Neuropharmacology and analgesia

Involvement of GPR40, a long-chain free fatty acid receptor,in the production of central post-stroke pain after globalcerebral 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 Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe 650-8586, Japanb Biochemical Toxicology Laboratory, School of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe 650-8586, Japan

a r t i c l e i n f o

Article history:Received 7 May 2014Received in revised form22 September 2014Accepted 23 September 2014Available online 30 September 2014

Chemical compounds studied in this article:Docosahexaenoic acid (PubChem CID:6440152)

Keywords:Central post-stroke pain (CPSP)Global ischemia (BCAO)GPR40AstrocyteDocosahexaenoic acid (DHA)

a b s t r a c t

Central post-stroke pain (CPSP), one of the complications of cerebral ischemia and neuropathic painsyndrome, is associated with specific somatosensory abnormalities. Although CPSP is a serious problem,detailed underlying mechanisms and standard treatments for CPSP are not well established. In thisstudy, 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 ofpolyunsaturated fatty acids, which act as potential GPR40 ligands. The aim of this study was todetermine the interactions between CPSP and astrocyte/GPR40 signaling. Male ddY mice were subjectedto 30 min of bilateral carotid artery occlusion (BCAO). The development of hind paw mechanicalhyperalgesia was measured after BCAO using the von Frey test. Neuronal damage was estimated byhistological analysis on day 3 after BCAO. The thresholds for hind paw mechanical hyperalgesia weresignificantly decreased on days 1–28 after BCAO when compared with those of pre-BCAO assessments.BCAO-induced mechanical hyperalgesia was significantly decreased by intracerebroventricular injectionof docosahexaenoic acid or GW9508, a GPR40 agonist; furthermore, these effects were reversed byGW1100, a GPR40 antagonist. The expression levels of glial fibrillary acidic protein, an astrocytic marker,and some free fatty acids were significantly decreased 5 h after BCAO, although no effects of BCAO werenoted on hypothalamic GPR40 protein expression. Our data show that BCAO-induced mechanicalhyperalgesia is possible to be regulated by astrocyte activation and stimulation of GPR40 signaling.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

Cerebral stroke is the primary cause of disability and one of themost common causes of death (Moskowitz et al., 2010). In strokesurvivors, several factors may adversely influence quality of life,such as motor impairment, depression, and disability. Theseconditions impair activities of daily living and negatively influencethe rehabilitation process (Klamroth-Marganska et al., 2014;Lambiase et al., 2014; Langhorne et al., 2011).

Patients with stroke may additionally suffer from several typesof pain, including articular pain, musculoskeletal pain, painfulspasticity, headache, and neuropathic central post-stroke pain

(CPSP). CPSP was first described by Dejerine and Roussy in 1906as a spontaneous pain experienced after a thalamic stroke (Dejerineand Roussy, 1906), but is now also associated with extrathalamiclesions because the symptoms and severity of CPSP in thalamicversus extrathalamic stroke are identical (Misra et al., 2008). That is,CPSP can be defined as a central neuropathic pain conditionoccurring after stroke in which the affected area is the body partscorresponding to a cerebrovascular lesion of the somatosensorysystem (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) thanmost other tissues (Contreras et al., 2000). PUFAs are involved inmany physiological functions, including brain and retinal develop-ment, aging, memory, synaptic membrane function, photoreceptorfunction, and neuroprotection (Bazan, 2003, 2006; Eady et al., 2014;Hong et al., 2014). Recently, PUFAs such as DHA have been reportedto interact with some pain conditions (Figueroa et al., 2013; Torres-Guzman et al., 2014). We recently reported that DHA and/or

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European Journal of Pharmacology

http://dx.doi.org/10.1016/j.ejphar.2014.09.0360014-2999/& 2014 Elsevier B.V. All rights reserved.

Abbreviations: BCAO, bilateral carotid artery occlusion; CNS, central nervoussystem; 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.: þ81 78 974 1551; fax: þ81 78 974 4780.E-mail address: stoku@pharm.kobegakuin.ac.jp (S. Tokuyama).

European Journal of Pharmacology 744 (2014) 115–123

GW9508, a GPR40 agonist, have suppressive effects on painbehavior elicited by formalin, acetic acid and a complete Freund'sadjuvant. These effects appear to be mediated through the releaseof β-endorphin, an endogenous opioid peptide, which is stimulatedby GPR40 signaling in the supraspinal region (Nakamoto et al., 2010,2011, 2012, 2013). GPR40 is a member of the large family of seven-transmembrane receptors referred to as G-protein-coupled recep-tors, and is known to be activated by long-chain fatty acids such asDHA and eicosapentaenoic acid (EPA) (Hirasawa et al., 2008). It hasbeen reported that DHA has a relatively higher affinity for GPR40than other fatty acids (Itoh et al., 2003). DHA is also known toimprove behavioral deficits, and reduce infarct volumes and edemaafter experimental focal cerebral ischemia, presumably by activatingsignaling cascades that increase cell survival, tipping the overallcellular 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 viaeffects on PUFA/GPR40 signaling (Eady et al., 2014; Hong et al.,2014). The aim of the present study was to determine theinvolvement of GPR40 in the development of CPSP in an animalmodel of global cerebral ischemia.

