血管内皮细胞糖萼结构状态与脓毒症小鼠病理状态相关：使用电镜和活体荧光显微技术分析糖萼结构和功能的综合方法
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Structural Behavior of the Endothelial Glycocalyx Is Associated With Pathophysiologic Status in Septic Mice: An Integrated Approach to Analyzing the Behavior and Function of the Glycocalyx Using Both Electron and Fluorescence Intravital Microscopy
背景与目的
内皮表层（ESL）调节血管通透性维持血流动力学稳定。糖萼（GCX）是一种复杂而脆弱的超微结构，是内皮表层（ESL）的重要组成部分。糖萼（GCX）的异常被认为可触发病理性渗透增加。在本实验中，我们对重要器官中糖萼（GCX）的形态和功能特性进行综合分析。
方 法
我们使用电镜（EM）和活体显微镜检（IVM）分析了ESL和GCX的结构。我们还比较了糖苷酶处理组和对照组小鼠皮肤ESL的形态学变化。使用综合方法分析了脂多糖诱导脓毒症小鼠内皮的形态和功能、白细胞-内皮相互作用的病理生理特征、血管通透性。
结 果
我们使用活体镜检将ESL的明亮部分确定为GCX，并使用形态学和生化方法证实了我们的观察。我们发现在脓毒症小鼠中，EM成像分析（0.98±2.08nm对70.68±36.36nm，P <0.001）和IVM成像分析（0.36±0.15μm对1.07±1.0μm）中，GCX比非脓毒症对照组更薄0.39μm，P <0.001）。与对照组相比（低于检测值下限，P<0.001），在脓毒症条件下，syndecan-1作为GCX代表性的核心蛋白以较高的速率释放到血清中（7.33±3.46ng / mL）。白细胞-内皮相互作用显著增多被定义为白细胞流动或黏附增多，在体内糖萼（GCX）脱落后也观察到组织间隙超渗透。
结 论
使用IVM，我们可看见ESL的明亮部分，随后使用EM确认为糖萼（GCX）。严重败血症引起糖萼（GCX）降解，伴随着syndecan-1核心蛋白的脱落和白细胞-内皮相互作用的增加影响血管通透性。我们在在体模型描述了一种解释糖萼（GCX）结构与功能关系的新方法。
原始文献摘要
Kataoka H, Ushiyama A, Akimoto Y, et al. Structural Behavior of the Endothelial Glycocalyx Is Associated With Pathophysiologic Status in Septic Mice: An Integrated Approach to Analyzing the Behavior and Function of the Glycocalyx Using Both Electron and Fluorescence Intravital Microscopy.[J]. Anesthesia & Analgesia, 2017, 125(3):874-883.
BACKGROUND: The endothelial surface layer (ESL) regulates vascular permeability to maintain fluid homeostasis. The glycocalyx (GCX), which has a complex and fragile ultrastructure, is an important component of the ESL. Abnormalities of the GCX have been hypothesized to trigger pathological hyperpermeability. Here, we report an integrated in vivo analysis of the morphological and functional properties of the GCX in a vital organ.
METHODS: We examined the behavior of the ESL and GCX, using both electron microscopy (EM) and intravital microscopy (IVM). We also compared morphological changes in the ESL of mouse skin in a glycosidase-treated and control group. Combined approaches were also used to examine both morphology and function in a lipopolysaccharide-induced septic model and the pathophysiological features of leukocyte–endothelial interactions and in vivo vascular permeability.
RESULTS: Using IVM, we identified an illuminated part of the ESL as the GCX and confirmed our observation using morphological and biochemical means. In septic mice, we found that the GCX was thinner than in nonseptic controls in both an EM image analysis (0.98 ± 2.08 nm vs 70.68 ± 36.36 nm, P < .001) and an IVM image analysis (0.36 ± 0.15 μm vs 1.07 ± 0.39 μm, P < .001). Under septic conditions, syndecan-1, a representative core protein of the GCX, was released into the blood serum at a higher rate in septic animals (7.33 ± 3.46 ng/mL) when compared with controls (below the limit of detection, P < .001). Signifcant increases in leukocyte–endothelial interactions, defned as the numbers of rolling or firm-sticking leukocytes, and molecular hyperpermeability to the interstitium were also observed after GCX shedding in vivo.
CONCLUSIONS: Using IVM, we visualized an illuminated part of the ESL layer that was subsequently confirmed as the GCX using EM. Severe sepsis induced morphological degradation of the GCX, accompanied by shedding of the syndecan-1 core protein and an increase in leukocyte–endothelial interactions affecting the vascular permeability. Our in vivo model describes a new approach to deciphering the relationship between structural and functional behaviors of the GCX.
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