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SUMATRIPTAN-INDUCED SENSITIZATION OF THE
TRIGEMINAL SYSTEM TO CORTICAL SPREADING
DEPRESSION (CSD) IS BLOCKED BY TOPIRAMATE
By
Pengfei Gu
______________________
A Thesis Submitted to the Faculty of the
DEPARTMENT OF MEDICAL PHARMACOLOGY
In Partial Fulfillment of the Requirements
For the Degree of
MASTER OF SCIENCE
In the Graduate College
THE UNIVERSITY OF ARIZONA
2012
2
STATEMENT BY AUTHOR
This thesis has been submitted in partial fulfillment of requirements for
an advanced degree at The University of Arizona and is deposited in the
University Library to be made available to borrowers under rules of the Library.
Brief quotations from this thesis are allowable without special
permission, provided that accurate acknowledgment of source is made.
Requests for permission for extended quotation from or reproduction of this
manuscript in whole or in part may be granted by the head of the major
department or the Dean of the Graduate College when in his or her judgment
the proposed use of the material is in the interests of scholarship. In all other
instances, however, permission must be obtained from the author.
SIGNED: Pengfei Gu
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
___________________________________ 07/30/2012
Frank Porreca Date
Professor of Medical Pharmacology
3
ACKNOWLEDGMENTS
First and foremost, I would like to thank my advisor Dr. Frank Porreca
who made this great opportunity possible, and showed tremendous support,
leadership and patience throughout my graduate studies. I would also like to
thank my other committee members, Dr. Michael Ossipov and Dr. Gregory
Dussor for their critical review of this thesis and for guidance throughout my
studies.
I would like to further acknowledge each and every member in Dr.
Porreca’s lab who always offered me a helping hand.
In addition, I would like to acknowledge all faculty members in the
department of Medical Pharmacology as well as the rest of the staff and the
students who made this graduate career a truly valuable academic experience.
4
DEDICATION
I dedicate this dissertation to my wife Weixi Kong and my parents Chunxin Gu
and Lianzhen Liu.
5
TABLE OF CONTENTS
LIST OF FIGURES .......................................................................................... 7
LIST OF TABLES ............................................................................................. 9
ABSTRACT ................................................................................................... 10
1 INTRODUCTION ..................................................................................... 12
1.1 Overview of migraine headache .................................................... 12
1.2 Possible mechanisms of migraine.................................................. 15
1.3 Treatment of migraine .................................................................... 22
1.4 Statement of problem and research hypothesis ............................. 26
2 METHODS AND MATERIALS ................................................................. 29
2.1 Chemicals ...................................................................................... 29
2.2 Animals .......................................................................................... 29
2.3 Behavioral testing protocols ........................................................... 30
2.4 Drug infusion .................................................................................. 31
2.5 Implantation of electrodes .............................................................. 32
2.6 Electrophysiological recording of CSD event ................................. 33
2.7 Immunolabeling.............................................................................. 43
2.8 Determination of c-Fos labeled cells .............................................. 44
2.9 Bright light stress ........................................................................... 44
2.10 Statistical analysis .......................................................................... 46
3 RESULTS ................................................................................................ 47
3.1 Persistent sumatriptan exposure produces generalized tactile
allodynia ................................................................................................. 47
3.2 Measurement of electrical stimulated Cortical Spreading Depression
(CSD) ..................................................................................................... 49
3.3 Evaluation of CSD threshold .......................................................... 54
6
3.4 CSD is associated with the enhanced C-Fos expression in the
trigeminal nucleus cardalis (TNC) .......................................................... 57
3.5 Topiramate reverses environmental stress induced allodynia in
persistent sumatriptan exposed rats ....................................................... 61
3.6 Topiramate attenuates the enhanced expression of c-Fos in TNC of
saline and sumatriptan infused rats ........................................................ 65
4 DISCUSSION AND CONCLUSIONS ...................................................... 67
REFERENCES .............................................................................................. 75
7
LIST OF FIGURES
Figure 1.1 Sensory pathways from periphery to cortex ................................. 18
Figure 2.1 Schematic of skull indicating positions of electrodes and ECoG/ DC
traced from anesthetized rats after receiving a electrical stimulation ............. 35
Figure 2.2 A stimulating electrode used in the experiment to deliver current into
cortex. ............................................................................................................ 36
Figure 2.3 A representative picture of the post-surgery rat ............................ 37
Figure 2.4 A picture of the entire system of electrical stimulation and recording
....................................................................................................... 38
Figure 2.5 The oscilloscope was used to verify the duration and intensity of
current delivered to cortex ............................................................................. 39
Figure 2.6 CSD parameters recorded during a CSD event. .......................... 41
Figure 3.1Continuous infusion of sumatriptan (0.6 mg/kg/day) decreased
withdrawal thresholds to light tactile stimuli applied to the hind paws of rats . 48
Figure 3.2 Reduction of ECoG amplitude during the CSD event measured in
frontal and parietal lobes of saline or sumatripan infused rats which pre-treated
with or without topiramate .............................................................................. 50
Figure 3.3 DC-shift measured in frontal and parietal lobes of saline or
sumatripan infused rats with or without topiramate injection ......................... 51
Figure 3.4 CSD propagation speed measured in saline and sumatripan treated
rats with or without topiramate pretreatment at day 20 .................................. 52
Figure 3.5 CSD duration measured in saline and sumatripan treated rats with
or without topiramate pretreatment at day 20 ................................................ 53
Figure 3.6 Evaluation of electrical stimulation threshold of CSD in saline and
sumatripan infused rats with or without topiramate pretreatment .................. 56
Figure 3.7 Evaluation of c-Fos expression on both the ipsilateral and
8
contralateral side of TNC slices ..................................................................... 59
Figure 3.8 Electrical stimulated CSD is correlated to the enhanced c-Fos
expression in both saline and sumatripan infused rats .................................. 60
Figure 3.9 Persistent sumatriptan exposure induced latent sensitization in rats
....................................................................................................... 63
Figure 3.10 Environmental stress-induced allodynia is blocked by a single
dose of topiramate (80 mg/kg, i.p.) ................................................................ 64
Figure 3.11 Pretreatment of topiramate (80 mg/kg, i.p.) attenuates c-Fos
expression in both saline and sumatriptan exposed rats in TNC following bright
light exposure ................................................................................................ 66
9
LIST OF TABLES
Table 2.1 Electrical stimulation protocol to produce a CSD ........................... 42
Table 2.2 Experimental flow of bright light exposure. ..................................... 45
10
ABSTRACT
The studies in this thesis research were conducted to investigate if
sensitivity to induced cortical spread depression (CSD) or the consequence of
a CSD event is affected by sumatriptan induced latent sensitization. Previous
studies in our lab showed persistent exposure of sumatripan to rats produced
a latent state of sensitization. Using persistent sumatripan exposed rats as a
model for medication overuse headache, behavior, electrical stimulation
threshold to provoke a CSD event and the immunoreactivity of c-Fos in the
trigeminal nucleus caudalis (TNC) were characterized. Current results showed
no statistical difference of electrically induced CSD thresholds in anesthetized
rats measured at day 20 in sumatripan exposed rats compared with saline
treated rats. Topiramate (80 mg/kg, i.p.) used clinically for prophylaxis of
migraine headache significantly increased CSD threshold in both saline and
sumatriptan infused rats. CSD events appear to be associated with trigeminal
vascular system activation in TNC because c-Fos expression significantly
enhanced in rats with electrically stimulated CSD events. As compared to
saline treated rats, sumatriptan-exposed rats demonstrated a significantly
higher number of c-Fos positive cells following the electrically stimulated CSD
event. Under environmental stress (bright light), sumatripan exposed rats
11
demonstrated decreased response thresholds to periorbital and hindpaw
tactile stimuli (i.e., allodynia) and enhanced c-Fos expression in TNC. A single
dose of topiramate (80 mg/kg, i.p.) reversed environmental stress induced
allodynia and c-Fos over-activity. Taken together, these results suggest that
latent sensitization induced by persistent sumatripan exposure seems not
correlated to the threshold of electrically stimulated CSD in current model.
However, CSD enhanced the responses of trigeminal system in rats with
sumatriptan-induced latent sensitization. The protective effects of topiramate
shown in this model may be related to blocking the initiation of CSD events
resulting from environmental stimulation as well as inhibiting the
consequences of CSD events in primary afferents. These findings correlate
with clinical observations of protective effects of topiramate for migraine
prophylaxis.
12
1 INTRODUCTION
1.1 Overview of migraine headache
1.1.1 Symptoms and prevalence
Migraine headache is a complex paroxysmal neurological disorder
characterized as an episodic, unilateral throbbing cephalic pain (De Felice et
al., 2010b; Mehrotra et al., 2008; Rasmussen et al., 1991).
