Neuropharmacology and AnalgesiaNeuroprotective effect of forsythiaside against transient cerebral global ischemia in gerbilJong Min Kim a ,Sunho Kim a ,1,Dong Hyun Kim a ,Choong Ho Lee d ,Se Jin Park a ,Ji Wook Jung e ,Kwang Ho Ko f ,Jae Hoon Cheong g ,Seung Ho Lee d ,⁎,Jong Hoon Ryu a ,b ,c ,⁎⁎aDepartment of Life and Nanopharmaceutical Sciences,College of Pharmacy,Kyung Hee University,Hoeki-dong,Dongdaemoon-Ku,Seoul 130-701,Republic of Korea bDepartment of Oriental Pharmaceutical Science and,College of Pharmacy,Kyung Hee University,Hoeki-dong,Dongdaemoon-Ku,Seoul 130-701,Republic of Korea cKyung Hee East-West Pharmaceutical Research Institute,College of Pharmacy,Kyung Hee University,Hoeki-dong,Dongdaemoon-Ku,Seoul 130-701,Republic of Korea dCollege of Pharmacy,Yeungnam University,Gyeongsan 712-749,Republic of Korea eDepartment of Herbal Medicinal Resource,College of Health and Welfare,Daegu Haany University,Gyeongsan 712-715,Republic of Korea fDepartment of Pharmacology,College of Pharmacy,Seoul National University,San 56-1,Shillim-Dong,Kwanak-Gu,Seoul 151-742,Republic of Korea gDepartment of Pharmacy,Sahmyook University,Nowon-goo,Seoul 139-742,Republic of Koreaa b s t r a c ta r t i c l e i n f o Article history:Received 25March 2010Received in revised form 7March 2011Accepted 28March 2011Available online 9April 2011Keywords:Forsythiaside Global ischemia Y-maze task Microglia AstrocyteForsythiaside,a phenylethanoside,has been reported to have anti-oxidative activity and memory ameliorating effects against a scopolamine-induced memory de ficit model.The aim of this study was to determine whether forsythiaside has neuroprotective activity on transient cerebral global ischemia in gerbil.Transient cerebral ischemia was induced by bilateral common carotid artery occlusion for 5min and followed by reperfusion for 7days.Oral administration of forsythiaside was conducted immediately after reperfusion and once a day over the next 7days.The forsythiaside administration signi ficantly increased the number of viable neurons detected by neuronal nuclei immunostaining and decreased degenerating neuronal cells detected by Fluoro-Jade B staining in the hippocampal CA1region,at the 7th day post-ischemia (P b 0.05).Forsythiaside also signi ficantly decreased the number of ionized calcium-binding adaptor molecule-1-detected activated microglia and glial fibrillary acidic protein-detected astrocytes,both of which were increased after ischemic insults,and decreased interleukin-1βand tumor necrosis factor-αexpression levels,which were also increased after the insults (P b 0.05).In addition,forsythiaside signi ficantly improved ischemia-induced cognitive impairments in the Y-maze task (P b 0.05).These results suggest that forsythiaside exhibits neuroprotective properties,which are,in part,mediated by its anti-in flammatory activities supported by forsythiaside-induced reductions of activated glial cells and expression levels of interleukin-1βand tumor necrosis factor-α.©2011Elsevier B.V.All rights reserved.1.IntroductionBrain ischemia occurs when cerebral blood flow is reduced to a low level by certain pathological conditions,such as stroke or cardiac arrest (Nedergaard,1988;Neigh et al.,2004;White et al.,1993),which then induces oxygen and glucose deprivation in brain tissues.Transient global ischemia induced by bilateral common carotid artery occlusion (BCCAO)in gerbil causes delayed neuronal cell death in the pyramidal neurons in the CA1hippocampal region,whereas the neighboring CA3and dentate neurons are spared (Antonawich et al.,2004).This vulnerability involves multiple mechanisms,including glutamateneurotoxicity,the expression of pro-apoptotic and anti-apoptotic genes,mitochondrial dysfunction,oxygen free radicals and in flamma-tion (Kirino,2000;Nakamura et al.,1999;Nitatori et al.,1995;Ouyang et al.,2007;Won et al.,2001).Although several attempts to block these detrimental events have been applied,such as N-methyl-D -aspartate (NMDA)antagonists,free