research article effect of electroacupuncture on rat...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 878521 12 pageshttpdxdoiorg1011552013878521

Research ArticleEffect of Electroacupuncture on Rat Ischemic Brain InjuryImportance of Stimulation Duration

Fei Zhou12 Jingchun Guo3 Jieshi Cheng3 Gencheng Wu3 and Ying Xia45

1 Shanghai Research Center for Acupuncture and Meridians Shanghai 201203 China2 Gongli Hospital Pudong New District Shanghai 200135 China3 Shanghai Medical College of Fudan University Shanghai 200032 China4The University of Texas Medical School at Houston Houston TX 77030 USA5 Yale University School of Medicine New Haven CT 06520 USA

Correspondence should be addressed to Ying Xia yingxiauthtmcedu

Received 23 August 2012 Revised 10 November 2012 Accepted 13 December 2012

Academic Editor Di Zhang

Copyright copy 2013 Fei Zhou et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We explored the optimal duration of electroacupuncture (EA) stimulation for protecting the brain against ischemic injury Theexperiments were carried out in rats exposed to right middle cerebral artery occlusion (MCAO) for 60min followed by 24-hrreperfusion EA was delivered to ldquoShuigourdquo (Du 26) and ldquoBaihuirdquo (Du 20) acupoints with sparse-dense wave (520Hz) at 10mAfor 5 15 30 and 45min respectivelyThe results showed that 30min EA starting at 5 minutes after the onset of MCAO (EA duringMCAO) or 5 minutes after reperfusion (EA after MCAO) significantly reduced ischemic infarct volume attenuated neurologicaldeficits and decreased death rate with a larger reduction of the ischemic infarction in the former group Also in the group of EAduring MCAO this protective benefit was positively proportional to the increase in the period of stimulation that is increasedprotection in response to EA from 5- to 30-min stimulation In all groups EA induced a significant increase in cerebral bloodflow and promoted blood flow recovery after reperfusion and both blood flow volume and blood cell velocity returned to thepreischemia level in a short period of time Surprisingly EA for 45min did not show reduction in the neurological deficits orthe infarct volume and instead demonstrated an increase in death rate in this group Although EA for 45min still increased theblood flow during MCAO it led to a worsening of perfusion after reperfusion compared to the group subjected only to ischemiaThe neuroprotection induced by an ldquooptimalrdquo period (30min) of EA was completely blocked by Naltrindole a 120575-opioid receptor(DOR) antagonist (10mgkg iv)These findings suggest that earlier EA stimulation leads to better outcomes and that EA-inducedneuroprotection against ischemia depends on an optimal EA-duration viamultiple pathways includingDOR signaling while ldquoover-lengthrdquo stimulation exacerbates the ischemic injury

1 Introduction

Ischemichypoxic brain injury such as stroke leads to seriousand complex pathophysiological changes affecting multiplelevels of the brain and sports a high global mortality rateas the leading cause of neurological disability [1ndash9] Inspite of extensive research conducted in the past severaldecades limited therapeutic options are available againstischemichypoxic brain injury till date The vast physicalemotional andfinancial tolls that stroke inflicts uponpatientsand their families imposes a daunting and continuous chal-lenge to the medical community [5ndash9] Previous studies at

our laboratory and at those of others have shown that elec-troacupuncture (EA) at appropriate acupoints with suitablestimulation parameters significantly attenuates neurologicaldeficits infarct volume and mortality in animal modelsexposed to ischemic insults [10ndash17] which may potentiallyshed a new light on developing a better modality for ischemicbrain injury Indeed there is substantial clinical evidencedemonstrating beneficial effects of acupuncture on strokepatients [18ndash21]

Because of inadequate control poor methodologicalquality and small samples seen in previous studies theefficacy of acupuncture on hypoxicischemic injury is still

2 Evidence-Based Complementary and Alternative Medicine

unproven in clinical settings In factmajor controversies existwith regards to the effectiveness of acupuncture in strokepatients [22ndash24] These may at least partially be attributedto the varied approaches adopted in different studies andsince acupuncture induces complex changes atmultiple levelsin the central nervous system the outcomes also differwith varying acupunctural methods [25ndash30] Therefore itis extremely important to define what optimal parametersare required for acupuncture treatment in ischemichypoxicbrain injury

Towards this goal our serial studies have establishedthat EA treatment for acute stroke in experimental animalsis critically dependent upon specific acupoints along withstimulation currents of specific intensities and frequencies[11ndash15 17] Since the timing and duration of medical treat-ment has a huge impact on patients with acute stroke [7ndash9]we conducted this work to address two fundamental issuesthat is (1) when should EA be applied to induce optimalprotection during or after ischemia and (2) what durationof EA application is optimal to induce maximal beneficialeffect on the ischemic brain

2 Materials and Methods

21 Animals Adult male Sprague-Dawley rats (240ndash270 g)were used for the experiments All animals were purchasedwith permission from the Experimental Animal Center forthe Shanghai Chinese Academy of ScienceThe animals werehoused at an ambient temperature of 24 plusmn 1∘C and wereprovided with free access to food and water All surgicalprocedures were approved by the Animals Care and UseCommittees of Fudan University Shanghai Medical CollegeShanghai China and were performed under anesthesia(chlorate hydrate 400mgkg ip)

22 Cerebral Blood Flow Monitoring The cerebral bloodflow was measured using laser-Doppler perfusion monitor(LDPM PeriFlux5000 Perimed Sweden) The major stepsof the procedure were as follows first a small hole wasdrilled in the right parietal bone at a point 15mm posteriorto the bregma and 5mm lateral to the sagittal suture aspreviously described [14 17 31]The superficial micro-vesselsof cerebral pia mater were accessed using a laser Dopplerprobe (045mm diameter) inserted into the hole at a depthof 2mm and fixed to the skull bone The probe was usedto measure the blood perfusion to the cortex to recordthe cerebral blood flow Continuous monitoring of cerebralblood flow was performed beginning 5 minutes prior tothe induction of cerebral ischemia until at least 15 minutesafter reperfusion As per the manufacturerrsquos guidelines themeasured change in perfusion values was dependent onthe number of blood cells present and their velocity in thearea illuminated by the tip of the probe The changes inthe cerebral blood flow of a localized area recorded as ameasure of the dynamic changes in Perfusion Units (PU)concentration of the moving blood cells (CMBC) and thevelocity of blood cells (Velocity) were constantly monitoredin real-time using LDPM PU is an index of cerebral blood

flow and is expressed as the product of the number of movingblood cells and their relative velocities

23 Method for Inducing Cerebral Ischemia We followed themethods described by Longa et al [32] that are also detailedin our previous publications [14 17] for creating a focalcerebral ischemia model by middle cerebral artery occlusion(MCAO) Animals were taken under anesthesia to surgicallyexpose their right common carotid external carotid andinternal carotid arteries After ligation of the distal endof the right external carotid artery it was incised and a4ndash0 monofilament nylon suture (30mm length 018mmdiameter with a 024mm diameter round tip MONOSOFSN-1699G USA) was introduced to the right external carotidartery and further into the right internal carotid artery forsim20mm up till the origin of the right middle cerebral arteryThe blood flow to the right middle cerebral artery was thenoccluded at this level

We constantly monitored the blood flow in all animals toensure that a uniform cerebral ischemia level was maintainedacross all groups in a standard fashion The blood flowwas controlled by adjusting the suture in the artery for theinduction of ischemiaThe ischemic rats that showed a stabledrop of sim85 in PU compared to the baseline level (beforeMCAO) that is reaching a level of sim15 of the baseline PUwere used for further experimentation The PU was kept at alow level with minor fluctuations during the entire ischemicperiod without any significant change except during EAapplication After the occlusion reperfusion of the ischemicarea was allowed by withdrawing the suture from the rightexternal carotid artery Cortical blood flow changes weremonitored in all animals beginning 5 minutes prior to theinduction of cerebral ischemia until at least 15 minutes afterreperfusion This duration was inclusive of the entire lengthof MCAO and of EA as well