According to the International Headache Society, the criteria for
migraine headache is repeated episodic headache (4 – 72 hours) with the
following features of any two of symptoms such as unilateral, throbbing,
nausea, vomiting, photophobia and phonophobia (Olesen et al., 2004). These
symptoms will be worse with physical movement (Ferrari et al., 1993; Olesen
et al., 1994).
As a worldwide disease, the prevalence of migraine does not
discriminate between economic and race background. However, it does
appear to have strong association with age, sex and genetics. Generally,
females are more susceptible to migraine attack than males (15~18% vs. 6%)
(Stewart et al., 1992). Also, people who have one or more relatives suffering
from migraine are more likely to experience this disease during their lifetime
than those without family history. Based on several sources, migraine
headache most commonly starts between 15 and 24 years of age and occurs
13
most frequently in those 35 to 45 years of age (Bartleson et al., 2010).
Approximately 4 - 5% of children aged under 12 suffer from migraine but no
apparent differences occur between boys and girls (Mortimer et al., 1992). A
rapid growth in incidence of migraine is seen amongst girls occurs after
puberty, which continues throughout most of the adult life. After menopause,
attacks in women tend to decline dramatically, so that after the age over 50,
approximately equal numbers of males and females will experience migraine
headache (Lipton et al., 1993; Stovner et al., 2006).
In addition to human suffering, migraine headache causes a
substantial economic and societal burden. Since it generally affects the people
in the age range of 15 - 50, these people are most industrious in term of
societal influence and contribution. Also, migraine attack will disrupt the ability
to work, care for families, or meet social obligations. Days lost from work
represent a societal and indirect economic loss from migraine. Translated into
dollars, estimations of the cost of lost productivity range from $ 1.2 billion to
$ 17.2 billion per year (Andlin-Sobocki et al., 2005).
1.1.2 Classification of migraine and aura
Migraine headache could be classified into two main subtypes,
migraine with aura and migraine without aura. Aura is characterized as a
transient episode of focal neurologic phenomena that precedes or
14
accompanies the migraine attack. It is caused by an imbalance between
excitatory and inhibitory neuronal activity at different levels in the central
nervous system and about 15% - 30% of migraine patients may have aura at
some time (D'Andrea et al., 2011). Aura appears gradually over 5 to 20 min
and generally last less than 1 h (Kallela et al., 2012). The headache phase of
the migraine attack usually begins within 60 min of the end of the aura phase,
but sometimes delays up to several hours, or it may miss completely. (Kallela
et al., 2012; Panayiotopoulos, 2012; Tfelt-Hansen, 2010).
Symptoms of migraine aura can be sensory or motor in nature. Visual
aura is the most common aura that can occur without any headache. Visual
aura is a disturbance of vision consisting of unformed flashes of white or black,
or formations of dazzling zigzag lines often arranged like the battlement of a
castle. The other form of aura is somatosensory aura. It may perform like a
tingling feeling in the hand and arm. This paresthesia may migrate from the
arm and then extend to the face, lips and tongue. Other symptoms of the aura
phase include auditory, gustatory or olfactory hallucinations, temporary
aphasia, vertigo, and hypersensitivity to touch (Panayiotopoulos, 2012).
1.1.3 Triggers and four phases of migraine
Many triggers are known to induce migraine headache such as light,
stress, emotion, altered sleep pattern, menses, and etc. A trigger may occur up
15
to 24 h prior to the onset of symptoms (Bartleson et al., 2010). Once a migraine
headache is induced, four phases of this disease occur, although not all the
phases are necessarily experienced. The first phase is prodrome that occurs
days or hours before migraine attack in 40 - 60% of migraine patients. In this
phase, patients will experience symptoms, such as depression or euphoria,
light sensitivity, yawning and etc. The second phase is aura (the details see
above). The third phase is a pain phase, usually starts within 60 min of aura
and occurs on one side of the brain. This throbbing pain might be associated
with nausea, vomiting, photophobia, phonophobia, and cutaneous
hypersensitivity throughout the body. Those symptoms usually last from 4 to
72 h. The last phase is postdrome with symptoms like fatigue, changes in
mood or behavior. About 25% of migraine patients will experience this last
phase (Bartleson et al., 2010; Kelman, 2006). It is necessary to point out that
the migraine phases and symptoms experienced can be different from one
migraine attack to another even in a same person (2004).
1.2 Possible mechanisms of migraine
Migraine is a form of sensory disturbance in the central nerve system
(CNS). Two major hypotheses, vascular hypothesis and neurogenic
hypothesis, have been proposed for the mechanism of migraine headache.
16
Vascular hypothesis explained the pain of migraine is caused by dilation of
meningeal vessels. It is first proposed by Willis in1664 and best articulated by
Wolff in 1948 (Goadsby, 2009). Neurogenic hypothesis proposed that
vasodilation is only an epiphenomenon for migraine attack but activation of
certain receptors in trigeminal vascular system is the key event during the
occurrence of migraine pain (Schoonman et al., 2008). In this hypothesis,
cortical spreading depression (CSD) is believed to activate trigeminal vascular
system to send pain signals to the trigeminal nucleus in the brain stem. The
trigeminal nucleus conveys the signals to the thalamus, which relays them to
the sensory cortex that involved in the sensation of pain (Ayata, 2009; Dalkara
et al., 2006).
Unfortunately, the available data are not fully support neither of these
hypotheses alone (Bergerot et al., 2006; Goadsby, 2007). It is important to
note that migraine is a complex disorder. Any unitary theory seems a long way
off to clearly elucidate the formation of this disease. The bright side for
migraine headache study is that more and more researchers agree that
dysregulation of trigeminal nerve system is a key component for migraine pain.
The trigeminal, or the fifth cranial nerve (V), is a nerve responsible for
sensation in the face and certain motor functions such as biting, chewing, and
swallowing. The trigeminal nerve has three major branches: the ophthalmic
17
nerve (V1), the maxillary nerve (V2), and the mandibular nerve (V3). The
ophthalmic and maxillary nerves are purely sensory. The mandibular nerve
has both sensory and motor functions. All together, these three main branches
provide somatosensory innervation to distinct regions of the cranium. The dura
is the outermost of the three layers of the meninges surrounding the brain and
spinal cord. It consists of dense vascular system and is innervated by a large
quantity of sensory neurons. Part of the sensory neurons are also referred as
nociceptive fibers which can sense the dilation and constriction of vessels and
can be activated by pro-nociceptive peptides such as CGRP (calcitonin
gene-related peptide), substance P and histamine (Goadsby et al., 1990; Ma et
al., 2001; Potrebic et al., 2003; Schwenger et al., 2007). The major function of
these sensory neurons is to collect sensory information and convey
information to upper level of neurons. Sensory pathways from periphery to
cortex are illustrated below:
18
Figure 1.1 Sensory pathways from periphery to cortex. Figure is modified
based on the illustration obtained from the following source
http://www.scientificamerican.com/article.cfm?id=the-root-of-migraine-pain
Sensory information collected from primary afferents travel to the
trigeminal nuclei located in the brainstem. The trigeminal nucleus caudalis
(TNC) is believed to be the most relevant trigeminal nucleus for transmission
of headache related information (Buzzi, 2001). Primary afferent terminate in
the TNC where they synapse with the second-order neurons that project to the
thalamus. The thalamus conveys the information to certain somatosensory
cortex (i.e., insula and cingulate cortex) via the third-order neurons, where the
brain will analyze this information and make the corresponding response.
Among all the proposed mechanisms of migraine, Cortical Spreading
Depression (CSD) has received attention recently, although it has been
described decades ago (Kunkler et al., 2003; Lauritzen et al., 1982; Milner,
1958). The phenomenon of CSD was first reported by Dr. Leão in rabbit (Leao,
19
1944). It is characterized as a transient wave of neuronal and glial
depolarization, followed by long-lasting suppression of neuronal activity. CSD
will propagate in all directions at a rate of 3 - 6 mm/min and last approximately
1 min once it is triggered. The critical event in the generation and propagation
of CSD is a significant decrease in neuronal membrane resistance associated
with a massive increase in extracellular K+ and neurotransmitters, as well as
an increase in intracellular Na+ and Ca2+. These ionic shifts produce the
characteristic slow DC potential shift that can be recorded extracellularly.
Several different stimuli can trigger CSD including direct cortical trauma,
exposure to high concentration of K+, and direct electrical stimulation (Ayata et
al., 2006; Bergerot et al., 2006; Sanchez-Del-Rio et al., 2006).