radical scavengers and caspase inhibitors,clinical trials have generally been unsuccessful.Accumulating evidences indicate that glial cells (i.e.,astrocytes and microglial cells)are actively involved in the pathogenesis of ischemic stroke (Gehrmann et al.,1992;Sugawara et al.,2002).Microglial cells,which are the immunocompetent cells of the brain,typically respond to neuronal cell damage and release proin flammatory cytokines including interleukin (IL)-1α,IL-1βand tumor necrosis factor (TNF)-αwithin the brain tissue (Streit et al.,1988;Zielasek and Hartung,1996),and they serve as neuropathological sensors (Ling et al.,2001).Astrocytes are also activated swiftly by ischemic insult (Ling et al.,2001;Wu and Ling,1998).Both microglial and astrocytic reactions have been used as markers for global cerebral ischemia-induced neuro-in flammatory reactions.Previous reports suggested that minocycline,a tetracyclineEuropean Journal of Pharmacology 660(2011)326–333⁎Correspondence author.Tel.:+82538102818;fax:+82538104654.⁎⁎Correspondence to:J.H.Ryu,Department of Oriental Pharmaceutical Science and,College of Pharmacy,Kyung Hee University,Hoeki-dong,Dongdaemoon-Ku,Seoul 130-701,Republic of Korea.Tel.:+8229619230;fax:+8229663885.E-mail addresses:seungho@yu.ac.kr (S.H.Lee),jhryu63@khu.ac.kr (J.H.Ryu).1Present address:Herbal Medicine Research Division,National Institute of Food and Drug Safety Evaluation,Seoul 122-704,Republic ofKorea.0014-2999/$–see front matter ©2011Elsevier B.V.All rights reserved.doi:10.1016/j.ejphar.2011.03.051Contents lists available at ScienceDirectEuropean Journal of Pharmacologyj o u r n a l h o me p a g e :w w w.e l sev i e r.c om /l oc a te /e j p h a rderivative,is neuroprotective against various neurodegenerative diseases including ischemia,Parkinson's disease and Alzheimer's disease by inhibiting the activation of microglia(Faust et al.,2009; Seabrook et al.,2006;Tikka et al.,2001).Accordingly,a potent inhibitor of glial activation may be a good candidate for these neurodegenerative diseases(Ransohoff and Perry,2009).Forsythiaside is a phenylethanoid glycoside isolated from Forsythia suspensa Vahl(Oleaceae)that had been widely used as an anti-inflammatory,antipyretic and antidotal agent for the treatment of infections,such as acute nephritis,erysipelas and ulcers(Guo et al., 2007;Schinella et al.,2002).Previous reports have revealed that forsythiaside has anti-oxidative effects and non-competitively inhibits the formation of5-hydroxy-6,8,11,14-eicosatetraenoic acid,which contributes to inflammatory responses through the leukotriene synthesis pathway(Kimura et al.,1987;Qu et al.,2008).In addition, we reported that forsythiaside attenuates cognitive dysfunction induced by scopolamine through its anti-oxidative activity(Kim et al.,2009).No attempts,however,have been conducted to investigate whether forsythiaside has neuroprotective effects during the course of neurode-generative diseases.The purpose of this study was to investigate the neuroprotective effect of forsythiaside against transient global ischemic cell death induced by BCCAO in gerbil.Pathological changes in the transient global ischemic gerbil hippocampus with or without forsythiasidewere investigated using immunohistochemical methods.In addition, the ameliorating effect of forsythiaside on ischemia-induced memory impairment was investigated using the Y-maze task.2.Materials and methods2.1.AnimalsGerbils(60–80g body weight)were purchased from the Orient Co.,Ltd,which is a branch of Charles River Laboratories(Seoul,Korea). Animals were housed5per cage,allowed access to water and food ad libitum,and maintained under constant temperature(23±1°C)and humidity(60±10%)with a12-h light/dark cycle(light on 07:30–19:30).Behavioral tasks were conducted between10:00and 16:00.Animal treatment and maintenance were conducted in accordance with the Principles of Laboratory Animal Care(NIH publication No.85–23,revised1985)and the Animal Care and Use Guidelines of Kyung