The Endocannabinoid System Is Involved In The Ischemic Tolerance Induced By Pretreatment With Electroacupuncture

Cerebral Ischemia is one of the most common causes of disability and death. Clinical treatment of this debilitating disorder is inadequate. To explore an effective, less harmful and more acceptable pretreatment is of great significance. As a product of integration of traditional acupuncture and modern electrical stimulation technique, electroacupuncture possesses a number of advantages such as simple operation, safety, reliability and acceptability. Its therapeutic and protective effect of cerebral ischemic injury has been proved by clinical and animal experiments. Our previous study has demonstrated that pretreatment with electroacupuncture (EA) at the Baihui acupoint has significant neuroprotective effect on cerebral ischemic injury. It can protect the brain from the subsequent cerebral ischemic injury, significantly reduce brain infarct volumes and greatly improve the neurological function scores of rats, and this neuroprotective effect is possibly mediated by the release of enkephalins, which may bindδ- andμ-opioid receptors to induce the tolerance against focal cerebral ischemia. The interaction between endocannabinoid and opioid neurotransmission has been reported. They share a similar pharmacological profile: both induce analgesia, hypothermia, hypotension, immunosuppression and so on. Besides, previous studies demonstrated that pretreatment with either 2-AG or AEA before ischemia mimicked preconditioning inasmuch as it protected the ischemic insults in a similar fashion in rat isolated hearts. Synthetic and endocannabinoids exert neuroprotective effects in animals as well as in vitro models of various forms of acute neuronal injury. These findings indicate that the endocannabinoid system participates in the ischemic tolerance which induced by electroacupuncture. Thus, the present study is designed to explore whether the endocannabinoid system are involved in the ischemic tolerance induced by EA pretreatment at the Baihui acupoint.Part 1 The effect of EA pretreatment at the Baihui acupoint on the endocannabinoid systemMethods1. The effect of pretreatment with single electroacupuncture on the endocannabinoid system Male SD rats weighing 280-320 g were randomly assigned to 3 groups: Sham, SP and EA groups. The rats in Sham group did not receive any treatment. The rats in SP group only received anesthesia with intraperitoneal injection (ip) of 40 mg/kg sodium pentobarbital; rats in EA group were anesthetized with 40 mg/kg sodium pentobarbital (ip) and received EA stimuli for 30 min. At 2 h after the end of EA pretreatment, CB1 receptor expression was examined and the endocannabinoid content was determined; at 30 min, 1 h and 2 h after EA pretreatment, expression of both CB1 receptor mRNA and protein was determined.2. The effect of pretreatment with repeated electroacupuncture on the endocannabinoid systemMale SD rats weighing 280-320 g were randomly assigned to 4 groups: Sham-2, EA-2, Sham-24 and EA-24 groups. Rats in Sham-2 group and Sham-24 group were anesthetized with sodium pentobarbital (ip) once a day for 5 consecutive days, rats in EA-2 group and EA-24 group were anesthetized with sodium pentobarbital 40 mg/kg (ip) and received electroacupuncture pretreatment at the Baihui acupoint 30 min a day for 5 consecutive days. Rats in Sham-2 group and EA-2 group were decapitated 2 h after last pretreatment; rats in Sham-24 group and EA-24 group were decapitated 24 h after last pretreatment. The endocannabinoid content was determined.Results1. The effect of pretreatment with single electroacupuncture on the endocannabinoid system1.1. Double immunofluorescence for CB1 receptor and NeuNThe CB1 receptor-like immunoreactivity in the ipsilateral hemisphere was significantly increased by EA pretreatment. Double immunofluorescence for CB1 receptor and NeuN demonstrated that CB1 receptor was mainly localized in neurons. The CB1 receptor immunoreactivities were increased and colocalized with the neuronal immunoreactivities at 2 h after EA pretreatment in the ipsilateral hemisphere.1.2 CB1 receptor mRNA expressionThe expression of CB1 receptor mRNA significantly increased at 30 and 60 min after EA pretreatment compared with that in Sham or SP group while it was similar at 120 min after EA pretreatment.1.3 CB1 receptor protein expressionThe analysis of Western blot bands indicated that EA pretreatment significantly increased CB1 receptor protein expression level compared with that in Sham or SP group at 120 min after EA pretreatment. 1.4. The content of endocannabinoidPretreatment with EA increased the brain tissue content of the endocannabinoid 2-AG and AEA.2. The effect of pretreatment with repeated electroacupuncture on the endocannabinoid systemPretreatment with repeated electroacupuncture increased the brain tissue content of the endocannabinoid 2-AG and AEA. The content of AEA was significantly increased in the EA-2 group and EA-24 group. The content of 2-AG was increased in the EA-2 group but decreased in the EA-24 group.Part 2The effect of the endocannabinoid system on the ischemic tolerance induced by EA pretreatmentMethods1. CB1 Receptor is involved in the ischemic tolerance induced by pretreatment with single electroacupuncture Male SD rats weighing 280-320 g were anesthetized with sodium pentobarbital (ip) at 2.5 h before induction of focal cerebral ischemia, and were randomly assigned to 5 groups: Con, EA-MCAO, AM-EA-MCAO, v-EA-MCAO and AM-MCAO groups. The rats in Con group only received MCAO and the rats in EA-MCAO group received MCAO at 2 h after EA pretreatment for 30 min. Rats in AM-MCAO group were