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Abstract

Background

In recent years, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptides (PACAPs) have gained special interest in headache science. VIP and PACAPs (two isoforms, PACAP27 and PACAP38) are related in structure and function, as are their receptors, but they show differences in vasodilating- and headache-inducing properties. Intravenous infusion of PACAP27 or PACAP38, but not VIP, induces a long-lasting dilation of cranial arteries and delayed headache. The relationship between the long-lasting cranial vasodilation and headache development is not fully clarified.

Methods

In a double-blinded, placebo-controlled, crossover study in 12 healthy volunteers, diameter changes of cranial arteries, occurrence of headache and the parasympathetic system were examined before, during and after a 2-hour continuous intravenous infusion of VIP and placebo. Primary endpoints were the differences in area under the curve for the superficial temporal artery diameter and headache intensity scores, as well as in headache incidence, between VIP and placebo.

Results

The superficial temporal artery diameter was significantly larger on the VIP day compared to placebo (p < 0.001) and the dilation lasted for more than 2 h. The incidence of headache was higher (p = 0.003) on the VIP day compared to the placebo day. The difference in headache intensity scores was more evident in the post-infusion period (120–200 min, p = 0.034) and in the post-hospital phase (4–12 h, p = 0.025). Cranial parasympathetic activity, measured through the production of tears, was higher during VIP compared to placebo (p = 0.033).

Conclusion

Continuous intravenous infusion of VIP over 2 h induced a long-lasting cranial vasodilation, activation of the cranial parasympathetic system, and delayed mild headaches in healthy volunteers.

Trial Registration: The study is registered at ClinicalTrials.gov (NCT03989817).

Keywords

Migraine VIP parasympathetic system PACAP27 PACAP38

Introduction

The vasoactive intestinal polypeptide (VIP) is a neurotransmitter (1) and neuromodulator (2) with many physiological functions, particularly in the parasympathetic nervous system. It is involved in vasodilator, lacrimal and secretory responses. It is closely related in structure and function to pituitary adenylate cyclase-activating polypeptide-27 (PACAP27) and pituitary adenylate cyclase-activating polypeptide-38 (PACAP38), and acts on the same receptor family. VIP and PACAPs receptors belongs to a family of G-protein coupled receptors; vasoactive intestinal polypeptide receptor 1 (VPAC₁) and vasoactive intestinal polypeptide receptor 2 (VPAC₂), both activated by VIP and PACAP, and the PACAP type I receptor (PAC1), being 1000-fold more sensitive for PACAP compared to VIP. Comparable doses of all three peptides increase intracellular cAMP concentration (3) and dilate cranial arteries (4 –6). Infusion of PACAP27 or PACAP38 over 20 min induces extracranial vasodilation for more than 4 h, whereas the vasodilation induced by VIP normalises within 2 h (6,7). PACAP-induced prolonged dilation is associated with a delayed headache in healthy volunteers (4,5). Conversely, VIP-induced headache is negligible and monophasic (i.e. delayed headache is not reported) (6). The discrepancy between VIP and the PACAPs may be due to the differential activation of PAC1 receptors, as well as to the duration of the vasodilatory response. Interestingly, PAC1 receptors are not expressed in smooth muscle cells (8) and monoclonal antibody against PAC1 failed in migraine prevention (9). Thus, the relationship between a prolonged dilation of cranial arteries and headache is not yet fully understood. Hence, whether a long-lasting infusion of VIP will lead to a prolonged cranial vasodilation and biphasic (delayed) headache response has never been studied. As a first step in investigating the relationship between a long-lasting cranial vasodilation and headache, we studied arterial dilation, headache-inducing and parasympathetic properties of a 2 h infusion of VIP in healthy volunteers. We hypothesised that a long-lasting administration of VIP will cause a prolonged dilation of cranial arteries and delayed headache in healthy volunteers. In addition, we investigated the cranial parasympathetic activation through lacrimal secretion, measurable by collecting tears.

