| Cerebral ischemic and reperfusion (I/R) injuries often occur in clinicalpractice and are associated with high morbidity and mortality. It is important toimprove the ability of brain to tolerate I/R injury. There are different ways to dealwith I/R injury, for example, ischemic preconditioning (IPC). Short ischemicepisodes could induce protection against a subsequent lethal ischemic iujury. Thisphenomenon is known as IPC and ischemic tolerance. Up to now, a number ofmeasures and drugs have been proved to induce ischemic tolerance in brain andprotect neurons from ischemia/reperfusion injury.Previous investigations performed in our laboratory have demonstrated thatpretreatment with electroacupuncture (EA) at Baihui acupoint 30 min a day for 5days could induce the ischemic tolerance in rat. Recently, we furtherdemonstrated that EA pretreatment upregulated the endocannabinoid 2-AG andAEA of brain in rat. Meanwhile, EA pretreatment produced an evident upregulationof CB1 receptor (CB1R) in the ipsilateral hemisphere and the cerebral ischemic tolerance induced by EA pretreatment can be reversed by interventionwith the CB1R antagonist AM251. Those data suggested that EA pretreatmentinduced ischemic tolerance to focal cerebral ischemia through regulation ofendocannabinoid system. However, which signal pathway mediated in EApreconditioning through CB1 receptor is unclear.It is now well demonstrated that IPC shows the tolerance of brain to asubsequent I/R injury. Although IPC has successfully attenuated I/R injury, itsutilization as clinical strategy is largely limited because we cannot predict thebeginning of ischemia. However, the onset of reperfusion is more predictable.Ischemic postconditioning (PostC), which is defined as rapid intermittentinterruptions of blood flow in the early phase of reperfusion and mechanicallyalters the hydrodynamics of reperfusion, are verified to protect tissues from I/Rinjury. The clinical applicability of PostC is limited because PostC can interruptintermittent reperfusion of vital organs required mechanical intervention, which isimprobable in acute coronary syndromes, and implausible in stroke. Recently, theconcept of ischemic PostC has been extended to remote ischemicpostconditioning (RPostC), which was induced by cycles of few minutes of I/Rapplied to a distal artery territory (femoral or renal artery) either immediatelyafter the beginning of heart reperfusion or just few minutes before heartreperfusion. RPostC has greater potential for clinical application thanconventional ischemic PostC, since it can be performed in a non-vital organ,avoiding the high risk of inducing ischemia as PostC in the vital organ, such as thebrain or the heart. RPostC is a simple and harmless method which provides a newtool to protect organs from I/R injury. In the latest years, the protection inducedby RPostC has been the topic of intensive interest in cardiac research, in whichRPostC protects the heart against acute myocardial infarction in rabbits, pigs, and rats. It was confirmed that RPostC induced by transient limb ischemia can preventendothelial I/R injury in humans. However, the protective effect of RPostCagainst focal cerebral ischemiaand its potential protective mechanisms have notbeen established.The Protein kinase C (PKC) family of serine/threonine kinases consists of atleast 11 different isozymes. The expression ofαPKC、β1PKC、β2PKC、γPKC、εPKC、δPKC、ηPKC、θPKC andξPKC mRNA and protein are detected in centralnerve system. Increased evidence indicated that PKC plays a central role inmediating ischemic and reperfusion damage in multiple tissues, including thebrain. Although individual PKC isozymes mediate different and sometimesopposing functions after activation by the same stimulus, most of studiessupported that activation of epsilon PKC (εPKC) before ischemia protects theheart by mimicking preconditioning, whereas inhibition of delta PKC (δPKC)during reperfusion protects the heart from reperfusion-induced damage.Thus, in the present study, we investigated whether RPostC could confer theneuroprotective effect against cerebral I/R injury. Furthermore, we investigatedthe role ofεPKC andδPKC in neuroprotection of EA pretreatment and RPostCusing a rat model of focal cerebral ischemia.ParPartⅠActivation of epsilonPKC-mediated anti-apoptosis is involved inelectroacupuncture pretreatment through cannabinoid CB1receptor ExperimenExperimentⅠActivation ofεPKC in the rats’brain is mediated byelectroacupuncture pretreatmentObjective To assess effect of EA pretreatment on theεPKC activation priorto ischemia. Methods