超声刺激诱导M2型小胶质细胞外泌体促进卒中后脑重塑的机制研究

Ultrasound brain stimulation is a non-invasive, safe and simple novel stimulation technique. Studies found that ultrasound brain stimulation regulates neural activity of different brain areas in both human and animals, and also promotes hippocampus neurogenesis in normal mice. These studies indicate that ultrasound brain stimulation could modulate brain remodeling. Our previous study demonstrated that low frequency ultrasound stimulation increased neurogenesis and improved motor function of ischemic mice. However, the underlying mechanism of ultrasound stimulation mediated-brain plasticity modulation remains largely unknown. Emerging data suggest that exosomes contribute to brain functional repair and remodeling by regulating intercellular communication between source and target cells in ischemic brain. Our further study revealed that the number of microglia was also increased after ultrasound stimulation. Microglia plays important roles in every stage of neurogenesis during cerebral ischemia. Given this, we hypothesize that ultrasound stimulation could increase exosome release from M2 type microglia in ischemic brain, which further regulates brain remodeling. In the present study, we aim to investigate the effects of ultrasound brain stimulation on modulating brain plasticity of ischemic mice. Using molecular biology and microimaging techniques, we will explore: 1) whether ultrasound brain stimulation promotes neurogenesis via increasing exosome release from M2 type microglia; 2) whether transplantation of exosome derived from ultrasound stimulated M2 microglia could promote neurogenesis and improve the neurological functions. The data of this study will provide theoretical basis for using ultrasound brain stimulation to promote brain repair and remodeling after ischemic stroke.

超声脑刺激是一种非侵入，安全易行的新型刺激技术。研究发现其可调节人和动物不同脑区的神经活动并能促进小鼠海马神经再生，提示超声刺激可调控脑可塑性。申请者前期研究发现低频超声刺激可促进脑缺血小鼠神经再生，改善神经功能，但超声通过何种机制调控脑重塑尚不清楚。研究提示外泌体可通过调控源细胞和靶细胞间信息交流促进缺血后脑功能修复和重塑。我们前期研究还发现超声可增加小胶质细胞数量，而小胶质细胞在卒中后神经再生的每一阶段都扮演重要角色，因此我们假设超声可诱导缺血脑M2型小胶质细胞释放外泌体，进而调控脑重塑。本课题拟建立小鼠局灶性脑缺血模型，应用低频超声刺激、分子生物学和显微成像等技术，探索：1）超声刺激是否诱导缺血脑M2型小胶质细胞释放外泌体，促进神经再生；2）超声刺激M2型小胶质细胞来源的外泌体移植是否可促进神经再生和脑重塑，改善神经功能。本课题研究将为超声促进卒中后脑修复/功能重塑提供理论基础。

与其他无创脑刺激技术如经颅磁刺激和经颅直流电刺激相比，超声脑刺激具有刺激深度深、空间分辨率高、成本低等优点，是一种极具临床应用前景的新型脑刺激技术。研究发现其可促进小鼠海马神经再生，提示超声刺激可增强脑可塑性。但较少有人研究其对卒中后神经再生的作用及其机制。申请者建立了小鼠局灶性脑缺血模型，应用低频超声对小鼠大脑进行刺激，并通过分子生物学、显微成像和神经行为学等进行分析，发现其可促进脑缺血小鼠神经再生，改善神经功能，说明超声可调控缺血脑可塑性。通过免疫组化及流式细胞分析，我们发现，超声刺激增加了M2型小胶质细胞，说明超声促进了小胶质细胞向保护性M2型转化。通过蛋白和基因测试，我们还发现超声上调了IL10及其受体的表达，说明超声可能通过调控M2型小胶质细胞，进而调控IL10信号通路起作用。通过细胞学实验，我们发现超声可诱导M2型小胶质细胞释放外泌体，通过miRNA 芯片分析，我们发现了外泌体内高表达与IL10通路有关的，并且与神经再生相关的miRNAs如miRNA-124，-106，27a。综上所述，在脑缺血条件下，超声可促进小胶质细胞向M2型转化，并诱导M2型小胶质细胞释放外泌体，通过miRNA调节IL10信号通路，从而促进神经再生，进一步增强脑功能重塑，最终促进神经功能的恢复。为超声脑刺激的基础研究增添了证据，同时鉴于超声刺激的安全性和有效性，为其进一步的临床应用奠定了基础。