| Part I Research on the TheoryCerebrovascular disease is nervous system diseases that occur commonly and frequently, those death rate are about 10% of all diseases, and is currently one of three major causes of death of human diseases, including ischemic stroke which accounts for about 70% of all stroke. Cerebral ischemia-reperfusion injury is a very complex pathophysiological process, and it is important to explore the mechanism and treatment methods of ischemia-reperfusion injury, to know how to resume blood flow of ischemic tissue as soon as possible and reduce or prevent the occurrence of ischemia-reperfusion injury.Acupuncture is one part of the heritage of Chinese medicine that is unique in China, and a national medical approach. EA technology is to combine traditional manual acupuncture with electronic technology in order to acupuncture is more easy to implement than before. It is well known that EA has shown good results in treatment of cerebral ischemic disease, could improve the prognosis of those patients, and promote their rehabilitation. The present researches presume that EA has a good therapeutic effect to cerebral ischemia. EA also has a multilevel, multichannel and multitarget protective effect and interfering role to cerebral ischemic injury. Du, Chinese medicine said, is Yang pulse of the sea. Line at the back center, yang is the confluence of various yang of human body. brain and kidney has a very close relationship with it, so Du is first choice to treat cerebral ischemic disease.As acupuncture was used to treat diseases, mostly chronic diseases, it often caused pain. Owing to be afraid of pain, some patients, especially children, usually give up the treatment, which is not conducive to the rehabilitation of the disease. During needle insertion and electric stimulation, the major pain comes from the skin, because the skin epidermis of the germinal layer, especially in thick 1～2mm of the dermis, is rich in sensory nerve endings. When some parts of the body especially the skin nerve endings were stimulated by the superpnormal stimuli, it would cause the body reaction to pain. The level of pain was relevant to stimulus intensity, stimulation time and stimulation frequency.The pain originated from acupuncture often make patients feel panic, and provoke stress reaction. oxidative stress plays a key role on ischemical reperfusion injury, and is one of the important factors that resulting in irreversible cerebral ischemical reperfusion injury. Reperfusion injury results in oxidative stress, calcium overload and mass production of inflammatory factor. Calcium overload and the production of inflammatory factor both have mutual promotion relationship with oxidative stress. Calcium overload and oxidative stress attacked nerve cell directly that resulted in brain tissue damage.Now propofol has been the most widely used intravenous anesthetic due to its property of fast and complete anesthesia onset and recovery. EA treatment under propofol anesthesia, not only relieved patient's pain and fear to it, but also eased stress reaction, so that it became possible that the acupuncture of higher intensities would be applied to patients. Acupuncture in various dosages may have a different treatment effect. In the early nerve damage, EA can promote tissue expression of IGF-1 to increase, thereby enhancing the survival of neurons and promote axonal regeneration and synapse formation, is conducive to nerve regeneration and repair.Cerebral ischemia injury decreased cerebral blood flow,which reduced the supply of oxygen and glucose, and triggerred energy metabolism disorder of tissues, for instance the activating anaerobic glycolysis and inhibiting aerobic oxidation. These changes led to lactic acid piling up and the production of ATP decreasing in tissues that induced tissue acidosis and energy exhaustion. Lactic acid is specific product, the change of which may reflect the state of oxadation metabolism and the extent of acidosis.Mitochondria of Cerebral tissue has always been known as the center of energy transformation in neuron. The latest research indicated mitochondria played an important role in regulation of neuron apoptosis. Cerebral cells energy metabolism disorder and ATP exhausted after cerebral ischemia-perfusion became main reason of decrease in ATPase activity. If the decrease of ATP content and ATPase activity could be prevented and relieved, preventing nervous plasmalemma function from exhaustion and inhibiting development of cerebral ischemia-reperfusion could be achieved.The previous part of our research to observe the protective effects of electroacupuncture which was applied to Baihui(GV20), Mingmen(GV4) and Zusanli(ST36)on cerebral four-vessel occlusion and reperfusion injury of the rats under chloral hydrate or propfol anesthesia.The