animal migraine models for drug development: status and future perspectives

REVIEW ARTICLE

Animal Migraine Models for Drug Development: Statusand Future Perspectives

Inger Jansen-Olesen • Peer Tfelt-Hansen •

Jes Olesen

Published online: 14 November 2013

� Springer International Publishing Switzerland 2013

Abstract Migraine is number seven in WHO’s list of all

diseases causing disability and the third most costly neu-

rological disorder in Europe. Acute attacks are treatable by

highly selective drugs such as the triptans but there is still a

huge unmet therapeutic need. Unfortunately, drug devel-

opment for headache has almost come to a standstill partly

because of a lack of valid animal models. Here we review

previous models with emphasis on optimal characteristics

of a future model. In addition to selection of animal spe-

cies, the method of induction of migraine-like changes and

the method of recording responses elicited by such mea-

sures are crucial. The most naturalistic way of inducing

attacks is by infusion of endogenous signaling molecules

that are known to cause migraine in patients. The most

valid response is recording of neural activity in the tri-

geminal system. The most useful headache related

responses are likely to be behavioral, allowing multiple

experiments in each individual animal. Distinction is made

between acute and prophylactic models and how to validate

each of them. Modern insight into neurobiological mech-

anisms of migraine is so good that it is only a question of

resources and efforts that determine when valid models

with ability to predict efficacy in migraine will be

available.

1 Introduction

Migraine is number seven in WHO’s list of all diseases

causing disability [1–3] and it is the third most costly

neurological disorder [4]. Even if the triptans revolution-

ized the acute treatment of migraine, a huge unmet need for

better or different acute treatments exists [5]. The majority

of prophylactic drugs for migraine were not developed for

this indication but their efficacy was discovered by seren-

dipity [6–8]. The need for new specific prophylactic drugs

is thus much higher than for acute drugs [5, 9].

In general, animal models are often of uncertain

validity in predicting efficacy of new drugs. Multiple

models of stroke in rodents which seemingly mimic the

human condition exactly have for example shown efficacy

of new drugs but this was not translated into efficacy in

patients [10–12]. Many other disappointments can be

mentioned, but the fact remains that the pharmaceutical

industry normally requires positive effects in animal

models before moving novel chemical entities into clini-

cal trials. Migraine drug development is currently

severely hampered by a lack of generally accepted animal

models with ability to predict efficacy of anti-migraine

drugs.

Several thorough reviews of migraine animal models

have already been published [13–15]. They have carefully

evaluated the published literature but the scope of the

present review is different. We do not review existing

models one by one, but we discuss them and possible future

models in relation to a number of positive characteristics of

an ideal model. We focus on the following major issues:

(1) type of model (2) animal species (3) methods for pro-

voking migraine-like responses (4) characterization of a

migraine-like response (5) how to show response to acute

anti-migraine drugs and (6) how to show response to

I. Jansen-Olesen � P. Tfelt-Hansen � J. Olesen (&)

Department of Neurology, Faculty of Health and Medical

Sciences, Danish Headache Center, Glostrup Hospital,

University of Copenhagen, Nordre Ringvej 57, 2600 Glostrup,

Denmark

e-mail: [email protected]

CNS Drugs (2013) 27:1049–1068

DOI 10.1007/s40263-013-0121-7

prophylactic migraine drugs. Finally, we present a cross-

cutting discussion of all these issues.

2 Different Models have Different Virtues

Modern drug development begins with a target, usually a

receptor, an enzyme or an ion channel. Typically such

targets have been identified by academia in human or

animal experimental studies. Several novel targets for

migraine treatment have been identified and validated in

human experimental models [16]. But the industry has been

reluctant to pursue these targets. One reason is undoubtedly

the absence of evidence from animal models. Targets, such

as receptors and ion channels can be expressed in cell lines

or in other in-vitro systems allowing high throughput

screening. However, the predictive power of high

throughput is limited to the effect on the particular target

and does not reliably bespeak efficacy in migraine. Other

in-vitro models such as the study of isolated cranial blood

vessels also allow a relatively high throughput and are

somewhat closer to the disease target [17–20]. A third class

of model is in-vivo animal experimentation. This can for

example be the open or closed cranial window models to

view dural and pial artery diameter after administration of

vasoactive substances [21, 22], neurophysiological models

with recording of provoked or spontaneous responses in the

trigeminal ganglion [23] or the trigeminal nucleus caudalis

[23–26] or histological measurements of neuronal activa-

tion markers in areas involved in pain after administration

of migraine provoking agents [27, 28]. Some of these

models are rather close to migraine but the throughput is

low as every animal has to be euthanized after the exper-

iment while the number of recordings varies from one in

immunohistochemistry to several in neurophysiological

studies. The ideal animal model is obviously one that does

not require sacrificing the animal and therefore can be used

over and over again allowing crossover experiments and

evaluation of several compounds or multiple doses. Such

models would have to be behavioral. Behavioral models

have been attempted [29–31], but it remains unknown

whether rats and other animals ever experience headache

and, if so, what the behavioral correlate would be. A lot

more work therefore remains before an optimal behavioral

model has been developed and validated. Fortunately, it is

not difficult to see how such developmental work should be

done. This is discussed in the following sections.

3 Desirable Characteristics of Experimental Animals

The ideal animal model would be one with proven

migraine attacks, however; so far no natural migraine

model exists. Furthermore, we still lack the knowledge of

how to construct such a model by genetic manipulation.

For reasons of availability, generalizability and price, the

choice of species is in reality restricted to mice and rats.

Both have been used in previous studies. For many pur-

poses mice are too small and too difficult to work with.

