Name: A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System (Video) | JoVE Journal
Uploaded: 2016-05-13T13:00:00.000+00:00
Duration: 8 min 9 s
Description: Learn a reproducible ex ovo banding technique to alter cardiac hemodynamics in developing chicken embryos. This method enables study of genetic pathways responding to hemodynamic changes in cardiovascular development.

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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.

<ol><li>Neeb, Z., Lajiness, J., Bolanis, E., Conway, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+outflow+tract+anomalies%5BTitle%5D)+AND+%22Wiley+Interdiscip+Rev+Dev+Biol%22%5BJournal%5D)">Cardiac outflow tract anomalies.</a> Wiley Interdiscip Rev Dev Biol. 2 (4), 499-530 (2013).</li>
<li>Combs, M., Yutzey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+valve+development%3A+regulatory+networks+in+development+and+disease%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Heart valve development: regulatory networks in development and disease.</a> Circ Res. 105 (5), 408-421 (2009).</li>
<li>Hinton, R., Yutzey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+valve+structure+and+function+in+development+and+disease%5BTitle%5D)+AND+%22Annu+Rev+Physiol%22%5BJournal%5D)">Heart valve structure and function in development and disease.</a> Annu Rev Physiol. 73, 29-46 (2011).</li>
<li>von Gise, A., Pu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endocardial+and+epicardial+epithelial+to+mesenchymal+transitions+in+heart+development+and+disease%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Endocardial and epicardial epithelial to mesenchymal transitions in heart development and disease.</a> Circ Res. 110 (12), 1628-1645 (2012).</li>
<li>Person, A., Klewer, S., Runyan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+biology+of+cardiac+cushion+development%5BTitle%5D)+AND+%22Int+Rev+Cytol%22%5BJournal%5D)">Cell biology of cardiac cushion development.</a> Int Rev Cytol. 243, 287-335 (2005).</li>
<li>de Vlaming, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atrioventricular+valve+development%3A+new+perspectives+on+an+old+theme%5BTitle%5D)+AND+%22Differentiation%22%5BJournal%5D)">Atrioventricular valve development: new perspectives on an old theme.</a> Differentiation. 84 (1), 103-116 (2012).</li>
<li>Butcher, J., McQuinn, T., Sedmera, D., Turner, D., Markwald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transitions+in+early+embryonic+atrioventricular+valvular+function+correspond+with+changes+in+cushion+biomechanics+that+are+predictable+by+tissue+composition%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Transitions in early embryonic atrioventricular valvular function correspond with changes in cushion biomechanics that are predictable by tissue composition.</a> Circ Res. 100 (10), 1503-1511 (2007).</li>
<li>Hove, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracardiac+fluid+forces+are+an+essential+epigenetic+factor+for+embryonic+cardiogenesis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis.</a> Nature. 421 (6919), 172-177 (2003).</li>
<li>Tan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluid+flow+forces+and+rhoA+regulate+fibrous+development+of+the+atrioventricular+valves%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Fluid flow forces and rhoA regulate fibrous development of the atrioventricular valves.</a> Dev Biol. 374 (2), 345-356 (2013).</li>
<li>Biechler, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+impact+of+flow-induced+forces+on+the+morphogenesis+of+the+outflow+tract%5BTitle%5D)+AND+%22Front+Physiol%22%5BJournal%5D)">The impact of flow-induced forces on the morphogenesis of the outflow tract.</a> Front Physiol. 5, (2014).</li>
<li>Hu, N., Clark, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hemodynamics+of+the+stage+12+to+stage+29+chick+embryo%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Hemodynamics of the stage 12 to stage 29 chick embryo.</a> Circ Res. 65 (6), 1665-1670 (1989).</li>
<li>Hogers, B., DeRuiter, M., Gittenberger-de Groot, A., Poelmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unilateral+vitelline+vein+ligation+alters+intracardiac+blood+flow+patterns+and+morphogenesis+in+the+chick+embryo%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Unilateral vitelline vein ligation alters intracardiac blood flow patterns and morphogenesis in the chick embryo.</a> Circ Res. 80 (4), 473-481 (1997).</li>
<li>Hogers, B., DeRuiter, M., Gittenberger-de Groot, A., Poelmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extraembryonic+venous+obstructions+lead+to+cardiovascular+malformations+and+can+be+embryolethal%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">Extraembryonic venous obstructions lead to cardiovascular malformations and can be embryolethal.</a> Cardiovasc Res. 41 (1), 87-99 (1999).</li>
<li>Reckova, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hemodynamics+is+a+key+epigenetic+factor+in+development+of+the+cardiac+conduction+system%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Hemodynamics is a key epigenetic factor in development of the cardiac conduction system.</a> Circ Res. 93 (1), 77-85 (2003).</li>
<li>Stekelenburg-de Vos, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acutely+altered+hemodynamics+following+venous+obstruction+in+the+early+chick+embryo%5BTitle%5D)+AND+%22J+Exp+Biol%22%5BJournal%5D)">Acutely altered hemodynamics following venous obstruction in the early chick embryo.</a> J Exp Biol. 206 (pt 6), 1051-1057 (2003).</li>
<li>Lucitti, J., Tobita, K., Keller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arterial+hemodynamics+and+mechanical+properties+after+circulatory+intervention+in+the+chick+embryo%5BTitle%5D)+AND+%22J+Exp+Biol%22%5BJournal%5D)">Arterial hemodynamics and mechanical properties after circulatory intervention in the chick embryo.</a> J Exp Biol. 208 (pt 10), 1877-1885 (2005).</li>
<li>Menon, V., Eberth, J., Goodwin, R., Potts, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+Hemodynamics+in+the+Embryonic+Heart+Affects+Outflow+Valve+Development%5BTitle%5D)+AND+%22J.+Cardiovasc.+Dev.+Dis%22%5BJournal%5D)">Altered Hemodynamics in the Embryonic Heart Affects Outflow Valve Development.</a> J. Cardiovasc. Dev. Dis. 2 (2), 108-124 (2015).</li>
<li>Hamburger, V., Hamilton, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+series+of+normal+stages+in+the+development+of+the+chick+embryo%5BTitle%5D)+AND+%22J.+Morphol%22%5BJournal%5D)">A series of normal stages in the development of the chick embryo.</a> J. Morphol. 88, 473-481 (1951).</li>
<li>Midgett, M., Goenezen, S., Rugonyi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood+flow+dynamics+reflect+degree+of+outflow+tract+banding+in+Hamburger-Hamilton+stage+18+chicken+embryos%5BTitle%5D)+AND+%22J+R+Soc+Interface%22%5BJournal%5D)">Blood flow dynamics reflect degree of outflow tract banding in Hamburger-Hamilton stage 18 chicken embryos.</a> J R Soc Interface. 11 (100), (2014).</li>
</ol>

