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Original Article
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Increased VIP levels in peripheral blood ! International Headache Society 2014
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DOI: 10.1177/0333102414535111
cep.sagepub.com
biomarker of cranial parasympathetic
activation in chronic migraine

Eva Cernuda-Morollón1, Pablo Martı́nez-Camblor2,

Rocı́o Alvarez1, Davinia Larrosa1, César Ramón1 and J Pascual1

Abstract
Aim: The aim of this article is to determine vasoactive intestinal peptide (VIP) levels outside migraine attacks in peripheral
blood as a potential biomarker for chronic migraine (CM).
Methods: Women older than 17 and diagnosed as CM were recruited. Matched healthy women with no headache history
and women with episodic migraine (EM) served as control groups, together with a series of patients with episodic cluster
headache in a pain-free period. VIP levels were determined in blood samples obtained from the right antecubital vein by
ELISA outside a migraine attack, the patients having taken no symptomatic medication the day before. For ethical
reasons, preventives were not stopped.
Results: We assessed plasma samples from 119 women with CM, 33 healthy women, 51 matched women with EM and 18
patients (16 males) with cluster headache matched for age. VIP levels were significantly increased in CM (165.1 pg/ml) as
compared to control healthy women (88.5 pg/ml) and episodic cluster headache patients (101.1 pg/ml). VIP levels in EM
(134.9 pg/ml) were significantly higher compared to controls and numerically lower than those of CM. Thresholds of 71.8
and 164.5 pg/ml optimized the sensitivity and specificity to differentiate CM from healthy controls and EM, respectively.
Variables such as age, CM duration, the presence of aura, analgesic overuse, depression, fibromyalgia, vascular risk
factors, history of triptan consumption or kind of preventive treatment did not significantly influence VIP levels.
Conclusion: Increased interictal VIP level measured in peripheral blood could be a biomarker helping in CM diagnosis,
though it does not clearly differentiate between EM and CM.

Keywords
Chronic migraine, cluster headache, VIP, migraine
Date received: 24 February 2014; revised: 14 April 2014; accepted: 15 April 2014