This seems for example to be the case with the closed

cranial window model where a large percent of operated

mice were not valid for further experimentation and pre-

constriction of the dural arteries with endothelin-1 was

necessary before studying the effect of vasorelaxing agents

[32]. Most neurophysiological and other studies have also

been done in rats for practical purposes. Mice are however,

attractive because of the possibility of using genetically

modified animals. In fact genetically modified mice

expressing a CACNA1A gene mutation of familial hemi-

plegic migraine type 1 (FHM1) have been developed [33,

34]. The R192Q mutation of the CACNA1A causes a mild

form of FHM1 while the S218L mutation is more severe

and often lethal. These models have been used in several

physiological and behavioral experiments with consider-

able success [35]. In R192Q mutated mice a reduced

threshold and increased velocity of cortical spreading

depression (CSD) has been found [34]. Furthermore, the

model show enhanced synaptic acetylcholine release under

conditions in which the synaptic Ca2? sensors are not

saturated [34]. It also seems that the R192Q mutation has

an effect on the trigeminal system as calcitonin gene-

related peptide (CGRP) immunoreactivity was decreased in

thoracic ganglia and in the superficial lamina of the trige-

mino cervical complex in R192Q knock in mice [36]. A

major problem with the CACNA1A knock in mice seems

to be the validity of these animals for the prevalent types of

migraine, migraine without aura (MO) and migraine with

typical aura (MA). In humans it has been shown that FHM

differs from MA and MO not only in genetics but also in

the response to migraine provoking agents [37–39]. Per-

haps the FHM mice are not useful for the prevalent types of

migraine. CGRP release from the trigeminal ganglion was

increased in R192Q knock in mice as compared to wild

type mice [40]. No change in CGRP release was found in

dura mater between mutated and wild type mice [40].

During restraint these animals seem to spontaneously

express changes in facial expression that were reversed by

rizatriptan [30]. Another type of genetically modified mice

has elevated expression of human receptor activity-modi-

fying protein 1 (hRAMP1), a subunit of the CGRP receptor

[41]. These transgenic mice display light-aversive behavior

that is greatly enhanced by intracerebroventricular injec-

tion of CGRP and blocked by co-administration of the

CGRP receptor antagonist olcegepant [42]. It is still

unknown if migraine triggering substances other than

CGRP are able to enhance the light aversive behavior in

1050 I. Jansen-Olesen et al.

these mice. Thus, validity of this model for the develop-

ment of new anti-migraine substances is still unknown.

Recently, a rat exhibiting episodes of spontaneous tri-

geminal allodynia (STA) has been presented as the spon-

taneous trigeminal allodynia (STA) rat model of primary

headache [43]. This STA rat has five features in common

with migraineurs: (1) Episodically changing trigeminal

thresholds to von Frey filaments, possibly reflecting tri-

geminal hypersensitivity. (2) The STA trait is inherited. (3)

The STA rats have increased sensitivity to sound as com-

pared to normal rats. (4) Increased sensitivity to the

headache triggers GTN and CGRP. (5) The STA rats

respond to commonly used acute and preventive migraine

therapeutics [43]. The model is interesting in the sense that

it is spontaneous rather than provoked and does not need

any manipulations in order to show the desired phenotype.

To further validate and increase face-and construct validity

of the STA model it must be shown that it is in fact having

episodes of spontaneous migraine and not just allodynia.

This can be done through testing of spontaneous behaviors

detecting clinical manifestations of headache. One major

disadvantage using a spontaneous model compared to a

provocation model is the episodic and unpredictable nature

of allodynia. Therefore the animals need to be tested every

day in order to determine whether they have allodynia

or not.

Male animals have generally been preferred in previous

migraine models to avoid variability of the female cycle.

However, migraine patients are mostly female and

migraine drugs are expected to work throughout the cycle.

Future migraine models should therefore explore the use of

female animals and the role of menstrual cycle on the

effects explored. Animals should be in the reproductive age

but relatively young as a mimic of the human situation. As

migraines do not occur as frequently during a stable low or

a stable high concentration of estrogen, we suggest that

experiments should be performed in animals that are not

ovariectomized and not treated with estrogen. However,

the status of the hormonal cycle could be investigated

during or immediately after experimentation.

4 Inducing a Migraine-like Response in Animals

A number of procedures have been used to induce a

response that may be related to migraine. The so-called

neurogenic inflammation model was among the first animal

models of migraine [44, 45]. Strong electrical stimulation

is applied to the trigeminal ganglion (0.6–3.0 mA with

square pulses of 5 ms duration for 5 min) [46, 47]. This

results in retrograde activity in trigeminal nerve fibers and

liberation of a number of signaling molecules that increase

permeability for plasma protein of the vessels in the dura

mater [46]. The consequent increase in water content can

easily be recorded with 125I-BSA or Evans blue. This

model has been extensively characterized in a series of

excellent experimental studies (Fig. 1; Table 1). For some

time it looked promising in relation to drug development

for migraine as drugs for migraine treatment were effective

in blocking this response (Fig. 1) [47–51]. Unfortunately, it

turned out that many drugs work in this model but have

absolutely no efficacy in migraine [52–59] (Table 1). It

remains possible, however, that this model is sensitive to

acute antimigraine drugs albeit not specific.

In the genuine closed cranial window model the dural

artery is dilated by release of CGRP evoked by a much

milder electrical stimulation (50–300 lA for 10 s). Using

this model a more close relation to the clinical situation is

found, as neurokinin1 (NK1) receptor antagonists and the

endothelin antagonist bosentan, ineffective in migraine, do

not inhibit dural artery dilatation due to electrical stimu-

lation or CGRP release (Fig. 2) [60, 61]. Drugs effective in

the acute treatment of migraine such as sumatriptan, the

CGRP receptor antagonists CGRP8–37 and olcegepant and

dihydroergotamine cause inhibition [60–62] (Fig. 2;

Table 2).

Another classical inducing procedure is cortical

spreading depression (CSD) [63]. It is more and more

likely that CSD is the mechanism underlying the migraine

aura [64]. Furthermore, a drug developed for its ability to

inhibit cortical spreading depression, tonabersat (Fig. 3)

[65], has prophylactic efficacy against migraine with aura

Fig. 1 The effect of sumatriptan on plasma extravasation produced in

the dura mater of the rat by electrical stimulation (0.6 mA, 5 ms,

5 Hz for 5 min) of the right trigeminal ganglion. Data is expressed as

the ratio of extravasation on the stimulated/unstimulated sides.

Histograms show effects of pretreatment (i.v.) with vehicle (control,

distilled H2O, n = 10), sumatriptan (1–1,000 lg/kg, n = 9–10).

