| Objective:Cerebrovascular disease has become one of the major diseases that may harm the health and life of Chinese middle-aged and aged people. Acute ischemic cerebral vascular disease takes up 43%～65% of cerebrovascular diseases, with a fatality rate of 15%～5%. Its high incidence, high mortality and high morbidity brings our country and patients heavy economic and social burdens. Due to the first aid is not timely during disease ultra-early phase, many of the clinical patients with cerebral ischemia have their brain cells suffer irreversible damagein after short-term ischemia and hypoxia, ultimately resulting in death or serious sequelae. There will be a positive impact on patients' prognosis, if safe, effective and simple first aid measures are used to effectively protect brain tissue during shortest possible time after the onset. Drug therapy is currently one of the effective means for ischemic brain injury but with a certain toxic and side effects, while acupuncture therapy has been widely used for cerebral vascular disease recovery treatment, and has been become increasingly common treatment to the acute stage of ischemic stroke. An immediate acupuncture treatment given after ischemia gives ischemic cerebral blood flow significant increase and maintain effective local blood supply against ischemia-induced damage, so that makes it possible for re-function after reperfusion; If acupuncture therapy is given during reperfusion period, ischemic cerebral blood supply can also increase and infarct size will significantly reduced so that neurological function was effectively protected. In this study, on the basis of pre-microPET observation that ultra-early acupuncture improves ischemic cerebral glucose metabolism, we are conducting further exploration on the mechanism of ultra-early acupuncture therapy against cerebral ischemia injury.Methods:140 female Sprague-Dawley (SD) rats (specific-pathogen free (SPF) grade) of 3 months old, weighing 200-250g, were randomly divided into 7 groups (20 rats in each group):normal group, acupoint 2h group, non-acupoint 2h group, model 2h group, acupoint 24h group, non-acupoint 24h group, model 24h group.10 rats brains in each group were removed after perfusion, with the other 10 were sampled for fresh brain tissue. Acute right middle cerebral artery ischemia was produced in rats with craniotomy and eletrocoagulation on middle cerebral artery. Acupoint groups were treated by acupuncture at "Baihui"and"Shuigou", using reducing method, with 120 twisting per minute, one time every 5 minutes, one minute every time, retaining the needles for 30 minutes. Non-acupoint groups were respectively treated by acupuncture at 5mm away on the left side of "Baihui"and"Shuigou". Acupoint 24h group and non-acupoint 24h group were treated by acupuncture once again after 24 hours.10 rats in each group were examined ATP, ADP, AMP contents in ischemic brain tissue with high-performance liquid chromatography (HPLC), and colorimetry on Na+-K+-ATP enzyme, Ca2+-ATP enzyme levels, and real-time PCR on HIF-1αmRNA, EPOmRNA gene expression, and immunohistochemical to GLUT1, GLUT3 expression of the other 10 rats of each group. Then we conducted a comprehensive statistical analysis of these results. Results:1. Compared with normal group, ATP, ADP contents in rats'brain tissue after cerebral ischemia for 2h decreased significantly (P<0.01), and the model 24h group's ATP and ADP content decreased significantly. And compared with model 2h group, acupoint 2h group's ATP, ADP content increased significantly (P<0.01), and compared with non-acupoint 2h group, there was a extremely significant difference (P<0.01). But no significant difference was observed between non-acupoint 2h group and model 2h group (P>0.05). Compared with model 24h group and non-acupoint 24h group, acupoint 24h group's ATP, ADP content significantly higher (P<0.01), but no significant difference was observed between non-acupoint 24h group and model 24h group(P>0.05). Compared with normal group, AMP concent in rats' brain tissue after cerebral ischemia for 2h significantly increased significantly (P<0.01), and the model 24h group's AMP content increased more significantly. And compared with model 2h group, acupoint 2h group's AMP content decreased significantly (P<0.01), and compared with non-acupoint 2h group, there was a significant difference (P<0.01). But no significant difference was observed