For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).
The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.
Abnormally formed outflow valves are the most common type of congenital heart defects 1. However, defective cardiac valve structure and function, even though present at birth, may become symptomatic only in adulthood. In fact, several adult valve diseases can be attributed to a congenital origin. Treatment of such patients often involves replacing defective valves, and, importantly, replaced aortic valves have been shown to have congenital anomalies 2. Given the fact that critical processes involved in valve development begin early during embryogenesis, the importance of better understanding the mechanisms that regulate these events is highlighted.
The primitive heart tube, which is the first functioning organ in an embryo, exhibits two distinct layers – an endothelial endocardium surrounded by myocardium – separated by extracellular matrix (cardiac jelly) which is mostly produced and secreted by the myocardium 3-5. As development continues, valve primordia (endocardial cushions) are formed, after rightward looping of the embryonic heart, by local expansion of the cardiac jelly at the atrioventricular (AV) canal and the outflow tract (OFT) 4,6. This expansion is mediated by the highly regulated process of epithelial-mesenchymal transition (EMT), during which the cardiac jelly becomes populated by endocardially-derived mesenchymal cells 6. In addition to the mesenchymal population derived through EMT, neural crest cells are also involved in valvulogenesis of the OFT 3.
Hemodynamic stimuli, such as shear stress, are important epigenetic factors that regulate heart development in the embryo 7,8. Using a 3D in vitro system, we have previously shown shear stress to be a factor influencing the expression and deposition of fibrous extracellular matrix (ECM) proteins in AV and OFT cushions 9,10. Moreover, studies carried out by several researchers have demonstrated that altered blood flow leads to improper valves and septa formation 11-16. Recently, using the novel banding procedure presented here, we have shown that changing hemodynamics in the embryonic chick heart affects the early processes involved in OFT valve formation 17.
The technique described here provides a novel model for altering hemodynamics in the developing chick heart by partially constricting the OFT ex ovo. This reproducible procedure is relatively quick and allows researchers to obtain a sufficient number of embryos/whole hearts/OFT tissue, etc. for downstream analyses including gene expression studies. Moreover, this new model can be used to study ‘chronic’ effects of altered hemodynamics on OFT valve development.
这种技术是相对快速和容易执行,然而某些关键点需要牢记以便获得准确的下游结果。胚胎应保持前卵在包含蒂罗德缓冲以提供足够的补液的培养皿。同样重要的是水合蛋黄手术后与台氏缓冲液,以确保孵育室充分水合。手术不应该在胚胎如有出血可见或蛋黄甚至略有破损进行。这将影响胚胎存活率,特别是对于长期的实验。杀伤力的胚胎另一个潜在原因,是各地OFT乐队是否太紧时首次应?…
The authors have nothing to disclose.
The authors would like to acknowledge Dr. Robert Price and the staff of the Instrumentation Resource Facility at the University of South Carolina School of Medicine. This work was partially supported by a SPARC Graduate Research Grant from the Office of the Vice President for Research at the University of South Carolina (JDP/VM). In addition this work was supported by Cook Biotech research agreement (JDP) and by FirstString Research Inc (JDP) and NIH 2 P20-RR016434-06 (JDP). In addition, NIH IDeA Networks of Biomedical Research Excellence (INBRE) grant for South Carolina P20GM103499 (JE)
Fertilized Bovan chicken eggs | Clemson University, Clemson, SC | ||
11/0 Nylon suture | Ashaway | S30001 | UV sterilize knots before surgery |
100 x 26 mm petri dish | VWR | 25387-030 | |
Transfer pipettes | Thermo Scientific | 232-20S | |
Scalpel handle #3 | Fine Science Tools | 91003-12 | |
Straight scissor | Roboz | RS-6702 | |
Dumont #5 fine forceps | Fine Science Tools | 11254-20 | |
Tyrodes buffer | Sigma-Aldrich | 2145-10L | Filter sterlize before use |
Sodium bicarbonate | Fisher Scientific | S233-500 | |
Vevo 770 Ultrasound Imaging system | VisualSonics, Inc. | VS-11392 | |
708 Ultrasound transducer | VisualSonics, Inc. | VS-11171 |