Body temperature was maintained at 365∘C plusmn 05∘Cthroughout the surgical procedures till the animal recoveredfrom anesthesia After the reperfusion the animals werehoused for 24 hours at an ambient temperature of 24 plusmn 1∘CThe same surgical procedures were performed on the animalsin the sham-operation group excluding MCAO

24 Electroacupuncture Methods WHO standards were fol-lowed for the name and localization of acupoints [33 34]ldquoShuigou (Du 26)rdquo and ldquoBaihui (Du 20)rdquo are acupointslocated on the head and face Shuigou (Du 26) is located at apoint on the midline of the upper lip 23rds from the mouthto the nose Baihui (Du 20) is located at a point on themidlineof the head approximatelymidway on the line connecting theapices of both auricles

To obtain optimal EA effect that induces maximum pro-tection against cerebral ischemia we applied EA at ldquoShuigourdquo(Du 26) and ldquoBaihuirdquo (Du 20) with 520Hz sparse-densefrequency at a constant intensity of sim10mA in all EAgroups beginning at 5min after the onset of MCAO or 5minafter MCAO The selection of the acupoints and stimulationparameters was based on the results of our serial studies inthe past [14 17]

Evidence-Based Complementary and Alternative Medicine 3

In determination of effects of different EA periods oncerebral ischemia EA was applied using varying lasting-durations that is beginning at 5min after the onset ofMCAOand continued up to 5 minutes 15 minutes 30 minutes and45 minutes respectively Five different groups were studiedin the present study with the numbers in each group rangingfrom 16 to 45 MCAO only (Ischemia 119899 = 45) MCAO plusEA for 5min (EA-5min 119899 = 18) MCAO plus EA for 15min(EA-15min 119899 = 16) MCAO plus EA for 30min (EA-30min119899 = 30) andMCAO plus EA for 45min (EA-45min 119899 = 30)

EA was delivered through stainless steel filiform needles(15mm in length and 03mm in diameter Suzhou MedicalApparatus Limit Co China) by an EA apparatus (ModelG-6805-II Shanghai Medical Instruments High-Tech CoChina) The needle on Du 26 is inserted 1mm deep verticalto the plane of the skin At Du 20 the needle is insertedobliquely and to a depth of 2mm [14 17 25 33 34] Theintensity and frequency of the output waves with a negative-going pulse on the posterior border (pulse width = 05ms plusmn01ms component of direct current = Zero) were monitoredon a general oscillograph (Model XJ4210A Shanghai XinJianInstrument and Equipment Co China)

25 120575-Opioid Receptors Blockade The rats were randomlyassigned to one of the four experimental groups (1) Ischemiaalone (119899 = 7) (2) Ischemia + EA at ldquoShuigourdquo (DU 26) andldquoBaihuirdquo (DU 20) (119899 = 8) (3) Ischemia + Naltrindole (119899 = 8)in which rats were administered Naltrindole (10mgkg iv)5min before the onset of MCAO and (4) Ischemia + EA +Naltrindole (119899 = 10) in which the animals were treatedwith both EA and Naltrindole EA was started 5min afterischemia and continued to 35min after ischemia in this setof experiments

26 Mortality and Neurological Deficits Monitoring Someischemic rats died between 2 and 20 hrs after reperfusionThe death rates were reported for this period based on thenumber of dead rats and the total number of the rats allocatedto the given group A myriad of factors could be implicatedas the cause of death as for example hemorrhage Howeverinvestigating the precise cause of death was not our aim inthis work

Assessments on neurological deficits were made on ani-mals in all groups The rats that died within 24 h afterMCAO were excluded Assessments were made 24 h afterreperfusion just before the sampling of brain tissue was doneThe assessments on neurological deficits were blinded thatis the person evaluating and scoring the neurological deficitsbased on pre-set criteria had no prior knowledge on thegroups and treatments The degree of neurological deficitswas graded from 0ndash7 grades [14 17] The criteria were set asfollows Grade 0mdashldquonormalrdquo symmetrical movement withoutany abnormal sign Grade 1mdashincomplete stretch of the leftanterior limb when the tail was lifted up Grade 2mdashdodderycrawl along with the signs of Grade 1 Grade 3mdashkept the leftanterior limb close to the breast when the tail was lifted upGrade 4mdashleft turnwhen crawlingGrade 5mdashleft anterior claw

pushed backward along with the signs of Grade 4 Grade 6mdashrepeated rotational motion with an immotile posterior leftlimb and Grade 7mdashleft recumbent position because of bodysupporting incapability

27 Cerebral Infarct Measurement After the assessment ofneurological deficits animals were sacrificed under anesthe-sia and samples of brain were taken The brain slices wereprepared as 20mm sections (119899 = 12ndash16) and were incu-bated in a solution of triphenyltetrazolium chloride (TTC20 gL) for 30 minutes at 37∘C before being transferred intoparaformaldehyde solution (40 gL) for fixing the infarctedarea The infarct region appeared white or pale while theldquonormalrdquo tissue appeared red [35 36] Pictures were takenof the brain slices with a digital camera and the volume ofinfarct was analyzed using a computer-assisted image systemwith ACT-2U software (Nikon) Relative infarct ratio wascalculated using the following equation [14 17 31] (2 lowastleft hemisphere area (non-ischemic side) minus non-infarct areaof whole brain slices)(2 lowast left hemisphere areas) lowast 100This equation excludes the factors that could result in aninaccurate calculation of the infarct volume (such as edema)

28 Data Analysis The cerebral blood flow was calculatedfrom PU CMBC and velocity measurements All individualmeasurements were compared to the baseline values beforeMCAO (control level) in each animal The grouped valueswere compared between different groups An average gradeof neurological deficits per group was used to make compar-isons between groups Cerebral infarct volume was expressedas a percent fraction of the entire cerebral volume

All data is presented as mean plusmn SD and subjected tostatistical analysis The rate of death was compared using theChi-square test All other data was subjected to Analysis ofvariance (ANOVA) t-test Rank-Sum test andor Chi-squaretest The changes were considered to be significant if the 119875value was less than 005

3 Results

31 Different Effects Induced by EA Starting after the Onsetof MCAO versus Post-MCAO In the group of Ischemia(119899 = 45) with 1 hr MCAO and 24 hr reperfusion about18 (845) of the rats died within 2ndash20 hours after the onsetof reperfusion At 24 hours of reperfusion the living ratsdisplayed serious neurological deficits (Grade 60 plusmn 05 119899 =37) TTC staining showed that the volume of cerebral infarctwas about one third of the whole brain (328plusmn37 119899 = 16)(Table 1 Figure 1(a)) The infarct areas were mainly localizedin the frontoparietal lobes of the cortex and striatum in theright hemisphere (ischemic side Figure 1(a))

EA starting at 5min after the onset of MCAO induceda marked protection against cerebral ischemia leading to asignificant reduction of infarct volume neurological deficitsand death rate (Table 1 Figures 1(b)ndash1(d)) EA starting at5min afterMCAO induced a similar outcome in the ischemicrats (refer to Figure 6) However it seemed that EA induceda larger reduction of the infarct volume in the former than


Table 1 EA period-dependent protection against ischemic injury

Groups Neurological deficit Infarct volume Death rateIschemia 60 plusmn 05 (5sim 7) (119899 = 37) 328 plusmn 37 (119899 = 16) 18 (8 out of 45)EA-5min 50 plusmn 05 (4sim 6) (119899 = 16)lowast998771 256 plusmn 53 (119899 = 12)lowast998771 11 (2 out of 18)lowastlowast998771

EA-15min 30 plusmn 05 (2sim 4) (119899 = 15)lowastlowast998771 154 plusmn 42 (119899 = 12)lowastlowast998771 6 (1 out of 16)lowastlowast998771


EA-45min 70 plusmn 00 (sim7) (119899 = 12)lowast 343 plusmn 24 (119899 = 12) 60 (18 out of 30)lowastlowastlowast

