BACKGROUND Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive treatment for ischemic stroke. Astrocytes regulation has been suggested as one mechanism for rTMS effectiveness. But how rTMS regulates astrocytes remains largely undetermined. There were neurotoxic and neuroprotective phenotypes of astrocytes (also denoted as classically and alternatively activated astrocytes or A1 and A2 astrocytes) pertaining to pro- or anti-inflammatory gene expression. Pro-inflammatory or neurotoxic polarized astrocytes were induced during cerebral ischemic stroke. The present study aimed to investigate the effects of rTMS on astrocytic polarization during cerebral ischemic/reperfusion injury. METHODS Three rTMS protocols were applied to primary astrocytes under normal and oxygen-glucose deprivation/reoxygenation (OGD/R) conditions. Cell survival, proliferation, and phenotypic changes were assessed after 2-day treatment. Astrocytes culture medium (ACM) from control, OGD/R, and OGD/R + rTMS groups were mixed with neuronal medium to culture neurons for 48 h and 7 days, in order to explore the influence on neuronal survival and synaptic plasticity. In vivo, rats were subjected to middle cerebral artery occlusion (MCAO), and received posterior orbital intravenous injection of ACM collected from different groups at reperfusion, and at 3 days post reperfusion. The apoptosis in the ischemic penumbra, infarct volumes, and the modified Neurological Severity Score (mNSS) were evaluated at 1 week after reperfusion, and cognitive functions were evaluated using the Morris Water Maze (MWM) tests. Finally, the 10 Hz rTMS was directly applied to MCAO rats to verify the rTMS effects on astrocytic polarization. RESULTS Among these three frequencies, the 10 Hz protocol exerted the greatest potential to modulate astrocytic polarization after OGD/R injury. Classically activated and A1 markers were significantly inhibited by rTMS treatment. In OGD/R model, the concentration of pro-inflammatory mediator TNF-α decreased from 57.7 to 23.0 рg/mL, while anti-inflammatory mediator IL-10 increased from 99.0 to 555.1 рg/mL in the ACM after rTMS treatment. The ACM collected from rTMS-treated astrocytes significantly alleviated neuronal apoptosis induced by OGD/R injury, and promoted neuronal plasticity. In MCAO rat model, the ACM collected from rTMS treatment decreased neuronal apoptosis and infarct volumes, and improved cognitive functions. The neurotoxic astrocytes were simultaneously inhibited after rTMS treatment. CONCLUSION Inhibition of neurotoxic astrocytic polarization is a potential mechanism for the effectiveness of high-frequency rTMS in cerebral ischemic stroke.

中文翻译：

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高频重复经颅磁刺激可通过抑制缺血大鼠星形胶质细胞的神经毒性极化来改善功能恢复。

背景技术重复经颅磁刺激（rTMS）是用于缺血性中风的非侵入性治疗。星形胶质细胞调节已被建议作为rTMS有效性的一种机制。但是，rTMS如何调节星形胶质细胞尚不清楚。有星形胶质细胞（也称为经典和替代激活星形胶质细胞或A1和A2星形胶质细胞）的神经毒性和神经保护表型，与促炎或抗炎基因表达有关。在脑缺血性中风期间会诱发促炎或神经毒性的极化星形胶质细胞。本研究旨在研究rTMS对脑缺血/再灌注损伤中星形胶质细胞极化的影响。方法在正常和氧葡萄糖剥夺/复氧（OGD / R）条件下，将三种rTMS方案应用于原代星形胶质细胞。细胞存活 治疗2天后评估增殖和表型变化。将对照组，OGD / R和OGD / R + rTMS组的星形胶质细胞培养基（ACM）与神经元培养基混合，以培养神经元48 h和7天，以研究其对神经元存活和突触可塑性的影响。在体内，对大鼠进行大脑中动脉闭塞（MCAO），并在再灌注时和再灌注后3天接受从不同组收集的ACM眼眶后静脉注射。在再灌注后1周评估缺血半影的细胞凋亡，梗塞体积和改良的神经系统严重程度评分（mNSS），并使用莫里斯水迷宫（MWM）测试评估认知功能。最后，将10 Hz rTMS直接应用于MCAO大鼠，以验证rTMS对星形细胞极化的影响。结果在这三个频率中，10 Hz协议在OGD / R损伤后发挥了最大的潜力来调节星形细胞极化。rTMS处理可显着抑制经典激活和A1标记。在OGD / R模型中，rTMS治疗后ACM中促炎介质TNF-α的浓度从57.7降至23.0рg/ mL，而抗炎介质IL-10从99.0升高至555.1рg/ mL。从经rTMS处理的星形胶质细胞收集的ACM显着减轻了OGD / R损伤诱导的神经元凋亡，并促进了神经元可塑性。在MCAO大鼠模型中，从rTMS治疗中收集的ACM减少了神经元凋亡和梗塞体积，并改善了认知功能。rTMS处理后，神经毒性星形胶质细胞同时被抑制。结论抑制神经毒性星形细胞极化是高频rTMS在脑缺血性卒中中发挥作用的潜在机制。