doi:10.1093/brain/aws249
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BRAIN
A JOURNAL OF NEUROLOGY
A translational in vivo model of trigeminal
autonomic cephalalgias: therapeutic
characterization
Simon Akerman, Philip R. Holland,* Oliver Summ,† Michele P. Lasalandra and Peter J. Goadsby
Headache Group, Department of Neurology, University of California, San Francisco, San Francisco, CA 94158, USA
*Present address: Centre for Neuroregeneration, University of Edinburgh, Edinburgh, EH16 4SB, UK
†
Present address: Department of Neurology, University Hospital Münster, 48149 Münster, Germany
Correspondence to: Dr Simon Akerman,
Headache Group,
Department of Neurology,
University of California,
San Francisco,
675 Nelson Rising Lane,
San Francisco,
CA 94158, USA
E-mail: [email protected]
Trigeminal autonomic cephalalgias are highly disabling primary headache disorders, characterized by severe unilateral head pain
and associated ipsilateral cranial autonomic features. There is limited understanding of their pathophysiology and how and
where treatments act to reduce symptoms; this is significantly hindered by a lack of animal models. We have developed the first
animal model to explore trigeminal autonomic cephalalgias, using stimulation within the brainstem, at the level of the superior
salivatory nucleus, to activate the trigeminal autonomic reflex arc. Using electrophysiological recording of neurons of the
trigeminocervical complex and laser Doppler blood flow changes around the ipsilateral lacrimal duct, superior salivatory nucleus
stimulation exhibited both neuronal trigeminovascular and cranial autonomic manifestations. These responses were specifically
inhibited by the autonomic ganglion blocker hexamethonium bromide. These data demonstrate that brainstem activation may be
the driver of both sensory and autonomic symptoms in these disorders, and part of this activation may be via the parasympathetic outflow to the cranial vasculature. Additionally, both sensory and autonomic manifestations were significantly inhibited
by highly effective treatments for trigeminal autonomic cephalalgias, such as oxygen, indomethacin and triptans, and some part
of their therapeutic action appears to be specifically on the parasympathetic outflow to the cranial vasculature. Treatments more
used to migraine, such as naproxen and a calcitonin gene-related peptide receptor inhibitor, olcegepant, were less effective in
this model. This is the first model to represent the phenotype of trigeminal autonomic cephalalgias and their response to
therapies, and indicates the parasympathetic pathway may be uniquely involved in their pathophysiology and targeted to relieve
symptoms.
Keywords: trigeminal autonomic cephalalgias; brainstem; superior salivatory nucleus; parasympathetic outflow; triptan
Abbreviations: 5-HT = 5-hydroxytryptamine; TAC = trigeminal autonomic cephalalgias
Received March 19, 2012. Revised May 24, 2012. Accepted July 13, 2012. Advance Access publication October 11, 2012
ß The Author (2012). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: [email protected]
Rodent model of trigeminal autonomic cephalalgias
Introduction
The trigeminal autonomic cephalalgias (TACs) (Goadsby and
Lipton, 1997), including cluster headache, are highly disabling primary headache disorders characterized by ‘severe unilateral head
pain, described by females as a pain worse than childbirth’, occurring in association with ipsilateral cranial autonomic features
(Headache Classification Committee of the International
Headache Society, 2004). An understanding of their pathophysiology involves three major clinical features: unilateral trigeminal
distribution of pain, cranial autonomic features and individually
highly characteristic attack patterns (Goadsby, 2002). It is the
combination of the attack pattern and phenotype that differentiates these disorders from migraine. Cluster headache attacks tend
to have the longest duration with lower attack frequency (May,
2005); paroxysmal hemicrania has an intermediate duration and
attack frequency (Cittadini et al., 2008); short-lasting unilateral
neuralgiform headache attacks with conjunctival injection and
tearing have the shortest duration and many discrete attacks per
day (Cohen et al., 2006); and hemicrania continua presents with
unremitting pain (Cittadini and Goadsby, 2010).
The basic anatomy and physiology of TACs are beginning to be
understood, based mainly on clinical research. One of the major
symptoms of TACs is the excruciating pain, which is most likely a
consequence of activation of the trigeminovascular system
(Goadsby et al., 2002). This includes the peripheral nerve fibres
that innervate the pain-producing cranial vessels and dura mater,
and the centrally projecting fibres that synapse in the trigeminocervical complex. Animal models have demonstrated activation of
the trigeminocervical complex by stimulation of dural structures
(Kaube et al., 1993; Hoskin et al., 1999). Stimulation of these
same structures also causes neuronal activity in the superior salivatory nucleus within the pons (Knight et al., 2005), which is the
origin of cells for the cranial parasympathetic autonomic vasodilator pathway (Spencer et al., 1990). This efferent projection is predominantly through the greater petrosal nerve, a branch of the
facial (VIIth) cranial nerve, and its projection through the sphenopalatine ganglion (pterygopalatine ganglion in humans, Fig. 1). It
is thought that this autonomic activation leads to the second important symptom in TACs: cranial autonomic features, which include lacrimation, conjunctival injection and nasal congestion and
a local third-order sympathetic lesion due to carotid swelling. TACs
and other primary headaches may share a common activation
through the trigeminovascular system, and all primary headaches
can exhibit autonomic symptoms to some degree, through reflex
activation of the cranial autonomic outflow. However, in primary
headaches where activation of the trigeminal autonomic reflex
contributes far more significantly to their pathophysiology and as
a consequence their major symptoms, they are conveniently classified together as TACs.
TACs each have unique therapeutic responses that differentiate
them one from another, and from migraine (Goadsby et al., 2008).
