European Journal of Pharmacology 744 (2014) 115123

Contents lists available at ScienceDirect

European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Involvement of GPR40, a long-chain free fatty acid receptor,

in the production of central post-stroke pain after global
cerebral ischemia
Shinichi Harada a, Yuka Haruna a, Fuka Aizawa a, Wataru Matsuura a, Kazuo Nakamoto a,
Takuya Yamashita b, Fumiyo Kasuya b, Shogo Tokuyama a,n
a
b

Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe 650-8586, Japan
Biochemical Toxicology Laboratory, School of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe 650-8586, Japan

art ic l e i nf o

a b s t r a c t

Article history:
Received 7 May 2014
Received in revised form
22 September 2014
Accepted 23 September 2014
Available online 30 September 2014

Central post-stroke pain (CPSP), one of the complications of cerebral ischemia and neuropathic pain
syndrome, is associated with specic somatosensory abnormalities. Although CPSP is a serious problem,
detailed underlying mechanisms and standard treatments for CPSP are not well established. In this
study, we assessed the role of GPR40, a long-chain fatty acid receptor, showing anti-nociceptive effects,
in CPSP. We also examined the role of astrocytes in CPSP due to their effects in mediating the release of
polyunsaturated fatty acids, which act as potential GPR40 ligands. The aim of this study was to
determine the interactions between CPSP and astrocyte/GPR40 signaling. Male ddY mice were subjected
to 30 min of bilateral carotid artery occlusion (BCAO). The development of hind paw mechanical
hyperalgesia was measured after BCAO using the von Frey test. Neuronal damage was estimated by
histological analysis on day 3 after BCAO. The thresholds for hind paw mechanical hyperalgesia were
signicantly decreased on days 128 after BCAO when compared with those of pre-BCAO assessments.
BCAO-induced mechanical hyperalgesia was signicantly decreased by intracerebroventricular injection
of docosahexaenoic acid or GW9508, a GPR40 agonist; furthermore, these effects were reversed by
GW1100, a GPR40 antagonist. The expression levels of glial brillary acidic protein, an astrocytic marker,
and some free fatty acids were signicantly decreased 5 h after BCAO, although no effects of BCAO were
noted on hypothalamic GPR40 protein expression. Our data show that BCAO-induced mechanical
hyperalgesia is possible to be regulated by astrocyte activation and stimulation of GPR40 signaling.
& 2014 Elsevier B.V. All rights reserved.

Chemical compounds studied in this article:

Docosahexaenoic acid (PubChem CID:
6440152)
Keywords:
Central post-stroke pain (CPSP)
Global ischemia (BCAO)
GPR40
Astrocyte
Docosahexaenoic acid (DHA)

1. Introduction
Cerebral stroke is the primary cause of disability and one of the
most common causes of death (Moskowitz et al., 2010). In stroke
survivors, several factors may adversely inuence quality of life,
such as motor impairment, depression, and disability. These
conditions impair activities of daily living and negatively inuence
the rehabilitation process (Klamroth-Marganska et al., 2014;
Lambiase et al., 2014; Langhorne et al., 2011).
Patients with stroke may additionally suffer from several types
of pain, including articular pain, musculoskeletal pain, painful
spasticity, headache, and neuropathic central post-stroke pain

Abbreviations: BCAO, bilateral carotid artery occlusion; CNS, central nervous

system; CPSP, central post-stroke pain; DHA, docosahexaenoic acid; DMSO,
dimethyl sulfoxide; EPA, eicosapentaenoic acid; GPR40, free fatty acid receptor 1
(FFA1); PUFA, polyunsaturated fatty acid; PWT, paw withdrawal threshold
n
Corresponding author. Tel.: 81 78 974 1551; fax: 81 78 974 4780.
E-mail address: stoku@pharm.kobegakuin.ac.jp (S. Tokuyama).
http://dx.doi.org/10.1016/j.ejphar.2014.09.036
0014-2999/& 2014 Elsevier B.V. All rights reserved.

