Activation of Epsilon Protein Kinase C-Mediated Anti ...€¦ · Activation of Epsilon Protein Kinase C-Mediated Anti-Apoptosis Is Involved in Rapid Tolerance Induced by Electroacupuncture

Activation of Epsilon Protein Kinase C-MediatedAnti-Apoptosis Is Involved in Rapid Tolerance Induced by

Electroacupuncture Pretreatment Through CannabinoidReceptor Type 1

Qiang Wang, MD, PhD*; Xuying Li, MD*; Yanke Chen, PhD*; Feng Wang, MD; Qianzi Yang, MD;Shaoyang Chen, MD; Yuyuan Min, MD; Xin Li, MD; Lize Xiong, MD, PhD

Background and Purpose—Our previous study has demonstrated that the rapid tolerance to cerebral ischemia byelectroacupuncture (EA) pretreatment was possibly mediated through an endocannabinoid system-related mechanism.The purpose of this study was to investigate whether activation of epsilon protein kinase C (�PKC) was involved in EApretreatment-induced neuroprotection via cannabinoid receptor type 1 in a rat model of transient focal cerebral ischemia.

Methods—The activation of �PKC in the ipsilateral brain tissues after EA pretreatment was investigated in the presenceor absence of cannabinoid receptor antagonists. At 2 hours after the end of EA pretreatment, focal cerebral ischemia wasinduced by middle cerebral artery occlusion for 120 minutes in rats. The neurobehavioral scores, infarction volumes,neuronal apoptosis, and the expression of Bcl-2 and Bax were evaluated after reperfusion in the presence or absence of�PKC-selective peptide inhibitor (TAT-�V1–2) or activator (TAT–��RACK).

Results—EA pretreatment enhanced �PKC activation. Systemic delivery of TAT–��RACK conferred neuroprotectionagainst a subsequent cerebral ischemic event when delivered 2 hours before ischemia. Pretreatment with EA reducedinfarct volumes, improved neurological outcome, inhibited neuronal apoptosis, and increased the Bcl-2-to-Bax ratio afterreperfusion, and the beneficial effects were attenuated by TAT-�V1–2. In addition, the blockade of cannabinoid receptortype 1, but not cannabinoid receptor type 2 receptor, reversed the increase in �PKC activation and neuroprotectioninduced by EA pretreatment.

Conclusion—EA pretreatment may activate endogenous �PKC-mediated anti-apoptosis to protect against ischemicdamage after focal cerebral ischemia via cannabinoid receptor type 1, which represents a new mechanism of EApretreatment-induced rapid tolerance to focal cerebral ischemia in rats. (Stroke. 2011;42:389-396.)

Key Words: apoptosis � cerebral ischemia � electroacupuncture � pretreatment � protein kinase C

Our previous studies have demonstrated that pretreatmentwith electroacupuncture (EA) induces rapid tolerance to

cerebral ischemic insult, which appears at 2 hours afterpretreatment,1 and that the rapid ischemic tolerance is possi-bly mediated through an endocannabinoid system-relatedmechanism in which EA pretreatment increases the produc-tion of the endocannabinoid 2-arachidonylglycerol andN-arach-idonoylethanolamine-anandamide, which elicit pro-tective effects against transient cerebral ischemia via canna-binoid receptor type 1 (CB1) receptors.2 However, the factorslinking EA pretreatment with the development of ischemictolerance are complex and unclear.

The resultant activation of the CB1 receptor triggers signaltransduction events that can influence compensatory responses.3

Cellular responses that elicit neuroprotection may involveCB1 receptors and their link to a variety of signalingelements, including the Gi/Go family of G-proteins, mitogen-activated protein kinase, and its substrate extracellular signal-regulated kinase.4,5 Previous studies have demonstrated thatthe protein kinase C (PKC) signaling pathway was implicatedin cerebral ischemic preconditioning via the adenosine A1receptor and adenosine-induced preconditioning in vitro.6,7

PKC is a widely expressed family of serine/threoninekinases. It has been demonstrated that the activation of PKCin the central nervous system may play a key role inmediating both rapid and delayed preconditioning.8–11 Inter-estingly, among the different PKC isozymes, multiple studieshave now demonstrated that epsilon PKC (�PKC) was a key

Received July 27, 2010; accepted September 27, 2010.From the Department of Anesthesiology (Q.W., X.L., F.W., Q.Y., S.Y., Y.M., L.X., L.X.), Xijing Hospital, Fourth Military Medical University, Xi’an,

China; Center for Biomedical Research on Pain (Y.C.), College of Medicine, Xi’an Jiaotong University, Xi’an, China.*Q.W., X.L., and Y.C. contributed equally to this work.The online-only Data Supplement is available at http://stroke.ahajournals.org/cgi/content/full/STROKEAHA.110.597336/DC1.Correspondence to Qiang Wang and Lize Xiong, Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032,

Shaanxi Province, China. E-mail [email protected] or [email protected]© 2011 American Heart Association, Inc.

Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.110.597336

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player in the induction of ischemic tolerance in variousmodels of preconditioning.12–15 Furthermore, a recent studyreported that �PKC confers acute tolerance to cerebral ische-mic/reperfusion injury.10 Based on these findings, the presentstudy was undertaken to test the hypothesis that an activationof �PKC was involved in EA pretreatment-induced neuropro-tection via CB1 receptors in rat model of transient focalcerebral ischemia.

Materials and MethodsAnimal CareThe experimental protocol used in this study was approved by theEthics Committee for Animal Experimentation of the Fourth Mili-tary Medical University and was conducted according to the Guide-lines for Animal Experimentation of the Fourth Military MedicalUniversity (Xi’an, China). Male Sprague-Dawley rats, weighing 280to 320 grams, were provided by the Experimental Animal Center ofthe Fourth Military Medical University (Xi’an, China) and housedunder controlled conditions with a 12-hour light/dark cycle, atemperature of 21°C�2°C, and humidity of 60% to 70% for at least1 week before drug treatment or surgery. The rats were allowed freeaccess to standard rodent diet and tap water.

Peptide Preparation and DrugsThe ��RACK (receptor for activated C kinase, an �PKC activatorpeptide, �PKC85–92, C-HDAPIGYD) or �V1–2 (an �PKC inhibitorpeptide, �PKC14–21, C-EAVSLKPT) was synthesized as previ-ously described.16 The protein transduction domain of the transacti-vator of transcription (TAT) protein (C-YGRKKRRQRRR), wasconjugated to the �PKC peptide via a disulfide conjugation throughfree cysteines at the N-terminus.17 The new fusion protein wasnamed TAT–��RACK or TAT–�V1–1, respectively. The TAT–�-galactosidase (TAT–�-Gal) fusion protein, as a control protein, wasobtained as previously described.2,18 The �PKC isozyme-selectiveactivity of TAT–��RACK (��RACK) and TAT–�V1–2 (�V1–2)previously has been demonstrated in both in vitro and in vivomodels14,19,20 and, in particular, has been shown to alter �PKCactivity in the brain after systemic intraperitoneal delivery.19 Peptidedose (0.2 mg/kg) was based on previous studies.19–21 The AM251 (aCB1 receptor antagonist) and AM630 (a CB2 receptor antagonist)were purchased from Tocris Bioscience and were dissolved inDMSO and Tween-80 and then diluted with saline (DMSO:Tween-80:saline�1:1:18) before use. The dose of AM251 or AM630 (1mg/kg) in this experiment was chosen based on previous studies.2

First ExperimentTo assess effect of EA pretreatment on the �PKC activation beforeischemia, rats were randomly divided into 3 groups: naive, sham, andEA groups.

Second ExperimentTo determine whether activation of �PKC confers rapid toleranceagainst ischemic injury, the rats were randomly divided into 3groups: middle cerebral artery occlusion (MCAO), TAT–��RACK�MCAO, and TAT–�-Gal�MCAO groups.

Third ExperimentTo evaluate the effect of TAT–�V1–2 on neuroprotection induced byEA pretreatment, male rats were randomly assigned to 5 groups:MCAO, EA�MCAO, TAT–�V1–2�EA�MCAO, TAT–�-Gal�EA�MCAO, and TAT–�V1–2�MCAO groups.

Fourth ExperimentTo test the regulatory effect of �PKC on neuronal apoptosis, ratswere randomly divided into 5 groups: control, MCAO, EA�MCAO,TAT–�V1–2�EA�MCAO, and TAT–��RACK�MCAO groups.

Fifth ExperimentTo explore the role of cannabinoid receptors in neuroprotectioninduced by EA pretreatment, rats were randomly divided into 6groups: MCAO, EA�MCAO, AM251�EA�MCAO, AM251�MCAO, AM630�EA�MCAO, and AM630�MCAO groups. Tofurther investigate the regulatory effect of cannabinoid receptorson activation of �PKC after EA pretreatment, rats were randomlydivided into 6 groups: sham, EA, AM251�EA, AM251,AM630�EA, and AM630 groups. Details of the experimentalgrouping and protocols are included in the online SupplementaryData (please see http://stroke.ahajournals.org).

EA PretreatmentEA pretreatment was performed at the acupoint Baihui (GV 20) ofrats under anesthesia with 40 mg/kg sodium pentobarbital (intraperi-toneal), as described in our previous studies.1,2 The detailed meth-odology for EA pretreatment is described in the online Supplemen-tary Data.

