电针结合重复经颅磁刺激对脑缺血大鼠星形胶质细胞激活和海马突触可塑性的研究
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摘要
脑梗死是常见的脑血管疾病，严重危害我国中、老年人身体健康。脑梗死所致功能障碍给患者、家庭和社会带来了沉重的负担。一直以来，脑梗死后神经系统的可塑性和相关的神经康复倍受专注。探索最大限度地激活脑梗死患者大脑的对抗损伤及重塑潜能的康复方法，以使患者获得更好功能恢复，提高其生存质量显得非常有意义。
目的
本研究提出脑缺血后EA+rTMS治疗可活化AST，再通过后者有效地促进海马突触结构和功能的重塑，从而促进脑缺血后学习记忆功能恢复的假说，希望通过此项研究能揭示EA+rTMS治疗缺血性卒中的可能机制，为进一步的完善、丰富针灸学原理和磁刺激作用机理提供科学实验依据。
方法
本研究以MCAO模型为研究对象，将Wistar大鼠随机分为正常组、模型组、EA组、rTMS组和EA+rTMS组，根据不同时间点每个组又细分为7d、14d和28d组3个亚组，分别给予EA、rTMS、EA+rTMS干预。在不同时间点处死动物取材，首先采用免疫荧光标记方法观察不同时间点、不同干预后梗塞灶周围及缺血侧海马CA3区AST特异性标志物GFAP蛋白及缺血侧海马CA3区突触前膜蛋白SYN的表达，免疫组化方法考察缺血侧海马突触后膜蛋白PSD-95的时间分布规律；然后运用蛋白印迹技术定量分析不同时间点、不同组别之间缺血侧海马SYN表达的差异；最后采用Morris Water Maze实验评估脑缺血28d大鼠学习记忆功能的变化，透射电镜观察缺血侧海马CA3区突触形态结构的变化。
结果
1、脑缺血7d时，各组大鼠脑梗塞病灶周围及脑缺血侧海马CA3区均可见到AST的活化，主要表现为细胞胞体肥大、突起增多、荧光强度增加，3个康复干预组AST活化更加明显。14d时3个康复干预组GFAP阳性细胞进一步增多(P＜0.05)，28d时与14d时比较GFAP的表达减少，差异没有统计学意义。7d和28d时EA+rTMS组缺血侧海马CA3区GFAP的表达高于EA组和rTMS组(P＜0.05)。
2、脑缺血7d时，海马DG区和CA3区PSD-95蛋白的表达下降。经过14d和28d的康复干预，各组大鼠海马DG区和CA3区PSD-95蛋白的表达与模型组比较均增加，至28d时差异有统计学意义。28d时，EA+rTMS组大鼠海马CA3区PSD-95蛋白的表达高于EA组和rTMS组(P＜0.05)。
3、EA、rTMS和EA+rTMS都能显著上调3个时间点海马SYN的表达。从时间上看，随着治疗时间的增加，SYN的表达逐步上调；不同组间比较，28d时rTMS和EA+rTMS海马SYN的表达最显著。
4、在Morris Water Maze训练的第1d-3d，5组大鼠的潜伏期均逐渐缩短，各康复干预组与模型组比较差异不明显。在训练的第4d、5d，3个康复干预组潜伏期明显降低，与模型组比较差异有统计学意义(P＜0.05或P＜0.01)。在测试中，模型组大鼠的逃避潜伏期比正常组明显延长；60s内跨越平台次数减少，tP／tT和dP／dT下降，均提示模型组大鼠存在一定的学习记忆功能障碍。3个康复干预组大鼠的逃避潜伏期缩短，60s内跨越平台次数增加，tP／tT和dP／dT上升，与模型组比较差异有显著性意义(P＜0.05或P＜0.01)，提示经过康复干预之后，脑缺血大鼠的学习记忆功能明显改善。3个康复干预组间比较，EA+rTMS组大鼠的逃避潜伏期和60s内跨越平台次数与EA组和rTMS组比较差异有统计学意义(P＜0.05)。
5、模型组大鼠海马CA3区突触多为对称性突触，突触小泡明显减少，线粒体变性明显。EA组、rTMS组和EA+rTMS组突触多表现为凹型或凸型的不对称性突触，突触小泡明显增多，细胞浆内线粒体双层结构完整，线粒体内嵴清楚。突触形态图像分析统计结果显示：各组间突触间隙宽度无显著性差异。3个康复干预组PSD厚度、穿孔性突触百分率比模型组显著增加，3组间比较无显著差异。3个康复干预组CA3区突触的突触界面曲率显著增加，EA+rTMS组大于EA组和rTMS组，差异有统计学意义。
结论
1、EA、rTMS和EA+rTMS均可活化脑缺血大鼠梗塞灶周围和缺血侧海马CA3区AST，这种活化不是单方向的上调。
2、随着干预时间的延长，EA、rTMS和EA+rTMS均可逐步增加脑缺血海马SYN和PSD-95的表达。
3、EA、rTMS和EA+rTMS可提高脑缺血大鼠学习记忆功能和改善脑缺血大鼠海马突触的超微结构。
4、EA+rTMS在促进AST活化和海马突触重塑中优于EA和rTMS。EA+rTMS提高学习记忆能力与其促进海马突触可塑性有关。
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.
