Effects Of Electroacupuncture On Apoptosis In The Cerebral Cortex Of Rats With Cerebral Ischemia/Reperfusion

ObjectiveTo investigate the effects of electroacupuncture (EA) on neuronal apoptosis and caspase-3 expression in the cerebral cortex of rats with acute focal cerebral ischemia/reperfusion (I/R), and discuss the function of mitochondrial-mediated apoptosis pathway in ischemic brain injury and the protective mechanism of acupuncture intervention so as to supply experimental basis for treating cerebrovascular diseases with electroacupuncture.Methods(1) Male Sprague-Dawley (SD) rats were anesthetized with 10% chloral hydrate. The right common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA) were isolated via a cervical midline incision. A nylon suture was introduced into ECA lumen and advanced into the ICA in order to block the origin of the middle cerebral artery. After 30 minutes of ischemia, the nylon suture was withdrawn to establish reperfusion for 24 hours. In sham–operation group, the nylon suture was placed around the ECA approaching the ICA branch without occlusion.(2) Electroacupuncture (EA, 2～15 Hz, 1 mA) was applied to"Shuigou"(GV 26) and"Baihui"(GV 20) for 30 minutes after reperfusion.(3) Male SD rats were evenly randomized into sham operation control (sham), I/R and I/R+ EA groups. After cerebral ischemia for 30 minutes and reperfusion for 24 hours, the rats were killed rapidly by decapitation for collecting the cerebral cortex tissue and processing into homogenate and single-cell suspension (106 cells/ml). The cell suspension (100μl/tube) was added separately with Rhodamine 123 and Annexin V-FITC, followed by detecting the mitochondrial membrane potential and the number of neuronal apoptosis with flow cytometer (FCM).(4) Caspase-3 positive neurons'average fluorescent intensity in cerebral cortex tissue of each group was observed by two channel scanning of confocal laser scanning microscope (CLSM) after cerebral ischemia for 30 minutes followed by reperfusion for 24 hours.(5) The expression of cerebral cortex tissue caspase-3 mRNA in cerebral cortex tissue of each group was detected by reverse transcription-poly-merase chain reaction (RT-PCR) at 24 hours after reperfusion.Results(1) Compared with the I/R group, the mitochondrial membrane potential was significantly increased (P﹤0.01) after electroacupuncture treatment, while the potential was still lower than sham group (P﹤0.01).(2) The number of neuronal apoptosis in cerebral cortex tissue was significantly increased at 24 hours after ischemia/reperfusion. EA could decrease the number of apoptosis obviously (P﹤0.01) in comparison with I/R group.(3) In I/R and EA groups, the expression of caspase-3 positive protein was scattered, most in cytoplasm and a little in nucleus. There were few caspase-3 positive neuron in the sham group. The average fluorescent intensity in EA group was significantly lower than that of I/R group ( P﹤0.01) but higher than that of the sham group, which is statistically significant.(4) Compared with the sham group, the level of caspase-3 mRNA in cerebral cortex was significantly increased after ischemia/reperfusion (P﹤0.01). After electroacupuncture treatment, the level of caspase-3 mRNA was decreased significantly (P﹤0.01).ConclusionThis study investigated that electroacupuncture can reduce the neuronal apoptosis and inhibit the expression of caspase-3 in the rats with focal cerebral ischemia/reperfusion. The mechanism has a connection with mitochondrial-mediated apoptosis pathway. Electroacupuncture can alleviate cerebral ischemia/reperfusion injury partly by up-regulating the mitochondrial membrane potential and decreasing the expression of caspase-3, this maybe one of mechanisms for electroacupuncture to suppress apoptosis and protect ischemic cerebral neurons.