Methods

Recruitment of participants

Healthy volunteers were recruited via advertisement on social media and among participants from previous studies who were interested in future studies. All interested participants received written information material and were invited for a screening visit. The experiment was postponed if the patient had experienced any type of headache 48 h before the start of the study. Inclusion criteria were age between 18 and 45 years, weight between 50 and 95 kg and secure prevention for fertile women (oral contraception or intrauterine device). Exclusion criteria were a history of migraine or any other type of headache (except tension-type headache no more than once a month in the last year); first-degree relatives diagnosed with migraine or any other type of headache; anamnestic and/or clinical signs of cardiovascular diseases, mental illness, deviations of the nasal septum, nasal polyps or other anatomical restrictions of the upper airways; substance abuse or smoking; daily intake of any medicine other than oral contraceptives; pregnant or breastfeeding women.

Pilot studies

First, an open-label pilot experiment was conducted to evaluate optimal duration time of the infusion with respect to feasibility. Six participants received a continuous intravenous infusion of VIP (8 pmol/kg per min) for 1.5 h and three participants received a continuous intravenous infusion of VIP (8 pmol/kg per min) for 2 h. A second open-label pilot experiment was performed to explore the effects of a long-lasting infusion of VIP on intracranial and extracranial vessels. In six participants, the diameter of the superficial temporal artery (STA) was measured by a high-resolution ultrasonographic unit during continuous infusion of VIP (8 pmol/kg per min). In the other three participants, circumferences of the middle cerebral artery (MCA), middle meningeal artery (MMA) and superficial temporal artery (STA) were investigated, at baseline and repeatedly during continuous infusion of VIP (8 pmol/kg per min). High resolution magnetic resonance angiography (MRA) technique using a 3.0 Tesla Philips Achieva Scanner (Philips Medical Systems, Best, Netherlands) was used to obtain three-dimensional time-of-flight angiograms. The acquired images were analyzed by LKEB-MRA vessel wall analysis software program (4,10).

Main study

We recruited healthy volunteers and randomly allocated them to receive VIP (8 pmol/kg per min, manufactured by Bachem, Bubendorf, Switzerland) or placebo (isotonic saline) over 2 h on two different days, separated by at least one week. The study drug was prepared by the Capital Region Pharmacy and randomisation was performed by medical staff not involved in the study. The randomisation code remained in the hospital during the study and was not available to the investigators until the study was completed and data were analysed. All participants arrived non-fasting and headache free at the clinic. The intake of tobacco, alcohol, coffee, tea, cocoa or other methylxanthine-containing beverages or foods, was not allowed for at least 8 h before the start of the study. All procedures were performed in a quiet room at a temperature of 25°C. The participant was placed in the supine position and two venous catheters were placed into the left and right forearm for drug infusion and blood sampling. The participants then rested for 30 min before baseline measurements were performed. Headache intensity, accompanying symptoms including nausea, vomiting, photophobia and phonophobia, adverse events (AEs) and vital signs were recorded at T_-10, T₀ and every 10 min after the start of infusion, until T₂₀₀. STA diameter measurements were performed at T₀, T₁₀, T₃₀ and every 30 min, until T₁₈₀. We conducted Schirmer’s test during a kinetic oscillation stimulation (KOS) procedure at the baseline, T₃₀ and T₉₀. Cranial autonomic parasympathetic symptoms (CAPS) scale was recorded at the baseline, infusion and post-infusion periods. The participants were discharged after finishing the measurements and asked to complete a headache diary every hour, until 12 h after the start of the infusion. The diary included headache characteristics, accompanying symptoms (nausea, vomiting, photophobia and phonophobia), any medication taken and AEs.

Diameter of the superficial temporal artery (STA)

Diameter of the frontal branch of the STA was measured by a high-resolution ultrasonographic unit (Dermascan C; Cortex Technology, Hadsund, Denmark), as previously described (11,12).

Headache recordings

Headache intensity was scored on a numerical rating scale (NRS) verbally declared from 0 to 10, where 0 is no headache; 1 is a very mild headache, including a feeling of pressing or throbbing; 5 is a moderate headache; 10 is the worst imaginable headache.

