期刊
MICROCIRCULATION
卷 27, 期 7, 页码 -出版社
WILEY
DOI: 10.1111/micc.12643
关键词
electron microscopy; endothelial glycocalyx; high-pressure freezing; soft x-ray imaging
资金
- Fonden for Laegevidenskabens Fremme and Forskningsudvalget (Copenhagen University Hospital)
- Lundbeckfonden (Research Initiative in Brain Barrier Drug Delivery)
- Independent Research Fund Denmark (FSS) [6110-00554]
- EMBO [ALTF 461-2013]
- Carlsbergfonden [CF15-0644]
Objective The endothelial glycocalyx covers the luminal surface of the endothelium and plays key roles in vascular function. Despite its biological importance, ideal visualization techniques are lacking. The current study aimed to improve the preservation and subsequent imaging quality of the endothelial glycocalyx. Methods In mice, the endothelial glycocalyx was contrasted with a mixture of lanthanum and dysprosium (LaDy). Standard chemical fixation was compared with high-pressure frozen specimens processed with freeze substitution. Also, isolated brain microvessels and cultured endothelial cells were high-pressure frozen and by transmission soft x-rays, imaged under cryogenic conditions. Results The endothelial glycocalyx was in some tissues significantly more voluminous from chemically fixed specimens compared with high-pressure frozen specimens. LaDy labeling introduced excessive absorption contrast, which impeded glycocalyx measurements in isolated brain microvessels when using transmission soft x-rays. In non-contrasted vessels, the glycocalyx was not resolved. LaDy-contrasted, cultured brain endothelial cells allowed to assess glycocalyx volume in vitro. Conclusions Both chemical and cryogenic fixation followed by dehydration lead to substantial collapse of the glycocalyx. Cryogenic fixation without freeze substitution could be a way forward although transmission soft x-ray tomography based solely on amplitude contrast seems unsuitable.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据