Transient Focal Cerebral IschemiaFocal cerebral ischemia was induced by MCAO in rats using anintraluminal filament technique as described previously.2,18 Regionalcerebral blood flow (rCBF) was monitored through a disposablemicrotip fiber optic probe (diameter, 0.5 mm) connected through aMaster Probe to a laser Doppler computerized main unit (PeriFlux5000; Perimed AB). The MCAO was considered adequate if rCBFshowed a sharp decrease to 20% of baseline (before ischemia) level;otherwise, animals were excluded. Reperfusion was accomplished bywithdrawing the suture after 120 minutes of ischemia, rCBF recov-ered up to �80% of baseline, and then wounds were sutured.

Neurobehavioral Evaluation andInfarct AssessmentAt 72 hours after reperfusion, an 18-point scoring system reported byGarcia et al22 with modifications was used for neurological assess-ment by a blinded observer. Then, animals (n�8 for each group)were decapitated and 2-mm-thick coronal sections from throughoutthe brain were stained with 2% 2,3,5-triphenyltetrazolium chloride(Sigma-Aldrich) to evaluate the infarct volume, as describedpreviously.2,18

TUNEL StainingSamples from 5 groups (n�5 for each group) in experiment 4 wereused for experiments. At 24 hours after reperfusion, neuronal cellapoptosis in the ischemic penumbra was assessed in situ by TUNELstaining as described in our previous studies.2,18 The TUNELstaining was quantitatively evaluated with the method described byWang et al.23 Briefly, 32 pixels of 0.10 mm2 were placed by lightmicroscope with 100� magnification, and then the total number ofpositively stained cells in these pixels was counted and expressed ascells/mm2.

Western Blot AnalysisFor translocation of �PKC, the brain tissues from identical ipsilateralarea to ischemic penumbra were quickly isolated and snap-frozen.Tissue fractionation was performed to collect soluble (cytosolic) andparticulate (membrane) fractions, as previously described.24 Tocompare �PKC concentration in each fraction, total protein concen-tration was assessed using Bradford reagent, and 20 �g of total lysatefrom each fraction was subjected to gel electrophoresis (12%bisacrylamide gel) and transferred to nitrocellulose membrane. Blotswere blocked in 3% milk Tris-buffered saline Tween, probed with ananti-�PKC (C-15) rabbit polyclonal antibody (1:500 dilution; SantaCruz Biotechnology) in 2% milk Tris-buffered saline Tween andprobed with an anti-rabbit secondary antibody.

For Bcl-2 and Bax, at 24 hours after reperfusion the ipsilateralischemic penumbra (n�6 per group) was dissected and then homog-enized and lysed on ice in RIPA buffer containing protease inhibi-tors. The supernatant was collected by centrifugation, and protein

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concentration was quantified by Bradford reagent. The Western blotwas performed as previously described.2 The rat anti-Bcl-2 and ratanti-Bax antibodies (1:1000 dilution; Santa Cruz Biotechnology)were used in this study as primary antibodies. Appropriate secondaryantibody was applied.

Semiquantitative analysis of the blots was performed using den-sitometry followed by quantification with the NIH image program(NIH image version 1.61). Each sample was subjected to immuno-blotting 3 times, and the final optical density value (relative to thatfor the internal standard) represents the average of these 3 separateanalyses. Ischemic core and penumbra were dissected according towell-established protocols in rodent models of unilateral proximalMCAO.25,26

Statistical AnalysisBrain sections were examined by 2 independent and blinded inves-tigators. The software (SPSS 13.0 for Windows; SPSS) was used toconduct statistical analyses. All values, except for neurologicalscores, are presented as mean�SEM and were analyzed by 1-wayanalysis of variance, and between-group differences were detectedwith post hoc Student-Newman-Keuls test. The neurological deficitscores were expressed as median (range) and were analyzed withKruskal-Wallis test, followed by the Mann-Whitney U test withBonferroni correction. Values of P�0.05 were considered as statis-tically significant.

ResultsPhysiological ParametersPhysiological parameters of animals in the period of EApretreatment and transient focal cerebral ischemia and reper-fusion were analyzed. There were no significant differencesfor the variables during EA pretreatment (at the onset of EA,15 minutes after EA, and the end of EA) and surgery (at theonset of ischemia, 60 minutes after ischemia, and 30 minutesafter reperfusion). Arterial blood gases (PO2, PCO2, pH), meanarterial pressure, body temperature, and plasma glucoseremained in the normal range during the experimental periodobserved. The rCBF was monitored in the period of transientfocal cerebral ischemia and reperfusion. No significance wasobserved in the rCBF changes at different time points.Physiological parameters and rCBF changes are summarizedin the online Supplementary Data.