Vital signs

Blood pressure and HR were measured at the baseline and every 10 min using an auto-inflatable cuff (Microlife AG, Switzerland). Blood pressure was registered as mean arterial pressure (MAP), equal to diastolic blood pressure + 1/3 (systolic blood pressure – diastolic blood pressure). An ECG was recorded on paper at the baseline and then monitored on an LCD screen until the end of the experiment.

Kinetic oscillation stimulation (KOS) procedure

KOS (Chordate Medical AB, Stockholm, Sweden) in the nasal cavity was performed using similar equipment to that described in previous studies (13 –15). A mechanical device was used to stimulate the nasal mucosa and elicit cranial parasympathetic activity for 5 min, quantifiable via lacrimation. A balloon catheter with an inflatable tip was coated with paraffin and inserted into the participant’s dominant nostril. Prior to the experimental procedure, it was securely fixed to prevent it moving and left in position for one minute to allow for habituation to the mechanical perception. During stimulation, the catheter was firstly inflated to 80 mbar and an oscillation at a frequency of 85 Hz was switched on, which activates the cranial parasympathetic system.

Schirmer’s test

Lacrimation utilised as a quantifiable measure of cranial parasympathetic activation was recorded using standardised Schirmer’s tests II. Three tests were applied: At baseline, at T₃₀ and T₉₀. Sterile tear strips (Haag-Streit Clement Clarke, UK) were placed inside the lower eyelids and lacrimation was recorded during a total time of 5 min. Schirmer’s test performed on the side stimulated by the KOS procedure was identified as the test run on the “KOS eye”. The Schirmer’s test performed on the contralateral eyelid was identified as the test performed on the “free eye”.

Statistics

All absolute values are presented as mean ± standard deviation (SD), except headache scores, which are presented as medians and quartiles. The highest variation of STA diameter and HR are reported as the highest mean percentage increase and 95% confidence interval (CI). Primary endpoints were a) difference in the area under the curve (AUC) for STA diameter from baseline to the last measurement (T_180min) and b) difference in the AUC for headache intensity scores, as well as headache incidence, between VIP and placebo during the entire observational period (T₀–T_200min). Secondary endpoints were a) difference in the AUC for both KOS-eye and free-eye between VIP and placebo from baseline to the last measurement (T_90min); b) difference in the AUC for headache intensity scores between VIP and placebo during immediate (T₀–T_120min), delayed (T_120min–T_200min) and post-hospital (T_4h–T_12h) phases; c) difference in the AUC_MAP between VIP and placebo during the entire observational period (T₀–T_200min) and d) difference in the AUC_HR between VIP and placebo during the entire observational period (T₀–T_200min). CAPS scores are displayed in the Supplementary table.

According to the trapezium rule (16), we calculated AUC to obtain summary measures and to analyse the differences in response between VIP and placebo. All statistical analyses were conducted between paired samples (e.g. within participants). Baseline differences, as well as AUC values for STA diameter (mm), KOS eye (mm), free eye (mm), MAP (mmHg) and HR (bpm) were compared by a paired two-way t-test or Wilcoxon matched-pairs signed rank test, according to the normal distribution of data. AUC values for headache intensity were compared by the Sign’s test for paired observations. Occurrence of headache was tested with McNemar’s test.

Statistical analysis and graphs were performed using GraphPad Prism 8.3.0 (San Diego, CA, USA). Level of significance at five percent (p < 0.05, two-tailed) was accepted for all tests. Nominal p-values were reported without adjusting for multiplicity.

Results

Pilot studies – dose finding and arterial responses

The first pilot study was conducted in nine healthy volunteers (three women and six men, mean age 27.7 years, range: 23–43 years; mean weight: 72.2 kg, range: 65–90 kg). Dosages were well tolerated, with only mild AEs such as flushing, palpitations and warm sensations. No difference in AEs between 1.5 h and 2 h was observed.