(CPSP). CPSP was rst described by Dejerine and Roussy in 1906

as a spontaneous pain experienced after a thalamic stroke (Dejerine
and Roussy, 1906), but is now also associated with extrathalamic
lesions because the symptoms and severity of CPSP in thalamic
versus extrathalamic stroke are identical (Misra et al., 2008). That is,
CPSP can be dened as a central neuropathic pain condition
occurring after stroke in which the affected area is the body parts
corresponding to a cerebrovascular lesion of the somatosensory
system (Klit et al., 2009, 2011; Kumar and Soni, 2009). However,
there is no standard treatment for CPSP at the present time.
The brain is more highly enriched in polyunsaturated fatty acids
(PUFAs) such as docosahexaenoic acid (DHA, an n-3 PUFA) than
most other tissues (Contreras et al., 2000). PUFAs are involved in
many physiological functions, including brain and retinal development, aging, memory, synaptic membrane function, photoreceptor
function, and neuroprotection (Bazan, 2003, 2006; Eady et al., 2014;
Hong et al., 2014). Recently, PUFAs such as DHA have been reported
to interact with some pain conditions (Figueroa et al., 2013; TorresGuzman et al., 2014). We recently reported that DHA and/or

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GW9508, a GPR40 agonist, have suppressive effects on pain

behavior elicited by formalin, acetic acid and a complete Freund's
adjuvant. These effects appear to be mediated through the release
of -endorphin, an endogenous opioid peptide, which is stimulated
by GPR40 signaling in the supraspinal region (Nakamoto et al., 2010,
2011, 2012, 2013). GPR40 is a member of the large family of seventransmembrane receptors referred to as G-protein-coupled receptors, and is known to be activated by long-chain fatty acids such as
DHA and eicosapentaenoic acid (EPA) (Hirasawa et al., 2008). It has
been reported that DHA has a relatively higher afnity for GPR40
than other fatty acids (Itoh et al., 2003). DHA is also known to
improve behavioral decits, and reduce infarct volumes and edema
after experimental focal cerebral ischemia, presumably by activating
signaling cascades that increase cell survival, tipping the overall
cellular fate towards survival and long-term repair (Belayev et al.,
2009; Eady et al., 2013).
We hypothesized that it may be possible to regulate CPSP via
effects on PUFA/GPR40 signaling (Eady et al., 2014; Hong et al.,
2014). The aim of the present study was to determine the
involvement of GPR40 in the development of CPSP in an animal
model of global cerebral ischemia.

Unique Medical, Osaka, Japan). The bilateral common carotid arteries

were occluded for 30 min with standard surgical aneurysm clips
(Mizuho Ikakogyo Co., Ltd., Tokyo, Japan). Sham-operated mice were
subjected to the same procedures without the occlusion.

2.5. Analysis of pyknotic cell death

Pyknotic cell death was evaluated by hematoxylineosin (HE)
staining as previously reported (Tamiya et al., 2013). Briey, mice
were decapitated on days 1, 3, 7, 14, and 28 after BCAO, and their
brains were dissected immediately. The brain slices were incubated in ice-cold phosphate-buffered 4% paraformaldehyde
(pH 7.4) overnight at 4 1C. Using a sliding microtome, parafnxed brain tissue was sectioned (thickness, 6 m), deparafnized
with xylene and ethanol, and then stained with HE dyes (Carrazzi's
hematoxylin solution, Sakura Finetek Japan Co., Ltd., Tokyo, Japan)
for histological study. After HE staining, we counted the shrunken
neurons with pyknotic nuclei to assess neuropathy.

2.6. Learning and memory tests

2. Material and methods
2.1. Animals
The present study was conducted in accordance with the Guiding
Principles for the Care and Use of Laboratory Animals, as adopted by
the Japanese Pharmacological Society. In addition, all experiments
were approved by the Ethical Committee for Animals of Kobe
Gakuin University (approval number: A13-25). All experiments were
performed on male ddY mice (5 weeks old, 2530 g) obtained from
SLC (Shizuoka, Japan). The animals were housed at 2324 1C with a
12 h lightdark cycle (lights on 8:00 a.m. to 8:00 p.m.). Food and
water were available ad libitum.
2.2. Drugs
The selective GPR40 agonist, GW9508 (0.1, 1, and 10 mg/mouse;
Cayman Chemical Co., Ann Arbor, MI, USA), DHA (25, 50, and 100 mg/
mouse; Ikeda Tohka Industries Co., Ltd., Fukuyama, Japan), and the
GPR40 antagonist GW1100 (10 and 50 mg/mouse; Cayman Chemical
Co.) were initially dissolved in 1% DMSO (Sigma-Aldrich Japan, K.K.,
Ishikari, Japan); the solutions were diluted with saline before each
study. The doses of GW9508, DHA, and GW1100 were chosen based
upon our previous reports (Nakamoto et al., 2012, 2013).
2.3. Administration schedule for drugs
Mice received i.c.v. administration of GW9508 (0.1 or 1 mg/mouse),
DHA (25, 50, 100 mg/mouse) or vehicle on day 3 after BCAO (Fig. 3).
GW9508 (10 mg/mouse) was i.c.v. administered in some animals 5 h
after BCAO (Fig. 5). All i.c.v. administrations were performed as
previously described (Harada et al., 2011). GPR40 antagonist pretreatment with GW1100 was administered 10 min before GW9508 or DHA
administration.
2.4. Animal models of global cerebral ischemia
Transient global cerebral ischemia was induced by occlusion of
the bilateral carotid arteries in mice as described previously (Harada
et al., 2013b; Tamiya et al., 2013). Briey, mice were anesthetized
with pentobarbital (60 mg/kg). Rectal temperature was maintained
at 3770.5 1C with the use of a heating blanket (FH-100, Unique
Medical, Osaka, Japan) and a small animal heat controller (ATC-101B,