EA Pretreatment Enhances �PKC ActivationUsing a time course of EA pretreatment, �PKC transloca-tion was assessed in the brain tissues from identicalipsilateral area to ischemic penumbra. EA pretreatmentproduced an evident activation of �PKC as revealed byWestern blot (Figure 1B). The semiquantitative analysis ofWestern blot indicated that at 30 minutes after the end ofEA pretreatment, the proportion of �PKC in themembrane-bound fraction was significantly higher thanthat in the sham animals (96% increase; P�0.05). Thistranslocation peaked at 60 minutes (134% increase;P�0.01) and was maintained at 120 minutes after the endof EA pretreatment (59% increase; P�0.05). However,there was no difference in �PKC translocation between thesham and naive control groups at 30, 60, and 120 minutesafter the end of EA pretreatment (Figure 1C).

Activation of �PKC Conferred NeuroprotectionAgainst Ischemic InjuryAs noted in Figure 2, at 72 hours after reperfusion pretreat-ment with TAT–��RACK significantly improved the neuro-

logical scores and reduced the infarction volumes(33.5�1.8%) compared with those of the MCAO group(46.1�2.0%; P�0.000 and P�0.011, respectively). Therewere no statistical differences between MCAO and TAT–�-Gal�MCAO groups (45.5�2.1%; P�0.844 and P�0.971,respectively).

EA Pretreatment-Induced Neuroprotection WasAlleviated by TAT–�V1–2 InterventionAs shown in Figure 3B, EA�MCAO group showed a smallerbrain infarct volume (24.1%�2.8%) compared with MCAOgroup (45.1%�2.9%; P�0.000). The infarct volume of theTAT–�V1–2�EA group (36.3%�2.7%) was smaller thanthat of the MCAO group (P�0.025) and was larger thanthat of the EA�MCAO group (P�0.003). However, theinfarct volume of the TAT–�-Gal�EA group(26.6%�2.3%) was still significantly different from that ofthe MCAO group (P�0.000) and was similar to that of theEA�MCAO group (P�0.513). The result of the TAT–�V1–2�MCAO group (47.1�2.6%) was not significantlydifferent from that of the MCAO group (P�0.612).

Similar changes were observed in neurological scores,except the neurological score of the TAT–�V1–2�EAgroup was similar to that of the MCAO group (P�0.579;Figure 3A).

EA Pretreatment-Induced Reduction of NeuronalApoptosis Is Attenuated by TAT–�V1–2No positive TUNEL staining (brown) was detected in the brainsections of control animals at 24 hours after reperfusion. How-

Figure 1. Electroacupuncture (EA) pretreatment activated epsi-lon protein kinase C (�PKC) isoform translocation to membranefraction (n�5). A, The gray region (I) is defined as the ischemicpenumbra and the black area (region II) is defined as the ische-mic core. B, Representatives of 5 separate experiments for�PKC isoform. C, Correspondent membrane-to-cytosol ratios asindex of �PKC isoform translocation. *P�0.05 vs sham group.

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ever, a large number of TUNEL-positive cells in the ischemicpenumbra of rat brain were seen in the MCAO, TAT–�V1–2�EA, and TAT–�V1–2�MCAO groups, whereas, in contrast,only small amounts of TUNEL-positive cells in theEA�MCAO and TAT–�-Gal�EA groups were observed (Fig-

ure 4A). The quantitative analysis of the number of TUNEL-positive cells in the ischemic penumbra of rats showed that thepretreatment with EA significantly reduced the number ofTUNEL-positive cells (24.7�2.0) at 24 hours after reperfusioncompared to the MCAO (51.0�2.3), TAT–�V1–2�EA(46.3�1.6), and TAT–�V1–2�MCAO (21.7�3.4) groups(P�0.05). There was no difference among the MCAO, TAT–�V1–2�EA, and TAT–�V1–2�MCAO groups (Figure 4B).

Expression of Bcl-2 and Bax Proteins in theIschemic PenumbraAs shown in Figure 5, at 24 hours of reperfusion the levels ofBcl-2 proteins in the ischemic penumbra of rats were higherthan in sham-operated animals (P�0.05; MCAO vs control).Compared with rats only subjected to MCAO, EA or TAT–��RACK pretreatment markedly upregulated the Bcl-2 levels(P�0.05 vs MCAO) in the ischemic penumbra at 24 hoursafter reperfusion, whereas TAT–�V1–2 intervention beforeEA stimulus clearly suppressed the increase in Bcl-2 proteincontents by EA pretreatment (P�0.05; TAT–�V1–2�EA vsEA�MCAO). Focal cerebral ischemia/reperfusion signifi-cantly increased the Bax content in ischemic penumbra at 24hours after reperfusion (P�0.05; MCAO vs control). Inter-estingly, the upregulation of Bax in the ischemic penumbrawas markedly reduced by EA and TAT–��RACK pretreat-ment (P�0.05 vs MCAO). TAT–�V1–2 intervention beforeEA stimulus clearly reversed the reduction in Bax proteinlevels by EA pretreatment (P�0.05; TAT–�V1–2�EA vsEA�MCAO).

