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Hindawi Neural Plasticity Volume 2017, Article ID 9545646, 11 pages https://doi.org/10.1155/2017/9545646 Research Article Electroacupuncture Regulates Hippocampal Synaptic Plasticity via miR-134-Mediated LIMK1 Function in Rats with Ischemic Stroke WeilinLiu,1JieWu,2JiaHuang,1PeiyuanZhuo,2YunjiaoLin,2LuluWang,2 RuhuiLin,2LidianChen,1andJingTao1 1CollegeofRehabilitationMedicine,FujianUniversityofTraditionalChineseMedicine,Fuzhou,Fujian350122,China 2FujianKeyLaboratoryofRehabilitationTechnology,Fuzhou,Fujian350122,China CorrespondenceshouldbeaddressedtoLidianChen;[email protected];[email protected] Received26August2016;Accepted3November2016;Published2January2017 AcademicEditor:Cun-ZhiLiu Copyright©2017WeilinLiuetal. This is an open access article distributed under the Creative Commons Attribution License, whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. MircoRNAs(miRs)havebeenimplicatedinlearningandmemory,byregulatingLIMdomainkinase(LIMK1)toinducesynaptic- dendriticplasticity.ThestudyaimedtoinvestigatewhethermiRNAs/LIMK1signalingwasinvolvedinelectroacupuncture-(EA- ) mediated synaptic-dendritic plasticity in a rat model of middle cerebral artery occlusion induced cognitive deficit (MICD). Comparedtountreatmentornon-acupoint-EAtreatment,EAatDU20andDU24acupointscouldshortenescapelatencyand increasethefrequencyofcrossingplatforminMorriswatermazetest.T2-weightedimagingshowedthattheMICDratbrain lesionswerelocatedincortex,hippocampus,corpusstriatum,andthalamusregionsandinjuredvolumeswerereducedafterEA. Furthermore,wefoundthatthedensityofdendriticspineandthenumberofsynapsesinthehippocampalCA1pyramidalcells wereobviouslyreducedatDay14afterMICD.However,synaptic-dendriticlosscouldberescuedafterEA.Moreover,thesynaptic- dendriticplasticitywasassociatedwithincreasesofthetotalLIMK1andphospho-LIMK1levelsinhippocampalCA1region,wherein EAdecreasedtheexpressionofmiR-134,negativelyregulatingLIMK1toenhancesynaptic-dendriticplasticity.Therefore,miR-134- mediatedLIMK1wasinvolvedinEA-inducedhippocampalsynapticplasticity,whichservedasacontributortoimprovinglearning andmemoryduringtherecoverystageofischemicstroke. 1.Introduction after3years,andeachyearthemeangrowthrateisapproxi- mately8%[6].Inadditiontoconventionalcognitivetraining, Ischemicstrokeresultsinahighmortalityrateandincreased electroacupuncture (EA) is a stretch therapeutic method of disability rate all over the world [1]. Approximately 64% of acupuncture,whichistraditionalacupunctureincorporation strokepatientsareoftenfollowedwithcognitiveimpairment with modern electrotherapy. The clinical efficacy of EA on and 33% of them turn into dementia existing for several poststroke cognitive impairment has been widely demon- monthsduringdecubation[2].Cognitivedeficitsarisefrequ- strated [7, 8]. However, the functional mechanism of EA is entlyafterischemicstroke,whichcausedifficultieswithanal- farfrombeenfullyelucidated. ysis, concentration, organization, interpretation, and other Thehippocampusisapivotalstructureofthebrain;the abatesincognitivefunctionsthatbringaboutthelowquality areaplaysanimportantroleintheformationofacquisition, oflife[3,4].Thedysfunctionoflearningandmemoryisthe consolidation, and recognition of declarative and spatial cardinalsymptomofcognitiveimpairmentafterstrokeandis memory[9,10].Thelossofhippocampalsynapsesandneu- the main culprit of persistent sequelae [5]. A recent study ronsinpoststrokeinducescognitivedeficitsincludingspatial demonstrated that the incidence rate of poststroke mild reference learning and memory impairment [11, 12]. In the cognitiveimpairmentwasdiagnosedin24.4%ofindividuals formationofspatialreferencememoryiscloselyrelatedtothe 2 NeuralPlasticity plasticityofdendriticspinesandthemorphologicalchanges #FUTCM-2015008) and was performed in accordance with suchasexpansionandcontraction[13].Dendriticspinesalter the national guidelines for the care and use of laboratory their shape to make the information spreading more easily animals.Foreuthanasia,3%sodiumpentobarbital(40mg/kg and influence the synaptic efficacy (i.e., long-term potenti- bodyweight,i.p.)wasused.Middlecerebralarteryocclusion ation and long-term depression) [14, 15], which have been (MCAO) surgery was carried out under general anesthesia widelyconsideredasacellularmechanismforlearningand (1.5%isofluranein68.5%N2Oand30%O2).Alleffortswere memory [16]. LIM domain kinase (LIMK1) is enriched in madetominimizesuffering. both axonal and dendritic growth cones of hippocampal pyramidalneuronsinrats[17].LIMK1encodesaserine/thre- 2.2.ExperimentalProcedures. TheMCAO-inducedcognitive onineproteinkinasethatregulatestheactincytoskeletonby deficit(MICD)modelwasestablishedaspreviouslydescribed phosphorylatingandinactivatingtheactindepolymerization [5,30].Ratswererandomlyassignedtofourgroupsaccording factor(ADF)/cofilin[18].Inaddition,LIMK1isalsoknown totherandomnumbertable(𝑛 = 24ratseachgroup)asfol- as having an important role in synapse and dendritic spine lows:(1)Shamgroup,(2)MICDgroup,(3)MICD+EAgroup, function.Ithasbeenreportedthattheknockoutmicelacking and (4) MICD+non-EA group. The left middle cerebral LIMK1areseverelyimpairedindendriticspinemorphology arterywasoccludedbya4-0nylonmonofilament(0.23mm and hippocampal long-term potentiation [19, 20]. Evidence in diameter, Jialing-bio, China). Focal cerebral ischemia showed that LIMK1 regulated long-term memory (LTM) was monitored using transcranial temporal laser Doppler andlonglastingsynapticplasticitythroughinteractingwith (BIOPACSystems,Goleta,CA,USA)andan80%decreasein andactivatingcyclicAMPresponseelement-bindingprotein bloodflowaftertheocclusionwasrecorded.After90minof (CREB)[21]. occlusion, reperfusion was achieved by pulling out the fila- Inaddition,apotentialroleformicroRNAs(miRNAsor menttorestorebloodflow.Sham-operatedratsoftheSham miRs) in synaptic function has been particularly intriguing groupunderwentthesameprocedure,butarterialocclusion giventheevidencethatabrain-specificmiRNAcontributes wasnotperformed. tosynapticdevelopment,maturation,and/orplasticity[22]. After24hoursofMCAOsurgery,ratsoftheShamgroup miRNAsareendogenous,noncodingRNAsthatmediatethe and the MICD group received no treatment. Rats of the posttranscriptionalregulationofgeneexpressionmainlyby MICD+EAgroupweregivenEAtreatmentfor30minperday 󸀠 binding to the 3 -untranslated region of messenger RNAs for 14 consecutive days. The EA needles (diameter, 0.3mm, (mRNAs) [23]. A number of miRNAs have been isolated needlepurchasedfromHualunacupunctureofSuzhouCo., from nervous system, and a recent study has demonstrated Ltd.,SuzhouChina)wereinsertedatadepthof2-3mminto a crucial role for dynamically regulating synaptic plasticity the Baihui (DU20, located in the median of frontalis) and [24,25].Moreover,miRNAshavebeenimplicatedinhippo- Shenting(DU24,locatedinthemedianoftheparietalbone) campus-dependentfunction,whichhaveasignificantpoten- acupoints[31].StimulationwasthengeneratedusingtheEA tial in learning and memory formation, regulating LIMK1 apparatus[ModelG6805,ShanghaiHuayi(Group)Company, expressiontoinducesynaptic-dendriticplasticity[22].Den- Ltd.,Shanghai,China]andthestimulationparameterswere dritic mRNAs encode diversified functionalities in hippo- setasfollows:dilatationalwavesof1∼20Hz(adjustedtothe campal pyramidal neurons and play an important role in muscle twitch threshold), peak voltage of 6V, and 0.2mA synapticplasticity,aswellaslearningandmemory[26]. intensity[31].RatsoftheMICD+non-EAgroupweregiven Therefore,miRNA-LIMK1canbeconsideredasatarget EA treatment at the bilateral nonacupoints (located in the