Shear stress, a mechanical force created by blood flow, is known to affect the developing cardiovascular system. Shear stress is a function of both shear rate and viscosity. While established techniques for measuring shear rate in embryos have been developed, the viscosity of embryonic blood has never been known but always assumed to be like adult blood. Blood is a non-Newtonian fluid, where the relationship between shear rate and shear stress is nonlinear. In this work, we analyzed the nonNewtonian behavior of embryonic chicken blood using a microviscometer and present the apparent viscosity at different hematocrits, different shear rates, and at different stages during development from 4 days (Hamburger-Hamilton stage 22) to 8 days (about HamburgerHamilton stage 34) of incubation. We chose the chicken embryo since it has become a common animal model for studying hemodynamics in the developing cardiovascular system. We found that the hematocrit increases with the stage of development. The viscosity of embryonic avian blood in all developmental stages studied was shear rate dependent and behaved in a non-Newtonian manner similar to that of adult blood. The range of shear rates and hematocrits at which nonNewtonian behavior was observed is, however, outside the physiological range for the larger vessels of the embryo. Under low shear stress conditions, the spherical nucleated blood cells that make up embryonic blood formed into small aggregates of cells. We found that the apparent blood viscosity decreases at a given hematocrit during embryonic development, not due to changes in protein composition of the plasma but possibly due to the changes in cellular composition of embryonic blood. This decrease in apparent viscosity was only visible at high hematocrit. At physiological values of hematocrit, embryonic blood viscosity did not change significantly with the stage of development. microelectromechanical systems; hematocrit; shear rate; shear stress; hemodynamic; rouleaux; vascular development HEMODYNAMICS, or blood fluid dynamics, are important not only for cardiovascular function but also for the development of the cardiovascular system. Blood flow creates a force called shear stress. Chronic changes in shear stress levels lead to a remodeling of the vasculature that normalizes the level of shear stress in the adult (20). Shear stress has also been found to be important during cardiovascular development, affecting heart formation (17), vascular remodeling (25, 36), arterial-venous differentiation (21), and hematopoiesis by the vascular endothelium (1, 29). For these reasons, there has been a significant effort in recent years to measure the shear stress levels during early embryonic development and link specific flow patterns or levels of shear stress to events in vascular development.Shear stress is a function of the shear rate and the viscosity of the fluid. The development of flow visualization techniques with micrometer-scale resolution, such as Doppler optical coherence tomography (12) and microparticle ima...

The glycocalyx, and the thicker endothelial surface layer (ESL), are necessary both for endothelial barrier function and for sensing mechanical forces in the adult. The goal of this study is to use a combination of imaging techniques to establish when the glycocalyx and endothelial surface layer form during embryonic development and to determine the biological significance of the glycocalyx layer during vascular development in quail embryos. Using transmission electron microscopy, we show that the glycocalyx layer is present as soon as blood flow starts (14 somites). The early endothelial glycocalyx (14 somites) lacks the distinct hair-like morphology that is present later in development (17 and 25 somites). The average thickness does not change significantly (14 somites, 182 nm ± 33 nm; 17 somites, 218 ± 30 nm; 25 somites, 212 ± 32 nm). The trapping of circulating fluorescent albumin was used to evaluate the development of the ESL. Trapped fluorescent albumin was first observed at 25 somites. In order to assess a functional role for the glycocalyx during development, we selectively degraded luminal glycosaminoglycans. Degradation of hyaluronan compromised endothelial barrier function and prevented vascular remodeling. Degradation of heparan sulfate down regulated the expression of shear-sensitive genes but does not inhibit vascular remodeling. Our findings show that the glycocalyx layer is present as soon as blood flow starts (14 somites). Selective degradations of major glycocalyx components were shown to inhibit normal vascular development, examined through morphology, vascular barrier function, and gene expression.

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Endothelial cells have the capacity to sense hemodynamic forces through the process of mechanotransduction. When blood flow starts in the embryo, many of the components required for sensing hemodynamic forces in the adult have not yet formed. We recently showed that hemodynamic forces were required for normal vascular development. In this work, we have been focusing on the development of a mechano‐sensory complex by studying the development of the glycocalyx. The glycocalyx forms extensions into the vascular lumen that are anchored directly to actin stress fibres of the cytoskeleton and transmit hemodynamic signals. By electron microscopy and immunohistochemical staining, we show that the glycocalyx is present as soon as blood flow starts. We then used selective enzymatic degradation to look at whether the glycocalyx has a functional role during vascular development. We found that during development, hyaluronan, but not other glycocalyx components, affected vascular permeability. We also found that degradation of heparan sulfate resulted in decreased expression of a subset of mechanotransduced genes.