Hee University,Seoul,Korea.2.2.MaterialsForsythiaside(β-(3′,4′-dihydroxyphenyl)ethyl-O-β-D-apiofurano-syl-(1→6)-α-l-rhamnopyranosyl(1→3)-β-D-(4-O-caffeoyl)-gluco-pyranoside;Fig.1)was isolated as described elsewhere(Kim et al., 2009).MK-801(5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohep-ten-5,10-imine;an NMDA receptor antagonist),bovine serum albumin (BSA)and3,3′-diaminobenzidine tetrahydrochloride(DAB)were purchased from Sigma-Aldrich(St.Louis,MO).Zoletil50®was purchased from Virbac Laboratory(06516Carros,France).The goat anti-ionized calcium-binding adaptor molecule-1(Iba-1),rabbit anti-IL-1βand rabbit anti-TNF-αantibodies were purchased from Abcam (Cambridge,UK).The mouse anti-glialfibrillary acidic protein(GFAP) and anti-neuronal nuclei(a neuron-specific protein)antibodies and Fluoro-Jade B were purchased from Chemicon(Temecula,CA).An avidin-biotin peroxidase complex(ABC)kit was purchased from Vector Laboratories(Burlingame,CA).Fluorescein isothiocyanate(FITC)-conjugated donkey anti-rabbit or goat,and Cy3-conjugated donkey anti-rabbit or mouse IgG were purchased from Jackson ImmunoRe-search(West Grove,PA).All other materials were of the highest grade and quality available and were obtained from normal commercial sources.2.3.Surgeries and drug administrationGerbils were anesthetized with2.0%isoflurane and70%nitrous oxide in oxygen and subjected to transient global ischemia as described elsewhere(Kim et al.,2006),with minor modifications.Transient global ischemia was induced by occluding both common carotid arteries with aneurysm clips for5min.Circulation was restored by removing the clips.Gerbils that received the same surgical operation without clipping of the carotid arteries served as sham-operated controls.Body temperatures were maintained at37±0.5°C throughout surgery with a heating pad(Biomed S.L.,Spain).Regional cerebral bloodflow was monitored using laser Dopplerflowmetry(LDF;Perimed,PF5010, JarFalla,Sweden)from the time of anesthetic induction to10min after reperfusion.Any gerbil showing under20%of baseline of regional cerebral bloodflow was used for further study.Gerbils were orally treated with forsythiaside(1,3or10mg/kg) suspended in a10%Tween80solution immediately after reperfusion and once a day via an oral Zonde needle over the next7days to investigate the neuroprotective effect of forsythiaside.A group intraperitoneally treated with3mg/kg of MK-801,an NMDA receptor antagonist,dissolved in a0.9%saline solution immediately after reperfusion was used as a positive control group.In the sham-operated and the vehicle-treated ischemic control groups,both vehicle solutions, 10%Tween80(for forsythiaside)and0.9%saline(for MK-801),were administered using the same time schedule,volume and route as the forsythiaside or MK-801treatments.2.4.The Y-maze taskThe Y-maze was a horizontal maze(40cm long and3cm wide with walls12cm high)with three arms(labeled A,B and C)at120°angles(Sarter et al.,1988).The mazefloor and walls were constructed from dark opaque polyvinyl plastic.The gerbil was placed initially within one arm,and the sequence(e.g.,ABCCAB)and the number of arm entries were recorded manually for each gerbil over an8min period as described elsewhere(Kim et al.,2006).An alternation was defined as entry into all three arms consecutively(i.e.,ABC,CAB or BCA,but not BAB).One hour before the test,the last administration of forsythiaside(10mg/kg)or vehicle was completed.The maze arms were thoroughly cleaned with water spray between tests to remove residual odors.The percentage of alternation was determined by the following equation:%Alternation=[(Number ofalternations)/(TotalFig.1.Structure of forsythiaside.327J.M.Kim et al./European Journal of Pharmacology660(2011)326–333arm entries−2)]×100.The total number of arm entries served as an indicator of locomotor activity.2.5.Tissue preparationGerbils were anesthetized with an intramuscular injection of Zoletil50®(10mg/kg)immediately after the Y-maze task and perfused