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

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's buffer to be poured atop the yolk.
A single 1 cm thr...

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 ...

The overall goal of this Ex Ovo banding procedure is to create a reproducible, nonlethal alteration in cardiac hemodynamics to study the resulting genetic consequences. This method can help answer key questions in the cardiovascular development field, such as the development of cardiac valves, and associated disease states. The power of this system is that it can be used to study genetic pathways that respond to changing hemodynamics in the developing heart and valves and also in help finding a tipping point at which rescuing a heart does not have any long-term deleterious effects.So demonstrating the procedure will be Lorain Junor, a technician from my laboratory, and Mr.Vinal Menon, a PhD student in the laboratory, who will be demonstrating the handling of the eggs and the use of the ultrasound machine. Incubate 80 to 90 fertilized, Bovan chicken eggs with their blunt ends up on plastic egg trays in a humidified rocker incubator set to 40 degrees Celsius. Let them develop for approximately 72 hours to HH 17.Once at Stage 17, pour about 20 milliliters of warm Tyrode's Buffer, supplemented with sodium bicarbonate into a 100 millimeter by 26 millimeter dish. Next, remove an egg from the incubator and sterilize it with 70%ethanol. Then, gently crack the shell using a scalpel handle and carefully release its contents into the dish.Now, check the embryo under a dissection microscope. Discard the embryo if it appears abnormal, such as being at the wrong developmental stage, being improperly oriented on the yolk, or if there is any bleeding. Be sure to look for the appropriate folding and bending of the vasculature.Keep some embryos not subjected to OFT Banding at 40 degrees Celsius as control embryos. In preparation, tweeze out individual one centimeter long threads from a single 11 to zero nylon surgical suture and form them into loose knots. UV sterilize all the preformed knots three times at 1, 200 by 100 micro joules.Under a dissection microscope, visually ensure that the heart of the embryo is beating at a normal rate, and if not, discard the embryo. Just before the surgery, pipet five to six milliliters of warm Tyrode's Buffer onto the embryo's surface. Then, pass one free end of a preformed knot under the OFT.Position the suture at the OVJ or any other desired location and pass the free end into the knot to constrict the OFT, including the membranes surrounding the heart. Then, wet the surface of the yolk with Tyrode's Buffer, as before. Transfer all the manipulated embryos to the developmental incubator, along with the non-banded control embryos, and let them develop for a set amount of time.Later, excise the embryo from the yolk using straight scissors. Then, dissect the heart with fine forceps and proceed with downstream studies, such as examination of the cardiac morphology. The parse constriction caused by the banding intervention leads to an increase in blood flow at the OVJ.This hemodynamic parameter is conveniently assessed using 2D ultrasound imaging. After removing a single embryo from the incubator for ultrasound imaging, place its dish on a heating pad set to 40 degrees Celsius that is on an adjustable stand. Then, slowly and carefully fill the dish to the brim with warm Tyrode's Buffer.Do not disturb the yolk. If the yolk is compromised, discard the embryo. Now, orient the embryo to the ultrasound probe by adjusting the stand.Orient the embryo such that the central axis of the embryo is perpendicular to the ultrasound probe. With the ultrasound machine operating in B mode, obtain a 2D image of the beating heart. Then adjust the position of the embryo so that the OFT, ventricle, and OVJ are clearly visible in the ultrasound.Next, switch to the pulse wave mode, set the desired pulse repetition frequency, and collect velocity data exactly at the OVJ. If the heart rate of the embryo decreases during imaging, velocity data 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.Embryos were developed to HH 17 and a suture was placed at the OVJ as described. 48 hours later, the embryos were examined with 2D ultrasound to measure changes in blood flow velocity at the OVJ. As expected, the velocity at the OVJ is higher in the banded embryo relative to the control.Once mastered, this technique can be done in less than five minutes, from cracking the egg to banding of the embryonic heart, if it is performed properly. While attempting this procedure, it's important to remember to have the band already created and to be careful in not damaging surrounding tissue when placing the band around the heart. Following this procedure, other methods like real-time PCR, Immunohistochemistry, and Insitu Hybridization can be performed to answer additional questions, like how are key genes and proteins affected by the altered hemodynamics.

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Learn a reproducible ex ovo banding technique to alter cardiac hemodynamics in developing chicken embryos. This method enables study of genetic pathways responding to hemodynamic changes in cardiovascular development.

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

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