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Abstract

The role of the parasympathetic nervous system in the pathogenesis of migraine is disputed. The headache-eliciting effect of the parasympathetic neurotransmitter, vasoactive intestinal polypeptide (VIP), and its effect on cerebral arteries and brain haemodynamics has not been systematically studied in man. We hypothesized that infusion of VIP might induce headache in healthy subjects and cause changes in cerebral haemodynamics. VIP (8 pmol/kg per min) or placebo (0.9± saline) was infused for 25 min into 12 healthy young volunteers in a crossover, double-blind design. Headache was scored on a verbal rating scale from 0 to 10, regional cerebral blood flow (rCBF) was measured with single-photon emission computed tomography and ¹³³Xe inhalation and mean flow velocity in the middle cerebral artery (V_meanMCA) was measured with transcranial Doppler ultrasonography. The headache was very mild with a maximum score of 2 and described as a pressing or throbbing sensation. Five participants developed headache during VIP and one during placebo. During the infusion, a significant drop in V_meanMCA was seen for VIP compared with placebo (P < 0.001), but the effect quickly waned and no difference was found when comparing the time between 30 and 120 min. In addition, no significant difference in the diameter of the MCA could be found during the infusion. No significant differences in rCBF (P = 0.10) were found between VIP and placebo. A marked dilation of the superficial temporal artery was seen (P = 0.04) after VIP in the first 30 min but no difference was found when comparing the time between 30 and 120 min. We found no difference in mean arterial blood pressure between VIP and placebo days but the heart rate increased significantly on a VIP day compared with a placebo day (AUC_0–30min, P < 0.001). Plasma VIP was significantly higher on a VIP day compared with placebo (AUC_0–80min, P < 0.001). These results show that VIP causes a decrease in V_meanMCA without affecting rCBF. In spite of a marked vasodilator effect in the extracranial vessels and increased plasma VIP, healthy subjects developed only a very mild headache.

Keywords

Cerebral blood flow experimental headache models headache healthy volunteers intracranial hemodynamics transcranial doppler vasoactive intestinal polypeptide

Introduction

Animal (1–3) and human (4–6) studies have suggested that activation and sensitization of nociceptors around extra- and intracranial vessels may be a primary source of pain in migraine. The parasympathetic nervous system has for a long time been implicated in the pathogenesis of migraine (7). There has recently been increasing focus on the role of the parasympathetic system in migraine (8–10). It has been suggested that parasympathetic outflow to cephalic vasculature may trigger activation and sensitization of perivascular sensory afferents (10) and thereby migraine pain.

Parasympathetic efferent nerves release various neuropeptides such as pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) to regulate cerebrovascular tone and haemodynamics of the brain (11). Immunohistochemical studies have shown that VIP is located in the cerebral, meningeal and temporal arteries in close relation to the adventitia and adventitia–medial borders of the vessel wall (12–15). Furthermore, in vitro and in vivo studies have demonstrated that VIP acts as a powerful vasodilator in various species (16), including man (16–19). VIP may be increased in the cranial venous blood during attacks in a subgroup of patients with migraine associated with prominent autonomic symptoms (20) and in patients with cluster headache (21). It remains unknown, however, whether VIP may cause headache and its effect on brain haemodynamics has not been systematically studied in humans. We hypothesized that infusion of VIP may induce headache in healthy subjects and that VIP-induced headache may be associated with dilation of intra- and extracerebral blood vessels. To test this hypothesis, we performed a double-blind, placebo-controlled, crossover study in normal human volunteers and studied the effect on headache and haemodynamic variables.

Design and methods

Pilot experiment

Before the main study, we conducted an open-label pilot study in three healthy subjects(two male and one female) to find the optimal dose of VIP with respect to tolerable adverse events and detectable changes in mean blood flow velocity of the middle cerebral artery (V_meanMCA) measured with transcranial Doppler. The subjects received intravenous VIP in stepwise increasing doses of 6, 8 and 10 pmol/kg per min. Each infusion lasted 25 min followed by a 60-min wash-out. The results of the pilot study showed that a dosage of 8 pmol/kg per min was well tolerated and a led to a decrease in V_meanMCA. Therefore, we chose this dose for the main study.

Main experiment

We recruited 14 healthy subjects (seven male, seven female), mean age 24 years (range 22–28 years). Exclusion criteria were: a history of migraine or any other type of headache (except episodic tension-type headache more than once a month); any daily medication apart from oral contraceptives; serious somatic or psychiatric diseases. The study was approved by the Ethics Committee of the County of Copenhagen (KA03099) and was undertaken in accordance with the Helsinki Declaration of 1964, as revised in Edinburgh in 2000. All subjects gave informed consent to participate in the study.

Experimental design

In a double-blind, placebo-controlled, crossover design, the subjects were randomly allocated to receive 8 pmol/kg per min VIP or placebo (isotonic saline) over 25 min on 2 days separated by at least 1 week. All subjects reported to the laboratory at 08.00 h headache free. The intake of coffee, tea, cocoa or other methylxanthine-containing foods or beverages was not allowed for the last 8 h before the start of the study. All procedures were performed in a quiet room at a temperature of 25°C. The subjects were placed in the supine position and a venous catheter (Venflon^®) was inserted into the right and left antecubital vein for blood sampling and drug infusion. The participant then rested for 30 min before baseline measurements of blood pressure, heart rate (HR) and ECG were performed and the infusion started, using a time and volume controlled infusion pump (Braun Perfusor, Melsungen, Germany). Headache intensity, V_meanMCA, superficial temporal artery diameter, end-tidal partial pressure of CO₂ (P_etCO₂), adverse events and vital signs were recorded at T₋₁₀, T₀ and then every 10 min until 120 min after the start of infusion. Cerebral blood flow (CBF) measurements by single photon emission computed tomography (SPECT) were performed at T_0, T₂₀ and T₆₀. The subjects were discharged from the hospital after finishing the measurements and asked to complete a headache diary every hour until 10 h after discharge. The diary included headache characteristics and accompanying symptoms according to the International Headache Society (22), any rescue medication taken and adverse events. Subjects were allowed to take rescue medication of their own choice at any time.