forcognitivedeficit.OurpreviousstudyhasshownthatEA costalregionand10mmdistaltotheiliaccrest)for30minper at Baihui (DU20) and Shenting (DU24) acupoints could dayfor14consecutivedays[32].TheEAneedlesandstimula- improve cognitive impairment through Rho GTPases to tionparametersofthebilateralnonacupointswereconsistent enhancedendriticplasticityinratswithischemicstroke[27]. withtheEAtreatment.Manipulatorswereexperiencedand Interestingly,ithasbeensuggestedthattheactivationofthe blindedtotherat’sgroup. Rho GTPases signaling is essential for LIMK1 activation by phosphorylationonthreonine508,whichiswidelyknownas 2.3.BehavioralAssessment. Behavioraltestingwasconducted masterregulatorofactindynamics[28,29].Thus,thestudy byresearcherswhowereblindedtotherat’sgroup.At10days aimed to elucidate whether EA at the DU20 and DU24 afterEA,alltheratsweresubjectedtotheMorriswatermaze acupoints could improve the cognitive deficits in rats with test (Shanghai Xinruan Information Technology Co., Ltd, ischemicstrokeviamiRNA-LIMK1-mediatedsynapticplas- Shanghai, China) to evaluate spatial reference learning and ticitytoenhancespatialreferencelearningandmemory. memory(Figure1).TheMorriswatermazeconsistedofacir- cularpool(diameter150cm,height60cm)filledwithwater 2.MaterialsandMethods (depth of 30cm and temperature of 25 ± 2∘C). A circular escape platform (diameter 12cm, height 29cm) was sub- 2.1. Animal Ethics Statement. 96 male Sprague-Dawley rats merged 2cm below the water surface, in the middle of the (weight,250±20g)wereobtainedfromShanghaiSLACLab- thirdquadrantofthepoolandthereferenceobjectsaround oratoryAnimalCo.,Ltd.(Shanghai,China,SCXK2013-0005), thepoolwereplaced.Morriswatermazetasksmainlyinclude and housed under pathogen-free conditions with a 12h orientation navigation and space exploration trials. During light/darkcycle.ThestudywasapprovedbytheCommitteeof thefirstsetoftrials,eachratwasplacedinthewaterateach FujianUniversityofTraditionalChineseMedicine(protocol of the four equidistant locations to the platform. When the NeuralPlasticity 3 Electroacupuncture T2WI Baseline: T2WI Morris water test 0 24h 1d 7d 10d 14d Sham or MCAO surgery Sacrificed Figure1:Experimentaldesignusedinthestudy. ratsarrivedattheplatformwithinthe90sectimerestriction in a mixture of A and B solutions from the kit and stored and remained on it for 3sec, they were considered to have inthedarkatroomtemperature,followingwhichtheywere ∘ foundtheplatformandwerescoredbythetimetaken/length transferredintosolutionCfromthekit,storedat4 Cfor7 oftheroute.Whentheratswereunabletofindtheplatform days. Finally, brains were then frozen and coronal sections within90sec,theywereplacedontheplatformfor10secand (150𝜇m)weremadeusingacryostat(LeicaCM3050S,Leica thetimescorewas90sec.Thetimetakenandthelengthofthe Microsystems K. K., Tokyo, Japan). The sections incubated routebywhicheachratfoundthesafeplatformwererecorded in a mixture of D and E solutions from the kit and then bythecomputer.Theaverageofthetimetakenandthelength dehydratedinalcohol(50,70,90,and100%for5mineach), oftherouteforthefourquadrantsasaresultofeachratwere clearedinxylene,andwerecover-slipped.Theimagesfinally assessedeveryday.Thedurationofthefirstsetoftrialswas were viewed under a microscope (Leica DM6000 B, Leica performedonDays10to13afterEA.Thesecondpartofthe Microsystems,Wetzlar,Germany).Theslideswerereviewed experiment was performed on Day 14, to examine the time bytwoorthreepathologistsblindtothestudy. in which rats found the location of the platform within the 90sectimerestriction,whichtestedtheirabilitytoremember 2.6. Transmission Electron Microscopy. Four rats in each the position of the platform. After all trials, the rats were groupwereanesthetizedandtheleftventriclewasperfused driedthoroughlywithahairdrierandreturnedtotheircages with200mLofsalinefollowedby400mL4%paraformalde- (GuangzhouRiboBioCo.,Ltd.,Guangzhou,China).Morris hyde (pH 7.4). The tissue was taken from the left ischemic watermazetestwasrepeatedthreetimes. 3 hippocampus, cut into 1mm size cubes, and fixed in 1% paraformaldehydewith1%lanthanumnitratetracerfor24h 2.4.MeasurementofBrainLesions. AnimalMRIscanswere followedbyfixationwith3%glutaraldehydefor24h.Samples performed on a 7.0T MRI scanner (70/20USR BioSpec, werefixedin1%osmiumtetroxidefor2handdehydratedin BrukerBiospinGmbls,Germany)usinga38mmbirdcagerat gradedethanol1%lanthanumnitratetracer(LNT)solution brainquadratureresonatorforradiofrequencytransmission andembeddedinaraldite.UltrathinhippocampalCA1slices andreception.Animalswereanesthetizedwithisoflurane/O2 were obtained (90nm); then they were stained with uranyl (with 3% induction for 5min and 1.2–1.5% maintenance in acetate and lead citrate and observed under TEM (H-7650; order to let the rats in the depth of anesthesia state) and Hitachi, Ltd., Tokyo, Japan). The photos were obtained on kept warm with circuit. After anesthesia, the rat was put hp digital CCD camera (SIS4 million voxel). Images were in prone position on a custom-made holder to minimize acquireddigitallyfromarandomlyselectedpoolof10to15 headmotion,setthelocationofheadposition,andperform fieldsundereachcondition. real-time monitor of the breathing rate and maintained in 40breaths/min.Rat’stemperatureismaintainedat33±2∘C intheprocessofscanningholdertominimizeheadmotion 2.7. Western Blotting. Isolated left hippocampus tissue was whilerespirationwasmaintained. lysedin100𝜇Lradioimmunoprecipitationassay(RIPA)buf- T2-weighted imaging (T2WI) in three planes with a fer(Beyotime,Haimen,Jiangsu,China)plusproteaseinhibi- fast spin echo (FSE) pulse sequence was first acquired to tors. Total protein (50𝜇g) was loaded into 10% SDS-PAGE controlratheadpositioning.T2WIscanwasacquiredusing gels,electrophoresed,andthentransblottedontopolyvinyli- a Rapid Acquisition with Relaxation Enhancement (RARE) dene difluoride membranes (Immobilon-P; Millipore, Bil- pulsesequencewiththefollowingparameters:fieldofview= lerica, MA) in a Tris-glycine transfer buffer. After being 32mm×32mm,matrixsize=256×256,repetitiontime(TR) blocked in 5% milk in PBST for 1h at room temperature =4200ms,echotime(TE)=35ms,slicethickness=1.0mm, with shaking, membranes were incubated with antibodies andslicegap=0mm. overnightat4∘Cagainstthefollowing:anti-LIMK1antibody Image J analysis and processing system was applied for (dilution, 1:1000; ab81046, Abcam) and anti-LIM kinase T2W images, the percentage of the brain lesions = brain 1 antibody (phospho-Thr508) (dilution, 1:1000; ab131341, lesionsvolume/wholebrainvolume×100%. Abcam) and 𝛽-actin (dilution, 1:1000; ab189073; Abcam). The following day, membranes were incubated in 5% milk 2.5. Golgi Staining. Golgi staining of brain was performed (in TBST) with an anti-goat or anti-mouse IgG antibody using an FD Rapid GolgiStain Kit following the manufac- (dilution,1:5000;PerkinElmerLifeSciences,Waltham,MA) turer’sinstructions(FDNeurotechnologies,Inc.,Columbia, for1hatroomtemperaturewithshaking.Membraneswere MD, USA). The removed rat brains were placed incubated washedaminimumoffourtimes(10minperwash)inPBST 4 NeuralPlasticity betweeneachantibodytreatment.Detectedbandswerevisu- whereas in the MICD+EA group the times where the rats alizedusingenhancedchemiluminescenceandimageswere crossedthepositionoftheplatformweresignificantlyincrea- captured using a Bio-Image system (Bio-Rad Laboratories, sed compared with the MICD group and the MICD+non- Inc., Hercules, USA). Western blotting was repeated three EA group (𝑝 < 0.01, 𝑝 < 0.05, Figure 2(d)). As illustrated times. inFigures2(e)and2(f),thepercentageoftimespentinthe target quadrant was used for statistical analysis during the