To assess learning and memory, a one-trial step-throughtype passive avoidance learning test was used as described
previously (Harada et al., 2009). The apparatus (Ohara Co., Ltd., Tokyo,
Japan) consisted of illuminated and dark compartments (each
4 cm  13 cm  10 cm) adjoining each other through a small
gate (3 cm in diameter) and with a grid oor composed of
2.5 mm stainless steel rods set 7 mm apart. On the training
trial (on days 2, 6, 13 or 27 after BCAO), mice were placed in the
illuminated compartment facing away from the dark compartment. When the mice entered the dark compartment, an
electric shock (50 V, 3 s duration) was delivered. Mice were
then conned to the dark compartment for 5 s, after which
they were carried back to the home cage. In the test trial, 24 h
after the training trial (on days 3, 7, 14, or 28 after BCAO), the
mice were again placed in the illuminated compartment and
the latency times for the mice to enter the dark compartment
(maximum 600 s) were measured.

2.7. Assessment of mechanical hyperalgesia

Assessment of the PWT in response to mechanical stimulation
was performed using von Frey laments (North Coast Medical,
Inc., CA, USA) as described in our previous reports (Takami et al.,
2011; Tamiya et al., 2013). Mice were placed on a 5 mm  5 mm
wire mesh grid oor and covered with an opaque cup to avoid
visual stimulation. They were allowed to remain in this condition
over a 3 h adaptation period prior to testing. During the test, mice
were stimulated on the plantar surface of the paw. Each stimulus
was repeated ve times (at intervals of 10 s). The PWT was
determined to be the lowest force that evoked a withdrawal
response to at least three of the ve stimulations. As using above
method, we assessed study of Figs. 2A and B, and 5A and B.
The assessment of withdrawal response times for the 10-timesstimulation von Frey lament test (0.4 g) was conducted as
described previously (Nakamoto et al., 2013). Mechanical hyperalgesia was dened as an increase in the number of withdrawal
responses to stimulation. As using above method, we assessed
study of Figs. 2C, 3, and 5C and D. To test the effects of GW9508,
DHA and GW1100 on mechanical hyperalgesia on day 3 after
BCAO, the von Frey test was performed at 10, 20, 30, and 60 min
after GW9508, DHA or GW1100 i.c.v. injection (Fig. 3).

S. Harada et al. / European Journal of Pharmacology 744 (2014) 115123

2.8. Western blotting analysis

Western blotting was done as previously described with some
modications (Harada et al., 2013a; Nakamoto et al., 2013). Briey,
the hypothalamus was homogenized in homogenization buffer
and diluted with an equal volume of 2X sodium dodecyl sulfate
(SDS) sample buffer (0.5 M TrisHCl [pH 6.8], 10% SDS, 12% mercaptoethanol, 20% glycerol, and 1% bromophenol blue). Each
sample was heated for 3 min at 97 1C and protein samples (20 g)
were separated via electrophoresis on 15% SDS-polyacrylamide gel
and then transferred onto nitrocellulose membranes (BioRad,
Hercules, CA, USA) at 15 V for 50 min. Membranes were blocked
(60 min at room temperature) in Tris-buffered saline (TBS) (pH
7.6) with 0.1% Tween 20, and either 5% bovine serum albumin
(BSA) (Sigma-Aldrich) for GPR40 or 5% skim milk for GFAP and
GAPDH (loading control) (Wako Pure Chemical Industries, Inc.,
Osaka, Japan). Membranes were incubated with the primary
antibodies (in their corresponding blocking solution, overnight at
4 1C). GPR40 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA,
USA) was then assessed using rabbit polyclonal primary antibodies, and glial brillary acidic protein (GFAP 1:1000; Millipore
Corp., Billerica, MA, USA) was detected using mouse monoclonal
primary antibodies. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was detected using primary antibodies (1:20,000; Chemicon International Inc., Temecula, CA, USA). Blots were then
incubated (for 1 h at room temperature) in HRP-conjugated
secondary antibodies:anti-rabbit IgG (1:1000, KPL, Guildford, UK)
for GPR40, and anti-mouse IgG (1:10,000, KPL) for GFAP and
GAPDH. Immunoreactive bands were visualized with enhanced
chemiluminescence western immunoblotting substrate (Pierce;
Thermo Scientic, Rockford, IL, USA) followed by a Light-Capture
instrument (AE-6981; ATTO, Tokyo, Japan). The signal intensity of