Headache intensity

Headache intensity was recorded on a verbal rating scale (VRS) from 0 to 10 [0, no headache; 1, a very mild headache (including a feeling of pressing or throbbing); 5, moderate headache; 10, worst imaginable headache] (23).

Cerebral haemodynamics

Middle cerebral artery blood flow velocity

V_meanMCA was recorded bilaterally by transcranial Doppler (TCD) with hand-held 2-MHz probes (Multidop X; DWL, Sipplingen, Germany). Fixed probes were not used since they may cause discomfort and even headache (24). A time-averaged mean over 4 s or approximately four cardiac cycles was used as final measure for each time point. Identification of the MCA and marking of reproducible fix points were done as previously described (25). All recordings were done by the same skilled technician. PetCO₂ was recorded simultaneously with the TCD measurements using an open mask that caused no respiratory resistance (ProPac Encore^®; Welch Allyn Protocol, Beaverton, OR, USA). The measurements at T₀, T₂₀ and T₆₀ were performed immediately after regional cerebral blood flow (rCBF) measurements to obtain corresponding values. Flow in the territory of the MCA (rCBF_MCA) is proportional to the product of V_meanMCA and cross-sectional area of the MCA. Therefore changes in cross-sectional area could be calculated as ΔA = (rCBF_MCA(2) · V_meanMCA(2) ⁻¹) · (V_meanMCA(1) · rCBF_MCA(1) ⁻¹) − 1 (26).

Diameter of the superficial temporal artery

Diameter of the frontal branch of the superficial temporal artery (STA) was measured by a high-resolution ultrasonography unit (Dermascan C; Cortex Technology, Hadsund, Denmark: 20 MHz, bandwidth 15 MHz) as previously described (27, 28).

Cerebral blood flow

The examination was performed with the subject in the supine position, in quiet surroundings with eyes closed and ears unplugged. Four markers were drawn on the skin to ensure accurate positioning in each acquisition. P_etCO₂ was measured during each examination (Datex Normocap 200, Roedovre, Denmark). CBF was measured with ¹³³Xe inhalation (Ceretronix XAS SM 32C, Randers, Denmark) and SPECT, using a brain-dedicated gamma camera (Ceraspect; DSI, Waltham, MA, USA). The system uses a stationary annular NaI crystal and a fast rotating collimator. Flow was calculated in each pixel based on the clearance curve, output was the k_i value (29). To obtain CBF values, a partition coefficient (λ) of 0.85 was used. Calculation of flow in the perfusion territories of the major cerebral arteries was performed by fitting standard vascular regions of interest (ROI) on transactional slices of the brain. rCBF_MCA was calculated as mean of the right and left side, since there was no statistically significant difference between the sides.

Vital signs

HR and blood pressure were measured every 10 min using an auto-inflatable cuff (ProPac Encore^®; Welch Allyn Protocol). ECG (Cardiofax V; Nihon-Kohden, Shinjuku-ku, Tokyo, Japan) was monitored on an LCD screen and recorded on paper every 10 min.

Plasma concentration of VIP

Blood samples for VIP measurement were collected at baseline and 20, 25, 60 and 80 min after start of infusion in ice-chilled tubes containing 50 IU of heparin and 500 kIU aprotinin (Trasylol, Bayer, Germany) per ml blood. Plasma was separated by centrifugation (1500 g ) at 4°C within half an hour and immediately frozen at −20°C. The VIP radioimmunoassay was performed as previously described using antiserum 5603–6 at a final titre of 1.2 × 10⁶ in a total volume of 0.8 ml/tube (30, 31). This antiserum recognizes the mid- and C-terminal region of the VIP molecule (sequence 11–24) and displays no cross-reactivity with other known gastrointestinal peptides or neuropeptides. The label has a specific radioactivity of 0.92 nCi/fmol (∼34 Bq/fmol). The IC₅₀ value (the concentration of VIP giving 50% displacement of label) was 24 pmol/l and the assay could detect changes of 3 pmol/l with 95% confidence.

Data analysis and statistics

We defined an immediate phase as a period from 0 to 30 min (0–30 min) and a postinfusion phase as a period from 30 to 120 min (30–120 min) based on pharmakinetics of VIP. VIP has a short half-life in plasma of only a few minutes (32). The binding of VIP to one of the VIP receptors results in the activation of adenylate cyclase (AC), thereby causing marked increases in cellular cAMP production (33). Even though VIP may be present in plasma for only a short time, it may also have long-term effects after it has been eliminated from plasma. This seems to be the case for the effect of VIP on glycogen metabolism in astrocytes, where the effect is divided in two: an early action (within minutes) caused by cAMP and a delayed phase, caused by gene transcription (34).

The primary end-points were differences in area under the curve (AUC) for headache score (AUC_{headache score}), V_meanMCA (AUC_VMCA) and STA (AUC_STA) between groups in the period 0–30 min. Additional explorative end-points were differences in AUC_{headache score}, AUC_MCA and AUC_STA between groups in the period 30–120 min and differences in peak response between groups at time of maximum plasma concentration of VIP. The secondary end-points were differences in AUC_{heart rate} and AUC_MAP between groups in the period 0–30 min. Additional explorative end-points were differences in AUC_{heart rate} and AUC_MAP between groups in the period 30–120 min and differences in peak response (HR and MAP) between groups at time of maximum plasma concentration of VIP. Changes in P_etCO₂ was evaluated over 0–120 min, because V_MCA should be corrected with e^0.034 for each mmHg change in P_etCO₂ (35).