However, there is still little known about what causes TACs, and
how and where treatments act to reduce symptoms, which substantially limits attempts to develop new treatments. The lack of good
experimental models, which respond preferentially to the different
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therapies for TACs, has hindered progress in understanding their
pathophysiology, reduced our ability to determine the locus of
action of existing therapies and hindered the development of
novel treatment strategies. Our aim was to develop an in vivo
model combining trigeminovascular activation common to models
of migraine, with significant activation of the parasympathetic outflow to mimic cranial autonomic symptoms, using stimulation of the
trigeminal autonomic reflex. This may provide a basis to understand
better the pathophysiology of TACs, and provide an opportunity to
explore current strategies and develop new therapies. This in vivo
model would preferentially be responsive to therapies that are effective in the various TACs, and be less responsive to specific migraine therapies. Therefore, it would need to use a different
mechanism of activation to demonstrate that these therapies are
acting on alternative pathways. To do this, we stimulated the superior salivatory nucleus to activate directly the trigeminal autonomic reflex arc, including the greater petrosal branch of the VIIth
nerve, predicting this efferent activation would excite trigeminal
afferents to the trigeminocervical complex, as well as demonstrating
a parasympathetic component of symptomology.
Materials and methods
All experiments were conducted under license and approved by ethical
review of the University of California, San Francisco Institutional
Animal Care and Use Committee, conforming to the National
Institute of Health Guide for the Care and Use of Laboratory
Animals and adhering to the guidelines of the Committee for
Research and Ethical Issues of the International Association for the
Study of Pain (Zimmermann, 1983).
Surgical preparation
Male Sprague Dawley rats (n = 136, 290–375 g) were anaesthetized
with sodium pentobarbitone (60 mg/kg, intraperitoneal), and anaesthesia was maintained with propofol (15–20 mg/kg/h intravenous infusion). During the dural electrical stimulation, electrophysiological
recording, animals were paralysed with pancuronium bromide
(Pavulon, Organon), 0.4 mg initially and maintained with 0.2 mg every
35 min, as described previously (Akerman et al., 2007). To maintain full
physiological functionality of the outflow from the superior salivatory
nucleus, which is partly via the facial nerve and predominantly the
greater petrosal nerve, this subgroup of animals were not paralysed.
Animals were placed in a stereotaxic frame and continuously monitored
for blood pressure and body temperature; the animals were artificially
ventilated, and expired CO2 was measured and kept between 3.5 and
4.5%. A sufficient depth of anaesthesia was judged by the absence of
paw withdrawal and corneal blink reflex, and during muscular paralysis
by fluctuations of blood pressure and changes to expired CO2.
Electrophysiological recording in the
trigeminocervical complex
Methods for electrophysiological recording in the trigeminocervical complex have been published previously (Holland et al., 2006; Akerman
et al., 2007). Briefly, a C1 hemilaminectomy was performed and the
dura mater incised to expose the brainstem at the level of the caudal
medulla. A tungsten recording electrode (0.5 MV, tip diameter 0.5 mm)
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S. Akerman et al.
Figure 1 Overview of the major anatomical and physiological pathways believed to be involved in TACs. Pain afferents from the
trigeminovascular system traverse the ophthalmic division of the trigeminal nerve, taking signals from the cranial vessels and dura mater.
These inputs synapse in the trigeminocervical complex (TCC, black neuron) and project to higher brain structures such as the thalamus and
cortex (blue neurons), resulting in pain perception. Activation of the trigeminovascular system by stimulation of dural structures also
causes neuronal activation in the superior salivatory nucleus (SuS, via the grey neuron) within the pons, which is the origin of cells for the
cranial parasympathetic autonomic vasodilator pathway. In TAC pathophysiology, there is activation of this parasympathetic reflex via the
outflow from the superior salivatory nucleus, predominantly through the greater petrosal nerve (green neuron) and its relay with the
sphenopalatine ganglion (SPG), but also via the facial (VIIth cranial) nerve (purple neuron). Activation of both trigeminal and autonomic
nerves defines the trigeminal autonomic reflex arc and is thus integral to the pathophysiology of TACs. Additionally, it is thought that there
is descending control of the trigeminocervical complex and superior salivatory nucleus through the periaqueductal grey (PAG), locus
coeruleus (LC), nucleus raphe magnus (NRM) and hypothalamus (red projections). Tg = trigeminal ganglion.
was lowered into the brainstem with a piezoelectric motor controller.
Extracellular recordings were made from neurons in the trigeminocervical
complex and identified as having cutaneous facial receptive fields, and by
superior salivatory nucleus or dura mater stimulation (see Fig. 2A for
experimental set-up). The signal was amplified (total gain 20 000–
30 000) and passed through filters (300 Hz–20 KHz) and a 60-Hz noise
eliminator to a second-stage amplifier. This signal was fed to a gated
amplitude discriminator and analogue-to-digital converter (Cambridge
Electronic Design) and to a microprocessor-based computer for analysis
using Spike2 v5.
Evoked neuronal responses in the trigeminocervical complex were
established via stimulation of either the dura mater or the superior
salivatory nucleus. To stimulate the dura mater, a bipolar stimulating
electrode was placed on the dura mater adjacent to or on either side
of the middle meningeal artery, and electrical stimulus was applied
(0.6 Hz, 0.1–0.5 ms, 8–20 V) to activate trigeminal afferents. To stimulate the superior salivatory nucleus, a concentric bipolar tungstenstimulating electrode (impedance 10–15 KV, tip diameter 3–4 mm)
was stereotaxically positioned into the superior salivatory nucleus (anterior–posterior 10.20–10.80 mm from bregma or 1.2–1.8 mm from
interaural, dorsal–ventral 9.2–9.6 mm and medial–lateral 2.1–2.4 mm;
Paxinos and Watson, 2004) (Fig. 2B). Constant current stimulus was
applied (0.5 Hz, 150-ms duration and 20–50 mA), and neuronal responses in the trigeminocervical complex were recorded. Lesion
marks were made post-experiment, and the brain was removed and
post-fixed to confirm stimulation location (Fig. 2C). Neurons within the
trigeminocervical complex were first characterized for their cutaneous
and deep receptive field, assessed through all three territories of the
trigeminal nerve, and both noxious and non-noxious responses were
observed. Neurons sensitive to stimulation of the ophthalmic dermatome of the trigeminal nerve were then tested for convergent input
from either the dura mater or superior salivatory nucleus. Baseline
responses consisted of trains of 20 stimuli delivered at 5-min intervals,
stimulating either dura mater or superior salivatory nucleus. Reliable
electrically evoked responses with durations in the A-fibre or C-fibre
range, and also cutaneous receptive field, were tested pharmacologically with therapeutics for the various TACs, as well as specific
anti-migraine drugs, to characterize the reliability of the assay in predicting clinical therapeutic efficacy.