119875 lt 005 versus Ischemia lowastlowast119875 lt 001 versus Ischemia 998771119875 lt 001 versus EA-45min Note that EA significantly reduced the infarct volume neurologicaldeficit and death rate and this protective effect became better and better when the length of EA increased from 5 to 30 minutes However EA for 45mins didnot improve the neurological deficit and infarct volume and even increased the death rate

(a) Ischemia (MCAO-60min)

(b) Ischemia + EA-5min (c) Ischemia + EA-15min (d) Ischemia + EA-30min (e) Ischemia + EA-45min

Figure 1 EA-induced changes in cerebral infarct size in a time-dependent manner The brain slices were subjected to TTC staining andthe ischemic infarct volume was quantified by a computerized image system The slices on the right of each column show the backside ofthe left slices Note that the infarct region (pale-white portion) was mainly located in the striatum and the frontoparietal cortex in the righthemisphere The MCAO-induced infarction (a) was significantly reduced by EA at Du 20 and Du 26 acupoints for 5min (b) 15min (c) and30min (d) In contrast EA for 45min (e) enabled no protection against the cerebral infarction

the later group EA after the onset of MCAO reduced theinfarct volume by sim85 (Table 1 and Figure 1) while EA afterMCAO bysim45 (refer to Figure 6)These results suggest thatearly institution of EA stimulation induces better protectionagainst cerebral ischemic injury

32 Increased EA Protection with Increased Periods of theStimulation from 5 to 30mins In the group of MCAO plusEA for 5min (EA-5min 119899 = 18) except for 2 rats that diedat 5 and 15 hours after the onset of reperfusion (11 218119875 lt 001 versus Ischemia) the degree of average neurologicaldeficits in the living ischemic rats was slightly improved(Grade 50 plusmn 05 119899 = 16 119875 lt 005 versus Ischemia) Theinfarct volume was slightly reduced (256 + 53 119899 = 12119875 lt 005 versus Ischemia) (Table 1 and Figure 1(b)) In thegroup ofMCAOplus EA for 15min (EA-15min 119899 = 16) onlyone rat died at sim3 hours after the onset of reperfusion (6116 119875 lt 001 versus Ischemia) with a greater improvementin average neurological deficits (Grade 30 plusmn 05 119899 = 15119875 lt 001 versus Ischemia) along with a significant reductionin infarct volume (154 plusmn 42 119899 = 12 119875 lt 001 versusIschemia) (Table 1 and Figure 1(c)) In the group of MCAOplus EA for 30 min (EA-30min 119899 = 30) the neurological

deficits were greatly attenuated (Grade 10 plusmn 05 119899 = 28119875 lt 001 versus Ischemia) and a significant decrease in deathrate (7 230 119875 lt 001 versus Ischemia) was noted Theinfarct volume was reduced by 85 (49 plusmn 12 119899 = 12119875 lt 001 versus Ischemia) (Table 1 and Figure 1(d)) Incomparison to the groups of EA for 5min and EA for 15minEA for 30min induced more beneficial effects in all aspectsincluding neurological deficits ischemic infarct and deathrate (Table 1 Figures 1(b)ndash1(d))These results suggest that theEA protection is dependent on an appropriate EA duration

33 Exacerbation of Ischemic Injury by ldquoOver-Lengthrdquo Stimula-tion of EA We investigated to see if the EA protection wouldbe further enhanced by a longer duration of EA stimulationTherefore we assigned a few rats to a new group withMCAOplus EA for 45 minutes (EA-45min 119899 = 30) To our surpriseEA for 45min significantly increased the mortality in thisgroup of ischemic rats More than half of the animals in thisgroup (60 1830) died within 05 to 10 hours after the onsetof reperfusion All of the dying rats manifested symptomssuch as convulsions tumbling piloerection and perspiration(wet feathers) and other abnormalities When compared theIschemia group the death rate increased by 3 folds (60 18


out of 30 119875 lt 001 versus Ischemia) in this group Althoughthe remaining 12 living rats survived for 24 hours after thereperfusion they suffered from severe neurological deficits(Grade 7) and were even worse than the group with onlyIschemia (Grade 60 + 05 with range of 5sim 7 119875 lt 005) Interms of the infarct volume EA for 45min did not reduce theinfarct volume at all (343 plusmn 24 119899 = 12 119875 gt 005 versusIschemia) (Table 1 Figure 1(e)) These results suggest thatan increased duration of EA stimulation application furtherexacerbates the ischemic insult instead of conferring anyprotection

34 EA-Induced Increase in Cerebral Blood Flow duringMCAO Using a Laser-Doppler Perfusion Monitor we con-tinuously monitored the real time changes in cerebral bloodflow in all the groups beginning at 5min prior to MCAOtill 15min after the onset of reperfusion (suture withdrawal)and compared differences in the cerebral blood flow at thepre-MCAO level during MCAO withwithout EA and earlystages of reperfusion between various groups (Figures 2 3 4and 5)

Consistent with our previous studies [14 17] after theinsertion of a nylon suture into the right middle cerebralartery the blood perfusion (PU) to the monitored corteximmediately decreased by sim85 (ie reaching the level atsim15 of the pre-MCAO level) During the entire period ofMCAO the local blood flow was maintained at this low levelwith a minor fluctuation CMBC dropped by sim80 of thebase-value (ie reaching a level at sim20 of the pre-MCAOlevel) with a slight deceleration of blood cell velocity (reducedby sim25 of the pre-MCAO level) These changes indicatedthat MCAO induced a greater reduction in the blood volumeas compared to the velocity of blood cells (Figures 2(a) and3ndash5)

In all the EA groups EA induced an instant and signif-icant increase in the blood flow of the ischemic brain Theblood flow changed isochronously with the current impulsein response to EA stimulation (Figures 2(b)ndash2(e) and 3ndash5)EA induced an increase in PU to sim30 of the control (119875 lt001 versus MACO without EA) (Figure 3) and CMBC toover 65 of the control (119875 lt 001 versus MCAO withoutEA) (Figure 4) with a simultaneous decrease in the bloodcell velocity to sim55 of the control (119875 lt 005 versus MCAOwithout EA) (Figure 5) These results suggest that bothldquoappropriaterdquo and ldquooverrdquo periods of EA stimulation increasethe blood flow during MCAO

35 Differential Recovery of the Cerebral Blood Flow afterReperfusion among EA Groups In the Ischemia group (119899 =30) following the withdrawal of the nylon suture after 1 hMCAO PU gradually increased to sim25 of the control level(pre-MCAO) (119875 lt 005 versus during MCAO) and CMBCincreased to sim50 of the control (119875 lt 001 versus duringMCAO) with the velocity of blood cells being reduced fromsim75 to sim55 (119875 lt 005 versus duringMCAO) in the first 15minutes after the blood reperfusion (Figures 2(a) and 3ndash5)

EA changed the pattern of the blood reperfusion Therewas however a significant difference among different EA

groups which was evident in the early stage of reperfusionfor instance during the first 15 minutes after MCAO In thegroups of EA-5min (119899 = 14) EA-15min (119899 = 12) andEA-30min (119899 = 30) blood perfusion (PU) increased toover 90 of the base-value (preischemia) (Figure 3 119875 lt001 versus the ischemia group under reperfusion) CMBCrecovered to gt80 of the preischemia level (Figure 4 119875 lt001 versus the ischemia group under reperfusion) and theVelocity increased to gt80 of the control level (Figure 5 119875 lt005 versus the ischemia group under reperfusion) within 15minutes after reperfusion

In sharp contrast the EA-45min group (119899 = 18) showeda totally different pattern For example PU maintained ata similar level as that during MCAO (sim15) that is afterreperfusion its level was even lower than those of Ischemiaalone (sim25 119875 lt 005 versus that during reperfusion inthe group of Ischemia plus EA for 45 minutes) (Figure 3)Similarly CMBC still maintained at a low level (sim20)after reperfusion and the level was even lower than that(50) of the Ischemia group during reperfusion (119875 lt 001Figure 4) These findings suggest that EA for 30 minutes orless increases the blood flow duringMCAO and promotes therecovery of the blood flowduring reperfusion while an ldquoover-lengthrdquo (45min) stimulation though increasing the bloodflow during MCAO retards the recovery of the blood flowto the brain after MCAO (Figures 2(e) and 3ndash5)

