Name: a novel ex ovo banding technique to alter intracardiac hemodynamics in an embryonic chicken system
Uploaded: 2016-05-13T13:00:00
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)

Fågelembryon inte anses ryggradsdjur enligt IUCAC regler. 1. Skaffa embryon för kirurgi <ol><li> Inkubera 80 - 90 befruktade Bovan hönsägg (trubbiga änden upp) i en fuktad (60%) rocker inkubator vid 40 ° C i ungefär 72 h för erhållande av embryon vid Hamilton och Hamburger (HH) stadium 17. Bestäm exakta antal ägg baserat på effektanalys och överlevnaden av embryon 17. Använd plast äggbrickor för inkubation för att tillåta tillräcklig luftcirkulation. </li><li>...

<ol>
	<li>Neeb, Z., Lajiness, J., Bolanis, E., Conway, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+outflow+tract+anomalies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+regulatory+networks+in+development+and+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+structure+and+function+in+development+and+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocardial+and+epicardial+epithelial+to+mesenchymal+transitions+in+heart+development+and+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+cardiac+cushion+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrioventricular+valve+development:+new+perspectives+on+an+old+theme.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transitions+in+early+embryonic+atrioventricular+valvular+function+correspond+with+changes+in+cushion+biomechanics+that+are+predictable+by+tissue+composition.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracardiac+fluid+forces+are+an+essential+epigenetic+factor+for+embryonic+cardiogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+flow+forces+and+rhoA+regulate+fibrous+development+of+the+atrioventricular+valves.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+flow-induced+forces+on+the+morphogenesis+of+the+outflow+tract.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+of+the+stage+12+to+stage+29+chick+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilateral+vitelline+vein+ligation+alters+intracardiac+blood+flow+patterns+and+morphogenesis+in+the+chick+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraembryonic+venous+obstructions+lead+to+cardiovascular+malformations+and+can+be+embryolethal.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+is+a+key+epigenetic+factor+in+development+of+the+cardiac+conduction+system.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acutely+altered+hemodynamics+following+venous+obstruction+in+the+early+chick+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+hemodynamics+and+mechanical+properties+after+circulatory+intervention+in+the+chick+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Hemodynamics+in+the+Embryonic+Heart+Affects+Outflow+Valve+Development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+dynamics+reflect+degree+of+outflow+tract+banding+in+Hamburger-Hamilton+stage+18+chicken+embryos.">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>

Denna teknik är relativt snabb och enkel att utföra, men vissa viktiga punkter måste hållas i minnet, så att få korrekta resultat nedströms. Embryon bör bibehållas ex ovo i en petriskål som innehåller Tyrodes buffert för att ge adekvat rehydrering. Det är också viktigt att hydratisera gulan post-kirurgi med Tyrodes buffert och för att se till inkubationskammaren är tillräckligt hydrerad. Operationer bör inte utföras på ett embryo om någon blödning är synlig eller äggula är ännu något ...

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.

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			<td height="105" style="height:105px;width:216px;">Fertilized Bovan chicken eggs</td>
			<td style="width:71px;">Clemson University, Clemson, SC</td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;">11/0 Nylon suture</td>
			<td style="width:71px;">Ashaway</td>
			<td style="width:119px;">S30001</td>
			<td>UV sterilize knots before surgery</td>
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			<td height="21" style="height:21px;width:216px;">100 x 26 mm petri dish</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">25387-030</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Transfer pipettes</td>
			<td style="width:71px;">Thermo Scientific&#160;</td>
			<td style="width:119px;">232-20S</td>
			<td></td>
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			<td height="63" style="height:63px;width:216px;">Scalpel handle #3</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">91003-12</td>
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			<td height="21" style="height:21px;width:216px;">Straight scissor</td>
			<td style="width:71px;">Roboz</td>
			<td style="width:119px;">RS-6702</td>
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			<td height="63" style="height:63px;width:216px;">Dumont #5 fine forceps</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tyrodes buffer</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">2145-10L</td>
			<td>Filter sterlize before use&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Sodium bicarbonate</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">S233-500</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vevo 770 Ultrasound Imaging system</td>
			<td style="width:71px;">VisualSonics, Inc.</td>
			<td style="width:119px;">VS-11392</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">708 Ultrasound transducer&#160;</td>
			<td style="width:71px;">VisualSonics, Inc.</td>
			<td style="width:119px;">VS-11171</td>
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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.

Visas i figur 1 rekommenderas instrument som behövs för OFT bandning. Petriskålen (Figur 1A) som innehåller embryot ex ovo bör vara tillräckligt djupt för att inte förstöra embryot när den är täckt med locket. Djupa petriskålar (Figur 1C) bör också användas för ultraljudsundersökningar för att möjliggöra en tillräcklig volym Tyrodes buffert för att hällas ovanpå äggulan. <p class="jove_content" fo:k...

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).

En novell<em&gt; Ex Ovo</em&gt; Banding Teknik Alter Intrakardiell Hemodynamik i en embryonala kyckling 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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