Abstract
Mainly constituted of glycosaminoglycans and proteoglycans, the glycocalyx is anchored in the plasma membrane, covering, in particular, the extracellular face of the arterial endothelium. Due to its complex three-dimensional (3D) architecture, the glycocalyx interacts with a wide variety of proteins, contributing to vascular permeability, the flow of mechanotransduction, and the modulation of local inflammatory processes. Alterations of glycocalyx structure mediate the endothelial dysfunction and contribute to the aggravation of peripheral vascular diseases. Therefore, the exploration of its ultrastructure becomes a priority to evaluate the degree of injury under physiopathological conditions and to assess the impact of therapeutic approaches. The objective of this study was to develop innovative approaches in electron microscopy to visualize the glycocalyx at the subcellular scale. Intravenous perfusion on rats with a fixing solution containing aldehyde fixatives enriched with lanthanum ions was performed to prepare arterial samples. The addition of lanthanum nitrate in the fixing solution allowed the enhancement of the staining of the glycocalyx for transmission electron microscopy (TEM) and to detect elastic and inelastic scattered electrons, providing complementary qualitative information. The strength of scanning electron microscopy (SEM) was used on resin-embedded serial sections, allowing rapid and efficient large field imaging and previous correlative TEM observations for ultrastructural fine details. To demonstrate the dynamic feature of the glycocalyx, 3D tomography was provided by dual-beam focus-ion-beam-SEM (FIB-SEM). These approaches allowed us to visualize and characterize the ultrastructure of the pulmonary artery glycocalyx under physiological conditions and in a rat pulmonary ischemia-reperfusion model, known to induce endothelial dysfunction. This study demonstrates the feasibility of combined SEM, TEM, and FIB-SEM tomography approaches on the same sample as the multiscale visualization and the identification of structural indicators of arterial endothelial glycocalyx integrity.
Highlights
In recent years, the endothelial glycocalyx gained interest since many studies proved its fundamental role in the maintenance of vascular physiology and endothelial response to pathophysiological events
Higher resolution images are provided by transmission electron microscopy (TEM) observations (Figures 3C–E) supporting a well-developed filamentous glycocalyx emerging from the endothelium to the luminal arterial space, and present on red blood cells (Figure 3D) and platelet surfaces (Figure 3E)
Another example consisting of STEM-energy loss spectroscopy (EELS) application proved its usefulness in quantitatively evaluating the subcellular distribution of lanthanum
Summary
The endothelial glycocalyx gained interest since many studies proved its fundamental role in the maintenance of vascular physiology and endothelial response to pathophysiological events. The objective of this study was to propose innovative approaches in EM to visualize the vascular glycocalyx at the subcellular level and to use these approaches to assess the impact of a pathological condition known to profoundly alter vascular function and structure, i.e., ischemiareperfusion (IR) associated with cardiopulmonary bypass (CPB) which go along with lung transplantation procedures. For this purpose, we develop a workflow based on a single sample preparation protocol applied to SEM observations, (S)TEM chemical analysis, and tomography FIB3D. To demonstrate the dynamic feature of the glycocalyx, we provided 3D tomography by dual-beam focus-ion-beam-SEM (FIB-SEM) based on the principle of “slice and view.” This study presents the feasibility of combined SEM, TEM, and FIB-SEM tomography approaches for visualizing the vascular glycocalyx on the same artery sample
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