latter part of research is to duplicate ischemia-reperfusion rat model by cerebral four-vessel occlusion with electroacupuncture puncturing Baihui(GV20), Mingmen(GV4) and Zusanli(ST36) after CI/R,then to oxplore the effect of electroacupuncture of different intensities on the ultrastructure of cerebral cortex, the cerebral glycometabolism and energy metabolism, the function of pancreatic islet and the mitochondrial function of brain cells in rats with cerebral ischemia reperfusion injury. PartII Experimental research Charter 1:Anti-oxidative effects of propofol during treatment of electroacupuncture on cerebral ischemia-reperfusion injury in RatsReactive Oxygen Species remain to be a major mechanism, and it is crucial significance to search an effective protectant for cerebral ischemia-reperfusion injury.As an intravenous anesthetics,propofol exerts an inhibitory action to oxidative stress induced by various kinds of factors, by which relieving cerebral ischemia-reperfusion injury. The aim of this section was to observe the changes of superoxide dismutase(SOD), malonyldialdehyde(MDA) and PPAR-ymRNA expression in the brain tissues of rats with cerebral ischemia-reperfusion injury and the effects of electroacupuncture on the levels of SOD, MDA and PPAR-ymRNA expression under different anesthesia and to explore the anti-oxidative effects of propofol during treatment of electroacupuncture on cerebral ischemia-reperfusion injury in rats. Forty male SD rats were uniformly randomized into sham operation (SH), CL+CI/R, CL+CI/R+EA, P+CI/R and P+CI/R+EA groups with eight rats in each group. Transient general brain ischemia was induced by four-vessel occlusion and reperfusion. EA (30 to 50 Hz,1 mA) was applied to Baihui(GV20), Mingmen(GV4) and Zusanli(ST36) for 20 min after CI/R. The content of SOD and MDA and PPAR-ymRNA expression in the brain tissues were measured at the postischemia 24h. The results shows that SOD levels in CL+CI/R group was significantly lower,while MDA levels and PPAR-ymRNA expression was significantly higher than SH group (P<0.05,P<0.01,respectively); SOD levels in CL+CI/R+EA and P+CI/R+EA group were found to be increased, while MDA levels and PPAR-ymRNA expression in P+CI/R and P+CI/R+EA group were found to be decreased in comparison with SH group (P<0.05,P<0.01,respectively); SOD levels in P+CI/R+EA group were found to be increased in comparison with P+CI/R group (P<0.05); MDA levels and PPAR-ymRNA expression in P+CI/R and P+CI/R+EA group were found to be decreased in comparison with CL+CI/R+EA group (P<0.01). These findings revealed Electroacupuncture combined with propofol could protect the brain of cerebral ischemia-reperfusion rat by electroacupuncture improving the activity of SOD and propofol decreasing MDA content in the brain.Chapter 2:Anti-inflammation effects of propofol during treatment of electroacupuncture on cerebral Ischemia-reperfusion injury in rats Postischemia inflammatory reaction was an important component of pathologic processes in ischemic cerebrovascular disease. Overproduced Overdose inflammatory factor and white blood cell infiltration results in inflammatory reaction that exacerbates brain injuries. Existing research shows that propofol possesses a protective effect on cerebral ischemia-reperfusion injury,which could be induced by Regulating Cytokine. The aim of this section was to observe the changes of tumor necrosis factor (TNF), MPO and PPAR-ymRNA expression in the brain tissues of rats with cerebral ischemia-reperfusion injury and the effects of electroacupuncture on the levels of TNF, MPO and PPAR-ymRNA expression under propofol anesthesia,and to explore the anti-inflammation effects of propofol during treatment of electroacupuncture on cerebral ischemia-reperfusion injury in rats. The results shows that compared with SH group, The levels of TNF, MPO activity and PPAR-ymRNA expression in CL+CI/R and CL+CI/R+EA group was significantly higher (P<0.05 and P<0.01,respectively), while MPO and PPAR-ymRNA was significantly lower in P+CI/R group (P<0.05 and P<0.01,respectively); the levels of TNF, MPO activity and PPAR-ymRNA expression in P+CI/R groups and MPO activity and PPAR-ymRNA expression in P+CI/R+EA groups were found to be decreased in comparison with A group(P<0.05 and P<0.01,respectively); compared with CL+CI/R+EA, the levels of TNF, MPO and PPAR-ymRNA was significantly lower in P+CI/R+EA group (P<0.05 and P<0.01, respectively); the MPO activity and PPAR-ymRNA expression in P+CI/R+EA group were found to be increased in comparison with P+CI/R group (P<0.01 and P<0.05,respectively). The MPO activity had a significantly a positive relationship with the PPAR-ymRNA expression (r= 0.564, P<0.01)。