[1]王雪,南希,杜星月,等.脑血管内皮细胞线粒体原位结构分析体系建立与应用[J].卒中与神经疾病杂志,2024,31(05):482-489.[doi:10.3969/j.issn.1007-0478.2024.05.011]
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脑血管内皮细胞线粒体原位结构分析体系建立与应用()
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《卒中与神经疾病》杂志[ISSN:1007-0478/CN:42-1402/R]

卷:
第31卷
期数:
2024年05期
页码:
482-489
栏目:
论著
出版日期:
2024-10-20

文章信息/Info

文章编号:
1007-0478(2024)05-0482-08
作者:
王雪南希杜星月刘玉峰李霞张云峰
226000 南通大学特种医学研究院,南通大学附属医院卒中中心(王雪); 徕卡显微系统(上海)贸易有限公司(南希); 南通大学特种医学研究院[杜星月 李霞(通信作者)]; 南通市第二人民医院(刘玉峰); 南通大学附属医院卒中中心[张云峰(通信作者)]
关键词:
神经血管单元 线粒体 超微结构 聚焦离子束扫描电镜成像 机器学习
分类号:
R743.3
DOI:
10.3969/j.issn.1007-0478.2024.05.011
文献标志码:
A
摘要:
目的 旨在建立脑组织样本神经血管单元中内皮细胞线粒体快速可视化分割的方法和应用。方法 取假手术组纹状体和缺血再灌注组纹状体半暗区组织,采用锇-硫代碳酰肼-锇制样技术(Osmium-thiocarbohydrazide-osmium,OTO)包埋前块染色技术,结合聚焦离子束扫描电子显微镜(Focused ion beam scanning electron microscope,FIB/SEM)和人工智能可视化图像分析软件(Artificial intelligence-visualization-image analysis,AIVIA)机器学习图像分割技术,对神经血管单元中内皮细胞线粒体进行大尺度成像和快速分割分析。结果 本研究成功建立基于OTO-FIB/SEM-AIVIA(OFA)的脑组织块样本线粒体快速分割流程,实现微米级神经血管单元中特定细胞类型线粒体纳米级分辨率的快速可视化; 该流程可以提供脑组织样本线粒体数量和形态变化的准确信息; 同时,训练出的模型可以拓展应用于相同图像采集参数的不同样本数据,提高图像分割速度和数据处理效率; 基于这一方法,本研究发现相较于假手术组,脑缺血再灌注组纹状体半暗区内神经血管单元完整线粒体的平均体积变小且形态变圆; 本研究还进一步提取并三维重建了内皮细胞中的线粒体并发现其形态和数量的改变。结论 OFA流程可以快速获取神经血管单元或其中某个特定细胞类型,提供内皮细胞完整线粒体数量和体积的准确信息,为功能研究提供更准确全面的结构学证据。

参考文献/References:

[1] Iadecola C.The neurovascular unit coming of age:a journey through neurovascular coupling in health and disease[J].Neuron,2017,96(1):17-42. [2] Wang LY,Xiong AA,Zhang LY,et al.Neurovascular unit:a critical role in ischemic stroke[J].CNS Neurosci Ther,2021,27(1):7-16. [3] Obermeier B,Daneman R,Ransohoff RM.Development, maintenance and disruption of the blood-brain barrier[J].Nat Med,2013,19(12):1584-1596. [4] Sweeney MD,Zhao Z,Montagne A,et al.Blood-brain barrier:from physiology to disease and back[J].Physiol Rev,2019,99(1):21-78. [5] Ni XC,Wang HF,Cai YY,et al.Ginsenoside Rb1 inhibits astrocyte activation and promotes transfer of astrocytic mitochondria to neurons against ischemic stroke[J].Redox Biol,2022,54:102363. [6] Hayakawa K,Esposito E,Wang XH,et al.Transfer of mitochondria from astrocytes to neurons after stroke[J].Nature,2016,535(7613):551-555. [7] Lai YX,Lin PQ,Chen ML,et al.Restoration of L-OPA1 alleviates acute ischemic stroke injury in rats via inhibiting neuronal apoptosis and preserving mitochondrial function[J].Redox Biol,2020,34:101503. [8] Abdullahi W,Tripathi D,Ronaldson PT.Blood-brain barrier dysfunction in ischemic stroke: targeting tight junctions and transporters for vascular protection[J].Am J Physiol Cell Physiol,2018,315(3):C343-C356. [9] Abbott NJ,Patabendige AAK,Dolman DEM,et al.Structure and function of the blood-brain barrier[J].Neurobiol Dis,2010,37(1):13-25. [10] D'Souza A,Burch A,Dave KM,et al.Microvesicles transfer mitochondria and increase mitochondrial function in brain endothelial cells[J].J Control Release,2021,338:505-526. [11] Vangindertael J,Camacho R,Sempels W,et al.An introduction to optical super-resolution microscopy for the adventurous biologist[J].Methods Appl Fluoresc,2018,6(2):022003. [12] Brown TA,Tkachuk AN,Shtengel G,et al.Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction[J].Mol Cell Biol,2011,31(24):4994-5010. [13] Kizilyaprak C,Stierhof YD,Humbel BM.Volume microscopy in biology:FIB-SEM tomography[J].Tissue Cell,2019,57:123-128. [14] Laundon D,Katsamenis OL,Thompson J,et al.Correlative multiscale microCT-SBF-SEM imaging of resin-embedded tissue[J].Methods Cell Biol,2023,177:241-267. [15] Elizar E,Zulkifley MA,Muharar R,et al.A review on multiscale-deep-learning applications[J].Sensors(Basel),2022,22(19):7384. [16] Franke M,Bieber M,Kraft P,et al.The NLRP3 inflammasome drives inflammation in ischemia/reperfusion injury after transient middle cerebral artery occlusion in mice[J].Brain Behav Immun,2021,92:223-233. [17] Xu XM,Zhu LQ,Xue K,et al.Ultrastructural studies of the neurovascular unit reveal enhanced endothelial transcytosis in hyperglycemia-enhanced hemorrhagic transformation after stroke[J].CNS Neurosci Ther,2021,27(1):123-133. [18] Courson JA,Landry PT,Do T,et al.Serial block-face scanning electron microscopy(SBF-SEM)of biological tissue samples[J].J Vis Exp,2021(169):10.3791/62045. [19] Kizilyaprak C,Bittermann AG,Daraspe J,et al.FIB-SEM tomography in biology[J].Methods Mol Biol,2014,1117:541-558. [20] Cretoiu D.The third dimension of telocytes revealed by FIB-SEM tomography[J].Adv Exp Med Biol,2016,913:325-334. [21] Salvador E,Shityakov S,Förster C.Glucocorticoids and endothelial cell barrier function[J].Cell Tissue Res,2014,355(3):597-605. [22] Salmina AB,Kharitonova EV,Gorina YV,et al.Blood-brain barrier and neurovascular unit in vitro models for studying mitochondria-driven molecular mechanisms of neurodegeneration[J].Int J Mol Sci,2021,22(9):4661. [23] 张宝月,刘艾林,杜冠华.能量代谢紊乱对疾病的影响及其潜在药物靶点[J].药学学报,2019,54(8):1372-1381.

备注/Memo

备注/Memo:
基金项目:国家自然科学基金面上项目(81870941); 南通市社会民生面上项目(MS22022058); 南通市基础研究和民生科技计划指导性项目(JCZ2110T)
更新日期/Last Update: 2024-10-20