| After Kitagawa and co-workers first reported the ischemic preconditioning-induced tolerance against ischemic injury in the brain in 1990, a number of studies have been done to clarify its mechanisms and pursue the new preconditioning stimuli that can be accepted in clinic. Our laboratory was the first to provide the evidence that the repeated electroacupuncture (EA) at the Baihui acupoint 30 min a day for 5 days could induce cerebral ischemic tolerance (IT) in rats. The different parameters of electroacupuncture and their combinations made great effects on cerebral ischemic tolerance induced by EA. The present study was designed to investigate the role of different parameters including electric current, frequency and wave in IT to focal cerebral. ischemia induced by preconditioning with EA in rats, and to obtain the best combination of stimulating parameters; the other goal was to test the hypothesis that the endogenous opioid peptides,especially the enkephalin, and opioid receptors are involved in IT induced by repeated EA in rats.Objectives1. To investigate the roles of different parameters including electric current, frequency and wave in IT to focal cerebral ischemia induced by preconditioning with repeated EA in rats, and to obtain the best combination of stimulating parameters by nested design.2. To test the hypothesis that the opioid receptors and endogenous opioid peptides, especially the enkephalin, are involved in IT induced by repeated EA in rats.3. To prove the repeated EA (2/15Hz, 1mA, 30min/d, for 5ds) at Baihui acupoint can stimulate the release of met-enkephalin in rats by immunohistochemistry.Method1. Effects of three stimulating parameters and their various combinations on cerebral ischemic tolerance induced by preconditioning with repeated electroacupuncture in rats210 male SD rats were randomly divided into 35 groups(n=6 each): control group, pentobarbital group, 33 electroacupuncture groups including lmA+2Hz , lmA+15Hz , lmA+30Hz , 1mA+100Hz ,2mA+2Hz , 2mA+15Hz , 2mA+30Hz , 2mA+100Hz , 3mA+2Hz , 3mA+15Hz , 3mA+30Hz , 3mA+100Hz ; 1mA+2/0Hz , lmA+15/0Hz , 1mA+30/0Hz , 1mA+100/0Hz , 2mA+2/0Hz , 2mA+15/0Hz , 2rnA+30/0Hz, 2mA+100/0Hz, 3mA+2/0Hz, 3mA+15/0Hz, 3mA+30/0Hz , 3mA+100/0Hz ; lmA+2/15Hz , lmA+2/30Hz , 1mA+2/100Hz ,2mA+2/15Hz , 2mA+2/30Hz , 2mA+2/100Hz , 3mA+2/15Hz , 3mA+2/30Hz and 3mA+2/100Hz. 24 hours after the last treatment, the right middle cerebral artery was occluded for 120min, and the neurological deficit scores (NDS) were evaluated at 24h after reperfusion, and then the infarct volume was determined with TTC staining.2. Cerebral ischemic tolerance induced by repeated electroacupuncture in rats involves the opioid receptor42 male SD rats weighing 280-320g were randomly divided into 7 groups(n=6 each): control group, pentobarbitone group, EA group, EA+ naltrindole(NTI, a highly selectiveS-opioid receptor antagonist) group, EA+ ttor-binaltorphimine (wor-BNI, a highly selective-Kopioid receptor antagonist) group, NTI group and rcor-BNI group. In control group, saline was intraperitoneally administered (4mL/kg/d) for 5 days, while in all the other groups, 1% sodium pentobarbitone was intraperitoneally administered (40mg/kg/d) for 5 days; EA, EA+NTI andgroups received electroacupuncture at the Baihui acupoint for 30 min a day for 5 days; NTI (Img/kg) and rcor-BNI (2.5mg/kg) were intraperitoneally administered before the electroacupuncture stimulation in EA+NTI group and EA+nor-BNI group respectively. The animals in NTI group and wor-BNI group only received NTI (Img/kg) and nor-BNI (2.5mg/kg) respectively. 24 hours after the last treatment, the right middle cerebral artery was occluded for 120min, and the NDS were evaluated at 24h after reperfusion. Then the animals were sacrificed and the infarct volume of the brain was determined with TTC staining.3. Effects of repeated EA on the expression of met-enkephalin (MENK) in the brain of rats12 male SD rats weighing 280-320g were randomly...
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