probetrial.Thedateshowedthattheratsspentmoretimein 2.8.RealTimeQuantitativeRT-PCR. TheexpressionofmiR- thetargetquadrantcomparedtootherquadrants(𝑝 < 0.01; NAswasdeterminedbyrealtimequantitativereversetran- Figure 2(e)). The MICD group spent less time in the target scription polymerase chain reaction (RT-PCR). Total RNA quadrant compared with the other groups (𝑝 < 0.01; Fig- was extracted from hippocampal region of the ipsilateral ure2(e)).TheMICD+EAgroupspentmoretimeinthetarget lesion tissue using the TRIzol Reagent (Life Technologies quadrantcomparedwiththeMICDgroupandMICD+non- (AB & Invitrogen), Carlsbad, USA). Then extracted total EAgroup(𝑝 < 0.01,𝑝 < 0.05,Figure2(f)).Therefore,these RNA was reverse transcribed to generate cDNA accord- results suggest that acquisition or retention of spatial refer- ing to manufacturer instructions of Revert AidTM First- encelearningandmemorywasamelioratedinMICDratsby Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Bei- EAtreatment. jing, China). The reverse transcription reaction was ampli- fied using a Bio-Rad CFX96 Detection System (Bio-Rad, Hercules, CA, USA) with the Plexor(cid:2) One-Step qRT- 3.2.EAAttenuatedLeftCortex,Hippocampus,CorpusStria- PCR System (Promega, Madison, WI, USA). For miRNA tum,andThalamusLesionsinMICDRats. Thebrainlesions amplification, the following primers were used: rno-miR- were determined by T2-weighted imaging (T2WI) (Figures 134 (RmiRQP0168, GeneCopoeia, Guangzhou, China). The 3(a)and3(c))beforeandafterEAtreatment.Thevolumesof fold change in relative mRNA and miRNA expression was brainlesionsthatincludedleftcortex,hippocampus,corpus determinedusingthe2−ΔΔCtmethodasdescribedpreviously striatum, and thalamus regions in the all groups had no [33]andU6snRNA(RQP047936,GeneCopoeia)asmRNA significantdifferencebeforeEAtreatment(𝑝>0.05,Figures andmiRNAinternalcontrol,respectively. 3(a)and3(b)). Therewasmildspontaneousrecoveryfor14 days in the brain lesions of the MICD group. The volumes of left cortex, hippocampus, corpus striatum, and thalamus 2.9. Statistical Analysis. Statistical analysis was performed lesionsshowedcomprisingapproximately23%ofthewhole with the SPSS package for Windows statistical analysis brainintheMICDgroup,whereasthebrainlesionssizeswere software (Version 18.0, SPSS, Inc., Chicago, IL, USA). The significantly reduced to 15% in the MICD+EA group (𝑝 < data from all groups were determined by one-way analysis ofvariance(ANOVA)andStudent’s𝑡-tests.Thehomogeneity 0.01, Figures 3(c) and 3(d)). The difference of brain lesions between the MICD+EA and the MICD+non-EA group of variance was analyzed using the least significant differ- showed significant changes at Day 14 after EA (𝑝 < 0.05, encemethodandmissingvarianceusingtheGames-Howell Figures3(c)and3(d)). method.Intergroupcomparisonsofthebrainlesionsvolume atdifferenttimepointswereperformedwithpaired-samples 𝑡-tests.Alldataarepresentedasmean±standarderror,and 3.3.EAIncreasedtheDensityofDendriticSpinesintheHip- the significance was regarded as at least 𝑝 < 0.05. All final pocampusofMICDRats. ToinvestigatethefunctionofEAon resultswereanalyzedinablindedmanner. synapticplasticity,dendriticspinedensityinthehippocampal neurons was analyzed by the primary basilar dendrites of Golgi-stainedpyramidalneuronsatDays14afterEA.Golgi- 3.Results Coxstainingclearlyfilledthedendriticshaftsandthespines ofneuronsfrompyramidalneurons(Figure4(a)).Hereby,the 3.1. EA Improved Cognitive Impairment in MICD Rats. To representativehippocampusshowedthatthedensityofden- assess the effect of EA on spatial reference learning and driticspineswasreducedindifferentdegreebymacroscopic memoryimpairmentsinMICDrats,theMorriswatermaze examinationintheMICDgroup,theMICD+EAgroup,and (MWM)testwasperformed.Allgroupsofratslearnedtofind MICD+non-EA group, and the loss of dendritic spines in theplatform,andthelatencytimetoreachtheplatformwas hippocampalCA1wasobviousintheMICDgroup.Thus,as reducedinthefourtrainingdays.However,learningability illustrated in Figures 4(b) and 4(c), the density of selected was significantly reduced in MICD rats compared with the Shamgroup(𝑝 < 0.01,Figure2(a));theMICDratstreated dendritic spines that derived from hippocampal CA1 was significantly decreased in the MICD group compared with withEAsignificantlytooklesstimetofindtheplatformcom- theShamgroup(𝑝<0.01);however,thedensityofdendritic paredwiththeMICDgroupandtheMICD+non-EAgroup (𝑝 < 0.01or𝑝 < 0.05,Figure2(a)).Therewasnosignificant spinesofthehippocampalCA1intheMICD+EAgroupwas difference about path length among the four groups (𝑝 > morethanthatoftheMICDgroupandtheMICD+non-EA 0.05, Figure 2(b)). As illustrated in Figures 2(c) and 2(d), group (𝑝 < 0.01). In brief, EA treatment triggered large- scaleremodelingofdendritesinthehippocampalareaCA1. tracingimagesfromtheMWMtestshowedthatinthespace explorationtestwheretheplatformwasremovedtheMICD rats passed through the original position of the platform 3.4.EAEnhancedtheNumberofHippocampusCA1Synapses fewer times than the Sham group (𝑝 < 0.01, Figure 2(d)), inMICDRats. Furthermore,weobservedtheeffectofEAon NeuralPlasticity 5 100 1200 ∗∗ 90 80 m) 1000 atency (s) 567000 #∧# ∗∗∧ ∗∗ ∗∗ avelled (c 680000 Escape l 342000 ## #∧# #∧# Distance tr 420000 10 0 0 Day 10 Day 11 Day 12 Day 13 Day 10 Day 11 Day 12 Day 13 Days after MICD Training time Sham MICD+EA Sham MICD+EA MICD MICD+non-EA MICD MICD+non-EA (a) (b) Sham MICD 7 II I II I 6 5 ## e III IV III IV m 4 e ti ∧ b 3 Pro ∗∗ II I II I 2 1 III IV III IV 0 Sham MICD MICD+EA MICD+ MICD+EA MICD+non-EA non-EA (c) (d) drant 60 adrant 3450 ∧ nt in each qua 4200 ∗∗ ## ∧ nt in target qu 31225050 ∗∗ ## pe pe 10 me s me s 5 % ti 0 % ti 0 ppositedjacentTargetdjacent ppositedjacentTargetdjacent ppositedjacentTargetdjacent ppositedjacentTargetdjacent Sham MICD MICD+EA nMoInC-DEA+ Oa a Oa a Oa a Oa a Left Right Left Right Left Right Left Right Sham MICD+EA MICD MICD+non-EA (e) (f) Figure2:EvaluationofspatialreferencelearningandmemorybyMorriswatermazetestatDays10–14afterEA.(aandb)Meanescape latencytimeandpathlengthduringtheorientationnavigationtestonDays10–13afterEAtreatment.(candd)Timestheratscrossedover previousplatformlocationonDay14afterEAtreatmentduringthespatialmemorytestindifferentgroups.(e)Thepercentageoftimesspent ineachquadrantinallprobetrialsisshownonDay14afterEAtreatment.(f)ThetimesofpassingthehiddenplatformpositiononDay14 afterEAtreatment(eachgroup;∗∗𝑝 < 0.01versustheShamgroup;##𝑝 < 0.01,#𝑝 < 0.05versustheMICDgroup;∧∧𝑝 < 0.01,∧𝑝 < 0.05 versustheMICD+EAgroup).EA:electroacupuncture.MICD:middlecerebralarteryocclusioninducedcognitivedeficits. 6 NeuralPlasticity Sham 40 %) MICD es ( 30 m u ol v 20 MICD+EA ons Lesi 10 MICD+non-EA 0 MICD MICD+EA MICD+ non-EA (a) (b) 30 Sham ∧ %) MICD es ( 20 m olu ## v MICD+EA ns o 10 esi L MICD+non-EA 0 MICD MICD+EA MICD+ non-EA (c) (d) Figure3:T2WIsignalchangesbeforeandafterEA(aandc).ThebrainlesionsweremeasuredbyT2WI(slicesfrom11to13)intheShamgroup, theMICDgroup,theMICD+EAgroup,andtheMICD+non-EAgroupbefore(a)andafter(c)EA.(bandd)Lesionvolumeisrepresented asapercentageofthetotalbrainvolumeanddataarepresentedasthemean±standarddeviationfrom12individualratsineachgroup. (∗∗𝑝 < 0.01versustheShamgroup;##𝑝 < 0.01,#𝑝 < 0.05versustheMICDgroup;∧∧𝑝 < 0.01,∧𝑝 < 0.05versustheMICD+EAgroup). T2WI:T2-weightedmagneticresonanceimaging;MICD:middlecerebralarteryocclusioninducedcognitivedeficits. ultrastructural morphology of hippocampal CA1 pyramidal increasedintheMICD+EAgroupcomparedwiththeMICD neurons. Obviously, the density of synapses in the MICD groupandtheMICD+non-EAgroup(𝑝<0.01,Figures6(a) group was decreased compared with the Sham group (𝑝 < and6(c)). 