Name: Author Spotlight: Effect of Left Atrial Ligation on Avian Embryonic Hearts and HLHS Implications
Uploaded: 2023-06-16T15:00:00
Description: Watch this Scientific Journal Video about Effect of Left Atrial Ligation on Avian Embryonic Hearts and HLHS Implications at JoVE.com

We acknowledge Tubitak 2247A lead researcher award 120C139 providing funding. The authors would also like to thank PakTavuk G&#305;da. A. S., Istanbul, Turkey, for providing fertile eggs and supporting the cardiovascular research.

Fertile white Leghorn eggs are obtained from trusted suppliers and incubated according to university-approved guidelines. Chick embryos, stages 18 (day 3) to 24 (day 4) (the stages presented in this paper) are not considered live vertebrate animals by the European Union (EU) directive 2010/63/EU and the institutional animal care and use committee (IACUC) guidelines in the US. Chick embryos are considered &#34;live animals&#34; after day 19 of incubation according to US laws, but not for the EU. Each egg is labeled with t...

Avian Embryonic Left Atrial Ligation Model for HLHS and Cardiovascular Research

<ol>
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S., Pekkan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft-tissue+material+properties+and+mechanogenetics+during+cardiovascular+development.">Soft-tissue material properties and mechanogenetics during cardiovascular development.</a> Journal of Cardiovascular Development and Disease. 9 (2), 64 (2022).</li><li>Pesevski, Z., 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=Endocardial+fibroelastosis+is+secondary+to+hemodynamic+alterations+in+the+chick+embryonic+model+of+hypoplastic+left+heart+syndrome.">Endocardial fibroelastosis is secondary to hemodynamic alterations in the chick embryonic model of hypoplastic left heart syndrome.</a> Developmental Dynamics. 247 (3), 509-520 (2018).</li><li>Hu, N., 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=Dependence+of+aortic+arch+morphogenesis+on+intracardiac+blood+flow+in+the+left+atrial+ligated+chick+embryo.">Dependence of aortic arch morphogenesis on intracardiac blood flow in the left atrial ligated chick embryo.</a> Anatomical Record. 292 (5), 652-660 (2009).</li><li>Lashkarinia, S. 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H., Hof, R. B., Rudolph, A. M., Heymann, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+congenital+heart+disease+in+fetal+lambs.">Models of congenital heart disease in fetal lambs.</a> Circulation. 58 (2), 354-364 (1978).</li><li>Wong, F. 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In HLHS, blood flow is altered due to structural defects, leading to abnormal morphology on the left side4,6. The present model provides a practical experimental system to better understand the progression of HLHS and may even mimic its pathogenesis8. However, establishing a fully clinically equivalent HLHS animal model is a challenging task. In addition to the avian LAL model presented here, recent studies in mice, fetal sheep, and frogs ...

Congenital heart defects (CHDs) are structural disorders that occur due to abnormal embryonic development1. In addition to genetic conditions, the pathogenesis is influenced by altered mechanical loading2,3. Hypoplastic left heart syndrome (HLHS), a congenital heart disease, results in an underdeveloped ventricle/aorta at birth4 with a high rate of mortality5,6. Despite the recent advances in its clinical management, the vascular growth and development dynamics of HLHS are still unclear7. In normal embryonic development, the left ventricle (LV) endocardium and myocardium originate from cardiac progenitors as the early embryonic heart tube formation progresses. The gradual presence of myocardial trabeculation, thickening layers, and cardiomyocyte proliferation is reported2. For HLHS, altered trabecular remodeling and left ventricular flattening are observed, further contributing to myocardial hypoplasia due to abnormal cardiomyocyte migration2,8,9,10
Among the widely used model organisms to study heart development and understand hemodynamic conditions11, the avian embryo is preferred due to its four-chambered mature heart and its ease of culture11,12,13,14. On the other hand, advanced imaging access of zebrafish embryos and transgenic/knockout mice provide distinct advantages11,12. Various mechanical interventions have been tested for the avian embryo that alter the intramural pressure and wall shear stress in developing cardiovascular components. These models include left vitelline ligation, conotruncal banding15, and left atrial ligation (LAL)11,12,16. The resulting phenotype due to the altered mechanical loading can be observed approximately 24-48 h after the surgical intervention in studies focusing on early prognosis11,13. The LAL intervention is a popular technique to narrow the functional volume of the left atrium (LA) by placing a suture loop around the atrioventricular opening. Likewise, microsurgical interventions have also been performed that target right atrial ligation (RAL)17,18. Similarly, some researchers target the left atrial appendage (LAA) using micro clips to reduce the volume of the LA19,20. In some studies, a surgical nylon thread is applied to the atrioventricular node19,21. One of the interventions used is LAL, which can mimic HLHS but is also the most difficult model to perform, with very small sample yields due to the extremely fine microsurgical operations required. In our laboratory, LAL is performed in ovo between Hamburger-Hamilton (HH) stages 20 and 21, before the common atrium is fully septate6,14,22,23. A surgical suture is placed around the LA, which alters the intracardiac blood flow streams. In LAL models of HLHS, increased ventricle wall stiffness, altered myofiber angles, and decreased LV cavity size are observed14,24.
In this video article, a detailed protocol and approach for in ovo LAL is provided. Briefly, the fertilized avian embryos were incubated for microsurgery, the eggshell was cracked open, and the outer and inner membranes were cleared. The embryo was then slowly rotated so that the LA was accessible. A 10-0 nylon surgical suture was tied to the atrial bud, and the embryo was returned to its original orientation, completing the LAL procedure25. LAL and normal ventricles are compared for tissue compaction and ventricle volume via optical coherence tomography and basic histology.
A successfully executed LAL model pipeline, as described here, will contribute to basic studies focusing on the embryonic development of cardiovascular components. This model can also be used together with genetic manipulations and advanced imaging modalities. Likewise, the acute LAL model is a stable source of diseased vascular cells for tissue culture experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10-0 nylon surgical suture</td>
			<td>Ethicon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Elastica van Gieson staining kit</td>
			<td>Sigma-Aldrich</td>
			<td>115974</td>
			<td>For staining connective tissues in histological sections</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Interlab</td>
			<td>64-17-5</td>
			<td>For the sterilization step, 70% ethanol was obtained by diluting absolute ethanol with distilled water.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>KUHL, Flemington, New Jersey-U.S.A</td>
			<td>AZYSS600-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Interlab</td>
			<td>080.65.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>World Precision Instruments (WPI), Sarasota FL</td>
			<td>555640S</td>
			<td>Vannas STR 82 mm</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Sigma-Aldrich</td>
			<td>P7793-1EA</td>
			<td>Sealing stage for egg reincubation</td>
		</tr>
		<tr>
			<td>Paraplast Bulk</td>
			<td>Leica Biosystems&#160;</td>
			<td>39602012</td>
			<td>Tissue embedding medium</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Zeiss Stemi 508</td>
			<td>&#160;Stemi 508</td>
			<td>Used at station 1</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Zeiss Stemi 2000-C</td>
			<td>Stemi 2000-C</td>
			<td>Used at station 2</td>
		</tr>
		<tr>
			<td>Tweezer (Dumont 4 INOX #F4)</td>
			<td>Adumont &#38; Fils, Switzerland</td>
			<td></td>
			<td>Used to return the embryo</td>
		</tr>
		<tr>
			<td>Tweezer (Super Fine Dumont #5SF)&#160;</td>
			<td>World Precision Instruments (WPI), Sarasota FL</td>
			<td>501985</td>
			<td>Used to remove the membranes on the embryo</td>
		</tr>
	</tbody>
</table>

