Table Of Content

HindawiPublishingCorporation Evidence-BasedComplementaryandAlternativeMedicine Volume2013,ArticleID878521,12pages http://dx.doi.org/10.1155/2013/878521 Research Article Effect of Electroacupuncture on Rat Ischemic Brain Injury: Importance of Stimulation Duration FeiZhou,1,2JingchunGuo,3JieshiCheng,3GenchengWu,3andYingXia4,5 1ShanghaiResearchCenterforAcupunctureandMeridians,Shanghai201203,China 2GongliHospital,PudongNewDistrict,Shanghai200135,China 3ShanghaiMedicalCollegeofFudanUniversity,Shanghai200032,China 4TheUniversityofTexasMedicalSchoolatHouston,Houston,TX77030,USA 5YaleUniversitySchoolofMedicine,NewHaven,CT06520,USA CorrespondenceshouldbeaddressedtoYingXia;[email protected] Received23August2012;Revised10November2012;Accepted13December2012 AcademicEditor:DiZhang Copyright©2013FeiZhouetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,which permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. Weexploredtheoptimaldurationofelectroacupuncture(EA)stimulationforprotectingthebrainagainstischemicinjury.The experimentswerecarriedoutinratsexposedtorightmiddlecerebralarteryocclusion(MCAO)for60minfollowedby24-hr reperfusion.EAwasdeliveredto“Shuigou”(Du26)and“Baihui”(Du20)acupointswithsparse-densewave(5/20Hz)at1.0mA for5,15,30,and45min,respectively.Theresultsshowedthat30minEA,startingat5minutesaftertheonsetofMCAO(EAduring MCAO)or5minutesafterreperfusion(EAafterMCAO),significantlyreducedischemicinfarctvolume,attenuatedneurological deficits,anddecreaseddeathratewithalargerreductionoftheischemicinfarctionintheformergroup.AlsointhegroupofEA duringMCAO,thisprotectivebenefitwaspositivelyproportionaltotheincreaseintheperiodofstimulation,thatis,increased protectioninresponsetoEAfrom5-to30-minstimulation.Inallgroups,EAinducedasignificantincreaseincerebralblood flowandpromotedbloodflowrecoveryafterreperfusion,andbothbloodflowvolumeandbloodcellvelocityreturnedtothe preischemialevelinashortperiodoftime.Surprisingly,EAfor45mindidnotshowreductionintheneurologicaldeficitsor theinfarctvolumeandinsteaddemonstratedanincreaseindeathrateinthisgroup.AlthoughEAfor45minstillincreasedthe bloodflowduringMCAO,itledtoaworseningofperfusionafterreperfusioncomparedtothegroupsubjectedonlytoischemia. Theneuroprotectioninducedbyan“optimal”period(30min)ofEAwascompletelyblockedbyNaltrindole,a𝛿-opioidreceptor (DOR)antagonist(10mg/kg,i.v.).ThesefindingssuggestthatearlierEAstimulationleadstobetteroutcomes,andthatEA-induced neuroprotectionagainstischemiadependsonanoptimalEA-durationviamultiplepathwaysincludingDORsignaling,while“over- length”stimulationexacerbatestheischemicinjury. 1.Introduction ourlaboratoryandatthoseofothershaveshownthatelec- troacupuncture(EA),atappropriateacupointswithsuitable Ischemic/hypoxicbraininjury,suchasstroke,leadstoserious stimulationparameters,significantlyattenuatesneurological and complex pathophysiological changes affecting multiple deficits, infarct volume and mortality in animal models levels of the brain, and sports a high global mortality rate exposed to ischemic insults [10–17], which may potentially as the leading cause of neurological disability [1–9]. In shedanewlightondevelopingabettermodalityforischemic spite of extensive research conducted in the past several brain injury. Indeed, there is substantial clinical evidence decades, limited therapeutic options are available against demonstrating beneficial effects of acupuncture on stroke ischemic/hypoxic brain injury till date. The vast physical, patients[18–21]. emotional,andfinancialtollsthatstrokeinflictsuponpatients Because of inadequate control, poor methodological andtheirfamiliesimposesadauntingandcontinuouschal- quality and small samples seen in previous studies, the lenge to the medical community [5–9]. Previous studies at efficacy of acupuncture on hypoxic/ischemic injury is still 2 Evidence-BasedComplementaryandAlternativeMedicine unproveninclinicalsettings.Infact,majorcontroversiesexist flowandisexpressedastheproductofthenumberofmoving with regards to the effectiveness of acupuncture in stroke bloodcellsandtheirrelativevelocities. patients [22–24]. These may, at least partially, be attributed to the varied approaches adopted in different studies, and 2.3.MethodforInducingCerebralIschemia. Wefollowedthe sinceacupunctureinducescomplexchangesatmultiplelevels methodsdescribedbyLongaetal.[32]thatarealsodetailed in the central nervous system, the outcomes also differ in our previous publications [14, 17] for creating a focal with varying acupunctural methods [25–30]. Therefore, it cerebralischemiamodelbymiddlecerebralarteryocclusion is extremely important to define what optimal parameters (MCAO).Animalsweretakenunderanesthesiatosurgically arerequiredforacupuncturetreatmentinischemic/hypoxic expose their right common carotid, external carotid and braininjury. internal carotid arteries. After ligation of the distal end Towards this goal, our serial studies have established of the right external carotid artery, it was incised and a that EA treatment for acute stroke in experimental animals 4–0 monofilament nylon suture (30mm length, 0.18mm is critically dependent upon specific acupoints along with diameter with a 0.24mm diameter round tip, MONOSOF, stimulation currents of specific intensities and frequencies SN-1699G,USA)wasintroducedtotherightexternalcarotid [11–15, 17]. Since the timing and duration of medical treat- artery and further into the right internal carotid artery for menthasahugeimpactonpatientswithacutestroke[7–9], ∼20mmuptilltheoriginoftherightmiddlecerebralartery. weconductedthisworktoaddresstwofundamentalissues, Thebloodflowtotherightmiddlecerebralarterywasthen that is, (1) when should EA be applied to induce optimal occludedatthislevel. protection,duringorafterischemia?,and(2)whatduration Weconstantlymonitoredthebloodflowinallanimalsto of EA application is optimal to induce maximal beneficial ensurethatauniformcerebralischemialevelwasmaintained effectontheischemicbrain? across all groups in a standard fashion. The blood flow was controlled by adjusting the suture in the artery for the 2.MaterialsandMethods inductionofischemia.Theischemicratsthatshowedastable drop of ∼85% in PU compared to the baseline level (before 2.1. Animals. Adult male Sprague-Dawley rats (240–270g) MCAO),thatis,reachingalevelof∼15%ofthebaselinePU, were used for the experiments. All animals were purchased wereusedforfurtherexperimentation.ThePUwaskeptata with permission from the Experimental Animal Center for lowlevelwithminorfluctuationsduringtheentireischemic theShanghaiChineseAcademyofScience.Theanimalswere period without any significant change except during EA housed at an ambient temperature of 24 ± 1∘C and were application.Aftertheocclusion,reperfusionoftheischemic provided with free access to food and water. All surgical area was allowed by withdrawing the suture from the right procedures were approved by the Animals Care and Use external carotid artery. Cortical blood flow changes were CommitteesofFudanUniversityShanghaiMedicalCollege, monitored in all animals beginning 5 minutes prior to the Shanghai, China, and were performed under anesthesia inductionofcerebralischemiauntilatleast15minutesafter (chloratehydrate,400mg/kg,i.p.). reperfusion.Thisdurationwasinclusiveoftheentirelength ofMCAOandofEAaswell. Body temperature was maintained at 36.5∘C ± 0.5∘C 2.2. Cerebral Blood Flow Monitoring. The cerebral blood throughoutthesurgicalprocedurestilltheanimalrecovered flow was measured using laser-Doppler perfusion monitor from anesthesia. After the reperfusion, the animals were (LDPM, PeriFlux5000, Perimed, Sweden). The major steps housedfor24hoursatanambienttemperatureof24±1∘C. of the procedure were as follows: first, a small hole was Thesamesurgicalprocedureswereperformedontheanimals drilledintherightparietalboneatapoint1.5mmposterior inthesham-operationgroup,excludingMCAO. to the bregma and 5mm lateral to the sagittal suture, as previouslydescribed[14,17,31].Thesuperficialmicro-vessels of cerebral pia mater were accessed using a laser Doppler 2.4.ElectroacupunctureMethods. WHOstandardswerefol- probe (0.45mm diameter) inserted into the hole at a depth lowed for the name and localization of acupoints [33, 34]. of 2mm and fixed to the skull bone. The probe was used “Shuigou (Du 26)” and “Baihui (Du 20)” are acupoints to measure the blood perfusion to the cortex to record locatedontheheadandface.Shuigou(Du26)islocatedata the cerebral blood flow. Continuous monitoring of cerebral pointonthemidlineoftheupperlip2/3rdsfromthemouth blood flow was performed beginning 5 minutes prior to tothenose.Baihui(Du20)islocatedatapointonthemidline the induction of cerebral ischemia until at least 15 minutes ofthehead,approximatelymidwayonthelineconnectingthe after reperfusion. As per the manufacturer’s guidelines, the apicesofbothauricles. measured change in perfusion values was dependent on ToobtainoptimalEAeffectthatinducesmaximumpro- the number of blood cells present and their velocity in the tectionagainstcerebralischemia,weappliedEAat“Shuigou” area illuminated by the tip of the probe. The changes in (Du 26) and “Baihui” (Du 20) with 5/20Hz sparse-dense the cerebral blood flow of a localized area, recorded as a frequency at a constant intensity of ∼1.0mA in all EA measure of the dynamic changes in Perfusion Units (PU), groups,beginningat5minaftertheonsetofMCAOor5min concentration of the moving blood cells (CMBC), and the afterMCAO.Theselectionoftheacupointsandstimulation velocityofbloodcells(Velocity),wereconstantlymonitored parameters was based on the results of our serial studies in in real-time using LDPM. PU is an index of cerebral blood thepast[14,17]. Evidence-BasedComplementaryandAlternativeMedicine 3 In determination of effects of different EA periods on pushedbackwardalongwiththesignsofGrade4;Grade6— cerebral ischemia, EA was applied using varying lasting- repeated rotational motion with an immotile posterior left durations,thatis,beginningat5minaftertheonsetofMCAO limb;andGrade7—leftrecumbentpositionbecauseofbody andcontinuedupto5minutes,15minutes,30minutes,and supportingincapability. 