Facial flow responses
Lacrimation and tearing are common symptoms that help define the
TACs (Headache Classification Committee of the International
Rodent model of trigeminal autonomic cephalalgias
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Figure 2 Experimental set-up and overview of stimulation sites. (A) Experimental set-up for stimulation of the superior salivatory nucleus
(SuS) and recording in the trigeminocervical complex. (B) Locations of lesion sites in the area of the superior salivatory nucleus where
stimulation took place. Stimulation sites in regions 1.32–1.56 mm behind the interaural point highlight that the superior salivatory nucleus
was located as the stimulation site in each case, and also the proximity of the facial nerve was 5250 mm from the stimulation site and
therefore likely to be simultaneously activated even if motor responses were not observed. (C) Example lesion mark in an area proximate
to the superior salivatory nucleus. (D) Experimental set-up for stimulation of the superior salivatory nucleus and measurement of changes
in blood flow of the lacrimal duct/sac using laser Doppler flowmetry. 4V = fourth ventricle; 7 n = VIIth nerve; g7 = genu of the facial
nerve; MMA = middle meningeal artery; Pr5VL = principle sensory trigeminal nucleus (ventrolateral part); sp5 = spinal trigeminal tract;
Sp5O = spinal trigeminal nucleus oralis.
Headache Society, 2004). They are a significant component of these
disorders and thought to be a consequence of cranial parasympathetic
nerve activation (Goadsby, 2002; May, 2006). Within our animal
model we wanted to demonstrate activation of the parasympathetic
outflow to the cranium, or the autonomic reflex, after stimulation of
the superior salivatory nucleus, and provide some evidence of a lacrimal response. Previously methods have used cannulation of arterial or
excretory supplies to the lacrimal duct as evidence of lacrimal gland
flow (Botelho et al., 1976; Botelho and Fuenmayor, 1981). However,
we wanted to take a less invasive approach, as the surgery itself could
affect responses, and so we measured changes in blood flow around
the lacrimal gland/duct using carefully positioned laser Doppler probes
as an indication of this response (Moor Instruments) (Fig. 2D). Two
baseline responses were performed in response to superior salivatory
nucleus stimulation (5 Hz, 500-ms duration and 40–70 mA for 10 s), and
the animals were given treatment and superior salivatory nucleus
stimulation repeated at 5-min intervals for 30 min.
At the end of each experiment, either electrophysiology or facial blood
flow, and after euthanasia, a lesion was made in the trigeminocervical
complex and superior salivatory nucleus so that the area of recording or
stimulation could be identified post-experiment. The brain and spinal
cord were removed and stored in 10% formalin for post-fixing for 24 h
and then cryopreserved in 30% sucrose. Sections of 30-mm thickness
were cut, slide mounted and contrastained with cresyl violet to highlight
and identify both the recording site in the trigeminocervical complex and
the stimulation site of the superior salivatory nucleus.
Data analysis
The data collected as post-stimulus histograms after electrical stimulation of the dura mater for A-fibres represent the number of cells fired
over a 10-ms period in the region 5–20 ms post-stimulation over the
20 collections. During superior salivatory nucleus stimulation, latency
periods for A-fibres were extended to 40 ms because of the greater
distance for the signal to travel and activation across multiple synapses. Spontaneous activity was measured in cell firings per second
(Hz). Facial flow was measured as an average increase (in arbitrary
units) over the 10 s from the baseline level.
All data are expressed as mean SEM. Statistical analysis was performed using an ANOVA for repeated measures with Bonferroni post
hoc correction for multiple comparisons used to measure the time
course of significant drug intervention, using a 95% confidence interval. If Mauchly’s test of sphericity was violated, we made appropriate
corrections to degrees of freedom according to Greenhouse–Geisser.
Student’s paired t-test for post hoc analysis was used to test for the
time points of significance, using the average of the two or three
baselines for comparison, again using the criteria of Bonferroni correction. In some cases when between-groups analysis was necessary, a
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mixed-design repeated-measures ANOVA was used. Statistical significance was set at P 5 0.05 (using SPSS 16.0 throughout).
Results
Electrophysiology
Recordings were made from 103 neurons responsive to either dural
stimulation (26 neurons in 26 animals) or stimulation of the superior
salivatory nucleus (77 neurons in 65 animals), and with a cutaneous
receptor field that included the first (ophthalmic) division of the
trigeminal nerve and occasionally the second (maxillary) division.
Neurons were located in deep layers (layers IV, V and VI) of the
dorsal horn of the trigeminocervical complex, which includes the
cervical C1 region of the spinal cord and its transition to the trigeminal nucleus caudalis, at range of depth of 450–955 mm. Neurons
responsive to dural stimulation had a latency of firing of 16 2 ms
and 51 2 ms, respectively, for A-fibre and C-fibre inputs, and the
baseline spontaneous firing rate was 42.5 2 Hz.