There is considerable evidence supporting CSD as a potential
mechanism in migraine headache. First of all, recent studies have established
a link between migraine aura and CSD using magnetoencephalography, or
highfield strength, high-resolution magnetic resonance imaging (Cao et al.,
1999; Hadjikhani et al., 2001), based on the fact that visual or somatosensory
symptoms of migraine aura propagate at a rate that corresponds well to the
propagation speed of CSD. The other evidence is that CSD is associated with
a brief hyperaemia that lasts a few minutes, followed by a hypoperfusion
phase lasting up to 1 h. A similar long-lasting hypoperfusion in magnitude and
20
duration has been detected during migraine attacks in a few studies after the
hyperaemia phase finished (Lauritzen et al., 1983a; Lauritzen et al., 1983b).
Further evidence comes from the genetic studies performed in migraine
patients with aura. Results showed that the patients who have mutations on
familial hemiplegic migraine (FHM) genes such as CACNA1A (Cav2.1),
ATP1A2 (Na/K ATPase), and/or SCN1A (Nav1.1) demonstrated decreased
CSD threshold (De Fusco et al., 2003; Dichgans et al., 2005; Tottene et al.,
2002; van den Maagdenberg et al., 2004). These patients with mutation
became more susceptible to CSD induced by either excessive synaptic
glutamate release or decreased removal of glutamate and potassium from the
synaptic cleft, or persistent sodium influx (Moskowitz et al., 2004). Moreover, a
recent study showed that chronic administration of prophylaxis medications
such as topiramate, valproate, propranolol, amitriptyline, or methysergide
significantly reduced the number of potassium chloride evoked CSD and
elevated the electrical stimulation threshold to evoke CSD in rats (Ayata et al.,
2006). Finally, as mentioned before, migraine is more prevalent in women than
in men during particular age (i.e. menarche to menopause). Recent studies
show that CSD threshold is significantly higher in female mice over male. More
interestingly, this sex difference on CSD threshold was abolished by
gonadectomy and ageing (Brennan et al., 2007; Eikermann-Haerter et al.,
21
2009).
Similar with other proposed mechanisms, CSD events have shown to
activate the trigeminovascular system (Bolay et al., 2002; Moskowitz, 1984;
Moskowitz et al., 1993b). It has resulted in elevated c-Fos expression in TNC
(Moskowitz et al., 1993b), dilation of meningeal arteries (Bolay et al., 2002),
plasma protein extravasation and inflammation (Buzzi et al., 1995), stimulation
the release of calcitonin gene-related peptide and nitric oxide, and activation of
perivascular nerves (Read et al., 1997; Reuter et al., 1998; Wahl et al., 1994).
However, it is still under debate whether CSD should be considered
as a mechanism of migraine headache. Aura occurs only in 15 - 30% of
migraine patients, and it is unclear whether CSD events occur in the migraine
patients without aura. Although a recent study showed the silent CSD exists in
migraine patients without aura, it still remains to determine whether CSD
relates to migraine (Geraud et al., 2005; Kruit et al., 2005; Kruit et al., 2004). In
addition, although the CSD theory of migraine can explain the mechanisms of
aura and headache, it does not account for the diverse premonitory symptoms
(e.g. fatigue, phonophobia, yawning, nausea, mood changes) that often occur
prior to a migraine attack by hours. Moreover, since chronic administration of
prophylaxis drugs such as topiramate, valproate, propranolol, amitriptyline, or
methysergide significantly reduced the number of potassium chloride evoked
22
CSD and elevated the electrical stimulation threshold to evoke CSD in rats
(Ayata et al., 2006), it is difficult to explain why the prophylaxis drugs are only
effective in a small part of the patients if CSD is the underlying mechanism of
migraine.
Taken together, it is plausible to consider CSD as a possible
mechanism of migraine headache based on current clinical and experimental
evidences. However, this hypothesis needs further investigation.
1.3 Treatment of migraine
Current treatments for migraine headache are not as effective as
expected due to the lack of understanding on the mechanisms of this disease.
The available treatments can only temporarily relieve the symptoms but do not
cure. To date, the pharmacological treatments for migraine headache can be
divided into two major categories: abortive and prophylactic therapy.
The goal of abortive therapy is to stop the migraine headache once it
starts. Abortive agents include NSAIDs (non-steroidal anti-inflammatory drugs),
opiates, ergot, triptans and CGRP (calcitonin gene-related peptide)
antagonists. These medications usually are taken during aura phase,
headache onset or in between as the drugs are more effective if taken earlier
in an attack. Otherwise their efficacy will decrease towards to relieve
23
symptoms (Bartleson et al., 2010). NSAIDs such as Ibuprofen, acetaminophen,
aspirin and naproxen are used to treat migraine headache due to their
anti-inflammatory effects. However, in most cases the NSAIDs are not effective
at relieving migraine pain and long term use will cause severe gastrointestinal
(GI) side effects, which limit the application of NSAIDs in migraine treatment
(Brandes et al., 2007; Derry et al., 2010; Kirthi et al., 2010; Rabbie et al., 2010).
Opioids decrease the transmission of painful information due to their functions
of suppressing neuronal excitability and neuropeptide release (Tepper, 2012;
Tepper et al., 2012). Morphine, as one of the representative drug of opioids,
activates the G-protein coupled opioid receptors located on sensory fibers
throughout the trigeminal vascular system (Kelley et al., 2012b; Taheraghdam
et al., 2011). However, opioids are not the common treatment for migraine
because FDA does not approve for this indication. They are only used for
severe migraine patients in the emergency room.
Triptans are the most commonly prescribed abortive therapies for
migraine headache. Also, they are the only drug family used specifically for
migraine. However, the mechanism(s) of triptans to relieve migraine pain is still
unclear (Johnston et al., 2010). Drugs in this category such as sumatriptan are
5-HT agonists (serotonergic agonists). Sumatriptan is selective for the 5-HT1B
and 5-HT1D receptor and decreases dural vasodilatation and neuronal
24
excitability (Classey et al., 2010; De Vries et al., 1999; Goadsby, 2000;
Goadsby et al., 2002; Moskowitz et al., 1993a). Due to the fact that 5-HT
receptors exist in coronary vasculature, triptans also cause vasoconstriction in
this system and lead to cardiac perfusion problems in patients with
cardiovascular disease (Johnston et al., 2010). In addition to triptans, the
therapeutic activity of ergot derivatives such as dihydroergotamine are also
shown to be mediated through activation of 5-HT receptors (Kelley et al.,
2012a).
Recently, CGRP (calcitonin gene related peptide) has been found to
play a role in the pathogenesis of the pain associated with migraine. This
finding is based on the evidence that CGRP levels are elevated in the blood of
migraine patients. Also, clinical studies showed CGRP administration will
trigger migraine attack in migraine patients (Lassen et al., 2002). Triptans as
one of the most effective treatments for migraine have demonstrated the
abilities to reduce CGRP release from trigeminal nerve endings and suppress
the vasodilation function of CGRP in meningeal blood vessels (Tepper et al.,
2008). CGRP receptor antagonists, specifically olcegepant and telcagepant,
are being investigated both in vitro and in vivo for the treatment of migraine.
Preclinical and clinical results showed both of them were effective in the acute
treatment of migraine headache (Durham et al., 2010).
25
The other major category for migraine treatment is prophylactic
therapy. It is recommended to reduce the frequency and intensity of migraine
headache when patients experience more than two attacks per month
(Brandes, 2005; Modi et al., 2006). The prophylactic medications are usually
given daily for months or years, however, treatment can be episodic, subacute,
or chronic. The classes of drugs belonging to this category include
antiepileptics, beta-blockers, calcium channel blockers and tricyclic
antidepressants. Most of migraine preventive medications are designed to
treat other medical disorders (e.g., propranolol for hypertension, topiramate
and valproate for epilepsy, etc.). However, they are found serendipitously to be
beneficial in migraine treatment. As a result, mechanism of prophylaxis is still
not well understood (Mannix, 2004).
Antiepileptic medications like topiramate and valproate have been
shown to decrease abnormal neuronal excitation in the brain (Brandes, 2005).
In addition, beta-blockers such as propranolol are able to decrease and
stabilize blood pressure and keep the blood vessels in a relaxed state thereby
decrease the intensity and duration of the migraine attacks (Silberstein, 2005).
If the patient is unresponsive to these first line preventive medications, calcium
channel blockers can also be used. Drugs in this category like flunarizine are
considered to prevent migraine by reducing reflex narrowing of the blood
26
vessels after vasodilatation during an attack (Silberstein, 2004). Tricyclic
antidepressants such as amitriptyline are also prescribed for migraine
headache when other treatments fail (Yaldo et al., 2008).