45 minutes, respectively. Five different groups were studied inthepresentstudywiththenumbersineachgroupranging 2.7. Cerebral Infarct Measurement. After the assessment of from16to45:MCAOonly(Ischemia,𝑛 = 45),MCAOplus neurologicaldeficits,animalsweresacrificedunderanesthe- EAfor5min(EA-5min,𝑛 = 18),MCAOplusEAfor15min sia and samples of brain were taken. The brain slices were (EA-15min,𝑛=16),MCAOplusEAfor30min(EA-30min, prepared as 2.0mm sections (𝑛 = 12–16) and were incu- 𝑛=30),andMCAOplusEAfor45min(EA-45min,𝑛=30). bated in a solution of triphenyltetrazolium chloride (TTC, EAwasdeliveredthroughstainlesssteelfiliformneedles ∘ 20g/L)for30minutesat37 Cbeforebeingtransferredinto (15mm in length and 0.3mm in diameter, Suzhou Medical paraformaldehyde solution (40g/L) for fixing the infarcted Apparatus Limit Co., China) by an EA apparatus (Model area. The infarct region appeared white or pale while the G-6805-II, Shanghai Medical Instruments High-Tech Co., “normal” tissue appeared red [35, 36]. Pictures were taken China).TheneedleonDu26isinserted1mmdeep,vertical of the brain slices with a digital camera and the volume of to the plane of the skin. At Du 20, the needle is inserted infarctwasanalyzedusingacomputer-assistedimagesystem obliquely and to a depth of 2mm [14, 17, 25, 33, 34]. The with ACT-2U software (Nikon). Relative infarct ratio was intensityandfrequencyoftheoutputwaveswithanegative- calculated using the following equation [14, 17, 31]: (2 ∗ goingpulseontheposteriorborder(pulsewidth=0.5ms± lefthemispherearea(non-ischemicside)−non-infarctarea 0.1ms;componentofdirectcurrent=Zero)weremonitored of whole brain slices)/(2 ∗ left hemisphere areas) ∗ 100%. onageneraloscillograph(ModelXJ4210A,ShanghaiXinJian This equation excludes the factors that could result in an InstrumentandEquipmentCo.,China). inaccuratecalculationoftheinfarctvolume(suchasedema). 2.5. 𝛿-Opioid Receptors Blockade. The rats were randomly 2.8. Data Analysis. The cerebral blood flow was calculated fromPU,CMBC,andvelocitymeasurements.Allindividual assignedtooneofthefourexperimentalgroups:(1)Ischemia alone(𝑛 = 7);(2)Ischemia+EAat“Shuigou”(DU26)and measurementswerecomparedtothebaselinevalues,before “Baihui”(DU20)(𝑛=8);(3)Ischemia+Naltrindole(𝑛=8), MCAO (control level), in each animal. The grouped values werecomparedbetweendifferentgroups.Anaveragegrade in which rats were administered Naltrindole (10mg/kg i.v.) ofneurologicaldeficitspergroupwasusedtomakecompar- 5minbeforetheonsetofMCAO;and(4)Ischemia+EA+ Naltrindole (𝑛 = 10), in which the animals were treated isonsbetweengroups.Cerebralinfarctvolumewasexpressed asapercentfractionoftheentirecerebralvolume. with both EA and Naltrindole. EA was started 5min after All data is presented as mean ± SD and subjected to ischemia and continued to 35min after ischemia in this set statisticalanalysis.Therateofdeathwascomparedusingthe ofexperiments. Chi-square test. All other data was subjected to Analysis of variance(ANOVA),t-test,Rank-Sumtest,and/orChi-square test. The changes were considered to be significant if the 𝑃 2.6. Mortality and Neurological Deficits Monitoring. Some valuewaslessthan0.05. ischemic rats died between 2 and 20hrs after reperfusion. The death rates were reported for this period based on the numberofdeadratsandthetotalnumberoftheratsallocated 3.Results tothegivengroup.Amyriadoffactorscouldbeimplicated asthecauseofdeath,asforexamplehemorrhage.However, 3.1. Different Effects Induced by EA Starting after the Onset investigating the precise cause of death was not our aim in of MCAO versus Post-MCAO. In the group of Ischemia thiswork. (𝑛 = 45), with 1hr MCAO and 24hr reperfusion, about Assessmentsonneurologicaldeficitsweremadeonani- 18%(8/45)oftheratsdiedwithin2–20hoursaftertheonset mals in all groups. The rats that died within 24h after of reperfusion. At 24 hours of reperfusion, the living rats MCAO were excluded. Assessments were made 24h after displayedseriousneurologicaldeficits(Grade6.0±0.5,𝑛 = reperfusionjustbeforethesamplingofbraintissuewasdone. 37).TTCstainingshowedthatthevolumeofcerebralinfarct The assessments on neurological deficits were blinded, that wasaboutonethirdofthewholebrain(32.8%±3.7%,𝑛=16) is,thepersonevaluatingandscoringtheneurologicaldeficits (Table1,Figure1(a)).Theinfarctareasweremainlylocalized based on pre-set criteria had no prior knowledge on the inthefrontoparietallobesofthecortexandstriatuminthe groups and treatments. The degree of neurological deficits righthemisphere(ischemicside,Figure1(a)). wasgradedfrom0–7grades[14,17].Thecriteriaweresetas EA starting at 5min after the onset of MCAO induced follows:Grade0—“normal”,symmetricalmovementwithout a marked protection against cerebral ischemia, leading to a any abnormal sign; Grade 1—incomplete stretch of the left significantreductionofinfarctvolume,neurologicaldeficits, anteriorlimbwhenthetailwasliftedup;Grade2—doddery and death rate (Table 1, Figures 1(b)–1(d)). EA starting at crawlalongwiththesignsofGrade1;Grade3—kepttheleft 5minafterMCAOinducedasimilaroutcomeintheischemic anteriorlimbclosetothebreastwhenthetailwasliftedup; rats(refertoFigure6).However,itseemedthatEAinduced Grade4—leftturnwhencrawling;Grade5—leftanteriorclaw a larger reduction of the infarct volume in the former than 4 Evidence-BasedComplementaryandAlternativeMedicine Table1:EAperiod-dependentprotectionagainstischemicinjury. Groups Neurologicaldeficit Infarctvolume Deathrate Ischemia 6.0±0.5(5∼7)(𝑛=37) 32.8%±3.7%(𝑛=16) 18%(8outof45) EA-5min 5.0±0.5(4∼6)(𝑛=16)∗󳵳 25.6%±5.3%(𝑛=12)∗󳵳 11%(2outof18)∗∗󳵳 EA-15min 3.0±0.5(2∼4)(𝑛=15)∗∗󳵳 15.4%±4.2%(𝑛=12)∗∗󳵳 6%(1outof16)∗∗󳵳 EA-30min 1.0±0.5(0∼2)(𝑛=28)∗∗󳵳 4.9%±1.2%(𝑛=12)∗∗󳵳 7%(2outof30)∗∗󳵳 EA-45min 7.0±0.0(∼7)(𝑛=12)∗ 34.3%±2.4%(𝑛=12) 60%(18outof30)∗∗ ∗𝑃< 0.05versusIschemia.∗∗𝑃< 0.01versusIschemia.󳵳𝑃< 0.01versusEA-45min.NotethatEAsignificantlyreducedtheinfarctvolume,neurological deficit,anddeathrateandthisprotectiveeffectbecamebetterandbetterwhenthelengthofEAincreasedfrom5to30minutes.However,EAfor45minsdid notimprovetheneurologicaldeficitandinfarctvolumeandevenincreasedthedeathrate. (a) Ischemia (MCAO- (b) Ischemia+EA-5min (c) Ischemia+EA-15min (d) Ischemia+EA-30min (e) Ischemia + EA- 60min) 45min Figure1:EA-inducedchangesincerebralinfarctsizeinatime-dependentmanner.ThebrainslicesweresubjectedtoTTCstainingand theischemicinfarctvolumewasquantifiedbyacomputerizedimagesystem.Theslicesontherightofeachcolumnshowthebacksideof theleftslices.Notethattheinfarctregion(pale-whiteportion)wasmainlylocatedinthestriatumandthefrontoparietalcortexintheright hemisphere.TheMCAO-inducedinfarction(a)wassignificantlyreducedbyEAatDu20andDu26acupointsfor5min(b),15min(c),and 30min(d).Incontrast,EAfor45min(e)enablednoprotectionagainstthecerebralinfarction. the later group. EA after the onset of MCAO reduced the deficits were greatly attenuated (Grade 1.0 ± 0.5, 𝑛 = 28, infarctvolumeby∼85%(Table1andFigure1),whileEAafter 𝑃<0.01versusIschemia)andasignificantdecreaseindeath MCAO,by∼45%(refertoFigure6).Theseresultssuggestthat rate (7%, 2/30, 𝑃 < 0.01 versus Ischemia) was noted. The earlyinstitutionofEAstimulationinducesbetterprotection infarct volume was reduced by 85% (4.9% ± 1.2%, 𝑛 = 12, againstcerebralischemicinjury. 𝑃 < 0.01 versus Ischemia) (Table 1 and Figure 1(d)). In comparisontothegroupsofEAfor5minandEAfor15min, 3.2. Increased EA Protection with Increased Periods of the EAfor30mininducedmorebeneficialeffectsinallaspects Stimulation from 5 to 30mins. In the groupof MCAO plus including neurological deficits, ischemic infarct and death EAfor5min(EA-5min,𝑛 = 18),exceptfor2ratsthatdied rate(Table1,Figures1(b)–1(d)).Theseresultssuggestthatthe at 5 and 15 hours after the onset of reperfusion (11%, 2/18, EAprotectionisdependentonanappropriateEAduration. 𝑃<0.01versusIschemia),thedegreeofaverageneurological deficits in the living ischemic rats was slightly improved 3.3.ExacerbationofIschemicInjuryby“Over-Length”Stimula- (Grade 5.0 ± 0.5, 𝑛 = 16, 𝑃 < 0.05 versus Ischemia). The tionofEA. WeinvestigatedtoseeiftheEAprotectionwould infarctvolumewasslightlyreduced(25.6%+5.3%,𝑛 = 12, befurtherenhancedbyalongerdurationofEAstimulation. 𝑃 < 0.05 versus Ischemia) (Table 1 and Figure1(b)). In the Therefore,weassignedafewratstoanewgroupwithMCAO groupofMCAOplusEAfor15min(EA-15min,𝑛=16),only plusEAfor45minutes(EA-45min,𝑛=30).Tooursurprise, oneratdiedat∼3hoursaftertheonsetofreperfusion(6%, EA for 45min significantly increased the mortality in this 1/16,𝑃 < 0.01versusIschemia),withagreaterimprovement groupofischemicrats.Morethanhalfoftheanimalsinthis in average neurological deficits (Grade 3.0 ± 0.5, 𝑛 = 15, group(60%,18/30)diedwithin0.5to10hoursaftertheonset 𝑃 < 0.01versusIschemia)alongwithasignificantreduction of reperfusion. All of the dying rats manifested symptoms in infarct volume (15.4% ± 4.2%, 𝑛 = 12, 𝑃 < 0.01 versus suchasconvulsions,tumbling,piloerection,andperspiration Ischemia)(Table1andFigure1(c)).InthegroupofMCAO (wetfeathers)andotherabnormalities.Whencomparedthe plus EA for 30 min (EA-30min, 𝑛 = 30), the neurological Ischemiagroup,thedeathrateincreasedby3folds(60%,18 Evidence-BasedComplementaryandAlternativeMedicine 5 outof30,𝑃 < 0.01versusIschemia)inthisgroup.Although groups,whichwasevidentintheearlystageofreperfusion, the remaining 12 living rats survived for 24 hours after the forinstance,duringthefirst15minutesafterMCAO.Inthe reperfusion, they suffered from severe neurological deficits groups of EA-5min (𝑛 = 14), EA-15min (𝑛 = 12), and (Grade 7) and were even worse than the group with only EA-30min (𝑛 = 30), blood perfusion (PU) increased to Ischemia(Grade6.0+0.5withrangeof5∼7,𝑃 < 0.05).In over 90% of the base-value (preischemia) (Figure 3, 𝑃 < termsoftheinfarctvolume,EAfor45mindidnotreducethe 0.01 versus the ischemia group under reperfusion), CMBC infarctvolumeatall(34.3% ± 