Characterization of two distinct
neuronal populations in the
trigeminocervical complex after superior
salivatory nucleus stimulation
Activation of the parasympathetic outflow to the cranial vasculature
was produced by stimulation of the superior salivatory nucleus, and
its predominant projection through the greater petrosal branch of the
VIIth (facial) cranial nerve and its projection to the sphenopalatine
ganglion (see Fig. 1 for anatomical overview). Using 20 animals, two
populations of neuronal responses in the trigeminocervical complex
were identified with different latencies of firing. They were characterized by their response to therapeutic intervention for cluster headache, 100% O2 treatment, as well as through blockade of the
parasympathetic outflow to the cranial vasculature using the specific
autonomic ganglion blocker, a nicotinic cholinergic receptor antagonist, hexamethonium bromide (3 mg/kg), which does not readily
cross the blood–brain barrier and blocks synaptic transmission of the
autonomic ganglia (Goadsby, 1991; Toda et al., 2000; Gottselig and
Messlinger, 2004). The first population of neurons fired with shorter
response latency, between 3 and 20 ms, with an average response of
12.1 1.0 ms. There was no significant effect of 15 min of 100% O2
treatment for 45 min, 1 l/min [F(7,77) = 1.42, P = 0.21, n = 12] or
hexamethonium bromide, 10 mg/kg [F(2,12) = 0.15, P = 0.9, n = 7]
on neurons firing with this short latency (Fig. 2A). The second population of neurons recorded had a much longer latency of firing, between 7 and 40 ms, with an average response of 20.4 0.8 ms.
These responses were significantly inhibited by treatment with
100% O2 by 430% [F(7,133) = 8.2, P = 0.0001, n = 20] and by
hexamethonium bromide by 425% [F(7,105) = 6.8, P = 0.0001,
n = 16] compared with controls (Fig. 2B). Baseline spontaneous
firing for the aforementioned studies was 29.3 2 Hz. In a separate
group of animals (n = 9), vehicle-treated longer latency responses
were unaltered [F(7,56) = 1.3, P = 0.29, n = 9].
S. Akerman et al.
As a comparison, studies were conducted using an established
method of trigeminovascular nociceptive activation, with dural
stimulation and recording in the trigeminocervical complex
(Bergerot et al., 2006). It was found that neither oxygen
[F(2,13) = 1.5, P = 0.27, n = 7], as has been demonstrated previously (Akerman et al., 2009), nor hexamethonium bromide
[F(3,24) = 0.8, P = 0.52, n = 8] significantly inhibited dural-evoked
responses compared with control responses (Fig. 2C).
Indomethacin and naproxen
hydrochloride have differential effects
on superior salivatory nucleus and
dural-evoked firing
Stimulation of the superior salivatory nucleus and activation of the
parasympathetic outflow to the cranial vasculature, measuring the
longer latency response, appears to be the first animal model to differentiate inhibitory effects of oxygen, compared with dural-evoked
stimulation. For this assay to represent a model of TACs, it should be
responsive to other appropriate treatments. Indomethacin, a
cyclo-oxygenase inhibitor, has such a highly characteristic effect on
paroxysmal hemicranias and hemicrania continua, whereas other
cyclo-oxygenase inhibitors, such as naproxen, are not consistently
effective (Goadsby et al., 2008). Using the longer latency responses
to superior salivatory nucleus stimulation, or dural electrical stimulation, and neuronal recording in the trigeminocervical complex, and
using a single dose per animal, it was shown that indomethacin
(5 mg/kg) inhibited superior salivatory nucleus-evoked firing
[F(2.7,30) = 6.3, P = 0.003, n = 12] by 30% after 15 min
(t11 = 3.4, P = 0.006), and dural-evoked responses [F(7,56) = 3.4,
P = 0.004, n = 9] by only 15% (t8 = 3.6, P = 0.007) after 10 min.
Naproxen hydrochloride (30 mg/kg) also significantly inhibited
both superior salivatory nucleus-evoked firing [F(7,49) = 3.3,
P = 0.006, n = 8] by 11% after 30 min (t7 = 3.5, P= 0.005) and
dural-evoked responses [F(7,56)= 2.8, P= 0.015, n= 9] maximally
by 17% after 25 min. When the two responses were compared,
there was no significant difference between the effects of naproxen
on superior salivatory nucleus versus dural-evoked firing
[F(1,15)= 0.62, P= 0.45]. When the superior salivatory nucleusevoked responses in the trigeminocervical complex for indomethacin
and naproxen were compared, there was a significant difference
between the groups [F(1,18)= 5.3, P= 0.033], whereas the response
between the two treatments after the dural-evoked stimulus was not
significantly different [F(1,17)= 1.3, P= 0.27] (Fig. 3A and B).
Triptans and calcitonin gene-related
peptide receptor antagonists
demonstrate differential effectiveness at
inhibiting superior salivatory
nucleus-evoked firing
Classic anti-migraine drugs, serotonin, 5-hydroxytryptamine
(5-HT)1B/1D receptor agonists, ‘triptans’ are extremely effective
in the treatment of migraine (Goadsby et al., 2002), as well as
being effective in animal models of durally evoked
Rodent model of trigeminal autonomic cephalalgias
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Figure 3 Response of 100% oxygen or hexamethonium bromide on dural-evoked and superior salivatory nucleus-evoked neuronal firing
in the trigeminocervical complex (TCC). (A) Stimulation of the superior salivatory nucleus (SuS) evoked two populations of neuronal firing
in the trigeminocervical complex, which has either a shorter or longer latency of firing (as indicated by the arrows). When 100% oxygen
treatment was given for 15 min, or after treatment with the autonomic nicotinic cholinergic receptor blocker, hexamethonium bromide,
there was no effect on the firing of these shorter latency responses (3–20 ms). It is believed that this evoked firing is a consequence of
antidromic stimulation of the trigeminal system within the brainstem (grey neuron). (B) Neurons that fired with a longer latency (9–40 ms)
were significantly inhibited by treatment with either 100% oxygen (1 l/min) or hexamethonium bromide (10 mg/kg). It is thought that this
evoked response is via a different mechanism, a result of direct stimulation of the parasympathetic outflow to the cranial vasculature
(green neuron), which activates trigeminal nerve endings that innervate the cranial vasculature (black neuron), resulting in neuronal
activation in the trigeminocervical complex. (C) Direct activation of trigeminal afferents through stimulation of the dural vasculature (black
neuron) results in A-fibre neuronal responses in the trigeminocervical complex, these responses were not inhibited with treatment of
either 100% oxygen or hexamethonium bromide. *P 5 0.005 compared with baseline control. ES = electrical stimulation.
trigeminovascular nociception. Triptans are also an extremely effective abortive treatment of cluster headache (Goadsby et al.,
2008). Calcitonin gene-related peptide receptor antagonists are a
new and effective class of acute migraine treatments (Olesen
et al., 2004), which, among other effects, inhibit neuronal firing
in the trigeminocervical complex in response to dural stimulation
(Storer et al., 2004); they are untried in TACs. Using a single
treatment per animal, naratriptan (10 mg/kg) significantly inhibited
the longer latency responses of superior salivatory nucleus-evoked
firing in the trigeminocervical complex [F(7,49) = 3.5, P5 0.004,
n = 8], maximally by 34% after 30 min (t7 = 4.1, P = 0.004).