transcardially with0.05M phosphate buffered saline(PBS) (pH7.4)followed by ice-cold4%paraformaldehyde for pre-fixation. The brains were removed and post-fixed in0.05M phosphate buffer (pH7.4)containing4%paraformaldehyde overnight.Afterfixation, the brains were immersed in a30%sucrose solution(in0.05M PBS), and stored at4°C until cryostatic sectioning.Frozen brains were coronally sectioned on a cryostat(Leica Microsystems AG,Wetzlar, Germany)at30μm intervals and then kept in storage solution at4°C.2.6.Immunohistochemistry and double immunofluorescenceFreefloating sections were incubated for24h in0.05M PBS containing goat anti-Iba-1(1:1000dilution),mouse anti-GFAP (1:1000dilution)or mouse anti-neuronal nuclei antibody(1:1000 dilution),and0.3%Triton X-100,1%BSA and1.5%normal rabbit serum (for Iba-1)or rat serum(for GFAP and neuronal nuclei)at4°C,as described elsewhere(Kim et al.,2006).Sections were then incubated for90min with a biotinylated secondary antibody(1:200dilution) and treated with ABC solution(1:100dilution)for1h at room temperature.They were then treated with0.02%DAB and0.01%H2O2 for3min.After each incubation step,the sections were washed three times with PBS for5min.Finally,they were mounted on gelatin-coated slides,dehydrated in an ascending alcohol series,and cleared in xylene.For double immunofluorescence labeling,sections were blocked in 20%normal donkey serum in PBS,followed by an overnight incubation with a rabbit anti-IL-1βantibody(1:200dilution)and a goat anti-GFAP antibody(1:1000dilution)or a rabbit anti-TNF-αantibody(1:100dilution)and goat anti-GFAP antibody(1:1000 dilution)in1%BSA diluted in PBS.After several washes,the sections were incubated with a secondary antibody conjugated to Cy3or FITC (1:1000dilution)for1h at room temperature,then mounted on glass slides using Vecta-shield mounting medium.Slice analysis was conducted by confocal microscopy(Zeiss Axiovert LSM510META, Germany).2.7.Fluoro-Jade B stainingFluoro-Jade B,a high affinityfluorescent marker for the localiza-tion of neuronal degeneration,histofluorescence staining was conducted as described elsewhere(Candelario-Jalil et al.,2003).In brief,the sections werefirst immersed in a solution containing1% sodium hydroxide in80%alcohol,and followed in70%alcohol.They were then transferred to a solution of0.06%potassium permanganate, and transferred to a0.0004%Fluoro-Jade B staining solution.After washing,the sections were placed on a slide warmer(approximately 50°C),and then examined using an epifluorescent microscope (Olympus Microscope System BX51;Olympus,Tokyo,Japan)with green excitation light(450–490nm)and a barrierfilter.2.8.Quantification and statisticsFor the quantification of immunostained cells,the cell counts in CA1of the hippocampal region were performed using a computerized image analysis system(Leica Microsystems AG,Wetzlar,Germany)by one person blind to the treatment group identities.The number of immunopositive cells was expressed using4gerbils from each group, the average of6sections(one of everyfive sections from−1.5mm of bregma)per gerbil was examined.Values are expressed as means±S.E.M.The results of the immuno-histochemical analysis and the Y-maze task were analyzed by one-way analysis of variance(ANOVA),and when results were found to be significant,treatment groups were compared using Tukey's post-hoc test.Statistical significance was accepted for P values of b0.05.3.Results3.1.Forsythiaside attenuates delayed neuronal cell death induced by transient global ischemiaRepresentative photomicrographs of the CA1region of the hippocampal formation are shown in Fig.2A.Marked cell losses in this subfield were observed in the vehicle-treated ischemic control group compared to the sham group as measured by neuronal nuclei immunostaining.Significant group effects on surviving neuronal cell number detected by neuronal nuclei were observed[F(5,18)=11.52, P b0.05,Fig.2A-B].Post-hoc test revealed that the mean number of neuronal nuclei-immunopositive