| Cerebral infarction is one of common cerebral vascular diseases and a significanthealth problem in the elderly and the wrinkly in China. The dysfunctions involved in it leadto heavy burden to patients, their families and society. The plasticity of nervous systemafter ischemia and neuro-rehabilitation concerned is always the hottest issues. To recoverbetter and improve quality of life, it's very meaningful to develop rehabilitation methods toactivate brain potentials of anti-injury and remodeling after cerebral ischemia.ObjectiveA hypothesis that electroacupuncture (EA) combined with repetitive transcranialmagnetic stimulation (rTMS) can activate astrocytes (AST) which can improve synapticremodeling, and then enhance the recovery of learning and memory after cerebral ischemia,was proposed in this study. This study was designed to investigate any possible therapeuticmechanisms about EA combined with rTMS after cerebral ischemia.MethodsHealthy male Wistar rats were divided into normal, model, EA, rTMS and EA +rTMS group randomly. Furthermore, rats in each group were divided into 7d, 14d and 28d,3 subgroups. Model of middle cerebral artery occlusion (MCAO) was established, followedby 7d, 14d or 28d of EA, rTMS and EA + rTMS treatment. Firstly, the expression of GFAPin periinfarct and area CA3 of the hippocampus of the ischemic cerebral hemisphere andthe expression of synaptophysin (SYN) were investigated in different time points anddifferent groups by immunofluorescence technique. The expression of postsynapticdensity-95 (PSD-95) was investigated by immunohistochemical technique. Secondly, semi-quantitative analysis of the expression of SYN in hippocampus of the ischemiccerebral hemisphere in different time points and different groups was processed by Westernblot. Finally. Morris water maze task and the ultrastructure of the synapses in area CA3 ofthe hippocampus of the ischemic cerebral hemisphere were investigated.Results1. The activation of AST in periinfarct and area CA3 of the hippocampus of theischemic cerebral hemisphere was observed at days 7 after ischemia, with manifestations ofhypertrophy somata, increased processes and increased fluorescence intensity. Moresignificant activation was detectived at three rehabilitation intervention groups. The Furtheractivation was detectived at three rehabilitation intervention groups at days 14 afterischemia (P＜0.05) but the decreased expression of AST at days 28 was detectivedcompared with days 14 (P＞0.05) . The EA+rTMS group was superior to the EA and rTMSgroups in the expression of AST in area CA3 of the hippocampus of the ischemic cerebralhemisphere at days 7 and 28 after ischemia.2. The decreased expression of PSD-95 in dentate gyrus (DG) and area CA3 of thehippocampus of the ischemic cerebral hemisphere was detectived at days 7 after ischemia.Compared with model group, the expression of PSD-95 in above zone increased at days 14and 28, especillay at days 28 with statistical differences. The EA+rTMS group was superiorto the EA and rTMS groups in the expression of PSD-95 in area CA3 of the hippocampusat days 28 after ischemia.3. The expression of SYN in all 3 rehabilitation intervention groups increasedsignificantlly at days of 7, 14 and 28. The longer treatment time, the more significantexpression of SYN and the expression in the EA+rTMS group was the most significant atdays of 28.4. The shortened latencies were detectived in all 5 groups during the first 3 days oftraining in Morris Water Maze and there were no statistical differences between 3rehabilitation intervention groups and model group. But compared with model group, the latencies of 3 rehabilitation intervention groups decreased significantly at days of 4 and 5(P＜0.05 or P＜0.01). In the place navigation trials, ischemia had a significant effect on watermaze learning, as demonstrated by prolonged latency in the model group compared with thenormal group. In the 3 intervention groups, the average latency was shorter than that of themodel group, especially in rats from the EA+rTMS group. In the spatial probe trials, thefrenquency of swimming across the platform site in the model group was lower than in thenomal group. After the interventions, the frequencies of the EA, rTMS and EA+rTMS ratswere significantly higher. The average frequency of EA+rTMS was significantly higherthan those of the other groups (P＜0.05).5. In model group, most synapses in the CA3 area of the hippocampus wereasymmetric synapses, with features of few synaptic vesicles and mitochondria degeneration.With more synaptic vesicles and complete structure of the mitochondria, most synapses ofEA, rTMS and EA+rTMS group are symmetric synapses. No significant differences wereobserved in the average width of the synaptic interspaces among the five groups. MCAOdecreased the average postsynaptic density in the ischemic hemisphere. EA, rTMS orEA+rTMS allsignificantly increased the postsynaptic density compared with the modelgroup, but no significant difference was observed among the three intervention groups orbetween the intervention groups and the normal group. Similar results were founded interms of the average curvature of the synaptic interface. Ischemia was associated withreduced curvature, and each of the different interventions increased it. But on this measurethere was a significant difference between the EA+rTMS group and the other interventiongroups.Conclusions1. All EA, rTMS and EA+rTMS can activate the expression of AST in periinfarct and areaCA3 of the hippocampus of the ischemic cerebral hemisphere after ischemia. It isn'tsingle- direction activation.2. With the longer treatment time, EA, rTMS and EA+rTMS can gradually increase the expression of SYN and PSD-95 in the hippocampus of the ischemic cerebralhemisphere after ischemia.3. All three interventions can enhance learning and memory and improve synapticultrastructure in the hippocampus of the ischemic cerebral hemisphere after ischemia.4. EA+rTMS is superior to either EA or rTMS in the activation of AST and synapticremodeling. Enhancing learning and memory is related to improving synaptic plasticityin the hippocampus of the ischemic cerebral hemisphere after ischemia.
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