Olcegepant (900 mg/kg), a small molecule calcitonin gene-related
peptide receptor antagonist, also significantly inhibited superior
salivatory nucleus-evoked firing in the trigeminocervical complex
across the time course [F(7,49) = 2.5, P = 0.031, n = 8], but not
specifically at any time point (Fig. 3C).
Measuring cranial autonomic outflow
using lacrimal flow
TACs are characterized by cranial autonomic features, such as lacrimation. Therefore, changes in flow in the lacrimal gland were
monitored as an indication of activation of the parasympathetic
outflow to the cranium, the autonomic reflex component (Fig 5A).
Characteristic changes in flow in the lacrimal gland/duct were
observed in response to stimulation of the superior salivatory
nucleus/VIIth nerve. This effect was reproducible over 30 min
(Akerman et al., 2009). Forty-seven animals were included in
these studies, and hexamethonium was used in the oxygen
study after 60-min washout, to determine that they both affected
the same response, whereas the other treatment groups used a
fresh animal/treatment. When 100% O2 was given for 15 min,
there was a significant inhibition of the flow [F(6,48) = 3.25,
P = 0.009, n = 9], maximally by 26% at 15 min (t8 = 4.2,
P = 0.003), and when the autonomic ganglion blocker hexamethonium bromide (10 mg/kg) was used, there was a significant inhibition of flow [F(6,36) = 4.1, P = 0.003, n = 7], maximally by 36%
at 15 min (t6 = 4.2, P = 0.0005; Fig. 5B). When the cyclooxygenase inhibitors indomethacin and naproxen were used in
response to stimulation of the superior salivatory nucleus, there
was a differential effect. Indomethacin significantly inhibited superior salivatory nucleus-evoked flow changes [F(6,66) = 4.6,
P = 0.006, n = 12], maximally by 20% after 5 min (t11 = 3.8,
P = 0.003). However, naproxen did not affect lacrimal flow responses
after
superior
salivatory
nucleus
stimulation
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S. Akerman et al.
Figure 4 Response of cyclo-oxygenase inhibitors and anti-migraine drugs on dural-evoked and superior salivatory nucleus-evoked
neuronal firing in the trigeminocervical complex. (A) Cyclo-oxygenase inhibitors indomethacin (5 mg/kg) and naproxen (30 mg/kg) are
both able to inhibit the longer latency responses after stimulation of the superior salivatory nucleus (SuS); however, there was a significantly greater inhibition after indomethacin (30%) compared with naproxen (15%). (B) Indomethacin and naproxen were also both able
to inhibit dural-evoked neuronal firing in the trigeminocervical complex, but this inhibition was not significantly different (15% and 17%,
respectively) from each other. (C) Naratriptan, a 5-HT1B/1D receptor agonist, and the calcitonin gene-related peptide receptor antagonist,
olcegepant, both of which are known to inhibit dural-evoked firing in the trigeminocervical complex, were also used in the superior
salivatory nucleus stimulation assay. Both naratriptan (10 mg/kg) and olcegepant (900 mg/kg) were able to inhibit the long latency
response of neuronal firing in the trigeminocervical complex after superior salivatory nucleus stimulation, although naratriptan did inhibit
to a greater extent, 34% compared with 24% inhibition. *P 5 0.05 compared with baseline control.
[F(6,36) = 0.31, P = 0.93, n = 7] (Fig. 5C). There was a similar
differentiation of response to superior salivatory nucleus
stimulation after the anti-migraine drugs naratriptan and olcegepant. Naratriptan significantly inhibited lacrimal flow changes
[F(6,60) = 4.0, P = 0.002, n = 11], maximally by 29% after
15 min (t10 = 4.8, P = 0.001), whereas olcegepant had no effect
on flow response [F(6,42) = 0.69, P = 0.66, n = 8] (Fig. 5D).
Discussion
In this study, we developed and characterized an in vivo model of
the TACs, using electrical stimulation of the superior salivatory
nucleus to activate the trigeminal autonomic reflex arc. The
model addresses the combination of trigeminovascular neuronal
activation and the cranial autonomic symptoms that are distinctive
to TACs, and tests the broader question of an important role for
the brainstem in these disorders. These responses are different
from those generated by direct dural electrical stimulation. This
novel model is also preferentially responsive to treatments for
TACs over more specific migraine treatments and has allowed
us, for the first time, to begin to understand their locus of
action and more about the pathophysiology of TACs.
Superior salivatory nucleus stimulation produced a distinctive
electrophysiological response with two populations of neuronal
firing in the trigeminocervical complex, with differing latencies:
those with shorter latencies of 3–20 ms, and those with more
delayed latencies of 7–40 ms. These two populations were characterized by their response to the acute cluster headache treatment, 100% oxygen, followed—after 60-min washout—with
medical air, and by the nicotinic autonomic ganglion blocker hexamethonium bromide. Cluster headache, the most common TAC,
demonstrates a robust response to high-flow oxygen as an abortive therapy (Fogan, 1985; May et al., 2006; Cohen et al., 2009);
yet, in carefully studied cohorts of patients with paroxysmal
hemicrania (Cittadini et al., 2008) or short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing
(Cohen et al., 2006), not a single patient reported an adequate
response to oxygen. Oxygen thus seems a useful treatment with
which to test the new model. Oxygen specifically inhibited firing
of the longer latency neuronal responses but had no effect on the
shorter latency responses. Similarly, on the same neurons, hexamethonium bromide significantly inhibited the longer latency response but had no effect on the shorter latency response.
