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Abstract

Neurovascular coupling may be altered in migraneurs. Therefore, visual evoked potentials (VEP) and visually evoked cerebral blood flow velocity responses (VEFR) were simultaneously recorded in 30 healthy controls and 30 migraneurs interictally using a checkerboard stimulus with visual contrasts of 1%, 10% and 100%. The VEFR were measured in the posterior cerebral artery using transcranial Doppler and VEP were recorded from occipital leads. We found an increase in VEFR and VEP in both the healthy and migraneur groups (P < 0.01). VEFR were significantly higher in migraneurs (P < 0.01), while VEP did not significantly differ between the groups (P > 0.05). Regression showed a significant association between VEP and VEFR in both healthy controls (r = 0.66, P < 0.01) and migraneurs (r = 0.63, P < 0.01). The regression coefficient of migraneurs (b = 0.88, SE = 0.08) was significantly higher than that of healthy controls (b = 0.55, SE = 0.07) (P = 0.04). We conclude that neurovascular coupling is increased in migraneurs interictally.

Keywords

Visually evoked cerebral blood flow responses TCD Visual evoked potentials Neurovascular coupling Migraine

Introduction

The precise matching of local cerebral blood flow (CBF) to metabolic demand is a crucial process that sustains neuronal activity. Although this coupling between cortical activity and cerebral blood flow is an old concept (1), the underlying mechanism is still far from being completely understood (2). Recent animal studies, in which simultaneous measurements of neuronal activity and haemodynamic responses upon stimulation were made, demonstrated that the vascular responses directly reflect an increase in neuronal activity, correlating in particular with the local field potential, which represents the synchronized synaptic inputs of a given neuronal population (3). Similarly, a significant correlation between evoked potentials and CBF was found in animals (4) and humans using functional magnetic resonance (fMRI) (5) or near infrared spectroscopy (NIRS) (6).

Migraine is considered a neurovascular disorder, in which both vascular and neuronal components play a pathophysiological role (7). During visual stimulation, several functional neuroimaging methods, including fMRI, positron emission tomography (PET) and transcranial Doppler (TCD), show a localized increase in blood volume and blood flow velocity in the territory of the posterior cerebral artery (PCA) in patients with migraine during the headache-free period compared to healthy controls (8–10). Empirically, however, without a corresponding index of neuronal electrical activity, any increase in blood flow observed with neuroimaging methods following stimulation might have occurred through an increase in neuronal activity, the neurovascular coupling mechanism, or both. Electrophysiological studies, performed in recent years, have demonstrated dysfunctional cortical excitability in migraneurs during headache – free periods (11). Studies of cortical excitability using transcranial magnetic stimulation have yielded conflicting results (12). However, results obtained using habituation of pattern-reversal visual evoked potentials to explore cortical excitability changes induced by repetitive transcranial magnetic stimulation suggest a decreased level of preactivation excitability rather than hyperexcitability (13).

These observations prompted us to hypothesize that the cerebral vascular response to stimulation is increased in migraneurs due to an increase in the neurovascular coupling mechanism. To test this hypothesis in patients with migraine during the headache-free period visual evoked potentials (VEP) and visually evoked cerebral blood flow velocity responses (VEFR) were simultaneously recorded and their relationship analysed.

Methods

Thirty patients with migraine, aged 36.6 ± 0.4 years (range 19–51 years; 20 women, 10 men), and 30 healthy volunteers, aged 38.0 ± 9.6 years (range 22–55 years; 22 women, 8 men), were included in the study. The group of patients with migraine was further divided into subgroups of patients without aura (MwA) and with aura (MA). The first subgroup consisted of 16 subjects (5 male, 11 female) with a mean age of 35.2 ± 10.7 years, while the second subgroup consisted of 14 subjects (5male, 9 female) with a mean age of 38.2 ± 10.1 years. The diagnosis of migraine was made, independently of the neurosonologist, by a neurologist according to the International headache society (IHS) criteria (14). Written informed consent was obtained from the patients and controls. The study was approved by the Medical Ethics Committee of the Republic of Slovenia. Patients with migraine answered a short questionnaire concerning duration of migraine, number of attacks per month, number of days since the last attack, presence of aura, prophylactic treatment and characteristics of attacks. Disability was assessed using the MIDAS disability scale.