Statistical analysis was performed by unpaired t-test; *P \ 0.05 from

vehicle, bars SEM (adapted from Shepheard et al. [59])

Animal Migraine Models 1051

Table 1 Neurogenic

inflammation model: effect of

different inhibitors on dural

plasma protein extravasation

Treatment Dose (i.v) Inhibition of dural

plasma protein

extravasation

References

Electrical stimulation 1.2 mA Indomethacin 1 mg/kg ?? [50]

2 mg/kg ??

Acetylsalicylic acid 10 mg/kg ?

50 mg/kg ??

Dexamethasone 1 mg/kg ?

Electrical stimulation 1.2 mA Sumatriptan 30 lg/kg – [51]

100 lg/kg ???

300 lg/kg ?

Electrical stimulation 1.2 mA a-methylhistamine 5 lmol/kg – [165]

15 lmol/kg ???

SMS 2111-905 (somatostatin agonist) 0.1 lmol/kg –

0.3 lmol/kg –

1 lmol/kg ???

UK-14,304 (a2 receptor agonist) 34 nmol/kg –

100 nmol/kg ?

340 nmol/kg ??

Electrical stimulation 1.2 mA Sodium valproate 3 mg/kg ? [49]

10 mg/kg ??

30 mg/kg ??

100 mg/kg ??

Electrical stimulation 1.2 mA Muscimol 10 lg/kg ? [49]

100 lg/kg ??

1,000 lg/kg ??

Baclofen 10 lg/kg –

100 lg/kg –

1 mg/kg –

10 mg/kg –

Electrical stimulation 1.2 mA GR82334 (NK1 receptor antagonist) 0.02 mg/kg ? [166]

0.2 mg/kg ?

SR 48968 (NK2 receptor antagonist) 1 mg/kg –

CGRP (8–37) 0.1 mg/kg –

Electrical stimulation 1.2 mA Bosentan (mixed ET receptor

antagonist)

10 mg/kg ?? [52]

30 mg/kg ??

BQ-123 (ETA receptor antagonist) 10 mg/kg –

Ro-46-8443 (ETB receptor antagonist) 10 mg/kg ???

Electrical stimulation 1.2 mA Sumatriptan 1 lg/kg – [167]

10 lg/kg –

100 lg/kg ?

1 mg/kg ?

CP 122,288 (5-HT1B/1D receptor

agonist)

1 pg/kg –

10 pg/kg –

100 pg/kg ?

10 lg/kg –

100 lg/kg –

CP 93,129 (5-HT1B/1D receptor

agonist)

1 mg/kg ?

3 mg/kg ?

Electrical stimulation 1.2 mA 100 % oxygen 200 mmHg ? [47]

300 mmHg ??

400 mmHg ??


Table 1 continuedTreatment Dose (i.v) Inhibition of

dural

plasma protein

extravasation

References

Electrical stimulation 0.6 mA CP 99,994 (NK1 receptor antagonist) 10 lg/kg – [59]

100 lg/kg ?

1 mg/kg ?

3 mg/kg ?

Sumatriptan 10 lg/kg –

100 lg/kg ?

1 mg/kg ?

Electrical stimulation 0.6 mA Rizatriptan 1 lg/kg – [136]

10 lg/kg –

100 lg/kg ?

1,000 lg/kg ?

Electrical stimulation 1.2 mA (guinea

pig)

SMS 2111–905 (somatostatin agonist) 0.1 lmol/kg – [165]

0.3 lmol/kg ?

1 lmol/kg ???


pig)

GR82334 (NK1 receptor antagonist) 0.02 mg/kg – [166]

0.2 mg/kg ?

CGRP (8–37) 0.1 mg/kg ?


pig)

PNU 10929 (5-HT1D agonist) 0.24 nmol/

kg

– [168]

2.4 nmol/kg ??

7.3 nmol/kg ??

24.4 nmol/

kg

??

73.3 nmol/

kg

??

Substance P (1 nmol/kg) Indomethacin 2 mg/kg – [50]

10 mg/kg ?

Acetylsalicylic acid 10 mg/kg –

50 mg/kg ?

Dexamethasone 1 mg/kg –

Substance P (1 nmol/kg) Sodium valproate 3 mg/kg ? [49]

10 mg/kg ?

30 mg/kg ?

100 mg/kg ?

Muscimol 0.3 mg/kg ?

1 mg/kg ?

30 mg/kg ?

Substance P (1 nmol/kg) Sumatriptan 100 lg/kg – [51]

Substance P (0.3 nmol/kg) Sumatriptan 100 lg/kg – [51]

300 lg/kg –

Bradykinin (0.1 lmol/kg) Sumatriptan 10 lg/kg – [51]

30 lg/kg ?

100 lg/kg ?

Capsaicin (1 lmol/kg) Sumatriptan 30 lg/kg – [51]

100 lg/kg ?

Capsaicin (0.5 lmol/kg) (guinea pig) Sumatriptan 10 lg/kg – [51]

30 lg/kg ?

Capsaicin (1 lmol/kg) a-Methylhistamine (histamine H3

receptor agonist)

15 lmol/kg ?? [165]

SMS 201–995 (somatostatin

analogue)

1 lmol/kg ???

UK-14,304 (alpha-adrenoceptor

agonist)

100 nmol/kg ??

Capsaicin (0.37 mg/kg) Bosentan (mixed ET receptor

antagonist)

10 mg/kg ?? [52]

30 mg/kg

Sumatriptan 300 lg/kg ??


[66], but not against migraine without aura [67]. Taken

together with extensive human brain blood flow studies it

seems overwhelmingly likely that cortical spreading

depression in animals is a valid model for the testing of

prophylactic drugs against migraine with aura [66, 68]. The

number of patients with a high attack frequency of

migraine with aura is, however, limited. Probably the small

market size for this indication is the reason why the pharma

industry has made little use of the cortical spreading

depression model for drug development. Whether this

model might also be relevant to migraine without aura is

debatable. From human brain blood flow studies there is no

indication of CSD in migraine without aura [69, 70], but

prophylactic migraine drugs seem to inhibit CSD in rats

when dosed for two weeks or more [71, 72]. Thus, CSD

models may perhaps predict efficacy of prophylactic drugs

not only for migraine with aura but also for migraine

without aura.