1.4 Statement of problem and research hypothesis
Although several treatment options exist, there are still over 50% of
migraine patients suffering from migraine headache. Even worse, the frequent
use of abortive migraine medications over an extended period of time may
result in medication overuse headache (MOH), in which the headaches
become more severe and more frequent. MOH may occur with opioids, ergot
alkaloids, serotonin 5-HT(1B/1D) receptor agonists ('triptans') and medications
containing barbiturates, codeine, caffeine, tranquillizers and mixed analgesics
(2004; Dowson et al., 2005). The International Headache Society defines MOH
as more than 15 headaches per month during the regular overuse (> 15 day
per month) of acute analgesics or symptomatic drugs for more than three
months (Ghiotto et al., 2009; Olesen et al., 2006; Silberstein et al., 2005). Since
frequent use of triptans has shown to cause MOH in migraine patients (more
than 10 doses of triptans per month for a period of 3 months) (Diener et al.,
2004; Ghiotto et al., 2009; Olesen et al., 2006; Silberstein et al., 2005),
attention was focused on triptan (sumatriptan) -induced MOH in this research
27
based on the previous studies in our lab.
Recently, latent sensitization as one of the potential mechanisms of
MOH attracts more and more attention (De Felice et al., 2010a; De Felice et al.,
2010b). One clinical study showed MOH patient demonstrated facilitation of
trigeminal and somatic parameters in the measurement of pain related cortical
potentials (Ayzenberg et al., 2006). Similar results were obtained from the
studies performed in our lab using rats with sustained or repetitive
administration of triptans (naratriptan or sumatriptan). Persistent sumatriptan
exposure elicited a time-dependent and reversible cutaneous tactile allodynia
and induced a state of latent sensitization that is sensitive to presumed
migraine triggers such as environmental stress (bright light stress) and NO
donor challenge (De Felice et al., 2010b). Cutaneous allodynia is accepted as
a marker of central sensitization (Burstein et al., 2000). The results suggested
that sensitization of primary afferent neurons may lead to further sensitization
of higher level neurons in the trigeminal nucleus caudalis and thalamus which
eventually result in cephalic and extracephalic cutaneous allodynia (Burstein et
al., 2004; De Felice et al., 2010b; Landy et al., 2004). Based on the previous
studies in our lab, sumatriptan over-exposed rats will be used as a “migraine
model” to investigate the correlation of persistent sumatriptan exposure
induced latent sensitization and CSD.
28
The hypothesis of this research is that persistent sumatriptan
exposure induced a state of latent sensitization that is associated with
increased responsiveness to electrically stimulated CSD or
environmental stress in rats. To support this hypothesis, three specific aims
were tested:
Specific aim 1: Develop a method to determine electrical stimulation threshold
to produce a CSD in anesthetized rats.
Specific aim 2: Determine if persistent sumatriptan exposure reduces the
stimulation threshold to produce a CSD and the effect of topiramate on CSD
threshold.
Specific aim 3: Determine the effects of topiramate in sumatripan induced
latent sensitization under bright light, i.e., environmental stress.
29
2 METHODS AND MATERIALS
2.1 Chemicals
Sumatriptan was purchased from GlaxoSmithKline (Philadelphia, PA,
USA). Topiramate tablets (100 mg/tablet, Johnson & Johnson) were obtained
from pharmacy. Isoflurane was purchased from Piramal Heathcare. Other
used chemicals including urethane, paraformaldehyde and general reagents
were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise,
and used without further purification.
2.2 Animals
Male Sprague-Dawley (SD) rats, 175 - 200 g at the time of arrival,
were obtained from Harlan laboratory Inc. (Indianapolis, IN). They were
housed in the University of Arizona Animal Care Facility (UAC) which is fully
accredited by the Association for Assessment and Accreditation of Laboratory
Animal Care (AAALAC). Upon receipt, the animals were taken to a designated
animal room where they were acclimated for 4 - 7 days in polyethylene cages
(three animals per cage) before being used in the experiments. The room
temperature was maintained between 20 - 25 °C and the relative humidity
between 40 - 60%. A light/dark cycle was maintained at 12 hour intervals.
30
Biannual testing for coliform and nitrates was performed by the UAC
Diagnostics Laboratory Medical Technologist. During the acclimation period,
rats were fed standard commercial diets (Harlan Teklad 4% rodent diet, Harlan,
Indianapolis, IN) and were allowed food and water ad libitum. All experimental
procedures were performed in accordance with the policies and
recommendations of the International Association for the Study of Pain, the
National Institutes of Health guidelines for the handling and use of laboratory
animals, and the Animal Care and Use Committees of the University of
Arizona.
2.3 Behavioral testing protocols
2.3.1 Periorbital sensory testing of non-noxious tactile stimuli in rats
Animals were acclimated to suspended plexiglass chambers (30 cm L
x 15 cm W x 20 cm H) with a wire mesh bottom (1 cm2) for 45 min. The animals
were allowed to freely move in their chambers during the entire testing protocol.
The response thresholds to tactile stimuli were determined in response to
probing the periorbital region with calibrated von Frey filaments (model 58011,
Stoelting). Each von Frey filament was applied for 3 to 6 sec, perpendicular to
the midline of the forehead, within a 3 mm diameter area at the level of the
eyes, until buckling slightly. A positive response was indicated by a sharp
withdrawal of the head, which sometimes included an attempt to grasp and/or
31
bite the filament. The withdrawal thresholds were determined by Dixon’s
up-down method (Chaplan et al., 1994; Dixon, 1980). Maximum filament
strength was 8 g.
2.3.2 Hindpaw sensory testing of non-noxious tactile stimuli in rats
Hindpaw measurements were determined in the same animals that
received the periorbital testing. The hindpaw withdrawal thresholds to tactile
stimuli were also determined in response to probing with calibrated Von Frey
filaments. Each Von Frey filament was applied perpendicularly to the plantar
surface of hindpaw until it buckled slightly, and was held for 3 to 6 sec. A
positive response was indicated by a sharp withdrawal of the hindpaw. The
withdrawal thresholds were determined by Dixon’s up-down method (Chaplan
et al., 1994; Dixon, 1980). Maximum filament strengths were 15 g for hindpaw.
2.4 Drug infusion
Alzet osmotic mini-pumps (Alzet, Cupertino CA, USA; model 2001) with
a nominal flow rate of 1 µL/h for 7 days were used for subcutaneous drug
infusion. The mini-pumps were implanted subcutaneously in rats under
anaesthesia with isoflurane (2% in air at 2 L/min). The day of the pump implant
was considered as day 0. Drug administered by infusion was sumatriptan (0.6
mg/kg/day; GlaxoSmithKline, Philadelphia, PA, USA). At day 6, minipumps
32
were removed under isoflurane anesthesia. Withdrawal thresholds were tested
at day 0, day 6 and day 20.
2.5 Implantation of electrodes
On day 20, rats were deeply anesthetized with urethane (1.2 g/kg, i.p.)
and fixed to a stereotaxic frame (Stoelting) to prepare the rats for electrical
stimulation and recording. Recording electrodes were made from 0.25 mm
diameter Ag wire (A-M Systems, Inc., Everett, WA). The wire was flamed to
produce spherical tips (1 mm diameter) and then coated with AgCl. Electrodes
were placed in burr holes through the skull made with electrical drills, and one
screw (#MPX-080-3F-1M, Small Parts Inc., Miami Lakes, FL) was placed over
the uninjured hemisphere (left) that served as a head-mount anchor.
Recording electrodes (electrode 1 and 2) were placed over frontal and parietal
cortices (2.0 mm lateral, 1.5 mm anterior and 2.5 mm posterior to bregma,
respectively) (Figure 2.1). A reference electrode was located posterior to
lambda (11.5mm from bregma). The wires at the free ends of the electrodes
were soldered to a multi-pin connector (Continental Connector, Hatfield, PA)
and the assembly was fixed to the skull with dental cement (Figure 2.3). An
additional burr hole (3.0 mm diameter) was made with an electrical drill to
expose the dura at 6.5 mm posterior and 3.0 mm lateral (right) to bregma. The
33
dura was carefully cut with a number 11 scalpel. A pair of electrodes, with tips
1 mm apart, (A-M systems, tungsten, 1 mm tip exposure, 20 degree,
WA,98382) (Figure 2.2) was inserted through the broken dura and placed 1.2
mm into cortical tissue.
2.6 Electrophysiological recording of CSD event
2.6.1 Electrophysiological recording
After surgery, rats were placed in suspended Faraday cages (40 cm L
x 49 cm W x 37 cm H) with wire mesh bottom (0.5 cm2) during the recording
period. A feedback-controlled heating pad (Harvard Apparatus, 110 v, 60 Hz)
was used to maintain body temperature of rats consistent at 37 °C during
recording (Figure 2.4). The multi-pin connector was attached to an
electro-cannular swivel (#CAY-675-6 commutator, Airflyte, Bayonne, NJ) fixed
to the chamber’s ceiling.