AM251, but not AM630, Inhibited Activation of�PKC by EA PretreatmentAs shown in Figure 6A, the EA�MCAO group (25.3�2.3%)showed a smaller brain infarct volume compared with theMCAO group (45.6%�2.5%; P�0.000). The infarct volumeof the AM251�EA�MCAO group (42.1%�2.7%) was

Figure 2. Delivery of ��RACK peptidereduced cerebral damages when deliv-ered before transient focal ischemia(n�10). A, Representative photographsof coronal sections of rat brain afterinfarction stained with 2,3,5-triphenyltet-razolium chloride. Red area is live tissue;white area is dead or dying tissue. B,Neurological scores at 72 hours afterreperfusion in the rats with 120 minutesof middle cerebral artery occlusion(MCAO). C, The bar graph showing thestatistical analysis for infarct volumes in3 groups. Infarct size represented aspercent infarct with respect to contralat-eral hemisphere. *P�0.01 vs MCAOgroup.

Figure 3. Effect of TAT–�V1–2 on electroacupuncture (EA)pretreatment-induced neuroprotection (n �10). A and B, Neuro-logical scores and infarct sizes at 72 hours after reperfusion inthe rats with 120 minutes of middle cerebral artery occlusion(MCAO), respectively. *P�0.01 vs MCAO; #P�0.01 vsEA�MCAO.

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larger than that of the EA�MCAO group (P�0.003),whereas the infarct volume of the AM630�EA�MCAOgroup (26.6%�3.0%) was still similar to that of theEA�MCAO group and smaller than that of the MCAOgroup. There was no difference among MCAO, AM251�MCAO (44.1%�2.9%), and the AM630�MCAO (43.6%�2.9%) groups. Similar changes were observed in neurologicalscores (Figure 6B).

Furthermore, there was no difference in translocation of�PKC among the sham, AM251, and AM630 groups. Theproportion of �PKC in the membrane-bound fraction in theEA and AM630�EA groups was significantly higher thanthat in sham group (P�0.01). However, AM251 inhibited theincrease in �PKC activation induced by EA pretreatment(P�0.05; AM251�EA vs EA) and had no effect on thetranslocation of �PKC when administered alone (P�0.05;AM251 vs sham; Figure 6C).

DiscussionAs one of the top killers of humans, cerebral ischemiaclaims hundreds of thousands of lives every year through-

out the world. To fulfill an increasing need for an effectiveand practical intervention strategy, it is important tounderstand the mechanisms underlying a potent neuropro-tection. Preconditioning, as a potent endogenous protectivemaneuver, activates several endogenous signaling path-ways that result in protection against ischemia.27,28 Iden-tification of these pathways and their targets will likelycontribute to the development of novel therapeutic con-cepts.29 We recently reported that EA pretreatment pro-duced rapid ischemic tolerance against lethal ischemia.1

However, the mechanisms responsible for ischemic toler-ance are complex and remain to be further elucidated, butthey appear to involve an early cellular response.30 Thesignaling mechanisms of rapid EA pretreatment remainobscure, except a potential involvement of adenosine A1receptor1 and endocannabinoid system.2

The PKC family of serine/threonine kinases consists of atleast 11 different isozymes. Importantly, PKC plays a poten-tial role in mediating ischemic and reperfusion damages inthe brain.30 However, individual PKC isozymes mediatedifferent and sometimes opposing functions after activationby the same stimulus.17,31 On stimuli, PKC isoforms translo-cate from the cytosol to subcellular membrane regions, aprocess associated with their activation. Such translocationhas been deemed as a hallmark of PKC activation.32 Activa-

Figure 4. Neuronal cell apoptosis at 24 hours after reperfusionin the rats with 120 minutes of middle cerebral artery occlusion(MCAO; n�5). A, Representative photomicrographs of TUNELstaining in the penumbral zone. The blue cells indicate viablecells and the brown cells indicate TUNEL-positive cells. Scalebars�20 �m. B, Quantitative analysis of the number of TUNEL-positive cells in the ischemic penumbra of rats in 5 groups.*P�0.01 vs MCAO; #P�0.01 vs EA�MCAO.

Figure 5. Western blot analysis of Bcl-2 and Bax protein in theischemic penumbra (n�5). A, Representative Western blotsshowed immunoreactivity of Bcl-2 and Bax proteins in the is-chemic penumbra at 24 hours after 120 minutes of middle cere-bral artery occlusion (MCAO). B, Relative changes in Bcl-2 andBax protein expression. Data are expressed as the ratio of is-chemic animals to controls. *P�0.01 vs control; #P�0.01 vsMCAO; †P�0.05 vs EA�MCAO.