Hexamethonium bromide is a specific nicotinic autonomic ganglion
blocker that inhibits synaptic transmission and neuronal responses
at the level of the autonomic ganglia and does not readily cross
the blood–brain barrier (Goadsby, 1991; Toda et al., 2000;
Gottselig and Messlinger, 2004). It is likely that hexamethonium
blocks the greater petrosal nerve projection to the craniovascular/
dural complex, at the level of the sphenopalatine ganglion (green
nerve projection in Figs 1 and 3B). Given that oxygen followed the
same pattern and level of response as hexamethonium, it is probable that the two treatments are acting on the same pathway. The
data imply that the longer latency firing is a consequence of activation of the parasympathetic outflow to the cranial vasculature,
which in turn activates the trigeminal projection to the trigeminal
nucleus caudalis (orthodromic stimulation of the trigeminal
nerve—black nerve fibre in Figs 1 and 3B), causing neuronal activation in the trigeminocervical complex. We believe that these
data demonstrate that activation of this autonomic projection to
the cranial vasculature, via the dura mater, can activate the trigeminovascular system and potentially contribute to the head pain
experienced during TACs. It also helps us to begin to understand
the locus of action of oxygen, demonstrating that it is acting on
this parasympathetic outflow to the cranial vasculature, and subsequent activation of the trigeminovascular system to exert its
therapeutic effects of reducing both autonomic symptoms and
nociceptive traffic to the trigeminocervical complex to reduce
pain symptoms.
Rodent model of trigeminal autonomic cephalalgias
Brain 2012: 135; 3664–3675
| 3671
Figure 5 Responses of treatments to stimulation of the superior salivatory nucleus and measurement of changes to lacrimal flow.
(A) Overview of the parasympathetic outflow to the cranial vasculature via the greater petrosal nerve (green neuron) and the facial (VIIth
cranial) nerve (purple neuron). It is thought that stimulation of the superior salivatory nucleus (SuS) causes activation of the parasympathetic outflow to the face and head, via the greater petrosal nerve and, to a lesser extent, the facial nerve, and is responsible for the flow
changes that occur in the lacrimal duct/gland and the autonomic symptoms seen in TACs. (B) Flow increases within the lacrimal duct/sac
caused by stimulation of the superior salivatory nucleus were significantly inhibited by treatment with both 100% oxygen (1 l/min) and
hexamethonium bromide (10 mg/kg), implying the response was driven solely by activation of the autonomic sphenopalatine ganglion.
(C) Flow increases caused by superior salivatory nucleus stimulation were also inhibited by the cytochrome c oxidase inhibitor, indomethacin, which has characteristic effects in TACs, (5 mg/kg) by 20%, but there was no inhibition by naproxen (30 mg/kg), which is
consistently ineffective as a treatment. (D) The anti-migraine treatment, 5-HT1B/1D receptor agonist, naratriptan (30 mg/kg) was also able
to inhibit lacrimal duct/gland flow by 29%, whereas the calcitonin gene-related peptide receptor antagonist, olcegepant, had no effect on
flow. *P 5 0.05 compared with baseline control.
The second population of neuronal firing in the trigeminocervical
complex was unresponsive to both oxygen treatment and antagonism of the autonomic ganglia with hexamethonium. Physiologically
the firing also differed because it tended to have a much shorter
latency of firing, with an average of 12.1 ms, whereas the longer
latency responses averaged 20.4 ms after the stimulus. It would
seem likely that these cells are not a response to activation of the
parasympathetic outflow to the cranium, but rather involve another
pathway. We know already that as part of the trigeminal autonomic
reflex arc there is likely to be a synaptic connection between the
trigeminocervical complex and the superior salivatory nucleus.
Indeed, it has been demonstrated that dural electrical stimulation,
which causes neuronal activation in the trigeminocervical complex,
also causes neuronal activation in the superior salivatory nucleus
(Knight et al., 2005). It is possible that this shorter latency response
is a consequence of antidromic stimulation of this synaptic connection (grey neuron in Figs 1 and 3A) causing firing in the trigeminocervical complex. This would also explain the lack of effect of the
autonomic ganglion blocker hexamethonium, as this autonomic
pathway is not involved. The shorter latency response is a consequence of a shorter distance and single synapse crossed in this
pathway, compared with the much longer parasympathetic pathway that crosses several synapses.
This may explain why the autonomic ganglion blocker hexamethonium has no effect on this neuronal response, but it does not
explain why oxygen has no effect on the trigeminal firing. it is
likely then that oxygen has no effects on the trigeminovascular
system per se, or specifically, the peripheral trigeminal fibre
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| Brain 2012: 135; 3664–3675
projections to the dural vasculature or the afferent projections to
the trigeminal nucleus caudalis. We were able to demonstrate that
oxygen has no effect on inhibiting responses to dural-evoked trigeminovascular activation, either in the periphery (Akerman et al.,
2009) or at the level of the trigeminocervical complex. Similarly,
hexamethonium was also ineffective at inhibiting the neuronal
firing in the trigeminocervical complex. The data combined indicate that the neurons with the shorter latency of firing followed a
different pathway to those with the longer latency, and it is likely
to be within the brainstem. The findings suggest that it is unlikely
that oxygen conveys any therapeutic benefit directly in the trigeminovascular system either at the level of the periphery or the
nucleus caudalis, but rather acts at the level of the parasympathetic outflow to the cranial vasculature.
