Pearls and pitfalls in human pharmacological models of migraine: 30 years' experience

Abstract

In vitro studies have contributed to the characterization of receptors in cranial blood vessels and the identification of new possible anti-migraine agents. In vivo animal models enable the study of vascular responses, neurogenic inflammation, peptide release and genetic predisposition and thus have provided leads in the search for migraine mechanisms. All animal-based results must, however, be validated in human studies because so far no animal models can predict the efficacy of new therapies for migraine.

Given the nature of migraine attacks, fully reversible and treatable, the headache- or migraine-provoking property of naturally occurring signaling molecules can be tested in a human model. If such an endogenous substance can provoke migraine in human patients, then it is likely, although not certain, that blocking its effect will be effective in the treatment of acute migraine attacks.

To this end, a human in vivo model of experimental headache and migraine in humans has been developed. Human models of migraine offer unique possibilities to study mechanisms responsible for migraine and to explore the mechanisms of action of existing and future anti-migraine drugs. The human model has played an important role in translational migraine research leading to the identification of three new principally different targets in the treatment of acute migraine attacks and has been used to examine other endogenous signaling molecules as well as genetic susceptibility factors. New additions to the model, such as advanced neuroimaging, may lead to a better understanding of the complex events that constitute a migraine attack, and better and more targeted ways of intervention.

Keywords

Migraine human models of migraine drug targets migraine provocation

Introduction

The recent tremendous progress in headache medicine and research is the result of translational work, taking ideas from bedside to bench – and back. Animal experiments are necessary to separate and analyze different components of the migraine attack such as the transmission of nociceptive impulses, vascular reactions and the role of inflammatory mechanisms. However, an animal model that is identical or almost identical to human migraine has not yet been developed, and eventually all predictions from animal studies need to be confirmed in a suitable human model.

Over the last 30 years the systematic use of human models of migraine has provided crucial data on mechanisms underlying migraine pathophysiology. Human experimental models of migraine allow the study of the pathophysiological events during an attack. This is not always possible during spontaneous migraine attacks because disability inhibits the migraineurs from traveling to the hospital. Furthermore, experimental models allow the study of migraine attacks under carefully controlled and monitored conditions, a definitive advantage from a scientific viewpoint (Figure 1). The model can therefore be used to identify and validate potential drug targets and study their effect in the ideal model: The patient. In the model, the headache-inducing effects of all naturally occurring endogenous substances can be studied. If a substance is found to induce migraine, antagonists to that specific molecule or receptor can then subsequently be studied.

Figure 1.

The human provocation model, modified from Olesen et al. (127) with permission. In the main version of this model, patients with migraine are randomly allocated to receive intravenous infusion (over 25 min) of ‘target substance’ or placebo (isotonic saline) in a double-blind, crossover design. Headache intensity is recorded on a verbal rating scale 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). The following haemodynamic variables are recorded at pre-defined intervals: mean velocity of blood flow in the middle cerebral artery by transcranial Doppler with hand-held 2-MHz probes; diameter of the frontal branch of the superficial temporal artery by a high-resolution ultrasonography unit. With the addition of other imaging modalities such as high-field magnetic resonance imaging (MRI) angiography, even more detailed information can be collected on the vascular response of the cephalic circulation. Heart rate and blood pressure are measured continuously throughout the study. The subjects are asked to complete a headache diary every hour until 10 hours after discharge. The diary included headache characteristics and accompanying symptoms necessary to classify the headache according to the second edition of the International Classification of Headache Disorders (ICHD-II).

Here we present pearls and pitfalls from the history of human provocation experiments. A number of pharmacological substances have been studied in humans. We will critically review the available human studies, with special emphasis on glyceryl trinitrate (GTN) since this has been studied in most detail.

The GTN model

It has been known for more than 100 years that GTN induces headache (1,2). GTN is a donor of nitric oxide (NO) and its headache-inducing effect was demonstrated in a provocation experiment (3).