2.4%,𝑛=12,𝑃>0.05versus recovered to >80% of the preischemia level (Figure 4, 𝑃 < Ischemia). (Table 1, Figure 1(e)). These results suggest that 0.01 versus the ischemia group under reperfusion) and the anincreaseddurationofEAstimulationapplicationfurther Velocityincreasedto>80%ofthecontrollevel(Figure5,𝑃< exacerbates the ischemic insult, instead of conferring any 0.05versustheischemiagroupunderreperfusion)within15 protection. minutesafterreperfusion. Insharpcontrast,theEA-45mingroup(𝑛 = 18)showed a totally different pattern. For example, PU maintained at 3.4. EA-Induced Increase in Cerebral Blood Flow during a similar level as that during MCAO (∼15%), that is, after MCAO. UsingaLaser-DopplerPerfusionMonitor,wecon- reperfusion,itslevelwasevenlowerthanthoseofIschemia tinuouslymonitoredtherealtimechangesincerebralblood alone (∼25%, 𝑃 < 0.05 versus that during reperfusion in flow in all the groups beginning at 5min prior to MCAO the group of Ischemia plus EA for 45 minutes) (Figure 3). till15minaftertheonsetofreperfusion(suturewithdrawal), Similarly, CMBC still maintained at a low level (∼20%) and compared differences in the cerebral blood flow at the after reperfusion and the level was even lower than that pre-MCAOlevel,duringMCAOwith/withoutEA,andearly (50%)oftheIschemiagroupduringreperfusion(𝑃 < 0.01, stagesofreperfusionbetweenvariousgroups(Figures2,3,4, Figure4).ThesefindingssuggestthatEAfor30minutesor and5). lessincreasesthebloodflowduringMCAOandpromotesthe Consistent with our previous studies [14, 17], after the recoveryofthebloodflowduringreperfusion,whilean“over- insertion of a nylon suture into the right middle cerebral length” (45min) stimulation, though increasing the blood artery, the blood perfusion (PU) to the monitored cortex flow during MCAO, retards the recovery of the blood flow immediately decreased by ∼85% (i.e., reaching the level at tothebrainafterMCAO(Figures2(e)and3–5). ∼15% of the pre-MCAO level). During the entire period of MCAO,thelocalbloodflowwasmaintainedatthislowlevel with a minor fluctuation. CMBC dropped by ∼80% of the 3.6. DOR Antagonist Attenuated EA-Induced Protection base-value (i.e., reaching a level at ∼20% of the pre-MCAO against Ischemic Infarction. We previously found that level)withaslightdecelerationofbloodcellvelocity(reduced microinjection of DOR antagonist into the lateral cerebral by ∼25% of the pre-MCAO level). These changes indicated ventricle largely attenuated the EA protection against thatMCAOinducedagreaterreductioninthebloodvolume cerebral ischemic injury [10]. To further reaffirm this ascomparedtothevelocityofbloodcells(Figures2(a)and observation,weappliedNaltrindoletotheischemicratsand 3–5). determined its effect on the EA-induced neuroprotection. InalltheEAgroups,EAinducedaninstantandsignif- The results showed that in the ischemia group (𝑛 = 7) icant increase in the blood flow of the ischemic brain. The the infarct size reduced by ∼40% (from 24.8% ± 4.1% to bloodflowchangedisochronouslywiththecurrentimpulse 14.7% ± 3.4%, 𝑃 < 0.05) following treatment with EA in response to EA stimulation (Figures 2(b)–2(e) and 3–5). beginning immediately after MCAO (𝑛 = 8). Although EAinducedanincreaseinPUto∼30%ofthecontrol(𝑃 < Naltrindole (10mg/kg, i.v.) application did tend to increase 0.01 versus MACO without EA) (Figure 3) and CMBC to theinfarctvolume,thechangewasnotstatisticallysignificant over 65% of the control (𝑃 < 0.01 versus MCAO without (30.4% ± 4.3%, 𝑛 = 8, 𝑃 > 0.05 versus Ischemia alone). EA) (Figure 4) with a simultaneous decrease in the blood However,itcompletelyabolishedtheEA-inducedprotection cellvelocityto∼55%ofthecontrol(𝑃 < 0.05versusMCAO againstischemicinfarction(23.6%±5.2%,𝑛 = 10,𝑃 > 0.05 without EA) (Figure 5). These results suggest that both versus Ischemia + EA) (Figure 6). These results indicate “appropriate”and“over”periodsofEAstimulationincrease that 𝛿-opioid receptor plays a very important role in the thebloodflowduringMCAO. EA-inducedprotectionagainstischemicinjury. 3.5. Differential Recovery of the Cerebral Blood Flow after 4.Discussion ReperfusionamongEAGroups. IntheIschemiagroup(𝑛 = 30), following the withdrawal of the nylon suture after 1h Thisisthefirststudytosystematicallydefineoptimalduration MCAO,PUgraduallyincreasedto∼25%ofthecontrollevel ofEAstimulationtoinducebeneficialeffectsagainstischemic (pre-MCAO) (𝑃 < 0.05 versus during MCAO) and CMBC infarction, neurological deficits and mortality. Our results increased to ∼50% of the control (𝑃 < 0.01 versus during show that EA given for 5–30 minutes significantly reduces MCAO)withthevelocityofbloodcellsbeingreducedfrom ischemic infarct volume and decreases neurological deficits ∼75%to∼55%(𝑃<0.05,versusduringMCAO)inthefirst15 andmortalityrate.Ontheotherhand,EAgivenfor45min minutesafterthebloodreperfusion(Figures2(a)and3–5). did not show reduction in the neurological deficits or the EAchangedthepatternofthebloodreperfusion.There infarctvolume,andratherdemonstratedanincreaseindeath was, however, a significant difference among different EA rate in this group. However, all groups showed an increase 6 Evidence-BasedComplementaryandAlternativeMedicine MCAO Rep. 100 U P 15 C 100 B M C 20 y ocit10705 Vel 50 5 min (a) Effectof60minMCAOonthebloodflow MCAO Rep. 100 EA U P 15 C 100 B M C 20 city10705 o Vel 50 5 min (b) EffectofEA-5minonthebloodflowunderischemia MCAO Rep. 100 U EA P 15 C100 B M C 20 city17050 o Vel 50 5 min (c) EffectofEA-15minonthebloodflowunderischemia MCAO Rep. 100 EA U P 15 C B100 M C 20 y cit100 o 75 Vel 50 5 min (d) EffectofEA-30minonthebloodflowunderischemia MCAO Rep. 100 U EA P 15 C B100 M C 20 y cit100 o 75 Vel 50 5 min (e) EffectofEA-45minonthebloodflowunderischemia Figure2:RepresentativetracerecordingsofthebloodflowintheratswithorwithoutEA.BloodPerfusion(PU),ConcentrationofMoving BloodCells(CMBC),andVelocityofBloodCells(Velocity)weremeasuredintheischemicratsbyaLaserDopplerPerfusionMonitorsystem. (a)EffectofMCAO-60minonCBFduringischemiaandafterreperfusioninIschemiagroup.(b)EffectofEAfor5minatacupointsofDu 20andDu26onCBF.(c)EffectofEAfor15minonCBF.(d)EffectofEAfor30minonCBF.(e)EffectofEAfor45minonCBF.Notethatthe PUandCMBCdecreasedimmediatelyafterMCAOandthebloodflowwasmaintainedatalowlevelwithsmallfluctuationsduringtheentire periodofMCAOwithaslightdecreaseinthevelocity.Aftertheonsetofreperfusion,PUandCMBCincreasedwithafurtherdecreaseinthe velocity.EAinducedanisochronousincreaseinPUandCMBCwithadecreaseinvelocityduringMCAO.Afterreperfusion,PU,CMBC, andvelocityallincreasedrapidlyandreachedthebaselinevalueswithin15minafterreperfusiononsetingroupsofEAfor5–30min.EAfor 45min,however,inducedagreaterdecreaseinPU,CMBC,andvelocitythanthechangesobservedintheIschemiagroupafterreperfusion. Evidence-BasedComplementaryandAlternativeMedicine 7 Relative changes in amount of blood perfusion (% of base-value before MCAO)1147659832110000000000000 MC∗AEOA∗ R#e#p#e#rfu𝛁#sion𝛁# Relative changes in amount of blood perfusion (% of base-value before MCAO)1110987653214000000000000 MC∗A∗O∗EA∗ ∗ #R#ep##erfu𝛁#sio𝛁n# −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (minutes) Time (minutes) Ischemia (𝑛=30) Ischemia (𝑛=30) Ischemia+EA-5 min (𝑛=14) Ischemia+EA-15 min (𝑛=12) (a) (b) n 110 n 110 o o perfusi AO)10900 #### # # perfusi O)19000 d C d A Relative changes in amount of bloo (% of base-value before M 87653214000000000 MC∗AO∗∗ ∗ ∗E∗A ∗ ∗ ∗ Reperf𝛁usio𝛁n Relative changes in amount of bloo (% of base-value before MC 87653214000000000 MC∗AO∗∗ ∗∗∗ ∗EA∗∗∗ ∗ ∗ ∗ Reperfu𝛁sio𝛁n −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (minutes) Time (minutes) Ischemia (𝑛=30) Ischemia (𝑛=30) Ischemia+EA-30 min (𝑛=30) Ischemia+EA-45 min (𝑛=18) (c) (d) Figure3:StatisticalsummaryofEAeffectsonPUintheischemicbrain.∗𝑃<0.01,Ischemia+EAversusIschemiagroupduringMCAO;∇𝑃< 0.05,underreperfusionversusduringMCAOintheIschemiagroup; #𝑃<0.01,Ischemia + EAversusIschemiagroupunderreperfusion; 󳶃𝑃 < 0.05,Ischemia+EAversusIschemiagroupunderreperfusion.NotethatMCAOsharplydecreasedPUto∼15%ofthebasalvalue (pre-MCAO),whileEAfor5–45minsignificantlyincreasedthebloodflowto∼30%ofthebaseline.Within10–15minutesafterreperfusion, PUgraduallyincreasedto∼25%ofthebaseline(pre-MCAO)intheIschemiagroup,whereasitrapidlyincreasedtoabout90%ofthebaseline intheEAfor5–30minutesgroups.Insharpcontrast,PUwasstillattheMCAOlevel,thatis,∼15%ofthebasalvalue,duringthesameperiod afterreperfusioninthegroupofEAfor45minutes. in blood flow after EA stimulation during the period of reduce the cerebral ischemic injury. Our previous studies MCAO. These findings are consistent with our preliminary concluded that EA for 30 minutes increases the blood flow observations[37]andsuggestthattheEAprotectionagainst duringMCAOandreducesischemicinsulttothebrain[11,12, ischemicinjuryisdependentonthelengthofEAstimulation, 14,17].Inextension,wespeculatedthatanextendedperiodof andthatan“over-length”stimulationcanpotentiallyaddto EAstimulationwouldproducebetteroutcomes.Apparently, theischemicinjuryinsteadofprotectingthebrainfromthe thiswasnotthecaseasweobservedoppositeresults;thatis,a injury. prolongedstimulationofEApotentiates,ratherthanprotects Thisobservationwasbeyondourexpectations.Sincethe against, cerebral damage during ischemic injury to brain. primarycauseofischemicbraininjuryisinsufficient blood Interestingly, Dr. Wang et al. [38] also observed a similar supplytothebrain,increasedbloodflowtothebrainshould phenomenonintheirstudiesonEA-inducedhypoalgesiain 8 Evidence-BasedComplementaryandAlternativeMedicine 110 110 100 100 Relative changes in CMBC (% of base-value before MCAO) 9876532400000000 ∗EA∗ ## # # Relative changes in CMBC % of base-value before MCAO) 9876532400000000 ∗ ∗∗EA∗ ∗ # ## # # ( 10 10 MCAO Reperfusion MCAO Reperfusion 0 0 −5 0 5 10 1520 25 30 35 40 45 50 55 60 65 70 75 80 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (minutes) Time (minutes) Ischemia (𝑛=30) Ischemia (𝑛=30) Ischemia+EA-5 min (𝑛=14) Ischemia+EA-15 min (𝑛=12) (a) (b) 110 110 100 100 Relative changes in CMBC (% of base-value before MCAO) 9876532400000000 ∗ ∗∗ ∗ ∗E∗A ∗ ∗ ∗ 𝛁#𝛁#𝛁# 𝛁# 𝛁# Relative changes in CMBC (% of base-value before MCAO) 9876532400000000 ∗ ∗∗ ∗ ∗∗ ∗EA∗ ∗∗ ∗ ∗ ∗ 𝛁##𝛁𝛁# 𝛁# #𝛁 10 10 MCAO Reperfusion MCAO Reperfusion 0 0 −5 0 5 10 15 2025 30 35 40 45 50 55 60 65 70 75 80 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (minutes) Time (minutes) Ischemia (𝑛=30) Ischemia (𝑛=30) Ischemia+EA-30 min (𝑛=30) Ischemia+EA-45 min (𝑛=18) (c) (d) Figure4:StatisticalsummaryofEAeffectsonconcentrationofMovingBloodCells.