Introduction
Migraine is considered to be a neurovascular disorder. Migraine can be divided into two types: episodic
Either changes in the modulating nociceptive inputs (EM) (fewer than 15 headache days per month) and
from the locus ceruleus and raphe brain stem nuclei chronic (CM) (15 or more headache days per month).
(1) or a cortical spreading depression phenomenon (2) The International Headache Society (IHS) defines CM
are thought to activate the trigemino-vascular system as 15 or more headache days per month for at least
(TVS), which releases vasoactive neuropeptides, mainly three months, with 8 days per month fulfilling
calcitonin gene-related peptide (CGRP) and vasoactive
intestinal peptide (VIP) around leptomeningeal and 1
Neuroscience Area, Service of Neurology, University Hospital Central
extracranial vessels (3,4). Cranial autonomic nervous de Asturias and Ineuropa, Asturias, Spain
2
system has for a long time been implicated in migraine Oficina de Investigación Biosanitaria OIB, Oviedo, Asturias, Spain
pathophysiology (4,5). VIP is a neurotransmitter, in
Corresponding author:
human cerebral parasympathetic perivascular nerve Julio Pascual, Service of Neurology, University Hospital Central de
fibers and the sphenopalatine ganglion, acting as a Asturias, Calle Celestino Villamil s/n, Oviedo 33006, Asturias, Spain.
powerful vasodilator (4,6). Email: juliopascual@telefonica.net
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migraine criteria, in the absence of medication overuse medication in the previous 24 hours. For ethical rea-
(7). Although the source of pain persistence in CM is sons, preventives were not withdrawn.
unknown, it is widely accepted that pain pathways Serum VIP levels were determined using a commercial
become sensitized by repeated episodes of activation enzyme-linked immunosorbent assay (ELISA) kit
of the two arms, sensory and parasympathetic, of the (USCN Life Science Inc., Hubei, China) strictly following
TVS (8,9). It seems reasonable to speculate that VIP the manufacturer’s instructions. Absorption levels were
could play a role in migraine chronification by inducing measured with a spectrophotometer from BioRad. The
sensitization of cranial perivascular nociceptors. detection limit of the assay was <2.34 pg/ml.
We determined interictal VIP levels in peripheral
blood in a series of women with CM as a potential
biomarker for a rather continuous TVS activation,
Statistical analysis
which is thought to be present in CM. VIP levels and age (years) were described by
mean  standard deviations. Absolute and relative fre-
quencies were used for describing categorical variables.
The general Bootstrap algorithm (gBA) and the Exact
Material and methods Chi-square test were used in order to compare, respect-
ively, the VIP levels and the categorical variables of the
Study participants different groups. The receiver operating characteristic
Females older than 17 years attending the headache (ROC) curve and the AUC (area under the ROC curve)
clinic at the University Hospital Central de Asturias were obtained to measure the discrimination capacity
who had been diagnosed by us as having CM according (CM vs controls and CM vs EM) of the VIP levels. The
to current IHS criteria were included in this study (7). point associated with the Youden index was chosen as
All patients fulfilling criteria for analgesic overuse had the threshold (TH) point. The confidence band for the
been detoxified at least once for a minimum of two ROC curves and 95% confidence intervals (CI) for both
months. As control groups, we recruited matched the AUC and the TH were computed by 10,000 iter-
healthy women (medical students, residents, nurses or ations of the Bootstrap method. Finally, to check the
physicians from our hospital) with no history of head- diagnostic capacity of the proposed criteria, the Leave-
ache and taking no medication and females also attend- one-out method was performed. P values below 0.05
ing our clinic and meeting IHS diagnostic criteria for were considered statistically different. Both the CI
EM diagnosis. As an ancillary control group we also and the statistical tests were not adjusted by
studied patients with episodic cluster headache always multiplicity.
outside a symptomatic period. All subjects underwent
a general physical and neurological examination.
Without exception, migraine (chronic or episodic) had
Results
at least a normal neuroimaging study (brain magnetic We assessed plasma samples from 119 women with CM
resonance imaging (MRI) in 81 cases and brain com- (mean age 44.4  12.2; range 17–63 years), 33 healthy
puted tomography (CT) with contrast in the remaining women with no headache history (39.8 44.4 the Boots–
89 cases). All episodic cluster headache cases had a 61 years), 51 women with EM (44.0  11.6 years; 17–66
normal brain MRI. Exclusion criteria were pregnant years) and 18 patients (16 males, 44.6  7.9) with epi-
or breast-feeding women, excessive use of alcohol, a sodic cluster headache. By history, the average time in
history of sinusitis and serious, active psychiatric and which patients had remained in a CM situation was
somatic diseases, including diabetes. The study was 9.5  8.4 years. Comorbidities and treatment of
approved by the ethics review board of our center women with CM enrolled in this study are illustrated
and all the participants gave written consent. in Table 1. VIP levels were in all cases above the limit of
detection.
VIP levels were significantly higher in women with
Laboratory determinations CM (165.1  125.4 pg/ml; range 20.6–668.2) as com-
Patients rested in supine position and blood samples pared to control women (88.6  62.3 pg/ml; range 5.1–
were obtained from the right antecubital vein between 256.11; p < 0.001) and patients with episodic cluster
9:30 a.m. and noon in a fasting condition. The blood headache (101.1  78.6, range 28.4–349.9) in an inter-
was collected, allowed to clot and serum was separated ictal period. VIP levels for EM (134.9  80.4 pg/ml;
after centrifugation for 10 minutes at 2000 g. Aliquots range 39.8–402.1) were numerically, but not statistically
were stored at –80 iquots were stored at condition. All different from those for CM, but were significantly
samples were obtained interictally, outside a migraine higher (p < 0.01) than those found in control women
attack the patients having taken no symptomatic (Figure 1). Both in the group of healthy controls and
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Cernuda-Morollón et al. 3

Table 1. Summary of clinical characteristics of our CM patients.