0.01,Figures5(a)and5(b)),whereastherewasamplifyingin the number of synapses in the MICD+EA group compared 3.6.EARegulatedmiR-134ExpressioninHippocampalCA1in with the MICD group and the MICD+non-EA group (𝑝 < MICDRats. Toidentifytheroleofsynaptic-dendrite-related 0.05or𝑝<0.01,Figures5(a)and5(b)). miR-134 in hippocampus CA1, the levels were detected. As Taken together, these results suggest that EA could showninFigure7,theexpressionofmiR-134wassignificantly improvesynaptic-dendriticplasticityinvivo. increasedintheMICDgroupcomparedwiththeShamgroup (𝑝 < 0.01). However, repeated EA treatment significantly 3.5. EA Promoted LIMK1 Expression and Phosphorylation of decreased the expression of miR-134 compared with the Hippocampus CA1 in MICD Rats. To explore the underly- MICDgroupandtheMICD+non-EAgroup(𝑝<0.05). ingmolecularmechanismofEA-inducedsynaptic-dendritic plasticity,thelevelsoftotalLIMK1andphospho-LIMK1(P- 4.Discussion LIMK1, Thr508) in hippocampal CA1 were investigated. As showninFigures6(a)and6(b),theexpressionoftotalLIMK1 Ischemic stroke leads to a high incidence of long-term ofhippocampalCA1wassignificantlydecreasedintheMICD cognitiveimpairment,whichisstronglyassociatedwithloss groupcomparedwiththeShamgroup(𝑝 < 0.01).However, of hippocampal neurons and synapses [34]. While review- the total LIMK1 level in the MICD+EA group was more ing ancient Chinese documents regarding acupuncture and thanthatoftheMICDgroupandtheMICD+non-EAgroup cognitive impairment-related terms, we discovered that the (𝑝 < 0.01,Figures6(a)and6(b)).Moreover,thechangesof DU20andDU24acupointswerethemostfrequentlyselected phospho-LIMK1(Thr508)ingroupsweresimilartothetotal acupointsforcognitiveimpairment-relatedrehabilitationin LIMK1 expression. The level of P-LIMK1 was significantly China.AnumberofstudieshaveshownthatEAcanimprove NeuralPlasticity 7 Sham MICD MICD+EA MICD+non-EA (a) Sham 20 m) MICD 10u 15 ## y/( ∗∗ ∧∧ nsit 10 MICD+EA de e n pi 5 S MICD+non-EA 0 Sham MICD MICD+EA MICD+ non-EA (b) (c) Figure4:Thestructuresanddensitiesofdendriticspineinthehippocampusofrats.(a)Representativeimagesofdendriticspineinthe hippocampusofeachgroup(Golgistaining,×50).(b)Representativeimagesofdendriticspinedensityandmorphologyfromthepyramidal celllayerofhippocampalCA1areaineachgroup(Golgistaining,×1000).(c)ThedensityofdendriticspinewasanalyzedinhippocampalCA1 pyramidalcellsineachgroup(𝑛 = 6/group;∗∗𝑝 < 0.01versustheShamgroup;##𝑝 < 0.01,#𝑝 < 0.05versustheMICDgroup;∧∧𝑝 < 0.01, ∧𝑝<0.05versustheMICD+EAgroup).Allexperimentswererepeatedthreetimes. learning and memory ability and stimulate consciousness lesionexhibitedamildspontaneousrecoveryperformancein [30,31,35].TheseresultsindicatedthatEAcouldbeacomple- MICDrats.However,thelesionregionscomprisingapproxi- mentary therapy for cognitive impairment after stroke. The mately23%ofthewholebrainvolumewerereducedto15%by presentstudyfoundthatEAattheDU20andDU24acupoints EAtreatment,indicatingthatEAcouldattenuatecortexand shortenedtimetofindtheplatformandincreasedthetimes hippocampus,corpusstriatum,andthalamusregionlesions ofcrossingthepositionoftheplatformcomparedtountreat- intheMICDrats.Someoftheseregions,suchasthehippoca- ment or nonacupoint EA (stimulation control) in Morris mpus and cortex, are essential for regulating learning and water maze test, suggesting that EA could improve spatial memorybehaviorsincludingspatialexploration[36].Studies reference learning and memory ability in MCAO-induced have confirmed that the hippocampus regions play a very cognitivedeficit(MICD)rats.Moreover,itisworthmention- important role in learning and memory through their spe- ingthatthepathlengthintheMorriswatermazeinthefour cific structure and location which connect with other brain groupsshowednosignificantdifference,indicatingthatthe regions together [37]. Furthermore, in order to identify the timespendingofMICDratstofindtheobjectwasnotaffected hippocampusfunctionincognitivedeficit,thehippocampal bythemotorfunction.However,themechanismsofcognitive morphologystainingwasobserved. treatmentinvolvedarefarfrombeingfullyelucidated. UsingGolgistainingandelectronmicroscopy,wedemo- Firstly, the cognitive deficit-related brain regions were nstratedthatthedensityofdendriticspineandthenumberof determined by a small animal MRI in rats with ischemic synapsesinhippocampalCA1pyramidalcellswereobviously stroke.T2-weightedimagingshowedthatMICDcausedthe reducedatDay14afterMICD.However,EAcanrescuethe lesions of left cortex, hippocampus, corpus striatum, and loss of dendritic spine and synapses in hippocampal CA1 thalamusregionsbeforeEA.Ithadbeennoticedthatthebrain region. Moreover, EA promoted synaptic-dendritic spine 8 NeuralPlasticity Sham MICD 10 ) 2 m c 8 0 1pses ( 6 # a of syn 4 ∗∗ ∧∧ er b m u 2 n e Th 0 Sham MICD MICD+EA MICD+ MICD+EA MICD+non-EA non-EA (a) (b) Figure5:ThenumberofsynapsesinhippocampalCA1pyramidalcells.(a)Representativeelectronmicrographicimagesofsynapses(arrows) intheCA1regionofeachgroup(magnification,×50000).(b)Histogramshowsthesignificantdifferenceinthemean±SEMofthenumber ofsynapsesineachgroup(𝑛 = 5/group;∗∗𝑝 < 0.01versustheShamgroup;##𝑝 < 0.01,#𝑝 < 0.05versustheMICDgroup;∧∧𝑝 < 0.01, ∧𝑝<0.05versustheMICD+EAgroup). MICD+ Sham MICD MICD+EA non-EA P-LIMK1 T-LIMK1 𝛽-Actin (a) 1.0 1.0 0.8 ## 0.8 actin 1MK 𝛽MK1/- 00..46 ∗∗ ∧∧ 1K/T-LI 00..46 ∗∗ ## ∧∧ LI M Total 0.2 P-LI 0.2 0.0 0.0 Sham MICD MICD+EA MICD+ Sham MICD MICD+EA MICD+ non-EA non-EA (b) (c) Figure6:EAamplifiedtheLIMK1levelinthehippocampusCA1pyramidalcells.(a)TheexpressionoftotalLIMK1andphosphorylated LIMK1wasevaluatedusingwesternblotting.(bandc)HistogramshowssignificantdifferenceofthelevelsofP-LIMK1andphosphor-LIMLK1 ineachgroup.Dataaremeans±SEM(𝑛 = 6/group;∗∗𝑝 < 0.01versustheShamgroup;##𝑝 < 0.01,#𝑝 < 0.05versustheMICDgroup; ∧∧𝑝<0.01,∧𝑝<0.05versustheMICD+EAgroup). NeuralPlasticity 9 12 ∗∗ plasticity of the hippocampal CA1 area, and the expression 6 U and phosphorylation of synaptic LIMK1 protein were acti- n to 10 vated to control dendritic spine number, size, and shape. pressio 8 ∧ FEuArtthreeartmmoernet,mthaeymbeecahssaoncisiamtedofwLiItMhmKi1Ri-n1c3r4e,awsehiicnhduiscleodcably- e ex 6 izedinhippocampalCA1,bynegativelyregulatingLIMK1to ativ enhancesynaptic-dendriticplasticityintherecoverystageof el 4 ischemicstroke. 4 r R-13 2 ## mi CompetingInterests 0 Sham MICD MICD+EA MICD+ The authors declare no financial or commercial conflict of non-EA interests. Figure 7: Changes of miR-134 expression in the hippocampus CA1cells.ThechangesofmiR-134expressionlevelweremeasured Authors’Contributions using quantitative RT-PCR. U6 was used as internal control for quantification of miRNAs. Data are mean ± SEM (𝑛 = 6/group; WeilinLiu,JieWu,andJiaHuangcontributedequallytothis ∗∗𝑝<0.01versustheShamgroup;##𝑝<0.01,#𝑝<0.05versusthe MICDgroup;∧∧𝑝<0.01,∧𝑝<0.05versustheMICD+EAgroup). work. Acknowledgments The present study was supported by the Natural Science regeneration. Dendritic spine is the tiny protrusions with FoundationofChina(no.81403450)andtheNaturalScience multi-densebodyandionchannelsonthesurfaceofvarious FoundationofFujianProvince(no.2016J01382). typesofneurons,whichserveascellularsubstratesofbrain connectivity and the major sites of information processing in the brain [38, 39]. Evidence is increasing that synaptic- References dendritic plasticity enables the encoding of memory [40]. 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Compared to untreatment or non-acupoint-EA treatment, EA at DU20 and DU24 . After 24 hours of MCAO surgery, rats of the Sham group brain quadrature resonator for radiofrequency transmission Detected bands were visu-.