Pearls

The important details of GTN-induced headache, such as headache time profile, pain characteristics and accompanying symptoms, were carefully investigated. Patients with migraine were hypersensitive to NO, i.e. migraineurs developed significantly stronger headache after GTN infusion than healthy subjects (4**). Furthermore, migraine without aura (MO) patients not only developed an immediate headache during GTN infusion but 80% of patients several hours after stopping the infusion developed a delayed headache fulfilling International Classification of Headache Disorders (ICHD-II) criteria for a migraine attack (5,6) (Figure 2). These data are based on intravenous GTN administration. Other routes of GTN administration (e.g. sublingual) can be used, but results are more variable and the rate of migraine induction is reduced (7); see Table 1(a).

Figure 2.

An illustrative example of the headache response curve after provocation, adapted from Thomsen et al. (6) with permission. The glyceryl trinitrate (GTN) experimental model of migraine showed that in migraine patients infusion of GTN causes an immediate headache during the infusion and a delayed headache that is maximal around six to seven hours after the infusion. This delayed headache has the characteristics of typical attacks of migraine without aura.

Table 1(a).

Percentages of migraine patients reporting migraine-like attacks in experimental studies.

Compound		Dose	Migraine-like attacks	Ref
Glyceryl trinitrate (GTN)	Migraine with aura	Intravenous 0.5 µg/kg/min	50%–67%	(12**,105)
	Sublingual 0.9 mg	40.9%	(7)
	Migraine without aura	Intravenous 0.5 µg/kg/min	80%–83%	(6,12**)
	Sublingual 0.9 mg	82.1%	(7)
Hemiplegic migraine	Intravenous 0.5 µg/kg/min	12.5%–30%	(118,122,123)
Histamine		Intravenous 0.5 µg/kg/min	70%	(48)
Histamine		Intravenous 0.1 mg	78%	(45)
Arterial 0.1 mg (carotid)	77%	(45)
Sildenafil		100 mg per os	83%	(71**)
Dipyridamole		Intravenous 0.142 mg/kg/min	50%	(73)
Calcitonin gene-related peptide (CGRP)	Migraine with aura	Intravenous 1.5 µg/min	57%	(61)
Calcitonin gene-related peptide (CGRP)	Migraine without aura	Intravenous 2 µg/min	33%–75%	(60)
Hemiplegic migraine	Intravenous 1.5 µg/min	9%–22%	(116**,117)
Vasoactive intestinal peptide (VIP)		Intravenous 8 pmol/kg/min	0%	(83)
Pituitary adenylate cyclase-activating polypeptide (PACAP-38)		Intravenous 10 pmol/ kg/min	66%	(85**)
Prostaglandins	Prostaglandin E2	Intravenous 0.4 µg/kg/min	58%	(95**)
Prostaglandin I2	Intravenous 10 ng/kg/min	50%	(92)

Table 1(b).

Percentages of healthy volunteers reporting headache in experimental studies.

Compound	Dose	Headache		Ref
Immediate	Delayed
Glyceryl trinitrate (GTN)	Intravenous 0.25–2.0 µg/kg/min	80%–90%	40%–75%	(3,4**,21)
Histamine	Inhalation, 2–64 mg/ml	47%	53%	(124)
Sildenafil	100 mg per os	∼60%	∼60%	(70)
Dipyridamole	Intravenous 0.142 mg/kg/min	66%	41%	(125)
Calcitonin gene-related peptide (CGRP)	Intravenous 1.5 µg/min	50%	30%	(63)
Vasoactive intestinal peptide (VIP)	Intravenous 8 pmol/kg/min	41%	25%	(82)
Pituitary adenylate cyclase-activating polypeptide (PACAP-38)	Intravenous 10 pmol/ kg/min	89%	100%	(85**,86)
Prostaglandin E2	Intravenous 0.4 µg/kg/min	75%–100%	12%–45%	(91,98)
Prostaglandin I2	Intravenous 10 ng/kg/min	92%	36%	(94)

Interestingly, positron-emission tomography (PET) studies showed brainstem activation during GTN-induced attacks (8,9), exactly as reported during spontaneous migraine attacks (10,11**). Furthermore, GTN induces premonitory symptoms (12**) and hypothalamic activation (13), thus providing an understanding of symptoms also reported during spontaneous migraine attacks (14).

Collectively, these studies clearly suggest that GTN may trigger a genuine migraine attack in migraine sufferers and that NO plays a crucial role in migraine pathogenesis.