**
n (%)
500
Aura 56
#
(47.06) 400
Comorbidities Arterial hypertension 17 *

VIP (pg/ml)
(14.28)
300
Hypercholesterolemia 21
(17.65)
200
Depression 62
(52.10)
Fibromyalgia 13 100

(10.92)
Others: Obesity 8 0
Asthma 6 Ct MC ME CH
Epilepsy 1
Preventives Topiramate 56 Figure 1. Vasoactive intestinal peptide (VIP) levels are
(47.06) increased in chronic migraine. Box plot for the VIP levels in
Beta-blockers 29 controls (Ct, gray), chronic migraine (CM), episodic migraine
(EM) and cluster headache (CH). **p < 0.001 vs Ct, #p < 0.01
(24.37)
vs Ct, *p < 0.01 vs CM.
Flunarizine 1
(0,84)
Amitriptyline 41 Bootstrap CI: 71.7–273.2) correctly classified 74.5%
(34.45)
of the EM patients and 44.2% of the CM patients
SSRIs 14 (Figure 2(b)).
(11,76)
Fifty-six women (47%) with CM had a history of
ACEIs 22 migraine with aura attacks. There were only four
(18.49)
women in this group experiencing aura at least once
Zonisamide 18 per month in the previous year. Within the sample of
(15.13)
women with EM, 26 (54%) also had a history of
Valproic acid 8 migraine with aura attacks, though there were just
(6.72)
two EM patients who had only attacks of migraine
Polytherapy 66 with aura. VIP levels were not different in patients
(2 preventives) (55.46)
with CM or EM having an aura history
Symptomatic treatment Triptans 92 (188.4  161.7 and 125.9  63.6) as compared with
(77.31)
those without aura (144.4  76.0 and 144.3  95.2). In
NSAIDs 86 the group of CM women, VIP levels were not signifi-
(72.27)
cantly different in patients meeting criteria for analgesic
overuse (44 patients, 40.0%) vs those without analgesic
CM: chronic migraine; SSRIs: selective serotonin uptake inhibitors; ACEIs: overuse (161.4 vs 167.2 pg/ml). VIP levels were not stat-
angiotensin-converting enzyme inhibitors; NSAIDs: nonsteroidal
anti-inflammatory drugs.
istically different in patients using triptans as symptom-
atic medication compared to those who were not taking
in the CM group VIP was not influenced by age in the triptans. VIP levels were not statistically different in
range of our study. patients taking preventives in monotherapy vs polither-
In order to evaluate the VIP concentration as a apy or in those taking topiramate vs those who were
potential marker for CM, the ROC curve and the not using topiramate. In this group, VIP levels did not
AUC were measured. The Youden index was achieved change significantly in the presence/absence of a history
for a VIP concentration of 71.8 pg/ml (95% Bootstrap of depression, fibromyalgia and/or arterial hyperten-
CI: 50.5–170.5, Figure 2(a)) with an AUC of 0.713 sion or depending on CM duration.
(95% Bootstrap CI: 0.659–0.841). For this TH 79.7%
of patients and 61.3% of the controls would be cor-
Discussion
rectly classified. The same discriminatory capacity was
obtained by applying the Leave-one-out method. When The main finding of this study was that, compared to
this methodology was applied to discriminate CM healthy women without a history of headache, interictal
and EM, a VIP concentration of 164.5 pg/ml (95% VIP levels were significantly increased in peripheral
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(a) 0.010 1.0

0.008 0.8

True-positive rate
0.006 0.6
Density

0.004 0.4

0.002 0.2

0.000 0.0
0 200 400 600 0.0 0.2 0.4 0.6 0.8 1.0
VIP levels False-position rate

(b) 0.010 1.0

0.008 True-positive rate 0.8

0.006 0.6
Density

0.004 0.4

0.002 0.2

0.000 0.0

0 200 400 600 0.0 0.2 0.4 0.6 0.8 1.0

VIP levels False-position rate

Figure 2. ROC curve threshold for VIP. (a) Left panel: Kernel density estimations for the VIP levels in CM (continuous line) and
controls (dotted line). The vertical line stands for the threshold that achieves the Youden index. In gray, a 95% Bootstrap confidence
interval. Right panel: Respective ROC curve with 95% Bootstrap confidence band. Highlighted, the results of the threshold. (b) Similar
to (a) for CM (continuous line) and EM (dotted line). ROC: receiver operating characteristics; VIP: vasoactive intestinal peptide;
CM: chronic migraine; EM: episodic migraine.