cells in the CA1region in the vehicle-treated ischemic control group was significantly lower than that in the sham group(P b0.05).In contrast,subchronic treatment with forsythiaside at10mg/kg for7days significantly attenuated the ischemia-induced loss of neuronal nuclei-immunopositive cells (P b0.05).Similar results were also obtained in the positive control group treated with MK-801(an NMDA receptor antagonist)at 3mg/kg.Moreover,there were significant group effects on degen-erating cell number detected by Fluoro-Jade B staining[F(5,18)= 9.426,P b0.05,Fig.2A and C].Degenerating cells were significantly increased in the vehicle-treated ischemic control group compared to sham group(P b0.05),and the administration of forsythiaside at 10mg/kg or MK-801at3mg/kg significantly suppressed the increase of degenerating cells induced by ischemic insults(P b0.05).No other doses of forsythiaside(1or3mg/kg)showed any significant effects on both surviving and degenerating neuronal cell numbers(Fig.2B-C). Based on these results,the most effective dose of forsythiaside (10mg/kg)was used for further mechanistic and behavioral studies.3.2.Forsythiaside inhibits activation of microglial cell and astrocyte induced by transient global ischemiaThere were significant group effects on the activated microglial cell number detected by Iba-1immunostaining[F(3,12)=9.216,P b0.05, Fig.3A-B].In the sham group,microglial cells immunostained with the anti-Iba-1antibody were scattered and had a ramified form,indicating an inactivate state(Fig.3A).In the vehicle-treated ischemic control group, microglial cells in the CA1region were condensed and hypertrophied, indicating an active state,and these numbers of activated microglial cells were significantly increased compared to sham group(P b0.05,Fig.3A-B). These activated microglial cells were significantly decreased by sub-chronic forsythiaside treatment(10mg/kg,for7days,P b0.05).Similarly to microglial cells,there were significant group effects on the activated astrocytes immunostained with the anti-GFAP antibody[F(3,12)=12.29, P b0.05,Fig.3A and C].Post-hoc test revealed that activated astrocytes rarely observed in the sham group were significantly increased in the CA1 region after transient global ischemia(P b0.05).The same forsythiaside treatment(10mg/kg,for7days)also significantly reduced these activated astrocytes(P b0.05).In addition,the effects of MK-801 (3mg/kg,an NMDA receptor antagonist)on the increase of activated microglial cells and astrocytes were almost the same as those of forsythiaside(Fig.3B-C).3.3.Forsythiaside inhibits IL-1βand TNF-αexpressions induced by transient global ischemiaSignificant group effects on the IL-1β-immunopositive cells were observed in different treatment groups[F(3,12)=7.293,P b0.05,328J.M.Kim et al./European Journal of Pharmacology660(2011)326–333Fig.4A-B].Post-hoc test revealed that the number of IL-1β-immunopo-sitive cells was signi ficantly increased in the vehicle-treated ischemic control group,whereas it was rarely observed in the sham group (P b 0.05).These IL-1β-immunopositive cells were signi ficantly decreased by subchronic administration of forsythiaside (P b 0.05).In addition,there were signi ficant group effects on the number of TNF-α-immunopositive cells [F (3,12)=12.29,P b 0.05,Fig.4A and C].TNF-α-immunopositive cells were signi ficantly increased by the ischemic insults (P b 0.05),and these cells were decreased by subchronic administration of forsythiaside (P b 0.05).Furthermore,the effects of MK-801(3mg/kg,an NMDA receptor antagonist)on the increase of IL-1β-and TNF-α-immunopositive cells were almost the same as those of forsythiaside.Double immuno fluorescence labeling revealed that these IL-1β-and TNF-α-immunopositive cells were also immunos-tained to anti-GFAP antibody (Fig.4D-E)and were scarcely positive to anti-Iba-1antibody (data not shown).3.4.Forsythiaside ameliorates memory impairment induced by transient global ischemiaA signi ficant group effect was