The clinical and neurological examinations, as well as the ultrasound examination of cerebral and precerebral arteries, were normal in all subjects. Visual acuity was investigated with Snellen cards, with the subjects with refractive errors wearing their glasses during the recordings. All subjects were instructed not to drink caffeine-containing beverages and to refrain from smoking on the day of testing. Volunteers taking any medication were excluded. Examinations were performed during headache-free intervals in a dark, quiet room at the same time of day, after an adaptation of at least 10 min. Before the actual testing, the research protocol was explained to the subjects, who were also asked to breathe regularly during the experiment. They were seated comfortably and fixed their eyes on a small spot of red light in the centre of the computer screen.

Stimulus paradigm

The visual stimulation was applied using a checkerboard on-set paradigm that was presented on the computer screen. The distance between the subjects and the screen was 1 meter, subtended 22 degrees of the visual angle. The stimulus consisted of white and black checks arranged in a checkerboard pattern with a spatial frequency of 1.6 cycles per degree. The mean luminance of the visual stimulus was 28 cd/m². The luminance (L) of the checks was changed in order to obtain visual contrasts of 100%, 10% and 1%. The visual contrast (C) was defined according to the formula:

C = (L_max − L_min)/(L_max + L_min)

where L_max was maximal luminance and L_min was minimal luminance. The mean luminance of the checkerboard at different visual contrasts remained unchanged.

The experimental session consisted of stimulus eyes-open and stimulus eyes-closed periods. The stimulus eyes-open period lasted 70 s, during which the visual stimuli, a pattern on-set stimulus, in which the checkerboard pattern interchanged with a diffuse white stimulus of equal mean luminance, were presented. The duration of the checkerboard pattern appearance was 200 ms and that of the diffuse white stimulus 500 ms. The stimulus frequency was 1.4 Hz. Therefore, exactly 100 VEP were obtained during the stimulus eyes-open period. The stimulus eyes-open period was repeated five times in a row at each visual contrast in order to obtain approximately 500 VEP for each visual contrast. The stimulus eyes-closed period lasted 30 s. The order of the stimulus conditions was randomly varied for each subject.

VEFR recording

A multimodal recording of arterial blood pressure, heart rate, end-tidal carbon dioxide and arterial blood flow velocity was performed. The arterial pressure was continuously monitored with a blood pressure monitor (Colin 7000, Komaki-City, Japan) and the end-tidal carbon dioxide was monitored with an infrared capnograph (Capnodig, Draeger: Lübeck, Germany). The heart rate was determined by processing the Doppler signal with the TCD8 commercial software (Multidop X4/TCD8; DWL Electronische System GmbH, Sipplingen, Germany). The arterial blood velocity was recorded in the left middle cerebral artery (MCA) and in the right posterior cerebral artery (PCA) through temporal acoustic windows with a Multi-Dop X4 (DWL, Sipplingen, Germany), using 2 MHz transducers. The vessels were identified according to the criteria described elsewhere (15). To confirm the localization of vessels we considered anatomic landmarks, the direction of flow and compression maneuvers. The criterion for successful PCA recording was a clear-cut increase in flow velocity during the period when the subjects had their eyes opened as opposed to the period when their eyes were closed. The P2 segment of the PCA was always insonated since it proved to have a higher visual response than the P1 segment (16). The MCA and the PCA were monitored at typical depths of 54 mm and 64 mm, respectively. We attempted to maintain a 10 mm difference in depth between the MCA and the PCA.

VEP recording

The cerebral evoked activity was recorded from the scalp by means of silver-silver chloride cup electrodes (10 mm diameter), fixed by contact paste. The resistance was kept below 5 kΩ. The electrodes were placed according to the recommendations of the International Society for Clinical Electrophysiology of Vision (ISCEV) (17). Three active electrodes were used, i.e. the Oz designated electrode placed 10% of the inion-nasion distance above the inion, and the O1 and O2 designated electrodes placed 10% of the head circumference laterally to the left and right, respectively. The reference electrode designated Fz was placed 20% of the inion-nasion distance anterior to the vertex. The VEP activity was fed to an amplifier system with a linear frequency response from 1 to 250 Hz, and displayed on an oscilloscope for continual observation. The analysis time was 600 ms, and the sensitivity 10 µV per division. The signal was subsequently fed to a computer system, which also served as an on-line averager. Five hundred responses were commonly averaged and presented on the screen of an oscilloscope prior to being recorded on disc memory.