between non-acupoint 2h group and model 2h group (P>0.05). Acupoint 24h group's AMP content was significantly lower than that in model 24h group and non-acupoint 24h group (P<0.01), but no significant difference was observed between non-acupoint 24h group and model 24h group(P>0.05).2. Compared with normal group, TAN concent in rats' brain tissue after cerebral ischemia for 2h significantly decreased significantly (P<0.01). and the model 24h group's TAN content decreased significantly. And compared with model 2h group, acupoint 2h group's TAN content increased significantly (P<0.01), and compared with non-acupoint 2h group, there was a significant difference (P<0.01). But no significant difference was observed between non-acupoint 2h group and model 2h group (P>0.05).Compared with model 24h group and non-acupoint 24h group separately, acupoint 24h group's TAN content significantly highered (P<0.01 and P<0.05), but no significant difference was observed between non-acupoint 24h group and model 24h group(P>0.05).3. Compared with normal group, EC concent in rats' brain tissue after cerebral ischemia for 2h significantly decreased significantly (P<0.01). and the model 24h group's EC content decreased significantly. And compared with model 2h group, acupoint 2h group's EC content increased significantly (P<0.01), and compared with non-acupoint 2h group, there was a significant difference (P<0.01). But there was no significant difference between non-acupoint 2h group and model 2h group (P>0.05). Compared with model 24h group and non-acupoint 24h group separately, acupoint 24h group's EC content significantly highered (P<0.05 and P<0.01), but no significant difference was observed between non-acupoint 24h group and model 24h group(P>0.05).4. Compared with normal group, Na+-K+-ATP enzyme concent in rats' brain tissue after cerebral ischemia for 2h significantly decreased significantly (P<0.01). and the model 24h group's Na+-K+-ATP enzyme content decreased significantly. And compared with model 2h group, acupoint 2h group's Na+-K+-ATP enzyme content increased significantly (P<0.01), and compared with non-acupoint 2h group, there was a significant difference (P<0.05). And there was significant difference between non-acupoint 2h group and model 2h group (P<0.05). Acupoint 24h group's Na+-K+-ATP enzyme content was significantly higher than model 24h group and non-acupoint 24h group (P<0.01), but no significant difference was observed between non-acupoint 24h group and model 24h group(P>0.05).5. Compared with normal group, Ca2+-ATP enzyme concent in rats' brain tissue after cerebral ischemia for 2h significantly decreased significantly (P<0.01). and the model 24h group's Ca2+-ATP enzyme content decreased significantly. And compared with model 2h group, acupoint 2h group's Ca2+-ATP enzyme content increased significantly (P<0.01), and compared with non-acupoint 2h group, there was a significant difference (P<0.01). But there was no significant difference between non-acupoint 2h group and model 2h group (P>0.05). Acupoint 24h group's Ca2+-ATP enzyme content was significantly higher than model 24h group and non-acupoint 24h group (P<0.01), but no significant difference was observed between non-acupoint 24h group and model 24h group(P>0.05).6. GLUT1 immunoreactive material MOD value of each group's ischemic cortex regionalin significantly higher than that in the normal group (P<0.05).And compared with model 2h group, acupoint 2h group's positive expression increased significantly (P<0.05), and compared with non-acupoint 2h group, there was a significant difference (P<0.05). But there was no significant difference between non-acupoint 2h group and model 2h group (P>0.05). And model 24h groupgroup's positive expression was higher than that of model 2h group (P<0.01). And acupoint 24h group's positive expression significantly higher than model groups and non-acupoint 24h group (P<0.01). These results indicate that GLUT1 expression increased gradually after cerebral ischemia for 2h～24h; Acupoint groups can significantly enhance positive expression of GLUT1 in cerebral cortex, rather than a slight increase in expression of that of non-acupoint groups, but there was no significant difference between them.7. GLUT3 immunoreactive material MOD value of each group's ischemic cortex regionalin significantly higher than the normal group (P<0.05). And compared with model 2h group, acupoint 2h group's