2. Material and methods

2.1. Animals

The present study was conducted in accordance with the GuidingPrinciples for the Care and Use of Laboratory Animals, as adopted bythe Japanese Pharmacological Society. In addition, all experimentswere approved by the Ethical Committee for Animals of KobeGakuin University (approval number: A13-25). All experiments wereperformed on male ddY mice (5 weeks old, 25–30 g) obtained fromSLC (Shizuoka, Japan). The animals were housed at 23–24 1C with a12 h light–dark cycle (lights on 8:00 a.m. to 8:00 p.m.). Food andwater 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 theGPR40 antagonist GW1100 (10 and 50 mg/mouse; Cayman ChemicalCo.) were initially dissolved in 1% DMSO (Sigma-Aldrich Japan, K.K.,Ishikari, Japan); the solutions were diluted with saline before eachstudy. The doses of GW9508, DHA, and GW1100 were chosen basedupon 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 hafter BCAO (Fig. 5). All i.c.v. administrations were performed aspreviously described (Harada et al., 2011). GPR40 antagonist pretreat-ment with GW1100 was administered 10min before GW9508 or DHAadministration.

2.4. Animal models of global cerebral ischemia

Transient global cerebral ischemia was induced by occlusion ofthe bilateral carotid arteries in mice as described previously (Haradaet al., 2013b; Tamiya et al., 2013). Briefly, mice were anesthetizedwith pentobarbital (60 mg/kg). Rectal temperature was maintainedat 3770.5 1C with the use of a heating blanket (FH-100, UniqueMedical, Osaka, Japan) and a small animal heat controller (ATC-101B,

Unique Medical, Osaka, Japan). The bilateral common carotid arterieswere occluded for 30 min with standard surgical aneurysm clips(Mizuho Ikakogyo Co., Ltd., Tokyo, Japan). Sham-operated mice weresubjected to the same procedures without the occlusion.

2.5. Analysis of pyknotic cell death

Pyknotic cell death was evaluated by hematoxylin–eosin (HE)staining as previously reported (Tamiya et al., 2013). Briefly, micewere decapitated on days 1, 3, 7, 14, and 28 after BCAO, and theirbrains were dissected immediately. The brain slices were incu-bated in ice-cold phosphate-buffered 4% paraformaldehyde(pH 7.4) overnight at 4 1C. Using a sliding microtome, paraffin-fixed brain tissue was sectioned (thickness, 6 μm), deparaffinizedwith xylene and ethanol, and then stained with HE dyes (Carrazzi'shematoxylin solution, Sakura Finetek Japan Co., Ltd., Tokyo, Japan)for histological study. After HE staining, we counted the shrunkenneurons with pyknotic nuclei to assess neuropathy.

2.6. Learning and memory tests

To assess learning and memory, a one-trial step-through-type passive avoidance learning test was used as describedpreviou-sly (Harada et al., 2009). The apparatus (Ohara Co., Ltd., Tokyo,Japan) consisted of illuminated and dark compartments (each4 cm�13 cm�10 cm) adjoining each other through a smallgate (3 cm in diameter) and with a grid floor composed of2.5 mm stainless steel rods set 7 mm apart. On the trainingtrial (on days 2, 6, 13 or 27 after BCAO), mice were placed in theilluminated compartment facing away from the dark compart-ment. When the mice entered the dark compartment, anelectric shock (50 V, 3 s duration) was delivered. Mice werethen confined to the dark compartment for 5 s, after whichthey were carried back to the home cage. In the test trial, 24 hafter the training trial (on days 3, 7, 14, or 28 after BCAO), themice were again placed in the illuminated compartment andthe 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 stimulationwas performed using von Frey filaments (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 mmwire mesh grid floor and covered with an opaque cup to avoidvisual stimulation. They were allowed to remain in this conditionover a 3 h adaptation period prior to testing. During the test, micewere stimulated on the plantar surface of the paw. Each stimuluswas repeated five times (at intervals of 10 s). The PWT wasdetermined to be the lowest force that evoked a withdrawalresponse to at least three of the five stimulations. As using abovemethod, we assessed study of Figs. 2A and B, and 5A and B.

The assessment of withdrawal response times for the 10-times-stimulation von Frey filament test (0.4 g) was conducted asdescribed previously (Nakamoto et al., 2013). Mechanical hyper-algesia was defined as an increase in the number of withdrawalresponses to stimulation. As using above method, we assessedstudy of Figs. 2C, 3, and 5C and D. To test the effects of GW9508,DHA and GW1100 on mechanical hyperalgesia on day 3 afterBCAO, the von Frey test was performed at 10, 20, 30, and 60 minafter GW9508, DHA or GW1100 i.c.v. injection (Fig. 3).

S. Harada et al. / European Journal of Pharmacology 744 (2014) 115–123116

2.8. Western blotting analysis

Western blotting was done as previously described with somemodifications (Harada et al., 2013a; Nakamoto et al., 2013). Briefly,the hypothalamus was homogenized in homogenization bufferand diluted with an equal volume of 2X sodium dodecyl sulfate(SDS) sample buffer (0.5 M Tris–HCl [pH 6.8], 10% SDS, 12% β-mercaptoethanol, 20% glycerol, and 1% bromophenol blue). Eachsample was heated for 3 min at 97 1C and protein samples (20 μg)were separated via electrophoresis on 15% SDS-polyacrylamide geland 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) (pH7.6) with 0.1% Tween 20, and either 5% bovine serum albumin(BSA) (Sigma-Aldrich) for GPR40 or 5% skim milk for GFAP andGAPDH (loading control) (Wako Pure Chemical Industries, Inc.,Osaka, Japan). Membranes were incubated with the primaryantibodies (in their corresponding blocking solution, overnight at4 1C). GPR40 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA,USA) was then assessed using rabbit polyclonal primary antibo-dies, and glial fibrillary acidic protein (GFAP 1:1000; MilliporeCorp., Billerica, MA, USA) was detected using mouse monoclonalprimary antibodies. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) was detected using primary antibodies (1:20,000; Che-micon International Inc., Temecula, CA, USA). Blots were thenincubated (for 1 h at room temperature) in HRP-conjugatedsecondary antibodies:anti-rabbit IgG (1:1000, KPL, Guildford, UK)for GPR40, and anti-mouse IgG (1:10,000, KPL) for GFAP andGAPDH. Immunoreactive bands were visualized with enhancedchemiluminescence western immunoblotting substrate (Pierce;Thermo Scientific, Rockford, IL, USA) followed by a Light-Captureinstrument (AE-6981; ATTO, Tokyo, Japan). The signal intensity of

immunoreactive bands was analyzed using Cs-Analyzer software(Ver. 3.0) (ATTO) and then normalized to the respective valuefor GAPDH.