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Hindawi Neural Plasticity Volume 2017, Article ID 9545646, 11 pages https://doi.org/10.1155/2017/9545646 Research Article Electroacupuncture Regulates Hippocampal Synaptic Plasticity via miR-134-Mediated LIMK1 Function in Rats with Ischemic Stroke WeilinLiu,1JieWu,2JiaHuang,1PeiyuanZhuo,2YunjiaoLin,2LuluWang,2 RuhuiLin,2LidianChen,1andJingTao1 1CollegeofRehabilitationMedicine,FujianUniversityofTraditionalChineseMedicine,Fuzhou,Fujian350122,China 2FujianKeyLaboratoryofRehabilitationTechnology,Fuzhou,Fujian350122,China CorrespondenceshouldbeaddressedtoLidianChen;[email protected];[email protected] Received26August2016;Accepted3November2016;Published2January2017 AcademicEditor:Cun-ZhiLiu Copyright©2017WeilinLiuetal. This is an open access article distributed under the Creative Commons Attribution License, whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. MircoRNAs(miRs)havebeenimplicatedinlearningandmemory,byregulatingLIMdomainkinase(LIMK1)toinducesynaptic- dendriticplasticity.ThestudyaimedtoinvestigatewhethermiRNAs/LIMK1signalingwasinvolvedinelectroacupuncture-(EA- ) mediated synaptic-dendritic plasticity in a rat model of middle cerebral artery occlusion induced cognitive deficit (MICD). Comparedtountreatmentornon-acupoint-EAtreatment,EAatDU20andDU24acupointscouldshortenescapelatencyand increasethefrequencyofcrossingplatforminMorriswatermazetest.T2-weightedimagingshowedthattheMICDratbrain lesionswerelocatedincortex,hippocampus,corpusstriatum,andthalamusregionsandinjuredvolumeswerereducedafterEA. Furthermore,wefoundthatthedensityofdendriticspineandthenumberofsynapsesinthehippocampalCA1pyramidalcells wereobviouslyreducedatDay14afterMICD.However,synaptic-dendriticlosscouldberescuedafterEA.Moreover,thesynaptic- dendriticplasticitywasassociatedwithincreasesofthetotalLIMK1andphospho-LIMK1levelsinhippocampalCA1region,wherein EAdecreasedtheexpressionofmiR-134,negativelyregulatingLIMK1toenhancesynaptic-dendriticplasticity.Therefore,miR-134- mediatedLIMK1wasinvolvedinEA-inducedhippocampalsynapticplasticity,whichservedasacontributortoimprovinglearning andmemoryduringtherecoverystageofischemicstroke. 1.Introduction after3years,andeachyearthemeangrowthrateisapproxi- mately8%[6].Inadditiontoconventionalcognitivetraining, Ischemicstrokeresultsinahighmortalityrateandincreased electroacupuncture (EA) is a stretch therapeutic method of disability rate all over the world [1]. Approximately 64% of acupuncture,whichistraditionalacupunctureincorporation strokepatientsareoftenfollowedwithcognitiveimpairment with modern electrotherapy. The clinical efficacy of EA on and 33% of them turn into dementia existing for several poststroke cognitive impairment has been widely demon- monthsduringdecubation[2].Cognitivedeficitsarisefrequ- strated [7, 8]. However, the functional mechanism of EA is entlyafterischemicstroke,whichcausedifficultieswithanal- farfrombeenfullyelucidated. ysis, concentration, organization, interpretation, and other Thehippocampusisapivotalstructureofthebrain;the abatesincognitivefunctionsthatbringaboutthelowquality areaplaysanimportantroleintheformationofacquisition, oflife[3,4].Thedysfunctionoflearningandmemoryisthe consolidation, and recognition of declarative and spatial cardinalsymptomofcognitiveimpairmentafterstrokeandis memory[9,10].Thelossofhippocampalsynapsesandneu- the main culprit of persistent sequelae [5]. A recent study ronsinpoststrokeinducescognitivedeficitsincludingspatial demonstrated that the incidence rate of poststroke mild reference learning and memory impairment [11, 12]. In the cognitiveimpairmentwasdiagnosedin24.4%ofindividuals formationofspatialreferencememoryiscloselyrelatedtothe 2 NeuralPlasticity plasticityofdendriticspinesandthemorphologicalchanges #FUTCM-2015008) and was performed in accordance with suchasexpansionandcontraction[13].Dendriticspinesalter the national guidelines for the care and use of laboratory their shape to make the information spreading more easily animals.Foreuthanasia,3%sodiumpentobarbital(40mg/kg and influence the synaptic efficacy (i.e., long-term potenti- bodyweight,i.p.)wasused.Middlecerebralarteryocclusion ation and long-term depression) [14, 15], which have been (MCAO) surgery was carried out under general anesthesia widelyconsideredasacellularmechanismforlearningand (1.5%isofluranein68.5%N2Oand30%O2).Alleffortswere memory [16]. LIM domain kinase (LIMK1) is enriched in madetominimizesuffering. both axonal and dendritic growth cones of hippocampal pyramidalneuronsinrats[17].LIMK1encodesaserine/thre- 2.2.ExperimentalProcedures. TheMCAO-inducedcognitive onineproteinkinasethatregulatestheactincytoskeletonby deficit(MICD)modelwasestablishedaspreviouslydescribed phosphorylatingandinactivatingtheactindepolymerization [5,30].Ratswererandomlyassignedtofourgroupsaccording factor(ADF)/cofilin[18].Inaddition,LIMK1isalsoknown totherandomnumbertable(𝑛 = 24ratseachgroup)asfol- as having an important role in synapse and dendritic spine lows:(1)Shamgroup,(2)MICDgroup,(3)MICD+EAgroup, function.Ithasbeenreportedthattheknockoutmicelacking and (4) MICD+non-EA group. The left middle cerebral LIMK1areseverelyimpairedindendriticspinemorphology arterywasoccludedbya4-0nylonmonofilament(0.23mm and hippocampal long-term potentiation [19, 20]. Evidence in diameter, Jialing-bio, China). Focal cerebral ischemia showed that LIMK1 regulated long-term memory (LTM) was monitored using transcranial temporal laser Doppler andlonglastingsynapticplasticitythroughinteractingwith (BIOPACSystems,Goleta,CA,USA)andan80%decreasein andactivatingcyclicAMPresponseelement-bindingprotein bloodflowaftertheocclusionwasrecorded.After90minof (CREB)[21]. occlusion, reperfusion was achieved by pulling out the fila- Inaddition,apotentialroleformicroRNAs(miRNAsor menttorestorebloodflow.Sham-operatedratsoftheSham miRs) in synaptic function has been particularly intriguing groupunderwentthesameprocedure,butarterialocclusion giventheevidencethatabrain-specificmiRNAcontributes wasnotperformed. tosynapticdevelopment,maturation,and/orplasticity[22]. After24hoursofMCAOsurgery,ratsoftheShamgroup miRNAsareendogenous,noncodingRNAsthatmediatethe and the MICD group received no treatment. Rats of the posttranscriptionalregulationofgeneexpressionmainlyby MICD+EAgroupweregivenEAtreatmentfor30minperday 󸀠 binding to the 3 -untranslated region of messenger RNAs for 14 consecutive days. The EA needles (diameter, 0.3mm, (mRNAs) [23]. A number of miRNAs have been isolated needlepurchasedfromHualunacupunctureofSuzhouCo., from nervous system, and a recent study has demonstrated Ltd.,SuzhouChina)wereinsertedatadepthof2-3mminto a crucial role for dynamically regulating synaptic plasticity the Baihui (DU20, located in the median of frontalis) and [24,25].Moreover,miRNAshavebeenimplicatedinhippo- Shenting(DU24,locatedinthemedianoftheparietalbone) campus-dependentfunction,whichhaveasignificantpoten- acupoints[31].StimulationwasthengeneratedusingtheEA tial in learning and memory formation, regulating LIMK1 apparatus[ModelG6805,ShanghaiHuayi(Group)Company, expressiontoinducesynaptic-dendriticplasticity[22].Den- Ltd.,Shanghai,China]andthestimulationparameterswere dritic mRNAs encode diversified functionalities in hippo- setasfollows:dilatationalwavesof1∼20Hz(adjustedtothe campal pyramidal neurons and play an important role in muscle twitch threshold), peak voltage of 6V, and 0.2mA synapticplasticity,aswellaslearningandmemory[26]. intensity[31].RatsoftheMICD+non-EAgroupweregiven Therefore,miRNA-LIMK1canbeconsideredasatarget EA treatment at the bilateral nonacupoints (located in the forcognitivedeficit.OurpreviousstudyhasshownthatEA