36 DOR Antagonist Attenuated EA-Induced Protectionagainst Ischemic Infarction We previously found thatmicroinjection of DOR antagonist into the lateral cerebralventricle largely attenuated the EA protection againstcerebral ischemic injury [10] To further reaffirm thisobservation we applied Naltrindole to the ischemic rats anddetermined its effect on the EA-induced neuroprotectionThe results showed that in the ischemia group (119899 = 7)the infarct size reduced by sim40 (from 248 plusmn 41 to147 plusmn 34 119875 lt 005) following treatment with EAbeginning immediately after MCAO (119899 = 8) AlthoughNaltrindole (10mgkg iv) application did tend to increasethe infarct volume the change was not statistically significant(304 plusmn 43 119899 = 8 119875 gt 005 versus Ischemia alone)However it completely abolished the EA-induced protectionagainst ischemic infarction (236 plusmn 52 119899 = 10 119875 gt 005versus Ischemia + EA) (Figure 6) These results indicatethat 120575-opioid receptor plays a very important role in theEA-induced protection against ischemic injury

4 Discussion

This is the first study to systematically define optimal durationof EA stimulation to induce beneficial effects against ischemicinfarction neurological deficits and mortality Our resultsshow that EA given for 5ndash30 minutes significantly reducesischemic infarct volume and decreases neurological deficitsand mortality rate On the other hand EA given for 45mindid not show reduction in the neurological deficits or theinfarct volume and rather demonstrated an increase in deathrate in this group However all groups showed an increase


15

10020

100

7550

100

Velo

city

5 minCM

BCPU

MCAO Rep

(a) Effect of 60min MCAO on the blood flow

15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA

(b) Effect of EA-5min on the blood flow under ischemia

15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA

(c) Effect of EA-15min on the blood flow under ischemia

15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA

(d) Effect of EA-30min on the blood flow under ischemia

15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA

(e) Effect of EA-45min on the blood flow under ischemia

Figure 2 Representative trace recordings of the blood flow in the rats with or without EA Blood Perfusion (PU) Concentration of MovingBlood Cells (CMBC) andVelocity of Blood Cells (Velocity) weremeasured in the ischemic rats by a Laser Doppler PerfusionMonitor system(a) Effect of MCAO-60min on CBF during ischemia and after reperfusion in Ischemia group (b) Effect of EA for 5min at acupoints of Du20 and Du 26 on CBF (c) Effect of EA for 15min on CBF (d) Effect of EA for 30min on CBF (e) Effect of EA for 45 min on CBF Note that thePU and CMBC decreased immediately afterMCAO and the blood flowwasmaintained at a low level with small fluctuations during the entireperiod of MCAOwith a slight decrease in the velocity After the onset of reperfusion PU and CMBC increased with a further decrease in thevelocity EA induced an isochronous increase in PU and CMBC with a decrease in velocity during MCAO After reperfusion PU CMBCand velocity all increased rapidly and reached the baseline values within 15min after reperfusion onset in groups of EA for 5ndash30min EA for45min however induced a greater decrease in PU CMBC and velocity than the changes observed in the Ischemia group after reperfusion


110100

9080

80

70

70 75

60

60 65

50

50 55

30

30 35

20

20 25

10

10 150

40

40 45

EA

MCAO

minus5 50

Reperfusion

Time (minutes)Ischemia (119899 = 30)

lowast lowast120513120513

Relat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia + EA-5 min (119899 = 14)

(a)

110100

9080706050

302010

0

40

8070 7560 6550 5530 3520 2510 15 40 45minus5 50Time (minutes)

EAlowast lowastlowast lowast lowast

120513 120513

MCAO ReperfusionRelat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia (119899 = 30)Ischemia + 119899 = 12)EA-15 min (

(b)

110100

9080706050

302010

0

40


EAlowast lowastlowast lowast lowastlowast lowast lowast lowast

120513 120513


ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)


(c)

Ischemia (119899 = 30)

110100

9080706050

302010

0

40

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

EA

MCAO Reperfusion

Time (minutes)

Relat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia + 119899 = 18)EA-45 min (

lowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowast120513120513

(d)

Figure 3 Statistical summary of EA effects on PU in the ischemic brain lowast119875 lt 001 Ischemia+EAversus Ischemia group duringMCAOnabla119875 lt005 under reperfusion versus during MCAO in the Ischemia group

119875 lt 001 Ischemia + EA versus Ischemia group under reperfusion998787

119875 lt 005 Ischemia + EA versus Ischemia group under reperfusion Note that MCAO sharply decreased PU to sim15 of the basal value(pre-MCAO) while EA for 5ndash45min significantly increased the blood flow to sim30 of the baseline Within 10ndash15 minutes after reperfusionPU gradually increased to sim25 of the baseline (pre-MCAO) in the Ischemia group whereas it rapidly increased to about 90 of the baselinein the EA for 5ndash30 minutes groups In sharp contrast PU was still at the MCAO level that is sim15 of the basal value during the same periodafter reperfusion in the group of EA for 45 minutes

in blood flow after EA stimulation during the period ofMCAO These findings are consistent with our preliminaryobservations [37] and suggest that the EA protection againstischemic injury is dependent on the length of EA stimulationand that an ldquoover-lengthrdquo stimulation can potentially add tothe ischemic injury instead of protecting the brain from theinjury

This observation was beyond our expectations Since theprimary cause of ischemic brain injury is insufficient bloodsupply to the brain increased blood flow to the brain should

reduce the cerebral ischemic injury Our previous studiesconcluded that EA for 30 minutes increases the blood flowduringMCAOand reduces ischemic insult to the brain [11 1214 17] In extension we speculated that an extended period ofEA stimulation would produce better outcomes Apparentlythis was not the case as we observed opposite results that is aprolonged stimulation of EA potentiates rather than protectsagainst cerebral damage during ischemic injury to brainInterestingly Dr Wang et al [38] also observed a similarphenomenon in their studies on EA-induced hypoalgesia in


Ischemia (119899 = 30)Ischemia + EA-5 min (119899 = 14)

EAlowastlowast

110

100

90

80

70

60

50

30

20

10

0

40

noisufrepeROACM

80756555352510 15 40 45minus5 50Time (minutes)

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

20 30 50 60 70

(a)



110

100

90

80

70

60

50

30

20

10

0

40

EA

lowastlowast lowast lowastlowast

MCAO Reperfusion

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

(b)

EA

lowastlowastlowastlowastlowastlowastlowastlowastlowast

110

100

90

80

70

60

50

30

20

10

0

40

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)

120513120513120513120513120513

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)


(c)

110

100

90

80

70

60

50

30

20

10

0

40

Relat

ive c

hang

es in

CM

BC EA

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)

lowast lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

120513120513120513120513120513

( o

f ba

se-v

alue

bef

ore M

CAO

)


(d)

Figure 4 Statistical summary of EA effects on concentration of Moving Blood Cells lowast119875 lt 001 Ischemia+EA versus Ischemia group duringMCAO nabla119875 lt 001 under reperfusion versus during MCAO in Ischemia group

119875 lt 001 Ischemia + EA versus Ischemia group underreperfusion Note that the MCAO greatly reduced the concentration of Moving Blood Cells (CMBC) to sim20 of the baseline and CMBCquickly increased to sim50 of the baseline after reperfusion EA for 5ndash45 minutes significantly increased CMBC during MCAO to sim65 ofthe baseline Within 15 minutes after reperfusion CMBC further increased to sim80 of the baseline in the EA for 5ndash30 minutes groups butnot in the EA for 45 minutes group with its level being similar to that during MCAO

healthy volunteers The subjects were randomized to receivedifferent durations (0min 20min 30min or 40min) ofasynchronous EA stimulations and then subjected to the testof hypoalgesia using a human experimental cold thermal painthreshold model They found that 30 min of asynchronousEA stimulation resulted in the most significant hypoalgesiceffect comparedwith 0 20 or 40min stimulationsThereforeit seems that a 30min period is the most optimal duration forEA-induced analgesia and brain protection against ischemicinjury