Another major feature of TACs is their prominent cranial autonomic symptoms, such as lacrimation, conjunctival injection and
nasal congestion. To observe an autonomic response in our animals after superior salivatory nucleus stimulation, we measured
changes in blood flow around the lacrimal sac/gland as indicative
of lacrimation. Changes in the flow were observed contemporaneously with superior salivatory nucleus stimulation (Akerman et al.,
2009). These changes were significantly inhibited by both oxygen
and the autonomic ganglion blocker to a similar extent. With the
autonomic ganglion blocker also attenuating lacrimal flow in
response to superior salivatory nucleus, it substantiates the
hypothesis that the effects of oxygen are directly on the parasympathetic outflow to the cranial vasculature via the greater petrosal
nerve and the sphenopalatine ganglion (green nerve fibre in Figs 1
and 5A) and that oxygen is probably able to relieve directly the
autonomic symptoms via this mechanism.
The development of this new model and the results lend further
support to the theory that the TACs are CNS neurovascular disorders (Goadsby, 2002). This states that the vascular changes seen
in the cranial circulation are considered to be a response to activation of the trigeminal autonomic reflex arc. Therefore, these
changes serve as a marker of brain activation rather than the
driver of the headaches. This new model demonstrates that stimulation within the brainstem, at the level of the superior salivatory
nucleus, is able to cause both autonomic and trigeminovascular
activation, and may be a key nucleus in driving both pain and
autonomic symptoms during TACs. This is supported by data
showing that direct stimulation of the superior salivatory nucleus
(Nakai et al., 1993), sphenopalatine ganglion (Goadsby, 1990) or
activation of the VIIth cranial (facial) nerve (Goadsby, 1989) also
cause increases in cerebral blood flow, dilating blood vessels and
activating trigeminal nerve endings. This parasympathetic outflow
to the extracranial vessels is thought to use vasoactive intestinal
peptide as its primary transmitter (Goadsby and MacDonald,
1985). The release of vasoactive intestinal peptide after stimulation of the facial nerve (Goadsby and Shelley, 1990) and during
TACs, including cluster headache (Goadsby and Edvinsson, 1994a)
and chronic paroxysmal hemicrania (Goadsby and Edvinsson,
1996), is consistent with this pathway being activated. Indeed,
recent preliminary data indicate that stimulation of the sphenopalatine ganglion in patients implanted with a neurostimulator can
provide significant pain relief in patients with chronic cluster headache (Schoenen et al., 2011). This is another clear indication of
S. Akerman et al.
the importance of the parasympathetic pathway both in the
pathophysiology of these disorders and in their treatment.
Determining how the superior salivatory nucleus is activated
during TACs may help us to understand further the pathophysiology of TACs. Imaging studies have demonstrated activation in
the region of the posterior hypothalamus during spontaneous
attacks of cluster headache (May et al., 1998a), paroxysmal hemicrania (Matharu et al., 2006), short-lasting unilateral neuralgiform
headache attacks with conjunctival injection and tearing (May
et al., 1999; Sprenger et al., 2005) and hemicrania continua
(Matharu et al., 2004). PET studies during capsaicin-induced
pain in the ophthalmic (first) division of the trigeminal nerve demonstrate many of the vascular changes that occur in TACs but did
not demonstrate any hypothalamic activation (May et al., 1998b).
It was concluded that in TACs, the vascular changes are a direct
consequence of activation in the brain, or maybe specifically activation of the trigeminal autonomic reflex. It has been hypothesized further that dysfunction within the hypothalamus may be the
cause of TACs. There is evidence of anatomical connections between the hypothalamus and trigeminal nucleus (Malick and
Burstein, 1998; Malick et al., 2000; Benjamin et al., 2004), and
the hypothalamus and the superior salivatory nucleus and its autonomic projections (Hosoya et al., 1983, 1990), and through the
superior salivatory nucleus, may contribute to sustained activation
of the trigeminovascular system (Burstein and Jakubowski, 2005).
Also, direct hypothalamic stimulation offers relief in patients with
chronic intractable cluster headache (Leone et al., 2001). Perhaps
similar to theories of migraine (Goadsby et al., 2002), TACs may
be considered a consequence of dysfunction of the hypothalamic
descending control of trigeminal nociceptive inputs and autonomic
projections to the cranial vasculature. It is possible that during
cluster headache and other TACs, neurons at the level of the
trigeminocervical complex are activated not only by this direct
loss of descending control of trigeminal nociceptive input, but
also by a loss of control of the autonomic arm of the trigeminal
autonomic reflex arc.
This novel model of TACs demonstrates some of their pathophysiology and is beginning to help unravel the mechanism of
action of therapies such as high-flow oxygen treatment in cluster
headache. It demonstrates the importance of the superior salivatory
nucleus and activation of the parasympathetic outflow to the cranial
vasculature, to stimulate the trigeminovascular system. It is also able
to predict the effectiveness of oxygen as an abortive therapy, over
other migraine models, and is therefore unique in this respect.
However, given that TACs are classified because they are thought
to share a common pathophysiology, for this new assay to be considered more generically a model of TACs, it needs to demonstrate
its ability to predict for TAC treatment over other primary headaches. Therefore, we used other specific TAC treatments to assess
the predictability of this assay over dural electrical stimulation,
which is commonly used to predict anti-migraine efficacy.
Indomethacin, a cyclo-oxygenase inhibitor, has a robust effect
on paroxysmal hemicrania that is considered diagnostic (Cohen
et al., 2007). Other cyclo-oxygenase inhibitors, such as naproxen,
have no comparable efficacy. Using the longer-latency cell firing
as our model of activation of the trigeminal autonomic reflex arc,
after stimulation of the superior salivatory nucleus, it was
Rodent model of trigeminal autonomic cephalalgias
demonstrated that indomethacin significantly inhibited evoked
firing in the trigeminocervical complex by 30%. In the
dural-evoked model, indomethacin also inhibited responses, but
by only 15%, and there was a significant difference between the
responses. Naproxen also inhibited neuronal responses in the trigeminocervical complex after superior salivatory nucleus stimulation
and dural stimulation, but only by 13% and 17%, respectively.