Male SD rats were randomly divided into 3 groups (n = 5):Naive, Sham and EA groups. The rats in Naive group did not receive anytreatment. The animals in EA group only received EA stimuli for 30 min withoutsubjecting to MCAO. The rats in Sham group received an identical protocol asEA group without electrical stimulation. ForεPKC translocation assays, rats weresacrificed at 30 min, 60 min or 120 min after the end of EA pretreatment. Theipsilateral“penumbra”was harvested and snap frozen for tissue homogenization.Results EA pretreatment produced an evident activation ofεPKC in the ipsilateral“penumbra”of brains. The semi-quantitative analysis of Western blot indicatedthat at 30 min after the end of EA pretreatment, the proportion ofεPKC in themembrane-bound fraction was significantly higher than in the sham animals (44%increase, P<0.05). This translocation peaked at 60 min (75% increase, P<0.01),and was maintained at 120 min after the end of EA pretreatment (29% increase,P<0.05). However, there was no difference inεPKC translocation between theSham and Na?ve control groups at 30, 60 and 120 min after the end of EApretreatment. Conclusion The proportion ofεPKC in the membrane-boundfraction was significantly increased at 30 min after the end of EA pretreatment,and lasted at 120 min after the end of the pretreatment, the translocation peakedwas at 60 min after the end of the pretreatment. ExperimenExperimentⅡEffect ofεPKC Activation on cerebral ischemia/reperfusion injuryin ratsObjective To determine whether activation ofεPKC confer rapid toleranceagainst ischemic injury in rats. Methods Male SD rats were randomly divided into3 groups (n = 10): MCAO, TAT–ψεRACK+MCAO and TAT–β-Gal+MCAOgroups. The rats were intraperitoneally injected with 1 ml saline, 0.2 mg/kgTAT–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 evaluatedat 72 h after reperfusion. Results At 72 h after reperfusion, pretreatment withTAT–ψεRACK significantly improved the neurological scores and reduced theinfarction volumes compared with those of MCAO group (P=0.000, 0.011,respectively). There were no statistical differences between MCAO and TAT–β-Gal+MCAO groups (P=0.844, 0.971, respectively). Conclusion Systemicdelivery of TAT–ψεRACK (anεPKC-selective peptide activator) confers rapidtolerance against ischemic injury as well as EA pretreatment.ExperimentⅢEffect of TAT–εV1-2 on neuroprotection induced byEA pretreatment in ratsObjective To evaluate the effect of TAT–εV1-2 on neuroprotection inducedby EA pretreatment in rats. Methods Male SD rats were randomly divided into 5groups (n=10): MCAO, EA+MCAO, TAT–εV1-2+EA, TAT–β-Gal+EA andTAT–εV1-2+MCAO groups. All rats were anesthetized with 1% sodiumpentobarbital (SP, 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+MCAOgroup received EA pretreatment for 30 minutes at 2 h before induction of focalcerebral ischemia. Animals in TAT–β-Gal+EA group and TAT–εV1-2+EA groupwere pretreated with 0.2 mg/kg TAT–β-Gal and TAT–εV1-2 at 30 min prior to EApretreatment respectively. Rats in TAT–εV1-2+MCAO group wereintraperitoneally injected with 0.2 mg/kg TAT–εV1-2 at 3 h before MCAO. Theneurological scores and infarct volumes were evaluated at 72 h after reperfusion.ResultResults EA+MCAO group showed a smaller brain infarct volume and higherneurological scores compared with MCAO group (P=0.000). The infarct volumeof TAT–εV1-2+EA group was smaller than that of MCAO group (P=0.025) andwas larger than that of EA+MCAO group (P=0.003). The neurological scores ofTAT–εV1-2+EA group was higher than that of MCAO group and was lower thanthat of EA+MCAO group (P<0.05). But the infarct volume and neurologicalscores of TAT–β-Gal+EA group was still significantly different from that ofMCAO group (P<0.01) and was similar to that of EA+MCAO group (P>0.05).The result of TAT–εV1-2+MCAO group was not significantly different from thatof MCAO group (P>0.05). Conclusion The neuroprotection of EA pretreatmentcould be partly blocked with TAT-εV1-2, anεPKC-selective peptide antagonist.ExperimentⅣEffect ofεPKC activation on neuronal apoptosis aftercerebral ischemia/reperfusionObjective To test the regulatory effect ofεPKC activation on neuronalapoptosis after cerebral I/R injury in rats. Methods Male SD rats were randomlydivided into 5 groups (n = 5): Control, MCAO, EA+MCAO, TAT–εV1-2+EA andTAT–ψεRACK+MCAO groups. The rats in Control group received an identical protocol as MCAO group without MCAO. The animals in other groups receivedthe same procedure as described above. The neuronal apoptosis and Bel-2/Baxratio in the ischemic penumbra