聚焦超声定向开放血脑屏障及其在活体内视觉通路无创性标记中的应用

The optic nerve is an important structure which connected eyeball to the brain visual cortex, and it is hided and highly protected by the skull and blood brain barrier (BBB), this protection makes it extremely difficult to non-invasion study the optic nerve in vivo. Based on our experience that carried out the NSFC project and being a visiting scholar in Stanford University, this study aims to reversely open the blood brain barrier of the targeted optic relay nuclei (supraoptic nucleus and geniculate body) and the brain visual cortex in a tree shrew animal model. After being intravenous injected, the nanoscale fluorescence gold reagent is chosen as a tracer to detect the blood brain barrier opening in targeted areas, since the reagent (Fluoro-gold) could be stimulated to emit fluorescence but could not cross the integrated blood brain barrier. Furthermore, the Fluoro-gold could be uptaken by the second and third-order neurons, retrograde transferred through the synapse and retinal ganglion cells to the retina in the eyes. Finally, the visual pathway would be marked in vivo...Focused ultrasound is a promising technology that could open the blood brain barrier non-invasively, positioning accurately, provisionally and reversely. Our research is intended to use it to mark the area-oriented nerve pathway and to explore the neural transport mechanism of intra-axonal and trans-synaptic in vivo. Based on this, we could conduct more intensive researches on much more significant areas, such as the functional regulation of blood brain barrier, repair of the optic nerve after injury, brain targeted drug delivery.

视神经是连接眼球和大脑视觉中枢的重要结构，位置深在并受到颅骨及血脑屏障等结构的重重保护，在活体内对视神经进行无创伤性的检查和研究极其困难。基于申请人已结项国自然课题及赴美访学研究基础，本研究拟采用小动物聚焦超声设备及颅脑立体定位技术，定向开放实验动物（树鼩）视神经中继核团处的血脑屏障。利用纳米级荧光金试剂不能透过完整血脑屏障和受激发可产生荧光的特性，在静脉注射后将其作为检测血脑屏障局部开放的示踪剂。同时，透出局部开放的血脑屏障后，荧光金将被神经元摄取，经由跨突触间传递及节细胞的轴突逆行转运至眼球视网膜内，最终在活体内实现对视觉通路的标记。.本研究将运用聚焦超声作为无创性、可精确定位、暂时且可复性开放血脑屏障的技术，对活体内特定区域的神经通路进行无创性标记、对轴突内及跨突触间神经转运机制开展研究。以此为基础，我们将在血脑屏障功能调控、视神经损伤及修复、脑内定向药物投递等领域展开更深入探索。

视神经是连接眼球和大脑视觉中枢的重要结构，位置深在并受到颅骨及血脑屏障等结构的重重保护，一旦损伤则极难修复。在活体内对视神经进行无创伤性的检查和研究极其困难，目前在活体内对视觉通路进行标记和成像的方法，均未能满足医学基础和临床诊断的需求。2018年至2021年间，在国家自然科学基金（81760316）和云南省医学学科带头人项目（D2018010）的资助下，项目组集成现有的聚焦超声开放血脑屏障、合成磁共振成像等关键技术，围绕活体内视觉通路、神经损伤后监测等内容展开研究，完成了以下主要内容：.1、利用聚焦超声开放血脑屏障，经静脉注射荧光金后视网膜及脑内神经细胞可被标记。通过反复实验，最终确定聚焦超声照射最佳参数组合：能量2 w，作用时间45s-60s，微泡剂量0.1ml，可以无创性开放SD雄性幼鼠血脑屏障。随着时间延长，荧光金逐渐从上丘转运到视神经及视网膜内，清楚完整地对整个视觉通路进行逆向标记，整个标记时间段为7天-14天。.2、发现聚焦超声开放血脑屏障的具体机制，并将其用于脑内无创的、靶向基因传递。聚焦超声联合微泡会造成脑部紧密连接（tight junction, TJ）分子结构解体，导致脑微血管内皮连接屏障功能丧失。同时，静脉注射重组腺相关病毒(recombinant adeno-associated virus, rAAV) 可以在脑内提供有效的基因传递和表达，显示出非侵入性和靶向性基因传递技术对治疗中枢神经系统疾病具有巨大潜力。.3、阐明了以多模态磁共振成像为主的监测体系，对反映血脑屏障开放程度及神经受损情况具有的重要价值。在大鼠视神经定量钳夹损伤、大鼠脑部控制性皮层撞击损伤、周围/中枢神经早期放射性损伤等诸多实验中，项目组证实了影像定量参数可作为重要的损伤评价指标，可在某种程度上取代有创性的病理检查。以此为基础，项目组建议在临床推广脑部神经功能成像等技术，以提高诊断准确率和降低病人残疾率。