Other models use stimulation of cranial vascular structures,

primarily the sagittal sinus [24, 73–79] but also the middle

meningeal artery [80, 81] (Table 3). This is associated with

activation of areas of the central nervous system that are rel-

evant to headache perception. Thus, it induces c-Fos expres-

sion in the nucleus caudalis [24, 75, 80–83] and activation of

neurophysiological responses [77, 79, 81, 84]. This model

responds to some anti-migraine drugs (Fig. 4) [24, 77, 78, 82,

83, 85] and failed to respond to NK1 antagonists [86] and

5-HT1D receptor specific triptans [82, 87]. These drugs are

proven ineffective in migraine. Some models have used a so-

called inflammatory soup on the dura mater in acute studies

with recording of neural responses in the pain pathway [81, 88,

89]. A model was recently developed where the inflammatory

soup or other agents can be delivered supradurally to awake

freely moving rats [90]. However, it was not investigated if

this model responds to specific anti-migraine treatments.

Others have used more continuous stimulation of the dura

Fig. 2 Example traces showing

the effects of a the NK1 receptor

antagonist RP67580 (1 mg/kg,

iv) and b the CGRP receptor

antagonist human-aCGRP8–37

(0.3 mg/kg, iv) on the increase

in dural vessel diameter

produced by substance P (SP;

100 ng/kg, iv) or electrical

stimulation (ES; 50–300 lA,

5 Hz, 1 ms for 10 s) of the

cranial window. Upper trace is

blood pressure (mmHg) and

lower trace is dural vessel

diameter (arbitrary units)

(adapted from Williamson et al.

[22])

Table 1 continued

Significant effect ? P \ 0.05,?? P \ 0.01, ??? P \ 0.001

Treatment Dose (i.v) Inhibition of dural

plasma protein

extravasation

References

Substance P (1 nmol/kg) a-Methylhistamine (histamine H3

receptor agonist)

15 lmol/kg – [165]

SMS 201–995 (somatostatin

analogue)

1 lmol/kg –

UK-14,304 (alpha-adrenoceptor

agonist)

100 nmol/kg –

GTN (60 lg/kg iv) L-NMMA 20 mg/kg iv ? [113]

L-NIL (iNOS inhibitor) 4 mg/kg ip ?


Table 2 The genuine closed cranial window model: effect of dif-

ferent inhibitors on middle meningeal artery (MMA) diameter change

after transcranial electrical stimulation

Treatment Dose (i.v) Inhibition

of arterial

dilatation

References

Olcegepant (CGRP

antagonist)

3 lg/kg – [62]

10 lg/kg ?

30 lg/kg ?

100 lg/kg ?

300 lg/kg ?

Iberiotoxin (BKCa

channel blocker)

0.1 mg/kg – [169]

NOX-C89 (CGRP

binding Spiegelmer)

1 mg/kg – [170]

CGRP antibody 10 mg/kg –

Glibenclamide (KATP

channel blocker)

7 mg/kg ? [171]

20 mg/kg ??

30 mg/kg ??

Ketamine (NMDA

receptor antagonist)

10 mg/kg – [172]

18 mg/kg ?

30 mg/kg ?

MK801 (NMDA receptor

antagonist)

0.5 mg/kg –

1 mg/kg –

3 mg/kg ?

GYKI52466 (AMPA


0.5 mg/kg –

2 mg/kg –

5 mg/kg –

LY466195 (kainate


0.03 mg/kg –

0.1 mg/kg –

0.3 mg/kg –

Sumatriptan 1 mg/kg – [60]

3 mg/kg ?

10 mg/kg ?

RP67580 (NK1 receptor

antagonist)

1 mg/kg –

CGRP (8–37) 0.3 mg/kg ?

Rizatriptan 1 mg/kg – [136]

3 mg/kg ?

10 mg/kg ?

Nociceptin 1 nmol/kg ?? [173]

10 nmol/kg ??

100 nmol/kg ??

L-NAME 40 mg/kg ? [174]

Diphenylene-iodonium

(eNOS inhibitor)

0.1 mg/kg –

0.3 mg/kg –

SMTC (nNOS inhibitor) 1 mg/kg –

3 mg/kg –

10 mg/kg ?

SMT (iNOS inhibitor) 3 mg/kg –

10 mg/kg –

Table 2 continued


of arterial

dilatation

References

Sumatriptan 1 mg/kg – [167]

10 mg/kg ?

CP 122,288 (5-HT1B/1D

receptor agonist)

1 mg/kg –

10 mg/kg ?

CP 93,129 (5-HT1B

receptor agonist)

1 lg/kg –

10 lg/kg ?

100 lg/kg ?

1 mg/kg ?

Flunarizine 1 mg/kg – [175]

2.5 mg/kg –

Indomethacin 3 mg/kg –

10 mg/kg ?

Phenylephrine

(a1-adrenoceptor

agonist)

1 lg/kg – [176]

5 lg/kg –

Corynanthine (a1-

adrenoceptor

antagonist)

1 mg/kg –

UK 14,304

(a2-adrenoceptor

agonist)

2 mg/kg –

Yohimbine (a2-

adrenoceptor

antagonist)

5 lg/kg –

10 lg/kg –

1 mg/kg –

3 mg/kg –

Propranolol (b-

adrenoceptor

antagonist)

1 mg/kg –

3 mg/kg –

Mepyramine (H1-receptor

antagonist)

1 mg/kg – [177]

3 mg/kg –

10 mg/kg ?

Famotidine (H2 receptor

antagonist)

1 mg/kg –

3 mg/kg –

10 mg/kg –

Calciseptine (L-type

voltage dependent

calcium channel

blocker)

7 lg/kg – [178]

10 lg/kg –

20 lg/kg ?

x-Agatoxin-TK (P/Q-

type voltage-dependent

calcium channel

blocker)

3 lg/kg –

10 lg/kg ?

20 lg/kg ?

x-Agatoxin-IVA (P/Q-


calcium channel

blocker)

3 lg/kg ?

10 lg/kg ?

20 lg/kg ?

x-Conotoxin-GVIA (N-


calcium channel

blocker)

10 lg/kg ?

