Ultrasound and microbubble mediated blood brain barrier opening is a non-invasive and effective technique for drug delivery to targeted brain region. However, the exact mechanisms are not fully resolved. The influences of Ca2+ signaling on sonoporation and endothelial tight junctional regulation affect the efficiency and biosafety of the technique. Therefore, an improved understanding of how ultrasound evokes Ca2+ signaling in the brain endothelial monolayer, and its correlation to endothelial permeability change is necessary. Here, we examined the effects of SonoVue microbubbles or integrin-targeted microbeads on ultrasound induced bioeffects in brain microvascular endothelial monolayer using an acoustically-coupled microscopy system, where focused ultrasound exposure and real-time recording of Ca2+ signaling and membrane perforation were performed. Microbubbles induced robust Ca2+ responses, often accompanied by cell poration, while ultrasound with microbeads elicited reversible Ca2+ response without membrane poration. At the conditions evoking reversible Ca2+ signaling, intracellular Ca2+ release and reactive oxygen species played key roles for microbubbles induced Ca2+ signaling while activation of mechanosensitive ion channels was essential for the case of microbeads. Trans-well diffusion analysis revealed significantly higher trans-endothelial transport of 70 kDa FITC-dextran for both integrin-targeted microbeads and microbubbles compared to the control group. Further immunofluorescence staining showed disruption of cell junctions with microbubble stimulation and reversible remodeling of many cell junctions by ultrasound with integrin-targeted microbeads. This investigation provides new insights for ultrasound induced Ca2+ signaling and its influence on endothelial permeability, which may help develop new strategies for safe and efficient drug/gene delivery in the vascular system.

中文翻译：

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声泡和靶向微珠诱导的可逆 Ca2 + 信号传导和增强的内皮单层细胞旁转运


超声和微泡介导的血脑屏障开放是一种将药物输送到目标大脑区域的非侵入性有效技术。然而，确切的机制尚未完全解决。Ca2 + 信号对超声和内皮紧密连接调节的影响影响该技术的效率和生物安全性。因此，有必要更好地了解超声如何在大脑内皮单层中诱发 Ca2 + 信号传导，及其与内皮通透性变化的相关性。在这里，我们使用声学耦合显微镜系统检查了 SonoVue 微泡或整合素靶向微珠对超声诱导的脑微血管内皮单层生物效应的影响，其中进行了聚焦超声暴露和 Ca2+ 信号传导和膜穿孔的实时记录。微泡诱导了强烈的 Ca2 + 反应，通常伴有细胞穿孔，而使用微珠的超声引起了可逆的 Ca2 + 反应，没有膜孔。在诱发可逆 Ca2 + 信号传导的条件下，细胞内 Ca2 + 释放和活性氧对微泡诱导 Ca2 + 信号传导起关键作用，而机械敏感离子通道的激活对于微珠的情况至关重要。跨孔扩散分析显示，与对照组相比，70 kDa FITC-葡聚糖对整合素靶向微珠和微泡的跨内皮转运显着升高。进一步的免疫荧光染色显示微泡刺激破坏细胞连接，并使用整合素靶向微珠通过超声对许多细胞连接进行可逆重塑。 这项研究为超声诱导的 Ca2 + 信号传导及其对内皮通透性的影响提供了新的见解，这可能有助于开发在血管系统中安全有效的药物/基因递送的新策略。