Name: a novel ex ovo banding technique to alter intracardiac hemodynamics in an embryonic chicken system
Uploaded: 2016-05-13T13:00:00.000+00:00
Duration: 8 min 9 s
Description: Watch this Scientific Journal Video about A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System at JoVE.com

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)

Avian embryos are not considered vertebrate animals under IUCAC regulations.
1. Obtaining Embryos for Surgery
<ol>
	<li>Incubate 80 - 90 fertilized Bovan chicken eggs (blunt end up) in a humidified (60%) rocker incubator at 40 &#176;C for approximately 72 hr to obtain embryos at Hamilton and Hamburger (HH) stage 17. Determine exact number of eggs based on power analysis and survival rate of embryos 17. Use plastic egg trays for incubation to allow adequate air circulation.</li>
	<li>Once this desired developmental stage is attained, pour approximately 20 ml of warm (37 &#176;C) Tyrode&#39;s buffer (supplemented with sodium bicarbonate (1 g/L)) into a 100 mm x 26 mm petri dish.</li>
	<li>Sterilize an egg shell with 70% ethanol.</li>
	<li>Gently crack the shell with a scalpel handle and carefully release its contents into the dish containing Tyrode&#39;s buffer. Discard embryos if (i) they visually appear abnormal (ii) they are not at the right developmental stage (iii) they are improperly oriented on the yolk and/or (iv) any bleeding occurs. Retain any embryos not subjected to surgery immediately after being placed ex ovo at 40 &#176;C so as to not compromise development. 
	Note: Staging is performed based on Hamilton and Hamburger 18. Abnormality of chick embryos is determined by the naked eye and under the dissection scope. Ensure that the embryo has appropriate folding and bending and vasculature.</li>
</ol>
2. OFT Banding
<ol>
	<li>Tweeze out individual 1 cm long threads from a single 11/0 nylon surgical suture and form a loose knot. UV sterilize all preformed knots.</li>
	<li>Under a dissection microscope, visually ensure that the heart is beating at a normal rate (~ 120 beats/min). If not, discard the embryo.</li>
	<li>Just before surgery, apply 5 - 6 ml of warm (37 &#176;C) Tyrode&#39;s buffer to the embryo surface using a transfer pipette.</li>
	<li>Pass one free end of the preformed knot under the OFT, position the suture at the OFT/ventricle junction (OVJ) (or desired location), and pass the free end into the knot thereby constricting the OFT including the membranes surrounding the heart.</li>
	<li>After surgery, wet the surface of the yolk with 5 - 6 ml of warm (37 &#176;C) Tyrode&#39;s buffer using a transfer pipette.</li>
	<li>Maintain control embryos ex ovo as the banded embryos; however do not subject them to the surgical procedure.</li>
	<li>Incubate the embryos, for the desired amount of time, at 40 &#176;C in a humidified (60%) incubator.</li>
	<li>Once the desired time point is reached, excise the embryo from the yolk using straight scissors. Dissect the heart with fine forceps and use for several downstream studies such as changes in cardiac morphology due to altered hemodynamics 17.</li>
</ol>
3. Confirming that the Banding Intervention Causes a Change in Hemodynamics
Note: The partial constriction caused by the banding intervention leads to an increase in blood flow velocity at the OVJ. This hemodynamic parameter is conveniently assessed using 2D ultrasound imaging, which is performed at the desired time point of the experiment.
<ol>
	<li>Since only a single embryo can be imaged at a time, maintain all other embryos designated for ultrasound imaging in a 40 &#176;C incubator.</li>
	<li>Place the petri dish containing the embryo to be imaged on a heating pad set at 40 &#176;C.</li>
	<li>Fill the dish, up to the brim, with warm (37 &#176;C) Tyrode&#39;s buffer. Slowly pour the buffer solution into the dish so as to keep the yolk intact. If, however, the integrity of the yolk is compromised during this step, discard the embryo.</li>
	<li>Orient the embryo such that the central axis of the embryo is perpendicular to the ultrasound probe which is suspended from an adjustable stand.</li>
	<li>With the ultrasound machine operating in B-mode, obtain a 2D image of the beating heart on the screen and move the stage (heating pad) such that the OFT, ventricle and OVJ are clearly visible.</li>
	<li>Switch to the pulsed-wave (PW) mode, at the desired pulse repetition frequency (e.g., 20 kHz) and obtain velocity data exactly at the OVJ. A B-mode image of the beating heart is obtained on the screen.
	<ol>
		<li>Move the stage to see the OFT, ventricle and the OVJ. Using the ultrasound machine software, obtain velocity measurements exactly at the OVJ.</li>
	</ol>
	</li>
</ol>
Note: If the heart rate of an embryo decreases during imaging, velocity data thus obtained should not be used for analysis. All embryos used for velocity measurements should preferably not be used for any other experiments after ultrasound imaging.

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The authors have nothing to disclose.

This technique is relatively quick and easy to perform, however certain key points need to be kept in mind so as to get accurate downstream results. Embryos should be maintained ex ovo in a petri dish that contains Tyrode&#39;s buffer to provide adequate rehydration. It is also important to hydrate the yolk post-surgery with Tyrode&#39;s buffer and to make sure the incubation chamber is adequately hydrated. Surgeries should not be performed on an embryo if any bleeding is visible or the yolk is even slightly bro...

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.