All values are presented as mean ± SD, unless otherwise stated. We calculated AUC according to the trapezium rule (36) to obtain a summary measure and to analyse the differences in response (V_meanMCA, headache score, diameter of STA, mean blood pressure, HR, P_etCO₂, VIP_plasma) between VIP and placebo. Analysis was performed with a paired, two-way t-test, except headache scores, where data are presented as medians and quartiles and tested with Wilcoxon signed rank test. Baseline was defined as T₋₀ before the start of infusion of each dose.

Continuous variables were analysed for changes over time for each dose separately with univariate analysis of variance with the fixed factors volunteer and time. To reduce mass significance the following time points were selected for analysis (T₀, T₂₀, T₆₀). If overall differences were found, Dunnett's test was applied to characterize which time points were different from baseline. We tested for period and carry-over effects with the use of independent samples t-test on the difference and the sums, respectively, of the AUC of the first and the second treatment. Headache was tested for period effect and carry-over effect with Mann–Whitney test.

All analyses were performed with SPSS for Windows 12.0 (Chicago, IL, USA). Five percent (P < 0.05) was accepted as the level of significance.

Results

There were no differences at baseline for any variables (Table 1).

Table 1

Baseline values (±SD) in 12 healthy subjects [cerebral blood flow (CBF) data for 10 subjects) on two trial days: mean velocity of blood flow in the middle cerebral artery (V_MCA), global CBF (CBF) and regional CBF in the territory of the middle cerebral artery (rCBF_MCA), end-tidal partial pressure of CO₂ (P_etCO₂) measured during transcranial Doppler measurements, mean arterial blood pressure (MAP), heart rate (HR), plasma VIP and diameter of superficial temporal artery (STA)

	VIP	Placebo	P-value
V_MCA (cm/s)	76.0 ± 11.2	77.9 ± 12.4	0.25
CBF (ml/100 g per min)	53.0 ± 5.6	54.6 ± 4.5	0.234
rCBF_MCA (ml/100 g per min)	52.5 ± 5.6	54.3 ± 4.1	0.219
P_etCO₂ (mmHg)	33.1 ± 2.6	33.0 ± 2.8	0.93
MAP (mmHg)	79.8 ± 6.9	80.9 ± 9.7	0.52
HR (/min)	59.6 ± 7.8	58.9 ± 10.1	0.77
Plasma VIP (pmol/l)	9.3 ± 3.6	8.4 ± 1.5	0.20
STA (mm)	1.32 ± 0.25	1.42 ± 0.18	0.23

P-value: paired t-test.

Twelve subjects completed the study on both trial days. One subject was excluded because we could not obtain intravenous access and one subject was excluded on his second trial day because of a vasovagal reaction 5 min after start of VIP infusion. Due to data loss, the CBF data for one subject could not be included and the CBF data for another subject had to be excluded from statistical analysis because of inexplicably high CBF values on the placebo day, probably due to technical problems. Thus, 10 subjects were left for the CBF analysis. Seven blood samples for the analysis of plasma VIP were not done, one sample on a VIP day, six samples on a placebo day, due to either haemolysis or technical problems.

There was no carry-over or period effect for baseline values of headache, MCA, CBF, rCBF_MCA, STA, MAP, HR or plasma VIP.

Headache

The AUC_0−30min on the VIP day, 0 (0–23.8), was significantly higher than on the placebo day, 0 (0–0) (P = 0.04) (Fig. 1). During the immediate phase (0–30 min), five subjects reported headache on the VIP day and one subject on the placebo day. There was no difference in the AUC_30−120min recorded during the postinfusion period between VIP and placebo days (P = 0.72). During the postinfusion period (30–120 min), three subjects reported headache on the VIP day and two on the placebo day.

Figure 1

Individual and median headache scores on a verbal rating scale (VRS). There were significantly higher pain responses after vasoactive intestinal polypeptide (VIP) (▪) compared with placebo (□) (P = 0.04). Thick lines are median pain scores.

We found no difference in the AUC_{delayed headache} recorded from 120 min to 12 h after infusion between VIP and placebo days (P = 0.75). Delayed headache was reported by six subjects, three on each trial day (Table 2). On the placebo day, the delayed headache lasted 3–6 h and none of the subjects had either a history of frequent headache or family history of headaches. On the VIP day, the delayed headache lasted 1–10 h and none of the subjects had a history of frequent headaches or family history, except subject 2, who reported that her mother had had frequent headaches. One subject reported migraine-like delayed headache on a VIP day (Table 2).

