Post-stroke electroacupuncture promotes cerebrovascular repair in the peri‑infarct cortex of ischemic stroke mice

目的: 脑血管修复在脑卒中后神经功能恢复过程中发挥关键作用, 然而促进其修复的有效策略及潜在机制尚不明确.本研究旨在探讨脑卒中后电针(EA)干预是否能够促进脑血管修复并改善神经功能, 并进一步阐明其可能的作用机制. 方法: 8周龄雄性C57BL/6J小鼠通过光化学法制备脑缺血模型, 造模后随机分为模型组,连续波电针组EA(Co)及疏密波电针组EA(SD), 每组10只;另设假手术对照组(Control), 含小鼠10只.电针组小鼠接受为期4周的电针干预, 每周5次, 每次20 min;刺激穴位为双侧“内关(PC6)”与“三阴交(SP6)”, 其中患侧连接电针仪进行刺激.电刺激参数为电流1 mA, 疏密波频率2/10 Hz,连续波频率2 Hz或50 Hz.光化学栓塞模型制备24 h后, 通过激光散斑对比成像与TTC染色验证缺血模型的建立和脑损伤程度.电针干预4周后, 采用黏附去除实验,悬挂实验及高架十字迷宫实验评估各组实验小鼠的神经功能相关行为学表现;免疫荧光染色技术检测脑梗死周围区域神经元活性,脑微血管重塑,内皮细胞增殖,血管基底膜和内皮糖萼相关修复指标的变化. 结果: 与健侧脑区相比, 模型小鼠梗死侧脑区血流灌注更低( P < 0.05), TTC染色分析可见明显梗死灶( P < 0.05), 表明脑缺血模型成功建立.(1)黏附去除实验中, 模型组小鼠的黏附去除时间长于假手术组( P < 0.05);电针干预4周后, 电针组黏附去除时间较模型组短( P < 0.05), 但EA(Co)与EA(SD)组间差异无统计学意义( P > 0.05).(2)悬挂实验中, 模型组小鼠肌力表现显著低于假手术组( P < 0.05);电针干预提升了模型小鼠的肌力表现( P < 0.05), 且EA(Co)组的改善效果优于EA(SD)组( P < 0.05).(3)高架十字迷宫实验中, 与假手术组相比, 模型组小鼠的总运动距离,进入开放臂次数比例及停留时间均较少( P < 0.05);电针干预后, 上述指标均高于模型组( P < 0.05), 而EA(Co)与EA(SD)组间差异无统计学意义( P > 0.05).(4)小鼠脑梗死灶周围区神经元存活分析显示, 与假手术组相比, 模型组Nissl⁺/NeuN⁺ 共标的神经元密度较低( P < 0.05);电针干预后, 该指标较模型组高(均 P < 0.05), 但EA(Co)与EA(SD)组间差异无统计学意义( P > 0.05).(5)脑血管结构分析显示, 模型组梗死灶周围区脑血管体积,分支密度及长度均低于假手术组(均 P < 0.05);电针干预后, 上述参数均显著恢复(均 P < 0.05), 且EA(Co)组在脑血管结构修复方面的改善程度优于EA(SD)组( P < 0.05).(6)血管内皮细胞增殖方面, 与假手术组相比, 模型组在梗死灶周围皮质区的Ki67⁺/CD31⁺共标的细胞密度差异无统计学意义( P > 0.05);电针干预后, 该指标升高(均 P < 0.05), 但EA(Co)与EA(SD)组间差异无统计学意义( P > 0.05).(7)脑血管内皮糖萼(Lectin标记)及基底膜Ⅳ型胶原蛋白(Collagen IV)表达水平均较假手术组低(均 P < 0.05);电针干预后, 二者表达均较模型组高(均 P < 0.05), 但EA(Co)与EA(SD)组间差异无统计学意义( P > 0.05). 结论: 电针可通过调节脑微血管重构,内皮细胞增殖,血管基底膜及内皮糖萼完整性, 促进缺血性脑卒中后的脑血管修复及神经功能恢复.其中, 连续波电针在脑血管修复程度方面优于疏密波电针.本研究为电针作为卒中后康复的潜在疗法提供了机制依据, 并提示刺激波形优化可提升其疗效, 具有一定临床转化潜力. 关键词: 缺血性脑卒中;电针;脑血管;神经功能障碍 : Cerebrovascular repair plays a crucial role in promoting post-stroke neurological recovery. However, effective strategies to enhance cerebrovascular repair and the underlying mechanisms remain elusive. Here, we investigated whether post-stroke electroacupuncture (EA) treatment could promote cerebrovascular repair and thereby improve neurological function, as well as the potential mechanisms involved. : Eight-week-old male C57BL/6J mice were subjected to photothrombosis-induced brain ischemia. Following successful model induction, the mice were randomly assigned to the model group, the EA continuous wave group (EA[Co]), and the EA dense-sparse wave group (EA[SD]), with 10 animals in each group. A sham-operated group (n = 10) served as the control. The EA groups received a 4-week EA intervention targeting the acupoints "Neiguan(PC6)" and "Sanyinjiao(SP6)". EA was administered five times per week with bilateral stimulation, and the affected side was connected to the EA device. The EA(SD) group received stimulation at 2/10 Hz, whereas the EA(Co) group received continuous wave stimulation at 2 Hz or 50 Hz; both were delivered at an intensity of 1 mA for 20 min per session. Twenty-four hours after photothrombosis induction, laser speckle contrast imaging and 2,3,5-triphenyltetrazolium chloride staining were performed to confirm model establishment and cerebral ischemic injury. Neurological function was evaluated using behavioral tests, including the adhesive removal test, hanging wire test, and elevated plus maze test. Immunofluorescence staining was performed to assess neuronal viability, cerebral microvascular structure, endothelial cell proliferation, and vascular repair markers in the peri-infarct cortex. : Significant reductions in cerebral