| ObjectiveTo investigate the effect of electroacupuncture(EA) on neurogenesis and restoration of nerve function in the rats after acute focal cerebral ischemia/reperfusion(I/R),and discuss the function and possible mechanism of electroacupuncture in brain ischemic injury so as to provide the experiment basis for treating cerebrovascular diseases with electroacupuncture.Methods1. The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was established by the modified Longa occlusion method. 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 After 30 minutes of ischemia, the nylon suture was withdrawn to establish reperfusion for 3h,6h,12h and 24h. In sham-operation group, the nylon suture was placed around the ECA approaching the ICA branch without occlusion.2. To inject BrdU(50mg/kg) into abdominal cavity 3 days before diawing the meterials from them twice a day.3. Electroacupuncture (EA, 2～15Hz,1～5mA) was applied to"Shuigou"(GV 26) and"Baihui"(GV 20) for 30 minutes after reperfusion.4. We did TTC experiment after simple cerebral ischemia 30 minutes,45 minutes,1 hour and 24 hours,cerebral ischemia 45 minutes reperfusion 24 hours and 3 days,and cerebral ischemia 1 hour reperfusion 24 hours and 3 days.5. Focal cerebral ischermia model of rats were made by thread locking up the blood vessel for 2 hours. After reperfusion 1d, 3d and 7d, the rat was killed and its brain cells were made into single cell suspension. We detected the neogenetic cell by flow cytometer(FCM).6. The expression of Nestin in corpora striatum and cornu ammonis of rats was detected by Western blot after cerebral ischemia for 2h and reperfusion for 7 days.7. We did the following experiments by making frozen section after cerebral ischemia 2 hours and reperfusion 7 days.(1)BrdU positive average fluorescent intensity and it's location of neogenetic colloid cells were observed by microscope/confocal laser scanning microscope.(2)BrdU and GFAP positive average fluorescent intensity and their location of neogenetic colloid cells were observed by microscope/confocal laser scanning microscope.(3)BrdU and NF200 positive average fluorescent intensity and their location of neogenetic cells were observed by microscope/confocal laser scanning microscope.(4)BrdU and Ach positive average fluorescent intensity and their function of neogenetic cells were observed by microscope.8. Neurofunctional re-establishment: The instruction of water maze was carried out 7 days before operation. We chose the qualified rats after the last instruction and record a result for the preoperative result. The nerve function was detected and the result was recorded after cerebral ischemia 12h and reperfusion 7d,14d and 30d.Result1. The TTC result told us that the cerebral infarction area increased gradually following the time of cerebral ischemia. After the same time of operation,the cerebral infarction area of the cerebral ischemia reperfusion group was larger than the simple cerebral ischemia group. Following the reperfusion time longer, the cerebral infarction area decreased.2. The mean fluorescence intensity (MFI) of the ischemia brain tissue (304.00±6.97)became the highest after cerebral ischemia/reperfusion 7 days, and the MFI began to decrease after reperfusion 14 days. Compared with sham group(176.95±9.33)and I/R group (304.00±6.97), The MFI of I/R+EA group (362.87±16.08)increased obsviously(P<0.01).3. The expression of Nestin in Comu ammonis and Corpora striata of the I/R group was more than the sham group,and up-regulated obviously in the I/R+EA group.4. Expression of neogenetic cell:(1)Neogenesis cells were observed in each group and they concentrated in lateral cerebral ventricle. (2)The main expression of neogenesis cells was in lateral cerebral ventricle,the CA1area of comu ammonis, corpora striata,cavitas subarachnoidealis and the cortical layer of fronto-apical lobe.(3)The expression of neogenesis cells in corpora striata in the I/R+EA group was more than that in the sham group in injured side,and the result had the statistical significance(P<0.01).(4)The small amounts of expression of NF-200 in neogenetic cells concentrated in Latersl cerebral ventricle,Corpora striata and Cerebral cortex. The amounts of expression of NF200 in EA+I/R group was more than that in the I/R group.(5)The small amounts of expression of NF200 in neogenetic cells concentrated in Latersl cerebral ventricle,Corpora striata and Cerebral cortex in I/R+EA group and none in sham group and I/R group.(6)The small amounts of expression of Ach in neogenesis cells concentrated in Corpora striata.5. Escape latency(EL) of both cerebral ischemia/reperfusion group and acupuncture therapy group became the highest after reperfusion 7 days,and they became decreased after reperfusion 14 days,then they were the lowest after reperfusion 30 days. EL in rats of acupuncture therapy group was obviously more than that in cerebral ischemia/reperfusion group.Conclusion1. The amount of neogenetic cell increased after the acute focal cerebral ischemia reperfusion in rats, brain.2. After the acupuncyure the amount of neogenetic cell was increased and the new cells were differentiated. Some cells had the function of producing Ach.3. The acupuncture treatment can improve the recuperation of rats, neuro function after cerebral ischemia.
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