Table 2

Clinical characteristics in 12 healthy subjects

Patient	Time (to peak headache)		Headache (localization/intensity/quality/aggr.)	Associated symptoms (nausea/photo./phono./heartbeat/flushing/heat)	Migraine-like	Rescue medication
1	a		/–/–/–/No	No/No/No/Yes/Yes/Yes	No	–
	b		/–/–/–/No	No/No/No/–/–/–/	No	–
	c		/–/–/–/No	No/No/No/No/No/No	No	–
	d		/–/–/–/No	No/No/No/–/–/–/	No	–
2	a		/–/–/–/No	No/No/No/No/Yes/No	No	–
	b	4	Bilat./2/throb./Yes	No/Yes/No/–/–/–/	No	–
	c		/–/–/–/No	No/No/No/No/No/No	No	–
	d		/–/–/–/No	No/No/No/–/–/–/	No	–
3	a		Bioccip/1/pressing/No	No/Yes/No/Yes/Yes/Yes	No	–
	b		/–/–/–/No	No/No/No/–/–/–/	No	–
	c		/–/–/–/No	No/No/No/No/No/No	No	–
	d		/–/–/–/No	No/No/No/–/–/–/	No	–
4	a		Bioccip./1/pressing/Throb./Yes	No/Yes/No/Yes/Yes/Yes	No	–
	b		/–/–/–/No	No/No/No/–/–/–/	No	–
	c		Bilat./Temp/1/pressing/Yes	No/No/No/No/No/No	No	–
	d	3	Bilat./1/pressing/No	No/No/No/–/–/–/	No	–
5	a		/–/–/–/No	No/No/No/No/Yes/No	No	–
	b		/–/–/–/No	No/No/No/–/–/–/	No	–
	c		Bilat./Temp./1/Press./No	No/No/No/No/No/Yes	No	–
	d	12	/–/2/–/Yes	No/No/No/–/–/–/	No	–
6	a		/–/–/–/No	No/No/No/Yes/Yes/No	No	–
	b		/–/–/–/No	No/No/No/–/–/–/	No	–
	c		/–/–/–/No	No/No/No/No/No/Yes	No	–
	d	6	Bilat./1/Press./–/	No/No/No/–/–/–/	No	–
7	a		Diffuse/1/pres./Yes	No/No/No/No/Yes/Yes	No	–
	b		/–/–/–/No	No/No/No/–/–/–/	No	–
	c		/–/–/–/No	No/No/No/No/No/No	No	–
	d		/–/–/–/No	No/No/No/–/–/–/	No	–
8	a		BiTemp./1/Throb./pressing/Yes	No/No/No/Yes/Yes/Yes	No	–
	b		/–/–/–/No	No/No/No/–/–/–/	No	–
	c		/–/–/–/No	No/No/No/No/No/No	No	–
	d		/–/–/–/No	No/No/No/–/–/–/	No	–
9	a		BiTemp./1/pressing/Yes	Yes/No/No/Yes/Yes/Yes	No	–
	b	4	Bilat./1/Throb./pressing/Yes	No/Yes/Yes/–/–/–/	Yes	–
	c		/–/–/–/No	No/No/No/No/No/No	No	–
	d		/–/–/–/No	No/No/No/–/–/–/	No	–
10	a		/–/–/–/No	No/No/No/No/Yes/Yes	No	–
	b	8	Bilat./1/–/No	No/No/No/–/–/–/	No	–
	c		/–/–/–/No	No/No/No/No/No/No	No	–
	d		/–/–/–/No	No/No/No/–/–/–/	No	–
11	a		/–/–/–/No	No/No/No/Yes/Yes/Yes	No	–
	b		/–/–/–/No	No/No/No/–/–/–/	No	–
	c		/–/–/–/No	No/No/No/No/No/No	No	–
	d		/–/–/–/No	No/No/No/–/–/–/	No	–
12	a		/–/–/–/No	No/No/No/Yes/Yes/Yes	No	–
	b		/–/–/–/No	No/No/No/–/–/–/	No	–
	c		/–/–/–/No	No/No/No/Yes/Yes/Yes	No	–
	d		/–/–/–/No	No/No/No/–/–/–/	No	–

a, Peak headache during VIP infusion; b, peak headache post-VIP infusion; c, peak headache during placebo infusion; d, peak headache post placebo infusion; Migraine like, migraine-like headache.

Middle cerebral artery and regional cerebral blood flow

During the immediate phase (AUC_0−30min), V_MCA decreased significantly on the VIP day compared with the placebo day (P < 0.001) (Fig. 2). The peak decrease in V_MCA occurred 20 min after the start of VIP infusion. At the end of the infusion (T₂₀), the mean decrease in V_MCA was 14.4 ± 4.8% on the VIP day and 2.2 ± 6.0% on the placebo day compared with baseline (Fig. 2). The mean difference in response between VIP and placebo at T₂₀ was −11.3% (95% confidence interval −16, −6.6). There were no changes after VIP for global CBF (AUC_0−60min, P = 0.10) or for rCBF_MCA (AUC_0−60min, P = 0.10) when compared with placebo measured during CBF acquisition (Fig. 3). Analysis of variance (anova) showed no changes in CBF over time (VIP, P = 0.94, placebo P = 0.41). There were no changes between trial days in P_etCO₂ measured during CBF acquisition (AUC_0−60min, P = 0.70).

Figure 2

Individual and mean flow velocities in the middle cerebral arteries (V_MCA) assessed by transcranial Doppler ultrasonography. Infusion of vasoactive intestinal polypeptide (VIP) (▪) resulted in significant decrease in V_MCA during immediate phase (0–30 min) compared with placebo (□) (P < 0.001). Thick lines show mean values.

Figure 3

Individual and mean global cerebral blood flow (CBF) and regional flow in the territory of the middle cerebral artery (MCA) (rCBF_MCA) measured with ¹³³Xenon inhalation and single photon computed tomography in 10 healthy subjects. There were no changes in global CBF or rCBF_MCA between vasoactive intestinal polypeptide (VIP) (▪) and placebo (□) days (P > 0.05). Global CBF and rCBF_MCA remained unchanged over time on both trial days (P > 0.05). Thick lines show mean values.

Using the corresponding rCBF and V_meanMCA values, the changes in MCA diameter were calculated to be 8.4 ± 6.2% between baseline and T_20, 2.2 ± 5.2% between baseline and T₆₀ for VIP, 4.6 ± 9.3% between baseline and T₂₀ and 3.4 ± 4.7% between baseline and T₆₀ for placebo_. No significant difference was found between VIP and placebo (T₂₀, P = 0.29, T₆₀, P = 0.62).

During the postinfusion period (AUC_30−120min), there was no difference between VIP and placebo days (P = 0.17) and anova showed significant changes over time for V_MCA on both the VIP day (P = 0.0001) and the placebo day (P = 0.008). (Dunnett's t-test: VIP 20 min P < 0.001, VIP 60 min P = 0.009, placebo 20 min P = 0.456, placebo 60 min P = 0.004).