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tion of �PKC before ischemia protects the heart by mimick-ing preconditioning, whereas inhibition of �PKC duringreperfusion protects the heart from reperfusion-induced dam-age.33 The role for �PKC in cerebral tolerance has beenverified using various in vitro models in which �PKC wasactivated after a preconditioning stimulus, such as applicationof ischemia or adenosine or NMDA, and was required forpreconditioning-induced protection.12,15,20,34 Similar to thesestudies in vitro, using an in vivo animal model PKC isoform-specific membrane translocation and protein expression inbrain regions in intact mice with hypoxic preconditioning wasinvestigated. Those results demonstrated that the develop-ment of cerebral hypoxic preconditioning was accompaniedby an obvious increase in membrane translocation of �PKC inthe cortex and hippocampus, but no significant changes werenoted in the membrane translocation of other PKC isoformsor in the whole protein expression of all 11 PKC isoforms,suggesting that activation of �PKC might be involved in thedevelopment of cerebral hypoxic preconditioning of mice.8 Inaddition, �PKC conferred acute tolerance to cerebral ische-mic/reperfusion injury.10

For this reason, we examined the effect of EA pretreatmenton �PKC translocation before ischemia insults in intact rats.We found that at 30 minutes after the end of EA stimulus,�134% increase in translocation of �PKC to membranefraction was observed. �PKC activation at early time pointsafter the stimulus suggested that this isozyme was activatedafter even short periods of stress. Therefore, it was likely that�PKC was activated and may mediate the initial cellularresponse to exogenous stress, such as EA pretreatment in

vivo. Sustained �PKC activation at 120 minutes after EApretreatment also implied that �PKC activity contributed tothe development of ischemic tolerance against more severeischemic insults. These results were consistent with previousstudies in which �PKC was activated within 1 hour afterpretreatment stress.12

After our finding that �PKC was activated in response toEA stimuli, we further examined whether activation orinhibition of �PKC altered outcome after ischemic stroke.Systemic delivery of TAT–��RACK (an �PKC-selectivepeptide activator) with direct activation of �PKC beforeischemia conferred a significant reduction in infarct sizeand improvement of neurological function compared tocontrol fusion protein (TAT–�-Gal). These results wereconsistent with previous studies in which activation of�PKC specifically mediated protective signaling before orearly during ischemia; however, it may not be involved inpromoting cell survival during the reperfusion period.10,12

Further support for the important role of �PKC in EApretreatment emerged from this study showing that neuro-protection of EA pretreatment could be partly blocked withTAT-�V1–2, an �PKC-selective peptide antagonist. Theseresults indicated that activation of �PKC before ischemiaattack may act as an essential step to switch the cells to atolerant state from ischemia insults, and failure of suchtranslocation results in the loss of neuroprotection, as weobserved in the presence of �PKC antagonist.

Compelling evidence indicates that apoptosis may occur inthe ischemic penumbra after transient cerebral ischemia. Inthis study, we observed that the EA pretreatment significantlyreduced the neuronal apoptosis in the ischemic penumbra,whereas TAT–�V1–2 intervention before EA stimulus clearlyreversed the beneficial effect, suggesting EA pretreatmentalleviated neuronal apoptosis via �PKC activation. Extensiveevidence suggests that Bcl-2 family shows a complex net-work regulating apoptosis. Bcl-2 is an anti-apoptotic pro-tein, whereas Bax is pro-apoptotic. The balance betweenthese proteins is critical to turning on and off the cellularapoptotic machinery.35 Upregulation of Bcl-2 and atten-dant decrease of Bax-to-Bcl-2 ratio appear to have keyroles in protective ischemic preconditioning.36 Our presentstudy showed that ischemia significantly increased theratio of the pro-apoptotic Bax to the anti-apoptotic Bcl-2,which was consistent with the previous studies.36 How-ever, pretreatment with EA or TAT–��RACK reduced theexpression of Bax and increased the expression of Bcl-2,thereby ameliorating the ischemia-induced Bax-to-Bcl-2ratio elevation in the ischemic penumbra. Moreover, TAT–�V1–2 intervention before EA stimulus clearly reversedthe regulatory effect of EA pretreatment on Bax-to-Bcl-2ratio. The results further support that the neuroprotectiveeffect of EA pretreatment on ischemia-induced apoptosismight be, at least partly, mediated by regulating theexpression of Bax and Bcl-2 through �PKC activation.