117

immunoreactive bands was analyzed using Cs-Analyzer software

(Ver. 3.0) (ATTO) and then normalized to the respective value
for GAPDH.
2.9. LCESI-MS/MS analysis of free fatty acids (FFAs) in the mouse
hypothalamus
The FFAs were extracted from the hypothalamus. The weight of
tissues was determined and 10 mL of methanol was added to per 0.1 g
of the wet tissues. C19:0 (Tokyo Chemical Industory, Tokyo, Japan) was
added as an internal standard in methanol. The tissues were homogenized by Potter-Elvehjem PTFE pestle and glass tube. The mixture
was incubated for 30 min at room temperature. Furthermore, the
mixture was centrifuged at 15,000g for 15 min. Finally, the supernatant
was removed and ltrated in sample vial for LCMS/MS analysis.
HPLC separation was performed with Agilent 1290 Innity LC
(Agirent technologies, California, U.S.A.) having a CAPCELL PAK
UG120 column: 2.0 mm I.D.  150 mm (Shiseido, Tokyo, Japan).
The mobile phases were A, 10 mM ammonium formate (pH 3.5)
and B, acetonitrile. The eluting gradient was as follows: the
column was equilibrated with 13% A, 13% A for 5 min, 13% A to
5% A in 5 min, 5% A for 5 min, 5% A to 13% A in 5 min, 13% A for
5 min. The ow rate was 0.2 mL/min. Quantitation was carried out
on a QTRAP 4500 (AB SCIEX, Massachusetts, U.S.A.). A mass
spectrometer was operated in the negative-ion mode. The free
fatty acids were quantied by selective multireaction monitoring
(MRM) with a negative ionization mode. The ion spray voltage was
 4500 V, the source temperature was 300 1C, the declustering
potential ranged from 70 to  105 V and collision energy ranged
from  10 to  22 eV for the fragment ions. The peak of each free
fatty acids was monitored by the product ion obtained from
[MH]  ion (i.e., m/z 255-m/z 255 for C16:0, m/z 279-m/z 279

Fig. 1. The development of neuronal damage and memory impairment after global cerebral ischemia. (AH) HE-stained sections in the hippocampal CA3 region showing
shrunken neurons with pyknotic nuclei (black arrow) from one representative animal in each group. (AD) sham group, (EH) BCAO group. (A, E) Day 3; (B, F) day 7; (C, G)
day 14; and (D, H) day 28 after sham or BCAO operation. Scale bar 50 m. (I) Quantitative analysis of shrunken neurons with pyknotic nuclei. nnp o 0.01, Student's t-test.
Results are presented as the mean 7S.E.M., n 4. (J) Memory impairment after global ischemia. The boxes show the values of the 25th and 75th percentiles, the lines across
the boxes represent the medians, and the whiskers extend to the highest and lowest values. nnp o 0.01, WilcoxonMannWhitney U test, n4.

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S. Harada et al. / European Journal of Pharmacology 744 (2014) 115123

for C18:2, m/z 281- m/z 281 for C18:1, m/z 283-m/z 283 for
C18:0, m/z 298-m/z 298 for C19:0, m/z 303-m/z 303 for C20:4,
m/z 327-m/z 327 for C22:6.).
2.10. Statistical analysis
Pyknotic cell death data, withdrawal response times, protein
expression levels and FFAs levels were analyzed using unpaired
Student's t-tests and/or two-way analysis of variance followed by
Tukey's test. Data from the one-trial step-through-type passive
avoidance learning test and PWT assessments were analyzed using
the SteelDwass test, a post-hoc nonparametric multiple comparisons test. Data are presented as medians (25th75th percentile).