There were no differences in the P_etCO₂ recordings during TCD scans between VIP and placebo days, either during the immediate (AUC_0−30min, P = 0.82) or postinfusion phases (AUC_30−120min, P = 0.63).

Superficial temporal artery

During immediate phase (AUC_0−30min), the diameter of STA increased significantly on the VIP day compared with the placebo day (P = 0.04) (Fig. 4) and anova showed significant changes over time on the VIP day (P < 0.001) but not on the placebo day (P = 0.70). The peak increase in diameter of STA occurred 20 min after the start of VIP infusion and was a mean 27.8 ± 12.1% compared with baseline (Fig. 4). During the postinfusion period (AUC_30−120min), there was no difference between VIP and placebo days (P = 0.52).

Figure 4

Individual and mean diameter of the superficial temporal artery (STA) assessed by a high-resolution ultrasonography. During the immediate phase (0–30 min), the diameter of the STA increased significantly after vasoactive intestinal polypeptide (VIP) (▪) compared with placebo (□) (P = 0.04). Thick lines show mean values.

Mean arterial blood pressure and heart rate

We found no difference in MAP between VIP and placebo days during either the immediate phase (AUC_0−30min, P = 0.08) or the postinfusion phase (AUC_30−120min, P = 0.65) (Fig. 5). anova showed significant changes in MAP over time on the VIP day (P < 0.001) but not on the placebo day (P = 0.17). HR increased significantly on the VIP day compared with the placebo day (AUC_0−30min, P < 0.001). Peak increase in HR occurred 20 min after start of VIP infusion and was a mean 51 ± 19.2% compared with baseline (Fig. 5). There was no difference in HR between two trial days in the following 90 min (AUC_30−120min, P = 0.90). anova showed significant changes in HR over time on the VIP day (P < 0.001) but not on the placebo day (P = 0.89).

Figure 5

Mean percent change from baseline in mean arterial blood pressure (MAP) and heart rate (HR) during and after infusion of vasoactive intestinal polypeptide (VIP) (▪, MAP; ○, HR) and placebo (□, MAP; ▵, HR). MAP tended to decrease on the VIP day compared with the placebo day, but not statistically significantly (P = 0.08). A post hoc analysis anova showed significant changes in MAP over time on the VIP day (P < 0.001). Heart rate changed significantly over time on the VIP day (P < 0.001) and peak increase occurred 20 min after the start of VIP infusion and was a mean 51 ± 19.2% compared with baseline.

Plasma VIP

Plasma VIP was significantly higher on VIP days compared with placebo (AUC_0−80min, P < 0.001) (Fig. 6). anova showed significant changes over time on the VIP day (P < 0.001) but not on the placebo day (P = 0.36). Peak plasma concentration of VIP was measured at the end of the infusion (T_25min) on the VIP day and was 111.4 ± 47.8 pmol/l.

Figure 6

Individual (black) and mean (red) plasma concentration of vasoactive intestinal polypeptide (VIP) after infusion of VIP (▪) and placebo (□). Infusion of VIP resulted in a significant increase in plasma VIP compared with placebo (P < 0.001). The peak plasma concentration of VIP was measured at the end of the infusion (T_{25 min}) and was (±SD) 111.4 ± 47.8 pmol/l. Thick lines show mean concentrations.

Adverse events

One subject was excluded on his second trial day because of a vasovagal reaction 5 min after the start of VIP infusion. He reported a feeling of dizziness, heat sensation and mild dyspnoea. The blood pressure dropped and the HR rose but without crossing any of the protocol safety limits. ECG and auscultation of heart and lungs were unremarkable. Immediately after cessation of infusion he felt better and 5 min later his blood pressure and HR returned to baseline values. He was observed for an additional 60 min and discharged. There were more reported adverse events such as flushing, palpitation, heat sensation and headache on VIP days than on placebo days (Table 3).

Table 3

Adverse events recorded and reported during 0–30 min and 30–120-min periods

	Placebo	VIP	P
Symptoms (0–30 min)
Flushing	0	12	0.001
Palpitation	1	6	0.06
Heat sensation	3	10	0.04
Headache	1	5	0.13
Symptoms (30–120 min)
Flushing	0	7	0.02
Palpitation	0	2	0.50
Heat sensation	0	5	0.06
Headache	2	3	1.00

Groups compared and tested with McNemar test.

Discussion

The major outcome of the present study is that systemic administration of VIP induces only a very mild and short-lasting headache, a long-lasting dilation of the extracerebral superficial temporal artery and a short-lasting increase in the diameter of the middle cerebral artery when calculated from the decreased blood flow velocity and unchanged global and regional CBF.

Can VIP induce headache?

Experimental vasodilation of the internal carotid artery and MCA may induce focal head pain in humans (37). In migraine patients, an attack was associated with MCA dilation and both pain and dilation were reversed by sumatriptan (38). A model has been proposed in which migraine is the result of activation of postganglionic parasympathetic neurons in the sphenopalatine ganglion, resulting in vasodilation and local release of inflammatory molecules that activate meningeal nociceptors (39). Activity in the parasympathetic nervous system decreases cerebrovascular tone (11, 40) and one of the most abundant peptides in the parasympathetic efferents, VIP, acts as a powerful vasodilator in animals and humans (16, 18, 41, 42). Immunohistochemical studies have shown that VIP is present in the parasympathetic nerve fibres surrounding human temporal (43–45) and middle cerebral artery (19). VIP receptors are also found in these arteries (46, 47). The binding of VIP to its receptors activates the adenylate cyclase, increases cellular cAMP (33) and causes vasodilation (48). However, VIP-induced vasodilation may not always be caused exclusively by cAMP (49). In addition to its direct effect, nitric oxide may contribute to parasympathetic vasodilation by regulating VIP release (50, 51), possibly acting as a second messenger system for VIP and acetylcholine (52). Another possible mechanism of generating headache is a direct activation and sensitization of sensory afferents around cerebral blood vessels by exogenous VIP. VIP has been found in rat spinal ganglia (53) and VIP receptors occur in the trigeminal ganglia (54). VIP may play an important role in sensory transmission in the dorsal root ganglia of the spinal cord (55) and in spinal dorsal horn neurons (56). Intrathecal administration of VIP facilitates a spinal nociceptive reflex in the rat (57) and VIP antagonists decrease the firing rate of DRG cells in animal models of neuropathic pain (58). Based on these data, we speculated that systemic administration of VIP might cause headache by inducing vasodilation and activation or sensitization of nociceptors. The present study shows, however, that although VIP induced a marked dilation of extracerebral arteries and a non-significant dilation of the MCA, it causes only a very mild and short-lasting headache. These results raise the question, why did VIP fail to induce significant dilation of the MCA and caused so little headache?