The second pilot study was performed in another nine healthy volunteers. In six participants (one woman and five men, mean age 28.9 years, range: 23–43; mean weight: 75.0 kg, range: 68–90 kg), STA diameter was measured with Dermascan C and showed a mean increase between 39% and 76% during the infusion period. The other three participants (two women and one man, mean age 23.3 years, range: 23–24 years; mean weight: 66.7 kg, range: 65–70 kg) were investigated with the MRA technique, showing an increase in the circumference of STA and MMA, but not of the MCA (Figure 1).

Figure 1.

Mean circumference changes (percentage) of the middle cerebral artery (MCA), middle meningeal artery (MMA) and superficial temporal artery (STA) after 2-h infusion of VIP in three healthy volunteers. The circumference changes were assessed by MR angiography.

Main study

Twelve healthy volunteers (six women and six men; mean age: 25.8 years, range: 23–29 years; mean weight: 69.5 kg, range: 59–84 kg) completed the study on both experimental days (Figure 2). One Schirmer’s test at T₃₀ and four tests at T₉₀ were not performed, thus comparisons were made with eight participants. All other data were included in statistical analyses and comparisons between other variables were performed with 12 participants. At the baseline, there were no differences for any variable (Table 1).

Figure 2.

Twelve healthy volunteers were randomly allocated to receive a 2-hour infusion of VIP and/or placebo in a randomised, double blind, placebo-controlled, crossover trial.

Table 1.

Baseline values of 12 healthy participants on two trial days.

	VIP	Placebo	p values
MAP (mmHg)	85.9 ± 11.0	85.9 ± 5.3	0.52
HR (bpm)	66.0 ± 10.6	69.9 ± 13.3	0.15
STA diameter (mm)	1.02 ± 0.14	1.08 ± 0.16	0.27
Schirmer’s test – KOS eye	32.3 ± 10.9	30.6 ± 13.1	0.11
Schirmer’s test – free eye	23.6 ± 13.9	20.3 ± 10.6	0.36

Note: All values are presented as mean ± SD.

MAP: mean arterial pressure; HR: heart rate; STA: superficial temporal artery; KOS: kinetic oscillation stimulation; p value: paired t-test or Wilcoxon signed-rank test, according to the normal distribution of data.

Superficial temporal artery diameter

The STA diameter increased significantly during the VIP day compared to the placebo day (AUC_0–180min, 194.2 ± 18.4 vs. 147.8 ± 19.2, p < 0.001) (Figure 3). The peak increase in diameter of STA occurred 60 min after the start of VIP infusion, with a mean percentage increase of 52.2% (CI: 45.1–59.3%). In the post-infusion period, the percentage increase did not exceed 15% with respect to the baseline.

Figure 3.

Individual and mean change of the superficial temporal artery (STA) diameter assessed by a high-resolution ultrasonography. Dotted black lines represent individuals; averages are depicted in red for VIP and in light blue for placebo.

Headache

During the observational period (0–200 min), eight out of 12 participants (67%) reported headache on the VIP day, compared to one out of 12 (8%) during placebo (p = 0.023) (Table 2). AUC_0–200min for headache score was larger on the VIP day (0.125 [0–0.4]) compared to the placebo day (0 [0–0]) (p = 0.005). In the infusion period, AUC_0–120min between VIP (0 [0–0.19]) and placebo day (0 [0–0]) was significantly different (p = 0.046). During that time, five participants reported headache during the VIP day, and one participant during placebo. A significant difference (p = 0.009) was also found in the post-infusion period (AUC_{120min–200min}) between VIP (0.155 [0–0.935]) and placebo (0 [0–0]) (p = 0.034). During this period, seven participants reported headache on the VIP day, and one participant on placebo day. In the post-hospital phase (from 4 h to 12 h after infusion), five participants reported headache after VIP administration and none after placebo (Table 2). AUC_4h–12h for headache intensity scores were different in the post-hospital phase (p = 0.025). Overall sums of AUC headache scores are displayed in Figure 4. Three participants reported a headache that fulfilled criteria C and D of the International Classification (17) for migraine without aura, 20 min, 2 h and 5 h after the start of VIP administration.