2.9. LC–ESI-MS/MS analysis of free fatty acids (FFAs) in the mousehypothalamus

The FFAs were extracted from the hypothalamus. The weight oftissues was determined and 10mL of methanol was added to per 0.1 gof the wet tissues. C19:0 (Tokyo Chemical Industory, Tokyo, Japan) wasadded as an internal standard in methanol. The tissues were homo-genized by Potter-Elvehjem PTFE pestle and glass tube. The mixturewas incubated for 30min at room temperature. Furthermore, themixture was centrifuged at 15,000g for 15 min. Finally, the supernatantwas removed and filtrated in sample vial for LC–MS/MS analysis.

HPLC separation was performed with Agilent 1290 Infinity LC(Agirent technologies, California, U.S.A.) having a CAPCELL PAKUG120 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: thecolumn was equilibrated with 13% A, 13% A for 5 min, 13% A to5% A in 5 min, 5% A for 5 min, 5% A to 13% A in 5 min, 13% A for5 min. The flow rate was 0.2 mL/min. Quantitation was carried outon a QTRAP 4500 (AB SCIEX, Massachusetts, U.S.A.). A massspectrometer was operated in the negative-ion mode. The freefatty acids were quantified by selective multireaction monitoring(MRM) with a negative ionization mode. The ion spray voltage was�4500 V, the source temperature was 300 1C, the declusteringpotential ranged from �70 to �105 V and collision energy rangedfrom �10 to �22 eV for the fragment ions. The peak of each freefatty acids was monitored by the product ion obtained from[M–H]� 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. (A–H) HE-stained sections in the hippocampal CA3 region showingshrunken neurons with pyknotic nuclei (black arrow) from one representative animal in each group. (A–D) sham group, (E–H) 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 bar¼50 μm. (I) Quantitative analysis of shrunken neurons with pyknotic nuclei. nnpo0.01, Student's t-test.Results are presented as the mean7S.E.M., n¼4. (J) Memory impairment after global ischemia. The boxes show the values of the 25th and 75th percentiles, the lines acrossthe boxes represent the medians, and the whiskers extend to the highest and lowest values. nnpo0.01, Wilcoxon–Mann–Whitney U test, n¼4.

S. Harada et al. / European Journal of Pharmacology 744 (2014) 115–123 117

for C18:2, m/z 281- m/z 281 for C18:1, m/z 283-m/z 283 forC18:0, 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, proteinexpression levels and FFAs levels were analyzed using unpairedStudent's t-tests and/or two-way analysis of variance followed byTukey's test. Data from the one-trial step-through-type passiveavoidance learning test and PWT assessments were analyzed usingthe Steel–Dwass test, a post-hoc nonparametric multiple compar-isons test. Data are presented as medians (25th–75th percentile).

3. Results

3.1. The development of neuronal damage and memory impairmentafter global cerebral ischemia

In the bilateral carotid artery occlusion (BCAO) group, pyknoticcell death (arrows) was observed in the hippocampal CA3 region ondays 3, 7, 14 and 28 after BCAO (Fig. 1A–H). The number of cellsundergoing pyknotic cell death was significantly increased in theBCAO group compared with that of the sham group at each timepoint after BCAO, reaching a maximum on day 7 after BCAO (Fig. 1I).

There was a significant decrease in response latencies in thepassive 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 thevalues exhibited by the sham-treated group (Fig. 1J).

3.2. Development of mechanical hyperalgesia after global cerebralischemia

In the sham group, there were no changes in the paw with-drawal threshold (PWT) in response to mechanical stimulation atany time point following the sham operation (Fig. 2A). In contrast,the PWT in the 30-min BCAO group was significantly decreased ondays 1–28 after BCAO as compared with baseline values (Fig. 2B).The response times for 10-times-stimulation using the von Freyfilament test (0.4 g) were significantly increased for both paws onday 3 after BCAO as compared to those of the sham group (Fig. 2C).

3.3. Effects of GW9508 and DHA on the development of mechanicalhyperalgesia on day 3 after global cerebral ischemia

On day 3 after BCAO, incremented response times for 10-times-stimulation using the von Frey filament test (0.4 g) were signifi-cantly and dose-dependently suppressed by treatment with eitherGW9508 (a GPR40 agonist) or DHA (Fig. 3A and B). These effects,which peaked at 10 min and continued for at least 20 min, werefurthermore inhibited by GW1100 (a GPR40 antagonist) (Fig. 3Aand B). When 1% dimethyl sulfoxide (DMSO) was given as acontrol (vehicle) treatment or GW1100 was used alone, there wereno effects on BCAO-induced mechanical hyperalgesia (Fig. 3C).

3.4. Changes in GPR40 and glial fibrillary acidic protein expressionlevels after global cerebral ischemia

Compared with the sham group, the expression of GPR40 proteinin hypothalamus was not significantly changed after BCAO (Fig. 4A).On the other hand, GFAP protein expression was significantlydecreased 5 h after BCAO, but not between 12 h and day 7 afterBCAO, as compared with that of the sham group (Fig. 4B).