3. Results
3.1. The development of neuronal damage and memory impairment
after global cerebral ischemia
In the bilateral carotid artery occlusion (BCAO) group, pyknotic
cell death (arrows) was observed in the hippocampal CA3 region on
days 3, 7, 14 and 28 after BCAO (Fig. 1AH). The number of cells
undergoing pyknotic cell death was signicantly increased in the
BCAO group compared with that of the sham group at each time
point after BCAO, reaching a maximum on day 7 after BCAO (Fig. 1I).
There was a signicant decrease in response latencies in the
passive avoidance test, on days 3 and 7 after BCAO. On day 14,
response latencies showed only a trend toward reduced values,
and by day 28, latencies of the BCAO group had recovered to the
values exhibited by the sham-treated group (Fig. 1J).
3.2. Development of mechanical hyperalgesia after global cerebral
ischemia
In the sham group, there were no changes in the paw withdrawal threshold (PWT) in response to mechanical stimulation at
any time point following the sham operation (Fig. 2A). In contrast,
the PWT in the 30-min BCAO group was signicantly decreased on
days 128 after BCAO as compared with baseline values (Fig. 2B).
The response times for 10-times-stimulation using the von Frey
lament test (0.4 g) were signicantly increased for both paws on
day 3 after BCAO as compared to those of the sham group (Fig. 2C).
3.3. Effects of GW9508 and DHA on the development of mechanical
hyperalgesia on day 3 after global cerebral ischemia
On day 3 after BCAO, incremented response times for 10-timesstimulation using the von Frey lament test (0.4 g) were signicantly and dose-dependently suppressed by treatment with either
GW9508 (a GPR40 agonist) or DHA (Fig. 3A and B). These effects,
which peaked at 10 min and continued for at least 20 min, were
furthermore inhibited by GW1100 (a GPR40 antagonist) (Fig. 3A
and B). When 1% dimethyl sulfoxide (DMSO) was given as a
control (vehicle) treatment or GW1100 was used alone, there were
no effects on BCAO-induced mechanical hyperalgesia (Fig. 3C).
3.4. Changes in GPR40 and glial brillary acidic protein expression
levels after global cerebral ischemia
Compared with the sham group, the expression of GPR40 protein
in hypothalamus was not signicantly changed after BCAO (Fig. 4A).
On the other hand, GFAP protein expression was signicantly
decreased 5 h after BCAO, but not between 12 h and day 7 after
BCAO, as compared with that of the sham group (Fig. 4B).

3.5. Changes in hypothalamic FFAs levels at 5 h after global cerebral

ischemia
At 5 h after BCAO, some FFAs (palmitate, stearate, oleinic acid,
linoleic acid, arachidonic acid and DHA) were signicantly decreased
as compared with sham group (Fig. 5).
3.6. Effect of GW9508 administered 5 h after global cerebral
ischemia on the development of mechanical hyperalgesia
On day 1 after BCAO, the signicantly decreased average hind
paw PWT in the BCAO group was eliminated by GW9508 treatment
5 h after BCAO; however, this effect of GW9508 treatment was not
apparent on day 3 after BCAO (Fig. 6A and B). Similarly, BCAOinduced incremented response times for 10-times-stimulation using
the von Frey lament test (0.4 g) were signicantly suppressed on
day 1 but not day 3 by GW9508 treatment (Fig. 6C and D). Both
of these effects of GW9508 on day 1 were inhibited by GW1100
(Fig. 6A and C).
3.7. Effect of GW9508 administered at 5 h after global cerebral
ischemia on the development of neuronal damage
The increases in pyknotic cell death (arrows) observed in the
hippocampal CA3 region on days 1 and 3 after BCAO were not
affected by GW9508 treatment administered 5 h after BCAO (Fig. 7).