Evaluation of data on multimodal recording

The multimodal recording (Fig. 1) consisted of the basal period before stimulation and of the stimulation periods. The mean amplitudes of the arterial blood flow velocities in the MCA (v_mMCA) and the PCA (v_mPCA), as well as the mean amplitudes of arterial pressure (MAP), heart rate (HR) and end tidal carbon dioxide (Et-CO₂), for the basal state (before stimulation with eyes closed) and for each stimulus eyes-open period at 1%, 10% and 100% visual contrasts were determined. The mean amplitudes were calculated with the TCD8 software, using a commercial algorithm according to the formula:

Figure 1

Multimodal recording of v_mMCA, v_mPCA, MAP, HR, and Et-CO₂ at three different visual contrasts. During each visual contrast, five consecutive eyes-open and eyes-closed periods are seen. The upper multimodal recording was obtained in a healthy 31-year-old subject. The lower multimodal recording was obtained in a 28-year-old migraine patient. v_mMCA, mean arterial velocity in the left middle cerebral artery; v_mPCA, mean arterial velocity in the right posterior cerebral artery; MAP, mean arterial pressure; HR, heart rate; Et-CO₂, end-tidal carbon dioxide.

A_m = ∫vd_t/(t₀ − t₁)

where A_m represents the mean amplitude of the variables (v) included in the measurements and t₀-t₁ the time interval in which variable was integrated. The VEFR were defined as the differences between the v_mPCA under basal conditions and the v_mPCA during the stimulus eyes-open periods at 1%, 10% and 100% visual contrasts. According to the VEFR, we defined the δv_mMCA, δMAP, δHR and δ Et-CO₂ as differences between the v_mMCA, MAP, HR and Et-CO₂ in the basal states, and the v_mMCA, MAP, HR and Et-CO₂ at the stimulus eyes-open periods at 1%, 10% and 100% visual contrasts. We then calculated the mean of five successive VEFR, δv_mMCA, δMAP, δHR and δ Et-CO₂.

VEP data evaluation

Analysis of the averaged responses of the on-set VEP, as detected from the active electrode at O1 position, was made off-line on a computer system by measurement of the amplitudes in the interval of interest. We designed software that enabled us to calculate the mean absolute amplitude A of the VEP according to the formula:

| A | = S | A | / n,

where A is the amplitude of the sample and n the number of samples during the chosen period. Amplitudes of the samples were measured from the baseline. The sampling frequency was 1.67 sample/ ms. The mean absolute amplitude was calculated for the interval from 50 to 200 ms, which is a typical interval in which the early on-set VEP response occurs (18).

Statistical methods

To analyse the differences between more than two measuring points in the same group, anova for repeated measures was used. If a model showed significant variances, a paired t-test with Bonferroni correction was used to locate the point of significance. In order to evaluate relationships, a model of linear regression was applied. All statistical analyses were performed using SPSS statistical software. The differences were considered significant when P < 0.05. In order to analyse the difference between two slopes of the regression curves, a t-test was applied (19).

Results

The characteristics of patients with migraine were as follows: duration of migraine was 14 ± 9.2 years, frequency of attacks was 2.8 ± 1.2 per month, number of days since the last attack was 6.7 ± 2.9 days. All patients reported photophobia and nausea, 87% phonophobia and 73% vomiting. None had received prophylactic treatment. Visual aura was reported in 14 subjects. All subjects reported a high disability grading of III and IV according to the MIDAS questionnaire. We found no significant differences between the groups, either in age (P > 0.58) or in the proportion of women (P = 0.97).