blood in a large sample of women with CM. In fact, as parasympathetic innervation (3,4,6). Activation and
an average, VIP levels in CM were double those of sensitization of nociceptors around extra- and intracra-
healthy controls. To a lesser degree, VIP levels were nial vessels is a primary source of pain in migraine. It
also significantly elevated in females with EM vs has been proposed that parasympathetic outflow to
healthy controls. The presence of aura or a variety of cephalic vasculature may trigger activation and sensi-
clinical comorbidities did not correlate with a change in tization of perivascular sensory afferents and thereby
VIP levels. Concurring with our CGRP results (10), migraine pain (19). The parasympathetic innervation
VIP levels were almost identical in CM with and with- of the cerebral circulation, arising from the superior
out analgesic overuse. salivary nucleus and passing out of the brain in the
There are several arguments strongly supporting an facial nerve, represents the most powerful of the
involvement of the parasympathetic arm of the TVS in neural vasodilator influences on that bed. The parasym-
migraine pathophysiology (4,5). Cranial autonomic pathetic system contains and releases a number of
parasympathetic symptoms, such as lacrimation, rhi- transmitter or neuromodulator substances including
norrhea and eyelid edema, do appear, depending on acetylcholine and several peptides, mainly peptide his-
criteria and study design, in 27% to 73% of migraine tidine methionine, pituitary adenylate cyclase-activat-
patients (11–18). Meningeal blood vessels receive dense ing polypeptide (PACAP) and VIP, which contribute
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Cernuda-Morollón et al.
to regulating cerebrovascular tone and brain hemo-
dynamics. VIP is a 28-amino-acid base polypeptide
belonging to a structural superfamily of peptides
along with glucagon, secretin and gastrin inhibitory
peptide (4,6). It has been clearly established that both
the large cerebral and cortical pial vessels have a rich
VIPergic innervation (20). Experimental studies have
demonstrated that VIP acts as a powerful vasodilator
in various species, including humans. In a well-vali-
dated model of acute cutaneous pain, intradermal injec-
tion of VIP in the forearm increased skin blood flow,
wheal and flare more than placebo and even PACAP
(21), which strongly supports a role in nociceptive
transmission.
Plasma VIP levels have been shown to be increased
during cluster headache attacks translating the intense
parasympathetic activation (22), but there are very few
studies testing VIP levels in migraine, a condition in
which up to three-quarters of patients show some
symptoms or signs of autonomic parasympathetic acti-
vation. Although, in contrast to CGRP, VIP levels were
in the range of controls in a series of migraine patients,
in the same study plasma VIP levels were elevated in the
cranial circulation in the subgroup of migraineurs with
pronounced autonomic symptoms (22). VIP levels, also
measured in the external jugular vein, were significantly
reduced in five migraine patients with autonomic symp-
toms after successful treatment with rizatriptan in
another study (23). Similar findings have been reported
in VIP saliva values after sumatriptan in a small group
of EM patients (24). There are no data on VIP levels in
patients with CM. Our finding of increased peripheral
VIP levels in CM patients outside migraine attacks and
in the absence of symptomatic medication for at least
24 hours could be interpreted as a distant sign of ‘‘per-
manent’’ activation of the parasympathetic arm of the
TVS. There are at least three possible immediate con-
sequences of these results. First, as happens with CGRP
and always carefully considering the clinical data, per-
ipheral VIP levels also seem to be a biomarker for CM,
though contrary to CGRP (10), VIP levels were not so
consistent and did not clearly differentiate between CM
and EM. It seems logical to propose that VIP levels
would be increased in those up to three-quarters of
patients who express parasympathetic symptoms
during migraine attacks. It is even possible that other
peptides involved in TVS activation, such as PACAP,
could also serve as migraine biomarkers. Second, in
theory, peripheral VIP levels could also serve to object-
ively monitor migraine status and response to prevent-
ive treatments, which could be of help, for instance, for
a more objective follow-up of patients included in clin-
ical trials. Although we are aware that the fact that, for
ethical reasons, most of our patients with migraine were
receiving daily preventives could be considered as a
[1–7]
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5
limitation of this study, it is also true that this could
make our results even more relevant as these drugs
should tone down TVS activation. As an example of
this, 19 out of the 51 women in this study in a current
EM situation had in the past met criteria for CM,
which was reversed by preventives. Mean VIP levels
in this subset of patients was 143.6 pg/ml, that is, in
the range of EM. Third, could these increased VIP
levels found in CM patients as a reflection of cranial
parasympathetic activation contribute to the develop-
ment of CM? The role of this peptide in the pathophysi-
ology of individual migraine attacks is controversial as,
contrary to CGRP and in spite of being one of the most
potent cranial vasodilators, intravenous VIP does not
consistently trigger migraine attack, but only a delayed
mild headache in some migraineurs (25,26). Other stu-
dies, however, have shown that neuromodulating the
sphenopalatine ganglion, the main origin of parasym-
pathetic fibers innervating cerebral vessels (6), might be
effective in the treatment of migraine attacks and espe-
cially in patients with cranial autonomic symptoms
(19). We still do not know the exact mechanisms that
lead to transformation from EM to CM. It is well
established, however, that activation of the TVS
system has a crucial role and leads to afferent
(CGRP) and efferent (VIP and others) release of neuro-
peptides. This facilitates leptomeningeal vasodilating
and inflammatory responses and induces activation of
second-order neurons involved in pain transmission.
VIP causes neurogenic inflammation and mast cell
degranulation via VIP and PACAP (VPAC) receptor
activation (21). Persistent release of VIP could contrib-
ute to the appearance of sensitization of central
trigeminal neurons (5,8,9,19), which underlies patho-
physiology of chronic pain conditions, such as CM.
In fact, indirect modulation of central sensory struc-
tures, including the trigeminal nucleus caudalis, has
been proposed as the mechanism explaining the pos-
sible efficacy of sphenopalatine stimulation in the treat-
ment of CM (27). This possible VIP-mediated
participation in the transformation from EM to CM
is not mutually exclusive, but rather additive, to the
more documented role of the CGRP-mediated sensory
arm of the TVS system.
Our study has several limitations. First, even though
interictal VIP levels in episodic cluster headache out of
a symptomatic period were below those of CM, to dem-
onstrate true specificity it would be necessary to test
VIP levels in conditions such as stroke, subarachnoid
hemorrhage, sinusitis and other trigemino-autonomic
cephalalgias in which parasympathetic activation
occurs (6). Second, the data shown here come from a
headache clinic population with still a limited and
selected number of cases. Finally, the real value of
VIP levels for monitoring patient status and response
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6 Cephalalgia 0(0)