The stimulating electrodes were connected to an electrical stimulator
(A-M systems, isolated pulse stimulator, Model 2100). The duration and
intensity of the current was monitored and verified by an oscilloscope (Figure
2.5). Baseline ECoG (Electrocorticogram) and DC (Direct Current) recordings
were obtained for 0.5 h before any electrical stimulation was initiated. Only rats
with stable electrical recordings were included in the experiment. Signals were
recorded through shielded cables, input to separate channels for amplification
34
of DC and AC currents with a Grass (West Warwick, RI) Model 15 amplifier
system (15A12 DC and 15A54AC amplifiers), digitized at 100 Hz, and
collected with EEG recording analysis software Gamma v.4.9 (Astro-Med, Inc.
West Warwick, RI).
35
Figure 2.1 Schematic of skull indicating positions of electrodes and ECoG/DC
traced from anesthetized (Urethane 1.2 g/kg) rats after receiving a electrical
stimulation (horizontal arrow). The electrophysiological recording clearly
demonstrated a wave of CSD moving from electrode 2 (DC 2) in the parietal
region to electrode 1 (DC 1) located at the frontal cortex.
Stimulating
electrode
36
Figure 2.2 A stimulating electrode which is formed as a pair of tungsten
electrodes with tips 1 mm apart, (A-M systems, 1 mm tip exposure, 20 degree,
WA, 98382) used in the experiment to deliver current into cortex. It was
inserted through the broken dura and placed 1.2mm into the cortex.
37
Figure 2.3 A representative picture of the post-surgery rat. The free ends of
the electrode wires were soldered to a multi-pin connector and the assembly
was fixed to the skull with dental cement. Via the multi-pin connector implanted
on the head, rats were attached to an electro-cannular swivel (the green part).
The stimulating electrode (the white part) was connected with an electrical
stimulator (A-M systems, isolated pulse stimulator, Model 2100) which delivers
current into cortex through the stimulating electrode.
38
Figure 2.4 A picture of the entire system of electrical stimulation and recording.
Recording were performed in an isolated, quiet room. The rat was placed on
the pad of a homeothermic monitor (Harvard Apparatus, 110 v, 60 Hz) to keep
the body temperature at 37 °C during the recording.
39
Figure 2.5 The oscilloscope was used to verify the duration and intensity of
current delivered into cortex. Here it indicates a current pulse of 300
millisecond (msec) duration at an intensity of 2 milliampere (mA).
40
2.6.2 CSD parameters and criteria to define a CSD event
As shown in Figure 2.6 A, DC shift is defined as the voltage difference
measured at parietal lobe (DC2) or frontal lobe (DC1) during a CSD event.
CSD duration is the time difference of a CSD passing through electrode 2 to
electrode 1. To measure CSD duration, the start points are the time (unit:
seconds) when the DC currents start to decrease (shown as two vertical
arrows in Figure 2.6 B). Electrocorticogram (ECoG) amplitude was recorded
with 1 min time frame before and during the occurrence of a CSD (Figure 2.6
C).
Two criteria are used to define the electrical recording is a CSD event:
DC shift and reduction of ECoG amplitude. First, CSD is recognized by a
negative deflection of the monitored DC current occurring first in the parietal
lobe (DC2), and then the frontal lobe (DC1) following an effective electrical
stimulation (Figure 2.6 B). Second, the amplitude of the ECoG trace will
decrease during a CSD event compared with the amplitude measured before
the CSD event (Figure 2.6 B).
41
A
.
B
.
C
.
Figure 2.6 CSD parameters recorded during a CSD event. (A) DC shift (B)
CSD duration (C) ECoG amplitude.
42
2.6.3 CSD threshold measurement
The stimulation threshold to produce a CSD event (CSD threshold)
was determined by electrical stimulation of the cortex applied through the
pair of stimulating electrodes with 1 mm apart, placed 1.2 mm into cortical
tissue. Square wave pulses of increasing intensity (10 to 4,000 µCoulombs)
were applied at 4 min intervals by adjusting the current and duration of
stimulus (Table 2.1) until a CSD was generated. The CSD threshold was
determined as total charge (µCoulombs), and calculated using the following
equation:
CSD threshold (μC) = Current (mA) Duration (mS)
Table 2.1 Electrical stimulation protocol to produce a CSD
2.6.4 CSD propagation speed calculation
CSD propagation speed was calculated based on the CSD duration
and the distance between two recorder electrodes (electrode 1 and electrode
Stimulation protocol
Current (mA) 1 2 3 4
Duration (mS) 10 25 50 100 200 300 400 300 400 500 400 400 500 1000
Charge (C) 10 25 50 100 200 300 400 600 800 1000 1200 1600 2000 4000
43
2). As the distance between these two electrodes is 4 mm, the propagation
speed of CSD (mm/s) can be calculated by the following equation:
CSD propagation speed (mm/s) = Distance (4 mm) / CSD duration (s)
2.7 Immunolabeling
Rats were deeply anesthetized with 100 mg/kg of an 80 : 12 mixture of
ketamine and xylazine and perfused transcardially with 250 ml of
phosphatebuffered saline (PBS; 0.1 M; pH 7.4), followed by 4%
paraformaldehyde in 0.1 M PBS for 20 min. The brainstem was removed,
postfixed in 4% paraformaldehyde overnight, and cryoprotected in 30%
sucrose for 48 h at 4 C. Transverse sections (30 mm thick) were cut from 2 - 4
mm caudal to obex and collected for immunohistochemistry (IHC) labeling.
Sections were first incubated in blocking buffer (0.1 M PBS, 0.05% triton X 100,
1% BSA and 5% goat serum) for 90 min following 10 min permeabilization (0.1
M PBS, 0.2% triton X 100). Then, sections were immunoblotted at room
temperature for c-Fos primary antibody overnight (rabbit polyclonal anti c-Fos
sc-52, dilution 1 : 20000, Santa Cruz Biotechnology) and biotinylated goat
anti-rabbit IgG for 90 min (dilution 1 : 600, Vector Laboratories, Burlingame,
CA). Next, tissue sections were quenched by endogenous peroxidases with
0.1 M PBS, 10% methanol and 0.3% H2O2 for 30 min and subjected to ABC
44
complex (Vector Laboratories, Burlingame, CA) incubation for 60 min. At the
end, sections were soaked in TSA (Tyramide Signal Amplification, dilution 1 :
50, Perkinelmer, Inc., Waltham, MA) solution, air dried for 30 min and then
cover-slipped with Vectashield (Vector Laboratories, Burlingame, CA) for
future study. A three-time of buffer wash (0.1 M PBS, 0.05% Triton X 100) was
required in the interval when the incubation solution changed in the IHC study.
2.8 Determination of c-Fos labeled cells
The sections of trigeminal nucleus caudalis (TNC) were examined
using an Olympus microscope (BX51) equipped with a digital Hamamatsu
C4742-95 color camera along with Wasabi software (version 1.5) for
fluorescence imaging. A digital Infinity 3 color camera with Infinity Capture
Application (version 3.7.5.) was used for light imaging. Following a systematic
sampling through the extension of the trigeminal nucleus caudalis, most c-Fos
immunoreactivity was identified in laminae I and II, 2 - 4 mm below the obex.
Thus cell counts were obtained from the same area for all testing groups. Both
sides of each slice were counted (Fioravanti et al., 2011).
2.9 Bright light stress
In this experiment, 30 adult male Sprague Dawley rats (175 - 250 g)
were selected based on the behavior results obtained from periorbital region
45
and hindpaw withdrawal thresholds tested using Von Frey (VF) filaments at
day 0, day 6 and day 20 (Section 2.2, 2.3 and 2.4). On day 20, these rats were
divided into 4 groups: saline-infused with saline injection (6 rats);
saline-infused with topiramate injection (6 rats); sumatriptan-infused with
saline injection (9 rats) and sumatriptan-infused with topiramate injection (9
rats). To induce an environmental stress, rats were placed in an open box (20 -
30 cm) and exposed to bright light for 1 h. The light was positioned to avoid
temperature changes in the box. Periorbital and hindpaw allodynia are
measured at 1 h intervals for 6 h, starting 1 h after the end of the light stress
stimulus exposure. This stress was repeated 24 h after the first exposure.
Saline or topiramate (80 mg/kg, i.p.) was injected 30 min before the
environmental stress. The flow of this experiment is shown in Table 2.2.