was assessed at 24 h after reperfusion. ResultResultsNo positive TUNEL staining was detected in the brain sections of control animalsat 24 h after reperfusion. However, a large number of TUNEL positive cells in theischemic penumbra of rat brain were seen in the MCAO, TAT–εV1-2+EA andTAT–εV1-2+MCAO groups. While in contrast, only small amount of TUNELpositive cells in the EA+MCAO and TAT–β-Gal+EA groups were observed. Thequantitative analysis of the number of TUNEL positive cells in the ischemicpenumbra of rats showed that the pretreatment with EA significantly reduced thenumber of TUNEL positive cells at 24 h after reperfusion, compared to MCAO,TAT–εV1-2+EA and TAT–εV1-2+MCAO groups (P<0.01). There was nodifference among the MCAO, TAT–εV1-2+EA and TAT–εV1-2+MCAO groups.At 24 h of reperfusion, the levels of Bcl-2 proteins in the ischemic penumbra ofrats were higher than in sham-operated animals (P<0.05, MCAO vs Control).Compared with rats only subjected to MCAO, EA or TAT–ψεRACK pretreatmentmarkedly up-regulated the Bcl-2 levels (P<0.05 vs MCAO) in the ischemicpenumbra at 24 h after reperfusion while TAT–εV1-2 intervention prior to EAstimulus clearly suppressed the increase in Bcl-2 protein contents by EApretreatment (P<0.05, TAT–εV1-2+EA vs EA+MCAO). Focal cerebralischemia/reperfusion significantly increased the Bax content in ischemicpenumbra at 24 h after reperfusion (P<0.05 MCAO vs Control). Interestingly, theup-regulation of Bax in the ischemic penumbra was markedly reduced by EA andTAT–ψεRACK pretreatment (P<0.05 vs MCAO). TAT–εV1-2 intervention priorto EA stimulus clearly reversed the reduction in Bax protein levels by EApretreatment (P<0.05, TAT–εV1-2+EA vs EA+MCAO). Conclusion The neuroprotective effect of EA pretreatment on ischemia-induced apoptosis mightbe, at least partly, mediated by regulating the expression of Bax and Bcl-2 throughεPKC activation.ExperimenExperimentⅤEffect of cannabinoid receptor antagonists on neuroprotectioninduced by EA pretreatmentObjective To evaluate the effect of cannabinoid receptor antagonists onneuroprotection induced by EA pretreatment in rats. Methods Male SD rats wererandomly divided into 7 groups (n = 10): MCAO, EA+MCAO, AM251+EA,AM251+MCAO, AM630+EA, AM630+MCAO and Vehicle+EA groups. All ratswere anesthetized with 1% sodium pentobarbital (SP, 40 mg/kg, i.p.) at 3 h beforeinduction of focal cerebral ischemia. The animals in MCAO group only receivedMCAO and the rats in EA+MCAO group received EA pretreatment for 30minutes at 2 h before induction of focal cerebral ischemia. Animals in AM251+EAgroup, AM630+EA group and Vehicle+EA group were pretreated with 1 mg/kgAM251，AM630 or 3ml/kg Vehicle at 30 min prior to EA pretreatmentrespectively. Animals in AM251+MCAO group and AM630+MCAO group werepretreated with 1 mg/kg AM251 and AM630 at 3 h before induction of focalcerebral ischemia respectively. The neurological scores and infarct volumes wereevaluated at 72 h after reperfusion. Results At 72 h after reperfusion, comparedwith MCAO group, EA+MCAO group showed a smaller brain infarct volume andhigher neurological scores (P<0.05). Systemic delivery of AM251 reversed theneuroprotection induced by EA pretreatment (P<0.05). While systemic deliveryof AM630 had no effect on the neuroprotection induced by EA pretreatment(P>0.05). AM251 and AM630 had no effect on cerebral ischemia/reperfusion (P>0.05). ConclusiConclusion The neuroprotection of EA pretreatment could be blockedwith AM251 but not AM630.ExperimentⅥEffects of cannabinoid receptor antagonists on the activationofεPKC followed by EA pretreatmentObjective To investigate the regulatory effect of cannabinoid receptors onactivation ofεPKC following EA pretreatment in rats. Methods Male SD ratswere randomly divided into 6 groups (n = 5): Sham, EA, AM251+EA, AM251,AM630+EA and AM630 groups. The animals in Sham and EA groups receivedthe same procedure as in experiment I. The rats in AM251+EA and AM630+EAgroups were injected intraperitoneally 1 mg/kg AM251 or AM630 at 30 min priorto the beginning of EA pretreatment respectively. The animals in AM251 andAM630 groups were intraperitoneally injected with 1 mg/kg AM251 or AM630 at3 h before simple harvested. The ipsilateral“penumbra”was harvested and snapfroze forεPKC translocation assays at 60 min after the end of EA pretreatment.Results There was no difference in translocation ofεPKC among Sham, AM251and AM630 groups. The proportion ofεPKC in the membrane-bound fraction inEA and AM630+EA groups was significantly higher than that in Sham group(P<0.01). However, AM251 inhibited the