Objective
A 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.
Methods
Healthy 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.
Results
1. 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.
Conclusions
1. 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.
目的
本研究提出脑缺血后EA+rTMS治疗可活化AST，再通过后者有效地促进海马突触结构和功能的重塑，从而促进脑缺血后学习记忆功能恢复的假说，希望通过此项研究能揭示EA+rTMS治疗缺血性卒中的可能机制，为进一步的完善、丰富针灸学原理和磁刺激作用机理提供科学实验依据。
方法
本研究以MCAO模型为研究对象，将Wistar大鼠随机分为正常组、模型组、EA组、rTMS组和EA+rTMS组，根据不同时间点每个组又细分为7d、14d和28d组3个亚组，分别给予EA、rTMS、EA+rTMS干预。在不同时间点处死动物取材，首先采用免疫荧光标记方法观察不同时间点、不同干预后梗塞灶周围及缺血侧海马CA3区AST特异性标志物GFAP蛋白及缺血侧海马CA3区突触前膜蛋白SYN的表达，免疫组化方法考察缺血侧海马突触后膜蛋白PSD-95的时间分布规律；然后运用蛋白印迹技术定量分析不同时间点、不同组别之间缺血侧海马SYN表达的差异；最后采用Morris Water Maze实验评估脑缺血28d大鼠学习记忆功能的变化，透射电镜观察缺血侧海马CA3区突触形态结构的变化。
结果
1、脑缺血7d时，各组大鼠脑梗塞病灶周围及脑缺血侧海马CA3区均可见到AST的活化，主要表现为细胞胞体肥大、突起增多、荧光强度增加，3个康复干预组AST活化更加明显。14d时3个康复干预组GFAP阳性细胞进一步增多(P＜0.05)，28d时与14d时比较GFAP的表达减少，差异没有统计学意义。7d和28d时EA+rTMS组缺血侧海马CA3区GFAP的表达高于EA组和rTMS组(P＜0.05)。
2、脑缺血7d时，海马DG区和CA3区PSD-95蛋白的表达下降。经过14d和28d的康复干预，各组大鼠海马DG区和CA3区PSD-95蛋白的表达与模型组比较均增加，至28d时差异有统计学意义。28d时，EA+rTMS组大鼠海马CA3区PSD-95蛋白的表达高于EA组和rTMS组(P＜0.05)。
3、EA、rTMS和EA+rTMS都能显著上调3个时间点海马SYN的表达。从时间上看，随着治疗时间的增加，SYN的表达逐步上调；不同组间比较，28d时rTMS和EA+rTMS海马SYN的表达最显著。
4、在Morris Water Maze训练的第1d-3d，5组大鼠的潜伏期均逐渐缩短，各康复干预组与模型组比较差异不明显。在训练的第4d、5d，3个康复干预组潜伏期明显降低，与模型组比较差异有统计学意义(P＜0.05或P＜0.01)。在测试中，模型组大鼠的逃避潜伏期比正常组明显延长；60s内跨越平台次数减少，tP／tT和dP／dT下降，均提示模型组大鼠存在一定的学习记忆功能障碍。3个康复干预组大鼠的逃避潜伏期缩短，60s内跨越平台次数增加，tP／tT和dP／dT上升，与模型组比较差异有显著性意义(P＜0.05或P＜0.01)，提示经过康复干预之后，脑缺血大鼠的学习记忆功能明显改善。3个康复干预组间比较，EA+rTMS组大鼠的逃避潜伏期和60s内跨越平台次数与EA组和rTMS组比较差异有统计学意义(P＜0.05)。
5、模型组大鼠海马CA3区突触多为对称性突触，突触小泡明显减少，线粒体变性明显。EA组、rTMS组和EA+rTMS组突触多表现为凹型或凸型的不对称性突触，突触小泡明显增多，细胞浆内线粒体双层结构完整，线粒体内嵴清楚。突触形态图像分析统计结果显示：各组间突触间隙宽度无显著性差异。3个康复干预组PSD厚度、穿孔性突触百分率比模型组显著增加，3组间比较无显著差异。3个康复干预组CA3区突触的突触界面曲率显著增加，EA+rTMS组大于EA组和rTMS组，差异有统计学意义。
结论
1、EA、rTMS和EA+rTMS均可活化脑缺血大鼠梗塞灶周围和缺血侧海马CA3区AST，这种活化不是单方向的上调。
2、随着干预时间的延长，EA、rTMS和EA+rTMS均可逐步增加脑缺血海马SYN和PSD-95的表达。
3、EA、rTMS和EA+rTMS可提高脑缺血大鼠学习记忆功能和改善脑缺血大鼠海马突触的超微结构。
4、EA+rTMS在促进AST活化和海马突触重塑中优于EA和rTMS。EA+rTMS提高学习记忆能力与其促进海马突触可塑性有关。
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.
Objective
A 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.
Methods
Healthy 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.
Results
1. 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.
Conclusions
1. 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.
引文
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