Chronic imaging of mitochondria in the murine cerebral vasculature using in vivo two-photon microscopy.
Chronic imaging of mitochondria in the murine cerebral vasculature using in vivo two-photon microscopy.
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使用体内双光子显微镜对小鼠脑血管系统中的线粒体进行慢性成像。
DOI：
10.1152/ajpheart.00751.2019
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Busija,DavidW
中科院分区：
文献类型：
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作者：
Rutkai,Ibolya;Evans,WesleyR;Bess,Nikita;Salter-Cid,Tomas;Čikić,Siniša;Chandra,ParthaK;Katakam,PrasadVG;Mostany,Ricardo;Busija,DavidW
线粒体是健康和疾病中脑血管功能的重要调节因子，但由于方法上的局限性，对其作用的理解进展受阻。我们报告了第一个在体内成像的线粒体特定的脑内皮细胞在真实的时间在同一小鼠的延长期。通过饲养PhAM-floxed和Tie 2-Cre小鼠产生在脑血管内皮中的线粒体（mito-Dendra 2）中表达Dendra 2荧光蛋白的小鼠。我们使用mito-Dendra 2表达，颅窗植入和双光子显微镜观察小鼠脑血管内皮细胞中的线粒体。免疫组织化学和线粒体染色用于证实线粒体信号定位于内皮细胞和mito-Dendra 2对线粒体的特异性。Mito-Dendra 2和罗丹明B结合的葡聚糖允许在同一小鼠中重复地同时测定线粒体密度、血管直径、面积和线粒体与血管的比例。在体外脑切片和主动脉上证实了mito-Dendra 2的内皮表达。此外，我们观察到一个重叠的mito-Dendra 2和Chromeo线粒体染色培养的脑微血管内皮细胞。对同一小鼠脑微循环中相同位置的重复成像证明了mito-Dendra 2的稳定性。虽然整体线粒体信号随时间推移是稳定的，但同一内皮细胞内的线粒体是移动的。总之，我们的研究结果表明，mito-Dendra 2信号和血管参数是适合于实时和纵向检查线粒体在体内的脑血管mice.NEW & NOTEWORTHWe介绍了一种创新的体内方法来研究线粒体在脑循环中的生理环境，通过展示长期成像和三维重建的可行性。我们推测，适当的组合Cre/Lox系统和双光子显微镜将有助于更好地了解线粒体的作用，不仅在内皮细胞，而且在不同的细胞类型的脑循环。
Mitochondria are important regulators of cerebral vascular function in health and disease, but progress in understanding their roles has been hindered by methodological limitations. We report the first in vivo imaging of mitochondria specific to the cerebral endothelium in real time in the same mouse for extended periods. Mice expressing Dendra2 fluorescent protein in mitochondria (mito-Dendra2) in the cerebral vascular endothelium were generated by breeding PhAM-floxed and Tie2-Cre mice. We used mito-Dendra2 expression, cranial window implantation, and two-photon microscopy to visualize mitochondria in the cerebral vascular endothelium of mice. Immunohistochemistry and mitochondrial staining were used to confirm the localization of the mitochondrial signal to endothelial cells and the specificity of mito-Dendra2 to mitochondria. Mito-Dendra2 and Rhodamine B-conjugated dextran allowed simultaneous determinations of mitochondrial density, vessel diameters, area, and mitochondria-to-vessel ratio in vivo, repeatedly, in the same mouse. Endothelial expression of mito-Dendra2 was confirmed in vitro on brain slices and aorta. In addition, we observed an overlapping mito-Dendra2 and Chromeo mitochondrial staining of cultured brain microvascular endothelial cells. Repeated imaging of the same location in the cerebral microcirculation in the same mouse demonstrated stability of mito-Dendra2. While the overall mitochondrial signal was stable over time, mitochondria within the same endothelial cell were mobile. In conclusion, our results indicate that the mito-Dendra2 signal and vascular parameters are suitable for real-time and longitudinal examination of mitochondria in vivo in the cerebral vasculature of mice.NEW & NOTEWORTHYWe introduce an innovative in vivo approach to study mitochondria in the cerebral circulation in their physiological environment by demonstrating the feasibility of long-term imaging and three-dimensional reconstruction. We postulate that the appropriate combination of Cre/Lox system and two-photon microscopy will contribute to a better understanding of the role of mitochondria in not only endothelium but also the different cell types of the cerebral circulation.