These findings revealed that Propofol can protect the brain of cerebral ischemia-reperfusion rat by decreasing the levels of TNF, MPO activity and PPAR-γmRNA expression in the brain, however, electroacupuncture has not present an antiinflammatory effect on cerebral ischemia-reperfusion injury.Chapter 3:Effects of electroacupuncture on cerebral glycometabolism and equilibrium of oxygen supply and demand of rats with cerebral ischemia-reperfusion injury under different anesthesiaThe oxygen supply and demand disbalance and glycometabolism disorder after cerebral ischemia-reperfusion injury are one of the main reasons for patient's death or disability in an early stage. Acupuncture could effectively decrease blood glucose and promote brain cells to absorb and utilize glucose. The aim of this section was to observe the change of the jugular venous blood glucose, blood lactate acid and saturation of blood oxygen of rats with cerebral ischemia-reperfusion injury and the effects of electroacupuncture on levels of the jugular venous blood glucose, blood lactate acid and saturation of blood oxygen of the rats under propofol anesthesia, and to explore the effects of electroacupuncture on cerebral energy metabolism and equilibrium of oxygen supply and demand of rats with cerebral ischemia-reperfusion injury under different anesthesia. The results showed that the levels of the jugular venous blood glucose and saturation of blood oxygen after ischemia-reperfusion in CL+CI/R group increased while lactate acid decreased significantly than that in sham group (P<0.01); the levels of the jugular venous blood glucose and saturation of blood oxygen in P+CI/R group increased (P<0.05) while lactate acid decreased significantly than that in sham group (P<0.01); compared with CL+CI/R group, the levels of the jugular venous blood glucose decreased (P<0.01) while lactate acid increased remarkably in CL+CI/R+EA group and P+CI/R+EA group (P<0.01); compared with P+CI/R group, the levels of the jugular venous blood glucose decreased (P<0.01) while lactate acid increased remarkably in P+CI/R+EA group (P<0.05);the levels of the jugular venous blood glucose in P+CI/R group is significantly lower than that in CL+CI/R group (P<0.05).These findings revealed that the treatment with EA can improve the glycometabolism of rats with cerebral ischemia-reperfusion injury and facilitate the brain cell to absorb and utilize glucose, the different intravenous anesthetics have no effect on conduction of electroacupuncture signs.Chapter 4:Effects of electroacupuncture of different intensities on cerebral glycometabolism and equilibrium of oxygen supply and demand of rats with cerebral ischemia-reperfusion injuryUnder propofol anesthesia, EA present a satisfactory therapeutic effect on cerebral ischemia-reperfusion injury, especially on cerebral glycometabolism and equilibrium of oxygen supply and demand. Because the stimulus parameters and quantity of EA were important factors that affect acupuncture effect, as well as there was larger range for the selection of EA intensity under propofol anesthesia, the study on how to select the suitable stimulus intensity of EA was very important to improve therapeutic effect and standardise therapeutic method. The aim of this section was to observe the change of the jugular venous blood glucose, blood lactate acid and saturation of blood oxygenof rats with cerebral ischemia-reperfusion injury and the effects of electroacupuncture of different intensities on levels of the jugular venous blood glucose, blood lactate acid and saturation of blood oxygen of the rats, and to explore the effects of electroacupuncture of different intensities on cerebral glycometabolism and equilibrium of oxygen supply and demand of rats with cerebral ischemia-reperfusion injury. Forty male SD rats were uniformly randomized into sham operation group(SH), CI/R group, CI/R+5mA EA, CI/R+3mA EAand CI/R+1mA EA groups with eight rats in each group. Transient general brain ischemia was induced by four-vessel occlusion and reperfusion. Baihui(GV20), Mingmen(GV4) and Zusanli(ST36) of the rats in CI/R+5mA EA, CI/R+3mA EA and CI/R+1mA EA group respectively were punctured and stimulated with the same intermittent and rarefaction-dense wave (30 to 50Hz) and different electric current intensities:5mA,3mA and 1 mA for 20 min after CI/R. The treatment was given again after 12 hours. Right jugular vein was cannulated to withdraw 0.5 mL blood samples for analysis blood gas, lactate acid and saturation of blood oxygenat the postischemia 24h. The results showed that the levels of the jugular venous blood glucose after ischemia-reperfusion in CL+CI/R group increased(P<0.01), while lactate acid decreased significantly than that in SH group (P<0.05); compared with SH group, the levels of the jugular venous saturation of blood oxygen in CI/R+5mA EA group decreased (P<0.05),and the levels of the jugular venous