positive expression increased significantly (P<0.01), and compared with non-acupoint 2h group, there was a significant difference (P<0.05). But no significant difference was observed between non-acupoint 2h group and model 2h group (P>0.05).And model 24h groupgroup's positive expression higher than that in model 2h group (P<0.01). And acupoint 24h group's positive expression was significantly higher than that in model groups and non-acupoint 24h group (P<0.05). These results indicate that GLUT3 expression increased gradually after cerebral ischemia for 2h～24h; Acupoint groups can significantly enhance positive expression of GLUT3 in cerebral cortex, rather than a slight increase in expression of non-acupoint groups, but there was no significant difference between them.8. HIF-1αmRNA gene expression of each group's ischemic cortex region was significantly higher than the normal group (P<0.01).And compared with model 2h group, acupoint 2h group's gene expression increased significantly (P<0.01), and compared with non-acupoint 2h group, there was significant difference (P<0.05). But there was no significant difference between non-acupoint 2h group and model 2h group (P>0.05).And model 24h group's gene expression higher than model 2h group (P<0.01). And acupoint 24h group's gene expression was significantly higher than model groups and non-acupoint 24h group (P<0.05). These results indicate that HIF-1αmRNA gene expression increased gradually after cerebral ischemia for 2h～24h; Acupoint acupuncture can significantly enhance expression of HIF-1αmRNA gene expression in cerebral cortex, rather than a slight increased in expression of non-acupoint groups, but there was no significant difference between them.9. EPOmRNA gene expression of each group's ischemic cortex region was significantly higher than the normal group (P<0.01).And compared with model 2h group, acupoint 2h group's gene expression increased significantly (P<0.01), and compared with non-acupoint 2h group, there was a significant difference(P<0.05). But there was no significant difference between non-acupoint 2h group and model 2h group (P>0.05).And model 24h group's gene expression higher than model 2h group (P<0.01). And acupoint 24h group's gene expression significantly higher than model groups and non-acupoint 24h group (P<0.05). These results indicate that EPOmRNA gene expression increased gradually after cerebral ischemia for 2h～24h; Acupoint acupuncture significantly enhanced expression of EPOmRNA gene expression in cerebral cortex, rather than a slight increase in expression of non-acupoint groups, but there was no significant difference between them.Conclusion:1. ATP production and utilization in rats' brain tissue were reduced after acute cerebral ischemia, and TAN and EC decreased, indicating an energy metabolism dysfunction, however, situration can be significantly improved by acupoint acupuncture, which plays the role against cerebral ischemia injury.2. Acupuncture can improve the energy metabolism of ischemic brain tissue, thus restore the Na+-K+-ATP and Ca2+-ATP enzyme activity, and help to reduce the intracellular Na+ load, reduce Ca2+ overload, maintaining Na+, Ca2+ inside and outside cells homeostatic state, which could protect the brain by reducing intracellular edema, affecting synaptic excitement, conduction and transmitter release, inhibiting the release of toxic substances, such as excitatory amino acids, etc.3. Acupuncture can up-regulate brain GLUT1 and GLUT3 protein expression, increase glucose transport into the brain through the blood-brain barrier, improve the efficiency of glucose transporter in the brain, continue the brain energy supply under hypoxic-ischemic, slow down the energy depletion against hypoxic-ischemic positive response to reduce brain damage caused by ischemia and hypoxia.4. Acupuncture further induced HIF-1αmRNA gene expression in ischemic area, thereby enhanced expression of target genes such as EPO, GLUT1, etc, so as to play the role against cerebral ischemia injury. 5. Acupoint acupuncture can adjust the content of HIF-1α,GLUT1,GLUT3 and EPO in ischemic area, so as to improve the energy metabolism and play the role against cerebral ischemia at Super Early Stage.6. Acupoint acupuncture regulation of energy metabolism of ischemic brain tissue and the relevant indicators was better than non-acupoint acupuncture.
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