271 Imaging the placental glycocalyx

Abstract

Introduction Glycocalyx is a gel-like mesh which lines the luminal surface of vascular endothelium and has an important role in vessel function and permeability. Disruption of glycocalyx has been implicated in the endothelial dysfunction in pre-eclampsia. There is increasing evidence for a significant glycocalyx present at the maternal-fetal interface of the human placenta. Due to the unstable nature of glycocalyx ex-vivo it has previously been difficult to demonstrate and study. Objectives To confirm the presence of the placental glycocalyx of the fetal capillary endothelium and syncytiotrophoblast and to develop a reliable imaging protocol to observe this ex-vivo. Methods Term placentae were collected from uncomplicated pregnancies at caesarean section and processed in one of two ways. 1) Electron Microscopy: placental glycocalyx was labelled using perfusion or immersion with Alcian Blue in glutaraldehyde. Samples were post fixed, embedded and sectioned for imaging with transmission electron microscopy (TEM). Glycocalyx depth was measured perpendicular to the phospholipid bilayer. 2) Lectin Staining. Biopsies were fixed in 4% paraformaldehyde, paraffin embedded and sectioned. Glycocalyx was labelled with FITC-conjugated wheat germ agglutinin (WGA) and membrane labelled with octadecyl rhodamine B chloride (R18) and imaged using confocal microscopy. The distance between the peak signals of FITC-WGA and R18 was used to measure glycocalyx depth. Results presented as mean glycocalyx thickness ±SEM. Results TEM demonstrated glycocalyx depth of 57.1 ± 5.8 nm at the fetal capillary and 62.2 ± 3.7 nm at the syncytiotrophoblast. Glycocalyx depth using lectin staining was significantly greater 279.3 ± 42.6 nm and 893.7 ± 150.1 nm at the capillary and syncytiotrophoblast respectively. Conclusion The placenta maintains a significant glycocalyx at the syncytiotrophoblast and fetal capillary endothelium in term uncomplicated pregnancy. The absolute depth of glycocalyx varied depending on the imaging modality used. Correlative fluorescence and electron microscopy techniques would help further define this.