20 lg/kg ?

40 lg/kg ?


mater for example with endotoxin resulting in a chronic

inflammation [29]. This model has responded to anti-migraine

drugs but the relevance of such strong inflammation to

migraine is otherwise improbable. Pain stimulation in the extra

cranial facial tissues such as injection of nociceptive agents in

the jaw joint or injection into chewing muscles of formalde-

hyde have also been used [91]. Since migraine patients do not

normally encounter pain in facial structures, this mode of

stimulation may not be close enough to migraine pathology.

5 Pharmacological Migraine Provocation in Man

and Animal

In relation to behavioral animal models it may be an

advantage to mimic human experimental models. Such

models have been developed over the last 20 years and

have been extensively characterized in normal individuals

and migraine sufferers. A number of naturally occurring

signaling substances or drugs interacting with known

pathological pathways are able to induce headache in

normal volunteers and migraine attacks in migraine suf-

ferers [92–102]. These human models should be further

developed to be practical in the testing of new drugs. But,

they will never replace the need for animal models.

However, it should be noted that the translational value of

such animal models is diminished by the differences

between migraine sufferers and normal animals. The most

extensively studied human model uses nitroglycerin,

glyceryl trinitrate (GTN) [92, 93, 96]. It has been mim-

icked in numerous animal experimental studies (Table 4).

In rats it is believed that increase in Fos expression within

the dorsolateral lamina 1 and 2 of the caudal region of the

trigeminal nucleus caudalis (TNC) may indicate activation

of the trigeminal vascular system [103, 104]. A dose

approximately 1,000 times the human dose has unfortu-

nately been used in most animal studies [28, 105–111]. It

causes depression of blood pressure and activates c-Fos

also in areas of the brain that are not related to pain [28,

109]. Other variations of this model have used anaesthe-

tized animals [112–116]. Anesthesia and surgery by

themselves affect the expression of c-Fos [117, 118]. In

anaesthetized rats GTN-induced hypotension which by

itself causes c-Fos expression [119]. Therefore, c-Fos

expression has been confounded by a number of unspecific

factors, which indicate a major deviation from studies in

awake human subjects. A more naturalistic model in the rat

has recently been presented [27]. GTN was administered to

awake freely moving rats in a dose (4 lg/kg/min for 20

min) 8 times the human dose, probably the equivalent of

Fig. 3 Box plot of number of cortical extracellular field potential

depolarisations. Four experimental groups were utilised; vehicle

(n = 3) (methylcellulose 1 ml/kg i.p. ? saline 1 ml/kg i.v.), suma-

triptan (n = 3) (300 lg/kg i.v.), tonabersat (n = 3) (10 mg/kg i.p.)

and sham (n = 3). Sham experiments included all surgery excepting

initiation of CSD. Data represented as medians and 25–75 % range.

Significant differences between groups were analysed by Kruskal–

Wallis and Mann–Whitney U test, **P \ 0.01 versus vehicle

(adapted from Read et al. [160])

Table 2 continued


of arterial

dilatation

References

LY334370 (5-HT1F

receptor agonist)

3 mg/kg – [179]

10 mg/kg –

Morphine 100 lg/kg – [180]

1 mg/kg ?

DAGO (l-opioid receptor

antagonist)

1 lg/kg ?

10 lg/kg ?

100 lg/kg ?

Butorphanol 1 mg/kg ?

10 mg/kg ?

DPDPE (j-opioid


1 mg/kg –

U 50,488 (d-opioid


100 lg/kg –

Orexin A 3 lg/kg – [181]

10 lg/kg –

30 lg/kg ?

Orexin B 3 lg/kg –

10 lg/kg –

30 lg/kg –

Anandamide 1 mg/kg ? [182]

3 mg/kg ?

Topiramate 10 mg/kg – [183]

30 mg/kg ?

GR79236 (adenosine A1


1 lg/kg – [184]

3 lg/kg –

10 lg/kg ??

Significant effect ? P \ 0.05, ?? P \ 0.01, ??? P \ 0.001


the human dose in rats. A significant increase in c-Fos

mRNA expression was observed in the trigeminal nucleus

caudalis at 30 min and 2 h that was followed by an

increase in Fos protein in the trigeminal nucleus caudalis at

2 and 4 h after GTN infusion (Fig. 5). Treatment with

sumatriptan and non-selective NOS inhibitor L-NAME as

well as pre-treatment with the CGRP receptor antagonist

olcegepant attenuated the activation of c-Fos at 4 h

Table 3 Stimulation of cat superior sagittal sinus: the table shows different effects observed after electrical stimulation of the superior sagittal

sinus and the effect of different treatments on these effects

Effect observed Treatment Effect studied by

treatment

References

Electrical stimulation of SSS

0.3 Hz 120 min

3 Hz 45 min

Increase of c-Fos in laminae I/II ofTNC, C1–C3

No treatment [185]

Electrical stimulation

0.3 Hz 120 min

Increase of c-Fos in laminae I/II of

TNC, C1–C2

L-NAME, 100 mg/kg ? [83]

Electrical stimulation

0.3 Hz 120 min

Increase of c-Fos in laminae I/II of

TNC, C1–C2

Eletriptan, 100 ng/kg – [82]

CP122,288, 0.5 mg/kg (5-HT1B/1D

receptor agonist)?


150 V, 250 ls duration, 0.3 Hz

Single unit activity in dorsolateral C2

spinal cord.

Sumatriptan – [85]

Sumatriptan ? mannitol

(disruption of BBB)

?


150 V, 250 ls duration, 0.3 Hz

Single unit activity in dorsolateral C2spinal cord

Zolmitriptan [82]

30 lg/kg ?

100 lg/kg ?


150 V, 500 ls duration, 10 Hz

Release of vaso-active peptides into

external jugular vein

[186]

CGRP Intact TG ?

SP –

VIP ?

NPY –

CGRP Bilateral trigeminal ablation –

SP –

VIP –

NPY –

Electrical stimulation of SSS Release of CGRP into external

jugular vein

Avitriptan (50 lg/kg) ? [187]

150 V, 500 ls duration, 10 Hz CP122, 288 (0.1 lg/kg) –

Release of CGRP into externaljugular vein

4991W93 (Zolmitriptananalogue)

[87]

0.1 lg/kg –

10 lg/kg –

100 lg/kg ?