Table 2.2 Experimental flow of bright light exposure
Day 0 Day 6 Day 20 Day 21
Baseline VF thresholds
Implant minipumps
VF thresholds
Remove minipumps
miminipumps
Topiramate (30 min before light)
Bright light (1h); VF thresholds
46
2.10 Statistical analysis
All data were expressed as mean ± SEM. Comparisons among
multiple means or treatment groups were determined by one-way ANOVA
followed by the post hoc Student-Newman-Keuls test. In case of comparison
between two groups, t-test is performed. A p-value < 0.05 was considered
significant and is indicated by an asterisk (*). A p-value < 0.001 was indicated
by two asterisk (**).
47
3 RESULTS
3.1 Persistent sumatriptan exposure produces generalized
tactile allodynia
To confirm our migraine pain model that using persistent sumatriptan
exposure produces tactile allodynia，SD rats were infused with sumatriptan
(0.6 mg/kg/day) or saline by osmotic minipump for 6 days. At day 6, the
minipumps were removed. The hindpaw withdrawal thresholds were measured
at day 0 prior to and on days 6 and 20 following minipump implantation.
Results showed persistent sumatriptan exposure significantly reduced paw
withdrawal thresholds at day 6 from 15.0 0.0 g to 8.5 1.5 g compared to
saline group (Figure 3.1). Removal of the minipump resulted in a return of
withdrawal thresholds to pre-implantation baseline levels by day 20.
48
Figure 3.1 Sustained infusion of sumatriptan (0.6 mg/kg/day) decreased
withdrawal thresholds to light tactile stimuli applied to the hindpaw of rats.
Sumatriptan or saline was continuously administered through an osmotic
minipump for 6 days, after which the minipumps were removed. Data are
shown as mean SEM. N=10, * p < 0.05 versus saline infused rats.
Von Frey
Day0 Day6 Day200
3
6
9
12
15
Saline
Sumatriptan
*
Pump implatation time
Pa
w W
ith
dra
wa
l T
hre
sh
old
(g
)
49
3.2 Measurement of electrical stimulated Cortical Spreading
Depression (CSD)
At day 20 after minipump implantation, CSD threshold was measured
in both saline and sumatriptan infused rats by the methods described in
section 2.5 and 2.6. According to the two criteria, two blocks of ECoG
recordings (1 min duration) were measured before and during CSD events and
used for data analysis. The results showed ECoG amplitude was significantly
reduced during CSD occurrence as compared to that recorded before CSD
(Figure 3.2). This reduction was measured in both parietal lobe (DC2) and
frontal lobe (DC1) in all treatment groups.
DC shifts measured in parietal lobes (DC2) and frontal lobes (DC1) are
shown in Figure 3.3. Statistical analysis indicated no significant difference of
DC-shift among all groups. Similarly, no significant difference of the other two
CSD parameters, CSD propagation speed and CSD duration, were detected
among all groups (Figure 3.4 and 3.5).
50
A ECoG amplitude of saline treated rats
Parieta
l-B-S
Parieta
l-D-S
Fronta
l-B-S
Fronta
l-D-S
Parieta
l-B-T
Parieta
l-D-T
Fronta
l-B-T
Fronta
l-D-T
0
200
400
600
800
** * *
Mic
rovo
lt
B ECoG amplitude of sumatriptan treated rats
Parieta
l-B-S
Parieta
l-D-S
Fronta
l-B-S
Fronta
l-D-S
Parieta
l-B-T
Parieta
l-D-T
Fronta
l-B-T
Fronta
l-D-T
0
200
400
600
800
1000
**
**Mic
rovo
lt
Figure 3.2 Reduction of ECoG amplitude during the CSD event measured in
frontal and parietal lobes of saline or sumatripan infused rats which pre-treated
with or without topiramate. Data are expressed at mean ± SEM. N= 7 - 9, * p <
0.05 versus ECoG amplitude recorded before CSD occurrence.
51
Figure 3.3 DC-shift measured in (A) frontal and (B) parietal lobes of saline or
sumatriptan infused rats with or without topiramate injection. Data are
expressed at mean ± SEM. N=8 - 10.
A Frontal Volt difference
Saline-S Saline-T Suma-S Suma-T0
1
2
3
4M
illi
vo
lt d
iffe
ren
ce
B Pareital Volt difference
Saline-S Saline-T Suma-S Suma-T0
1
2
3
4
Milliv
olt
dif
fere
nc
e
52
CSD propagation speed at day20
Saline-S Saline-T Suma-S Suma-T0
2
4
6
8
10
Mm
/Min
Figure 3.4 CSD propagation speed measured in saline and sumatripan
treated rats with or without topiramate pretreatment at day 20. Data are
expressed as mean ± SEM. N = 8 - 10.
53
Saline Saline-T Sumatriptan Sumatriptan-T0
20
40
60
80 CSD duration time (Second)C
SD
du
rati
on
tim
e (
Seco
nd
)
Figure 3.5 CSD duration measured in saline and sumatripan treated rats with
or without topiramate pretreatment at day 20. Data are expressed as mean ±
SEM. N = 8 - 10.
.
54
3.3 Evaluation of CSD threshold
Since persistent sumatripan exposure caused sensitization to tactile
stimuli in behavior testing (shown in Figure 3.1) , it would be interest to know if
sumatripan exposed rat demonstrates increased responsiveness to electrical
stimulated CSD events. Therefore, electrical stimulation threshold of CSD was
evaluated in our migraine model at day 20 following sumatripan minipump
implantation (14 days after removal of minipumps). Also, the protective effects
of topiramate, a prophylaxis of migraine headache used in clinic, were
investigated at the same time. Rats were divided into 4 groups at day 20
following the minipumps implantation: saline-infused rats with saline injection
(Saline-S); saline-infused rats with topiramate injection (Saline-T); sumatriptan
infused rats with saline injection (Suma-S) and sumatriptan-infused rats with
topiramate injection (Suma-T). Saline or topiramate (80 mg/kg, i.p.) was
injected 30 min before CSD testing. The results showed that there is no
statistically significant difference of CSD thresholds between Saline-S rats and
Suma-S rats (Figure 3.6). However, topiramate injection significantly increased
CSD thresholds in both saline and sumatriptan infused rats (p < 0.05) in
contrast to the rats with saline injection. CSD threshold increased from 1687.5
± 535.8 to 3100.0 ± 621.7 C after topiramate injection (80 mg/kg; i.p.) in
saline infused rats. In sumatriptan infused rats, CSD threshold increased from
55
1288.9 ± 334.5 to 3000.0 ± 641.7 C after topiramate injection.
56
CSD threshold at Day20
Saline-S Saline-T Suma-S Suma-T0
1000
2000
3000
4000
5000
**
Mic
roco
ulo
mb
s
Figure 3.6 Evaluation of electrical stimulation threshold of CSD in saline and
sumatripan infused rats with or without topiramate pretreatment. A single dose
injection of topiramate (80 mg/kg, i.p.) increased CSD thresholds measured in
both saline and sumatriptan treated rats at day 20. Data are shown as mean ±
SEM. N= 8 - 10, * p < 0.05 versus saline injection.
57
3.4 CSD is associated with the enhanced c-Fos expression in
the trigeminal nucleus cardalis
To determine if electrical stimulated CSD event is associated with the
neuron activation in the trigeminal nucleus caudalis (TNC) of rats, the
expression of c-Fos as a biomarker of neuron activation was evaluated via
immunohistochemistry. Brainstems were harvested for immunofluorescence
staining from all treatment groups: saline or sumatriptan-infused rats without
any stimulation; saline or sumatriptan-infused rats with electrical stimulated
CSD events; saline or sumatriptan-infused rats with electrical stimulated CSD
events and topiramate injection (80 mg/kg, i.p.) 30 min prior to CSD testing.
Due to our surgery is performed on the right side of the brain, c-Fos expression
on both sides of TNC slice was analyzed to discriminate the position difference.
Results indicated no significant difference on c-Fos expression between
ipsilateral and contralateral side of TNC slices (Figure 3.7). Then, c-Fos
expression on both sides of the slices were combined and shown in Figure 12.
Results showed that c-Fos expression significantly enhanced in both saline
and sumatriptan-infused rats with electrical stimulated CSD events. Compared
with rats without any stimulation, the number of c-Fos positive cells increased
from 1.0 0.6 to 7.5 2.3 in saline-infused rats and from 1.1 0.6 to 14.3 3.1
in sumatriptan-infused rats following electrical stimulation (p < 0.001).
58
Interestingly, sumatriptan infused rats demonstrated significant higher number
of c-Fos positive cells (14.3 3.1) than that in saline-infused rats (7.5 2.3)
following the same electrical stimulated CSD event. Injection of topriamate 30
minutes before electrical stimulation did not significantly reduce the number of
c-Fos positive cells in the TNC of the rats (Figure 3.8).