∗𝑃<0.01,Ischemia+EAversusIschemiagroupduring MCAO;∇𝑃 < 0.01,underreperfusionversusduringMCAOinIschemiagroup; #𝑃 < 0.01,Ischemia+EAversusIschemiagroupunder reperfusion.NotethattheMCAOgreatlyreducedtheconcentrationofMovingBloodCells(CMBC)to∼20%ofthebaselineandCMBC quicklyincreasedto∼50%ofthebaselineafterreperfusion.EAfor5–45minutessignificantlyincreasedCMBCduringMCAOto∼65%of thebaseline.Within15minutesafterreperfusion,CMBCfurtherincreasedto∼80%ofthebaselineintheEAfor5–30minutesgroups,but notintheEAfor45minutesgroupwithitslevelbeingsimilartothatduringMCAO. healthyvolunteers.Thesubjectswererandomizedtoreceive At present, the mechanism behind this phenomenon is different durations (0min, 20min, 30min, or 40min) of not known. In this work, however, it is noteworthy that asynchronousEAstimulationsandthensubjectedtothetest EAfor5–30minutessignificantlyimprovesthereperfusion ofhypoalgesiausingahumanexperimentalcoldthermalpain after MCAO and favors quick recovery of blood flow to threshold model. They found that 30 min of asynchronous the pre-ischemic level, in addition to increasing blood flow EA stimulation resulted in the most significant hypoalgesic duringMCAO.Insharpcontrast,EAfor45minutesdoesnot effectcomparedwith0,20,or40minstimulations.Therefore, promotetherecoveryofbloodflowafterMCAOatall,butto itseemsthata30minperiodisthemostoptimaldurationfor thecontrary,furtherworsensitasshowninourpresentwork. EA-inducedanalgesiaandbrainprotectionagainstischemic Thisphenomenonpartiallyaccountsforthedifferenceinthe injury. outcomesbetween“optimalperiod”and“over-length”inthe Evidence-BasedComplementaryandAlternativeMedicine 9 110 110 100 100 city AO) 90 EA ## # # # city AO) 90 EA ## # # # velo MC 80 velo MC 80 ge in blood alue before 765000 ∗ ∗ 𝛁𝛁𝛁 𝛁 𝛁 ge in blood alue before 765000 ∗∗∗ ∗ ∗ 𝛁𝛁𝛁 𝛁 𝛁 n v n v elative cha % of base- 324000 elative cha % of base- 324000 R ( R ( 10 10 MCAO Reperfusion MCAO Reperfusion 0 0 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 7075 80 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (minutes) Time (minutes) Ischemia (𝑛=30) Ischemia (𝑛=30) Ischemia+EA-5 min (𝑛=14) Ischemia+EA-15 min (𝑛=12) (a) (b) 110 110 100 100 city O) 90 EA ## # # # city AO) 90 EA ## # # # nge in blood velo alue before MCA 87650000 ∗ ∗∗ ∗ ∗∗ ∗ ∗ ∗ 𝛁𝛁𝛁 𝛁 𝛁 nge in blood velo value before MC 87650000 ∗ ∗∗ ∗ ∗∗ ∗ ∗ ∗ ∗ ∗ ∗ 𝛁𝛁 𝛁 𝛁 𝛁 Relative cha % of base-v 324000 Relative cha (% of base- 324000 ( 10 10 MCAO Reperfusion MCAO Reperfusion 0 0 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (minutes) Time (minutes) Ischemia (𝑛=30) Ischemia (𝑛=30) Ischemia+EA-30 min (𝑛=30) Ischemia+EA-45 min (𝑛=18) (c) (d) Figure5:StatisticalsummaryofEAeffectsonthevelocityofbloodcells.∗𝑃 < 0.05,Ischemia+EAversusIschemiagroupduringMCAO; ∇𝑃<0.05,underreperfusionversusduringMCAOinIschemiagroup; #𝑃<0.05,Ischemia+EAversusIschemiagroupunderreperfusion. NotethatMCAOreducedthevelocityofthebloodcellsto∼75%ofthebaseline,whichwasfurtherdecreasedto∼55%ofthebaselineafter reperfusion.InallEAgroups,EAduringMCAOimmediatelydecreasedthevelocityto∼55%ofthebaseline,andinducedasignificant increaseinitafterreperfusion.IncontrasttochangesinPUandCMBC,thevelocityincreasedto>80%afterreperfusioninallEAgroups includingEA-45min. EA treatment although the underlying mechanism remains mobilization of various survival signals and activation of unknown. protectivepathwaysintheischemicbrain. The fact that “over-length” of EA stimulation increases One of these pathways could involve DOR-mediated the blood flow under MCAO, but does not lead to any survival signaling. We have previously demonstrated that protection against ischemic injury strongly suggests that DORisimportantininducingneuroprotection[3,6].DOR optimal EA stimulation induces a multi-level regulation in activationattenuateshypoxic,ischemic,orexcitatoryinjury the brain via a complex mechanism. A single factor, such in the neurons and the brain [1, 3, 6]. Evidence shows as,anincreaseinthebloodflowunderischemicconditions, thatbothmanualacupunctureandEAenhancetheactivity may not be enough to induce protection against ischemia. of the endogenous opioid system, including DOR, in the Appropriatestimulationisextremelyimportantformaximal brain [26–29, 39]. Therefore, the DOR-mediated protection 10 Evidence-BasedComplementaryandAlternativeMedicine 40 duringMCAO,cansetthisbalanceofcombinedprotection off. 35 Moreover, our present results show that EA application during MCAO induces a stronger protection than EA after n) 30 MCAO,suggestingthatanearlyinstitutionofEAtreatment ai isrequiredtoachievebetterneuroprotectionagainstischemic br e injury.ThisissimilartothegoalsoftPAtreatmentinterms ol 25 h of the importance of the therapeutic window [7, 8]. An w of earlymobilizationofprotectivepowermaypreventneurons % 20 e ( ∗ frominitiatingtheprocessofapoptosis/necrosisinthebrain. m “Timeisbrain,”whichisalsotrueinthecaseofEAtreatment u ol 15 forischemicbraininjury. v ct Insummary,ourresultssuggestearlyEAtreatmentwith ar nf 10 optimalstimulationparametersforanappropriateperiodis I critical to achieve a therapeutic effect for neuroprotection againstischemicinsult.Thepreviouscontroversiesinthelit- 5 eratureonEAtreatmentforstrokecanbepartiallyattributed tothedifferencesinconditionsofthepatientsandEAparam- 0 eters.Furtherin-depthinvestigationsintothe“optimal”EA parametersanditsunderlyingmechanismscanhelpimprove clinicaloutcomesofpatientswithischemic/hypoxicinjuryin Ischemia Ischemia+N thebrain. Ischemia+EA Ischemia+EA+N Acknowledgments Figure6:EffectofNaltrindoleonEA-inducedprotectionagainst ischemicinfarctsize.Quantitativevolumesoftheischemicinfarct weredeterminedbasedonthepercentageofthewholebrainvolume. This work was supported by NSFC (30672721, 30873318), Note that the ischemia-induced infarct was reduced by the EA NBRP (2009CB522901; 2012CB518502), STCSM stimulation(∗𝑃 < 0.05versusischemiagroup),whichwasalmost (10DZ1975800), NIH (AT-004422 and HD-034852), and completelyreversedaftertreatmentwithNaltrindole(N,10mg/kg, VivianL.SmithNeurologicFoundation. i.v.)(∗𝑃<0.05versusIschemia+EA+Naltrindole). References [1] J. H. Sung, D. M. Chao, and Y. Xia, “Neuronal responses to might play a very important role in the EA-induced pro- hypoxia,” in New Frontiers in Neurological Research, W. Ying tection against ischemic injury. Indeed, we first reported andD.Wang,Eds.,pp.73–153,ResearchSignpost,Kerala,India, that microinjection of a DOR antagonist, Naltrindole, into 2008. the cerebral ventricle attenuated the EA-induced cerebral [2] Y. Luo, “Cell-based therapy for stroke,” Journal of Neural protection against ischemic infarction, suggesting DOR’s Transmission,vol.118,no.1,pp.61–74,2011. involvement in the EA-induced protection against brain [3] D. Chao and Y. Xia, “Ionic storm in hypoxic/ischemic stress: ischemia[10].Furthermore,ourstudiesandthoseofothers canopioidreceptorssubsideit?”ProgressinNeurobiology,vol. suggestthatEAsignificantlyincreasesthedensityofDORin 90,no.4,pp.439–470,2010. the ischemic cortex and that DOR signaling could mediate [4] K.Arai,J.Lok,S.Guo,K.Hayakawa,C.Xing,andE.H.Lo, theEAinducedneuroprotectionagainstischemicinjury[15, “Cellularmechanismsofneurovasculardamageandrepairafter 39, 40]. In the present study, we observed that intravenous stroke,”JournalofChildNeurology,vol.26,pp.1193–1198,2011. injection of Naltrindole almost completely blocked the EA- [5] J.H.Zhang,J.Badaut,J.Tang,A.Obenaus,R.Hartman,and inducedneuroprotectionagainstischemicinjury.Allofthese W.J.Pearce,“Thevascularneuralnetwork-anewparadigmin observations consistently favor the critical role of DOR in stroke pathophysiology,” Nature Review/Neurology, vol. 8, no. EA-induced neuroprotection against cerebral ischemia. On 12,pp.711–716,2012. theotherhand,EAcanactivateadditionalneurotransmitter [6] X. Z. He, H. K. Sandhu, Y. L. Yang et al., “Neuroprotection systemssuchasneurotrophicfactorsinthebrainthatenable againsthypoxia/ischemia:delta-opioidreceptor-mediatedcel- furtherprotectionunderischemicconditions[13]. lular/molecular events,” Cellular and Molecular Life Sciences, Thus,EAtargetsmultiplemechanismstoachieveapro- 2012. tective effect against ischemic insults, including an increase [7] R.J.Thurman,E.C.Jauch,P.D.Panagos,M.R.Reynolds,andJ. in the cerebral blood flow under ischemia, an improved Mocco,“Fourevolvingstrategiesintheemergenttreatmentof recoveryofthebloodflowafterreperfusion,upregulationof acuteischemicstroke,”EmergencyMedicinePractice,vol.14,no. the DOR pathway, and other survival signals. An optimal 7,pp.1–27,2012. duration of EA stimulation can combine and direct all of [8] K.A.WeantandS.N.Baker,“Newwindows,sameoldhouse: these protective pathways against ischemic stress, while an anupdateonacutestrokemanagement,”AdvancedEmergency over-stimulation of EA, despite increasing the blood flow NursingJournal,vol.34,pp.112–121,2012.

Description:
EA was delivered to “Shuigou” (Du 26) and “Baihui” (Du 20) acupoints with sparse-dense wave (5/20 Hz) at 1.0 mA for 5, 15 In all groups, EA induced a significant increase in cerebral blood 73–153, Research Signpost, Kerala, India, . Nomenclature for Acupuncture, World Health Organizatio
ebook img

Effect of Electroacupuncture on Rat Ischemic Brain Injury: Importance of Stimulation Duration PDF

13 Pages·2015·3.22 MB·English
by  
Save to my drive
Quick download
Download
Upgrade Premium
Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.