No significant difference in the basal velocity of blood flow in the PCA was found between the healthy controls and migraine patients. In all subjects, visual stimulation produced an immediate increase in v_mPCA with no adaptation. The pattern and absolute values of velocity changes on visual stimulation were not statistically significantly different between the controls and migraneurs (Fig. 2). On visual stimulation with 1%, 10% and 100% visual contrast, the v_mPCA increased in healthy controls by 3.1%, 8.4% and 10.2%, respectively, and in migraneurs by 6.3%, 11.1% and 16.3%, respectively. The increase in v_mPCA was statistically different at different contrasts of visual stimulus (1%, 10% and 100%) and between controls and migraneurs. The differences between the MwA and MA were not significant at any measuring point (P = 0.58) (Fig. 3).

Figure 2

Effect of visual contrast on arterial velocity in the posterior cerebral artery (v_mPCA). The visual contrast was transformed into the square root of visual contrast. The means and their standard deviations are presented at each visual contrast.

Figure 3

The dependence of visually evoked cerebral blood flow velocity responses (VEFR) on visual contrast for the group of patients with migraine with aura (MA) (—▴—), patients with migraine without aura (MwA) (—▵—) and healthy subjects (—○—).

On increase of visual contrast the amplitude of VEP increased in healthy controls, MwA and MA (Fig. 4). The increase in VEP amplitude was statistically different at different contrasts of visual stimulus (1%, 10% and 100%) in all groups (Fig. 5). The differences in the amplitude of VEP between the experimental groups were not statistically significant at any measuring point (P = 0.31), except that VEP was significantly higher in MA than in controls at 10% visual contrast (P = 0.03).

Figure 4

An example of visual evoked potentials (VEP) recordings in a 28-year-old migraine patient (a) and in a 31-year-old healthy subject (b) from occipital leads (O1, O2, Oz) at three different visual contrasts.

Figure 5

The dependence of visual evoked potentials (VEP) on visual contrast for the group of patients with migraine with aura (MA) (—▴—), patients with migraine without aura (MwA) (—▵—) and healthy subjects (—○—).

In order to examine neurovascular coupling we tested the relationship between the VEP and the VEFR in healthy controls and in patients with migraine (Fig. 6). Linear regression analysis showed a positive correlation between the VEP and the VEFR (r = 0.66, P < 0.01) in both healthy controls and in patients with migraine (r = 0.63, P < 0.01). The regression coefficient (slope) in the group of patients with migraine was 0.88 (SE = 0.08) and in healthy controls 0.55 (SE = 0.07), which was statistically significant (P = 0.04) (Table 1). We did not find any significant differences between regression coefficients in MwA compared to MA (P = 0.96).

Figure 6

The scatter plot between visually evoked cerebral blood flow velocity responses (VEFR) and visual evoked potentials (VEP) in patients with migraine (- - - -) and healthy subjects (——). The regression coefficients for the migraine group is b = 0.88 (standard error = 0.08; P < 0.01) and healthy subjects b = 0.55 (standard error = 0.07; P < 0.01).

Table 1

The correlation coefficients (r), regression coefficients (b), standard errors (SE), and significances (P) in healthy subjects, patients with migraine without aura (MwA) and migraine with aura (MA)

	r	b	SE	P-value
Healthy subjects	0.66	0.55	0.07	<0.01
MwA	0.64	0.76	0.15	<0.01
MA	0.68	0.77	0.14	<0.01

The other variables, i.e. δv_mMCA (P = 0.11), δMAP (P = 0.22), δEt-CO₂ (P = 0.18) and δHR (P = 0.17), did not show significant differences along the measuring points in healthy subjects (P = 0.11, P = 0.22, P = 0.18, P = 0.17, respectively) or in patients with migraine (P = 0.32, P = 0.42, P = 0.15, P = 0.26, respectively). The differences between the both subgroups (δv_mMCA: P = 0.54; δMAP: P = 0.42; δEt-CO₂: P = 0.38; δHR: P = 0.67) were also not significant.

Discussion

In the present study the simultaneous increase in VEFR and VEP on increasing the contrast of visual stimulus was found in healthy controls as well as in migraneurs interictally. The increase in VEFR was significantly higher in migraneurs than in controls, but no differences in the VEP responses were observed between the groups. In addition, VEFR correlated well with VEP in both groups, with the higher correlation coefficient between VEFR and VEP in migraneurs compared to controls suggested increased coupling between cortical activity and VEFR in migraneurs.