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

At 5 h after BCAO, some FFAs (palmitate, stearate, oleinic acid,linoleic acid, arachidonic acid and DHA) were significantly decreasedas compared with sham group (Fig. 5).

3.6. Effect of GW9508 administered 5 h after global cerebralischemia on the development of mechanical hyperalgesia

On day 1 after BCAO, the significantly decreased average hindpaw PWT in the BCAO group was eliminated by GW9508 treatment5 h after BCAO; however, this effect of GW9508 treatment was notapparent on day 3 after BCAO (Fig. 6A and B). Similarly, BCAO-induced incremented response times for 10-times-stimulation usingthe von Frey filament test (0.4 g) were significantly suppressed onday 1 but not day 3 by GW9508 treatment (Fig. 6C and D). Bothof 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 cerebralischemia on the development of neuronal damage

The increases in pyknotic cell death (arrows) observed in thehippocampal CA3 region on days 1 and 3 after BCAO were notaffected by GW9508 treatment administered 5 h after BCAO (Fig. 7).

4. Discussion

The occurrence of CPSP is reportedly 1–11% after stroke(Andersen et al., 1995; Bowsher, 2001; Hansen et al., 2012; Klitet al., 2011; Kumar and Soni, 2009). In addition, the importance ofCPSP, which typically develops 3–6 months after a stroke(Nasreddine and Saver, 1997), has been underestimated for anumber of years; however, it is now receiving considerableinterest. Although there is an increased need for the developmentof improved therapeutics, detailed mechanisms underlying thegeneration of CPSP are almost unknown. In previous reports,Wasserman and Koeberle established CPSP-like models usingthalamic hemorrhage in the rat (Wasserman and Koeberle,2009). Hanada et al. have also developed and characterized aCPSP-like model using a hemorrhagic stroke lesion with collage-nase in the ventral posterolateral nucleus of the thalamus (Hanadaet al., 2014). However, there has been little study of CPSP usingmodels of brain infarction rather than hemorrhagic stroke.Recently, we have successfully established a CPSP-like model byexperimentally inducing focal or global cerebral ischemia (Takamiet al., 2011; Tamiya et al., 2013). In the BCAO model of globalischemia used in the present study, pain thresholds were signifi-cantly decreased in response to mechanical and thermal stimula-tion to the hind limbs compared with those of sham-operatedanimals; moreover, this decrease in pain thresholds was associatedwith ischemic neuronal damage. Although pyknotic cell deathremained significantly increased, memory disturbance recoveredto the level of the sham groups by day 28 after BCAO. Liu et al.have reported that enhanced neurogenesis in the hippocampusmay be a compensatory adaptive response to ischemia-associatedinjury and can promote functional recovery after ischemic hippo-campal injury (Liu et al., 1998). The mechanism by which memorydeficits recovered after BCAO is still unknown, but it may involvethe development of neurogenesis in hippocampal regions afterBCAO. These results suggest that the development of CPSP directlyinvolves the response to neuronal damage in the brain. Wepreviously showed that functional alterations in some brainregions may play a role in the development of hypersensitization

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of primary afferent neurons (especially Aβ and C fibers) andmechanical hyperalgesia in this model (Tamiya et al., 2013).

In the present study, we focused on the role of GPR40, a long-chain fatty acid receptor, in the generation and development ofCPSP (Briscoe et al., 2003). Recently, it was reported that PUFA suchas DHA are involved in the regulation and development of somepain behaviors such as inflammatory and neuropathic pain(Goldberg and Katz, 2007; Nakamoto et al., 2013). We determinedthat these effects are in part mediated by β-endorphin release fromcells in the hypothalamus induced by GPR40 signaling (Nakamotoet al., 2012). In the present study, BCAO-induced mechanicalhyperalgesia was clearly suppressed by intracerebroventricular(i.c.v.) injection of DHA and a GPR40 agonist, GW9508. These resultssuggest that the activation of GPR40 may suppress the BCAO-induced pain signal and that GPR40 signaling may be a usefultarget of treatment strategies intended to reduce pain caused bycerebral ischemia. However, GPR40 expression levels in hypothala-mus were not affected by cerebral ischemia, suggesting that theGPR40-mediated suppressive effect on CPSP may involve changes ina GPR40 ligand such as DHA either upstream or downstream of

GPR40 signaling. Alternatively, GPR40 sensitivity may be affected bycerebral ischemia in the absence of changes in protein levels.

PUFAs such as DHA and arachidonic acid (AA) are important forcentral nervous system (CNS) function during development and invarious pathological states. Maintenance of the correct proportionsof n-3 and n-6 PUFAs in phospholipids associated with cellularmembranes has been shown to be crucial for normal functioning ofthe CNS (Farooqui and Horrocks, 2001; Jump, 2002). The content ofDHA and AA of phospholipids in the brain was significantly reducedin patients with various neuronal disorders and neurodegenerativediseases such as Alzheimer's disease (Markesbery, 1997; Pettegrewet al., 1995). In addition, DHA was shown to have neuroprotectiveproperties during ischemia and brain trauma (Begum et al., 2014;Zhang et al., 2014). In previous reports, astrocytes, a type ofneuroglia, have been found to have important roles in relation tothe biosynthesis of PUFAs, and to release PUFAs to neuronalpopulations (Moore, 1993; Williard et al., 2001). In the presentstudy, we found although that BCAO-induced mechanical hyper-algesia did not occur at 5 h after BCAO, astrocyte activation wassignificantly decreased. In addition, we have previously reported

Fig. 2. The development of mechanical hyperalgesia after global cerebral ischemia. The change in pain threshold was measured using the von Frey filament test to the lefthind 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 five times (at intervals of 10 s). ThePWT was determined to be the lowest force that evoked a withdrawal response to at least three of the five stimuli. (A) sham group, n¼8. (B) BCAO group, n¼9. nnpo0.01,Wilcoxon–Mann–Whitney U test. (C) Withdrawal responses following both hind paw stimulation were measured 10 times. The von Frey filament was applied to the middleof the planter surface of the hind paw with a weight of 0.4 g. nnpo0.01, Student's t-test. Results are presented as the mean7S.E.M., day 1: n¼5, and day 3: n¼8.