costalregionand10mmdistaltotheiliaccrest)for30minper at Baihui (DU20) and Shenting (DU24) acupoints could dayfor14consecutivedays[32].TheEAneedlesandstimula- improve cognitive impairment through Rho GTPases to tionparametersofthebilateralnonacupointswereconsistent enhancedendriticplasticityinratswithischemicstroke[27]. withtheEAtreatment.Manipulatorswereexperiencedand Interestingly,ithasbeensuggestedthattheactivationofthe blindedtotherat’sgroup. Rho GTPases signaling is essential for LIMK1 activation by phosphorylationonthreonine508,whichiswidelyknownas 2.3.BehavioralAssessment. Behavioraltestingwasconducted masterregulatorofactindynamics[28,29].Thus,thestudy byresearcherswhowereblindedtotherat’sgroup.At10days aimed to elucidate whether EA at the DU20 and DU24 afterEA,alltheratsweresubjectedtotheMorriswatermaze acupoints could improve the cognitive deficits in rats with test (Shanghai Xinruan Information Technology Co., Ltd, ischemicstrokeviamiRNA-LIMK1-mediatedsynapticplas- Shanghai, China) to evaluate spatial reference learning and ticitytoenhancespatialreferencelearningandmemory. memory(Figure1).TheMorriswatermazeconsistedofacir- cularpool(diameter150cm,height60cm)filledwithwater 2.MaterialsandMethods (depth of 30cm and temperature of 25 ± 2∘C). A circular escape platform (diameter 12cm, height 29cm) was sub- 2.1. Animal Ethics Statement. 96 male Sprague-Dawley rats merged 2cm below the water surface, in the middle of the (weight,250±20g)wereobtainedfromShanghaiSLACLab- thirdquadrantofthepoolandthereferenceobjectsaround oratoryAnimalCo.,Ltd.(Shanghai,China,SCXK2013-0005), thepoolwereplaced.Morriswatermazetasksmainlyinclude and housed under pathogen-free conditions with a 12h orientation navigation and space exploration trials. During light/darkcycle.ThestudywasapprovedbytheCommitteeof thefirstsetoftrials,eachratwasplacedinthewaterateach FujianUniversityofTraditionalChineseMedicine(protocol of the four equidistant locations to the platform. When the NeuralPlasticity 3 Electroacupuncture T2WI Baseline: T2WI Morris water test 0 24h 1d 7d 10d 14d Sham or MCAO surgery Sacrificed Figure1:Experimentaldesignusedinthestudy. ratsarrivedattheplatformwithinthe90sectimerestriction in a mixture of A and B solutions from the kit and stored and remained on it for 3sec, they were considered to have inthedarkatroomtemperature,followingwhichtheywere ∘ foundtheplatformandwerescoredbythetimetaken/length transferredintosolutionCfromthekit,storedat4 Cfor7 oftheroute.Whentheratswereunabletofindtheplatform days. Finally, brains were then frozen and coronal sections within90sec,theywereplacedontheplatformfor10secand (150𝜇m)weremadeusingacryostat(LeicaCM3050S,Leica thetimescorewas90sec.Thetimetakenandthelengthofthe Microsystems K. K., Tokyo, Japan). The sections incubated routebywhicheachratfoundthesafeplatformwererecorded in a mixture of D and E solutions from the kit and then bythecomputer.Theaverageofthetimetakenandthelength dehydratedinalcohol(50,70,90,and100%for5mineach), oftherouteforthefourquadrantsasaresultofeachratwere clearedinxylene,andwerecover-slipped.Theimagesfinally assessedeveryday.Thedurationofthefirstsetoftrialswas were viewed under a microscope (Leica DM6000 B, Leica performedonDays10to13afterEA.Thesecondpartofthe Microsystems,Wetzlar,Germany).Theslideswerereviewed experiment was performed on Day 14, to examine the time bytwoorthreepathologistsblindtothestudy. in which rats found the location of the platform within the 90sectimerestriction,whichtestedtheirabilitytoremember 2.6. Transmission Electron Microscopy. Four rats in each the position of the platform. After all trials, the rats were groupwereanesthetizedandtheleftventriclewasperfused driedthoroughlywithahairdrierandreturnedtotheircages with200mLofsalinefollowedby400mL4%paraformalde- (GuangzhouRiboBioCo.,Ltd.,Guangzhou,China).Morris hyde (pH 7.4). The tissue was taken from the left ischemic watermazetestwasrepeatedthreetimes. 3 hippocampus, cut into 1mm size cubes, and fixed in 1% paraformaldehydewith1%lanthanumnitratetracerfor24h 2.4.MeasurementofBrainLesions. AnimalMRIscanswere followedbyfixationwith3%glutaraldehydefor24h.Samples performed on a 7.0T MRI scanner (70/20USR BioSpec, werefixedin1%osmiumtetroxidefor2handdehydratedin BrukerBiospinGmbls,Germany)usinga38mmbirdcagerat gradedethanol1%lanthanumnitratetracer(LNT)solution brainquadratureresonatorforradiofrequencytransmission andembeddedinaraldite.UltrathinhippocampalCA1slices andreception.Animalswereanesthetizedwithisoflurane/O2 were obtained (90nm); then they were stained with uranyl (with 3% induction for 5min and 1.2–1.5% maintenance in acetate and lead citrate and observed under TEM (H-7650; order to let the rats in the depth of anesthesia state) and Hitachi, Ltd., Tokyo, Japan). The photos were obtained on kept warm with circuit. After anesthesia, the rat was put hp digital CCD camera (SIS4 million voxel). Images were in prone position on a custom-made holder to minimize acquireddigitallyfromarandomlyselectedpoolof10to15 headmotion,setthelocationofheadposition,andperform fieldsundereachcondition. real-time monitor of the breathing rate and maintained in 40breaths/min.Rat’stemperatureismaintainedat33±2∘C intheprocessofscanningholdertominimizeheadmotion 2.7. Western Blotting. Isolated left hippocampus tissue was whilerespirationwasmaintained. lysedin100𝜇Lradioimmunoprecipitationassay(RIPA)buf- T2-weighted imaging (T2WI) in three planes with a fer(Beyotime,Haimen,Jiangsu,China)plusproteaseinhibi- fast spin echo (FSE) pulse sequence was first acquired to tors. Total protein (50𝜇g) was loaded into 10% SDS-PAGE controlratheadpositioning.T2WIscanwasacquiredusing gels,electrophoresed,andthentransblottedontopolyvinyli- a Rapid Acquisition with Relaxation Enhancement (RARE) dene difluoride membranes (Immobilon-P; Millipore, Bil- pulsesequencewiththefollowingparameters:fieldofview= lerica, MA) in a Tris-glycine transfer buffer. After being 32mm×32mm,matrixsize=256×256,repetitiontime(TR) blocked in 5% milk in PBST for 1h at room temperature =4200ms,echotime(TE)=35ms,slicethickness=1.0mm, with shaking, membranes were incubated with antibodies andslicegap=0mm. overnightat4∘Cagainstthefollowing:anti-LIMK1antibody Image J analysis and processing system was applied for (dilution, 1:1000; ab81046, Abcam) and anti-LIM kinase T2W images, the percentage of the brain lesions = brain 1 antibody (phospho-Thr508) (dilution, 1:1000; ab131341, lesionsvolume/wholebrainvolume×100%. Abcam) and 𝛽-actin (dilution, 1:1000; ab189073; Abcam). The following day, membranes were incubated in 5% milk 2.5. Golgi Staining. Golgi staining of brain was performed (in TBST) with an anti-goat or anti-mouse IgG antibody using an FD Rapid GolgiStain Kit following the manufac- (dilution,1:5000;PerkinElmerLifeSciences,Waltham,MA) turer’sinstructions(FDNeurotechnologies,Inc.,Columbia, for1hatroomtemperaturewithshaking.Membraneswere MD, USA). The removed rat brains were placed incubated washedaminimumoffourtimes(10minperwash)inPBST 4 NeuralPlasticity betweeneachantibodytreatment.Detectedbandswerevisu- whereas in the MICD+EA group the times where the rats alizedusingenhancedchemiluminescenceandimageswere crossedthepositionoftheplatformweresignificantlyincrea- captured using a Bio-Image system (Bio-Rad Laboratories, sed compared with the MICD group and the MICD+non- Inc., Hercules, USA). Western blotting was repeated three EA group (𝑝 < 0.01, 𝑝 < 0.05, Figure 2(d)). As illustrated times. inFigures2(e)and2(f),thepercentageoftimespentinthe target quadrant was used for statistical analysis during the probetrial.Thedateshowedthattheratsspentmoretimein 