Arterial dilatation may cause headache (15), and GTN infusion causes a more pronounced dilation of extra- and intracerebral arteries in migraine patients than in controls (16); see Table 3. Long-term administration of isosorbide-5-mononitrate, a long-acting NO donor, induced vasodilation of the superficial temporal artery (STA) attenuated over seven days, which correlated with the disappearance of NO-induced headache (17). This was not observed for the middle cerebral artery (MCA), suggesting an important role for extracerebral arteries in GTN-induced headache.

The onset of the immediate GTN-induced headache in healthy volunteers is correlated to dilation of the MCA (18), but more studies found that vasodilation outlasted the headache (18,19). A post-hoc analysis of a series of human experiments found no linear relationship between experimental immediate headache and dilatation of the intra- and extracerebral arteries (20). In contrast to the indirect measurement of the vessels (18,19), magnetic resonance angiography (MRA) is a noninvasive and widely available technique used to visualize the intracerebral vessels directly. In a randomized, double-blind, crossover study of GTN infusion in healthy volunteers, 1.5T MRA showed a peak vasodilation of the MCA 10–15 minutes (min) after beginning the infusion and a normalization of the vascular diameters to baseline 60 min after starting the infusion (21). Collectively, these data speak against vasodilation per se as the primary source of pain in GTN-induced immediate headache.

Given the reliable and robust migraine-inducing effects of GTN, the antimigraine action of nonselective nitric oxide synthase (NOS) inhibitor, N(G)-mono-methyl-L-arginine (L-NMMA) was examined, and it was demonstrated that NOS inhibition was effective in treating spontaneous migraine attacks (22).

The human GTN model can also be used to test prophylactic anti-migraine drugs. Tvedskov and colleagues examined the effect of the prophylactic drug valproate (23). This study showed that pretreatment with valproat was better than placebo in preventing GTN-induced migraine.

Pitfalls

A number of observations have highlighted the limitations of the GTN model. Thus, despite the robust headache-inducing effects of GTN (6), a number of subjects do not report migraine attacks. This variation in GTN sensitivity is likely to reflect patient heterogeneity, which also results in differences in sensitivity to other migraine trigger factors (24,25). Furthermore, in a 3Tesla MRA study in migraineurs, no statistical significant vasodilation of cerebral or meningeal arteries was reported during GTN-provoked delayed migraine attacks in MO patients (26**). The lack of GTN-induced vasodilation is in conflict with Doppler studies demonstrating a slight but significant dilatation of MCA (27,28) during spontaneous migraine attacks. The discrepancy could be explained either by methodological issues in the MRA studies (26**,29**) or possible differences between GTN-induced and spontaneous migraine.

Based on the promising treatment result of nonselective NOS inhibitors (22), selective inhibition of one of the three NOS isoforms – endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) – seemed to be an attractive approach for the targeted treatment of migraine. The highly selective iNOS inhibitor GW274150 was tested both for acute (30) and prophylactic (31) migraine treatment. Surprisingly, and despite high selectivity and potency, GW274150 was not effective, suggesting that iNOS is unlikely to be a promising drug target. GTN infusion (and possibly migraine attacks) may induce the expression of nNOS (32), and selective nNOS inhibitors show promising results in preclinical pain trials (33). For now, NOS inhibitors still have to prove their usefulness in migraine treatment.

The GTN models have been used to test acute and preventive drugs. The effect of the specific antimigraine drug sumatriptan, a 5-HT_1B/1D receptor agonist, on GTN-induced headache has been examined in several studies (34). In a double-blind crossover study in 10 healthy subjects, 6 mg subcutaneous sumatriptan followed by GTN infusion reduced GTN-induced immediate headache and the temporal and radial artery diameter compared to placebo (35). Another study by Schmetterer et al. (36) in healthy subjects confirmed the efficacy of sumatriptan in GTN-induced headache and in addition demonstrated that sumatriptan also prevents GTN-induced dilation of the MCA. When aspirin and zolmitriptan were administered to healthy volunteers after GTN infusion, both drugs had no effect on continuing headache caused by a long-lasting infusion of a relatively low dose of GTN (37). This suggests that the active drug must then be given as pretreatment before GTN is administered and highlights the need to test treatment in the relevant target population. Tvedskov and colleagues (38) observed no clinical effect of propranolol on GTN-induced headache and migraine. These data suggested that the efficacy of future prophylactic drugs in the GTN model depends on their mechanism of action. There may also be a different mechanism involved in different patients, so new drugs may not be effective in all patients or all attacks depending on the causative mechanism. Another seemingly negative drug-screening result of the GTN model was the finding that calcitonin gene-related peptide (CGRP) receptor antagonists, which are proven effective in migraine treatment (see below), had no effect on GTN-induced vasodilation in healthy humans (39) and importantly, did not prevent GTN-induced migraine (40). This suggests that the migraine-inducing effect of GTN is not caused by CGRP liberation. In summary, the ineffectiveness of a well-proven propranolol and CGRP antagonism efficacy limits the usefulness of the model, and must be considered in future testing of new migraine prophylactic and acute drugs (41).

