Involvement Of Cannabinoid Receptors In The Ischemic Tolerance Of Different Phase Induced By Pretreatment With Electroacupuncture

Discovering safe,effective and easy measures for non-ischemic preconditioning is a hotspot of neuroprotective research. Our previous study has demonstrated that pretreatment with electroacupuncture (EA) at the Baihui acupoint (GV 20) induces rapid and delayed tolerance to cerebral ischemic insult. And EA has the best combination of parameters and point speciality【1,2】. Later we found thatδ-andμ-opioid receptor participate in the neuroprotection induced by EA, butδ-andμ-opioid receptor antagonist could not block the neuroprotection induced by EA. All the findings indicate that the neuroprotective effect may also be mediated by other active compounds and signaling pathways. CB1 receptor agonists have a neuroprotective effect, and CB1 receptors and opioid receptor have the effects of cooperation and interaction. All these indicate that endocannabinoids and CB1 receptor are possible to participating in the neuroprotection induced by EA. In the meanwhile, acupuncture stimuli might induce neuroprotection through repressing the excessive activation of glial cell and the anti-inflammatory reaction【3】. After cerebral ischemia, activation of glial cell will release inflammatory mediators and induce inflammatory, which will result in the second neuronal damage. As inflammatory response induces cerebral damage mainly in the late stage after ischemia, so inhibiting the activation of glial cells after ischemia and reducing the inflammatory response can play role in cerebral protection of the late stage after ischemia. Activation of CB2 receptor, which is expressed in microglia and astrocyte【4】, may repress inflammatory reaction and induce neuroprotection【5,6】. According to all these findings, we presumed that the endocannabinoid system is involved in the mediation of the neuroprotective effect of EA pretreatment. Thus, the present study was designed to explore whether the cannabinoid receptors are involved in the mediation of the rapid and delayed neuroprotection induced by EA pretreatment at the Baihui (GV 20) acupoint.Part 1 The effect of the cannabinoid receptors on rapid neuroprotection induced by EA pretreatmentMetheds1 The expression of cannabinoid receptors after the end of EA.Forty-two adult male SD rats weighing 280-320g were randomly divided into 3 groups: CON, SP and EA groups. The rats in CON group did not receive any treatments. The rats in SP group only received anesthesia with intraperitoneal injection (ip) of 40mg/kg sodium pentobarbital(SP). The rats in EA group were anesthetized with 40mg/kg SP intraperitoneal and received EA stimuli for 30 minutes. Respectively at 30 minutes, 60minutes and 120minutes after EA pretreatment, expressions of CB1 and CB2 receptors were detected.2 Involvement of CB1 receptor in the rapid tolerance to focal cerebral ischemia induced by EA pretreatment.Fifty-six adult male SD rats were randomly assigned to 7 groups (n=8): Sham, MCAO, EA,Vehicle + EA, AM630 + EA, AM251 + EA和AM630+AM251+EA groups. The rats in MCAO group only received MCAO and the rats in EA group received MCAO at 2 hours after the end of EA pretreatment for 30 minutes. The rats in Vehicle +EA, AM251+EA and AM630+AM251+EA groups were intraperitoneally administered with 1 ml Vehicle (DMSO:Tween80: saline=1:1:8) and 1 mg/kg AM251 at 30 minutes prior to the onset of EA pretreatment respectively, and then subjected to MCAO at 2 hours after the end of EA pretreatment. The rats in AM630+EA group and AM630+AM251+EA group were injected intraperitoneally with 1 mg/kg AM630 at 3 hours prior to the onset of EA pretreatment. The rats in Sham group had not been given insertion of nylon monofilament suture into the right MCA , and the remaining operations were similar to the MCAO group.Results1 The expression of cannabinoid receptors after the end of EA.1.1 CB1 receptor mRNA expressionThe expression of CB1 receptor mRNA significantly increased at 30 minutes and 60 minutes after the end of EA pretreatment compared with that in CON and SP group(P<0.01), while it was similar at 120 minutes after EA pretreatment. 1.2 CB1 receptor protein expressionThe analysis of western blot indicated that the expression of CB1 receptor protein significantly increased at 120 minutes after the end of EA pretreatment compared with that in CON and SP group(P<0.01).1.3 CB2 receptor mRNA and protein expressionThe RT-PCR and Western blot results indicated that all the groups had no differences at each time points.2 Involvement of CB1 receptor in the rapid tolerance to focal cerebral ischemia induced by EA pretreatment.2.1 Neurobehavioral scoreSeven-two hours after reperfusion, pretreatment with EA significantly improved the neurobehavioral scores compared with that of MCAO group (P<0.01, EA vs MCAO). The CB2 receptor antagonist AM630 did not reverse the rapid beneficial effect of EA pretreatment (P>0.05, AM630+EA vs EA), but the CB1 receptor antagonist AM251 did (P<0.01, AM251+EA vs EA). There were no statistical differences in the neurobehavioral scores