2.8.RealTimeQuantitativeRT-PCR. TheexpressionofmiR- thetargetquadrantcomparedtootherquadrants(𝑝 < 0.01; NAswasdeterminedbyrealtimequantitativereversetran- Figure 2(e)). The MICD group spent less time in the target scription polymerase chain reaction (RT-PCR). Total RNA quadrant compared with the other groups (𝑝 < 0.01; Fig- was extracted from hippocampal region of the ipsilateral ure2(e)).TheMICD+EAgroupspentmoretimeinthetarget lesion tissue using the TRIzol Reagent (Life Technologies quadrantcomparedwiththeMICDgroupandMICD+non- (AB & Invitrogen), Carlsbad, USA). Then extracted total EAgroup(𝑝 < 0.01,𝑝 < 0.05,Figure2(f)).Therefore,these RNA was reverse transcribed to generate cDNA accord- results suggest that acquisition or retention of spatial refer- ing to manufacturer instructions of Revert AidTM First- encelearningandmemorywasamelioratedinMICDratsby Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Bei- EAtreatment. jing, China). The reverse transcription reaction was ampli- fied using a Bio-Rad CFX96 Detection System (Bio-Rad, Hercules, CA, USA) with the Plexor(cid:2) One-Step qRT- 3.2.EAAttenuatedLeftCortex,Hippocampus,CorpusStria- PCR System (Promega, Madison, WI, USA). For miRNA tum,andThalamusLesionsinMICDRats. Thebrainlesions amplification, the following primers were used: rno-miR- were determined by T2-weighted imaging (T2WI) (Figures 134 (RmiRQP0168, GeneCopoeia, Guangzhou, China). The 3(a)and3(c))beforeandafterEAtreatment.Thevolumesof fold change in relative mRNA and miRNA expression was brainlesionsthatincludedleftcortex,hippocampus,corpus determinedusingthe2−ΔΔCtmethodasdescribedpreviously striatum, and thalamus regions in the all groups had no [33]andU6snRNA(RQP047936,GeneCopoeia)asmRNA significantdifferencebeforeEAtreatment(𝑝>0.05,Figures andmiRNAinternalcontrol,respectively. 3(a)and3(b)). Therewasmildspontaneousrecoveryfor14 days in the brain lesions of the MICD group. The volumes of left cortex, hippocampus, corpus striatum, and thalamus 2.9. Statistical Analysis. Statistical analysis was performed lesionsshowedcomprisingapproximately23%ofthewhole with the SPSS package for Windows statistical analysis brainintheMICDgroup,whereasthebrainlesionssizeswere software (Version 18.0, SPSS, Inc., Chicago, IL, USA). The significantly reduced to 15% in the MICD+EA group (𝑝 < data from all groups were determined by one-way analysis ofvariance(ANOVA)andStudent’s𝑡-tests.Thehomogeneity 0.01, Figures 3(c) and 3(d)). The difference of brain lesions between the MICD+EA and the MICD+non-EA group of variance was analyzed using the least significant differ- showed significant changes at Day 14 after EA (𝑝 < 0.05, encemethodandmissingvarianceusingtheGames-Howell Figures3(c)and3(d)). method.Intergroupcomparisonsofthebrainlesionsvolume atdifferenttimepointswereperformedwithpaired-samples 𝑡-tests.Alldataarepresentedasmean±standarderror,and 3.3.EAIncreasedtheDensityofDendriticSpinesintheHip- the significance was regarded as at least 𝑝 < 0.05. All final pocampusofMICDRats. ToinvestigatethefunctionofEAon resultswereanalyzedinablindedmanner. synapticplasticity,dendriticspinedensityinthehippocampal neurons was analyzed by the primary basilar dendrites of Golgi-stainedpyramidalneuronsatDays14afterEA.Golgi- 3.Results Coxstainingclearlyfilledthedendriticshaftsandthespines ofneuronsfrompyramidalneurons(Figure4(a)).Hereby,the 3.1. EA Improved Cognitive Impairment in MICD Rats. To representativehippocampusshowedthatthedensityofden- assess the effect of EA on spatial reference learning and driticspineswasreducedindifferentdegreebymacroscopic memoryimpairmentsinMICDrats,theMorriswatermaze examinationintheMICDgroup,theMICD+EAgroup,and (MWM)testwasperformed.Allgroupsofratslearnedtofind MICD+non-EA group, and the loss of dendritic spines in theplatform,andthelatencytimetoreachtheplatformwas hippocampalCA1wasobviousintheMICDgroup.Thus,as reducedinthefourtrainingdays.However,learningability illustrated in Figures 4(b) and 4(c), the density of selected was significantly reduced in MICD rats compared with the Shamgroup(𝑝 < 0.01,Figure2(a));theMICDratstreated dendritic spines that derived from hippocampal CA1 was significantly decreased in the MICD group compared with withEAsignificantlytooklesstimetofindtheplatformcom- theShamgroup(𝑝<0.01);however,thedensityofdendritic paredwiththeMICDgroupandtheMICD+non-EAgroup (𝑝 < 0.01or𝑝 < 0.05,Figure2(a)).Therewasnosignificant spinesofthehippocampalCA1intheMICD+EAgroupwas difference about path length among the four groups (𝑝 > morethanthatoftheMICDgroupandtheMICD+non-EA 0.05, Figure 2(b)). As illustrated in Figures 2(c) and 2(d), group (𝑝 < 0.01). In brief, EA treatment triggered large- scaleremodelingofdendritesinthehippocampalareaCA1. tracingimagesfromtheMWMtestshowedthatinthespace explorationtestwheretheplatformwasremovedtheMICD rats passed through the original position of the platform 3.4.EAEnhancedtheNumberofHippocampusCA1Synapses fewer times than the Sham group (𝑝 < 0.01, Figure 2(d)), inMICDRats. Furthermore,weobservedtheeffectofEAon NeuralPlasticity 5 100 1200 ∗∗ 90 80 m) 1000 atency (s) 567000 #∧# ∗∗∧ ∗∗ ∗∗ avelled (c 680000 Escape l 342000 ## #∧# #∧# Distance tr 420000 10 0 0 Day 10 Day 11 Day 12 Day 13 Day 10 Day 11 Day 12 Day 13 Days after MICD Training time Sham MICD+EA Sham MICD+EA MICD MICD+non-EA MICD MICD+non-EA (a) (b) Sham MICD 7 II I II I 6 5 ## e III IV III IV m 4 e ti ∧ b 3 Pro ∗∗ II I II I 2 1 III IV III IV 0 Sham MICD MICD+EA MICD+ MICD+EA MICD+non-EA non-EA (c) (d) drant 60 adrant 3450 ∧ nt in each qua 4200 ∗∗ ## ∧ nt in target qu 31225050 ∗∗ ## pe pe 10 me s me s 5 % ti 0 % ti 0 ppositedjacentTargetdjacent ppositedjacentTargetdjacent ppositedjacentTargetdjacent ppositedjacentTargetdjacent Sham MICD MICD+EA nMoInC-DEA+ Oa a Oa a Oa a Oa a Left Right Left Right Left Right Left Right Sham MICD+EA MICD MICD+non-EA (e) (f) Figure2:EvaluationofspatialreferencelearningandmemorybyMorriswatermazetestatDays10–14afterEA.(aandb)Meanescape latencytimeandpathlengthduringtheorientationnavigationtestonDays10–13afterEAtreatment.(candd)Timestheratscrossedover previousplatformlocationonDay14afterEAtreatmentduringthespatialmemorytestindifferentgroups.(e)Thepercentageoftimesspent ineachquadrantinallprobetrialsisshownonDay14afterEAtreatment.(f)ThetimesofpassingthehiddenplatformpositiononDay14 afterEAtreatment(eachgroup;∗∗𝑝 < 0.01versustheShamgroup;##𝑝 < 0.01,#𝑝 < 0.05versustheMICDgroup;∧∧𝑝 < 0.01,∧𝑝 < 0.05 versustheMICD+EAgroup).EA:electroacupuncture.MICD:middlecerebralarteryocclusioninducedcognitivedeficits. 6 NeuralPlasticity Sham 40 %) MICD es ( 30 m u ol v 20 MICD+EA ons Lesi 10 MICD+non-EA 0 MICD MICD+EA MICD+ non-EA (a) (b) 30 Sham ∧ %) MICD es ( 20 m olu ## v MICD+EA ns o 10 esi L MICD+non-EA 0 MICD MICD+EA MICD+ non-EA (c) (d) Figure3:T2WIsignalchangesbeforeandafterEA(aandc).ThebrainlesionsweremeasuredbyT2WI(slicesfrom11to13)intheShamgroup, theMICDgroup,theMICD+EAgroup,andtheMICD+non-EAgroupbefore(a)andafter(c)EA.(bandd)Lesionvolumeisrepresented asapercentageofthetotalbrainvolumeanddataarepresentedasthemean±standarddeviationfrom12individualratsineachgroup. (∗∗𝑝 < 0.01versustheShamgroup;##𝑝 < 0.01,#𝑝 < 0.05versustheMICDgroup;∧∧𝑝 < 0.01,∧𝑝 < 0.05versustheMICD+EAgroup). T2WI:T2-weightedmagneticresonanceimaging;MICD:middlecerebralarteryocclusioninducedcognitivedeficits. ultrastructural morphology of hippocampal CA1 pyramidal increasedintheMICD+EAgroupcomparedwiththeMICD neurons. Obviously, the density of synapses in the MICD groupandtheMICD+non-EAgroup(𝑝<0.01,Figures6(a) group was decreased compared with the Sham group (𝑝 < and6(c)). 