At present the mechanism behind this phenomenon isnot known In this work however it is noteworthy thatEA for 5ndash30 minutes significantly improves the reperfusionafter MCAO and favors quick recovery of blood flow tothe pre-ischemic level in addition to increasing blood flowduringMCAO In sharp contrast EA for 45 minutes does notpromote the recovery of blood flow after MCAO at all but tothe contrary further worsens it as shown in our present workThis phenomenon partially accounts for the difference in theoutcomes between ldquooptimal periodrdquo and ldquoover-lengthrdquo in the


110100

9080706050

302010

0

40


EA

lowastlowast

120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(a)

110100

9080706050

302010

0

40


EA

lowastlowastlowastlowastlowast

120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(b)

110100

9080706050

302010

0

40


EA

lowastlowastlowastlowastlowastlowastlowastlowastlowast 120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(c)

110100

9080706050

302010

0

40


EA

lowastlowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowast

120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(d)

Figure 5 Statistical summary of EA effects on the velocity of blood cells lowast119875 lt 005 Ischemia + EA versus Ischemia group during MCAOnabla119875 lt 005 under reperfusion versus during MCAO in Ischemia group

119875 lt 005 Ischemia + EA versus Ischemia group under reperfusionNote that MCAO reduced the velocity of the blood cells to sim75 of the baseline which was further decreased to sim55 of the baseline afterreperfusion In all EA groups EA during MCAO immediately decreased the velocity to sim55 of the baseline and induced a significantincrease in it after reperfusion In contrast to changes in PU and CMBC the velocity increased to gt80 after reperfusion in all EA groupsincluding EA-45min

EA treatment although the underlying mechanism remainsunknown

The fact that ldquoover-lengthrdquo of EA stimulation increasesthe blood flow under MCAO but does not lead to anyprotection against ischemic injury strongly suggests thatoptimal EA stimulation induces a multi-level regulation inthe brain via a complex mechanism A single factor suchas an increase in the blood flow under ischemic conditionsmay not be enough to induce protection against ischemiaAppropriate stimulation is extremely important for maximal

mobilization of various survival signals and activation ofprotective pathways in the ischemic brain

One of these pathways could involve DOR-mediatedsurvival signaling We have previously demonstrated thatDOR is important in inducing neuroprotection [3 6] DORactivation attenuates hypoxic ischemic or excitatory injuryin the neurons and the brain [1 3 6] Evidence showsthat both manual acupuncture and EA enhance the activityof the endogenous opioid system including DOR in thebrain [26ndash29 39] Therefore the DOR-mediated protection


40

35

30

25

20

15

10

5

0

Infa

rct v

olum

e (

of w

hole

bra

in)

Ischemia

Ischemia + EA

Ischemia + N

Ischemia + EA+ N

lowast

Figure 6 Effect of Naltrindole on EA-induced protection againstischemic infarct size Quantitative volumes of the ischemic infarctwere determined based on the percentage of thewhole brain volumeNote that the ischemia-induced infarct was reduced by the EAstimulation ( lowast119875 lt 005 versus ischemia group) which was almostcompletely reversed after treatment with Naltrindole (N 10mgkgiv) ( lowast119875 lt 005 versus Ischemia + EA + Naltrindole)

might play a very important role in the EA-induced pro-tection against ischemic injury Indeed we first reportedthat microinjection of a DOR antagonist Naltrindole intothe cerebral ventricle attenuated the EA-induced cerebralprotection against ischemic infarction suggesting DORrsquosinvolvement in the EA-induced protection against brainischemia [10] Furthermore our studies and those of otherssuggest that EA significantly increases the density of DOR inthe ischemic cortex and that DOR signaling could mediatethe EA induced neuroprotection against ischemic injury [1539 40] In the present study we observed that intravenousinjection of Naltrindole almost completely blocked the EA-induced neuroprotection against ischemic injury All of theseobservations consistently favor the critical role of DOR inEA-induced neuroprotection against cerebral ischemia Onthe other hand EA can activate additional neurotransmittersystems such as neurotrophic factors in the brain that enablefurther protection under ischemic conditions [13]

Thus EA targets multiple mechanisms to achieve a pro-tective effect against ischemic insults including an increasein the cerebral blood flow under ischemia an improvedrecovery of the blood flow after reperfusion upregulation ofthe DOR pathway and other survival signals An optimalduration of EA stimulation can combine and direct all ofthese protective pathways against ischemic stress while anover-stimulation of EA despite increasing the blood flow

during MCAO can set this balance of combined protectionoff

Moreover our present results show that EA applicationduring MCAO induces a stronger protection than EA afterMCAO suggesting that an early institution of EA treatmentis required to achieve better neuroprotection against ischemicinjury This is similar to the goals of tPA treatment in termsof the importance of the therapeutic window [7 8] Anearly mobilization of protective power may prevent neuronsfrom initiating the process of apoptosisnecrosis in the brainldquoTime is brainrdquo which is also true in the case of EA treatmentfor ischemic brain injury

In summary our results suggest early EA treatment withoptimal stimulation parameters for an appropriate period iscritical to achieve a therapeutic effect for neuroprotectionagainst ischemic insult The previous controversies in the lit-erature on EA treatment for stroke can be partially attributedto the differences in conditions of the patients and EA param-eters Further in-depth investigations into the ldquooptimalrdquo EAparameters and its underlying mechanisms can help improveclinical outcomes of patients with ischemichypoxic injury inthe brain

Acknowledgments

This work was supported by NSFC (30672721 30873318)NBRP (2009CB522901 2012CB518502) STCSM(10DZ1975800) NIH (AT-004422 and HD-034852) andVivian L Smith Neurologic Foundation

References

[1] J H Sung D M Chao and Y Xia ldquoNeuronal responses tohypoxiardquo in New Frontiers in Neurological Research W YingandDWang Eds pp 73ndash153 Research Signpost Kerala India2008

[2] Y Luo ldquoCell-based therapy for strokerdquo Journal of NeuralTransmission vol 118 no 1 pp 61ndash74 2011

[3] D Chao and Y Xia ldquoIonic storm in hypoxicischemic stresscan opioid receptors subside itrdquo Progress in Neurobiology vol90 no 4 pp 439ndash470 2010

[4] K Arai J Lok S Guo K Hayakawa C Xing and E H LoldquoCellularmechanisms of neurovascular damage and repair afterstrokerdquo Journal of Child Neurology vol 26 pp 1193ndash1198 2011

[5] J H Zhang J Badaut J Tang A Obenaus R Hartman andW J Pearce ldquoThe vascular neural network- a new paradigm instroke pathophysiologyrdquo Nature ReviewNeurology vol 8 no12 pp 711ndash716 2012

[6] X Z He H K Sandhu Y L Yang et al ldquoNeuroprotectionagainst hypoxiaischemia delta-opioid receptor-mediated cel-lularmolecular eventsrdquo Cellular and Molecular Life Sciences2012

[7] R JThurman E C Jauch P D Panagos M R Reynolds and JMocco ldquoFour evolving strategies in the emergent treatment ofacute ischemic strokerdquo EmergencyMedicine Practice vol 14 no7 pp 1ndash27 2012

[8] K A Weant and S N Baker ldquoNew windows same old housean update on acute stroke managementrdquo Advanced EmergencyNursing Journal vol 34 pp 112ndash121 2012


[9] M Mazighi E Meseguer J Labreuche and P AmarencoldquoBridging therapy in acute ischemic stroke a systematic reviewand meta-analysisrdquo Stroke vol 43 pp 1302ndash1308 2012

[10] P Zhao J Guo S S Hong A Bazzy-Asaad J S Cheng and YXia ldquoElectro- acupuncture and brain protection from cerebralischemia the role of delta-opioid receptorrdquo SfN AbstractProgram no 49013 2002