4. Discussion
The occurrence of CPSP is reportedly 111% after stroke
(Andersen et al., 1995; Bowsher, 2001; Hansen et al., 2012; Klit
et al., 2011; Kumar and Soni, 2009). In addition, the importance of
CPSP, which typically develops 36 months after a stroke
(Nasreddine and Saver, 1997), has been underestimated for a
number of years; however, it is now receiving considerable
interest. Although there is an increased need for the development
of improved therapeutics, detailed mechanisms underlying the
generation of CPSP are almost unknown. In previous reports,
Wasserman and Koeberle established CPSP-like models using
thalamic hemorrhage in the rat (Wasserman and Koeberle,
2009). Hanada et al. have also developed and characterized a
CPSP-like model using a hemorrhagic stroke lesion with collagenase in the ventral posterolateral nucleus of the thalamus (Hanada
et al., 2014). However, there has been little study of CPSP using
models of brain infarction rather than hemorrhagic stroke.
Recently, we have successfully established a CPSP-like model by
experimentally inducing focal or global cerebral ischemia (Takami
et al., 2011; Tamiya et al., 2013). In the BCAO model of global
ischemia used in the present study, pain thresholds were signicantly decreased in response to mechanical and thermal stimulation to the hind limbs compared with those of sham-operated
animals; moreover, this decrease in pain thresholds was associated
with ischemic neuronal damage. Although pyknotic cell death
remained signicantly increased, memory disturbance recovered
to the level of the sham groups by day 28 after BCAO. Liu et al.
have reported that enhanced neurogenesis in the hippocampus
may be a compensatory adaptive response to ischemia-associated
injury and can promote functional recovery after ischemic hippocampal injury (Liu et al., 1998). The mechanism by which memory
decits recovered after BCAO is still unknown, but it may involve
the development of neurogenesis in hippocampal regions after
BCAO. These results suggest that the development of CPSP directly
involves the response to neuronal damage in the brain. We
previously showed that functional alterations in some brain
regions may play a role in the development of hypersensitization

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119

Fig. 2. The development of mechanical hyperalgesia after global cerebral ischemia. The change in pain threshold was measured using the von Frey lament test to the left
hind paw. PWT: paw withdrawal threshold. Pre indicates measurement before BCAO. Data are shown as boxes plotting the values between the 25th and 75th percentiles,
the lines across the boxes represent the medians, and the whiskers extend to the highest and lowest values. Each stimulus was repeated ve times (at intervals of 10 s). The
PWT was determined to be the lowest force that evoked a withdrawal response to at least three of the ve stimuli. (A) sham group, n 8. (B) BCAO group, n 9. nnp o 0.01,
WilcoxonMannWhitney U test. (C) Withdrawal responses following both hind paw stimulation were measured 10 times. The von Frey lament was applied to the middle
of the planter surface of the hind paw with a weight of 0.4 g. nnp o 0.01, Student's t-test. Results are presented as the mean 7S.E.M., day 1: n 5, and day 3: n 8.

of primary afferent neurons (especially A and C bers) and

mechanical hyperalgesia in this model (Tamiya et al., 2013).
In the present study, we focused on the role of GPR40, a longchain fatty acid receptor, in the generation and development of
CPSP (Briscoe et al., 2003). Recently, it was reported that PUFA such
as DHA are involved in the regulation and development of some
pain behaviors such as inammatory and neuropathic pain
(Goldberg and Katz, 2007; Nakamoto et al., 2013). We determined
that these effects are in part mediated by -endorphin release from
cells in the hypothalamus induced by GPR40 signaling (Nakamoto
et al., 2012). In the present study, BCAO-induced mechanical
hyperalgesia was clearly suppressed by intracerebroventricular
(i.c.v.) injection of DHA and a GPR40 agonist, GW9508. These results
suggest that the activation of GPR40 may suppress the BCAOinduced pain signal and that GPR40 signaling may be a useful
target of treatment strategies intended to reduce pain caused by
cerebral ischemia. However, GPR40 expression levels in hypothalamus were not affected by cerebral ischemia, suggesting that the
GPR40-mediated suppressive effect on CPSP may involve changes in
a GPR40 ligand such as DHA either upstream or downstream of

GPR40 signaling. Alternatively, GPR40 sensitivity may be affected by

cerebral ischemia in the absence of changes in protein levels.
PUFAs such as DHA and arachidonic acid (AA) are important for
central nervous system (CNS) function during development and in
various pathological states. Maintenance of the correct proportions
of n-3 and n-6 PUFAs in phospholipids associated with cellular
membranes has been shown to be crucial for normal functioning of
the CNS (Farooqui and Horrocks, 2001; Jump, 2002). The content of
DHA and AA of phospholipids in the brain was signicantly reduced
in patients with various neuronal disorders and neurodegenerative
diseases such as Alzheimer's disease (Markesbery, 1997; Pettegrew
et al., 1995). In addition, DHA was shown to have neuroprotective
properties during ischemia and brain trauma (Begum et al., 2014;
Zhang et al., 2014). In previous reports, astrocytes, a type of
neuroglia, have been found to have important roles in relation to
the biosynthesis of PUFAs, and to release PUFAs to neuronal
populations (Moore, 1993; Williard et al., 2001). In the present
study, we found although that BCAO-induced mechanical hyperalgesia did not occur at 5 h after BCAO, astrocyte activation was
signicantly decreased. In addition, we have previously reported