observed in the spontaneous alternation in the Y-maze task [F (3,17)=7.96,P b 0.05,Fig.5A].Post-hoc test revealed that the spontaneous alternation of the vehicle-treated ischemic control group was signi ficantly shorter than that of the sham group (P b 0.05).Moreover,the shorter spontaneous alternation induced by ischemic treatment was signi ficantly reversed by subchronic treatment with forsythiaside (10mg/kg,P b 0.05).MK-801(3mg/kg,an NMDA receptor antagonist)-treated group also showed similar effect onspontaneous alternation induced by ischemic treatment as forsythia-side-treated group.No signi ficant differences in the total number of entries were observed among all groups (Fig.5B).4.DiscussionIschemic injury to neurons is primarily due to the interruption of blood flow by the ischemic event,resulting in a lack of oxygenation,and the subsequent re-oxygenation caused by reperfusion (Frantseva et al.,2001;Sims and Anderson,2002).In addition,various evidences suggest that excessive accumulation of glutamate in the extracellular space after ischemic stroke causes activation of its receptors,mainly the NMDA type of glutamate receptor (Castillo et al.,1997;Choi and Rothman,1990;Davalos et al.,1997).MK-801,an NMDA receptor antagonist,has neuroprotective effect against several ischemia models and has been widely used as positive control drug (Benquet et al.,2008;Park et al.,1988).In the present study,the administration of MK-801prevented the neuronal cell death in the hippocampal CA1region after ischemic insults con firmed by neuronal nuclei immunohistochemistry and Fluoro-Jade B staining.In addition,the forsythiaside-treated group showed similar results with MK-801-treated group in both neuronal nuclei immuno-histochemistry and Fluoro-Jade B staining when administered at 10mg/kg for 7days.Thus,subchronic administration of forsythiaside was demonstrated to have strong neuroprotective activity against transient global ischemia induced by BCCAO.While the subchronic administration of forsythiaside showed strong neuroprotective effect against transient global ischemia induced by BCCAO,a single adminis-tration of forsythiaside did not exert any signi ficantneuroprotectiveFig.2.The neuroprotective effects of forsythiaside.Forsythiaside (1,3or 10mg/kg,p.o.)or the same volume of vehicle solution was administered immediately after reperfusion and once a day for the next 7days.The MK-801(3mg/kg,i.p.,an NMDA receptor antagonist)-treated group was used as positive control group.A.Representative photomicrographs of neuronal nuclei (NeuN)-immunopositive cells (left and middle panels)and of Fluoro-Jade B staining (right panels)of CA1region.Magni fication:400X.Bar=50μm.B.Quanti fication of the neuronal nuclei-immunopositive cells observed in the middle panel of Figure A.C.Quanti fication of the Fluoro-Jade B (FJB)-positive cells observed in the right panel of Figure A.Data are presented as means ±S.E.M.from 6determinations of each region (n =4/group).*P b 0.05,compared with the sham group.#P b 0.05,compared with the vehicle-treated ischemic control group.329J.M.Kim et al./European Journal of Pharmacology 660(2011)326–333effects (data not shown).Although it is unclear why acute administra-tion of forsythiaside did not exhibit neuroprotective activity,we speculated that the single administration of forsythiaside is not enough to exert its anti-in flammatory effect.As our previous findings (Shin et al.,2010),it would be taken time for the in flammatory events including microglial activation after the global ischemia.Therefore,a drug which has anti-in flammatory activities needs to be administered until the in flammatory factors are activated or released.These speculations are supported by several previous reports that minocyline or cyclooxygenase inhibitors exerted anti-ischemic activities after their subchronic administration (Institoris et al.,2007;Tang et al.,2007).Recently,pharmacokinetic studies revealed that forsythiaside has a moderate binding