blood glucose and saturation of blood oxygen decreased (P<0.01) while lactate acid increased significantly in CI/R+3mA EA group than that in CI/R group (P<0.05); the levels of the jugular venous blood glucose and saturation of blood oxygen in CI/R+1mA EA group is also significantly lower than that in CI/R group (P<0.05). These findings revealed that the treatment with electropuncture can improve the glycometabolism of rats with cerebral ischemia-reperfusion injury and facilitate the brain cell to absorb and utilize glucose, The treatment effect of 3mA EA was better than that of 5mA EA and 1 mA EA.Chapter 5:Effects of electroacupuncture of different intensities on jugular venous blood glucose and the function of pancreatic isletβcells of rats with cerebral ischemia-reperfusion injuryIn cerebral ischemia diseases, autonomic nerve dysfunction and endocrine disorder lead to abnormal secretion of pancreatic islet that results in the imbalance between the levels of internal glucagon and insulin. This change has interfered peripheral glycometabolism. The aim of this section was to observe the change of the jugular venous blood glucose, and the function of pancreatic islet (3 cells rats with cerebral ischemia-reperfusion injury and the effects of electroacupuncture of different intensities on levels of the jugular venous blood glucose and the function of pancreatic isletβcells of the rats, and to explore the effects of electroacupuncture of different intensities on jugular venous blood glucose and the function of pancreatic isletβcells of rats with cerebral ischemia-reperfusion injury. The results showed that the levels of the jugular venous blood glucose after ischemia-reperfusion in CI/R group increased while insulin and C-peptide serum levels decreased significantly than that in SH组(P<0.01); Compared with CI/R group, insulin serum levels and the value of HOMA-IR both significantly decreased (P<0.01) in CI/R+5mA EA group, the levels of the jugular venous blood glucose significantly decreased (P<0.01) while C-peptide serum levels increased significantly(P<0.05) in CI/R+3mA EA group, and the levels of the jugular venous blood glucose significantly decreased (P<0.01) while C-peptide serum levels increased significantly in CI/R+1mA EA group(P<0.05).There was a significant correlation between C-peptide serum levels and C-peptide serum levels(r=-0.396, P<0.05). These findings revealed that The treatment with electropuncture can improve the function of pancreatic isletβcells of the rats with cerebral ischemia-reperfusion injury and decrease blood glucose. Electroacupuncture of different intensities all show a satisfactory treatment Effectiveness, but 5mA EA appeared strong stress reaction and insulin resistance.Chapter 6:Effects of electroacupuncture of different intensities on LDH,SDH and ATPase in brain tissue of rats with cerebral ischemia-reperfusion injuryThe brain cell glycometabolism and energy metabolism disorder after cerebral ischemia-reperfusion injury results in ATP depletion, which becomes the main reason for the decreased activities of APTase. If improving brain cell glycometabolism and energy metabolism could prevent or alleviate the decrease of ATP content and ATPase activities due to cerebral ischemia-reperfusion injury, the development of the functional failure of nerve cell membrane and the cerebral ischemia-reperfusion injury would be stoped. The aim of this section was to observe the effects of electroacupuncture of different intensities on LDH,SDH and ATPase in brain tissue of rats with cerebral ischemia-reperfusion injury so as to explore its mechanisms in improving total cerebral ischemia-reperfusion injury. The results showed that compared with the sham operation group, the activities of LDH was higher in the CI/R group (P<0.05),while the activities of Na+-K+-ATPase was lowerer in the CI/R group than those in the sham operation group. Compared with the CI/R group, the activities of LDH was lower in the CI/R+5mA EA group (P<0.05),while the activities of Na+-K+-ATPase and SDH was higher in the CI/R+5mA EA group than those in the CI/R group(P<0.05 and P<0.01 respectively). These findings revealed that the EA of higher intensities could strenthen aerobic metabolism by elevating the activities of SDH, and inhibiting anaerobic metabolism by decreasing the activities of LDH, meanwhile maintaining the ionic equilibrium in the exterior and interior of brain cell and relieving the cellular edema by reinforcing the activities of Na+-K+-ATPase.Chapter 7:Effect of electroacupuncture different intensities on cerebral cortex ultrastructure in rats with cerebral ischemia-reperfusion injuryThe different stimulus parameter and quantity of stimulus are important factors that affect therapeutic effect, and EA of different intensities also displays the