| 王钰宏,罗凯旋,李俊瑞,廖烨晖,陈茉弦,敖丽娟.聚焦超声联合微泡开放血脑屏障对小鼠紧密连接蛋白的影响[J].中国康复医学杂志,2023,(1):8~15 |
| 聚焦超声联合微泡开放血脑屏障对小鼠紧密连接蛋白的影响 点此下载全文 |
| 王钰宏 罗凯旋 李俊瑞 廖烨晖 陈茉弦 敖丽娟 |
| 昆明医科大学康复学院，云南省昆明市，650500 |
| 基金项目：国家自然科学基金项目（81960421）；昆明医科大学研究生创新基金项目（2021S025） |
| DOI：10.3969/j.issn.1001-1242.2023.01.002 |
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| 摘要： |
| 摘要
目的：探讨聚焦超声（focused ultrasound， FUS）联合微泡开放血脑屏障（blood-brain barrier， BBB）的安全性，并通过对FUS辐照后紧密连接蛋白的检测探究FUS开放BBB的可能机制。
方法：健康昆明小鼠70只，随机分为对照组、FUS联合微泡开放BBB组，并进一步分为FUS开放BBB1h、4h、24h组。采用Garcia量表评估小鼠的神经行为学变化；HE染色观察超声辐照脑区的出血情况；ELISA动态检测炎性因子：肿瘤坏死因子α（tumor necrosis factor alpha，TNF-α）、白细胞介素1β（interleukin 1 beta，IL-1β）、白细胞介素6（interleukin 6，IL-6）的表达水平。经尾静脉注射伊文思蓝（evans blue， EB）动态观察不同分组小鼠脑内EB的渗透情况。Western Blot检测水通道蛋白4（aquaporin-4，AQP4）表达量的变化分析脑组织的水肿情况；分别检测不同时段紧密连接蛋白occludin、claudin-5、紧密连接蛋白1（zonula occludens-1，ZO-1）的表达水平评估超声对紧密连接蛋白的影响。
结果：FUS诱导BBB开放后，小鼠不同时段的神经行为学评分均与对照组无明显差异（P>0.05）；HE染色显示FUS辐照后小鼠脑实质内无明显的红细胞渗出，炎性因子TNF-α、IL-1β、IL-6的表达水平在BBB开放后的1h、4h、24h均与对照组无明显差异（P>0.05）。在EB渗透实验中，1h组小鼠脑实质内有EB渗透，而其他分组均未观察到EB。不同时段AQP4蛋白表达量与对照组相比无显著性差异（P>0.05）。FUS辐照后1h，occludin（P=0.034）、claudin-5（P<0.001）、ZO-1（P=0.002）蛋白表达水平显著降低；4h后ZO-1、occludin蛋白水平逐渐上升至对照组水平（P>0.05）。而claudin-5蛋白表达量在24h仍低于对照组(P=0.002)。
结论：FUS可以安全有效的开放BBB，其机制可能是通过作用于紧密连接蛋白ZO-1、occludin、claudin-5实现的。 |
| 关键词：聚焦超声 微泡 血脑屏障 安全性 紧密连接蛋白 |
| Effects of focused ultrasound combined with microbubbles to open the blood-brain barrier on tight junction proteins Download Fulltext |
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| School of Rehabilitation， Kunming Medical University， Yunnan Province， 650500 |
| Fund Project: |
| Abstract: |
| Abstract
Objective：To investigate the safety of focused ultrasound (FUS) combined with microbubbles opening the blood-brain barrier (BBB)， and to explore the possible mechanism of BBB opening in FUS by detecting tight junction proteins after FUS irradiation.
Method：Seventy healthy Kunming mice were randomly divided into control group， FUS combined with microbubble open BBB group， and further divided into FUS open BBB 1h， 4h， 24h groups. The Garcia scale was used to evaluate the neurological behavioral changes of mice； HE staining was used to observe the bleeding in the brain area irradiated by ultrasound； ELISA dynamic detection of the expression levels of inflammatory factors tumor necrosis factor alpha (TNF-α)，interleukin 1 beta (IL-1β) and interleukin 6 (IL-6).Evans blue (EB) was injected through the tail vein to dynamically observe the infiltration of EB in the brains of mice in different groups. Western Blot detects changes in aquaporin-4(AQP4) expression to analyze brain tissue edema； detects the protein expression levels of occludin， claudin-5 and zonula occludens-1(ZO-1) at different time periods to evaluate the effect of ultrasound on tight junction proteins.
Result：After FUS induced BBB opening， the neurobehavioral scores of the mice at different time periods were not significantly different from those of the control group (P>0.05)； HE staining showed that there was no obvious red blood cell exudation in the brain parenchyma of the mice after FUS irradiation， and inflammation. The expression levels of TNF-α， IL-1β and IL-6 were not significantly different from those of the control group at 1h， 4h and 24h after BBB was opened (P>0.05).In the EB penetration experiment， there was EB penetration in the brain parenchyma of mice in the 1h group， but no EB was observed in other groups. Compared with the control group， the expression of AQP4 protein in different periods was not statistically different (P>0.05). 1h after FUS irradiation， the protein expression levels of occludin (P=0.034)， claudin-5 (P<0.001)， and ZO-1 (P=0.002) decreased significantly； 4h later， the protein levels of ZO-1 and occludin gradually increased to the control group Level (P>0.05). The expression of claudin-5 protein was still lower than that of the control group at 24h (P=0.002).
Conclusion：FUS can safely and effectively open BBB， and the mechanism may be realized by acting on tight junction proteins ZO-1， occludin and claudin-5. |
| Keywords:focused ultrasound microbubble blood-brain barrier safety tight junction protein |
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