Future perspectives: Pearls and pitfalls

The results of drug testing in the human model of migraine show major advantages as well as limitations of the GTN model. It is a definitive advantage that a potential prophylactic migraine agent can be tested at a very early stage of development with approximately two weeks’ toxicology. Furthermore, the model allows extensive testing of safety parameters such blood pressure, heart rate and effects of drugs on the cerebral circulation (41). GTN studies have also shown that the sensitivity of models could be improved with some changes in experimental design. Thus, previous studies underscore the need for well-powered studies to avoid possible type-II errors (23). Furthermore, the clinical difference could be increased by adapting less conservative migraine criteria, such as probable or migraine-like headache rather than the strict International Headache Society (IHS) criteria (5); see Table 2.

Table 2.

The modified migraine criteria for migraine-like attack in experimental studies.

Diagnose	Migraine-like attack after pharmacological provocation
Diagnostic criteria	Headache fulfilling either 1 or 2:
	1: Headache fulfilling ICHD-II criteria C and D for migraine without aura
	C: Headache has at least two of the following characteristics:
	1) Unilateral location
	2) Pulsating quality
	3) Moderate or severe pain intensity (>4 on VRS)
	4) Aggravation by cough (in-hospital phase) or causing avoidance of routine physical activity (out-hospital phase) (e.g. walking or climbing stairs)
	D: During headache at least one of the following:
	1) Nausea and/or vomiting
	2) Photophobia and phonophobia
2: Headache described as mimicking usual migraine attack and treated with a triptan

ICHD-II: Second edition, International Classification of Headache Disorders; VRS: verbal rating scale.

One of the limitations of the model is that a negative result does not prove that the drug does not work in spontaneous migraine (37). Thus, spontaneous migraine may be induced via a number of different mechanisms, whereas GTN induces migraine via one particular pathway (NO – cGMP) (42). Efficacy in the model would, on the other hand, imply a high likelihood of clinical efficacy. The GTN model in healthy volunteers is thus a good screening model that allows selection of compounds that should ultimately be tested in patients. Future studies may reveal whether other prophylactic migraine drugs work in the GTN model, whether the model can be used for dose finding in prophylactic studies and how such a dose finding study should best be designed.

Histamine model

Pickering and Hess (43) reported the first detailed study of the histamine-induced headache. These findings were confirmed in further studies (44 –46). It was later established that histamine, in a dose-dependent fashion, caused a more severe and more pulsating headache in migraine patients than in healthy subjects (47). In a double-blind study, MO patients were randomized to receive either mepyramine (an H1 receptor blocker) or placebo before histamine infusion. Patients who received both histamine and pretreatment with placebo developed a headache peak during infusion (immediate headache), a reduction in headache intensity for approximately two hours (intermediate phase) and a second headache peak several hours later (delayed headache). Seventy percent of patients experienced a migraine-like attack fulfilling the diagnostic criteria for MO (48). Interestingly, the time profile of the histamine-induced headache in patients is strikingly similar to the time profile of GTN-induced headache in MO patients (6) (Figure 2). Given that activation of endothelial H1-receptors induces the endogenous formation of NO, it is possible that histamine-induced migraine is caused by hypersensitivity to activation of the NO pathway.