among MCAO, AM251+EA and AM630+AM251+EA groups.2.2 Infarction volume percentageSeven-two hours after reperfusion, pretreatment with EA significantly reduced the infarction volume percentages compared with that of MCAO group (P<0.01, EA vs MCAO). The CB2 receptor antagonist AM630 did not reverse the rapid beneficial effect of EA pretreatment ( P>0.05, AM630+EA vs EA), but the CB1 receptor antagonist AM251 did (P<0.01, AM251+EA vs EA). There were no statistical differences in the infarction volume percentages among MCAO, AM251+EA and AM630+AM251+EA groups. Part 2 The effect of the cannabinoid receptors on delayed neuroprotection induced by EA pretreatmentMetheds1 The expression of CB receptor after the end of EA.Sixty-six adult male SD rats weighing 280-320g were randomly divided into 3 groups: CON, SP and EA groups. The rats in CON group did not receive any treatments. The rats in SP group only received anesthesia with intraperitoneal injection(ip) of 40mg/kg sodium pentobarbital(SP). The rats in EA group were anesthetized with 40mg/kg SP intraperitoneal and received EA stimuli for 30 minutes. Respectively at 2 hours,6 hours,12 hours,18 hours and 24 hours after EA pretreatment, expressions of CB1 and CB2 receptors were detected.2 Involvement of CB2 receptor in the delayed tolerance to focal cerebral ischemia induced by EA pretreatment.Fifty-six male SD rats randomly assigned to 7 groups: Sham, MCAO, EA, Vehicle+EA, AM630+EA, AM251+EA and AM630+AM251+EA groups (n=8). The rats in MCAO group only received MCAO and the rats in EA group received MCAO at 24 hours after the end of EA pretreatment for 30 minutes. The rats in Vehicle +EA, AM251+EA and AM630+AM251+EA groups were intraperitoneally administered with 1 ml Vehicle (DMSO:Tween80:saline=1:1:8) and 1 mg/kg AM251 at 30 minutes prior to the onset of EA pretreatment respectively, and then subjected to MCAO at 24 hours after the end of EA pretreatment. The rats in AM630+EA group and AM630+AM251+EA group were injected intraperitoneally with 1 mg/kg AM630 at 3 hours prior to the onset of EA pretreatment. The rats in Sham group had not been given insertion of nylon monofilament suture into the right MCA , and the remaining operations were similar to the MCAO group.Results1 The expression of CB receptor during 24h after the end of EA.1.1 CB1 receptor mRNA and protein expressionAll the groups had no differences in the results of RT-PCR at each time points. CB1 receptor protein significantly increased at 2 hours after the end of EA pretreatment compared with that in CON and SP group(P<0.01).1.2 CB2 receptor mRNA expressionThe expression of CB1 receptor mRNA significantly increased at 18 hours after the end of EA pretreatment compared with that in CON and SP group(P<0.01), while it was similar at 24 hours after EA pretreatment.1.3 CB2 receptor protein expressionThe analysis of western blot indicated that the expression of CB2 receptor protein significantly increased at 24 hours after the end of EA pretreatment compared with that in CON and SP group(P<0.01).2 Involvement of CB2 receptor in the delayed tolerance to focal cerebral ischemia induced by EA pretreatment.2.1 Neurobehavioral scoreSeven-two hours after reperfusion, pretreatment with EA significantly improved the neurobehavioral scores compared with that of MCAO group (P<0.01, EA vs MCAO). The CB1 receptor antagonist AM251 had no effect on reversing the protective effect (P>0.05, AM251+EA vs EA), but the CB2 receptor antagonist AM630 partly reversed the beneficial effect of EA pretreatment (P<0.05, AM630+EA vs EA). There were no statistical differences in the neurobehavioral scores between AM630+EA and AM251+AM630+EA groups.2.2 Infarction volume percentageSeven-two hours after reperfusion, pretreatment with EA significantly reduced the infarction volume percentages compared with that of MCAO group (P<0.01, EA vs MCAO). The CB1 receptor antagonist AM251 had no effect on reversing the protective effect (P>0.05, AM251+EA vs EA), but the CB2 receptor antagonist AM630 partly reversed the beneficial effect of EA pretreatment (P<0.05, AM630+EA vs MCAO and EA). There were no statistical differences in the infarction volume percentages between AM630+EA and AM251+AM630+EA groups.Summary1 EA pretreatment up-regulated the expression of CB1 receptor in brain tissue rapidly and in the same timepoints the expression of CB2 receptor had no differences. The CB1 receptor antagonist AM251 may reverse the rapid beneficial effect of EA pretreatment, but the CB2 receptor antagonist AM630 had not the same effect. These results indicate that CB1 receptor is involved in the rapid tolerance to focal cerebral ischemia induced by EA pretreatment.2 EA pretreatment delayedly up-regulates the expression of CB2 receptor and in the same timepoints the expression of CB1 receptor had no differences. The CB2 receptor antagonist AM630 partly reversed the beneficial effect of EA pretreatment, but the CB1 receptor antagonist AM251 had not the same effect. These results indicate that CB2 receptor is involved in the delayed tolerance to focal cerebral ischemia induced by EA pretreatment.