Preview Effect of Electroacupuncture on Rat Ischemic Brain Injury: Importance of Stimulation Duration

HindawiPublishingCorporation Evidence-BasedComplementaryandAlternativeMedicine Volume2013,ArticleID878521,12pages http://dx.doi.org/10.1155/2013/878521 Research Article Effect of Electroacupuncture on Rat Ischemic Brain Injury: Importance of Stimulation Duration FeiZhou,1,2JingchunGuo,3JieshiCheng,3GenchengWu,3andYingXia4,5 1ShanghaiResearchCenterforAcupunctureandMeridians,Shanghai201203,China 2GongliHospital,PudongNewDistrict,Shanghai200135,China 3ShanghaiMedicalCollegeofFudanUniversity,Shanghai200032,China 4TheUniversityofTexasMedicalSchoolatHouston,Houston,TX77030,USA 5YaleUniversitySchoolofMedicine,NewHaven,CT06520,USA CorrespondenceshouldbeaddressedtoYingXia;[email protected] Received23August2012;Revised10November2012;Accepted13December2012 AcademicEditor:DiZhang Copyright©2013FeiZhouetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,which permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. Weexploredtheoptimaldurationofelectroacupuncture(EA)stimulationforprotectingthebrainagainstischemicinjury.The experimentswerecarriedoutinratsexposedtorightmiddlecerebralarteryocclusion(MCAO)for60minfollowedby24-hr reperfusion.EAwasdeliveredto“Shuigou”(Du26)and“Baihui”(Du20)acupointswithsparse-densewave(5/20Hz)at1.0mA for5,15,30,and45min,respectively.Theresultsshowedthat30minEA,startingat5minutesaftertheonsetofMCAO(EAduring MCAO)or5minutesafterreperfusion(EAafterMCAO),significantlyreducedischemicinfarctvolume,attenuatedneurological deficits,anddecreaseddeathratewithalargerreductionoftheischemicinfarctionintheformergroup.AlsointhegroupofEA duringMCAO,thisprotectivebenefitwaspositivelyproportionaltotheincreaseintheperiodofstimulation,thatis,increased protectioninresponsetoEAfrom5-to30-minstimulation.Inallgroups,EAinducedasignificantincreaseincerebralblood flowandpromotedbloodflowrecoveryafterreperfusion,andbothbloodflowvolumeandbloodcellvelocityreturnedtothe preischemialevelinashortperiodoftime.Surprisingly,EAfor45mindidnotshowreductionintheneurologicaldeficitsor theinfarctvolumeandinsteaddemonstratedanincreaseindeathrateinthisgroup.AlthoughEAfor45minstillincreasedthe bloodflowduringMCAO,itledtoaworseningofperfusionafterreperfusioncomparedtothegroupsubjectedonlytoischemia. Theneuroprotectioninducedbyan“optimal”period(30min)ofEAwascompletelyblockedbyNaltrindole,a𝛿-opioidreceptor (DOR)antagonist(10mg/kg,i.v.).ThesefindingssuggestthatearlierEAstimulationleadstobetteroutcomes,andthatEA-induced neuroprotectionagainstischemiadependsonanoptimalEA-durationviamultiplepathwaysincludingDORsignaling,while“over- length”stimulationexacerbatestheischemicinjury. 1.Introduction ourlaboratoryandatthoseofothershaveshownthatelec- troacupuncture(EA),atappropriateacupointswithsuitable Ischemic/hypoxicbraininjury,suchasstroke,leadstoserious stimulationparameters,significantlyattenuatesneurological and complex pathophysiological changes affecting multiple deficits, infarct volume and mortality in animal models levels of the brain, and sports a high global mortality rate exposed to ischemic insults [10–17], which may potentially as the leading cause of neurological disability [1–9]. In shedanewlightondevelopingabettermodalityforischemic spite of extensive research conducted in the past several brain injury. Indeed, there is substantial clinical evidence decades, limited therapeutic options are available against demonstrating beneficial effects of acupuncture on stroke ischemic/hypoxic brain injury till date. The vast physical, patients[18–21]. emotional,andfinancialtollsthatstrokeinflictsuponpatients Because of inadequate control, poor methodological andtheirfamiliesimposesadauntingandcontinuouschal- quality and small samples seen in previous studies, the lenge to the medical community [5–9]. Previous studies at efficacy of acupuncture on hypoxic/ischemic injury is still 2 Evidence-BasedComplementaryandAlternativeMedicine unproveninclinicalsettings.Infact,majorcontroversiesexist flowandisexpressedastheproductofthenumberofmoving with regards to the effectiveness of acupuncture in stroke bloodcellsandtheirrelativevelocities. patients [22–24]. These may, at least partially, be attributed to the varied approaches adopted in different studies, and 2.3.MethodforInducingCerebralIschemia. Wefollowedthe sinceacupunctureinducescomplexchangesatmultiplelevels methodsdescribedbyLongaetal.[32]thatarealsodetailed in the central nervous system, the outcomes also differ in our previous publications [14, 17] for creating a focal with varying acupunctural methods [25–30]. Therefore, it cerebralischemiamodelbymiddlecerebralarteryocclusion is extremely important to define what optimal parameters (MCAO).Animalsweretakenunderanesthesiatosurgically arerequiredforacupuncturetreatmentinischemic/hypoxic expose their right common carotid, external carotid and braininjury. internal carotid arteries. After ligation of the distal end Towards this goal, our serial studies have established of the right external carotid artery, it was incised and a that EA treatment for acute stroke in experimental animals 4–0 monofilament nylon suture (30mm length, 0.18mm is critically dependent upon specific acupoints along with diameter with a 0.24mm diameter round tip, MONOSOF, stimulation currents of specific intensities and frequencies SN-1699G,USA)wasintroducedtotherightexternalcarotid [11–15, 17]. Since the timing and duration of medical treat- artery and further into the right internal carotid artery for menthasahugeimpactonpatientswithacutestroke[7–9], ∼20mmuptilltheoriginoftherightmiddlecerebralartery. weconductedthisworktoaddresstwofundamentalissues, Thebloodflowtotherightmiddlecerebralarterywasthen that is, (1) when should EA be applied to induce optimal occludedatthislevel. protection,duringorafterischemia?,and(2)whatduration Weconstantlymonitoredthebloodflowinallanimalsto of EA application is optimal to induce maximal beneficial ensurethatauniformcerebralischemialevelwasmaintained effectontheischemicbrain? across all groups in a standard fashion. The blood flow was controlled by adjusting the suture in the artery for the 2.MaterialsandMethods inductionofischemia.Theischemicratsthatshowedastable drop of ∼85% in PU compared to the baseline level (before 2.1. Animals. Adult male Sprague-Dawley rats (240–270g) MCAO),thatis,reachingalevelof∼15%ofthebaselinePU, were used for the experiments. All animals were purchased wereusedforfurtherexperimentation.ThePUwaskeptata with permission from the Experimental Animal Center for lowlevelwithminorfluctuationsduringtheentireischemic theShanghaiChineseAcademyofScience.Theanimalswere period without any significant change except during EA housed at an ambient temperature of 24 ± 1∘C and were application.Aftertheocclusion,reperfusionoftheischemic provided with free access to food and water. All surgical area was allowed by withdrawing the suture from the right procedures were approved by the Animals Care and Use external carotid artery. Cortical blood flow changes were CommitteesofFudanUniversityShanghaiMedicalCollege, monitored in all animals beginning 5 minutes prior to the Shanghai, China, and were performed under anesthesia inductionofcerebralischemiauntilatleast15minutesafter (chloratehydrate,400mg/kg,i.p.). reperfusion.Thisdurationwasinclusiveoftheentirelength ofMCAOandofEAaswell. Body temperature was maintained at 36.5∘C ± 0.5∘C 2.2. Cerebral Blood Flow Monitoring. The cerebral blood throughoutthesurgicalprocedurestilltheanimalrecovered flow was measured using laser-Doppler perfusion monitor from anesthesia. After the reperfusion, the animals were (LDPM, PeriFlux5000, Perimed, Sweden). The major steps housedfor24hoursatanambienttemperatureof24±1∘C. of the procedure were as follows: first, a small hole was Thesamesurgicalprocedureswereperformedontheanimals drilledintherightparietalboneatapoint1.5mmposterior inthesham-operationgroup,excludingMCAO. to the bregma and 5mm lateral to the sagittal suture, as previouslydescribed[14,17,31].Thesuperficialmicro-vessels of cerebral pia mater were accessed using a laser Doppler 2.4.ElectroacupunctureMethods. WHOstandardswerefol- probe (0.45mm diameter) inserted into the hole at a depth lowed for the name and localization of acupoints [33, 34]. of 2mm and fixed to the skull bone. The probe was used “Shuigou (Du 26)” and “Baihui (Du 20)” are acupoints to measure the blood perfusion to the cortex to record locatedontheheadandface.Shuigou(Du26)islocatedata the cerebral blood flow. Continuous monitoring of cerebral pointonthemidlineoftheupperlip2/3rdsfromthemouth blood flow was performed beginning 5 minutes prior to tothenose.Baihui(Du20)islocatedatapointonthemidline the induction of cerebral ischemia until at least 15 minutes ofthehead,approximatelymidwayonthelineconnectingthe after reperfusion. As per the manufacturer’s guidelines, the apicesofbothauricles. measured change in perfusion values was dependent on ToobtainoptimalEAeffectthatinducesmaximumpro- the number of blood cells present and their velocity in the tectionagainstcerebralischemia,weappliedEAat“Shuigou” area illuminated by the tip of the probe. The changes in (Du 26) and “Baihui” (Du 20) with 5/20Hz sparse-dense the cerebral blood flow of a localized area, recorded as a frequency at a constant intensity of ∼1.0mA in all EA measure of the dynamic changes in Perfusion Units (PU), groups,beginningat5minaftertheonsetofMCAOor5min concentration of the moving blood cells (CMBC), and the afterMCAO.Theselectionoftheacupointsandstimulation velocityofbloodcells(Velocity),wereconstantlymonitored parameters was based on the results of our serial studies in in real-time using LDPM. PU is an index of cerebral blood thepast[14,17]. Evidence-BasedComplementaryandAlternativeMedicine 3 In determination of effects of different EA periods on pushedbackwardalongwiththesignsofGrade4;Grade6— cerebral ischemia, EA was applied using varying lasting- repeated rotational motion with an immotile posterior left durations,thatis,beginningat5minaftertheonsetofMCAO limb;andGrade7—leftrecumbentpositionbecauseofbody andcontinuedupto5minutes,15minutes,30minutes,and supportingincapability. 