Electroacupuncture effect on synaptic ultrastructure in focal cerebral ischemia marginal zone of the rat

BACKGROUND：Synapses undergo high levels of plasticity within the nervous system, and cerebral ischemia induces synaptic plasticity changes.OBJECTIVE：To demonstrate the effects of electroacupuncture on ultrastructural synaptic changes in the focal cerebral ischemia marginal zone in rats using quantitative analysis of stereological measurement.DESIGN, TIME AND SETTING：A randomized, controlled, animal experiment was performed at the Experimental Animal Center and Laboratory of Electron Microscopy, Guangzhou University of Traditional Chinese Medicine from January 2008 to January 2009.MATERIALS：The G-6805 electric acupuncture apparatus was provided by Shanghai Huayi Instrument Factory, China.METHODS：A total of 90 male, Wistar rats were randomly assigned to sham-surgery, model, and electroacupuncture groups, with 30 animals in each group. Each group was subdivided into 1 hour, as well as 1, 3, 7, and 21 days post-surgery groups, with six animals assigned to each time point. Heat coagulation-induced occlusion of the middle cerebral artery was performed to establish a model of focal cerebral ischemia. Electroacupuncture was applied immediately following surgery to the electroacupuncture group [4/20 Hz, 2.0-3.0 V, 1-3 mA, to Baihui （GV 20） and Dazhui （GV 14）] for 30 minutes. Treatment was performed once a day, and experimental animals were sacrificed, at 1 hour, as well as 1, 3, 7 and 21 days post-surgery.MAIN OUTCOME MEASURES：At different time points after intervention, changes in synaptic ultrastructure, such as postsynaptic density thickness, synaptic cleft width, and synaptic interface curvature, were observed in the focal cerebral ischemia marginal zone in rats through the use of transmission electronic microscopy.RESULTS：Broken synapses were observed following cerebral ischemia, and the number of synapses was significantly decreased. Compared to the model group, synaptic ultrastructure was significantly improved in the electroacupuncture group. Compared to the sham-surgery group, postsynaptic density thickness was significantly decreased, as were synaptic cleft width and synaptic interface curvature in the electroacupuncture and model groups. However, compared to the model group, postsynaptic density thickness was significantly increased in the electroacupuncture group at the same time point post-surgery （P 〈 0.05 or P 〈 0.01）. In addition, synaptic cleft width and synaptic interface curvature were significantly increased with the passage of time （P〈 0.05 or P〈 0.01）.CONCLUSION：Electroacupuncture significantly ameliorated structural synapse lesion during the early stage of cerebral ischemic injury, promoted repair of synaptic structure, improved structural parameters of synapses, and increased synaptic structural plasticity, which suggested that the therapeutic effect of electroacupuncture was related to synaptic reorganization.

Study on the Correlation between Synaptic Reconstruction and Astrocyte after Ischemia and the Influence of Electroacupuncture on Rats

Objective： To observe the effects of electroacupuncture （EA） on the structure parameters of synapse and reactive changes of astrocyte in the marginal zone of focal cerebral ischemia in rats at different time zones so as to further explore its underlying mechanisms in the treatment of cerebral ischemia. Methods： Ninety male Wistar rats were randomly assigned to sham-operation, model, and EA groups, with 30 animals in each group. Each group was subdivided into 1 h, as well as 1, 3, 7, and 21 days post-operation groups, with 6 animals assigned to each time point subgroup. Heat coagulation-induced occlusion of the middle cerebral artery was performed to establish a model of focal cerebral ischemia. EA was applied immediately following surgery to the EA group [4/20 Hz, 2.0-3.0 V, 1-3 mA, to Baihui （GV20） and Dazhui （GV14）] for 30 min. Treatment was performed once a day, and experimental animals were sacrificed at 1 h, as well as 1, 3, 7 and 21 days postoperation. The ultrastructure changes in synapse and astrocytes were observed by using transmission electron microscopy. Glial fibrillary acidic protein （GFAP） expression and Ca2＋ of astrocytes were measured by using laser confocal scanning microscope. Excitatory amino acid transporters-2 （EAAT2） and connexin 43 （CX43） expressions were assayed with immunohistochemical method. Canonical correlation analysis was conducted between structure parameters of synapse and parameters of astrocyte in the same time and group. Results： Broken synapses were observed following cerebral ischemia, and the numbers of synapses were significantly decreased. Compared with the model group, synaptic ultrastructure was significantly improved in the EA group. Compared with the sham-operation group, synaptic number density was significantly decreased, as were postsynaptic density thickness, synaptic cleft width and synaptic interface curvature in the EA and model groups. However, compared with the model group, postsynaptic density thickness was significantly increased in the EA group at the same time points post-operation （P〈0.05, P〈0.01）. In addition, synaptic cleft width, synaptic number density and synaptic interface curvature were significantly increased with the passage of time （P〈0.05, P〈0.01）. The expression of GFAP in the EA group were significantly lower than those in the model group at all the time points （P〈0.05, P〈0.01）. OD values of EAAT2 in the EA group were significantly higher than those in the model group at the same time （P〈0.05, P〈0.01）. Compared with that in the model group, the expressions of CX43 in the EA group increased significantly at 3 days and 7 days （P〈0.05, P〈0.01）. Ca2＋ average fluorescence intensity of astrocytes in the EA group was significantly lower than those in the model group at 1 h, 1 day, 3 days and 7 days （P〈0.05, P〈0.01）. The changes in structure parameters of synapse were closely related to the changesof CX43, EAAT2, GFAP, Ca＋ of astrocytes by EA treatment at all the time points. Conclusions： EA is helpful for synaptic reorganization, which may be related to its effect on intervening the activation state of astrocytes and promoting the beneficial interaction between astrocytes and synapses. Acupuncture could start the adjustment of neuron-glial network so as to promote the synaptic reorganization, which may be the key mechanism of treating cerebral ischemia.