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S. Harada et al. / European Journal of Pharmacology 744 (2014) 115123

Fig. 3. The effect of GW9508 or DHA on the development of mechanical hyperalgesia on day 3 after global cerebral ischemia. The development of mechanical hyperalgesia
was analyzed using the von Frey test. The lament used was 0.4 g, and during each test trial, the left hind paw was stimulated 10 times for 6 s each. Pre indicates
measurement before BCAO. On day 3 after BCAO, either the GPR 40 agonist, GW9508 (A) or DHA (B) was i.c.v. administered 10 min before rst trial of the von Frey lament
test. Tests were performed at 10, 20, 30 and 60 min after GW9508 administration. In some animals, the GPR40 antagonist, GW1100 was administered 10 min before GW9508
treatment. Results are presented as the mean7 S.E.M. nnp o 0.01: compared with vehicle (veh-) sham group, ##p o 0.01: compared with veh-BCAO group, p o 0.01,

p o 0.05: compared with GW9508 1 g-BCAO group, Tukey's multiple comparison test. (A) veh-sham: n 8, GW9508 1 g-sham: n 6, veh-BCAO: n 8, GW9508 0.1 gBCAO: n 4, GW9508 1 g-BCAO: n 7, GW1100 10 g/GW9508 1 g-BCAO: n 7. (B) veh-sham: n8, DHA 100 g-sham: n 6, veh-BCAO: n 6, DHA 25 g-BCAO: n 3, DHA
50 g-BCAO: n 4, DHA 100 g-BCAO: n7, GW1100 10 g/DHA 100 g-BCAO: n 5, GW1100 50 g/DHA 100 g-BCAO: n 5. (C) The effect of GW1100 on the development of
mechanical hyperalgesia on day 3 after BCAO. Results are presented as the mean 7S.E.M. nnpo 0.01: compared with veh-sham group, Tukey's multiple comparison test, n 3.

Fig. 4. Changes in GPR40 and GFAP expression levels after global cerebral ischemia. Representative western blots of GPR40, GFAP and GAPDH levels. Relative levels were
analyzed by determining the ratio of (A) GPR40/GAPDH and (B) GFAP/GAPDH. Results are presented as the mean7 S.E.M. npo 0.05, Student's t-test. A: GPR40, sham: 5 h;
n 6, 12 h; n 6, 18 h; n 5, day 1; n 4, day 3; n 4, day 7; n 4, BCAO: 5 h; n 6, 12 h; n 6, 18 h; n 6, day 1; n5, day 3; n 5, day 7; n 5, B: GFAP: sham: 5 h; n 11,
12 h; n 6, 18 h; n 6, day 1; n9, day 3; n 6, day 7; n 4, BCAO: 5 h; n 10, 12 h; n 6, 18 h; n 6, day 1; n 10, day 3; n6, day 7; n4, GFAP: glial brillary acidic protein.

that double immunouorescence techniques revealed that GPR40

was co-localized on neurons, but not astrocytes (Nakamoto et al.,
2013). That is, activation of astrocytes may be involved indirectly

against GPR40 signaling. Ouyang et al. previously reported that

astrocytes displayed functional changes without any loss of viability
at early reperfusion times (5 h) after transient forebrain ischemia

S. Harada et al. / European Journal of Pharmacology 744 (2014) 115123

(Ouyang et al., 2007). By contrast, it has also been reported that

astrocytic processes were fragmented and mitochondria were
inhibited after 15 min of exposure to acidic conditions in hippocampal slice cultures (Hulse et al., 2001). Astrocytic demise was
found to precede delayed neuronal death after focal ischemia (Liu
et al., 1999) and was observed early after traumatic brain injury
(Zhao et al., 2003). Although these studies suggest that astrocytic
changes can precede neuronal death in some situations, there is
relatively little information currently available about differences in
astrocytic responses to ischemia. However, based on our results we
can hypothesize that a reduction of astrocytes in the early phase

Fig. 5. FFAs prole in the hypothalamus tissue at 5 h after global cerebral ischemia.
FFAs were analyzed with UHPLCMS/MS using MRM; FFA prole in the hypothalamus of sham or BCAO mice. nnp o 0.01, Student's t-test. Results are presented as
the mean7 S.E.M., n 6. (C16:0) palmitate, (C18:0) stearate, (C18:1) oleinic acid,
(C18:2) linoleic acid, (C20:4) arachidonic acid, and (C22:6) DHA.