The pitfall of histamine models is that an NOS inhibitor in the highest possible dose did not block the histamine-induced headache response or arterial dilatation (49). This suggests that histamine dilates arteries and causes headache via NO independent mechanisms. Based on these results, antihistamines should have a migraine-relieving effect, possibly by blocking mast cell degranulation (50).

Few high-quality clinical trials have examined the efficacy of antihistamines as single agents (51), but one small (sub)study examining hydroxyzine in patients with acute MO found no difference compared to placebo (52), and the prophylactic treatment effect of histamine blockers did not differ from placebo (53).

CGRP model

CGRP is present in the trigeminal ganglion and in nerve fibers around cerebral (54) and temporal arteries (55). In 1988 Goadsby et al. (56) reported that CGRP was increased in the extracerebral circulation of humans upon thermo coagulation of the trigeminal ganglion. Studies in migraine patients showed elevation of CGRP during (57) and outside of migraine attacks (58). However, Tvedskov et al. found no changes in plasma CGRP during migraine attacks compared to outside of attacks (59) and thus challenged earlier reports.

The importance of CGRP in migraine pathogenesis became firmly established using the experimental migraine model. Lassen et al. (60) conducted a double-blind cross-over study, where human α-CGRP (2 µg/min) or placebo was infused for 20 min in 12 MO patients. During the following 11 h all patients experienced headaches after human α-CGRP and only one patient after placebo, and in three patients the delayed headache fulfilled the IHS criteria for MO (60). Applying the criteria presented in Table 2, CGRP-induced migraine-like attacks in 75% of MO patients with (29) and 57% of patients with migraine with aura (MA) (61); see Table 1(a). Ultrasonography studies have shown that CGRP infusion and CGRP-induced headache and migraine were associated with just a modest immediate vasodilation (62,63); see Table 3. Hypersensitivity to CGRP infusion in migraineurs triggered interest in CGRP antagonism as a potential target for antimigraine drugs. Indeed, olcegepant, (BIBN 4096), a selective IV-administered CGRP antagonist, was effective in treating acute migraine attacks (65). The oral CGRP receptor antagonist telcagepant was also effective (65,66). Development of this drug was halted in phase III, but new formulations of oral CGRP receptor antagonists are currently in development (67).

Table 3.

Cerebral vascular changes after pharmacological provocation.

Compound	Dose	Imaging modality	Middle cerebral artery	Middle meningeal artery	Superficial temporal artery	Basilar artery	Ref.
GTN	Intravenous 0.5 µg/kg/min	MRA, TCD and Dermascan	+11.7%–25% (infusion) + 4% (migraine)	+16.4% (infusion) −1.78% (migraine)	+ 35%	+20.7% infusion	(21,26**,38)
Histamine	Intravenous 0.5 µg/kg/min	TCD and Dermascan	−26.0 ± 2.3% (mean blood flow velocity)	N/D	+58.7%	N/D	(49)
Sildenafil	100 mg per os	TCD and Dermascan	No change	N/D	No change	N/D	(71**)
Dipyridamole	Intravenous 0.142 mg/kg/min	TCD	−16.3% (mean blood flow velocity) + 5.6% (diameter change)	N/D	N/D	N/D	(73,125)
CGRP	Intravenous 1.5–2 µg/min	MRA, TCD and Dermascan	+7.5%–9.3% (TCD) 11.22% (MRA)	+9.24% (MRA)	+42.8% (Dermascan)	N/D	(29,62,63,116)
VIP	Intravenous 8 pmol/kg/min	TCD and Dermascan	−16.3% (mean blood flow velocity)	N/D	+45.9 %	N/D	(83)
PACAP-38	Intravenous 10 pmol/ kg/min	MRA, TCD and Dermascan	No change (MRA) 6%–9.5% (TCD)	+23.4% (MRA)	+50%	N/D	(85**,86,126)

GTN: glyceryl trinitrate; MRA: magnetic resonance angiography; TCD: transcranial Doppler; Dermascan: ultrasound; N/D: not determined; CGRP: calcitonin gene-related peptide; VIP: vasoactive intestinal peptide; PACAP-38: pituitary adenylate cyclase-activating polypeptide-38.