45 minutes, respectively. Five different groups were studied inthepresentstudywiththenumbersineachgroupranging 2.7. Cerebral Infarct Measurement. After the assessment of from16to45:MCAOonly(Ischemia,𝑛 = 45),MCAOplus neurologicaldeficits,animalsweresacrificedunderanesthe- EAfor5min(EA-5min,𝑛 = 18),MCAOplusEAfor15min sia and samples of brain were taken. The brain slices were (EA-15min,𝑛=16),MCAOplusEAfor30min(EA-30min, prepared as 2.0mm sections (𝑛 = 12–16) and were incu- 𝑛=30),andMCAOplusEAfor45min(EA-45min,𝑛=30). bated in a solution of triphenyltetrazolium chloride (TTC, EAwasdeliveredthroughstainlesssteelfiliformneedles ∘ 20g/L)for30minutesat37 Cbeforebeingtransferredinto (15mm in length and 0.3mm in diameter, Suzhou Medical paraformaldehyde solution (40g/L) for fixing the infarcted Apparatus Limit Co., China) by an EA apparatus (Model area. The infarct region appeared white or pale while the G-6805-II, Shanghai Medical Instruments High-Tech Co., “normal” tissue appeared red [35, 36]. Pictures were taken China).TheneedleonDu26isinserted1mmdeep,vertical of the brain slices with a digital camera and the volume of to the plane of the skin. At Du 20, the needle is inserted infarctwasanalyzedusingacomputer-assistedimagesystem obliquely and to a depth of 2mm [14, 17, 25, 33, 34]. The with ACT-2U software (Nikon). Relative infarct ratio was intensityandfrequencyoftheoutputwaveswithanegative- calculated using the following equation [14, 17, 31]: (2 ∗ goingpulseontheposteriorborder(pulsewidth=0.5ms± lefthemispherearea(non-ischemicside)−non-infarctarea 0.1ms;componentofdirectcurrent=Zero)weremonitored of whole brain slices)/(2 ∗ left hemisphere areas) ∗ 100%. onageneraloscillograph(ModelXJ4210A,ShanghaiXinJian This equation excludes the factors that could result in an InstrumentandEquipmentCo.,China). inaccuratecalculationoftheinfarctvolume(suchasedema). 2.5. 𝛿-Opioid Receptors Blockade. The rats were randomly 2.8. Data Analysis. The cerebral blood flow was calculated fromPU,CMBC,andvelocitymeasurements.Allindividual assignedtooneofthefourexperimentalgroups:(1)Ischemia alone(𝑛 = 7);(2)Ischemia+EAat“Shuigou”(DU26)and measurementswerecomparedtothebaselinevalues,before “Baihui”(DU20)(𝑛=8);(3)Ischemia+Naltrindole(𝑛=8), MCAO (control level), in each animal. The grouped values werecomparedbetweendifferentgroups.Anaveragegrade in which rats were administered Naltrindole (10mg/kg i.v.) ofneurologicaldeficitspergroupwasusedtomakecompar- 5minbeforetheonsetofMCAO;and(4)Ischemia+EA+ Naltrindole (𝑛 = 10), in which the animals were treated isonsbetweengroups.Cerebralinfarctvolumewasexpressed asapercentfractionoftheentirecerebralvolume. with both EA and Naltrindole. EA was started 5min after All data is presented as mean ± SD and subjected to ischemia and continued to 35min after ischemia in this set statisticalanalysis.Therateofdeathwascomparedusingthe ofexperiments. Chi-square test. All other data was subjected to Analysis of variance(ANOVA),t-test,Rank-Sumtest,and/orChi-square test. The changes were considered to be significant if the 𝑃 2.6. Mortality and Neurological Deficits Monitoring. Some valuewaslessthan0.05. ischemic rats died between 2 and 20hrs after reperfusion. The death rates were reported for this period based on the numberofdeadratsandthetotalnumberoftheratsallocated 3.Results tothegivengroup.Amyriadoffactorscouldbeimplicated asthecauseofdeath,asforexamplehemorrhage.However, 3.1. Different Effects Induced by EA Starting after the Onset investigating the precise cause of death was not our aim in of MCAO versus Post-MCAO. In the group of Ischemia thiswork. (𝑛 = 45), with 1hr MCAO and 24hr reperfusion, about Assessmentsonneurologicaldeficitsweremadeonani- 18%(8/45)oftheratsdiedwithin2–20hoursaftertheonset mals in all groups. The rats that died within 24h after of reperfusion. At 24 hours of reperfusion, the living rats MCAO were excluded. Assessments were made 24h after displayedseriousneurologicaldeficits(Grade6.0±0.5,𝑛 = reperfusionjustbeforethesamplingofbraintissuewasdone. 37).TTCstainingshowedthatthevolumeofcerebralinfarct The assessments on neurological deficits were blinded, that wasaboutonethirdofthewholebrain(32.8%±3.7%,𝑛=16) is,thepersonevaluatingandscoringtheneurologicaldeficits (Table1,Figure1(a)).Theinfarctareasweremainlylocalized based on pre-set criteria had no prior knowledge on the inthefrontoparietallobesofthecortexandstriatuminthe groups and treatments. The degree of neurological deficits righthemisphere(ischemicside,Figure1(a)). wasgradedfrom0–7grades[14,17].Thecriteriaweresetas EA starting at 5min after the onset of MCAO induced follows:Grade0—“normal”,symmetricalmovementwithout a marked protection against cerebral ischemia, leading to a any abnormal sign; Grade 1—incomplete stretch of the left significantreductionofinfarctvolume,neurologicaldeficits, anteriorlimbwhenthetailwasliftedup;Grade2—doddery and death rate (Table 1, Figures 1(b)–1(d)). EA starting at crawlalongwiththesignsofGrade1;Grade3—kepttheleft 5minafterMCAOinducedasimilaroutcomeintheischemic anteriorlimbclosetothebreastwhenthetailwasliftedup; rats(refertoFigure6).However,itseemedthatEAinduced Grade4—leftturnwhencrawling;Grade5—leftanteriorclaw a larger reduction of the infarct volume in the former than 4 Evidence-BasedComplementaryandAlternativeMedicine Table1:EAperiod-dependentprotectionagainstischemicinjury. Groups Neurologicaldeficit Infarctvolume Deathrate Ischemia 6.0±0.5(5∼7)(𝑛=37) 32.8%±3.7%(𝑛=16) 18%(8outof45) EA-5min 5.0±0.5(4∼6)(𝑛=16)∗󳵳 25.6%±5.3%(𝑛=12)∗󳵳 11%(2outof18)∗∗󳵳 EA-15min 3.0±0.5(2∼4)(𝑛=15)∗∗󳵳 15.4%±4.2%(𝑛=12)∗∗󳵳 6%(1outof16)∗∗󳵳 EA-30min 1.0±0.5(0∼2)(𝑛=28)∗∗󳵳 4.9%±1.2%(𝑛=12)∗∗󳵳 7%(2outof30)∗∗󳵳 EA-45min 7.0±0.0(∼7)(𝑛=12)∗ 34.3%±2.4%(𝑛=12) 60%(18outof30)∗∗ ∗𝑃< 0.05versusIschemia.∗∗𝑃< 0.01versusIschemia.󳵳𝑃< 0.01versusEA-45min.NotethatEAsignificantlyreducedtheinfarctvolume,neurological deficit,anddeathrateandthisprotectiveeffectbecamebetterandbetterwhenthelengthofEAincreasedfrom5to30minutes.However,EAfor45minsdid notimprovetheneurologicaldeficitandinfarctvolumeandevenincreasedthedeathrate. (a) Ischemia (MCAO- (b) Ischemia+EA-5min (c) Ischemia+EA-15min (d) Ischemia+EA-30min (e) Ischemia + EA- 60min) 45min Figure1:EA-inducedchangesincerebralinfarctsizeinatime-dependentmanner.ThebrainslicesweresubjectedtoTTCstainingand theischemicinfarctvolumewasquantifiedbyacomputerizedimagesystem.Theslicesontherightofeachcolumnshowthebacksideof theleftslices.Notethattheinfarctregion(pale-whiteportion)wasmainlylocatedinthestriatumandthefrontoparietalcortexintheright hemisphere.TheMCAO-inducedinfarction(a)wassignificantlyreducedbyEAatDu20andDu26acupointsfor5min(b),15min(c),and 30min(d).Incontrast,EAfor45min(e)enablednoprotectionagainstthecerebralinfarction. the later group. EA after the onset of MCAO reduced the deficits were greatly attenuated (Grade 1.0 ± 0.5, 𝑛 = 28, infarctvolumeby∼85%(Table1andFigure1),whileEAafter 𝑃<0.01versusIschemia)andasignificantdecreaseindeath MCAO,by∼45%(refertoFigure6).Theseresultssuggestthat rate (7%, 2/30, 𝑃 < 0.01 versus Ischemia) was noted. The earlyinstitutionofEAstimulationinducesbetterprotection infarct volume was reduced by 85% (4.9% ± 1.2%, 𝑛 = 12, againstcerebralischemicinjury. 𝑃 < 0.01 versus Ischemia) (Table 1 and Figure 1(d)). In comparisontothegroupsofEAfor5minandEAfor15min, 3.2. Increased EA Protection with Increased Periods of the EAfor30mininducedmorebeneficialeffectsinallaspects Stimulation from 5 to 30mins. In the groupof MCAO plus including neurological deficits, ischemic infarct and death EAfor5min(EA-5min,𝑛 = 18),exceptfor2ratsthatdied rate(Table1,Figures1(b)–1(d)).Theseresultssuggestthatthe at 5 and 15 hours after the onset of reperfusion (11%, 2/18, EAprotectionisdependentonanappropriateEAduration. 𝑃<0.01versusIschemia),thedegreeofaverageneurological deficits in the living ischemic rats was slightly improved 3.3.ExacerbationofIschemicInjuryby“Over-Length”Stimula- (Grade 5.0 ± 0.5, 𝑛 = 16, 𝑃 < 0.05 versus Ischemia). The tionofEA. WeinvestigatedtoseeiftheEAprotectionwould infarctvolumewasslightlyreduced(25.6%+5.3%,𝑛 = 12, befurtherenhancedbyalongerdurationofEAstimulation. 𝑃 < 0.05 versus Ischemia) (Table 1 and Figure1(b)). In the Therefore,weassignedafewratstoanewgroupwithMCAO groupofMCAOplusEAfor15min(EA-15min,𝑛=16),only plusEAfor45minutes(EA-45min,𝑛=30).Tooursurprise, oneratdiedat∼3hoursaftertheonsetofreperfusion(6%, EA for 45min significantly increased the mortality in this 1/16,𝑃 < 0.01versusIschemia),withagreaterimprovement groupofischemicrats.Morethanhalfoftheanimalsinthis in average neurological deficits (Grade 3.0 ± 0.5, 𝑛 = 15, group(60%,18/30)diedwithin0.5to10hoursaftertheonset 𝑃 < 0.01versusIschemia)alongwithasignificantreduction of reperfusion. All of the dying rats manifested symptoms in infarct volume (15.4% ± 4.2%, 𝑛 = 12, 𝑃 < 0.01 versus suchasconvulsions,tumbling,piloerection,andperspiration Ischemia)(Table1andFigure1(c)).InthegroupofMCAO (wetfeathers)andotherabnormalities.Whencomparedthe plus EA for 30 min (EA-30min, 𝑛 = 30), the neurological Ischemiagroup,thedeathrateincreasedby3folds(60%,18 Evidence-BasedComplementaryandAlternativeMedicine 5 outof30,𝑃 < 0.01versusIschemia)inthisgroup.Although groups,whichwasevidentintheearlystageofreperfusion, the remaining 12 living rats survived for 24 hours after the forinstance,duringthefirst15minutesafterMCAO.Inthe reperfusion, they suffered from severe neurological deficits groups of EA-5min (𝑛 = 14), EA-15min (𝑛 = 12), and (Grade 7) and were even worse than the group with only EA-30min (𝑛 = 30), blood perfusion (PU) increased to Ischemia(Grade6.0+0.5withrangeof5∼7,𝑃 < 0.05).In over 90% of the base-value (preischemia) (Figure 3, 𝑃 < termsoftheinfarctvolume,EAfor45mindidnotreducethe 0.01 versus the ischemia group under reperfusion), CMBC infarctvolumeatall(34.3% ± 2.4%,𝑛=12,𝑃>0.05versus recovered to >80% of