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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 ??
1052 I. Jansen-Olesen et al.
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 ???
Electrical stimulation 1.5 mA (guinea
pig)
GR82334 (NK1 receptor antagonist) 0.02 mg/kg – [166]
0.2 mg/kg ?
CGRP (8–37) 0.1 mg/kg ?
Electrical stimulation 1.5 mA (guinea
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 ??
Animal Migraine Models 1053
[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 ?
1054 I. Jansen-Olesen et al.
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
receptor antagonist)
0.5 mg/kg –
2 mg/kg –
5 mg/kg –
LY466195 (kainate
receptor antagonist)
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
Treatment Dose (i.v) Inhibition
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-
type voltage-dependent
calcium channel
blocker)
3 lg/kg ?
10 lg/kg ?
20 lg/kg ?
x-Conotoxin-GVIA (N-
type voltage-dependent
calcium channel
blocker)
10 lg/kg ?
20 lg/kg ?
40 lg/kg ?
Animal Migraine Models 1055
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
Treatment Dose (i.v) Inhibition
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
receptor antagonist)
1 mg/kg –
U 50,488 (d-opioid
receptor antagonist)
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
receptor antagonist)
1 lg/kg – [184]
3 lg/kg –
10 lg/kg ??
Significant effect ? P \ 0.05, ?? P \ 0.01, ??? P \ 0.001
1056 I. Jansen-Olesen et al.
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)?
Electrical stimulation of SSS
150 V, 250 ls duration, 0.3 Hz
Single unit activity in dorsolateral C2
spinal cord.
Sumatriptan – [85]
Sumatriptan ? mannitol
(disruption of BBB)
?
Electrical stimulation of SSS
150 V, 250 ls duration, 0.3 Hz
Single unit activity in dorsolateral C2spinal cord
Zolmitriptan [82]
30 lg/kg ?
100 lg/kg ?
Electrical stimulation of SSS
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 –
Significant effect ? P \ 0.05, ?? P \ 0.01, ??? P \ 0.001
Animal Migraine Models 1057
(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])
1058 I. Jansen-Olesen et al.
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
?
Animal Migraine Models 1059
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
Significant effect ? P \ 0.05, ?? P \ 0.01, ??? P \ 0.001
1060 I. Jansen-Olesen et al.
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
Animal Migraine Models 1061
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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