intraperitoneally injected with 1 mg/kg AM251 at 3 h before MCAO. Rats in v-EA-MCAO group and AM251-EA-MCAO group were administered with 3 ml/kg vehicle and 1 mg/kg AM251 at 30 min prior to the onset of EA pretreatment respectively, and then subjected to MCAO at 2 h after the end of EA pretreatment. At 24 h or 7 d after reperfusion, the neurological function scores were evaluated and the brain infarct volume, as a percentage of volume at normal cerebral hemisphere was then assessed.2. CB1 Receptor is involved in the ischemic tolerance induced by pretreatment with repeated electroacupunctureMale SD rats weighing 280-320 g were randomly assigned to 6 groups: Sham, Con, EA-MCAO, AM-EA-MCAO, v-EA-MCAO and AM-MCAO groups. All of the rats were anesthetized with intraperitoneal injection of 40 mg/kg sodium pentobarbital once a day for 5 consecutive days except those in Sham group. Rats in Sham group only received sham operation. Rats in Con group were only subjected to ischemia and reperfusion; rats in AM-MCAO group received 1 mg/kg AM251 (ip) once a day for 5 consecutive days; rats in EA-MCAO group received electroacupuncture at the Baihui acupoint for 30 min a day for 5 consecutive days; rats in v-EA-MCAO group and AM-EA-MCAO group received electroacupuncture at the Baihui acupoint for 30 min, and were respectively injected with 3 ml/kg vehicle and 1 mg/kg AM251 (ip) 30 min prior to the onset of electroacupuncture once a day for 5 consecutive days. 24 h after last treatment, each group except Sham group received the middle cerebral artery occlusion. At 24 h after reperfusion, the neurological function scores were evaluated and the brain infarct volume, as a percentage of volume at normal cerebral hemisphere was then assessed.3. The effect of pretreatment with repeated electroacupuncture on the endocannabinoid system after ischemia/reperfusion injuryMale SD rats weighing 280-320 g were randomly assigned to 5 groups: Sham, Con-2, EA-MCAO-2, Con-24 and EA-MCAO-24 groups. Rats in Sham group, Con-2 group and Con-24 group were anesthetized with sodium pentobarbital 40 mg/kg (ip) for 5 consecutive days; rats in EA-MCAO-2 group and EA-MCAO-24 group were anesthetized with sodium pentobarbital 40 mg/kg (ip) and then received electroacupuncture pretreatment at the Baihui acupoint 30 min a day for 5 consecutive days. 24 h after last treatment, each group except Sham group received the middle cerebral artery occlusion with 3-0 nylon monofilament. The rat were decapitated at 24 h after last treatment in Sham group, at 2 h after reperfusion in Con-2 group and EA-MCAO-2 group, 24 h after reperfusion in Con-24 group and EA-MCAO-24 group. The endocannabinoid content was determined.Results1. CB1 Receptor is involved in the ischemic tolerance induced by pretreatment with single electroacupuncture1.1. Neurologic Outcome24 h and 7 d after reperfusion, pretreatment with EA significantly improved the neurological function scores compared with that of Con group. The CB1 receptor antagonist AM251 had no effect on neurological function scores when administered alone but reversed the beneficial effect of EA pretreatment given 30 min before the onset of EA pretreatment.1.2. Infarct VolumeEA-MCAO group showed a smaller brain infarct volume at 24 h and 7 d after reperfusion compared with Con group, The CB1 receptor antagonist AM251 had no effect on infarct size when administered alone but reversed the beneficial effect of EA pretreatment given 30 min before the onset of EA pretreatment.2. CB1 Receptor is involved in the ischemic tolerance induced by pretreatment with repeated electroacupuncture2.1. Neurologic OutcomeIn sham group, no neurological deficits was observed. Pretreatment with EA significantly improved the neurological function scores at 24 h after reperfusion compared with that of Con group. The CB1 receptor antagonist AM251 had no effect on neurological function scores when administered alone but reversed the beneficial effect of EA pretreatment given 30 min before the onset of EA pretreatment. 2.2. Infarct VolumeIn sham group, no infarct volume was observed. EA-MCAO group showed a smaller brain infarct volume at 24 h after reperfusion compared with Con group, The CB1 receptor antagonist AM251 had no effect on infarct size when administered alone but reversed the beneficial effect of EA pretreatment given 30 min before the onset of EA pretreatment.3. The effect of pretreatment with repeated electroacupuncture on the endocannabinoid system after ischemia/reperfusion injuryThe brain tissue content of AEA was significantly increased in Con-2 group compared with that in Sham group, but decreased at 24 h after reperfusion. Compared with that in Sham group and Con-2 group, AEA content was significantly increased in EA-MCAO-2 group. In EA-MCAO-24 group, the AEA content was also significantly increased compared with that in Con-24 group. In EA-MCAO-2 group, the 2-AG content was significantly increased compared with that in Sham group and Con-2 group.Summary1. Pretreatment with single or repeated electroacupuncture at the Baihui acupoint increases the expression of CB1 receptor and the production of the endocannabinoid 2-AG and AEA. These results indicate that pretreatment with electroacupuncture activates the endocannabinoid system.2. Pretreatment with electroacupuncture increases the production of the endocannabinoid 2-AG and AEA in the brain after ischemia/reperfusion injury and the protective effect of EA pretreatment is reversed by CB1 receptor antagonist. These results indicate that pretreatment with electroacupuncture activates the endocannabinoid system which is involved in the ischemic tolerance induced by pretreatment with electroacupuncture.