blood perfusion ( P < 0.05) and non-infarct volume ( P < 0.05) were observed in the ipsilateral hemisphere of model mice compared with the contralateral hemisphere, confirming successful establishment of the photothrombosis-induced ischemia model. (1) In the adhesive removal test, the model group exhibited significantly prolonged adhesive removal latency compared with the control group ( P < 0.05). EA treatment shortened latency compared with the model group ( P < 0.05), with no statistically significant difference between the EA(Co) and EA(SD) groups ( P > 0.05). (2) In the wire hanging test, the model group exhibited significantly lower muscle strength scores than the sham group ( P < 0.05). EA treatment significantly enhanced muscle strength performance ( P < 0.05), and the improvement was greater in the EA(Co) group than in the EA(SD) group ( P < 0.05). (3) In the elevated plus maze test, the model group showed significant decreases in total distance traveled, percentage of open-arm entries, and time spent in open arms compared with the control group (all P < 0.05). Following EA treatment, all three parameters were significantly restored compared with the model group (all P < 0.05), with no statistically significant difference between the EA(Co) and EA(SD) groups ( P > 0.05). (4) Immunofluorescence staining showed a significantly reduced density of Nissl⁺/NeuN⁺ double-labeled neurons in the peri-infarct regions of the model group compared with the control group ( P < 0.05). Nissl⁺/NeuN⁺ neuronal density was significantly restored in the EA groups compared with the model group (all P < 0.05), with no statistically significant difference between the EA(Co) and EA(SD) groups ( P > 0.05). (5) In terms of cerebrovascular structure, the model group exhibited significantly reduced vascular volume, microvascular branching density, and microvascular length in the peri-infarct regions compared with the control group (all P < 0.05). EA treatment restored all three vascular parameters compared with the model group (all P < 0.05). Moreover, the EA(Co) group showed significantly greater enhancement of cerebrovascular structural integrity than the EA(SD) group ( P < 0.05). (6) Compared with the control group, the model group showed no significant change in the density of Ki67⁺/CD31⁺ double-labeled endothelial cells ( P > 0.05). Following EA treatment, this density was significantly increased compared with the model group ( P < 0.05), with no statistically significant difference between the EA(Co) and EA(SD) groups ( P > 0.05). (7) Compared with the control group, the model group exhibited significant reductions in lectin-labeled glycocalyx in vessels and basement membrane protein collagen IV expression (all P < 0.05), indicating disruption of cerebrovascular integrity. EA treatment significantly restored both indicators compared with the model group (all P < 0.05), with no statistically significant difference between the EA(Co) and EA(SD) groups ( P > 0.05). : This study demonstrates that EA promotes cerebrovascular repair and neurological recovery after ischemic stroke by enhancing microvascular remodeling, stimulating endothelial proliferation, and restoring basement membrane and glycocalyx integrity. EA(Co) is more effective than EA(SD) in repairing vascular structure. These findings provide mechanistic evidence that EA is a promising therapeutic approach for post-stroke rehabilitation and suggest that waveform optimization may enhance its clinical efficacy.