Table 2.

Clinical characteristics of headache and associated symptoms after VIP and placebo (0–12 h observational period).

Participant and administration	Time to peak headache (duration)	Headache characteristics^a	Associated symptoms^b	Migraine-like headache	Rescue medication
1
VIP	2 h (1 h and 30 min)	Unilateral/7/Throbbing/Yes	No/No/No/Yes/Yes/Yes	No	Yes
Placebo	No	–/–/–/No	No/No/No/No/No/No	No	–
2
VIP	No	–/–/–/No	No/No/No/Yes/Yes/Yes	No	–
Placebo	No	–/–/–/No	No/No/No/No/No/No	No	–
3
VIP	No	–/–/–/No	No/No/No/Yes/Yes/Yes	No	–
Placebo	No	–/–/–/No	No/No/No/No/No/No	No	–
4
VIP	2 h and 40 min (1 h)	Unilateral/1/Throbbing/No	Yes/No/No/Yes/Yes/Yes	Yes	Yes
Placebo	No	–/–/–/No	No/No/No/No/No/No	No	–
5
VIP	2 h and 40 min(1 h and 30 min)	Bilateral/3/Pressing/No	Yes/No/No/Yes/Yes/Yes	No	Yes
Placebo	No	–/–/–/No	No/No/No/No/No/No	No	–
6
VIP	10 min (6 h)	Bilateral/2/Pressing/Yes	No/No/No/Yes/Yes/Yes	No	Yes
Placebo	No	–/–/–/No	No/No/No/No/No/No	No	–
7
VIP	5 h (10 h)	Bilateral/5/Pressing/Yes	Yes/Yes/No/Yes/Yes/Yes	Yes	Yes
Placebo	No	–/–/–/No	No/No/No/No/No/No	No	–
8
VIP	2 h and 50 min(10 min)	Unilateral/2/Throbbing/No	No/No/No/Yes/Yes/Yes	No	No
Placebo	No	–/–/–/No	No/No/No/No/No/Yes	No	–
9
VIP	1 h and 50 min(20 min)	Unilateral/1/Throbbing/No	No/No/No/Yes/Yes/Yes	No	No
Placebo	No	–/–/–/No	No/No/No/No/No/No	No	–
10
VIP	No	–/–/–/No	No/No/No/Yes/Yes/Yes	No	–
Placebo	No	–/–/–/No	No/No/No/No/No/No	No	–
11
VIP	20 min (10 min)	Unilateral/2/Throbbing/Yes	Yes/No/No/Yes/Yes/Yes	Yes	No
Placebo	1 h and 30 min(10 min)	Unilateral/2/Pressing/Yes	No/No/No/No/No/No	No	No
12
VIP	No	–/–/–/No	No/No/No/Yes/Yes/Yes	No	–
Placebo	No	–/–/–/No	No/No/No/Yes/No/No	No	–

VIP: Vasoactive Intestinal Polypeptide; h: hours; min: minutes.

^alocalization, intensity, quality, aggravated by physical activity.

^bnausea, photophobia, phonophobia, flushing, heartbeat, heat sensations.

Figure 4.

Overall sums of summary measures of area under the curve (AUC) for headache scores. Bars are depicted in red for VIP and in light blue for placebo.

Mean arterial pressure and heart rate

We found no difference in MAP between VIP and placebo (AUC_0–200min, 82.4 ± 5.6 vs. 85.4 ± 5.4, p = 0.083) (Figure 5). Shortly after the start of the infusion, a decrease of MAP not exceeding 10% was observed during the VIP day. Afterwards, it stabilised until the end of the infusion. HR increased significantly during the VIP day compared to placebo (AUC_0–200min, 81.9 ± 7.3 vs. 68.8 ± 11.9, p < 0.001) (Figure 5). Peak increase in HR occurred 30 min after the start of the VIP infusion, equal to 41.1% (CI: 30.0–52.2%). During placebo, HR was steady throughout the observational period.