59
A
Naive CSD+Vehicle CSD+Topiramate0
3
6
9
12
15
18
Saline
Sumatriptan
Left TNC
#C
-Fo
s p
osit
ive c
ells in
TN
C
B
Naive CSD+Vehicle CSD+Topiramate0
3
6
9
12
15
18
Saline
Sumatriptan
Right TNC
#C
-Fo
s p
osit
ive c
ells/s
ecti
on
Figure 3.7 Evaluation of c-Fos expression on both the ipsilateral and
contralateral side of TNC slices. (A) Contralateral side (left TNC) (B) Ipsilateral
side (right TNC). Data is expressed as mean SEM. N=2 - 4. No significant
difference on c-Fos expression was measured between ipsilateral and
contralateral side of TNC slices.
60
Sal
ine
Sum
atripta
n
Sal
ine+
CSD
Sum
atripta
n+CSD
Sal
ine+
CSD+T
opiram
ate
Sum
atripta
n+CSD
+Topir
amat
e0
3
6
9
12
15
18
**
#C-Fos positive cells in TNC
**##
#C
-Fo
s p
osit
ive c
ells in
TN
C
Figure 3.8 Electrical stimulated CSD is correlated to the enhanced c-Fos
expression in both saline and sumatripan infused rats. Data are shown as
mean SEM, N=2 - 4. ** p < 0.001versus rats without CSD, ## p < 0.001
versus saline infused rats with CSD.
61
3.5 Topiramate reverses environmental stress induced
allodynia in persistent sumatriptan exposed rats
Our previous work has shown persistent sumatripan exposure
increased sensitivity to the traditional migraine triggers such as environmental
stress (De Felice et al., 2010a; De Felice et al., 2010b). To test if topiramate
could block this sensitization in current migraine pain model, the rats were
stressed by bright light for 1 h at day 20 (14 days after termination of
sumatriptan exposure). At the same day, rats were administrated with saline or
topiramate (80 mg/kg, i.p.) 30 min before bright light exposure. Results
showed the response threshold of sumatriptan exposed rats to periorbital
tactile stimuli significantly reduced from the baseline level (8.0 0.0 g) to 6.6
0.1 g. Similarly, the hindpaw withdrawal threshold reduced from the baseline
value (14.7 0.3 g) to 6.7 0.5g (Figure 3.9 A and 3.9 B). At the following day
(i.e. day 21, 15 days after termination of sumatriptan infusion), these rats were
exposed to bright light again for 1 h. Results showed the threshold of
periorbital withdrawal and hindpaw withdrawal in sumatripan exposed rats
significantly reduced to 2.9 0.3 g and 6.2 1.0 g respectively (Figure 3.9 C
and 3.9 D). More importantly, a single bolus injection of topiramate (80 mg/kg,
i.p.) given 30 min before the exposure to bright light almost completely blocked
environmental stress-induced cutaneous allodynia in periorbital region and the
62
hindpaw (Figure 3.10) on any days tested.
63
A B
C D
Figure 3.9 Persistent sumatriptan exposure induced latent sensitization in rats.
On Days 20 and 21 after pump implant, rats were injected topiramate(80
mg/kg, i.p.) or saline 30 min prior to bright light exposure. Sumatriptan
exposed rats demonstrated hind paw (A: Day 20, C: Day 21) and periorbital (B:
Day 20, D: Day 21) tactile allodynia. Data are expressed as mean SEM. N=4
- 5, * p < 0.05, versus saline infused rats.
Day 20 Hindpaw Allodynia
Baseline 1 2 3 4 5 60
3
6
9
12
15
Saline-saline
Suma-saline
Time(hours) after stress stimulusHin
d p
aw
wit
hd
raw
al th
resh
old
(g) Day 20 Periorbital Allodynia
Baseline 1 2 3 4 5 60
2
4
6
8
Saline-saline
Suma-saline
Time(hours) after stress stimulusPerio
rb
ital w
ith
draw
al th
resh
old
(g)
Day 21 Periorbital Allodynia
Baseline 1 2 3 4 5 60
2
4
6
8
Saline-salineSuma-saline
Time(hours) after stress stimulusPerio
rb
ital w
ith
draw
al th
resh
old
(g)Day 21 Hindpaw Allodynia
Baseline 1 2 3 4 5 60
3
6
9
12
15
Saline-salineSuma-saline
Time(hours) after stress stimulusHin
d p
aw
wit
hd
raw
al th
resh
old
(g)
64
A B
C D
Figure 3.10 Environmental stress-induced allodynia is blocked by a single
dose of topiramate (80 mg/kg, i.p.). Sumatripan infused rats were challenged
on Days 20 and 21 with a single dose injection of topiramate (80 mg/kg, i.p.) 30
min prior to bright light exposure. Topiramate (80 mg/kg, i.p.) blocked the
expression of hindpaw (A: Day 20, C: Day 21) and periorbital (B: Day 20, D:
Day 21) tactile allodynia in these rats. Data are shown as mean SEM. N=4 -
5.
Day 20 Periorbital Allodynia
Baseline 1 2 3 4 5 60
2
4
6
8
Saline-topiramate
Suma-topiramate
Time(hours) after stress stimulusPeri
orb
ital w
ith
dra
wal th
resh
old
(g)Day 20 Hindpaw Allodynia
Baseline 1 2 3 4 5 60
3
6
9
12
15
Saline-topiramate
Suma-topiramate
Time(hours) after stress stimulusHin
d p
aw
wit
hd
raw
al th
resh
old
(g)
Day 21 Hindpaw Allodynia
Baseline 1 2 3 4 5 60
3
6
9
12
15
Saline-topiramate
Suma-topiramate
Time(hours) after stress stimulusHin
d p
aw
wit
hd
raw
al th
resh
old
(g)
Day 21 Periorbital Allodynia
Baseline 1 2 3 4 5 60
2
4
6
8
Saline-topiramate
Suma-topiramate
Time(hours) after stress stimulusPeri
orb
ital w
ith
dra
wal th
resh
old
(g)
65
3.6 Topiramate attenuates the enhanced expression of c-Fos
in TNC of saline and sumatriptan infused rats
Two hour after the second bright light exposure on day 21 (15 days
after termination of sumatriptan and saline infusion), rats from all groups:
saline-infused with saline injection; saline-infused with topiramate injection;
sumatriptan-infused with saline injection and sumatriptan-infused with
topiramate injection were anesthetized and the brainstems were removed for
immunofluorescence staining. Results showed c-Fos immunoreactivity in the
superficial laminae of the TNC from sumatripan infused rats with saline
injection significantly increased (23.2 + 3.2) compared with saline-infused rats
with saline injection (15.3 1.5) (p < 0.05). Importantly, topiramate significantly
reduced c-Fos expression in both saline (10.7 1.9) and sumatriptan (14.4
1.6) infused rats. About 31% and 38% of c-Fos positive cells were decreased
by topiramate pretreatment in saline and sumatripan infused rats respectively
(Figure 3.11).
66
Saline-S Sumatriptan-S Saline-T Sumatriptan-T0
3
6
9
12
15
18
21
24
27 #
*
#C-Fos positive cells in TNC
*
#C
-Fo
s p
osit
ive c
ells in
TN
C
Figure 3.11 Pretreatment of topiramate (80 mg/kg, i.p.) attenuates c-Fos
expression in both saline and sumatriptan exposed rats in TNC following bright
light exposure. Data are expressed as mean + SEM. N= 2 - 4, * p< 0.05 versus
saline infused rat with saline injection, # p< 0.05 versus sumatripan infused
rats with saline injection.
67
4 DISCUSSION AND CONCLUSIONS
Migraine headache is a common and complex neurological disorder.
Although it is clear that head pain is a key manifestation of this disorder for
most patients, what drives the activation of neuronal pain pathways in migraine
patients is still not well understood. The main barriers for migraine pain study
are that such pain occurs without tissue damage and patients with this disease
perform normally between periods of migraine attack (De Felice et al., 2010b).
A relatively reliable model is pivotal for migraine research. So far,
several migraine models have been developed both in vivo and in vitro,
including vascular (vasodilation and vasoconstriction) model, neurovascular
model (plasma protein extravasation, activation of TNC, CSD, effects of NO
donors) and mutant mouse model (i.e., mutant Cav2.1 channels) (Bergerot et
al., 2006). In current research, sumatriptan over-exposed rats were used as a
“migraine model” which was originally developed in our lab. This model is
based on the clinical phenomenon that migraine patient with frequent use of
triptans will lead to medication overuse headache (MOH) during which
frequency of migraine attack increases (De Felice et al., 2010b; Diener et al.,
2004). Studies in our lab showed that persistent neural adaptation in trigeminal
ganglion cells was observed after repeated or sustained triptan administration.