It could be argued that the VIP dosage used in the present study was too low to elicit headache and marked vasodilation of the MCA. In the present study, the plasma concentration of VIP was monitored and found to be high compared with endogenous levels (mean 111.4 pmol/l) during infusion. The effect of exogenous VIP is mediated through VPAC₁ and VPAC₂ receptors, which have a binding affinity (IC₅₀) of 1–4 nm (59). We consider that the obtained VIP concentration is sufficient to activate the VIP receptors, which is supported by the high prevalence of systemic adverse events, such as a substantial increase in HR on the VIP day, facial flushing (92%), palpitations (67%) and feeling of warmth (75%) during infusion. These findings also indicate that the dose was close to the maximal tolerable.

Slow passage of VIP across the blood–brain barrier (BBB) is another possibility. We observed a mild and short decrease in V_MCA and no change in CBF, while the dilation of the STA was more pronounced and longer lasting. This difference in response between two vascular beds could be caused by the BBB since the STA is outside the barrier. If the BBB is bypassed and VIP injected intracisternally in dogs, a persistent vasodilation (120 min) is reported in the basilar artery (42). In mice, 0.15% of the injected VIP could be found in the brain 10 min after intravenous administration (60). In rats, after intracarotid infusion of a VIP analogue coupled to a transport vector, 3.2% of the perfusate concentration was found in the brain after 10 min (61). In baboons, intracarotid infusion of VIP after hypertonic opening of the BBB caused an increase in CBF of 37%, while no effect was seen under normal circumstances (62). In rats, VIP linked to a delivery vector increased CBF by 60% (63). Collectively, these data indicate that the BBB is very tight in arterioles, but less tight in the MCA. Dilation of STA seems without headache-inducing effect, as previously described (64, 65), while the mild and short-lasting effect on MCA corresponds to a mild and short-lasting headache.

In conclusion, this is the first double-blind cross-over study aimed at exploring the headache-eliciting effect of intravenous administration of the parasympathetic neurotransmitter VIP in healthy volunteers. In spite of marked vasodilator effect in the extracranial vessels and increased plasma VIP, healthy subjects developed only a very mild headache. Whether infusion of VIP may induce a more pronounced headache or migraine in migraineurs should be addressed in future studies.

Acknowledgements

The authors thank lab technicians Kirsten Brunsgaard, Lene Elkjær and Juliano Olsen for their dedicated and excellent assistance. The study was supported financially by University of Copenhagen, the Danish Headache Society and the Cool Sorption Foundation.

References

Strassman

Raymond

Burstein

Sensitization of meningeal sensory neurons and the origin of headaches. Nature 1996; 384 (6609):560–4.

Bove

Moskowitz

MA.

Primary afferent neurons innervating guinea pig dura. J Neurophysiol 1997; 77:299–308.

Bolay

Reuter

Dunn

Huang

Boas

Moskowitz

MA.

Intrinsic brain activity triggers trigeminal meningeal afferents in a migraine model. Nat Med 2002; 8:136–42.

Weissman-Fogel

Sprecher

Granovsky

Yarnitsky

Repeated noxious stimulation of the skin enhances cutaneous pain perception of migraine patients in-between attacks: clinical evidence for continuous sub-threshold increase in membrane excitability of central trigeminovascular neurons. Pain 2003; 104:693–700.

Burstein

Yarnitsky

Goor-Aryeh

Ransil

Bajwa

ZH.

An association between migraine and cutaneous allodynia. Ann Neurol 2000; 47:614–24.

Jensen

Extracranial blood flow, pain and tenderness in migraine. Clinical and experimental studies. Acta Neurol Scand 1993; 147 (Suppl.):1–27.

Möllendorff

W. Über hemikranie.

Arch Pathologische Anat Klinische Medizin 1867; 41:385–95.

Avnon

Nitzan

Sprecher

Rogowski

Yarnitsky

Different patterns of parasympathetic activation in uni- and bilateral migraineurs. Brain 2003; 126:1660–70.

Janig

Relationship between pain and autonomic phenomena in headache and other pain conditions. Cephalalgia 2003; 23 (Suppl. 1):43–8.

10.

Yarnitsky

Goor-Aryeh

Bajwa

Ransil

Cutrer

Burstein

. Wolff Award: Possible parasympathetic contributions to peripheral and central sensitization during migraine. Headache 2003; 43:704–14.

11.

Gulbenkian

Uddman

Edvinsson

Neuronal messengers in the human cerebral circulation. Peptides 2001; 22:995–1007.

12.

Jansen

Uddman

Ekman

Olesen

Ottosson

Edvinsson

Distribution and effects of neuropeptide Y, vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide in human middle meningeal arteries: comparison with cerebral and temporal arteries. Peptides 1992; 13:527–36.

13.

Edvinsson

Jansen

Cunha e Sa

Gulbenkian

Demonstration of neuropeptide containing nerves and vasomotor responses to perivascular peptides in human cerebral arteries. Cephalalgia 1994; 14:88–96.

14.