to treatment in migraine will be possible only after with long-duration daily or almost daily headaches and
longitudinal data are available. Taking into account a history of migraine, which both could be of help in
these limitations, interictal VIP levels seem to be a sharpening a CM diagnosis and supports a role of VIP
potential biomarker for CM in the context of a patient in the sensitization of pain circuits giving rise to CM.

Clinical implications
. Vasoactive intestinal peptide (VIP) levels are increased outside attacks in patients with chronic migraine as a
reflection of the activation of the parasympathetic arm of the trigemino-vascular system.
. Interictal VIP levels seem to be a potential biomarker for chronic migraine (CM) in the context of a patient
with long-duration daily or almost daily headaches and a history of migraine.
. These data support a role of VIP in the sensitization of pain circuits giving rise to CM.

Funding biomarker for chronic migraine. Neurology 2013; 81:

This work was supported by grant PI11/00889 FISSS 1191–1196.
(Fondos Feder, ISCIII, Ministry of Education, Spain) and 11. Barbanti P, Fabbrini G, Pesare M, et al. Unilateral cra-
by Allergan Eurasia (Institution Sponsored-Non- nial autonomic symptoms in migraine. Cephalalgia 2002;
Interventional Study MAF/ISS/NS/CM/003). PMC is sup- 22: 256–259.
ported by grant MTM2011-23204 of the Spanish Ministry 12. Avnon Y, Nitzan M, Sprecher E, et al. Different patterns
of Science and Innovation (FEDER support included). of parasympathetic activation in uni- and bilateral
migraineurs. Brain 2002; 126: 1660–1670.
13. Avnon Y, Nitzan M, Sprecher E, et al. Autonomic asym-
Conflict of interest
metry in migraine: Augmented parasympathetic activa-
None declared. tion in left unilateral migraineurs. Brain 2004; 127:
2099–2108.
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