Figure 5.

Individual and mean percentage change from baseline of mean arterial blood pressure (MAP) and heart rate (HR). Dotted black lines represent individuals; averages are depicted in red for VIP and in light blue for placebo.

Schirmer’s test

Figure 6 shows the mean changes (%) with respect to the baseline for both eyes, during VIP and placebo days. The AUC_0–90min did not differ between VIP and placebo for the KOS eye (ipsilateral to stimulation) (33.4 ± 9.3 vs. 29.6 ± 14.3, p = 0.095), but was significantly different for the free eye (contralateral to stimulation) (29.1 ± 10.8 vs. 20.5 ± 12.9, p = 0.033). On the KOS eye, the mean values at T₃₀ and T₉₀ were almost similar between the VIP day (33.6 and 35.4 mm) and placebo (32.6 and 32.3 mm). On the free eye, the mean values measured at T₃₀ and T₉₀ were increased during the VIP day (32.5 and 30.8 mm) compared to placebo (23.3 and 20.6 mm).

Figure 6.

Mean changes (percentage) and standard errors of the mean of Schirmer’s scores recorded from the KOS eye and free eye during experimental days. Mean changes are depicted in red for VIP and in light blue for placebo.

Adverse events

All AEs reported were transitory, mild or moderate in intensity (Table 3). Flushing, heat sensations and heart palpitations were more reported during VIP, compared to placebo. Two participants reported symptoms consistent with a drop in blood pressure (systolic blood pressure between 70 and 85 mmHg and diastolic blood pressure between 40 and 50 mmHg) without syncope. The infusion of VIP was temporarily stopped at T₉₀. They remained supine, in bed, with their legs elevated. Both reported feelings of dizziness, tiredness and pallor on the face, which disappeared in approximately 10 min. They avoided the KOS procedure at T₉₀. Another participant did not complete Schirmer’s tests at T₃₀ and T₉₀ because similar symptoms appeared at T₃₀. An additional participant did not complete Schirmer’s test at T₉₀ because similar symptoms appeared at T₉₀. The four participants recovered completely without any medical intervention. In the post-infusion period (120–200 min), only flushing was significantly more reported by those who performed the infusion of VIP.

Table 3.

Adverse events recorded and reported during 0–120 min (infusion period) and 121–200 min (post-infusion period).

	VIP (n = 12)	Placebo (n = 12)
Symptoms (0–120 min)
Flushing	12	1
Heart palpitations	12	0
Heat sensation	12	1
Weakness	5	0
Vasovagal reaction	4	0
Symptoms (121–200 min)
Flushing	9	0
Heart palpitations	2	0
Heat sensation	5	0
Weakness	3	0
Meteorism	2	0

Discussion

The main findings of the present study are that a long-lasting VIP infusion induced a prolonged extracranial vasodilation, increased cranial parasympathetic activity and caused a mild and delayed headache in healthy volunteers. Previous studies with VIP were performed with 20-minute infusions in healthy volunteers, causing only a mild headache and a short-lasting vasodilation of STA (6). In the current study, we extended the infusion from 20 minutes to 2 hours. According to the pharmacological properties of an intravenous infusion of VIP, we did not administer more VIP, but we administered the same amount of VIP over a longer period of time.

Effects on the cranial arteries

Main vasodilatory responses induced by VIP are mediated by two receptors, VPAC₁ and VPAC₂ (18,19). VPAC₁ is expressed on the endothelium, whereas VPAC₂ is located on the outer layers of the media arterial wall (8,20). Infusion of VIP in healthy humans resulted in a large and prolonged dilation of STA and MMA, but not MCA. To notice, a comparable extracranial vasodilation, but not cerebral, was obtained with a 20-minute infusion of PACAP27 and PACAP38 in healthy volunteers (4,5,21). Interestingly, the receptors for VIP and PACAPs are present on the MCA (22,23). In rats, VIP induced a vasodilatory response when applied to the abluminal side of the MCA and almost no response from the luminal side, suggesting that the MCA is protected by the BBB (24). In mice, VIP crossed the BBB by unidirectional transmembrane diffusion (25), but not in rats and baboons (26). In humans, infusion of VIP induced only a mild and short decrease of blood flow velocity of MCA and did not affect cerebral blood flow by single photon emission computed tomography (6).