Several TCD investigations established the changes in blood flow velocities in the PCA following visual stimulation to be rapid, highly reliable and fine-tuned (15, 20). The increase in blood flow velocity on visual stimulation was shown to be higher in more complex visual stimuli (16, 21). Although blood flow velocity cannot be used for the calculation of CBF, the changes in blood flow velocity have been found by various methods to correlate reliably with changes in CBF in the territory of the insonated artery, as long as the vessel diameter and the perfusion territory remain constant (22). Therefore our findings indicate that there is amplification of VEFR in the occipital lobe in migraneurs during the attack-free period as compared to healthy controls. This is in agreement with previous observations using TCD (8, 9) and fMRI (10). In addition, our findings suggest that amplification of VEFR on visual stimulation is contrast dependent.

In principle, increased cerebrovascular responsiveness to visual stimulation in migraneurs during an attack-free interval could be due to hyperexcitability of the cerebral cortex and/or hyperactive neurovascular coupling. In our study no differences were found in VEP amplitudes between healthy subjects and patients with migraine, which argue against the hypothesis of cortical hyperexcitability in migraneurs. The literature on VEP following visual stimulation is inconclusive. The amplitudes of the major positive waves of the VEP were shown to be greater in migraneurs between attacks than in the age and sex-matched controls, suggesting hyperexcitability in the visual pathways in migraneurs (23). On the other hand, some other studies failed to show differences in VEP latencies and amplitudes between migraneurs and healthy controls (24, 25). The discrepancies between the studies are possibly due to differences in the time of measurements with regard to attacks, as it is known that cortical excitability changes with duration of the headache-free period and normalizes before the next migraine attack (26). Studies of cortical excitability changes using transcranial magnetic stimulation have yielded conflicting results (12, 27). However, habituation of pattern-reversal VEP induced by repetitive transcranial magnetic stimulation suggests a decreased level of preactivation excitability, rather than hyperexcitability (13). Taken together, these results do not support the possibility of cortical hyperexcitability during an attack-free interval in migraneurs.

Simultaneous recording of VEFR and VEP in our study allowed testing of the relationship between VEP and VEFR in healthy subjects and patients with migraine. We found a linear relation between VEP and VEFR on visual stimulation in both groups of subjects. This is consistent with our recent observations on the association between VEP and VEFR following visual stimulation with increasing visual contrasts (28). Simultaneous measurements of neuronal activity and haemodynamic responses on stimulation in animals showed that the vascular responses directly reflect an increase in neuronal activity, correlating in particular with the local field potential, which represents the synchronized synaptic inputs of a given neuronal population (3, 29). Similar a significant correlation between evoked potentials and CBF was found in animals (4) and humans using fMRI BOLD (5) or NIRS (6).

To our knowledge, our study is the first to examine the relationship between vascular and neuronal activity in patients with migraine. We found a significantly higher regression coefficient (steeper slope) in patients with migraine compared to healthy subjects, suggesting higher neurovascular coupling in patients with migraine interictally. There were no differences in neurovascular coupling between MA and MwA patients.

The exact mechanism for increasing neurovascular coupling in migraneurs is not yet known. Nitric oxide, a potent vasodilator of cerebrovascular smooth muscle, plays an important role as a mediator or modulator in the coupling of blood flow to cortical activation (30), as well as in the pathogenesis of migraine (31). Therefore, it can be tentatively speculated that the effect of nitric oxide is intrinsically increased in migraneurs. Increased neurovascular coupling in migraneurs could also be associated with altered serotoninergic activity, which simultaneously affects both vascular and neuronal activity (32). On the other hand, genetic alterations of calcium channels could result in increased activity of neurovascular coupling (33).

We conclude that simultaneous recording of VEFR and VEP to graded visual contrasts enables assessment of neurovascular coupling in both healthy controls and patients with migraine. It seems that visual stimulation results in higher vascular responses in patients with migraine compared to healthy controls due to increased neurovascular coupling activity in migraneurs. This could be an important phenomenon in migraine pathophysiology that warrants further studies.

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