68
It was characterized as increased labeling for calcitonin gene related peptide
(CGRP) and neuronal nitric oxide synthase (nNOS) in identified rat trigeminal
dural afferents. In addition, a behavioral reaction of increased sensitivity to
presumed migraine triggers such as environmental stress and nitric oxide (NO)
donor was maintained for weeks following discontinuation of triptan
administration. This phenomenon is termed as “triptan-induced latent
sensitization” (De Felice et al., 2010a; De Felice et al., 2010b).
Results suggested that persistent sumatripan exposure induced latent
sensitization in rats mimic several common characteristics of clinical migraine.
First of all, in this model, no tissue injury was observed and normal sensory
threshold was maintained between periods of pain. These are consistent with
human migraine headache. Secondly, challenge of traditional migraine triggers
such as environmental stress (bright light stress) and NO donor reduced
sensory threshold and caused generalized tactile allodynia (defined as a
hypersensitivity of the skin to touch or mechanical stimuli that is considered
non-noxious under normal circumstances). Finally, after NO donor challenge,
CGRP level in blood was elevated. CGRP receptor, but not NK-1 (neurokinin-1)
antagonists can reverse allodynia caused by NO donor challenge (De Felice et
al., 2010b). Indeed, the results of current research confirmed our previous
observations (De Felice et al., 2010b). Current results showed that persistent
69
sumatriptan (0.6 mg/kg/day) infusion significantly reduced hindpaw withdrawal
threshold at day 6 (Figure 3.1) suggesting these rats exhibited tactile allodynia.
Removal of the sumatripan resulted in a return of withdrawal threshold to
pre-implantation baseline levels by day 20 (Figure 3.1).
Next, a stable electrical stimulation technique to evoke CSD events in
rats was established. Efforts were focused on finding a reliable electrode to
deliver current into the cortex of rats. This is a time consuming process in
which at least twelve electrodes in different material, size and form have been
tested. Eventually, a tungsten electrode was found to be the most reliable one
in terms of delivering the appropriate current stably in this study. Therefore,
this tungsten electrode was used in the following experiments.
Two criteria were used to define a CSD event. The first one is the
occurrence of DC shift that is caused by the ionic movement, i.e., Ca2+, Na+, K+.
During a CSD event, DC shift occurs first at parietal lobe (relatively close to
electrical stimulation site) then frontal lobe. The other criterion is the reduction
of ECoG amplitude during CSD development (Figure 3.2), which is exactly like
the name of CSD (spreading depression). These two criteria were used
throughout all experiments to confirm the occurrence of CSD event. Several
parameters of CSD such as DC shift, ECoG amplitude, propagation speed and
CSD duration were measured in this study to define if an electrical recording is
70
the same CSD event. Indeed, no significant difference on DC shift, ECoG
amplitude and DC duration were measured among all groups. Therefore, the
same CSD events were visualized, recorded and compared among all groups.
Because slowing of propagation speed has been associated with CSD
propagation failure (Akerman et al., 2005), the speed of propagation of CSD
were measured. Results showed no significant differences on CSD
propagation speed among all tested groups. Topiramate only slightly
decreased the propagation speed in sumatriptan and saline group (Figure 3.4)
This result is not consistent with the study reported by Ayata et al. that CSD
propagation speed was significantly reduced by topiramate (Ayata et al., 2006).
The possible reason for this discrepancy is the different duration of topiramate
treatment used in current study. In our study, one single dose of topiramate (80
mg/kg) was administrated Intraperitoneally instead of chronic administration
(60 - 80 mg/kg, i. p.) for 4 - 17 weeks in their study.
A slight decrease of CSD threshold in sumatriptan-exposed rats was
measured compared with saline treated rats (Figure 3.6). However, statistical
analysis did not show a significant difference between these two groups.
These results are unexpected. Our original expectation was that sumatriptan
exposure might decrease the stimulation threshold to produce a CSD. This
outcome suggests that increased migraine frequency after triptan exposure is
71
not likely due to an increase in susceptibility to generate CSD events. However,
a CSD may still trigger enhanced responses in the condition of latent
sensitization. Interestingly, a single dose of topiramate (80 mg/kg, i.p.)
elevated CSD threshold in both sumatriptan and saline treated rats (Figure
3.6). As a prophylaxis of migraine headache, chronic daily i.p. administration of
topiramate dose-dependently suppressed CSD frequency and increased the
electrically stimulated threshold of CSD in rats (Ayata et al., 2006). The other
study showed intravenous injection of topiramate (30 mg/kg) inhibited
occurrence of CSD evoked by a cortical needle plunge in rats (Akerman et al.,
2005). Our results are partly consistent with these reported studies. Our
interpretation for the results is that the elevated CSD threshold by topiramate
is probably due to its anti-epileptic menchanisms that can decrease abnormal
neuronal excitation in the brain (Brandes, 2005; Rogawski, 2012).
C-Fos immunoreactivity in TNC as a biomarker of the activation of
trigeminal vascular system has been widely used in migraine research
(Goadsby et al., 1997; Hoskin et al., 1999; Sugimoto et al., 1998). To determine
if electrical stimulation induced CSD is associated with the activation of TNC,
the expression of c-Fos in TNC was evaluated by immunohistochemistry.
Results showed c-Fos expression was significantly elevated in both
sumatriptan and saline treated rats following the occurrence of electrical
72
stimulated CSD, which indicated that CSD event can activate trigeminal
vascular system (Figure 3.8). This result is consistent with previous findings
showed that CSD provokes c-Fos expression within TNC (Bolay et al., 2002;
Moskowitz, 1984; Moskowitz et al., 1993b). More importantly, after CSD events,
the level of c-Fos expression in persistent sumatriptan exposed rats was
significantly higher than that measured in saline rats (Figure 3.8), regardless of
the c-Fos positive cells from the combination or separation of ipsilateral and
contralateral side of the TNC slices (Figure 3.7, 3.8). As observed in
sumatriptan-exposed rats, the enhanced expression of c-Fos is most likely
caused by sumatriptan induced latent sensitization. The previous data from
our lab have showed the primary afferents were sensitized after persistent
sumatriptan exposure. It was characterized as the increased labeling of CGRP
and nNOS in identified trigeminal dura afferents (De Felice et al., 2010a; De
Felice et al., 2010b). The sensitization of the 1st order neurons may lead to
sensitize the 2nd order neurons or even the 3rd order neurons (Burstein et al.,
2004; De Felice et al., 2010b; Landy et al., 2004). Here, further study is
necessary to investigate which level of neurons was sensitized to cause the
enhanced expression of c-Fos in sumatriptan exposed rats.
Unpublished data from our laboratories showed CSD event was
induced by bright light stress in sumatriptan exposed rats. Also because CSD
73
threshold was elevated by topiramate (Figure 3.6), next experiment was
designed to investigate whether preventive administration of topiramate could
block the allodynia in sumatriptan exposed rats following bright light stress.
The results showed that a single dose of topiramate (80 mg/kg, i.p.) decreased
trigeminal vascular sensitivity in sumatriptan exposure model as evidenced by
the reverse of bright light stress induced allodynia and attenuation of c-Fos
immunoactivity in TNC(Figure 3.9 and 3.10). This protective effects are similar
as observed in migraine patients who treated by topiramate (Brandes, 2005;
Krymchantowski et al., 2004; Silberstein, 2004). These results, coupled with
the results obtained in CSD threshold experiment, indicate topriamate
prevents the occurrence of sumatriptan induced latent sensitization possibly
via increasing the electrical stimulation threshold of CSD.
In summary, this study found that persistent sumatriptan exposure
does not increase responsiveness to electrically stimulated CSD in cortex,
since the stimulation thresholds were not decreased in sumatritan exposed
rats. However, the trigeminal system is still in a state of latent sensitization, as
indicated by the production of tactile allodynia after exposure to bright light
stress. This observation is supported by the enhanced c-Fos expression found
in the TNC of sumatriptan exposed animals after bright light stress. Therefore,
a normal CSD event could still trigger enhanced responses in the trigeminal
74
pathway, as shown by the increased c-Fos expression observed in
sumatriptan-exposed animals following CSD event. Topiramate may exert its
prophylactic effect by inhibiting the generation of CSD events, as indicated by
the increased stimulation thresholds to elicit a CSD. Moreover, we found that
topiramate produced a slight attenuation of the increased c-Fos expression in
TNC after CSD, and blocked the sumatriptan-induced increased c-Fos
expression after bright-light stress as well as the stress-induced tactile
allodynia. These observations suggest that the protective effects of topiramate
against medication overuse headache (MOH) are mediated by three possible
mechanisms: blocking the initiation of CSD events; inhibiting latent
sensitization or both. The relevance of these protective mechanisms of
topiramate to pathogenesis of medication overuse headache is remained to be
established.
75
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