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

13 Pages·2015·3.22 MB·English

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
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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).
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