[11] F Zhou J C Guo J S Cheng G C Wu and Y Xia ldquoElectro-acupuncture induced protection from cerebral ischemia isdependent on stimulation intensity and frequencyrdquo in Proceed-ings of the 3rd Cell Stress Society International Congress on StressResponses in Biology and Medicine and 2ndWorld Conference ofStress vol 3 p 250 Budapest Hungary 2007

[12] F Zhou J C Guo J S Cheng G C Wu and Y Xia ldquoElectro-acupuncture and brain protection from cerebral ischemiadifferential roles of acupointsrdquo in Proceedings of the 3rd CellStress Society International Congress on Stress Responses inBiology and Medicine and 2nd World Conference of Stress vol3 p 249 Budapest Hungary 2007

[13] J C Guo J S Cheng and Y Xia ldquoAcupuncture therapy forstrokerdquo in Acupuncture Therapy For Neurological Diseases ANeurobiological View Y Xia X Cao G-C Wu and J ChengEds pp 226ndash262 Springer-Tsinghua Press Beijing ChinaHeidelberg Germany London UK and New York USA 2010

[14] F Zhou J C Guo J S Cheng G C Wu and Y XialdquoElectroacupuncture increased cerebral blood flow and reducedischemic brain injury dependence on stimulation intensity andfrequencyrdquo Journal of Applied Physiology vol 111 pp 1877ndash18872011

[15] X Li P Luo Q Wang and L Z Xiong ldquoElectroacupuncturepretreatment as a novel avenue to protect brain against Ischemiaand reperfusion injuryrdquo Evidence-Based Complementary andAlternativeMedicine vol 2012 Article ID 195397 12 pages 2012

[16] M Xu L Ge and D Zhao ldquoElectro-acupuncture regula-tion of central monoamine neurotransmitters in Ischaemia-reperfusionrdquo in Current Research in Acupuncture Y Xia GDing and G C Wu Eds pp 401ndash430 Springer New YorkUSA Heidelberg Germany Dordrecht The Netherlands andLondon UK 2012

[17] F Zhou J CGuo J S ChengGCWu J Sun andYXia ldquoElec-troacupuncture and brain protection against cerebral ischemiaspecific effects of acupointsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2013 Article ID 804397 2013

[18] V Hopwood and G T Lewith ldquoDoes acupuncture help strokepatients becomemore independentrdquo Journal of Alternative andComplementary Medicine vol 11 no 1 pp 175ndash177 2005

[19] M H Rabadi ldquoRandomized clinical stroke rehabilitation trialsin 2005rdquo Neurochemical Research vol 32 no 4-5 pp 807ndash8212007

[20] Z X Yang and X M Shi ldquoSystematic evaluation of the thera-peutic effect and safety of Xingnao Kaiqiao needling method intreatment of strokerdquo Zhongguo Zhen Jiu vol 27 no 8 pp 601ndash608 2007

[21] P Wu E Mills D Moher and D Seely ldquoAcupuncture inpoststroke rehabilitation a systematic review andmeta-analysisof randomized trialsrdquo Stroke vol 41 no 4 pp e171ndashe179 2010

[22] M Liu ldquoAcupuncture for stroke in China needing more high-quality evidencerdquo International Journal of Stroke vol 1 no 1 pp34ndash35 2006

[23] Y Xie L Wang J He and T Wu ldquoAcupuncture for dysphagiain acute strokerdquo Cochrane Database of Systematic Reviews vol16 no 3 Article ID CD006076 2008

[24] X F Zhao Y Du P G Liu and S Wang ldquoAcupuncturefor stroke evidence of effectiveness safety and cost from

systematic reviewsrdquo Topics in Stroke Rehabilitation vol 19 pp226ndash233 2012

[25] F Zhou D K Huang and Y Xia ldquoNeuroanatomic basis ofacupuncture pointsrdquo in Acupuncture Therapy For NeurologicalDiseases A Neurobiological View Y Xia X Cao G-C Wuand J Cheng Eds pp 32ndash80 Springer-Tsinghua Press BeijingChina Heidelberg Germany London UK and New YorkUSA 2010

[26] G Q Wen Y L Yang Y Lu and Y Xia ldquoAcupuncture-inducedactivation of endogenous opioid systemrdquo in Acupuncture Ther-apy For Neurological Diseases A Neurobiological View Y XiaX Cao G-C Wu and J Cheng Eds pp 104ndash119 Springer-Tsinghua Press Beijing China Heidelberg Germany LondonUK and New York USA 2010

[27] G Q Wen X Z He Y Lu Y Xia and Y Xia ldquoEffect ofacupuncture on neurotransmitters modulatorsrdquo in Acupunc-ture Therapy For Neurological Diseases A Neurobiological ViewY Xia X Cao G-C Wu and J Cheng Eds pp 120ndash142Springer-Tsinghua Press Beijing China Heidelberg GermanyLondon UK and New York USA 2010

[28] J F Liang and Y Xia ldquoAcupuncture modulation of centralneurotransmittersrdquo in Current Research in Acupuncture Y XiaG Ding and G C Wu Eds pp 1ndash36 Springer New YorkUSA Heidelberg Germany Dordrecht The Netherlands andLondon UK 2012

[29] Y Xia X D Cao G C Wu and J S Cheng ldquoAcupuncturetherapy for strokerdquo in Acupuncture Therapy For NeurologicalDiseases A Neurobiological View Y Xia X Cao G-C Wuand J Cheng Eds pp 1ndash480 Springer-Tsinghua Press BeijingChina Heidelberg Germany London UK and New YorkUSA 2010

[30] Y Xia G H Ding and G C Wu Current Research inAcupuncture Springer New York USA Heidelberg GermanyDordrecht The Netherlands and London UK 2012

[31] Y Nishimura T Ito and J M Saavedra ldquoAngiotensin IIAT1 blockade normalizes cerebrovascular autoregulation andreduces cerebral ischemia in spontaneously hypertensive ratsrdquoStroke vol 31 no 10 pp 2478ndash2486 2000

[32] E Z Longa P R Weinstein S Carlson and R CumminsldquoReversible middle cerebral artery occlusion without craniec-tomy in ratsrdquo Stroke vol 20 no 1 pp 84ndash91 1989

[33] WHO Regional Office for the Western Pacific StandardizedNomenclature for Acupuncture World Health OrganizationManila Philippines 1993

[34] WHO Regional Office for the Western Pacific WHO StandardAcupuncture Point Locations in the Western Pacific RegionWorld Health Organization Manila Philippines 2008

[35] J B Bederson L H Pitts and S M Germano ldquoEvaluationof 235-triphenyltetrazolium chloride as a stain for detectionand quantification of experimental cerebral infarction in ratsrdquoStroke vol 17 no 6 pp 1304ndash1308 1986

[36] R A SwansonM TMorton andG Tsao-Wu ldquoSemiautomatedmethod formeasuring brain infarct volumerdquo Journal of CerebralBlood Flow ampMetabolism vol 10 pp 290ndash295 1990

[37] F Zhou J C Guo R Yang J S Cheng G C Wu and YXia ldquoElectro-acupuncture increases cerebral blood flow andprotects the rat brain from ischemic injuryrdquo inProceedings of the3rd Cell Stress Society International Congress on Stress Responsesin Biology andMedicine and 2ndWorld Conference of Stress vol3 pp 249ndash250 Budapest Hungary 2007

[38] S MWang E C Lin I Maranets and Z N Kain ldquoThe impactof asynchronous electroacupuncture stimulation duration oncold thermal pain thresholdrdquoAnesthesia and Analgesia vol 109no 3 pp 932ndash935 2009


[39] X S Tian F Zhou R Yang Y Xia G C Wu and J C GuoldquoElectroacupuncture protects the brain against acute ischemicinjury via up-regulation of delta-opioid receptor in ratsrdquo Journalof Chinese Integrative Medicine vol 6 no 6 pp 632ndash638 2008