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that double immunofluorescence techniques revealed that GPR40was 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 thatastrocytes displayed functional changes without any loss of viabilityat early reperfusion times (5 h) after transient forebrain ischemia

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 hyperalgesiawas analyzed using the von Frey test. The filament used was 0.4 g, and during each test trial, the left hind paw was stimulated 10 times for 6 s each. ‘Pre’ indicatesmeasurement 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 first trial of the von Frey filamenttest. 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 GW9508treatment. Results are presented as the mean7S.E.M. nnpo0.01: compared with vehicle (veh-) sham group, ##po0.01: compared with veh-BCAO group, ††po0.01,†po0.05: compared with GW9508 1 μg-BCAO group, Tukey's multiple comparison test. (A) veh-sham: n¼8, GW9508 1 μg-sham: n¼6, veh-BCAO: n¼8, GW9508 0.1 μg-BCAO: n¼4, GW9508 1 μg-BCAO: n¼7, GW1100 10 μg/GW9508 1 μg-BCAO: n¼7. (B) veh-sham: n¼8, DHA 100 μg-sham: n¼6, veh-BCAO: n¼6, DHA 25 μg-BCAO: n¼3, DHA50 μg-BCAO: n¼4, DHA 100 μg-BCAO: n¼7, GW1100 10 μg/DHA 100 μg-BCAO: n¼5, GW1100 50 μg/DHA 100 μg-BCAO: n¼5. (C) The effect of GW1100 on the development ofmechanical hyperalgesia on day 3 after BCAO. Results are presented as the mean7S.E.M. nnpo0.01: compared with veh-sham group, Tukey's multiple comparison test, n¼3.

Fig. 4. Changes in GPR40 and GFAP expression levels after global cerebral ischemia. Representative western blots of GPR40, GFAP and GAPDH levels. Relative levels wereanalyzed by determining the ratio of (A) GPR40/GAPDH and (B) GFAP/GAPDH. Results are presented as the mean7S.E.M. npo0.05, Student's t-test. A: GPR40, sham: 5 h;n¼6, 12 h; n¼6, 18 h; n¼5, day 1; n¼4, day 3; n¼4, day 7; n¼4, BCAO: 5 h; n¼6, 12 h; n¼6, 18 h; n¼6, day 1; n¼5, day 3; n¼5, day 7; n¼5, B: GFAP: sham: 5 h; n¼11,12 h; n¼6, 18 h; n¼6, day 1; n¼9, day 3; n¼6, day 7; n¼4, BCAO: 5 h; n¼10, 12 h; n¼6, 18 h; n¼6, day 1; n¼10, day 3; n¼6, day 7; n¼4, GFAP: glial fibrillary acidic protein.

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(Ouyang et al., 2007). By contrast, it has also been reported thatastrocytic processes were fragmented and mitochondria wereinhibited after 15 min of exposure to acidic conditions in hippo-campal slice cultures (Hulse et al., 2001). Astrocytic demise wasfound to precede delayed neuronal death after focal ischemia (Liuet al., 1999) and was observed early after traumatic brain injury(Zhao et al., 2003). Although these studies suggest that astrocyticchanges can precede neuronal death in some situations, there isrelatively little information currently available about differences inastrocytic responses to ischemia. However, based on our results wecan hypothesize that a reduction of astrocytes in the early phase

after ischemic stress may in part contribute to decreased GPR40signaling activation by free PUFA. This hypothesis is supported byour previous reports that free PUFA, including DHA, were sup-pressed 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 clearlydecreased by global cerebral ischemia in present study. Palmitate,stearate, oleinic acid, linoleic acid, arachidonic acid and DHA wereabundantly expressed in the hypothalamus as found in our previousstudy (Nakamoto et al., 2013). PUFAs are released from and takenup by astrocytes via transporters such as fatty acid binding protein,fatty acid translocase/CD36, and fatty acid transport proteins (Glatzet al., 2010). In particular, CD36, a class B scavenger receptor, hasbeen implicated in pathological conditions associated with inflam-mation including stroke and Alzheimer's disease (El Khoury et al.,2003; Febbraio et al., 2000; Kim et al., 2008). This receptor isexpressed in many different cell types such as microglia, astrocytes,microvascular endothelial cells, monocytes/macrophages, and pla-telets (Bao et al., 2012). It has been reported that the activation ofastrocytes may be regulated by the presence and function of thesetransporters (Glatz et al., 2010). Although the detailed mechanismsof these phenomena are still unclear, the reduced release of PUFAby astrocytes in the hypothalamus in the early phase of cerebralischemia may be involved in the suppression of GPR40 signalingobserved in the present study. This hypothesis was supported byour present results showing that treatment with GW9508 delayedthe development of BCAO-induced CPSP but did not affect thedevelopment of ischemic neuronal damage. These results suggest

Fig. 5. FFAs profile in the hypothalamus tissue at 5 h after global cerebral ischemia.FFAs were analyzed with UHPLC–MS/MS using MRM; FFA profile in the hypotha-lamus of sham or BCAO mice. nnpo0.01, Student's t-test. Results are presented asthe mean7S.E.M., n¼6. (C16:0) palmitate, (C18:0) stearate, (C18:1) oleinic acid,(C18:2) linoleic acid, (C20:4) arachidonic acid, and (C22:6) DHA.