capacity to plasma protein (Cheng et al.,2010).Plasma protein binding capacity of forsythiaside or its blood –brain barrier penetration may also affect its neuroprotective activities after subchronic administration.Further researches are needed to clarify these issues.On the other hand,transient cerebral ischemia induces the activation and proliferation of microglia and astrocytes (Denes et al.,2007;Gehrmann et al.,1992;Kato et al.,1994;Petito et al.,1990).These activated glial cells release various in flammatory and cytotoxic agents that also lead to neuronal injury (Boje and Arora,1992;Kato et al.,2003;Tichauer et al.,2007).Several recent studies have suggested that the inhibition of microglial activation using anti-in flammatory agents attenuates ischemic injury (Kim et al.,2005;Sironi et al.,2008;Yrjanheikki et al.,1998).In addition,activated microglial cells in ischemic brain can be observed by immunostaining with Iba-1which is speci fically expressed in microglia and is upregulated during its activation (Ito et al.,2001).And GFAP,an intermediate filament protein,is widely used as a speci fic marker for activated astrocyte and is associated with pathologic changes in various brain injuries,including cerebral ischemia (Eng et al.,2000;Petito and Halaby,1993).In this study,forsythiaside decreased the glial activation induced by transient global ischemia in gerbil.After the induction of transient ischemia,the number of activated microglial cells immunostained by the anti-Iba-1antibody and that of activated astrocytes immunostained by the anti-GFAP antibody in the hippocampal CA1region were signi ficantly higher in the untreated ischemic control group than those in the sham-operated control group.The subchronic administration of forsythiaside (10mg/kg)signi ficantly reduced the activation of microglial cells and astrocytes in hippocampal CA1regions on day 7,as shown by Iba-1and GFAP immunostaining,respectively.These changes in the CA1regions indicated that forsythiaside inhibits glial activation induced by transient global ischemia.Among the agents that activated glial cells release,IL-1βand TNF-α,the proin flammatory cytokines,are known to play key roles in the pathogenesis of ischemic stroke (Boutin et al.,2001;Hallenbeck,2002;Schielke et al.,1998).Besides attracting leukocytes into ischemic sites and promoting tissue necrosis,IL-1βand TNF-αcan disrupt the brain blood barrier and accelerate the formation of cerebral edema (Wang et al.,2007).It has been reported that the administration of IL-1βand TNF-αneutralizing antibody or their receptors'antagonists reduced brain injury after cerebral ischemia (Lavine et al.,1998;Wang and Shuaib,2002;Yang et al.,1997).Thus,in flammatory responses due to activated glial cells and proin flammatory cytokines released from these cells play causative roles in the neuronal damage resulting from ischemia.In the present study,subchoronic administration of forsythiaside (10mg/kg)signi ficantly decreased the number of IL-1β-and TNF-α-immunopositivecellsFig.3.The effect of forsythiaside on activation of microglia and astrocyte.Forsythiaside (10mg/kg,p.o.)or the same volume of vehicle solution was administered immediately after reperfusion and once a day for the next 7days.The MK-801(3mg/kg,i.p.,an NMDA receptor antagonist)-treated group was used as positive control group.A.Representative photomicrographs of Iba-1-and GFAP-immunopositive cells in the hippocampal CA1region.The inset photomicrographs display the magni fication of each rectangular region.Magni fication:400X.Bar=50μm.B.Quanti fication of the Iba-1-immunopositive cells observed in the left panel of Figure A.C.Quanti fication of the GFAP-immunopositive cells observed in the right panel of Figure A.Data represent means±S.E.M.for 6determinations of each region (n=4/group).*P b 0.05,compared with the sham group.#P b 0.05,compared with the vehicle-treated ischemic control group.330J.M.Kim et al./European Journal of Pharmacology 660(2011)326–333。