the preischemia level (Figure 4, 𝑃 < Ischemia). (Table 1, Figure 1(e)). These results suggest that 0.01 versus the ischemia group under reperfusion) and the anincreaseddurationofEAstimulationapplicationfurther Velocityincreasedto>80%ofthecontrollevel(Figure5,𝑃< exacerbates the ischemic insult, instead of conferring any 0.05versustheischemiagroupunderreperfusion)within15 protection. minutesafterreperfusion. Insharpcontrast,theEA-45mingroup(𝑛 = 18)showed a totally different pattern. For example, PU maintained at 3.4. EA-Induced Increase in Cerebral Blood Flow during a similar level as that during MCAO (∼15%), that is, after MCAO. UsingaLaser-DopplerPerfusionMonitor,wecon- reperfusion,itslevelwasevenlowerthanthoseofIschemia tinuouslymonitoredtherealtimechangesincerebralblood alone (∼25%, 𝑃 < 0.05 versus that during reperfusion in flow in all the groups beginning at 5min prior to MCAO the group of Ischemia plus EA for 45 minutes) (Figure 3). till15minaftertheonsetofreperfusion(suturewithdrawal), Similarly, CMBC still maintained at a low level (∼20%) and compared differences in the cerebral blood flow at the after reperfusion and the level was even lower than that pre-MCAOlevel,duringMCAOwith/withoutEA,andearly (50%)oftheIschemiagroupduringreperfusion(𝑃 < 0.01, stagesofreperfusionbetweenvariousgroups(Figures2,3,4, Figure4).ThesefindingssuggestthatEAfor30minutesor and5). lessincreasesthebloodflowduringMCAOandpromotesthe Consistent with our previous studies [14, 17], after the recoveryofthebloodflowduringreperfusion,whilean“over- insertion of a nylon suture into the right middle cerebral length” (45min) stimulation, though increasing the blood artery, the blood perfusion (PU) to the monitored cortex flow during MCAO, retards the recovery of the blood flow immediately decreased by ∼85% (i.e., reaching the level at tothebrainafterMCAO(Figures2(e)and3–5). ∼15% of the pre-MCAO level). During the entire period of MCAO,thelocalbloodflowwasmaintainedatthislowlevel with a minor fluctuation. CMBC dropped by ∼80% of the 3.6. DOR Antagonist Attenuated EA-Induced Protection base-value (i.e., reaching a level at ∼20% of the pre-MCAO against Ischemic Infarction. We previously found that level)withaslightdecelerationofbloodcellvelocity(reduced microinjection of DOR antagonist into the lateral cerebral by ∼25% of the pre-MCAO level). These changes indicated ventricle largely attenuated the EA protection against thatMCAOinducedagreaterreductioninthebloodvolume cerebral ischemic injury [10]. To further reaffirm this ascomparedtothevelocityofbloodcells(Figures2(a)and observation,weappliedNaltrindoletotheischemicratsand 3–5). determined its effect on the EA-induced neuroprotection. InalltheEAgroups,EAinducedaninstantandsignif- The results showed that in the ischemia group (𝑛 = 7) icant increase in the blood flow of the ischemic brain. The the infarct size reduced by ∼40% (from 24.8% ± 4.1% to bloodflowchangedisochronouslywiththecurrentimpulse 14.7% ± 3.4%, 𝑃 < 0.05) following treatment with EA in response to EA stimulation (Figures 2(b)–2(e) and 3–5). beginning immediately after MCAO (𝑛 = 8). Although EAinducedanincreaseinPUto∼30%ofthecontrol(𝑃 < Naltrindole (10mg/kg, i.v.) application did tend to increase 0.01 versus MACO without EA) (Figure 3) and CMBC to theinfarctvolume,thechangewasnotstatisticallysignificant over 65% of the control (𝑃 < 0.01 versus MCAO without (30.4% ± 4.3%, 𝑛 = 8, 𝑃 > 0.05 versus Ischemia alone). EA) (Figure 4) with a simultaneous decrease in the blood However,itcompletelyabolishedtheEA-inducedprotection cellvelocityto∼55%ofthecontrol(𝑃 < 0.05versusMCAO againstischemicinfarction(23.6%±5.2%,𝑛 = 10,𝑃 > 0.05 without EA) (Figure 5). These results suggest that both versus Ischemia + EA) (Figure 6). These results indicate “appropriate”and“over”periodsofEAstimulationincrease that 𝛿-opioid receptor plays a very important role in the thebloodflowduringMCAO. EA-inducedprotectionagainstischemicinjury. 3.5. Differential Recovery of the Cerebral Blood Flow after 4.Discussion ReperfusionamongEAGroups. IntheIschemiagroup(𝑛 = 30), following the withdrawal of the nylon suture after 1h Thisisthefirststudytosystematicallydefineoptimalduration MCAO,PUgraduallyincreasedto∼25%ofthecontrollevel ofEAstimulationtoinducebeneficialeffectsagainstischemic (pre-MCAO) (𝑃 < 0.05 versus during MCAO) and CMBC infarction, neurological deficits and mortality. Our results increased to ∼50% of the control (𝑃 < 0.01 versus during show that EA given for 5–30 minutes significantly reduces MCAO)withthevelocityofbloodcellsbeingreducedfrom ischemic infarct volume and decreases neurological deficits ∼75%to∼55%(𝑃<0.05,versusduringMCAO)inthefirst15 andmortalityrate.Ontheotherhand,EAgivenfor45min minutesafterthebloodreperfusion(Figures2(a)and3–5). did not show reduction in the neurological deficits or the EAchangedthepatternofthebloodreperfusion.There infarctvolume,andratherdemonstratedanincreaseindeath was, however, a significant difference among different EA rate in this group. However, all groups showed an increase 6 Evidence-BasedComplementaryandAlternativeMedicine MCAO Rep. 100 U P 15 C 100 B M C 20 y ocit10705 Vel 50 5 min (a) Effectof60minMCAOonthebloodflow MCAO Rep. 100 EA U P 15 C 100 B M C 20 city10705 o Vel 50 5 min (b) EffectofEA-5minonthebloodflowunderischemia MCAO Rep. 100 U EA P 15 C100 B M C 20 city17050 o Vel 50 5 min (c) EffectofEA-15minonthebloodflowunderischemia MCAO Rep. 100 EA U P 15 C B100 M C 20 y cit100 o 75 Vel 50 5 min (d) EffectofEA-30minonthebloodflowunderischemia MCAO Rep. 100 U EA P 15 C B100 M C 20 y cit100 o 75 Vel 50 5 min (e) EffectofEA-45minonthebloodflowunderischemia Figure2:RepresentativetracerecordingsofthebloodflowintheratswithorwithoutEA.BloodPerfusion(PU),ConcentrationofMoving BloodCells(CMBC),andVelocityofBloodCells(Velocity)weremeasuredintheischemicratsbyaLaserDopplerPerfusionMonitorsystem. (a)EffectofMCAO-60minonCBFduringischemiaandafterreperfusioninIschemiagroup.(b)EffectofEAfor5minatacupointsofDu 20andDu26onCBF.(c)EffectofEAfor15minonCBF.(d)EffectofEAfor30minonCBF.(e)EffectofEAfor45minonCBF.Notethatthe PUandCMBCdecreasedimmediatelyafterMCAOandthebloodflowwasmaintainedatalowlevelwithsmallfluctuationsduringtheentire periodofMCAOwithaslightdecreaseinthevelocity.Aftertheonsetofreperfusion,PUandCMBCincreasedwithafurtherdecreaseinthe velocity.EAinducedanisochronousincreaseinPUandCMBCwithadecreaseinvelocityduringMCAO.Afterreperfusion,PU,CMBC, andvelocityallincreasedrapidlyandreachedthebaselinevalueswithin15minafterreperfusiononsetingroupsofEAfor5–30min.EAfor 45min,however,inducedagreaterdecreaseinPU,CMBC,andvelocitythanthechangesobservedintheIschemiagroupafterreperfusion. Evidence-BasedComplementaryandAlternativeMedicine 7 Relative changes in amount of blood perfusion (% of base-value before MCAO)1147659832110000000000000 MC∗AEOA∗ R#e#p#e#rfu𝛁#sion𝛁# Relative changes in amount of blood perfusion (% of base-value before MCAO)1110987653214000000000000 MC∗A∗O∗EA∗ ∗ #R#ep##erfu𝛁#sio𝛁n# −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (minutes) Time (minutes) Ischemia (𝑛=30) Ischemia (𝑛=30) Ischemia+EA-5 min (𝑛=14) Ischemia+EA-15 min (𝑛=12) (a) (b) n 110 n 110 o o perfusi AO)10900 #### # # perfusi O)19000 d C d A Relative changes in amount of bloo (% of base-value before M 87653214000000000 MC∗AO∗∗ ∗ ∗E∗A ∗ ∗ ∗ Reperf𝛁usio𝛁n Relative changes in amount of bloo (% of base-value before MC 87653214000000000 MC∗AO∗∗ ∗∗∗ ∗EA∗∗∗ ∗ ∗ ∗ Reperfu𝛁sio𝛁n −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (minutes) Time (minutes) Ischemia (𝑛=30) Ischemia (𝑛=30) Ischemia+EA-30 min (𝑛=30) Ischemia+EA-45 min (𝑛=18) (c) (d) Figure3:StatisticalsummaryofEAeffectsonPUintheischemicbrain.∗𝑃<0.01,Ischemia+EAversusIschemiagroupduringMCAO;∇𝑃< 0.05,underreperfusionversusduringMCAOintheIschemiagroup; #𝑃<0.01,Ischemia + EAversusIschemiagroupunderreperfusion; 󳶃𝑃 < 0.05,Ischemia+EAversusIschemiagroupunderreperfusion.NotethatMCAOsharplydecreasedPUto∼15%ofthebasalvalue (pre-MCAO),whileEAfor5–45minsignificantlyincreasedthebloodflowto∼30%ofthebaseline.Within10–15minutesafterreperfusion, PUgraduallyincreasedto∼25%ofthebaseline(pre-MCAO)intheIschemiagroup,whereasitrapidlyincreasedtoabout90%ofthebaseline intheEAfor5–30minutesgroups.Insharpcontrast,PUwasstillattheMCAOlevel,thatis,∼15%ofthebasalvalue,duringthesameperiod afterreperfusioninthegroupofEAfor45minutes. in blood flow after EA stimulation during the period of reduce the cerebral ischemic injury. Our previous studies MCAO. These findings are consistent with our preliminary concluded that EA for 30 minutes increases the blood flow observations[37]andsuggestthattheEAprotectionagainst duringMCAOandreducesischemicinsulttothebrain[11,12, ischemicinjuryisdependentonthelengthofEAstimulation, 14,17].Inextension,wespeculatedthatanextendedperiodof andthatan“over-length”stimulationcanpotentiallyaddto EAstimulationwouldproducebetteroutcomes.Apparently, theischemicinjuryinsteadofprotectingthebrainfromthe thiswasnotthecaseasweobservedoppositeresults;thatis,a injury. prolongedstimulationofEApotentiates,ratherthanprotects Thisobservationwasbeyondourexpectations.Sincethe against, cerebral damage during ischemic injury to brain. primarycauseofischemicbraininjuryisinsufficient blood Interestingly, Dr. Wang et al. [38] also observed a similar supplytothebrain,increasedbloodflowtothebrainshould phenomenonintheirstudiesonEA-inducedhypoalgesiain 8 Evidence-BasedComplementaryandAlternativeMedicine 110 110 100 100 Relative changes in CMBC (% of base-value before MCAO) 9876532400000000 ∗EA∗ ## # # Relative changes in CMBC % of base-value before MCAO) 9876532400000000 ∗ ∗∗EA∗ ∗ # ## # # ( 10 10 MCAO Reperfusion MCAO Reperfusion 0 0 −5 0 5 10 1520 25 30 35 40 45 50 55 60 65 70 75 80 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (minutes) Time (minutes) Ischemia (𝑛=30) Ischemia (𝑛=30) Ischemia+EA-5 min (𝑛=14) Ischemia+EA-15 min (𝑛=12) (a) (b) 110 110 100 100 Relative changes in CMBC (% of base-value before MCAO) 9876532400000000 ∗ ∗∗ ∗ ∗E∗A ∗ ∗ ∗ 𝛁#𝛁#𝛁# 𝛁# 𝛁# Relative changes in CMBC (% of base-value before MCAO) 9876532400000000 ∗ ∗∗ ∗ ∗∗ ∗EA∗ ∗∗ ∗ ∗ ∗ 𝛁##𝛁𝛁# 𝛁# #𝛁 10 10 MCAO Reperfusion MCAO Reperfusion 0 0 −5 0 5 10 15 2025 30 35 40 45 50 55 60 65 70 75 80 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (minutes) Time (minutes) Ischemia (𝑛=30) Ischemia (𝑛=30) Ischemia+EA-30 min (𝑛=30) Ischemia+EA-45 min (𝑛=18) (c) (d) Figure4:StatisticalsummaryofEAeffectsonconcentrationofMovingBloodCells.