0.01,Figures5(a)and5(b)),whereastherewasamplifyingin the number of synapses in the MICD+EA group compared 3.6.EARegulatedmiR-134ExpressioninHippocampalCA1in with the MICD group and the MICD+non-EA group (𝑝 < MICDRats. Toidentifytheroleofsynaptic-dendrite-related 0.05or𝑝<0.01,Figures5(a)and5(b)). miR-134 in hippocampus CA1, the levels were detected. As Taken together, these results suggest that EA could showninFigure7,theexpressionofmiR-134wassignificantly improvesynaptic-dendriticplasticityinvivo. increasedintheMICDgroupcomparedwiththeShamgroup (𝑝 < 0.01). However, repeated EA treatment significantly 3.5. EA Promoted LIMK1 Expression and Phosphorylation of decreased the expression of miR-134 compared with the Hippocampus CA1 in MICD Rats. To explore the underly- MICDgroupandtheMICD+non-EAgroup(𝑝<0.05). ingmolecularmechanismofEA-inducedsynaptic-dendritic plasticity,thelevelsoftotalLIMK1andphospho-LIMK1(P- 4.Discussion LIMK1, Thr508) in hippocampal CA1 were investigated. As showninFigures6(a)and6(b),theexpressionoftotalLIMK1 Ischemic stroke leads to a high incidence of long-term ofhippocampalCA1wassignificantlydecreasedintheMICD cognitiveimpairment,whichisstronglyassociatedwithloss groupcomparedwiththeShamgroup(𝑝 < 0.01).However, of hippocampal neurons and synapses [34]. While review- the total LIMK1 level in the MICD+EA group was more ing ancient Chinese documents regarding acupuncture and thanthatoftheMICDgroupandtheMICD+non-EAgroup cognitive impairment-related terms, we discovered that the (𝑝 < 0.01,Figures6(a)and6(b)).Moreover,thechangesof DU20andDU24acupointswerethemostfrequentlyselected phospho-LIMK1(Thr508)ingroupsweresimilartothetotal acupointsforcognitiveimpairment-relatedrehabilitationin LIMK1 expression. The level of P-LIMK1 was significantly China.AnumberofstudieshaveshownthatEAcanimprove NeuralPlasticity 7 Sham MICD MICD+EA MICD+non-EA (a) Sham 20 m) MICD 10u 15 ## y/( ∗∗ ∧∧ nsit 10 MICD+EA de e n pi 5 S MICD+non-EA 0 Sham MICD MICD+EA MICD+ non-EA (b) (c) Figure4:Thestructuresanddensitiesofdendriticspineinthehippocampusofrats.(a)Representativeimagesofdendriticspineinthe hippocampusofeachgroup(Golgistaining,×50).(b)Representativeimagesofdendriticspinedensityandmorphologyfromthepyramidal celllayerofhippocampalCA1areaineachgroup(Golgistaining,×1000).(c)ThedensityofdendriticspinewasanalyzedinhippocampalCA1 pyramidalcellsineachgroup(𝑛 = 6/group;∗∗𝑝 < 0.01versustheShamgroup;##𝑝 < 0.01,#𝑝 < 0.05versustheMICDgroup;∧∧𝑝 < 0.01, ∧𝑝<0.05versustheMICD+EAgroup).Allexperimentswererepeatedthreetimes. learning and memory ability and stimulate consciousness lesionexhibitedamildspontaneousrecoveryperformancein [30,31,35].TheseresultsindicatedthatEAcouldbeacomple- MICDrats.However,thelesionregionscomprisingapproxi- mentary therapy for cognitive impairment after stroke. The mately23%ofthewholebrainvolumewerereducedto15%by presentstudyfoundthatEAattheDU20andDU24acupoints EAtreatment,indicatingthatEAcouldattenuatecortexand shortenedtimetofindtheplatformandincreasedthetimes hippocampus,corpusstriatum,andthalamusregionlesions ofcrossingthepositionoftheplatformcomparedtountreat- intheMICDrats.Someoftheseregions,suchasthehippoca- ment or nonacupoint EA (stimulation control) in Morris mpus and cortex, are essential for regulating learning and water maze test, suggesting that EA could improve spatial memorybehaviorsincludingspatialexploration[36].Studies reference learning and memory ability in MCAO-induced have confirmed that the hippocampus regions play a very cognitivedeficit(MICD)rats.Moreover,itisworthmention- important role in learning and memory through their spe- ingthatthepathlengthintheMorriswatermazeinthefour cific structure and location which connect with other brain groupsshowednosignificantdifference,indicatingthatthe regions together [37]. Furthermore, in order to identify the timespendingofMICDratstofindtheobjectwasnotaffected hippocampusfunctionincognitivedeficit,thehippocampal bythemotorfunction.However,themechanismsofcognitive morphologystainingwasobserved. treatmentinvolvedarefarfrombeingfullyelucidated. UsingGolgistainingandelectronmicroscopy,wedemo- Firstly, the cognitive deficit-related brain regions were nstratedthatthedensityofdendriticspineandthenumberof determined by a small animal MRI in rats with ischemic synapsesinhippocampalCA1pyramidalcellswereobviously stroke.T2-weightedimagingshowedthatMICDcausedthe reducedatDay14afterMICD.However,EAcanrescuethe lesions of left cortex, hippocampus, corpus striatum, and loss of dendritic spine and synapses in hippocampal CA1 thalamusregionsbeforeEA.Ithadbeennoticedthatthebrain region. Moreover, EA promoted synaptic-dendritic spine 8 NeuralPlasticity Sham MICD 10 ) 2 m c 8 0 1pses ( 6 # a of syn 4 ∗∗ ∧∧ er b m u 2 n e Th 0 Sham MICD MICD+EA MICD+ MICD+EA MICD+non-EA non-EA (a) (b) Figure5:ThenumberofsynapsesinhippocampalCA1pyramidalcells.(a)Representativeelectronmicrographicimagesofsynapses(arrows) intheCA1regionofeachgroup(magnification,×50000).(b)Histogramshowsthesignificantdifferenceinthemean±SEMofthenumber ofsynapsesineachgroup(𝑛 = 5/group;∗∗𝑝 < 0.01versustheShamgroup;##𝑝 < 0.01,#𝑝 < 0.05versustheMICDgroup;∧∧𝑝 < 0.01, ∧𝑝<0.05versustheMICD+EAgroup). MICD+ Sham MICD MICD+EA non-EA P-LIMK1 T-LIMK1 𝛽-Actin (a) 1.0 1.0 0.8 ## 0.8 actin 1MK 𝛽MK1/- 00..46 ∗∗ ∧∧ 1K/T-LI 00..46 ∗∗ ## ∧∧ LI M Total 0.2 P-LI 0.2 0.0 0.0 Sham MICD MICD+EA MICD+ Sham MICD MICD+EA MICD+ non-EA non-EA (b) (c) Figure6:EAamplifiedtheLIMK1levelinthehippocampusCA1pyramidalcells.(a)TheexpressionoftotalLIMK1andphosphorylated LIMK1wasevaluatedusingwesternblotting.(bandc)HistogramshowssignificantdifferenceofthelevelsofP-LIMK1andphosphor-LIMLK1 ineachgroup.Dataaremeans±SEM(𝑛 = 6/group;∗∗𝑝 < 0.01versustheShamgroup;##𝑝 < 0.01,#𝑝 < 0.05versustheMICDgroup; ∧∧𝑝<0.01,∧𝑝<0.05versustheMICD+EAgroup). NeuralPlasticity 9 12 ∗∗ plasticity of the hippocampal CA1 area, and the expression 6 U and phosphorylation of synaptic LIMK1 protein were acti- n to 10 vated to control dendritic spine number, size, and shape. pressio 8 ∧ FEuArtthreeartmmoernet,mthaeymbeecahssaoncisiamtedofwLiItMhmKi1Ri-n1c3r4e,awsehiicnhduiscleodcably- e ex 6 izedinhippocampalCA1,bynegativelyregulatingLIMK1to ativ enhancesynaptic-dendriticplasticityintherecoverystageof el 4 ischemicstroke. 4 r R-13 2 ## mi CompetingInterests 0 Sham MICD MICD+EA MICD+ The authors declare no financial or commercial conflict of non-EA interests. Figure 7: Changes of miR-134 expression in the hippocampus CA1cells.ThechangesofmiR-134expressionlevelweremeasured Authors’Contributions using quantitative RT-PCR. U6 was used as internal control for quantification of miRNAs. Data are mean ± SEM (𝑛 = 6/group; WeilinLiu,JieWu,andJiaHuangcontributedequallytothis ∗∗𝑝<0.01versustheShamgroup;##𝑝<0.01,#𝑝<0.05versusthe MICDgroup;∧∧𝑝<0.01,∧𝑝<0.05versustheMICD+EAgroup). work. Acknowledgments The present study was supported by the Natural Science regeneration. Dendritic spine is the tiny protrusions with FoundationofChina(no.81403450)andtheNaturalScience multi-densebodyandionchannelsonthesurfaceofvarious FoundationofFujianProvince(no.2016J01382). typesofneurons,whichserveascellularsubstratesofbrain connectivity and the major sites of information processing in the brain [38, 39]. Evidence is increasing that synaptic- References dendritic plasticity enables the encoding of memory [40]. 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Compared to untreatment or non-acupoint-EA treatment, EA at DU20 and DU24 . After 24 hours of MCAO surgery, rats of the Sham group brain quadrature resonator for radiofrequency transmission Detected bands were visu-.
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