[40] L Z Xiong J Yang Q Wang and Z H Lu ldquoInvolvement of120575-and 120583-opioid receptors in the delayed cerebral ischemic toler-ance induced by repeated electroacupuncture preconditioningin ratsrdquo Chinese Medical Journal vol 120 no 5 pp 394ndash3992007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


unproven in clinical settings In factmajor controversies existwith regards to the effectiveness of acupuncture in strokepatients [22ndash24] These may at least partially be attributedto the varied approaches adopted in different studies andsince acupuncture induces complex changes atmultiple levelsin the central nervous system the outcomes also differwith varying acupunctural methods [25ndash30] Therefore itis extremely important to define what optimal parametersare required for acupuncture treatment in ischemichypoxicbrain injury

Towards this goal our serial studies have establishedthat EA treatment for acute stroke in experimental animalsis critically dependent upon specific acupoints along withstimulation currents of specific intensities and frequencies[11ndash15 17] Since the timing and duration of medical treat-ment has a huge impact on patients with acute stroke [7ndash9]we conducted this work to address two fundamental issuesthat is (1) when should EA be applied to induce optimalprotection during or after ischemia and (2) what durationof EA application is optimal to induce maximal beneficialeffect on the ischemic brain

2 Materials and Methods

21 Animals Adult male Sprague-Dawley rats (240ndash270 g)were used for the experiments All animals were purchasedwith permission from the Experimental Animal Center forthe Shanghai Chinese Academy of ScienceThe animals werehoused at an ambient temperature of 24 plusmn 1∘C and wereprovided with free access to food and water All surgicalprocedures were approved by the Animals Care and UseCommittees of Fudan University Shanghai Medical CollegeShanghai China and were performed under anesthesia(chlorate hydrate 400mgkg ip)

22 Cerebral Blood Flow Monitoring The cerebral bloodflow was measured using laser-Doppler perfusion monitor(LDPM PeriFlux5000 Perimed Sweden) The major stepsof the procedure were as follows first a small hole wasdrilled in the right parietal bone at a point 15mm posteriorto the bregma and 5mm lateral to the sagittal suture aspreviously described [14 17 31]The superficial micro-vesselsof cerebral pia mater were accessed using a laser Dopplerprobe (045mm diameter) inserted into the hole at a depthof 2mm and fixed to the skull bone The probe was usedto measure the blood perfusion to the cortex to recordthe cerebral blood flow Continuous monitoring of cerebralblood flow was performed beginning 5 minutes prior tothe induction of cerebral ischemia until at least 15 minutesafter reperfusion As per the manufacturerrsquos guidelines themeasured change in perfusion values was dependent onthe number of blood cells present and their velocity in thearea illuminated by the tip of the probe The changes inthe cerebral blood flow of a localized area recorded as ameasure of the dynamic changes in Perfusion Units (PU)concentration of the moving blood cells (CMBC) and thevelocity of blood cells (Velocity) were constantly monitoredin real-time using LDPM PU is an index of cerebral blood

flow and is expressed as the product of the number of movingblood cells and their relative velocities

23 Method for Inducing Cerebral Ischemia We followed themethods described by Longa et al [32] that are also detailedin our previous publications [14 17] for creating a focalcerebral ischemia model by middle cerebral artery occlusion(MCAO) Animals were taken under anesthesia to surgicallyexpose their right common carotid external carotid andinternal carotid arteries After ligation of the distal endof the right external carotid artery it was incised and a4ndash0 monofilament nylon suture (30mm length 018mmdiameter with a 024mm diameter round tip MONOSOFSN-1699G USA) was introduced to the right external carotidartery and further into the right internal carotid artery forsim20mm up till the origin of the right middle cerebral arteryThe blood flow to the right middle cerebral artery was thenoccluded at this level

We constantly monitored the blood flow in all animals toensure that a uniform cerebral ischemia level was maintainedacross all groups in a standard fashion The blood flowwas controlled by adjusting the suture in the artery for theinduction of ischemiaThe ischemic rats that showed a stabledrop of sim85 in PU compared to the baseline level (beforeMCAO) that is reaching a level of sim15 of the baseline PUwere used for further experimentation The PU was kept at alow level with minor fluctuations during the entire ischemicperiod without any significant change except during EAapplication After the occlusion reperfusion of the ischemicarea was allowed by withdrawing the suture from the rightexternal carotid artery Cortical blood flow changes weremonitored in all animals beginning 5 minutes prior to theinduction of cerebral ischemia until at least 15 minutes afterreperfusion This duration was inclusive of the entire lengthof MCAO and of EA as well

Body temperature was maintained at 365∘C plusmn 05∘Cthroughout the surgical procedures till the animal recoveredfrom anesthesia After the reperfusion the animals werehoused for 24 hours at an ambient temperature of 24 plusmn 1∘CThe same surgical procedures were performed on the animalsin the sham-operation group excluding MCAO

24 Electroacupuncture Methods WHO standards were fol-lowed for the name and localization of acupoints [33 34]ldquoShuigou (Du 26)rdquo and ldquoBaihui (Du 20)rdquo are acupointslocated on the head and face Shuigou (Du 26) is located at apoint on the midline of the upper lip 23rds from the mouthto the nose Baihui (Du 20) is located at a point on themidlineof the head approximatelymidway on the line connecting theapices of both auricles

To obtain optimal EA effect that induces maximum pro-tection against cerebral ischemia we applied EA at ldquoShuigourdquo(Du 26) and ldquoBaihuirdquo (Du 20) with 520Hz sparse-densefrequency at a constant intensity of sim10mA in all EAgroups beginning at 5min after the onset of MCAO or 5minafter MCAO The selection of the acupoints and stimulationparameters was based on the results of our serial studies inthe past [14 17]











3 Results



























4 Discussion



15

10020

100

7550

100

Velo

city

5 minCM

BCPU

MCAO Rep


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA




110100

9080

80

70

70 75

60

60 65

50

50 55

30

30 35

20

20 25

10

10 150

40

40 45

EA

MCAO

minus5 50

Reperfusion



Relat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia + EA-5 min (119899 = 14)

(a)

110100

9080706050

302010

0

40



120513 120513


ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)


(b)

110100

9080706050

302010

0

40



120513 120513


ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)


(c)

Ischemia (119899 = 30)

110100

9080706050

302010

0

40

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

EA

MCAO Reperfusion

Time (minutes)

Relat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia + 119899 = 18)EA-45 min (


(d)









EAlowastlowast

110

100

90

80

70

60

50

30

20

10

0

40

noisufrepeROACM


Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

20 30 50 60 70

(a)



110

100

90

80

70

60

50

30

20

10

0

40

EA


MCAO Reperfusion

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

(b)

EA


110

100

90

80

70

60

50

30

20

10

0

40

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)

120513120513120513120513120513

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)


(c)

110

100

90

80

70

60

50

30

20

10

0

40

Relat

ive c

hang

es in

CM

BC EA

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)


120513120513120513120513120513

( o

f ba

se-v

alue

bef

ore M

CAO

)


(d)






110100

9080706050

302010

0

40


EA

lowastlowast

120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(a)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(b)

110100

9080706050

302010

0

40


EA


MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(c)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(d)








40

35

30

25

20

15

10

5

0

Infa

rct v

olum

e (

of w

hole

bra

in)

Ischemia

Ischemia + EA

Ischemia + N

Ischemia + EA+ N

lowast







Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results



























4 Discussion



15

10020

100

7550

100

Velo

city

5 minCM

BCPU

MCAO Rep


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA




110100

9080

80

70

70 75

60

60 65

50

50 55

30

30 35

20

20 25

10

10 150

40

40 45

EA

MCAO

minus5 50

Reperfusion



Relat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia + EA-5 min (119899 = 14)

(a)

110100

9080706050

302010

0

40



120513 120513


ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)


(b)

110100

9080706050

302010

0

40



120513 120513


ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)


(c)

Ischemia (119899 = 30)

110100

9080706050

302010

0

40

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

EA

MCAO Reperfusion

Time (minutes)

Relat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia + 119899 = 18)EA-45 min (


(d)









