Simultaneous imaging of blood flow dynamics and vascular remodelling during development

Abstract

Normal vascular development requires blood flow. Time-lapse imaging techniques have revolutionised our understanding of developmental biology, but measuring changes in blood flow dynamics has met with limited success. Ultrasound biomicroscopy and optical coherence tomography can concurrently image vascular structure and blood flow velocity, but these techniques lack the resolution to accurately calculate fluid forces such as shear stress. This is important because hemodynamic forces are biologically active and induce changes in the expression of genes important for vascular development. Regional variations in shear stress, rather than the overall level, control processes such as vessel enlargement and regression during vascular remodelling. We present a technique to concurrently visualise vascular remodelling and blood flow dynamics. We use an avian embryonic model and inject an endothelial-specific dye and fluorescent microspheres. The motion of the microspheres is captured with a high-speed camera and the velocity of the blood flow in and out of the region of interest is quantified by micro-particle image velocitymetry (µPIV). The vessel geometry and flow are used to numerically solve the flow physics with computational fluid dynamics (CFD). Using this technique, we can analyse changes in shear stress, pressure drops and blood flow velocities over a period of 10 to 16 h. We apply this to study the relationship between shear stress and chronic changes in vessel diameter during embryonic development, both in normal development and after TGFβ stimulation. This technique allows us to study the interaction of biomolecular and biomechanical signals during vascular remodelling using an in vivo developmental model.

Cell behaviors and dynamics during angiogenesis

“…Alternative models for angiogenesis are quail and chicken embryos because they develop ex utero (Ribatti et al, 1996;Ghaffari et al, 2015). Recently, transgenic quails expressing enhanced green fluorescent protein (EGFP) in ECs have been described (Sato, 2013;Sato et al, 2010) and they have helped researchers to study the sprouting behavior of ECs during the formation of the dorsal aortae in detail.…”

Flow dynamics control the location of sprouting and direct elongation during developmental angiogenesis

“…To study the role that flow dynamics play in the process of angiogenesis, we used a technique to simultaneously image vessel morphology and flow dynamics by time-lapse microscopy in the capillary plexus of avian embryos undergoing vascular remodelling (Ghaffari, et al, 2015). We injected Alexa Fluor 488-labelled acetylated low-density lipoprotein (AF488-AcLDL), which specifically labels endothelial cells and macrophages (Brown et al, 1980;Hallmann et al, 1987).…”

“…A detailed description of the methods was previously presented (Ghaffari, et al, 2015). Fertilised quail eggs (Cortunix japonica) were incubated at 37°C and ∼60% humidity until they reached a developmental stage of 12-14 somites or Hamburger Hamilton 11 (HH11).…”

“…A median filter was applied to the flattened image and then the image was thresholded to obtain a binary image used as the 'image mask' (for additional details, see Ghaffari et al, 2015). The image mask was applied to each individual frame of the microsphere motion.…”

“…The unsteady form of the momentum equations was used for flow inside the vessels to take into account the effects of blood flow pulsatility. The viscosity of blood was adjusted for embryonic stage, vessel diameter and shear rate as previously described (Ghaffari, et al, 2015). From N inlets and outlets, the flow rates at N−1 points were set as boundary conditions.…”