VIP-induced vasodilation appears to be intimately related to the presence of VIP in arteries; the increase of STA diameter occurred between 41.6% and 52.2% during the infusion. At the end of the infusion, STA normalised rapidly. The same trend was reported with 20 min infusion (6). On the other hand, 20 min infusion of PACAPs induced several hours of STA dilation. Considering the similar elimination half-life of VIP and PACAPs (27,28), long-lasting vasodilatory responses are most likely mediated by overlapping pathways, through different receptors and/or intracellular mechanisms (i.e. a mechanism or receptor activated more by PACAPs than VIP).

VIP-induced headache

Several vasoactive headache-inducing substances show a biphasic response, with an immediate mild headache as well as a delayed headache or a migraine attack (29). The majority of them, including VIP, act on G protein-coupled receptors. These receptors activate multiple intracellular signalling pathways, followed by immediate and time-consuming mechanisms (30). In a previous study, 20 min infusion of VIP induced very mild headache in 42% of healthy volunteers in the immediate phase, and in 25% of participants during the delayed phase (6). The immediate headache is usually very mild, which might be related to the changes detected in blood pressure and heart rate. In the present study, 2-hour infusion of VIP provoked mild headache in 67% of participants. The induced headache was mainly concentrated in the post-infusion period. It lasted between 10 min and 10 h (median 1 h), and was more often bilateral, pressing and of variable intensity. Interestingly, the delayed headache observed in healthy individuals (4,5) is suggestive of a certain migraine-inducing ability of PACAPs, once tested in migraine patients (10,31). In rats, PACAP38 is capable of inducing a delayed activation and sensitisation of central trigeminovascular neurons (19). However, it is unclear how PACAPs may act on human trigeminovascular neurons. There is insufficient evidence that both PACAPs cross the BBB (32). In mice, only a very small percentage (0.053%) of PACAP38 does cross the BBB after intravenous administration (33). Accordingly, PACAPs’ migraine-inducing ability may arise from peripheral mechanisms.

After the administration of VIP, three participants (25%) reported migraine-like attacks. In healthy volunteers, a migraine-like attack (8%) was previously reported after administering 20 min infusion of VIP (6). Even after 20 min infusion of PACAP38 and PACAP27, two participants (22%) and one participant (6%) respectively reported migraine-like attacks (4,5). The percentage of healthy volunteers who reported migraine-like attacks (6–25%) is relatively close to the prevalence of migraine in the young population (34). Considering the relatively young age of the volunteers involved in these studies, it is very likely that healthy volunteers reporting migraine-like attacks after PACAPs or VIP may have an underlying susceptibility for migraine.

Cranial parasympathetic system

VIP is recognised as a strong vasodilator and an important contributor of tear production in humans (35,36). Increased serum levels seem to reflect the activation of the cranial parasympathetic system (37,38). Preganglionic parasympathetic fibres originate in the lacrimatory nucleus and synapse in the sphenopalatine ganglion. Here, postganglionic fibres join branches of the maxillary nerve to reach the lacrimal glands (39). At that point, parasympathetic nerve endings contain the parasympathetic neurotransmitter acetylcholine and at least one biologically active peptide, VIP (40,41). Overall, the KOS procedure led to an increased production of tears, compared to the contralateral side. However, VIP did not exhibit any difference on the KOS side, compared to placebo. There was an incremental, significant effect of VIP only on the contralateral side. Presumably, five minutes of KOS over-stimulates the cranial parasympathetic system, and we were unable to see any difference on the KOS side. The confined effect of VIP at the neurovascular interface (27) appears in disagreement with a direct stimulation of parasympathetic neurons. However, it is possible that an indirect activation from perivascular sensory afferents occurred (42). In animals, intra-arterial injection of VIP stimulates lacrimal gland water, electrolyte and protein secretion (43). Vasodilator and secretory responses were well correlated, but they appeared to be mediated by different mechanisms (44).