Electrical stimulation of SSS Square wave pulsesevery 3 s, 130–150 V for 2 h

c-Fos positive neurons in: No treatment [188]

Rostral hypothalamus

Fornix –

Lat hypothalamic n. –

Ventromedial/ant. hypothalamus –

Supraoptic ?

Optic tract –

Paraventricular hypothalamus –

Caudal hypothalamus

Lat. hypothalamic n. –

Post. hypothalamus ??

Lat. mamillary –

Optic tract –

Supramamillary decussation –



(Fig. 5). However; a NK1 receptor antagonist that has no

efficacy in migraine also attenuated c-Fos expression [27].

Other migraine provoking agents such as CGRP, PACAP,

histamine, prostanoids and phosphodiesterase (PDE)

inhibitors have received limited or no study in this model.

Finally, it may be considered whether animals should be

sensitized in some way. GTN induces migraine in migraine

sufferers and a migraine-like relatively mild immediate

headache in normal volunteers [96]. If normal volunteers

are pre-treated for example with acetazolamide and then

receive GTN, roughly one fourth of the subjects develop a

migraine-like attack [120]. If a more reliable and stronger

sensitizing procedure could be developed, it might be

possible to induce a migraine in many more normal indi-

viduals. Perhaps sensitizing procedures could be developed

so that normal rats develop a migraine-like attack. Repe-

ated application of an inflammatory soup on the dura mater

produced a chronic state of trigeminal hypersensitivity that

potentiated the GTN evoked glutamate release in TNC

[121]. Daily treatment with sumatriptan over seven days

likewise caused allodynia that lasted for up to 14 days after

termination of sumatriptan exposure (Fig. 6) [31].

6 Recording the Response to Provocative Procedures

The responses to migraine provocation should obviously be

as close as possible to those occurring during a migraine

attack in humans. Pain is the dominant migraine symptom

and activation of the trigeminal afferent pain pathway is

therefore the most valid response. Neurophysiological

recordings in the trigeminal ganglion [122, 123] or the

trigeminal nucleus [85, 89, 123–125] or perhaps in even

higher centers in thalamus [124, 126, 127] or somatosen-

sory cortex can quantify such responses.

Activity in non-nociceptive afferent pathways can con-

found the picture and the methodology requires expertise.

The expression of c-Fos is often used as an indirect marker

of recent neuronal activity [128–130]. The up-regulated

protein expression of Fos and other biochemical markers

such as ERK, CREB and Jun reflect nociception if they are

located to areas involved in pain transmission [131, 132]. It

can be visualized by histological methods and has the

virtue of anatomical detail but the validity is not as high as

neurophysiological recordings. Furthermore, only one

measurement can be made in each animal using immuno-

histochemistry or other anatomical methods.

Extravasation in the dura mater, as in the neurogenic

inflammation model, is another possible way of recording a

response. As discussed above this may be sensitive but it is

certainly not specific to migraine. Increased dural and pial

arterial diameter or increased brain blood flow has been

used as a migraine-like response and in a human MR study

of 19 migraine patients during spontaneous migraine

attacks a slight increase in middle cerebral artery diameter,

but not of middle meningeal and extra cranial arteries were

found [133]. However, the importance of vasodilatation in

migraine has been under attack and been suggested to be an

epiphenomenon [134, 135]. However, some substances of

proven efficacy in the acute treatment of migraine such as

the triptans and olcegepant inhibit dural artery vasodilata-

tion due to electrical stimulation [60, 62, 136] (Table 2),

while a NK1 receptor antagonist that is not effective in

migraine treatment does not [61] (Table 2). For special

purposes liberation of CGRP from dura mater or other

migraine relevant tissues can be used as an outcome

parameter when testing drugs that target CGRP related

mechanism [60, 137, 138].

Recording behavioral responses to migraine provoking

stimuli is a whole issue of its own in need of much more

developmental work. However, a number of results have

already been obtained. In the genetically modified mouse

expressing a FHM1 mutation, a particular and quite

detailed facial expression that responds to a triptan has

been described to occur spontaneously [30].

Cutaneous allodynia is often present in migraineurs

[139–141] and has been induced in animals by direct

application of inflammatory mediators (inflammatory soup,

lipopolysaccharide, TNF-alpha) to the dura mater [29, 121,

142, 143]. It can also be induced by long term systemic

treatment with triptans [31]. The inflammatory response

causes increased sensibility of primary sensory nerve fibers

in dura mater which then respond to stimuli that under

normal condition would not cause activation [122]. Out-

comes were increased firing of primary or secondary tri-

geminal neurons [89, 122, 144], cutaneous allodynia

Fig. 4 Population effect of zolmitriptan on trigeminal evoked

potentials due to stimulation of SSS. The histogram illustrates the

trigeminal potential in the caudal trigeminal nucleus complex before

(control) and after 30 lg/kg (n = 5) or 100 lg (n = 4) of zolmitrip-

tan. There is a significant (* = p \ 0.05) effect on the evoked

potential at both doses. The ordinate is in lV (adapted from Goadsby

and Hoskin [74])


Table 4 Glyceryltrinitrate infusion studies: the table shows effects observed after infusion of GTN and the effect of different treatments on these

effects

Effect observed Treatment Inhibition of effect

studied by treatment

References

GTN 0.25 lg/kg/

min i.v. (cat)

Increase in pial artery diameter

Increase in rCBF

[114]

Anesthetized Increase in NO concentration

GTN 60 lg/kg over

30 min iv

Increase in NO concentration Sumatriptan ? [115]

Anesthetized Decrease in superoxide concentration Sumatriptan ?

Increase in rCBF Sumatriptan ?

