003eand E\u003csub\u003emax\u003c/sub\u003e values in response to CGRP and forskolin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Conclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur findings suggest that aging is linked to reduced CGRP release of the DM, potentially partly explaining the reduction in migraine attacks in elderly. This decreased CGRP release is not accompanied by altered peripheral vascular reactivity to CGRP.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Trigeminovascular Calcitonin Gene-Related Peptide Release and Peripheral Vascular Responses in a Mouse Model of Accelerated Aging: Implications for Migraine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-25 10:42:15","doi":"10.21203/rs.3.rs-9187264/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-07T15:27:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-01T10:27:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72541076678347439494391588771029265377","date":"2026-03-28T13:40:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64959534259958420348028133404758231367","date":"2026-03-26T14:32:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-25T16:38:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308231655664210807506984455877861105433","date":"2026-03-25T07:03:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211561340927480502507121261619856680115","date":"2026-03-24T23:57:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"85102693262241831621340560345579702274","date":"2026-03-24T06:23:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-23T13:32:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-23T13:28:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-23T11:16:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Journal of Headache and Pain","date":"2026-03-21T16:55:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-headache-and-pain","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tjhp","sideBox":"Learn more about [The Journal of Headache and Pain](https://thejournalofheadacheandpain.biomedcentral.com/)","snPcode":"10194","submissionUrl":"https://submission.nature.com/new-submission/10194/3","title":"The Journal of Headache and Pain","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"67858b98-46dd-42af-b0a9-a5d57f097f0f","owner":[],"postedDate":"March 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T15:41:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-25 10:42:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9187264","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9187264","identity":"rs-9187264","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}