【作者】 万鹏；

【导师】 俞婷婷； 朱(?)；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2024， 博士

【摘要】 糖尿病及并发症现已成为危害人类健康的世界公共卫生问题。研究表明,糖尿病会破坏血脑屏障结构的完整性、增加其通透性,进而影响大脑微环境及功能、甚至导致认知功能障碍等问题。目前研究者对于糖尿病导致的血脑屏障损伤情况及其潜在病理机制的理解相对有限,其主要原因在于,对血脑屏障多种结构的准确识别、以及糖尿病诱导不同脑区血脑屏障病理变化等存在技术挑战。针对以上问题,结合近年发展起来的组织光透明成像技术,围绕血脑屏障结构与功能的标记、透明和成像方法的建立而展开研究,在此基础上,探究糖尿病进展中小鼠血脑屏障结构与功能的损伤及其机制。其主要内容如下:(1)血脑屏障结构与功能整体成像方法研究:对几种可能用于反映血脑屏障渗透性的荧光染料进行成像测试,提出基于伊文思蓝的糖尿病鼠脑血脑屏障渗透性的表征方法;筛选可用于血脑屏障结构标记的抗体,引入电泳去脂优化免疫染色的促渗方案,构建针对大样本的血脑屏障紧密连接蛋白结构的整体免疫标记方法,实现了小鼠1.5 mm脑块和半脑的整体免疫标记;引入抗氧化试剂对现有的透明方法(FDISCO)进行优化,将光透明处理及样本长期保存的温度从低温(4℃-8℃)改为常温,进而发展出高效、荧光兼容的光透明方法FDISCO+;最后,利用所建立的免疫标记、透明方法,结合光片成像,成功获取大样本血脑屏障的整体结构信息。(2)糖尿病进展中小鼠血脑屏障结构与功能损伤研究:利用所建立的组织光透明成像方法,获取正常和糖尿病小鼠血脑屏障渗透性和紧密连接结构的成像数据,分析不同病程、不同脑区小鼠血脑屏障结构与功能变化。结果发现:糖尿病会造成SSp、CA、MBmot等多脑区的血脑屏障渗透性增大,血脑屏障紧密连接蛋白(Claudin-5蛋白和Occludin蛋白)表达水平会降低;且随着病程延长、血脑屏障渗透性增加、渗漏脑区的数量会进一步增多,同时,紧密连接蛋白的表达水平会进一步降低。以上说明糖尿病血脑屏障渗透性和紧密连接结构变化存在关联。(3)糖尿病诱导血脑屏障结构损伤的潜在机制研究:利用组织光透明成像获取正常和糖尿病小鼠不同脑区的小胶质细胞和血管的三维结构信息,利用定量聚合酶链反应(q PCR)等技术对各脑区小胶质细胞的极性变化进行研究,进一步探究小胶质细胞与血脑屏障损伤的关联性。结果发现:小鼠糖尿病进展的不同阶段,在不同脑区中小胶质细胞(VAM)的数量占比表现出不同程度的增加,这提示相应脑区的小胶质细胞向血管发生了异常迁移;糖尿病血脑屏障紧密连接蛋白的表达水平降低与VAM数量占比增加密切相关,在血脑屏障紧密连接结构发生明显损伤的脑区,往往伴随着VAM数量占比增加;糖尿病会诱导小胶质细胞极性发生变化,如皮层和纹状体的小胶质细胞主要表现为M1型(促炎型),而海马和中脑的小胶质细胞会同时表现M1型和M2型(抗炎型),这与血脑屏障紧密连接结构的损伤情况基本一致。综上,建立的适用于血脑屏障整体成像的标记与透明方法,为糖尿病小鼠血脑屏障三维结构的获取提供了重要的工具,不同病程中各脑区小鼠血管渗透性、血脑屏障紧密连接蛋白的变化为糖尿病进展中的血脑屏障病理特征提供了直接证据,对小胶质细胞极性的研究为理解糖尿病诱导血脑屏障结构损伤的潜在机制提供了重要参考。更多还原

【Abstract】 Diabetes and its complications have become a global public health problem that threatens human health.Studies have shown that diabetes can disrupt the integrity and permeability of the blood-brain barrier(BBB),affect the brain microenvironment and function,and even lead to cognitive dysfunction.Currently,researchers have a limited understanding of the extent of BBB damage caused by diabetes and its underlying pathological mechanisms.The main reason is that there are still technical difficulties in accurately identifying and labeling various structures of the BBB on a large scale and analyzing pathological changes of the BBB in different brain regions.To address these issues,this study combined the recently developed tissue optical clearing technology and established methods for labeling,clearing,and imaging of BBB structure and function.Based on these methods,the study explored the structural and functional damage to the BBB in diabetes progression in mice and its underlying mechanisms.The main contents are as follows:(1)Study of structure and function imaging methods of BBB:First,the Evans blue labeling method was selected for representing the BBB permeability in diabetic mice after testing several fluorescent dyes.Second,a global immunolabeling method for marking tight junction proteins structure of large samples was constructed by screening several antibodies that might be used as markers of BBB structure and introducing an electrophoretic lipid removal method to optimize the antibodies permeabilization,and achieved whole immunolabeling of 1.5 mm brain blocks and hemisphere of mice.Further,an efficient and fluorescence-compatible clearing method named as FDISCO+was developed by introducing antioxidant reagents into the clearing solution to increase the storage condition of samples from low temperature to room temperature.Finally,by using the established immunolabeling,clearing and imaging methods,the overall structural information in large samples was obtained successfully.(2)Study of the damage of structure and function in the mice BBB during the progression of diabetes:Based on the established overall imaging method of BBB structure and function,the imaging data of BBB permeability and tight junction proteins structure in different brain regions of normal and diabetic mice were obtained,and the damage rules of BBB structure and function in mice with different disease course were analyzed.The results showed that diabetes increased the BBB permeability in SSp,CA,MBmot and other brain regions,and decreased the expression level of Claudin-5 and Occludin proteins.With the prolongation of the disease course,the permeability of the BBB increased,the number of leaky brain regions increased,and the expression level of tight junction protein decreased.The above results suggested that the BBB permeability was associated with changes in tight junction structure in diabetes.(3)Study on the potential mechanism of the diabetes-induced BBB structure damage:The three-dimensional structure information of microglia and blood vessels in different brain regions of normal and diabetic mice was obtained by tissue clearing and imaging.Quantitative polymerase chain reaction(q PCR)and other techniques were used to study the polarity changes of microglia in different brain regions,so as to further explore the correlation between microglia and BBB injury.The results showed that the number ratio of the vessel-associated microglia(VAM)in some brain regions increased in different diabetes stages,indicating abnormal migration of microglia to blood vessels in corresponding brain regions.The decrease of BBB tight junction proteins expression was closely related to the increase of VAM number ratio.In the brain regions with significant damage of the tight junction structure,there was often an increase in the VAM number ratio.Diabetes also might induce changes in the polarity of microglia,for example,microglia in the cortex and corpus striatum mainly exhibited M1 type(pro-inflammatory type),while microglia in hippocampus and midbrain exhibited both M1 type and M2 type(anti-inflammatory type),which was basically consistent with the damage of the tight junction structure of the BBB.In summary,this paper established a method for labeling,clearing and imaging the structure and function of BBB,and obtained a data set of multiple structures of the mice BBB at different stages of diabetes,which provided a direct evidence for the pathological changes of the BBB in the progression of diabetes.Further,the role of microglia in the progression of BBB damage in diabetes was explored,which providing an important reference for understanding the potential mechanism of the diabetes-induced BBB damage.更多还原