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 GW9508was i.c.v. administered (10 μg/mouse). (A, B) The change in pain threshold was measured using the von Frey filament test at the left hind paw on day 1 (A) or day 3 (B) afterBCAO. PWT: paw withdrawal threshold. ‘Pre’ indicates measurement before BCAO. Data are shown as boxes plotting the values between the 25th and 75th percentiles, thelines across the boxes represent the medians, and the whiskers extend to the highest and lowest values. Each stimulus was repeated five times (interval of 10 s). The PWTwas determined to be the lowest force that evoked a withdrawal response to at least three of the five stimuli. npo0.05, Wilcoxon–Mann–Whitney U test. (C, D) Withdrawalresponses following left hind paw stimulation were measured 10 times on day 1 (C) or day 3 (D) after BCAO. The von Frey filament was applied to the middle of the plantersurface of the hind paw with a weight of 0.4 g. nnpo0.01, ##po0.01, ††po0.01, Tukey's multiple comparison test. (A, C) sham: n¼6, veh- or GW9508-BCAO: n¼5, GW1100/GW9508-BCAO: n¼4, (B, D) n¼8.

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that the dysfunctional astrocyte and GPR40 signaling mediated bycerebral ischemia may trigger the aggravation of CPSP.

In conclusion, our present study suggests that the BCAO modelmouse may be a useful animal model of CPSP and raises thepossibility that the regulation of BCAO-induced CPSP may involvealterations of astrocyte/GPR40 signaling. Furthermore, we canspeculate that GPR40 activation is a potential therapeutic targetin efforts to control the painful symptoms of CPSP.

Acknowledgments

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

References

Andersen, G., Vestergaard, K., Ingeman-Nielsen, M., Jensen, T.S., 1995. Incidence ofcentral post-stroke pain. Pain 61, 187–193.

Bao, Y., Qin, L., Kim, E., Bhosle, S., Guo, H., Febbraio, M., Haskew-Layton, R.E., Ratan, R.,Cho, S., 2012. CD36 is involved in astrocyte activation and astroglial scarformation. J. Cereb. Blood Flow Metab. 32, 1567–1577.

Bazan, N.G., 2003. Synaptic lipid signaling: significance of polyunsaturated fattyacids and platelet-activating factor. J. Lipid Res. 44, 2221–2233.

Bazan, N.G., 2006. Cell survival matters: docosahexaenoic acid signaling, neuro-protection and photoreceptors. Trends Neurosci. 29, 263–271.

Begum, G., Yan, H.Q., Li, L., Singh, A., Dixon, C.E., Sun, D., 2014. Docosahexaenoic acidreduces ER stress and abnormal protein accumulation and improves neuronalfunction following traumatic brain injury. J. Neurosci. 34, 3743–3755.

Belayev, L., Khoutorova, L., Atkins, K.D., Bazan, N.G., 2009. Robust docosahexaenoicacid-mediated neuroprotection in a rat model of transient, focal cerebralischemia. Stroke 40, 3121–3126.

Bowsher, D., 2001. Stroke and central poststroke pain in an elderly population.J. Pain 2, 258–261.

Briscoe, C.P., Tadayyon, M., Andrews, J.L., Benson, W.G., Chambers, J.K., Eilert, M.M.,Ellis, C., Elshourbagy, N.A., Goetz, A.S., Minnick, D.T., Murdock, P.R., Sauls Jr., H.R.,Shabon, U., Spinage, L.D., Strum, J.C., Szekeres, P.G., Tan, K.B., Way, J.M., Ignar, D.M.,Wilson, S., Muir, A.I., 2003. The orphan G protein-coupled receptor GPR40 isactivated by medium and long chain fatty acids. J. Biol. Chem. 278, 11303–11311.

Contreras, M.A., Greiner, R.S., Chang, M.C., Myers, C.S., Salem Jr., N., Rapoport, S.I.,2000. Nutritional deprivation of alpha-linolenic acid decreases but does notabolish turnover and availability of unacylated docosahexaenoic acid anddocosahexaenoyl-CoA in rat brain. J. Neurochem. 75, 2392–2400.

Dejerine, J., Roussy, G., 1906. Le syndrome thalamique. Rev. Neurol. 14, 521–532.Eady, T.N., Khoutorova, L., Anzola, D.V., Hong, S.H., Obenaus, A., Mohd-Yusof, A., Bazan, N.

G., Belayev, L., 2013. Acute treatment with docosahexaenoic acid complexed toalbumin reduces injury after a permanent focal cerebral ischemia in rats. PLoS One8, e77237.

Eady, T.N., Khoutorova, L., Obenaus, A., Mohd-Yusof, A., Bazan, N.G., Belayev, L.,2014. Docosahexaenoic acid complexed to albumin provides neuroprotectionafter experimental stroke in aged rats. Neurobiol. Dis. 62, 1–7.

El Khoury, J.B., Moore, K.J., Means, T.K., Leung, J., Terada, K., Toft, M., Freeman, M.W.,Luster, A.D., 2003. CD36 mediates the innate host response to beta-amyloid.J. Exp. Med. 197, 1657–1666.