Limitations

The AEs may have compromised blinding as both the investigator and the participants could notify AEs. However, AEs are part of the human physiological response to VIP and could not be avoided. We believe that the current double-blinded approach remains the preferred methodological option for studying the long-lasting effects of VIP in humans. Supine positioning throughout the entire experiment, as well as the regular monitoring of blood pressure, might reduce the incidence of such events. Moreover, we found an increased parasympathetic activity on the non-stimulated side, but not on the KOS-stimulated side. The observed discrepancy between the two sides is likely to be caused by a non-optimal methodological approach. Five minutes of KOS is perhaps too long. On the KOS-stimulated side, five out of eight patients (62.5%) produced enough tears to wet all the strips both at the baseline and in subsequent measurements, for both experimental days. Thus, no differences were observed on the KOS side between VIP and placebo. Regarding the non-stimulated side, this never happened.

Conclusion

The VIP infusion over 2 h promoted a long-lasting cranial vasodilation, cranial parasympathetic activity and a delayed mild headache. These findings suggest that prolonged cranial vasodilation is associated with delayed headaches in healthy volunteers, resembling the effect of two other well-known migraine causing substances (i.e. PACAP38 and PACAP27). Prolonged vasodilation may activate sensory nerve fibres innervating the cranial arteries, generating the pain sensation. Moreover, the current study provides a novel methodology to further investigate the ability of VIP to induce delayed migraine-like attacks in migraine patients.

Clinical implications

Two-hour infusion of VIP promotes a long-lasting extracranial arterial dilation and delayed headache in healthy volunteers.

The induced headache is mild in intensity and mainly concentrated in the post-infusive period.

The long-term impact of an intravenous VIP infusion in healthy volunteers resembles the effects of PACAPs, well known for causing migraine-like attacks in migraine patients.

Supplemental Material

sj-pdf-1-cep-10.1177_0333102420937655 - Supplemental material for Two-hour infusion of vasoactive intestinal polypeptide induces delayed headache and extracranial vasodilation in healthy volunteers

Supplemental material, sj-pdf-1-cep-10.1177_0333102420937655 for Two-hour infusion of vasoactive intestinal polypeptide induces delayed headache and extracranial vasodilation in healthy volunteers by Lanfranco Pellesi, Mohammad Al-Mahdi Al-Karagholi, Basit Ali Chaudhry, Cristina Lopez Lopez, Josefin Snellman, Jens Hannibal, Faisal Mohammad Amin and Messoud Ashina in Cephalalgia

Footnotes

Ethics or Institutional Review Board Approval

The study was approved and subsequently amended by the Capital Region Ethics Committee of Denmark (H-18050862), in accordance with the Helsinki Declaration, subsequently revised in 2008. All healthy volunteers gave informed consent in a written form prior to participate in the study. The study was also registered at ClinicalTrials.gov (NCT03989817).

Acknowledgements

The authors thank all participating healthy volunteers for their patience, and Dr. Hashmat Ghanizada and lab technicians Lene Elkjaer and Winnie Grønning for expert assistance.

Declaration of conflicting interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: MA reports personal fees from Allergan, Amgen, Alder, Eli Lilly, Lundbeck, Novartis and Teva. MA has no ownership interest and does not own stocks of any pharmaceutical company. MA serves as associate editor of Cephalalgia, co-editor of The Journal of Headache and Pain, and associate editor of Headache. MA is the current president of the International Headache Society. MMK has acted as an invited speaker for Novartis and received travel grant from ElectroCore. CLL and JS are full-time employees of Novartis Pharma AG, Basel, Switzerland. FMA reports personal fees from Novartis, Eli Lilly and Teva. JH was supported by the Danish Biotechnology Center for Cellular Communication. LP and BAC declare no actual or potential conflict of interest.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research has received a grant from Novartis.

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