EAlowastlowast

110

100

90

80

70

60

50

30

20

10

0

40

noisufrepeROACM


Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

20 30 50 60 70

(a)



110

100

90

80

70

60

50

30

20

10

0

40

EA


MCAO Reperfusion

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

(b)

EA


110

100

90

80

70

60

50

30

20

10

0

40

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)

120513120513120513120513120513

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)


(c)

110

100

90

80

70

60

50

30

20

10

0

40

Relat

ive c

hang

es in

CM

BC EA

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)


120513120513120513120513120513

( o

f ba

se-v

alue

bef

ore M

CAO

)


(d)






110100

9080706050

302010

0

40


EA

lowastlowast

120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(a)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(b)

110100

9080706050

302010

0

40


EA


MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(c)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(d)








40

35

30

25

20

15

10

5

0

Infa

rct v

olum

e (

of w

hole

bra

in)

Ischemia

Ischemia + EA

Ischemia + N

Ischemia + EA+ N

lowast







Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































4 Discussion



15

10020

100

7550

100

Velo

city

5 minCM

BCPU

MCAO Rep


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA




110100

9080

80

70

70 75

60

60 65

50

50 55

30

30 35

20

20 25

10

10 150

40

40 45

EA

MCAO

minus5 50

Reperfusion



Relat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia + EA-5 min (119899 = 14)

(a)

110100

9080706050

302010

0

40



120513 120513


ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)


(b)

110100

9080706050

302010

0

40



120513 120513


ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)


(c)

Ischemia (119899 = 30)

110100

9080706050

302010

0

40

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

EA

MCAO Reperfusion

Time (minutes)

Relat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia + 119899 = 18)EA-45 min (


(d)









EAlowastlowast

110

100

90

80

70

60

50

30

20

10

0

40

noisufrepeROACM


Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

20 30 50 60 70

(a)



110

100

90

80

70

60

50

30

20

10

0

40

EA


MCAO Reperfusion

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

(b)

EA


110

100

90

80

70

60

50

30

20

10

0

40

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)

120513120513120513120513120513

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)


(c)

110

100

90

80

70

60

50

30

20

10

0

40

Relat

ive c

hang

es in

CM

BC EA

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)


120513120513120513120513120513

( o

f ba

se-v

alue

bef

ore M

CAO

)


(d)






110100

9080706050

302010

0

40


EA

lowastlowast

120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(a)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(b)

110100

9080706050

302010

0

40


EA


MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(c)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(d)








40

35

30

25

20

15

10

5

0

Infa

rct v

olum

e (

of w

hole

bra

in)

Ischemia

Ischemia + EA

Ischemia + N

Ischemia + EA+ N

lowast







Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















15

10020

100

7550

100

Velo

city

5 minCM

BCPU

MCAO Rep


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA


15

10020

100

7550

100

Velo

city

5 min

CMBC

PU

MCAO RepEA




110100

9080

80

70

70 75

60

60 65

50

50 55

30

30 35

20

20 25

10

10 150

40

40 45

EA

MCAO

minus5 50

Reperfusion



Relat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia + EA-5 min (119899 = 14)

(a)

110100

9080706050

302010

0

40



120513 120513


ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)


(b)

110100

9080706050

302010

0

40



120513 120513


ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)


(c)

Ischemia (119899 = 30)

110100

9080706050

302010

0

40

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

EA

MCAO Reperfusion

Time (minutes)

Relat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia + 119899 = 18)EA-45 min (


(d)









EAlowastlowast

110

100

90

80

70

60

50

30

20

10

0

40

noisufrepeROACM


Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

20 30 50 60 70

(a)



110

100

90

80

70

60

50

30

20

10

0

40

EA


MCAO Reperfusion

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

(b)

EA


110

100

90

80

70

60

50

30

20

10

0

40

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)

120513120513120513120513120513

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)


(c)

110

100

90

80

70

60

50

30

20

10

0

40

Relat

ive c

hang

es in

CM

BC EA

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)


120513120513120513120513120513

( o

f ba

se-v

alue

bef

ore M

CAO

)


(d)






110100

9080706050

302010

0

40


EA

lowastlowast

120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(a)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(b)

110100

9080706050

302010

0

40


EA


MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(c)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(d)








40

35

30

25

20

15

10

5

0

Infa

rct v

olum

e (

of w

hole

bra

in)

Ischemia

Ischemia + EA

Ischemia + N

Ischemia + EA+ N

lowast







Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















110100

9080

80

70

70 75

60

60 65

50

50 55

30

30 35

20

20 25

10

10 150

40

40 45

EA

MCAO

minus5 50

Reperfusion



Relat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia + EA-5 min (119899 = 14)

(a)

110100

9080706050

302010

0

40



120513 120513


ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)


(b)

110100

9080706050

302010

0

40



120513 120513


ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)


(c)

Ischemia (119899 = 30)

110100

9080706050

302010

0

40

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

EA

MCAO Reperfusion

Time (minutes)

Relat

ive c

hang

es in

amou

nt o

f blo

od p

erfu

sion

( o

f ba

se-v

alue

bef

ore M

CAO

)

Ischemia + 119899 = 18)EA-45 min (


(d)









EAlowastlowast

110

100

90

80

70

60

50

30

20

10

0

40

noisufrepeROACM


Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

20 30 50 60 70

(a)



110

100

90

80

70

60

50

30

20

10

0

40

EA


MCAO Reperfusion

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

(b)

EA


110

100

90

80

70

60

50

30

20

10

0

40

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)

120513120513120513120513120513

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)


(c)

110

100

90

80

70

60

50

30

20

10

0

40

Relat

ive c

hang

es in

CM

BC EA

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)


120513120513120513120513120513

( o

f ba

se-v

alue

bef

ore M

CAO

)


(d)






110100

9080706050

302010

0

40


EA

lowastlowast

120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(a)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(b)

110100

9080706050

302010

0

40


EA


MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(c)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(d)








40

35

30

25

20

15

10

5

0

Infa

rct v

olum

e (

of w

hole

bra

in)

Ischemia

Ischemia + EA

Ischemia + N

Ischemia + EA+ N

lowast







Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















EAlowastlowast

110

100

90

80

70

60

50

30

20

10

0

40

noisufrepeROACM


Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

20 30 50 60 70

(a)



110

100

90

80

70

60

50

30

20

10

0

40

EA


MCAO Reperfusion

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)

(b)

EA


110

100

90

80

70

60

50

30

20

10

0

40

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)

120513120513120513120513120513

Relat

ive c

hang

es in

CM

BC(

of

base

-val

ue b

efor

e MCA

O)


(c)

110

100

90

80

70

60

50

30

20

10

0

40

Relat

ive c

hang

es in

CM

BC EA

MCAO Reperfusion

8070 7560 6550 5530 3520 2510 15 40 45minus5 50

Time (minutes)


120513120513120513120513120513

( o

f ba

se-v

alue

bef

ore M

CAO

)


(d)






110100

9080706050

302010

0

40


EA

lowastlowast

120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(a)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(b)

110100

9080706050

302010

0

40


EA


MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(c)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(d)








40

35

30

25

20

15

10

5

0

Infa

rct v

olum

e (

of w

hole

bra

in)

Ischemia

Ischemia + EA

Ischemia + N

Ischemia + EA+ N

lowast







Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















110100

9080706050

302010

0

40


EA

lowastlowast

120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(a)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(b)

110100

9080706050

302010

0

40


EA


MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(c)

110100

9080706050

302010

0

40


EA


120513120513120513120513120513

MCAO Reperfusion

Relat

ive c

hang

e in

bloo

d ve

loci

ty(

of

base

-val

ue b

efor

e MCA

O)


(d)








40

35

30

25

20

15

10

5

0

Infa

rct v

olum

e (

of w

hole

bra

in)

Ischemia

Ischemia + EA

Ischemia + N

Ischemia + EA+ N

lowast







Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















40

35

30

25

20

15

10

5

0

Infa

rct v

olum

e (

of w

hole

bra

in)

Ischemia

Ischemia + EA

Ischemia + N

Ischemia + EA+ N

lowast







Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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