Farooqui, A.A., Horrocks, L.A., 2001. Plasmalogens, phospholipase A2, and docosa-hexaenoic acid turnover in brain tissue. J. Mol. Neurosci. 16, 263–272 (discus-sion 279–284).

Febbraio, M., Podrez, E.A., Smith, J.D., Hajjar, D.P., Hazen, S.L., Hoff, H.F., Sharma, K.,Silverstein, R.L., 2000. Targeted disruption of the class B scavenger receptorCD36 protects against atherosclerotic lesion development in mice. J. Clin.Investig. 105, 1049–1056.

Fig. 7. Effect of GW9508 administered 5 h after global cerebral ischemia on the development of neuronal damage. (A–F) HE-stained sections in the hippocampal CA3 regionshowing shrunken neurons with pyknotic nuclei (black arrow) from one representative animal in each group together with HE-stained sections of sham or BCAO groups.(A–C) day 1, (D–F) 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 pyknoticnuclei. nnpo0.01, Tukey's multiple comparison test. Results are presented as the mean7S.E.M., n¼4. Scale bar¼50 μm.

S. Harada et al. / European Journal of Pharmacology 744 (2014) 115–123122

Figueroa, J.D., Cordero, K., Serrano-Illan, M., Almeyda, A., Baldeosingh, K., Almaguel, F.G.,De Leon, M., 2013. Metabolomics uncovers dietary omega-3 fatty acid-derivedmetabolites implicated in anti-nociceptive responses after experimental spinal cordinjury. Neuroscience 255, 1–18.

Glatz, J.F., Luiken, J.J., Bonen, A., 2010. Membrane fatty acid transporters asregulators of lipid metabolism: implications for metabolic disease. Physiol.Rev. 90, 367–417.

Goldberg, R.J., Katz, J., 2007. A meta-analysis of the analgesic effects of omega-3polyunsaturated fatty acid supplementation for inflammatory joint pain. Pain129, 210–223.

Hanada, T., Kurihara, T., Tokudome, M., Tokimura, H., Arita, K., Miyata, A., 2014.Development and pharmacological verification of a new mouse model ofcentral post-stroke pain. Neurosci. Res. 78, 72–80.

Hansen, A.P., Marcussen, N.S., Klit, H., Andersen, G., Finnerup, N.B., Jensen, T.S.,2012. Pain following stroke: a prospective study. Eur. J. Pain 16, 1128–1136.

Harada, S., Fujita, W.H., Shichi, K., Tokuyama, S., 2009. The development of glucoseintolerance after focal cerebral ischemia participates in subsequent neuronaldamage. Brain Res. 1279, 174–181.

Harada, S., Fujita-Hamabe, W., Tokuyama, S., 2011. Effect of orexin-A on post-ischemic glucose intolerance and neuronal damage. J. Pharmacol. Sci. 115,155–163.

Harada, S., Nakamoto, K., Tokuyama, S., 2013a. The involvement of midbrainastrocyte in the development of morphine tolerance. Life Sci. 93, 573–578.

Harada, S., Yamazaki, Y., Nishioka, H., Tokuyama, S., 2013b. Neuroprotective effectthrough the cerebral sodium-glucose transporter on the development ofischemic damage in global ischemia. Brain Res. 1541, 61–68.

Hirasawa, A., Hara, T., Katsuma, S., Adachi, T., Tsujimoto, G., 2008. Free fatty acidreceptors and drug discovery. Biol. Pharm. Bull. 31, 1847–1851.

Hong, S.H., Belayev, L., Khoutorova, L., Obenaus, A., Bazan, N.G., 2014. Docosahex-aenoic acid confers enduring neuroprotection in experimental stroke. J. Neurol.Sci. 338, 135–141.

Hulse, R.E., Winterfield, J., Kunkler, P.E., Kraig, R.P., 2001. Astrocytic clasmatoden-drosis in hippocampal organ culture. Glia 33, 169–179.

Itoh, Y., Kawamata, Y., Harada, M., Kobayashi, M., Fujii, R., Fukusumi, S., Ogi, K., Hosoya,M., Tanaka, Y., Uejima, H., Tanaka, H., Maruyama, M., Satoh, R., Okubo, S., Kizawa, H.,Komatsu, H., Matsumura, F., Noguchi, Y., Shinohara, T., Hinuma, S., Fujisawa, Y.,Fujino, M., 2003. Free fatty acids regulate insulin secretion from pancreatic beta cellsthrough GPR40. Nature 422, 173–176.

Jump, D.B., 2002. The biochemistry of n-3 polyunsaturated fatty acids. J. Biol. Chem.277, 8755–8758.

Kim, E., Tolhurst, A.T., Qin, L.Y., Chen, X.Y., Febbraio, M., Cho, S., 2008. CD36/fattyacid translocase, an inflammatory mediator, is involved in hyperlipidemia-induced exacerbation in ischemic brain injury. J. Neurosci. 28, 4661–4670.

Klamroth-Marganska, V., Blanco, J., Campen, K., Curt, A., Dietz, V., Ettlin, T., Felder,M., Fellinghauer, B., Guidali, M., Kollmar, A., Luft, A., Nef, T., Schuster-Amft, C.,Stahel, W., Riener, R., 2014. Three-dimensional, task-specific robot therapy ofthe arm after stroke: a multicentre, parallel-group randomised trial. LancetNeurol. 13, 159–166.

Klit, H., Finnerup, N.B., Jensen, T.S., 2009. Central post-stroke pain: clinicalcharacteristics, pathophysiology, and management. Lancet Neurol. 8, 857–868.

Klit, H., Finnerup, N.B., Andersen, G., Jensen, T.S., 2011. Central poststroke pain: apopulation-based study. Pain 152, 818–824.