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 

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

Our lab focuses on the effect of blood flow and pressure loading on the avian embryonic heart arches and ventricles. We aim to contribute to the complex biomechanical mechanisms that link vascular morphology to mechanical loading in the LL model. We also maintain a strong clinical and surgical bioengineering research program.In a recent collaborative study conducted by professorial ops group at Imperial College, image-based finite element modeling and single cell RNA sequencing were used to illustrate the increasing three dimensional myocardia strains in ventricles after left atrial ligation in stage 25. Such knowledge may be translated to fetal cardiovascular interaction in humans. A smaller left ventricular cavity and compression in the entire trabecula recesses were observed with LL intervention.Furthermore, LL altered the normal ventricular structure and reorientated the appearance of trabecula. In addition, the LL model caused right ventricular cavity enlargement, changes in trabecular structure, and increased myocardial volume load. The LL affects hemodynamic loading in the chick embryo, leading to hypoplastic left heart syndrome.The elasticity of the normally developing myocardium undergoes changes particularly in the HLHS phenotype. These findings can guide the development of potential treatments for clinical trials, and guide predictive competitional models.

Advanced time-resolved imaging techniques can be employed to observe the structural and morphological changes due to LAL intervention10. Furthermore, LAL samples are also amenable to molecular and biological methods19,28. In Table 1, sample studies that employed LAL model results are provided. In this context, LAL intervention was performed in chick embryos that reached HH20-21. Both control (healthy) and LAL hearts were r...

Watch this Scientific Journal Video about Effect of Left Atrial Ligation on Avian Embryonic Hearts and HLHS Implications at JoVE.com

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 ...

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Research

JoVE Journal

Developmental Biology

Left Atrial Ligation in Chick Embryos

Start by gently cleaning the eggshells of fertilized white leghorn chicken eggs with lint-free ethanol soaked wipes. Incubate the eggs blunt end up until the desired stage, usually HH20 to 21. Before starting the procedure, prepare the required number of knots by tying a loose overhand knot into a 1.5-centimeter-long 10-zero suture.Ensure that the knot is not tight and is large enough to fit over the atria. Crack open the eggshell gently with the reverse end of the tweezers, supporting the egg firmly to prevent unwanted cracks. Open a window from the blunt end of the egg and remove both the outer and inner membranes.Use micro scissors to remove only the necessary vitelline membrane. Once the embryo is free of the vitelline membrane, place the closed-tip tweezer under the dorsal segment of the embryo and gently flip it to expose the left side. Remove all membranes immediately around the atrial bud by removing the coarse membranes first, and then using finer tweezers for fine membrane removal.Position the pre-prepared knot close to the embryo. Correctly orient the open knot over the left atrial bud to execute the knot tightening. Using micro scissors, cut the excess suture ends as close as possible to the bud and remove the excess suture pieces with tweezers.Finally, using closed tweezers, return the embryo to its original position. After completing the microsurgery, cover the egg with a double-layer of param and return it to the incubator. The left atrial ligation, or LAL models, showed that a more compact myocardial structure was achieved with significant morphological changes compared to normal development.Extracellular matrix deposition was observed around the cardiac interstitium, resembling myocardial fibrosis. Morphometric porosity measurements revealed a smaller left ventricular cavity and trabecular compaction in the LAL models. At HH 25 of the LAL model, an enlarged right ventricular cavity, altered trabecular architecture, and myocardial volume were observed.Optical coherence tomography imaging of LAL models showed a significant reduction in left ventricular size and diameter compared to the control. LAL groups also exhibited an increase in wall thickness compared to control groups at HH 25.