The vascular effects of CGRP infusion in healthy volunteers was investigated with high-resolution 3T MRI. This study showed that CGRP caused significant dilation of the extracerebral middle meningeal artery (MMA) but not of the intracerebral MCA compared with placebo (68). Interestingly, the headache-aborting effect of sumatriptan was associated with constriction of the MMA but not MCA. Furthermore, in contrast to GTN-induced migraine (26**), CGRP-induced MO was associated with dilation of extra- and intracerebral arteries, and headache location corresponded to the location of vasodilation (29**). This suggests that vasodilation and perivascular release of vasoactive substances are an integral part of migraine pathophysiology.

Phosphodiesterase (PDE) inhibitor model

Several substances capable of inducing experimental vascular headache do so via NO or molecules in the cascade of intracellular reactions triggered by NO (69) and activating the NO-cyclic GMP pathway (42). A good example is sildenafil (Viagra™), a selective inhibitor of cGMP-hydrolyzing PDE5. Inhibition of this enzyme results in accumulation of cGMP, and the effect of sildenafil could therefore mimic the effects of NO, which increases cGMP formation via activation of soluble guanylate cyclase. When given to healthy volunteers in the human model, sildenafil caused significantly more headache than placebo, interestingly without cerebral arterial dilation (70). To verify this in migraine patients, 12 MO patients were included in a double-blind, placebo-controlled crossover study in which placebo or sildenafil 100 mg was administered orally on two separate days (71**). Sildenafil induced a migraine-like attack in 10 of 12 patients compared with a placebo response in two of 12 patients. The authors also found that blood flow velocity in the MCA, regional cerebral blood flow in the territory of the MCA and the diameter of the radial and temporal arteries were unaffected by sildenafil (71**). Thus, it appears that sildenafil triggered experimental migraine via a cGMP-dependent mechanism but without initial dilation of the MCA. The authors proposed that triggering mechanisms might reside within the perivascular sensory nerve terminals or the brainstem (71**). However, future studies are needed to explore the molecular sites of action responsible for sildenafil-induced experimental migraine, including MRA of the dural arteries. A functional magnetic resonance imaging (fMRI) study excluded that the headache-inducing properties of sildenafil were related to a general lowering of threshold for a neuronal or cerebrovascular response, at least in healthy volunteers (72).

Other PDE inhibitors have also been studied in humans. The migraine- and headache-eliciting effect of the PDE5 inhibitor dipyridamole was examined in a single-blind study, including 10 migraineurs and 10 healthy controls (73). Dipyridamole induced headache in all patients and migraine attack in 50% of patients (Table 1(a) and (b)). The headache-generating effect of cilostazol, an inhibitor of cAMP-degrading PDE3, has been studied in healthy subjects (74). The participants received either cilostazol 200 mg or placebo. The authors reported that cilostazol induced moderate headache in 11 of 12 participants with no family history of migraine. The headache in most cases had migraine-like features such as pulsating pain quality and aggravation by physical activity. In two participants, the symptoms fulfilled criteria for an attack of MO. Interestingly, cilostazol is one of the most important cAMP-degrading enzymes in cerebral arteries, and it has been suggested that not only cGMP but also cAMP-dependent pathways may be involved in the pathogenesis of head pain (74). The effect of cilostazol in migraine patients should be examined in future studies. The headache-eliciting effects of PDEs have not been tested in MA patients. In a case report, tadalafil was associated with visual aura symptoms (75).

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) models

VIP and PACAP are found in perivascular parasympathetic nerve fibers (76), and PACAP is also found in trigeminal nerve fibers surrounding cerebral blood vessels (77). Their release regulates cerebrovascular tone and hemodynamics of the brain (78). Two receptors, VPAC₁ (79) and VPAC₂ (80), are activated with equal affinity by PACAP and VIP, but a third receptor, PAC_1, is selectively activated by PACAP (81).

The systemic administration of VIP induces only a short-lasting and mild headache in healthy controls (82) and a very mild and short-lasting headache, and no migraine attacks in MO patients (83). VIP does not seem to be a trigger factor for headache and migraine, and VIP is probably not critically involved in migraine pathogenesis (84).

Interestingly, PACAP38 infusion led to sustained dilation of the superficial temporal artery (STA), and in contrast to VIP, PACAP induces more headache and often migraine-like attacks in MO patients (85**). In healthy volunteers, high-resolution MRA showed that PACAP38-induced headache was associated with prolonged dilation of the MMA but not of the MCA (86).