The mechanisms of �PKC activation induced by EApretreatment may be associated with upstream activators ormediators. In the central nervous system, cannabinoids actmainly via stimulation of the central CB1 receptor that ishighly localized in the basal ganglia, hippocampus, cortex,

Figure 6. Effect of electroacupuncture (EA) pretreatment ontranslocation of epsilon protein kinase C (�PKC) in the absenceand presence of a selective cannabinoid receptor antagonist(n�5). A and B, Neurological scores and infarct sizes at 72hours after reperfusion in the rats with 120 minutes of middlecerebral artery occlusion (MCAO), respectively. *P�0.01 vsMCAO; #P�0.01 vs EA�MCAO. C, Representatives of 5 sepa-rate experiments for �PKC isoform. Correspondent membrane-to-cytosol ratios as index of �PKC isoform translocation.AM251, but not AM630, blocked EA-induced �PKC transloca-tion and neuroprotective effects, suggesting that �PKC is down-stream to cannabinoid receptor type 1 (CB1) in the signalingpathway of EA. *P�0.05 vs sham group; #P�0.05 vs EA group.

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and molecular layer of the cerebellum.37 Our previous studyshowed that EA pretreatment upregulated the neuronal ex-pression of CB1 receptor in the rat brains. EA pretreatment-induced neuroprotective effects were attenuated by AM251or CB1 knockdown. Those findings indicated that EA pre-treatment elicited protective effects against transient cerebralischemia through CB1 receptors.2 In the present study,AM251, a selective antagonist that blocks anandamide and2-arachidonylglycerol from binding the CB1 receptors, inhib-ited EA pretreatment-induced �PKC activation and neuropro-tective effects, whereas AM630 (a selective CB2 antagonist)had no effect on the translocation of �PKC and beneficialeffects on EA pretreatment when administered before EApretreatment, indicating that the activation of �PKC involvedin EA pretreatment-mediated neuroprotection is dependent onCB1 receptor.

ConclusionTogether with previous work, the current results stronglysuggest that �PKC activation-mediated anti-apoptosis wasinvolved in EA pretreatment through CB1 receptor. Althougha further investigation is needed to elucidate the detailedsignal cascades underlined in the CB1 receptor–PKC path-way of the EA pretreatment, the present findings mayrepresent a novel mechanism for mechanism of pretreatmentwith EA-induced rapid tolerance to focal cerebral ischemia inrats and also provide considerable implication for otherPKC-related anti-ischemia interventions.

Sources of FundingThis work was supported by the National Natural Science Fundfor Distinguished Young Scholars (grant 30725039), the NationalNatural Science Foundation of China (grants 30873326,81072888, 30930091), and the major clinical project of XijingHospital (grant XJZT09Z05).

DisclosuresNone.

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Xin Li and Lize XiongQiang Wang, Xuying Li, Yanke Chen, Feng Wang, Qianzi Yang, Shaoyang Chen, Yuyuan Min,

Type 1Tolerance Induced by Electroacupuncture Pretreatment Through Cannabinoid Receptor

Activation of Epsilon Protein Kinase C-Mediated Anti-Apoptosis Is Involved in Rapid

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ONLINE SUPPLEMENT Supplemental Methods 1. Experimental protocols

Experiment I: To assess effect of EA pretreatment on the εPKC activation prior to ischemia, rats were randomly divided into 3 groups: Naive, Sham and EA groups. The rats in Naive control group did not receive any treatment. The animals in EA group only received EA stimuli for 30 min without subjecting to MCAO. The rats in Sham group received an identical protocol as EA group without electrical stimulation. For εPKC translocation assays, rats were sacrificed at 30 min, 60 min or 120 min after the end of EA pretreatment. The brain tissues from identical ipsilateral area to ischemic penumbra were harvested and snap frozen for tissue homogenization.

Experiment II: To determine whether activation of εPKC confer rapid tolerance against ischemic injury, the rats were randomly divided into 3 groups: MCAO, TAT–ψεRACK+MCAO and TAT–β-Gal+MCAO groups. The rats were intraperitoneally injected with 1 ml saline, 0.2 mg/kg TAT–ψεRACK, or 0.2 mg/kg TAT-β-Gal (all injections 0.2 mg/kg in 1 ml saline) at 2 h before MCAO. The neurological scores and infarct volumes were evaluated at 72 h after reperfusion.

Experiment III: To evaluate the effect of TAT–εV1-2 on neuroprotection induced by EA pretreatment, male rats were randomly assigned to 5 groups: MCAO, EA+MCAO, TAT–εV1-2+EA, TAT–β-Gal+EA and TAT–εV1-2+MCAO groups. All rats were anesthetized with 1% sodium pentobarbital (40 mg/kg, i.p.) at 3 h before induction of focal cerebral ischemia. The animals in MCAO group only received MCAO and the rats in EA+MCAO group received EA pretreatment for 30 min at 2 h before induction of focal cerebral ischemia. Animals in TAT–β-Gal+EA group and TAT–εV1-2+EA group were pretreated with 0.2 mg/kg TAT–β-Gal and TAT–εV1-2 at 30 min prior to EA pretreatment respectively. Rats in TAT–εV1-2+MCAO group were intraperitoneally injected with 0.2 mg/kg TAT–εV1-2 at 3 h before MCAO. The neurological scores and infarct volumes were evaluated at 72 h after reperfusion.