increase inεPKC activation induced byEA pretreatment (P<0.05, AM251+EA versus EA), and had no effect on thetranslocation ofεPKC when administered alone (P>0.05, AM251 versus Sham).Conclusion The activation ofεPKC was involved in EA pretreatment-mediatedneuroprotection in a manner dependent of cannabinoid CB1 receptor. PartⅡLimLimb remote postconditioning alleviates cerebral reperfusioninjury by inhibiting activation of delta protein kinase CExperimentⅠEffect of Limb remote postconditioning on cerebralischemia/reperfusion injury in ratsObjective To determine the protective effect of RPostC against cerebral I/Rinjury in rats. Methods Male SD rats were randomly divided into 3 groups (n =10): MCAO, SP, RPostC. The rats in the Control group were subjected to MCAOonly. The animals in the SP group were received anesthesia with 1% sodiumpentobarbital (SP, 40 mg/kg, i.p.) at the onset of reperfusion. The animals in theRPostC group were received RPostC under anesthesia with 1% SP (40 mg/kg,i.p.). The neurological scores and the infarct volumes were evaluated at 72 h afterreperfusion. Results At 72 h after reperfusion, compared with Control group,RPostC group had higher neurological scores and less infarction volumes(P<0.01). There were no statistical differences between Control group and SPgroups (P>0.05). Conclusion Three cycles of transient hindlimb I/R applied justat the onset of middle cerebral artery reperfusion inducing RPostC could improveneurologic outcome, reduce infarct size.ExperimentⅡEffect of Limb remote postconditioning onδPKC activation andneuronal apoptosis followed by cerebral ischemia/reperfusionObjective To assess the effect of RPostC onδPKC activation and neuronalapoptosis after reperfusion. Methods Male SD rats were randomly divided into 4 groups (n = 10): Naive, Control, SP, RPostC. The animals in the Naive groupreceived same anesthesia as Control group but no ischemia. The rats in the Controlgroup were subjected to MCAO only. The animals in the SP group were receivedanesthesia with 1% sodium pentobarbital (SP, 40 mg/kg, i.p.) at the onset ofreperfusion. The animals in the RPostC group were received RPostC underanesthesia with 1% SP (40 mg/kg, i.p.). Rats (n = 5 each group) were sacrificed at24 h after reperfusion for TUNEL staining. Animals (n = 5 each group) weresacrificed at 120 min after reperfusion. The ipsilateral peri-infarct samples wereharvested immediately and quickly frozen in liquid nitrogen forδPKCtranslocation assays by using western blot. ResultResults TUNEL staining was negativein the brain sections of Naive animals at 24 h after reperfusion. However, a largenumber of TUNEL positive cells in the ischemic penumbra of rat brain were seenin the control and SP groups while in contrast, only small TUNEL positive cells inthe RPostR group were observed. The RPostR significantly reduced the numberof TUNEL-positive cells at 24 h after reperfusion, compared to control group(P<0.01). However, the number of TUNEL-positive cells in SP group was similarto that of control group (P>0.05). There was no difference in translocation ofδPKC between SP and Na?ve control groups. The proportion ofδPKC in themembrane-bound fraction in control rats was significantly higher than in shamanimals (P<0.05). However, RPostC inhibited the increase in ofδPKC activationinduced by reperfusion (P<0.05, RPostC vs. Control). Conclusion RPostC appliedjust at the onset of middle cerebral artery reperfusion could inhibit neuronal cellapoptosis as well as suppress the activation ofδPKC.ExperimentⅢEffect of TAT-δV1-1 on cerebral ischemia/reperfusion injury ObjectivObjective To assess the effect of TAT-δV1-1 on cerebral I/R injury in rats.Methods Male SD rats were randomly divided into 3 groups (n = 10): Control,TAT–δV1-1 and TAT–β-Gal groups. The rats were intraperitoneally injected with1 ml saline, 0.2 mg/kg TAT–δV1-1, or 0.2 mg/kg TAT–β-Gal (all injections 0.2mg/kg in 1 ml saline) at the onset of reperfusion. The neurological scores andinfarct volumes were evaluated at 72 h after reperfusion. Results At 72 h afterreperfusion, intraperitoneally administration of TAT–δV1-1 at the onset ofreperfusion significantly improved the neurological scores and reduced theinfarction volumes compared with those of control group (P=0.000, 0.011,respectively). There were no statistical differences between control and TAT–β-Gal groups (P=0.844, 0.971, respectively). Conclusion Systemic delivery ofTAT–δV1-1 (anδPKC-selective peptide inhibitor) confers neuroprotection as wellas RPostC. |
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