These responses were not significant from each other. However,
when the responses of indomethacin and naproxen after superior
salivatory nucleus stimulation were compared, indomethacin
caused a significantly greater inhibition that came on more quickly.
These data point to the fact that indomethacin shows greater efficacy in the superior salivatory nucleus assay, compared with other
cyclo-oxygenase inhibitors and the dural electrical stimulation
assay, known to have greater predictability for anti-migraine
drugs. While it is likely that indomethacin is acting directly on the
neurons of the trigeminovascular system, it is also acting directly on
the parasympathetic outflow to the cranial vasculature. This point is
supported by the fact that lacrimal duct flow changes were inhibited by indomethacin, but there was no effect of naproxen. It
seems likely that both indomethacin and naproxen are acting in
some way within the trigeminocervical complex to inhibit neuronal
firing, but indomethacin also acts specifically on parasympathetic
projections from the superior salivatory nucleus to the cranium and
face, to further inhibit trigeminal responses and autonomic responses. This would again imply that superior salivatory nucleus
stimulation may more accurately represent the clinical phenotype
of TACs, and therefore help predict clinical therapeutic efficacy.
Classic anti-migraine drugs, 5-HT1B/1D receptor agonists, ‘triptans’, in addition to being extremely effective in the treatment of
migraine are also a highly effective abortive treatment of cluster
headache (Cohen et al., 2007). Calcitonin gene-related peptide receptor antagonists are a new class of compounds clinically effective
in the treatment of migraine (Olesen et al., 2004), but their effects
on TACs are unknown. However, it is known that calcitonin
gene-related peptide, alongside vasoactive intestinal peptide, is
released in patients during cluster headache and paroxysmal hemicrania (Goadsby and Edvinsson, 1994b, 1996); therefore, it might
be a new therapeutic option. Although both naratriptan and olcegepant significantly inhibited superior salivatory nucleus/VIIth
nerve-evoked neuronal firing in the trigeminocervical complex,
only naratriptan was able to inhibit lacrimal flow changes. Recent
evidence indicates that serotonin, 5-HT1D receptors in rat are present on nerve endings at the level of the sphenopalatine ganglion
and project to the lacrimal gland and nasal mucosa (Ivanusic et al.,
2011). This might indicate why triptans can preferentially inhibit
lacrimal flow after superior salivatory nucleus stimulation over calcitonin gene-related peptide receptor antagonists, as they are acting
directly on receptors at the sphenopalatine ganglion to inhibit craniovascular parasympathetic projections.
To validate superior salivatory nucleus stimulation as a novel model
of TACs, it was necessary to use a large number of subjects in this
study. Our aim was to demonstrate several characteristics of TACs,
observing both trigeminal and autonomic symptomology, using a
hypothesized pathophysiology of brainstem activation, and as
such, each observation had to be characterized separately. The
same animals were used to first illustrate a response to oxygen
Brain 2012: 135; 3664–3675
| 3673
during both neuronal responses and lacrimal changes, and followed
after suitable washout, with the autonomic ganglion blocker, to
ensure the same neuronal or lacrimal flow changes were being conducted on the same responses. To validate appropriately the response to specific TAC therapeutics in this assay, in the remaining
pharmacological studies, we used one drug per animal/assay, which
inevitably increases the numbers used. Additionally, when conducting repeated-measures studies, it is necessary to increase the number
of subjects to try to avoid violating the sphericity assumption. Finally,
to provide reliable pharmacological specificity of the model for TACs,
we believed it was necessary to compare with drugs of similar class
used for general primary headache that are more effective in the
dural-evoked assay, as well as suitable negative (vehicle) controls.
This meant up to seven different treatment interventions were necessary in some cases. To have conducted fewer studies with less
pharmacological validation would not have produced the reliability
of both pathophysiology and pharmacology we have demonstrated.
The overall data indicate that animal models of durally evoked
trigeminovascular nociception may have limited use at predicting
efficacy for treatments for TACs, with 100% oxygen treatment
being the clearest example of this failure, whereas the effects of
indomethacin are far more beneficial when studying the trigeminal
autonomic reflex. On the other hand, using stimulation of superior
salivatory nucleus/VIIth nerve not only do we see activation of the
parasympathetic outflow to the cranial vasculature, as demonstrated
by the facial blood flow changes, but this activation we believe is able
to activate trigeminal afferents and neuronal firing within the trigeminocervical complex, as described by the trigeminal autonomic
reflex arc. Indeed, two populations of neurons were characterized,
not only those believed to come from the parasympathetic outflow,
but also those that appear to be a result of antidromic stimulation of
the trigeminal nucleus within the brainstem itself. It seems that
studying the parasympathetic outflow from the superior salivatory
nucleus, and perhaps inputs that modulate the superior salivatory
nucleus, for example, the hypothalamus, may be integral in furthering our understanding of TACs and predicting treatment efficacy. It is
noteworthy that therapeutics specifically targeting the different
TACs appear to be equally effective at inhibiting both trigeminocervical complex and autonomic responses; yet, these treatments can be
considered exclusive to specific TACs. It is worth bearing in mind that
these disorders have been classified together because of their similar
symptomology and, to some extent, pathophysiology, with hypothalamic, trigeminovascular and cranial autonomic activation
demonstrated. We believe that the fact that oxygen, indomethacin
and the triptans are likely to act on the parasympathetic outflow
illustrates the importance of this locus in these disorders and illustrates the flexibility of the model in helping understand TACs more
generally. TACs are among the most disabling of the primary headache disorders, and thus, development of a model for the disorders is
of broad potential for translational studies to benefit these patients.
Acknowledgements
The authors thank Anna Andreou, Annabelle Charbit and James
Storer of the Headache Group at UCSF for their support and technical assistance during these experiments.
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| Brain 2012: 135; 3664–3675
Funding
The work has been supported by the Sandler Family Foundation.
OS received a fellowship in Neurology from Merck Sharp and
Dohme, Germany.
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