Experiment IV: To test the regulatory effect of εPKC activation on neuronal apoptosis, rats were randomly divided into 5 groups: Control, MCAO, EA+MCAO, TAT–εV1-2+EA and TAT–ψεRACK+MCAO groups. The rats in Control group received an identical protocol as MCAO group without MCAO. The animals in other groups received the same procedure as described above. The neuronal apoptosis and the expression of Bcl-2 and Bax in the ischemic penumbra were assessed at 24 h after reperfusion.

Experiment V: To explore the role of cannabinoid receptors in neuroprotection induced by EA pretreatment, rats were randomly divided into 6 groups: MCAO, EA+MCAO, AM251+EA+MCAO, AM251+MCAO, AM630+EA+MCAO and AM630+MCAO groups. All rats were anesthetized with 1% sodium pentobarbital (40 mg/kg, i.p.) at 3 h before induction of focal cerebral ischemia, received the same procedure as above experiment. The neurological scores and infarct volumes were

evaluated at 72 h after reperfusion. To further investigate the regulatory effect of cannabinoid receptors on activation

of εPKC following EA pretreatment, rats were randomly divided into 6 groups: Sham, EA, AM251+EA, AM251, AM630+EA and AM630 groups. The animals in Sham and EA groups received the same procedure as in experiment I. The rats in AM251+EA and AM630+EA groups were injected intraperitoneally 1 mg/kg AM251 or AM630 at 30 min prior to the beginning of EA pretreatment respectively. The animals in AM251 and AM630 groups were intraperitoneally injected with 1 mg/kg AM251 or AM630 at 2 h before tissue harvest respectively. The brain tissues from identical ipsilateral area to ischemic penumbra were harvested and snap froze for εPKC translocation assays at 60 min after the end of EA pretreatment. 2. Electroacupuncture Pretreatment

EA pretreatment was performed as described in our previous studies.1,2 Briefly, animals were anesthetized with 40 mg/kg sodium pentobarbital (i.p.), and inhaled oxygen by face mask at a flow rate of 1 L/min. The acupoint “Baihui (GV 20)”, which is located at the intersection of sagittal midline and the line linking two rat ears, was stimulated with the intensity of 1 mA and frequency of 2/15 Hz for 30 min by using the G6805-2 EA Instrument (Model No.227033, Qingdao Xinsheng Ltd., Qingdao, China). The core temperature of all the rats was maintained (Spacelabs Medical Inc., Redmond, WA) at 37.0°C±0.5°C during EA pretreatment by surface heating or cooling. The right femoral artery was cannulated for continuous monitoring of blood pressure and for arterial blood sampling. Arterial blood gases and plasma glucose were measured at the onset of EA, 15 min after EA and at the end of EA.

Supplemental Tables

S1. Physiologic variables during EA pretreatment. (n=5)

Arterial blood gases MAP(mm

Hg) pH PaO2 (mmHg) PaCO2 (mmHg)

Glucose

(mmol/L)

Onset of EA 102±3.1 7.40±0.02 132.6±4.8 35.4±1.7 6.33±0.23

15min after EA 106±3.8 7.39±0.03 135.2±5.1 35.9±1.6 6.87±0.21

End of EA 107±4.3 7.37±0.02 129.7±5.4 36.2±1.1 7.01±0.27

All the variables are presented in mean±SEM. MAP=mean arterial pressure; T=rectal

temperature; PaO2= arterial oxygen partial pressure; EA= electroacupuncture; PaCO2= arterial

CO2 partial pressure;

Supplemental Figures and Figure Legends

S2. Regional cerebral blood flow of ischemic hemisphere during surgery. (n=10) The initial rCBF before occlusion was recorded as 100% and subsequent flow changes are expressed relative to this value. During occlusion, the rCBF values were centralized and remained at <20% of baseline for all rats. At the onset of reperfusion, rCBF recovered up to >80% of baseline, and then returned to baseline within 30 min. As shown in S2, no significance was observed in the rCBF changes between MCAO group and EA+MCAO group. The animals in MCAO group only received MCAO and the rats in EA+MCAO group received EA pretreatment for 30 min at 2 h before induction of focal cerebral ischemia. Data represent mean ± SEM.

Supplemental References

1. Wang Q, Xiong L, Chen S, Liu Y, Zhu X. Rapid tolerance to focal cerebral ischemia in rats is induced by preconditioning with electroacupuncture: Window of protection and the role of adenosine. Neurosci Lett. 2005;381:158-162.

2. Wang Q, Peng Y, Chen S, Gou X, Hu B, Du J, Lu Y, Xiong L. Pretreatment with electroacupuncture induces rapid tolerance to focal cerebral ischemia through regulation of endocannabinoid system. Stroke. 2009;40:2157-2164.