121

after ischemic stress may in part contribute to decreased GPR40

signaling activation by free PUFA. This hypothesis is supported by
our previous reports that free PUFA, including DHA, were suppressed by the inhibition of astrocyte activation (Nakamoto et al.,
2013). In addition, some hypothalamic FFAs (palmitate, stearate,
oleinic acid, linoleic acid, arachidonic acid and DHA) were clearly
decreased by global cerebral ischemia in present study. Palmitate,
stearate, oleinic acid, linoleic acid, arachidonic acid and DHA were
abundantly expressed in the hypothalamus as found in our previous
study (Nakamoto et al., 2013). PUFAs are released from and taken
up by astrocytes via transporters such as fatty acid binding protein,
fatty acid translocase/CD36, and fatty acid transport proteins (Glatz
et al., 2010). In particular, CD36, a class B scavenger receptor, has
been implicated in pathological conditions associated with inammation including stroke and Alzheimer's disease (El Khoury et al.,
2003; Febbraio et al., 2000; Kim et al., 2008). This receptor is
expressed in many different cell types such as microglia, astrocytes,
microvascular endothelial cells, monocytes/macrophages, and platelets (Bao et al., 2012). It has been reported that the activation of
astrocytes may be regulated by the presence and function of these
transporters (Glatz et al., 2010). Although the detailed mechanisms
of these phenomena are still unclear, the reduced release of PUFA
by astrocytes in the hypothalamus in the early phase of cerebral
ischemia may be involved in the suppression of GPR40 signaling
observed in the present study. This hypothesis was supported by
our present results showing that treatment with GW9508 delayed
the development of BCAO-induced CPSP but did not affect the
development of ischemic neuronal damage. These results suggest

Fig. 6. Effect of GW9508 administered at 5 h after global cerebral ischemia on the development of mechanical hyperalgesia. At 5 h after BCAO, the GPR 40 agonist GW9508
was i.c.v. administered (10 g/mouse). (A, B) The change in pain threshold was measured using the von Frey lament test at the left hind paw on day 1 (A) or day 3 (B) after
BCAO. PWT: paw withdrawal threshold. Pre indicates measurement before BCAO. Data are shown as boxes plotting the values between the 25th and 75th percentiles, the
lines across the boxes represent the medians, and the whiskers extend to the highest and lowest values. Each stimulus was repeated ve times (interval of 10 s). The PWT
was determined to be the lowest force that evoked a withdrawal response to at least three of the ve stimuli. np o 0.05, WilcoxonMannWhitney U test. (C, D) Withdrawal
responses following left hind paw stimulation were measured 10 times on day 1 (C) or day 3 (D) after BCAO. The von Frey lament was applied to the middle of the planter
surface of the hind paw with a weight of 0.4 g. nnp o0.01, ##p o 0.01, p o 0.01, Tukey's multiple comparison test. (A, C) sham: n6, veh- or GW9508-BCAO: n 5, GW1100/
GW9508-BCAO: n 4, (B, D) n 8.

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S. Harada et al. / European Journal of Pharmacology 744 (2014) 115123

Fig. 7. Effect of GW9508 administered 5 h after global cerebral ischemia on the development of neuronal damage. (AF) HE-stained sections in the hippocampal CA3 region
showing shrunken neurons with pyknotic nuclei (black arrow) from one representative animal in each group together with HE-stained sections of sham or BCAO groups.
(AC) day 1, (DF) day 3 after sham or BCAO operation. (A, D) sham, (B, E) veh-BCAO, (C, F) GW9508-BCAO. (G) Quantitative analysis of shrunken neurons with pyknotic
nuclei. nnp o 0.01, Tukey's multiple comparison test. Results are presented as the mean7 S.E.M., n 4. Scale bar 50 m.

that the dysfunctional astrocyte and GPR40 signaling mediated by

cerebral ischemia may trigger the aggravation of CPSP.
In conclusion, our present study suggests that the BCAO model
mouse may be a useful animal model of CPSP and raises the
possibility that the regulation of BCAO-induced CPSP may involve
alterations of astrocyte/GPR40 signaling. Furthermore, we can
speculate that GPR40 activation is a potential therapeutic target
in efforts to control the painful symptoms of CPSP.

Acknowledgments
This study was supported by Grants-in-Aid and by special
coordination funds from Grants-in-Aid for Scientic Research (C)
(25462458) from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan.

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