Given that VIP infusion does not cause migraine, the shared VIP/PACAP receptors are unlikely to be causal for induction of migraine after PACAP-38 infusion. The migraine induction by PACAP-38, in contrast to VIP, might be due to the selective action of PACAP38 on the PAC₁ receptor (87). Further studies are warranted to investigate the pronociceptive mechanisms of PACAP that could involve central sensitization (88) or mast cell degranulation (89), which include secretion of histamine, serotonin, prostanoids and cytokines (90).

Prostaglandin model

Over the past years we have systematically investigated the role of prostaglandins (PGs) in the generation of headache in healthy volunteers and migraine sufferers. The intravenous administration of vasodilating PGs, such as PGI₂ (prostacyclin), PGE₂ and PGD₂, induced headache in healthy volunteers and PGI₂ triggered migraine-like attacks in migraine patients (91 –94). Interestingly, the infusion of PGE₂ caused immediate migraine-like attacks accompanied by vasodilation of the MCA and insignificant vasodilation of the STA in MO patients (95**). The fact that PGE₂ selectively triggers immediate migraine-like attacks is at odds with all other tested migraine-inducing substances; see Table 4. MRI verification of dilation of cerebral or extracerebral arteries in PG-induced headache or migraine is pending. Given that PGs are inflammatory mediators (96) capable of activating sensory afferents, it could be speculated that PG-induced vasodilation does not contribute to provoked headache. To investigate the role of inflammation and vasodilation in PG-induced headache, we investigated whether the pro-inflammatory and vasoconstricting prostanoid prostaglandin F_2α (PGF_2α) would cause headache in a human model of headache. PGF_2α, as opposed to PGE₂, PGI₂ and PGD₂, did not induce headache (97). The lack of a dilating effect of PGF_2α on cerebral arteries could explain the absence of headache. Based on these data we suggest that the vasodilating abilities of PGs are important in generating pain in healthy volunteers and are likely to play a role in the mechanisms of spontaneous migraine attacks.

Table 4.

Percentage of migraine patients reporting immediate and delayed migraine-like attacks after pharmacological provocation.

Compound	Immediate migraine-like attacks	Delayed migraine-like attacks
GTN (6)	10% (1/10)	80% (8/10)
PACAP-38 (86**)	9% (1/11)	55% (6/11)
PGI2 (93)	25% (3/12)	50% (6/12)
CGRP (62)	0% (0/9)	56% (6/9)
PGE2 (95**)	58% (7/12)	17% (2/12)

GTN: glyceryl trinitrate; PACAP-38: pituitary adenylate cyclase-activating polypeptide; PGI2: prostaglandin I2; CGRP: calcitonin gene-related peptide; PGE2: prostaglandin E2.

Pitfalls

The PGE₂ model of headache in drug testing was recently studied in healthy volunteers. A highly specific and potent EP₄ receptor antagonist, BGC20-1531, was not able to attenuate PGE₂-induced headache and vasodilation of intra- or extracerebral arteries (98). The missing attenuation of headache and vasodilation by BGC20-1531 suggests either that the EP₄ receptor is not involved or that a single receptor blockade may not be enough. It should be emphasized that a lack of efficacy of EP₄ receptor antagonist in the PGE₂ model in healthy volunteers does not exclude the possible efficacy of EP₄ antagonist as an acute or preventive drug in migraine.

The use of human models to explore migraine aura

Migraine aura is likely to be the symptom of cortical spreading depression (CSD) (99) originally described by Leão (100). In rats, CSD leads to activation of nociceptors that innervate the meninges (101) and it also activates central trigeminovascular neurons (102), thereby linking CSD to delayed activation of the trigeminovascular pathway and potentially to the development of headache (103).

