Background Low-intensity focused ultrasound stimulation (LIFUS) has been developed to enhance neurological repair and remodelling during the late acute stage of ischaemic stroke in rodents. However, the cellular and molecular mechanisms of neurological repair and remodelling after LIFUS in ischaemic stroke are unclear. Methods Ultrasound stimulation was treated in adult male mice 7 days after transient middle cerebral artery occlusion. Angiogenesis was measured by laser speckle imaging and histological analyses. Electromyography and fibre photometry records were used for synaptogenesis. Brain atrophy volume and neurobehaviour were assessed 0–14 days after ischaemia. iTRAQ proteomic analysis was performed to explore the differentially expressed protein. scRNA-seq was used for subcluster analysis of astrocytes. Fluorescence in situ hybridisation and Western blot detected the expression of HMGB1 and CAMK2N1. Results Optimal ultrasound stimulation increased cerebral blood flow, and improved neurobehavioural outcomes in ischaemic mice (p<0.05). iTRAQ proteomic analysis revealed that the expression of HMGB1 increased and CAMK2N1 decreased in the ipsilateral hemisphere of the brain at 14 days after focal cerebral ischaemia with ultrasound treatment (p<0.05). scRNA-seq revealed that this expression pattern belonged to a subcluster of astrocytes after LIFUS in the ischaemic brain. LIFUS upregulated HMGB1 expression, accompanied by VEGFA elevation compared with the control group (p<0.05). Inhibition of HMGB1 expression in astrocytes decreased microvessels counts and cerebral blood flow (p<0.05). LIFUS reduced CAMK2N1 expression level, accompanied by increased extracellular calcium ions and glutamatergic synapses (p<0.05). CAMK2N1 overexpression in astrocytes decreased dendritic spines, and aggravated neurobehavioural outcomes (p<0.05). Conclusion Our results demonstrated that LIFUS promoted angiogenesis and synaptogenesis after focal cerebral ischaemia by upregulating HMGB1 and downregulating CAMK2N1 in a subcluster of astrocytes, suggesting that LIFUS activated specific astrocyte subcluster could be a key target for ischaemic brain therapy. Data are available in a public, open access repository. All data relevant to the study are included in the article or uploaded as supplementary information. Data are available upon reasonable request.

中文翻译：

---

低强度聚焦超声刺激通过星形胶质细胞 HMGB1 和 CAMK2N1 促进小鼠中风恢复

背景低强度聚焦超声刺激（LIFUS）已被开发用于增强啮齿类动物缺血性中风急性期晚期的神经修复和重塑。然而，LIFUS治疗缺血性卒中后神经修复和重塑的细胞和分子机制尚不清楚。方法对成年雄性小鼠大脑中动脉短暂闭塞后7天进行超声刺激。通过激光散斑成像和组织学分析来测量血管生成。肌电图和纤维光度测量记录用于突触发生。缺血后 0-14 天评估脑萎缩体积和神经行为。进行 iTRAQ 蛋白质组分析以探索差异表达的蛋白质。scRNA-seq 用于星形胶质细胞的亚簇分析。荧光原位杂交和Western blot检测HMGB1和CAMK2N1的表达。结果 最佳超声刺激可增加缺血小鼠的脑血流量并改善神经行为结果（p<0.05）。iTRAQ蛋白质组学分析显示，超声治疗局灶性脑缺血后14天，同侧大脑半球HMGB1表达增加，CAMK2N1表达减少（p<0.05）。scRNA-seq 显示，这种表达模式属于缺血脑中 LIFUS 后的星形胶质细胞亚群。与对照组相比，LIFUS 上调 HMGB1 表达，并伴有 VEGFA 升高（p<0.05）。星形胶质细胞中 HMGB1 表达的抑制会减少微血管计数和脑血流量 (p<0.05)。LIFUS 降低了 CAMK2N1 表达水平，同时细胞外钙离子和谷氨酸突触增加 (p<0.05)。星形胶质细胞中 CAMK2N1 过度表达会减少树突棘，并加重神经行为结果 (p<0.05)。结论 我们的结果表明，LIFUS 通过上调星形胶质细胞亚群中的 HMGB1 和下调 CAMK2N1 来促进局灶性脑缺血后的血管生成和突触生成，表明 LIFUS 激活的特定星形胶质细胞亚群可能是缺血性脑治疗的关键靶点。数据可在公共、开放访问存储库中获取。与研究相关的所有数据都包含在文章中或作为补充信息上传。数据可根据合理要求提供。