distinct extent of cerebral protective effect. The cellular ultrastructure is the most intuitional index that reflcts cell damage and cytothesis. The aim of this section was to observe the effect of EA of different intensities on the ultrastructure of cerebral cortex in rats with cerebral ischemia reperfusion injury, and to explore the effect of electroacupuncture different intensities on cerebral cortex ultrastructure in rats with cerebral ischemia-reperfusion injury. The results showed that in A group, the anatomical structure of the cerebral cortex cells was normal basically; In CI/R group, the neuronal cellular structures were severely damaged, the neuronal mitochondria got swelling, the mitochondrial cristae were broken, the medullated nerve fibers were not integrated. In CI/R +5mA EA, CI/R+3mA EA and CI/R+1mA EA groups, the ultrastructure changes of impaired nerve cells in ectocinerea were slight or normal basically.The pathologic structures of impaired neuron were improved. These findings revealed that EA can reduce ischemic injury of the cerebral cortical neurons in rats with CI/R injury by improving the ultrastructure changes of impaired nerve cells. The treatment effect of 3mA and 1 mA EA was better than that of 5mA.SummaryThis study is to introduce propofol to electroacupuncture treatment,which assisting electroacupuncture to treat cerebral-ischemia injury and strengthening the treatment effectiveness of electroacupuncture.The research that focused on cerebral protective effect of electroacupuncture is to investigate whether the combination of anesthesia and electroacupuncture could inhibit oxidative stress, resist inflammation and improve the energy metabolism of brain cells, and be in favour of preferably bringing cerebral protective effect of electroacupuncture into full play.The previous part of the study is to duplicate cerebral ischemia-reperfusion injury model of rats by four-vessel occlusion and reperfusion and observe the protective effects of electroacupuncture was applied to Baihui(GV20), Mingmen(GV4) and Zusanli(ST36)on cerebral four-vessel occlusion and reperfusion injury of the rats under chloral hydrate or propfol anesthesia.The cerebral protective effects of electroacupuncture and the effect of anesthsia on the cerebral protective effects of electroacupuncture were checked from three aspects of cerebral glycometabolism, antiinflammatory effect and anti-oxidant stress effect. The latter part of the study is to oxplore the effect of electroacupuncture of different intensities on the ultrastructure of cerebral cortex, the cerebral glycometabolism and energy metabolism, the function of pancreatic islet and the mitochondrial function of brain cells in rats with cerebral ischemia reperfusion injury.The previous part of the experimental results indicates that electroacupuncture still has satisfactory cerebral protective effects especially on the regulation of the cerebral glycometabolism and energy metabolism,and propofol also shows a cerebral protective effects on antiinflammatory effect.Based on the The previous part of the experiment,the latter part of the experiment further investigated effect of electroacupuncture of different intensities on the cerebral glycometabolism and energy metabolism in rats with cerebral ischemia-reperfusion injury. The latter part of the experimental results indicates that there is different effect of electroacupuncture different intensities on the cerebral glycometabolism and energy metabolism in rats with cerebral ischemia-reperfusion injury. If the intensities of electroacupunture is too low, therapeutic action of electroacupuncture is weak and the effect is not good. However,if the intensities of electroacupunture is too high and strong, it will increase the stress effect of body, which affects the therapeutic action of electroacupuncture.The electroacupunture of 3mA intensity has a great regulatory effect on the cerebral glycometabolism and energy metabolism, and the function of pancreatic islet. The electroacupunture of 5mA intensities could strenthen aerobic metabolism and inhibit anaerobic metabolism by regulating energy metabolism relating enzymes in mitochondria of brain cells, and thus reduce the production of lactate acid, improve the energy metabolism of brain tissue and have a activating and prosthetic effect on the impaired brain cells, but increase the stress effect of body. The research also confirmed that propofol wouldn't have an influence on therapeutic action of electroacupunture, moreover there was a significant synergetic cerebral protective effect between propofol and electroacupunture if the electroacupunture under propofol anesthesia was applied to specified patients who suffered cerebral ischemia-reperfusion injury. |
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