<table><tbody><tr><td>Fertilized Bovan chicken eggs</td><td>Clemson University, Clemson, SC</td><td></td><td></td></tr><tr><td>11 / 0 Nylon suture</td><td>Ashaway</td><td>S30001</td><td>UV sterilize knots before surgery</td></tr><tr><td>100 x 26 mm petri dish</td><td>VWR</td><td>25387-030</td><td></td></tr><tr><td>Transfer pipettes</td><td>Thermo Scientific&amp;nbsp;</td><td>232-20S</td><td></td></tr><tr><td>Scalpel handle #3</td><td>Fine Science Tools</td><td>91003-12</td><td></td></tr><tr><td>Straight scissor</td><td>Roboz</td><td>RS-6702</td><td></td></tr><tr><td>Dumont #5 fine forceps</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>Tyrodes buffer</td><td>Sigma-Aldrich</td><td>2145-10L</td><td>Filter sterlize before use&amp;nbsp;</td></tr><tr><td>Sodium bicarbonate</td><td>Fisher Scientific</td><td>S233-500</td><td></td></tr><tr><td>Vevo 770 Ultrasound Imaging system</td><td>VisualSonics, Inc.</td><td>VS-11392</td><td></td></tr><tr><td>708 Ultrasound transducer&amp;nbsp;</td><td>VisualSonics, Inc.</td><td>VS-11171</td><td></td></tr></tbody></table>

a novel ex ovo banding technique to alter intracardiac hemodynamics in an embryonic chicken system

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.

Shown in Figure 1 are recommended instruments needed for OFT banding. The petri dish (Figure 1A) containing the embryo ex ovo should be deep enough so as to not destroy the embryo when covered with the lid. Deep petri dishes (Figure 1C) should also be used for ultrasound imaging to allow an adequate volume of Tyrode&#39;s buffer to be poured atop the yolk.
A single 1 cm...

Watch this Scientific Journal Video about A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System at JoVE.com

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

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).

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

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6.5K Views.  School of Medicine, University of South Carolina. 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). 

Video: A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

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Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

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Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Biology

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

Avian Embryonic Left Atrial Ligation Model for HLHS and Cardiovascular Research

Left Atrial Ligation in the Avian Embryo as a Model for Altered Hemodynamic Loading During Early Vascular Development

Due to its four-chambered mature ventricular configuration, ease of culture, imaging access, and efficiency, the avian embryo is a preferred vertebrate animal model for studying cardiovascular development. Studies aiming to understand the normal development and congenital heart defect prognosis widely adopt this model. Microscopic surgical techniques are introduced to alter the normal mechanical loading patterns at a specific embryonic time point and track the downstream molecular and genetic cascade. The most common mechanical interventions are left vitelline vein ligation, conotruncal banding, and left atrial ligation (LAL), modulating the intramural vascular pressure and wall shear stress due to blood flow. LAL, particularly if performed in ovo, is the most challenging intervention, with very small sample yields due to the extremely fine sequential microsurgical operations. Despite its high risk, in ovo LAL is very valuable scientifically as it mimics hypoplastic left heart syndrome (HLHS) pathogenesis. HLHS is a clinically relevant, complex congenital heart disease observed in human newborns. A detailed protocol for in ovo LAL is documented in this paper. Briefly, fertilized avian embryos were incubated at 37.5 &#176;C and 60% constant humidity typically until they reached Hamburger-Hamilton (HH) stages 20 to 21. The egg shells were cracked open, and the outer and inner membranes were removed. The embryo was gently rotated to expose the left atrial bulb of the common atrium. Pre-assembled micro-knots from 10-0 nylon sutures were gently positioned and tied around the left atrial bud. Finally, the embryo was returned to its original position, and LAL was completed. Normal and LAL-instrumented ventricles demonstrated statistically significant differences in tissue compaction. An efficient LAL model generation pipeline would contribute to studies focusing on synchronized mechanical and genetic manipulation during the embryonic development of cardiovascular components. Likewise, this model will provide a perturbed cell source for tissue culture research and vascular biology.

Author Spotlight: Effect of Left Atrial Ligation on Avian Embryonic Hearts and HLHS Implications 

Here, we present a detailed visual protocol for executing the left atrial ligation (LAL) model in the avian embryo. The LAL model alters the intracardiac flow, which changes wall shear stress loading, mimicking hypoplastic left heart syndrome. An approach to overcome the challenges of this difficult microsurgery model is presented.

Due to its four-chambered mature ventricular configuration, ease of culture, imaging access, and efficiency, the avian embryo is a preferred vertebrate ...

Generation of Hook Ischemia-Reperfusion Model using a Three-Day Developing Chick Embryo

Ischemia and reperfusion (I/R) disorders, such as myocardial infarction, stroke, and peripheral vascular disease, are a few of the leading causes of illness and death. Many in vitro and in vivo models are currently available for studying the I/R mechanism in disease or damaged tissues. However, to date, no in ovo I/R model has been reported, which would allow for a better understanding of I/R mechanisms and faster drug screening. This paper describes I/R modeling using a spinal needle customized hook in a 3-day chick embryo to understand I/R development and treatment mechanisms. Our model can be used to investigate anomalies at the DNA, RNA, and protein levels. This method is simple, quick, and inexpensive. The current model can be used independently or in conjunction with existing in vitro and in vivo I/R models.

This paper describes ischemia-reperfusion (I/R) modeling in a 3-day chick embryo using a spinal needle customized hook to better understand I/R development and treatment. This model is simple, quick, and inexpensive.

Ischemia and reperfusion (I/R) disorders, such as myocardial infarction, stroke, and peripheral vascular disease, are a few of the leading causes of ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

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An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In Ovo Intravascular Injection in Chicken Embryos

Left Atrial Ligation in the Avian Embryo as a Model for Altered Hemodynamic Loading During Early Vascular Development

Generation of Hook Ischemia-Reperfusion Model using a Three-Day Developing Chick Embryo