Larsson

Edvinsson

Fahrenkrug

Hakanson

Owman

Schaffalitzky de Muckadell

. Immunohistochemical localization of a vasodilatory polypeptide (VIP) in cerebrovascular nerves. Brain Res 1976; 113:400–4.

15.

Olesen

Gulbenkian

Valenca

Antunes

Wharton

Polak

. The peptidergic innervation of the human superficial temporal artery: immunohistochemistry, ultrastructure, and vasomotility. Peptides 1995; 16:275–87.

16.

Suzuki

McMaster

Lederis

Rorstad

OP.

Characterization of the relaxant effects of vasoactive intestinal peptide (VIP) and PHI on isolated brain arteries. Brain Res 1984; 322:9–16.

17.

White

RP.

Responses of human basilar arteries to vasoactive intestinal polypeptide. Life Sci 1987; 41:1155–63.

18.

Edvinsson

Ekman

Distribution and dilatory effect of vasoactive intestinal polypeptide (VIP) in human cerebral arteries. Peptides 1984; 5:329–31.

19.

Edvinsson

Ekman

Jansen

Ottosson

Uddman

Peptide-containing nerve fibers in human cerebral arteries: immunocytochemistry, radioimmunoassay, and in vitro pharmacology. Ann Neurol 1987; 21:431–7.

20.

Goadsby

Edvinsson

Ekman

Vasoactive peptide release in the extracerebral circulation of humans during migraine headache. Ann Neurol 1990; 28:183–7.

21.

Edvinsson

Goadsby

PJ.

Neuropeptides in migraine and cluster headache. Cephalalgia 1994; 14:320–7.

22.

22 IHS. The International Classification of Headache Disorders, 2nd Edition. Cephalalgia 2004; 24 (Suppl. 1):9–160.

23.

Iversen

Olesen

Tfelt-Hansen

Intravenous nitroglycerin as an experimental model of vascular headache. Basic characteristics. Pain 1989; 38:17–24.

24.

Thomsen

Ahlgren

Iversen

Olesen

Middle cerebral artery blood flow velocity increases during experimental pressure pain in the head. In: Olesen

, editor. Experimental headache models. New York: Raven Press 1995:337–9.

25.

Thomsen

Iversen

HK.

Experimental and biological variation of three-dimensional transcranial Doppler measurements. J Appl Physiol 1993; 75:2805–10.

26.

Dahl

Russell

Nyberg-Hansen

Rootwelt

Effect of nitroglycerin on cerebral circulation measured by transcranial Doppler and SPECT. Stroke 1989; 20:1733–6.

27.

Kruuse

Thomsen

Birk

Olesen

Migraine can be induced by sildenafil without changes in middle cerebral artery diameter. Brain 2003; 126:241–7.

28.

Iversen

Nielsen

Olesen

Tfelt-Hansen

Arterial responses during migraine headache. Lancet 1990; 336 (8719):837–9.

29.

Celsis

Goldman

Henriksen

Lassen

NA.

A method for calculating regional cerebral blood flow from emission computed tomography of inert gas concentrations. J Comput Assist Tomogr 1981; 5:641–5.

30.

Fahrenkrug

Schaffalitzky de Muckadell

OB.

Distribution of vasoactive intestinal polypeptide (VIP) in the porcine central nervous system. J Neurochem 1978; 31:1445–51.

31.

Fahrenkrug

Schaffalitzky de Muckadell

OV.

Radioimmunoassay of vasoactive intestinal polypeptide (VIP) in plasma. J Lab Clin Med 1977; 89:1379–88.

32.

Domschke

Bloom

Mitznegg

Mitchell

Lux

. Vasoactive intestinal peptide in man: pharmacokinetics, metabolic and circulatory effects. Gut 1978; 19:1049–53.

33.

McCulloch

MacKenzie

Johnson

Robertson

Holland

Ronaldson

. Additional signals from VPAC/PAC family receptors. Biochem Soc Trans 2002; 30:441–6.

34.

Magistretti

Cardinaux

Martin

JL.

VIP and PACAP in the CNS: regulators of glial energy metabolism and modulators of glutamatergic signaling. Ann NY Acad Sci 1998; 865:213–25.

35.

Markwalder

Grolimund

Seiler

Roth

Aaslid

Dependency of blood flow velocity in the middle cerebral artery on end-tidal carbon dioxide partial pressure—a transcranial ultrasound Doppler study. J Cereb Blood Flow Metab 1984; 4:368–72.

36.

Matthews

Altman

Campbell

Royston

Analysis of serial measurements in medical research. BMJ 1990; 300:230–5.

37.

Nichols

Mawad

3rd Mohr

Stein

Hilal

Michelsen

WJ.

Focal headache during balloon inflation in the internal carotid and middle cerebral arteries. Stroke 1990; 21:555–9.

38.

Friberg

Olesen

Iversen

Sperling

Migraine pain associated with middle cerebral artery dilatation: reversal by sumatriptan. Lancet 1991; 338 (8758):13–7.

39.

Burstein

Jakubowski

Unitary hypothesis for multiple triggers of the pain and strain of migraine. J Comp Neurol 2005; 493:9–14.

40.

Suzuki

Hardebo

JE.

The cerebrovascular parasympathetic innervation. Cerebrovasc Brain Metab Rev 1993; 5:33–46.

41.

Jansen-Olesen

Goadsby

Uddman

Edvinsson

Vasoactive intestinal peptide (VIP) like peptides in the cerebral circulation of the cat. J Auton Nerv Syst 1994; 49 (Suppl.):S97–103.

42.

Seki

Suzuki

Baskaya

Kano

Saito

Takayasu

. The effects of pituitary adenylate cyclase-activating polypeptide on cerebral arteries and vertebral artery blood flow in anesthetized dogs. Eur J Pharmacol 1995; 275:259–66.

43.