Attempts have been made to trigger aura in migraineurs using human models of migraine. Afridi et al. (104) reported that one out of 21 MA patients had an aura triggered on two separate occasions by GTN, and one during the second session only. Following sublingual GTN provocation, Sances et al. (7) reported that three of 22 (14%) developed a visual aura. Interestingly, CGRP infusion in 14 MA patients resulted in visual aura in four of 14 (29%) (61). However, given that relatively few MA patients experience aura during provocation studies with GTN and CGRP, these findings may be due to random occurrence of MA, and other methods of aura induction are needed. Using GTN, Christiansen et al. (105) demonstrated that 50% of the patients suffering exclusively from MA developed migraine headache with associated symptoms, but none developed migraine aura.

Two studies reported aura triggered by visual stimuli and vigorous physical activity in a few MA patients (106,107). Not all migraine patients, however, seem susceptible to these triggers (25). In patients undergoing conventional digital subtraction angiography (DSA), aura symptoms and possibly CSD were triggered shortly after the angiographic procedure (99). Animal studies have provided a number of possible CSD triggers (108,109) that eventually could be modified and tested in humans. We still need a reproducible and reliable experimental model to induce aura episodes in MA patients.

The use of human models to explore the contribution of genetic susceptibility

Familial hemiplegic migraine (FHM) is a rare, dominantly inherited subtype of MA in which hemiplegia occurs during the aura phase (5). The identification of FHM genes (110 –112) stimulated interest in the link between genotype and phenotype using molecular studies and animal models. Thus, FHM knock-in mice show increased susceptibility to CSD (113,114). The functional consequences of the FHM mutations in humans, however, are unknown, and the potential species differences should be considered. Genotyped FHM patients offer us the chance to study the interplay between genotype and phenotype and may be regarded as a valuable genetic migraine model (115). We therefore examined the relationship between the FHM phenotype, including patients with and without FHM mutations and the known migraine-inducing substances GTN and CGRP. To our surprise, both GTN (122,123) and CGRP (116**,117) failed to induce more migraine headache in FHM patients than in healthy controls, and neither induced aura in FHM patients. Our data indicate that the FHM genotype does not confer hypersensitivity to migraine triggers such as GTN and CGRP. We did, however, observe that the few FHM patients reporting migraine-like attacks after pharmacological provocation tended to be those who also had attacks of MA or MO. It might be important to distinguish between patients with the pure FHM phenotype and FHM patients with co-existing migraine. We therefore examined FHM with and without typical migraines. Our experiments showed that FHM patients with co-existing MA/MO attacks were more sensitive to GTN than patients with the pure FHM phenotype (118).

Collectively, these results suggest, but do not prove, that migraine-triggering mechanisms in FHM are distinct from those in MO and typical MA and question whether FHM is part of the spectrum of prevalent migraine disorders. This has implications for our understanding of migraine mechanisms and suggests that caution is warranted when extrapolating results from FHM studies to the prevalent types of migraine.

Open questions and future studies

When planning experiments using the human model, it should be kept in mind that migraine symptoms develop gradually (119); so that during the early stages of an attack these symptoms may not meet the criteria for definitive diagnosis of migraine (120). Patients should be asked to refrain from abortive medication until they fulfill the migraine criteria. We have developed criteria for a migraine-like attack in experimental studies (Table 2) and suggest that patient-reported migraine, or probable migraine rather than IHS migraine, should be chosen as the primary migraine-related outcome parameter.

Conclusion

The use of human models of migraine offer unique possibilities to understand the basic migraine mechanisms, including the very onset of symptoms (13), the mechanisms of antimigraine drugs (29**) and the difference between different migraine phenotypes (121).

Furthermore, the human models have contributed to the identification of new specific targets in the treatment of acute migraine attacks such as GTN and CGRP and the confirmed antimigraine action of antagonists to these migraine-inducing substances. The experimental migraine model is an important part of the translational research effort in migraine-drug development. New additions to the model, such as advanced imaging methods, may lead to a better understanding of the complex events that constitute a migraine attack and to better and more targeted ways of intervention.

Footnotes

Acknowledgments

We thank all patients and healthy volunteers who participated in the reported studies.

Funding

Studies included in this review were supported by grants from the University of Copenhagen, the Lundbeck Foundation through the Center for Neurovascular Signaling (LUCENS), The Research Foundation of the Capital Region of Denmark; Danish Council for Independent Research-Medical Sciences (FSS) (grant 271-08-0446); and The Novo Nordisk Foundation (R172-A14333).

Conflict of interest

None declared.

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