∗𝑃<0.01,Ischemia+EAversusIschemiagroupduring MCAO;∇𝑃 < 0.01,underreperfusionversusduringMCAOinIschemiagroup; #𝑃 < 0.01,Ischemia+EAversusIschemiagroupunder reperfusion.NotethattheMCAOgreatlyreducedtheconcentrationofMovingBloodCells(CMBC)to∼20%ofthebaselineandCMBC quicklyincreasedto∼50%ofthebaselineafterreperfusion.EAfor5–45minutessignificantlyincreasedCMBCduringMCAOto∼65%of thebaseline.Within15minutesafterreperfusion,CMBCfurtherincreasedto∼80%ofthebaselineintheEAfor5–30minutesgroups,but notintheEAfor45minutesgroupwithitslevelbeingsimilartothatduringMCAO. healthyvolunteers.Thesubjectswererandomizedtoreceive At present, the mechanism behind this phenomenon is different durations (0min, 20min, 30min, or 40min) of not known. In this work, however, it is noteworthy that asynchronousEAstimulationsandthensubjectedtothetest EAfor5–30minutessignificantlyimprovesthereperfusion ofhypoalgesiausingahumanexperimentalcoldthermalpain after MCAO and favors quick recovery of blood flow to threshold model. They found that 30 min of asynchronous the pre-ischemic level, in addition to increasing blood flow EA stimulation resulted in the most significant hypoalgesic duringMCAO.Insharpcontrast,EAfor45minutesdoesnot effectcomparedwith0,20,or40minstimulations.Therefore, promotetherecoveryofbloodflowafterMCAOatall,butto itseemsthata30minperiodisthemostoptimaldurationfor thecontrary,furtherworsensitasshowninourpresentwork. EA-inducedanalgesiaandbrainprotectionagainstischemic Thisphenomenonpartiallyaccountsforthedifferenceinthe injury. outcomesbetween“optimalperiod”and“over-length”inthe Evidence-BasedComplementaryandAlternativeMedicine 9 110 110 100 100 city AO) 90 EA ## # # # city AO) 90 EA ## # # # velo MC 80 velo MC 80 ge in blood alue before 765000 ∗ ∗ 𝛁𝛁𝛁 𝛁 𝛁 ge in blood alue before 765000 ∗∗∗ ∗ ∗ 𝛁𝛁𝛁 𝛁 𝛁 n v n v elative cha % of base- 324000 elative cha % of base- 324000 R ( R ( 10 10 MCAO Reperfusion MCAO Reperfusion 0 0 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 7075 80 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (minutes) Time (minutes) Ischemia (𝑛=30) Ischemia (𝑛=30) Ischemia+EA-5 min (𝑛=14) Ischemia+EA-15 min (𝑛=12) (a) (b) 110 110 100 100 city O) 90 EA ## # # # city AO) 90 EA ## # # # nge in blood velo alue before MCA 87650000 ∗ ∗∗ ∗ ∗∗ ∗ ∗ ∗ 𝛁𝛁𝛁 𝛁 𝛁 nge in blood velo value before MC 87650000 ∗ ∗∗ ∗ ∗∗ ∗ ∗ ∗ ∗ ∗ ∗ 𝛁𝛁 𝛁 𝛁 𝛁 Relative cha % of base-v 324000 Relative cha (% of base- 324000 ( 10 10 MCAO Reperfusion MCAO Reperfusion 0 0 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 −5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (minutes) Time (minutes) Ischemia (𝑛=30) Ischemia (𝑛=30) Ischemia+EA-30 min (𝑛=30) Ischemia+EA-45 min (𝑛=18) (c) (d) Figure5:StatisticalsummaryofEAeffectsonthevelocityofbloodcells.∗𝑃 < 0.05,Ischemia+EAversusIschemiagroupduringMCAO; ∇𝑃<0.05,underreperfusionversusduringMCAOinIschemiagroup; #𝑃<0.05,Ischemia+EAversusIschemiagroupunderreperfusion. NotethatMCAOreducedthevelocityofthebloodcellsto∼75%ofthebaseline,whichwasfurtherdecreasedto∼55%ofthebaselineafter reperfusion.InallEAgroups,EAduringMCAOimmediatelydecreasedthevelocityto∼55%ofthebaseline,andinducedasignificant increaseinitafterreperfusion.IncontrasttochangesinPUandCMBC,thevelocityincreasedto>80%afterreperfusioninallEAgroups includingEA-45min. EA treatment although the underlying mechanism remains mobilization of various survival signals and activation of unknown. protectivepathwaysintheischemicbrain. The fact that “over-length” of EA stimulation increases One of these pathways could involve DOR-mediated the blood flow under MCAO, but does not lead to any survival signaling. We have previously demonstrated that protection against ischemic injury strongly suggests that DORisimportantininducingneuroprotection[3,6].DOR optimal EA stimulation induces a multi-level regulation in activationattenuateshypoxic,ischemic,orexcitatoryinjury the brain via a complex mechanism. A single factor, such in the neurons and the brain [1, 3, 6]. Evidence shows as,anincreaseinthebloodflowunderischemicconditions, thatbothmanualacupunctureandEAenhancetheactivity may not be enough to induce protection against ischemia. of the endogenous opioid system, including DOR, in the Appropriatestimulationisextremelyimportantformaximal brain [26–29, 39]. Therefore, the DOR-mediated protection 10 Evidence-BasedComplementaryandAlternativeMedicine 40 duringMCAO,cansetthisbalanceofcombinedprotection off. 35 Moreover, our present results show that EA application during MCAO induces a stronger protection than EA after n) 30 MCAO,suggestingthatanearlyinstitutionofEAtreatment ai isrequiredtoachievebetterneuroprotectionagainstischemic br e injury.ThisissimilartothegoalsoftPAtreatmentinterms ol 25 h of the importance of the therapeutic window [7, 8]. An w of earlymobilizationofprotectivepowermaypreventneurons % 20 e ( ∗ frominitiatingtheprocessofapoptosis/necrosisinthebrain. m “Timeisbrain,”whichisalsotrueinthecaseofEAtreatment u ol 15 forischemicbraininjury. v ct Insummary,ourresultssuggestearlyEAtreatmentwith ar nf 10 optimalstimulationparametersforanappropriateperiodis I critical to achieve a therapeutic effect for neuroprotection againstischemicinsult.Thepreviouscontroversiesinthelit- 5 eratureonEAtreatmentforstrokecanbepartiallyattributed tothedifferencesinconditionsofthepatientsandEAparam- 0 eters.Furtherin-depthinvestigationsintothe“optimal”EA parametersanditsunderlyingmechanismscanhelpimprove clinicaloutcomesofpatientswithischemic/hypoxicinjuryin Ischemia Ischemia+N thebrain. Ischemia+EA Ischemia+EA+N Acknowledgments Figure6:EffectofNaltrindoleonEA-inducedprotectionagainst ischemicinfarctsize.Quantitativevolumesoftheischemicinfarct weredeterminedbasedonthepercentageofthewholebrainvolume. This work was supported by NSFC (30672721, 30873318), Note that the ischemia-induced infarct was reduced by the EA NBRP (2009CB522901; 2012CB518502), STCSM stimulation(∗𝑃 < 0.05versusischemiagroup),whichwasalmost (10DZ1975800), NIH (AT-004422 and HD-034852), and completelyreversedaftertreatmentwithNaltrindole(N,10mg/kg, VivianL.SmithNeurologicFoundation. i.v.)(∗𝑃<0.05versusIschemia+EA+Naltrindole). References [1] J. H. Sung, D. M. Chao, and Y. Xia, “Neuronal responses to might play a very important role in the EA-induced pro- hypoxia,” in New Frontiers in Neurological Research, W. Ying tection against ischemic injury. Indeed, we first reported andD.Wang,Eds.,pp.73–153,ResearchSignpost,Kerala,India, that microinjection of a DOR antagonist, Naltrindole, into 2008. the cerebral ventricle attenuated the EA-induced cerebral [2] Y. Luo, “Cell-based therapy for stroke,” Journal of Neural protection against ischemic infarction, suggesting DOR’s Transmission,vol.118,no.1,pp.61–74,2011. involvement in the EA-induced protection against brain [3] D. Chao and Y. Xia, “Ionic storm in hypoxic/ischemic stress: ischemia[10].Furthermore,ourstudiesandthoseofothers canopioidreceptorssubsideit?”ProgressinNeurobiology,vol. suggestthatEAsignificantlyincreasesthedensityofDORin 90,no.4,pp.439–470,2010. the ischemic cortex and that DOR signaling could mediate [4] K.Arai,J.Lok,S.Guo,K.Hayakawa,C.Xing,andE.H.Lo, theEAinducedneuroprotectionagainstischemicinjury[15, “Cellularmechanismsofneurovasculardamageandrepairafter 39, 40]. In the present study, we observed that intravenous stroke,”JournalofChildNeurology,vol.26,pp.1193–1198,2011. injection of Naltrindole almost completely blocked the EA- [5] J.H.Zhang,J.Badaut,J.Tang,A.Obenaus,R.Hartman,and inducedneuroprotectionagainstischemicinjury.Allofthese W.J.Pearce,“Thevascularneuralnetwork-anewparadigmin observations consistently favor the critical role of DOR in stroke pathophysiology,” Nature Review/Neurology, vol. 8, no. EA-induced neuroprotection against cerebral ischemia. On 12,pp.711–716,2012. theotherhand,EAcanactivateadditionalneurotransmitter [6] X. Z. He, H. K. Sandhu, Y. L. Yang et al., “Neuroprotection systemssuchasneurotrophicfactorsinthebrainthatenable againsthypoxia/ischemia:delta-opioidreceptor-mediatedcel- furtherprotectionunderischemicconditions[13]. lular/molecular events,” Cellular and Molecular Life Sciences, Thus,EAtargetsmultiplemechanismstoachieveapro- 2012. tective effect against ischemic insults, including an increase [7] R.J.Thurman,E.C.Jauch,P.D.Panagos,M.R.Reynolds,andJ. in the cerebral blood flow under ischemia, an improved Mocco,“Fourevolvingstrategiesintheemergenttreatmentof recoveryofthebloodflowafterreperfusion,upregulationof acuteischemicstroke,”EmergencyMedicinePractice,vol.14,no. the DOR pathway, and other survival signals. An optimal 7,pp.1–27,2012. duration of EA stimulation can combine and direct all of [8] K.A.WeantandS.N.Baker,“Newwindows,sameoldhouse: these protective pathways against ischemic stress, while an anupdateonacutestrokemanagement,”AdvancedEmergency over-stimulation of EA, despite increasing the blood flow NursingJournal,vol.34,pp.112–121,2012.

Description:
EA was delivered to “Shuigou” (Du 26) and “Baihui” (Du 20) acupoints with sparse-dense wave (5/20 Hz) at 1.0 mA for 5, 15 In all groups, EA induced a significant increase in cerebral blood 73–153, Research Signpost, Kerala, India, . Nomenclature for Acupuncture, World Health Organizatio
See more

The list of books you might like

Upgrade Premium
Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.
DMCA & Copyright: Dear all, most of the website is community built, users are uploading hundred of books everyday, which makes really hard for us to identify copyrighted material, please contact us if you want any material removed.
Copyright @ PDFdrive.to, 2022 | We love our users