Uddman

Edvinsson

Jansen

Stiernholm

Jensen

Olesen

. Peptide-containing nerve fibres in human extracranial tissue: a morphological basis for neuropeptide involvement in extracranial pain? Pain 1986; 27:391–9.

44.

Jansen

Uddman

Hocherman

Ekman

Jensen

Olesen

. Localization and effects of neuropeptide Y, vasoactive intestinal polypeptide, substance P, and calcitonin gene-related peptide in human temporal arteries. Ann Neurol 1986; 20:496–501.

45.

Edvinsson

Fahrenkrug

Hanko

Owman

Sundler

Uddman

VIP (vasoactive intestinal polypeptide)-containing nerves of intracranial arteries in mammals. Cell Tissue Res 1980; 208:135–42.

46.

Fahrenkrug

Hannibal

Tams

Georg

Immunohistochemical localization of the VIP1 receptor (VPAC1R) in rat cerebral blood vessels: relation to PACAP and VIP containing nerves. J Cereb Blood Flow Metab 2000; 20:1205–14.

47.

Amenta

Cavalotti

De Michele

De Vincentis

Rossodivita

Vasoactive intestinal polypeptide receptors in rat cerebral vessels: an autoradiographic study. J Auton Pharmacol 1991; 11:285–93.

48.

Paterno

Faraci

Heistad

DD.

Role of Ca(2+)-dependent K+ channels in cerebral vasodilatation induced by increases in cyclic GMP and cyclic AMP in the rat. Stroke 1996; 27:1603–7; discussion 1607–8.

49.

Yao

Sheikh

Ottesen

Jorgensen

JC.

Vascular effects and cyclic AMP production produced by VIP, PHM, PHV, PACAP-27, PACAP-38, and NPY on rabbit ovarian artery. Peptides 1996; 17:809–15.

50.

Kimura

Chang

Lee

TJ.

Segregation of VIPergic-nitric oxidergic and cholinergic-nitric oxidergic innervation in porcine middle cerebral arteries. Brain Res 1998; 801:78–87.

51.

Goadsby

Uddman

Edvinsson

Cerebral vasodilatation in the cat involves nitric oxide from parasympathetic nerves. Brain Res 1996; 707:110–8.

52.

Modin

Weitzberg

Lundberg

JM.

Nitric oxide regulates peptide release from parasympathetic nerves and vascular reactivity to vasoactive intestinal polypeptide in vivo . Eur J Pharmacol 1994; 261:185–97.

53.

Yaksh

Abay

2nd Go

VL.

Studies on the location and release of cholecystokinin and vasoactive intestinal peptide in rat and cat spinal cord. Brain Res 1982; 242:279–90.

54.

Chaudhary

Baumann

TK.

Expression of VPAC2 receptor and PAC1 receptor splice variants in the trigeminal ganglion of the adult rat. Brain Res Mol Brain Res 2002; 104:137–42.

55.

Liu

Morris

Vasoactive intestinal polypeptide produces depolarization and facilitation of C-fibre evoked synaptic responses in superficial dorsal horn neurones (laminae I–IV) of the rat lumbar spinal cord in vitro . Neurosci Lett 1999; 276:1–4.

56.

Jeftinija

Murase

Nedeljkov

Randic

Vasoactive intestinal polypeptide excites mammalian dorsal horn neurons both in vivo and in vitro . Brain Res 1982; 243:158–64.

57.

Cridland

Henry

JL.

Effects of intrathecal administration of neuropeptides on a spinal nociceptive reflex in the rat: VIP, galanin, CGRP, TRH, somatostatin and angiotensin II. Neuropeptides 1988; 11:23–32.

58.

Dickinson

Mitchell

Robberecht

Fleetwood-Walker

SM.

The role of VIP/PACAP receptor subtypes in spinal somatosensory processing in rats with an experimental peripheral mononeuropathy. Neuropharmacology 1999; 38:167–80.

59.

Harmar

Arimura

Gozes

Journot

Laburthe

Pisegna

. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol Rev 1998; 50:265–70.

60.

Dogrukol-Ak

Banks

Tuncel

Passage of vasoactive intestinal peptide across the blood–brain barrier. Peptides 2003; 24:437–44.

61.

Bickel

Yoshikawa

Landaw

Faull

Pardridge

WM.

Pharmacologic effects in vivo in brain by vector-mediated peptide drug delivery. Proc Natl Acad Sci USA 1993; 90:2618–22.

62.

McCulloch

Edvinsson

Cerebral circulatory and metabolic effects of vasoactive intestinal polypeptide. Am J Physiol 1980; 238:H449–56.

63.

Pardridge

WM.

Central nervous system pharmacologic effect in conscious rats after intravenous injection of a biotinylated vasoactive intestinal peptide analog coupled to a blood–brain barrier drug delivery system. J Pharmacol Exp Ther 1996; 279:77–83.

64.

Drummond

Lance

JW.

Extracranial vascular changes and the source of pain in migraine headache. Ann Neurol 1983; 13:32–7.

65.

Myers

Boone

Gregg

JM.

Superficial temporal arterial dilation without headache in extracranial-intracranial bypass patients. Headache 1982; 22:118–21.

Vasoactive Intestinal Polypeptide Evokes Only a Minimal Headache in Healthy Volunteers

Abstract

Keywords

Introduction

Design and methods

Pilot experiment

Main experiment

Experimental design

Headache intensity

Cerebral haemodynamics

Middle cerebral artery blood flow velocity

Diameter of the superficial temporal artery

Cerebral blood flow

Vital signs

Plasma concentration of VIP

Data analysis and statistics

Results

Headache

Middle cerebral artery and regional cerebral blood flow

Superficial temporal artery

Mean arterial blood pressure and heart rate

Plasma VIP

Adverse events

Discussion

Can VIP induce headache?

Acknowledgements

References