GTN 10 mg/kg s.c. Increase in TNC c-Fos expression [107]

Increase in TNC nNOS expression

GTN 10 mg/kg s.c. Decrease in TNC CGRP-IR expression [189]

Increase in TNC 5-HT-IR expression

GTN 10 mg/kg s.c. Increase in CamKII-IR in TNC – [190]

GTN 10 mg/kg i.p. Increase in TNC c-Fos expression Kynurenin (450 mg/kg) and

probenecid (200 mg/kg)

? [191]

GTN 10 mg/kg i.p. Increase in TNC c-Fos expression Kynurenic acid (1 mmol/kg i.p.) ??? [192]

SZR-72 (kynurenate analog)

(1 mmol/kg i.p.)

???

GTN 10 mg/kg i.p. Increase in c-Fos expression in TNC and

10 other brain nuclei

[28]

GTN 10 mg/kg i.p. Increase in c-Fos expression L-NAME (50 mg/kg i.p.) ? [110]

7-NI (20 mg/kg i.p.) ?

Ephedrine (25 mg/kg ip) ?

Indomethacin (5 mg/kg ip) ?

Capsaicin depletion ?

GTN 10 mg/kg i.p. Increase in cGMP in TNC lamina I/II [193]

GTN 10 mg/kg ip Increase in c-Fos expression Parthenolide

15 mg/kg for 6 days

? [194]

GTN 10 mg/kg ip Increase in c-Fos expression Anandamide

20 mg/kg ip

? [195]

GTN 10 mg/kg sc Increase in CamKII expression in TNC NS398 (COX-2 inhibitor) [196]

1 mg/kg –

3 mg/kg ?

5 mg/kg ??

SC 560 (COX-1 inhibitor)

1 mg/kg –

3 mg/kg –

5 mg/kg –

GTN 80 lg/kg over

20 min iv

Awake rats

Increase in c-Fos expression Sumatriptan

1.8 mg/kg iv

? [27]

GTN 80 lg/kg over

20 min iv

Awake rats

Increase in c-Fos expression L-NAME

40 mg/kg iv

? [157]

Olcegepant

1 mg/kg iv

?

L-733060 (NK1 receptor

antagonist)

1 mg/kg iv

?


located to the periorbital region of the head or to fore- or

hind paws [121, 142] and vocalization due to an air current

focused on the head of the rat [29]. While this certainly

indicates activation of the nociceptive system, the speci-

ficity to migraine is somewhat uncertain. Allodynia has

been recorded in mice after GTN challenge but with very

high doses (5–10 mg/kg) of GTN [105]. In humans without

migraine there was no change in thermal pain thresholds

and minor change in pressure pain thresholds after GTN

[145].

The migraine attack is associated with hypersensitivity

to light and sound and sometimes hypersensitivity to other

external stimuli. The amount of locomotion and grooming

of the head therefore seem relevant. Presence of photo-

phobia has been elegantly evaluated in an experimental

maze where one compartment was illuminated and animals

after GTN and CGRP avoided the light [42, 146]. The same

was demonstrated in the genetically modified ‘‘migraine’’

mouse [42]. Phonophobia has not yet been studied.

Migraine patients are also nauseated and anorexic. The

amount of food and water intake could thus be another

important parameter in a behavioural model of migraine.

Osmophobia is a specific but not very sensitive migraine

symptom [147–149]. Osmophobia could also be tested in

an animal maze. Migraine patients avoid physical activity

during attack [150]. Running wheel activity could possibly

be used as a marker of this. Little systematic work has been

done to validate different behaviors as migraine outcome

parameters. Hopefully this will happen in the near future.

7 Validating Models of Acute Migraine Treatment

We have already discussed a number of characteristics that

support the validity of experimental migraine models. The

final proof is, however, whether models respond to specific

acute anti-migraine drugs. Fortunately, there are now sev-

eral drugs with efficacy in migraine and no efficacy in any

other painful condition. This pertains to the 5-HT1B/D

receptor agonists, the triptans, CGRP receptor antagonists

and probably also for 5-HT1F receptor agonists [151, 152].

Because of their extreme receptor specificity these drugs

provide excellent validation of acute migraine models

[153]. Ergot alkaloids, ergotamine and dihydroergotamine,

are also specific for migraine [154, 155] but interact with a

multitude of receptors [155]. Thus, their mechanisms of

action are difficult to sort out and they are therefore of less

use. Non-steroidal anti-inflammatory drugs (NSAID) are

Table 4 continued

Effect observed Treatment Inhibition of effect

studied by treatment

References

GTN 80 lg/kg over

20 min iv

Awake rats

Increase in c-Fos expression in TNC

Increase in pERK in dura mater, TG, TNC

Increase CamKII in TNC

Increase in ATF1 in TNC

Increase in pCREB in TNC

[197]

GTN 10 lg/kg iv

over 20 min

Anesthetized

No effect on c-Fos expression in TNC [198]

GTN 10 mg/kg sc

(mice)

Increase in c-Fos expression in TNC [105]

Induces thermal allodynia Sumatriptan

Intrathecal 0.06 lg ?

ip 300 lg/kg –

ip 600 lg/kg ?

Induces mechanical allodynia Sumatriptan

Intrathecal 0.06 lg ?

ip 300 lg/kg –

ip 600 lg/kg –

No effect on CSD threshold

GTN 60 lg/kg iv

Anesthetized

Increase in IL1b expression in dura mater

Increase in iNOS expression in dura mater

Increase in mastcell degranulation

[113]

The experiment is performed in rats if species is not given



effective in migraine [156] but also have efficacy in all

other painful conditions and are thus not suitable for vali-

dating a migraine model. A valid animal model should be

sensitive and specific. As with diagnostic criteria for dis-

eases too rigorous criteria result in too low sensitivity but

high specificity. As a best compromise we suggest that a

useful model should respond to at least two of the three

classes of specific anti-migraine drugs. Alternatively, one

might require that all specific drugs should work in the

model and no unspecific drugs should work. This would be

a very strict requirement and it seems unlikely that any

animal model would be able to deliver such results. It is

more likely that a model would respond to one or two of

the specific drugs but not to the others and that a number of

different models must be used to test future anti-migraine

drugs. Depending on the type of new drug one model might

respond and another not. The existence of a number of such

models would allow a relatively precise estimation of new

drugs for the acute treatment of migraine.

