Electroacupuncture Regulates Hippocampal Synaptic Plasticity via miR-134-Mediated LIMK1 ...

12 Pages·2016·4.16 MB·English

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
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