Kumar, G., Soni, C.R., 2009. Central post-stroke pain: current evidence. J. Neurol. Sci.284, 10–17.

Lambiase, M.J., Kubzansky, L.D., Thurston, R.C., 2014. Prospective study of anxietyand incident stroke. Stroke 45, 438–443.

Langhorne, P., Bernhardt, J., Kwakkel, G., 2011. Stroke rehabilitation. Lancet 377,1693–1702.

Liu, D., Smith, C.L., Barone, F.C., Ellison, J.A., Lysko, P.G., Li, K., Simpson, I.A., 1999.Astrocytic demise precedes delayed neuronal death in focal ischemic rat brain.Brain Res. Mol. Brain Res. 68, 29–41.

Liu, J., Solway, K., Messing, R.O., Sharp, F.R., 1998. Increased neurogenesis in thedentate gyrus after transient global ischemia in gerbils. J. Neurosci. 18,7768–7778.

Markesbery, W.R., 1997. Oxidative stress hypothesis in Alzheimer's disease. FreeRadic. Biol. Med. 23, 134–147.

Misra, U.K., Kalita, J., Kumar, B., 2008. A study of clinical, magnetic resonanceimaging, and somatosensory-evoked potential in central post-stroke pain.J. Pain 9, 1116–1122.

Moore, S.A., 1993. Cerebral endothelium and astrocytes cooperate in supplyingdocosahexaenoic acid to neurons. Adv. Exp. Med. Biol. 331, 229–233.

Moskowitz, M.A., Lo, E.H., Iadecola, C., 2010. The science of stroke: mechanisms insearch of treatments. Neuron 67, 181–198.

Nakamoto, K., Nishinaka, T., Mankura, M., Fujita-Hamabe, W., Tokuyama, S., 2010.Antinociceptive effects of docosahexaenoic acid against various pain stimuli inmice. Biol. Pharm. Bull. 33, 1070–1072.

Nakamoto, K., Nishinaka, T., Ambo, A., Mankura, M., Kasuya, F., Tokuyama, S., 2011.Possible involvement of beta-endorphin in docosahexaenoic acid-inducedantinociception. Eur. J. Pharmacol. 666, 100–104.

Nakamoto, K., Nishinaka, T., Matsumoto, K., Kasuya, F., Mankura, M., Koyama, Y.,Tokuyama, S., 2012. Involvement of the long-chain fatty acid receptor GPR40 asa novel pain regulatory system. Brain Res. 1432, 74–83.

Nakamoto, K., Nishinaka, T., Sato, N., Mankura, M., Koyama, Y., Kasuya, F., Tokuyama,S., 2013. Hypothalamic GPR40 signaling activated by free long chain fatty acidssuppresses CFA-induced inflammatory chronic pain. PLoS One 8, e81563.

Nasreddine, Z.S., Saver, J.L., 1997. Pain after thalamic stroke: right diencephalicpredominance and clinical features in 180 patients. Neurology 48, 1196–1199.

Ouyang, Y.B., Voloboueva, L.A., Xu, L.J., Giffard, R.G., 2007. Selective dysfunction ofhippocampal CA1 astrocytes contributes to delayed neuronal damage aftertransient forebrain ischemia. J. Neurosci. 27, 4253–4260.

Pettegrew, J.W., Klunk, W.E., Kanal, E., Panchalingam, K., McClure, R.J., 1995.Changes in brain membrane phospholipid and high-energy phosphate meta-bolism precede dementia. Neurobiol. Aging 16, 973–975.

Takami, K., Fujita-Hamabe, W., Harada, S., Tokuyama, S., 2011. Abeta and Adelta butnot C-fibres are involved in stroke related pain and allodynia: an experimentalstudy in mice. J. Pharm. Pharmacol. 63, 452–456.

Tamiya, S., Yoshida, Y., Harada, S., Nakamoto, K., Tokuyama, S., 2013. Establishmentof a central post-stroke pain model using global cerebral ischaemic mice.J. Pharm. Pharmacol. 65, 615–620.

Torres-Guzman, A.M., Morado-Urbina, C.E., Alvarado-Vazquez, P.A., Acosta-Gonzalez, R.I., Chavez-Pina, A.E., Montiel-Ruiz, R.M., Jimenez-Andrade, J.M.,2014. Chronic oral or intraarticular administration of docosahexaenoic acidreduces nociception and knee edema and improves functional outcomes in amouse model of complete Freund's adjuvant-induced knee arthritis. ArthritisRes. Ther. 16, R64.

Wasserman, J.K., Koeberle, P.D., 2009. Development and characterization of ahemorrhagic rat model of central post-stroke pain. Neuroscience 161, 173–183.

Williard, D.E., Harmon, S.D., Preuss, M.A., Kaduce, T.L., Moore, S.A., Spector, A.A.,2001. Production and release of docosahexaenoic acid by differentiated ratbrain astrocytes. World Rev. Nutr. Diet. 88, 168–172.

Zhang, M., Wang, S., Mao, L., Leak, R.K., Shi, Y., Zhang, W., Hu, X., Sun, B., Cao, G., Gao, Y.,Xu, Y., Chen, J., Zhang, F., 2014. Omega-3 fatty acids protect the brain againstischemic injury by activating Nrf2 and upregulating heme oxygenase 1. J. Neurosci.34, 1903–1915.

Zhao, X., Ahram, A., Berman, R.F., Muizelaar, J.P., Lyeth, B.G., 2003. Early loss ofastrocytes after experimental traumatic brain injury. Glia 44, 140–152.

S. Harada et al. / European Journal of Pharmacology 744 (2014) 115–123 123