8 Validating Models of Prophylactic Migraine

Treatment

While models for the testing of acute migraine drugs are

available some of which have shown validity [27, 157], this

is not the case for models testing prophylactic anti-

migraine drugs. All available prophylactic compounds are

unspecific with multiple actions in the body and almost all

drugs have been developed for another indication and have

subsequently received migraine as a secondary indication

[6–8]. Furthermore, mechanisms of action of the prophy-

lactic anti-migraine drugs are highly variable and in gen-

eral not understood. Some antiepileptic drugs have for

example efficacy and others not. The same is true for anti-

hypersensitive drugs. Drugs that only have the migraine

indication such as flunarizine and pizotifen interact with

multiple receptor systems and their mode of action in

migraine is unknown [158, 159]. Adding to these problems

Fig. 5 Fos-positive cells in the superficial lamina (I/II) of rats treated

with saline, glyceryltrinitrate (GTN) and GTN in the presence of

sumatriptan (suma). Saline and one of the groups with GTN treated

rats were killed 2 h after the end of GTN infusion while the other

GTN treated group and the group with GTN treatment in the presence

of sumatriptan 0.6 mg/kg were killed at 4 h after termination of GTN

infusion. Six sections per animal evenly distributed from 0.8 to

5.12 mm distance from obex were counted for Fos-immunoreactivity.

Data are presented as mean ± SEM from 4–6 animals. Statistical

analysis using Kruskal–Wallis non parametric test **P \ 0.01 as

compared to saline treatment. Mann–Whitney U test was used

comparing the effect of sumatriptan on Fos activation induced by

GTN to GTN alone, #P \ 0.05 (adapted from Ramachandran et al.

[27])

Fig. 6 Sustained exposure to triptans reduces sensory thresholds to

light tactile stimuli applied to the periorbital region and the hind paws

of rats. Continuous infusion of sumatriptan (0.6 mg/kg/day sc)

decreased withdrawal thresholds to light tactile stimuli applied to

a the periorbital region or b the hind paws of rats. Sumatriptan or

saline was continuously administered through an osmotic minipump

for 6 days, after which the minipumps were removed. Withdrawal

responses to von Frey filaments were significantly (P \ 0.05) reduced

in a time dependent manner. BL baseline


is the relatively low efficacy of prophylactic anti-migraine

drugs. Fifty percent efficacy in fifty percent of the patients

is the standard, with a therapeutic gain of 25 %. No drug

has been shown to be more effective than another [6, 7,

156, 158, 159]. Thus, it is difficult to use existing pro-

phylactic drugs for the validation of animal models. The

one exception may be CSD which is a valid model of

migraine with aura but not necessarily of migraine without

aura. CSD is strongly inhibited by tonabersat [65, 160]

which has efficacy in the prophylactic treatment of

migraine with aura [66] but not migraine without aura [67].

We suggest that a model for the development of prophy-

lactic drugs should be tested with propranolol and valpro-

ate. Propranolol is one of the oldest prophylactic migraine

drugs [8] and represents the class of antihypertensive

agents while valproate is the oldest within the group of

antiepileptic drugs [7]. Both drugs can be given as injec-

tion. In the human GTN model there was no preventive

efficacy of propranolol [161] but valproate was effective

[162]. Provocation with GTN may, however, affect

migraine mechanisms deep in the cascade of events while

propranolol may work higher in the cascade and valproate

deeper in the cascade. Such is currently only speculation

but the human findings illustrate that a battery of animal

models may be necessary.

9 The Future of Animal Migraine Models

The future of animal models for migraine looks relatively

bright. There are already several models responding to

triptan treatment and the cortical spreading depression

model mimics migraine with aura. There is no doubt that

further development is needed and these needs have been

discussed above. One problem is that migraine research is

much more poorly funded than research into any of the

other major neurological disorders [163]. While it is easy

to see what needs to be done, it will take considerable

resources to develop a combination of models that is

generally accepted as predictive of efficacy of anti-

migraine drugs. A major EU program or NIH program

would enhance such development considerably. Hopefully,

this paper has demonstrated that there is a lot of knowledge

about migraine mechanisms that can be used in the

development of more predictive models of migraine.

Among the major future lines of development, genetics

holds promise. The so-called familial hemiplegic migraine

mouse expressing a CACNA1A gene mutation causing

familial hemiplegic migraine (FHM) has already been used

in a number of elegant experiments [30, 34, 164]. Even if

this mouse model may not predict efficacy in migraine

without aura and migraine with typical aura, there might be

new models developed as more and more variants are

described that cause an increased risk of the prevalent types

of migraine. Thirteen variants have already been associated

with an increased risk of migraine Anttila et al. [199, 200]

but the increase in risk may not be high enough for a

knock-in model to be useful. The genetic variability of

migraine may also prove too large for useful animal models

of genetically modified mice. Cell lines expressing known

and future genetic variants looks like a more promising

way forward and the development of induced pluripotent

stem cells from migraine patients is actually part of a huge

program under the Innovative Medicines Initiative in

Europe called STEMBANCC.

We favour the use of stimulation of animals with agents

known to induce migraine in migraine patients and the use

of behavioural responses as outcome parameters. The fact

that provoking agents have proven ability to cause

migraine in migraine sufferers provides validity and

recording behavioural responses is associated with a rela-

tively high throughput allowing multiple doses to be tested

in the same animal in cross-over experiments. Maybe the

discovery of animals with particular characteristics will

revolutionize the whole field in the future. This may be

particularly true for the testing of prophylactic anti-

migraine drugs.

10 Conclusion

Many animal models of migraine have been proposed.

Most have, however, been used in the analysis of migraine

pathophysiology. Others have responded to single anti

migraine treatments but none have been thoroughly vali-

dated. A dedicated effort should be made to develop

models that respond to at least two existing anti migraine

drugs and not to drugs proposed for migraine but subse-

quently proved not to be effective.

Acknowledgments Inger Jansen-Olesen’s salary is paid by funding

from The Lundbeck Foundation and Candy’s Foundation. No other

funding has been received for this paper. Dr Inger Jansen-Olesen, Dr

Peer Tfelt-Hansen and Professor Jes Olesen declare that they have no

conflicts of interest.

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animal migraine models for drug development: status and future perspectives

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