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 the hatching start date and is scheduled to hatch no later than the 10th day of incubation. After the eggs hatch, the chicks are removed from the incubator. The protocol is performed in two bench-top operation stations (Station 1 and Station 2), focusing on specialized model generation stages.
1. Preparation before microsurgery
<ol>
	<li>Obtain fertilized eggs either from a vaccine development center with a specific pathogen-free (SPF) grade or through a trusted commercial supplier farm by a fragile delivery courier in dry polystyrene containers. Before incubation, gently clean the eggshell with lint-free wipes soaked in 70% ethanol to remove contamination.</li>
	<li>Embryo inclusion/exclusion criteria
	<ol>
		<li>Do not incubate eggs that were cracked or damaged during transport.</li>
		<li>If bleeding is noted during the LAL procedure or following re-incubation, do not use the embryo.</li>
		<li>Do not use embryos developing in the left side-up position, as hemodynamic blood flow may differ from normal orientation.</li>
		<li>D&#305;scard embryos developing with congenital defects in both pre- and post-surgical procedures.</li>
		<li>Include embryos that reach the targeted stage developing in their original location, mimicking HLHS as a LAL model.</li>
	</ol>
	</li>
	<li>Incubate the fertilized white-Leghorn chicken eggs (Gallus gallus domesticus L.), blunt end-up, to the desired stage typically at HH20-2115 (37.5 &#176;C, 60% humidity, 3.5 days) (Figure 1). 
	NOTE: It is important to keep the eggs at a constant temperature and humidity to increase the yield. Depending on the incubator model, the addition of a pan filled with distilled water will maintain stable humidity. Blueprints of an additional/auxiliary temperature and humidity control system that would fit most incubators are developed by the authors and recommended. The electronics, hardware, and code details of this in-house-built sensor/control unit are provided in a data repository26. Continuous gentle shaking (rotating) of the eggs during incubation may allow optimal positioning of the embryo and thus lead to a higher percentage of &#34;operable&#34; embryos. Shaking can also work with incubators with this capacity and increase productivity further.</li>
	<li>Before starting the procedure, prepare the required number of knots by tying a loose overhand knot into a 1.5 cm long 10-0 suture. Be sure the knot is not tight and is large enough to fit over the atria easily during the operation (Figure 2). 
	NOTE: Tie the knots ahead of time and keep them in a sterile chick ringer solution before using. The knot-tying operation requires the use of two hands to operate the tweezers synchronously. Since this is a critical stage in the protocol, a model of the atrium can be made from putty for practicing this step (Figure 3). This will improve the three-dimensional microsurgery skills needed to perform step 3.2.3 at Station 2 (Figure 4).</li>
</ol>
2. Operations at Station 1 (Figure 4A)
<ol>
	<li>Open a window from the blunt end of the egg and remove both the outer and inner membranes15 (Figure 5A-D).</li>
	<li>Open the eggshell by cracking gently with the reverse end of the tweezers, with the free fingers firmly supporting the egg to reduce unwanted crack propagation.</li>
	<li>Since LAL is a long procedure, conserve the temperature and humidity of the embryo, as the heart rate depends on the temperature. Thus, ensure the initial shell window is created as small as possible, just enough to execute the operations. 
	NOTE: No humidity or temperature control systems are employed during the operation, but these systems, if available, would benefit the yield. If possible, the air conditioning system in the lab is shut-down, and the procedure is performed at the highest room temperature (RT) possible. Optimization of the embryonic heart rate, which can be controlled by the temperature, during the operation is also recommended. Some laboratories maintain the heart rate at slightly lower rates than 120 bpm via temperature control during the LAL operation. As such, humidity control employed around the surgical zone would increase the yield further. The eggshell windows are created as small as possible, just big enough to allow surgical access. This is also applicable to the thick outer membrane, which is typically smaller than the eggshell only to the extent of the embryo&#39;s circumference. These ensure to maintain the temperature and humidity of the embryo. While opening a window from the blunt end of the egg, the small shell fragments are cleaned so that these pieces do not damage the vitelline vascular integrity or lead to unwanted artifacts. In addition, other laboratories use curved micro-serrated scissors for making windows. Also, two widths of Scotch tape can be used to stabilize the eggshell to control cracking.</li>
	<li>Remove only the necessary vitelline membrane using micro scissors (Supplementary Video 1).</li>
	<li>Normal embryonic development is right side-up. Once the embryo is free of the vitelline membrane, place the tweezer with closed tips under the dorsal segment of the embryo and gently flip the embryo to expose the left side (i.e., left side-up configuration) (Figure 6A,B; Supplementary Video 2).</li>
	<li>Ensure that the left atrial bud is now exposed but still covered by a complex membrane system, typically consisting of a double layer of the pericardium.</li>
	<li>Remove the membranes, including the fine ones, immediately around the atrial bud. This is another critical stage; perform membrane removal from coarse membranes and progress to the fine ones around the left atrial bud. Reserve the finest tweezers for removal of the fine membrane (Supplementary Video 3).</li>
	<li>During the membrane removal process, orient the embryo in a left side-up position, so that the knot placement operation in step 3.2 can be performed without further repositioning. To achieve this, lift the embryo using the membranes in step 2.6 and hang it from the eggshell, ensuring the left side faces up. 
	&#8203;NOTE: Some embryos can be located close to the eggshell periphery and can be challenging to operate. Still, these embryos will most likely be oriented right side-up and display normal behavior, and can be included in the study. In these cases, if needed, the obscured atrium can be made more accessible by gently cleaning the pericardial membrane with #4 fine tweezers and removing the eggshell in the reverse direction (toward the shell opening). These embryos can also be lifted using parts of the extraembryonic membranes and fixed at the desired position by attaching one end of the membrane (the tweezer end) to the eggshell, using its natural stickiness. Additionally, the space between the head and spinal cord region of the embryo can be expanded with the help of tweezers to reveal the obscured atrium.</li>
</ol>
3. Operations at Station 2 (Figure 4B)
<ol>
	<li>Under the stereomicroscope, position the pre-prepared knot from step 1.4&#160;close to the embryo at an accessible location (Figure 6B). The atrial bud is now ready to be tied off (Supplementary Video 4).</li>
	<li>Retrieve the pre-prepared open knot and orient it over the left atrial bud. For LAL to work, place the egg in a uniquely inclined three-dimensional orientation.
	<ol>
		<li>Correctly orient the knot to execute the tightening process without damaging the embryos.</li>
		<li>Clear the fine membranes optimally in step 2.7 to reduce the effect of a beating heart.</li>
		<li>Tighten the suture (Figure 6C). For this step, practicing with the putty is very useful. For sham embryos, tighten the knot just enough to hold the knot.</li>
	</ol>
	</li>
	<li>Next, use the micro scissors to cut the excess ends of the suture as close as possible to the bud (Supplementary Video 5).</li>
	<li>Be very careful that the newly cut ends of the tied suture are not in a position to puncture nearby vessels during rotation or due to the heartbeat.</li>
	<li>Using the tweezers, remove the excess suture pieces cut away in step 3.3.</li>
	<li>Finally, using closed tweezers, return the embryo to its original position, as in step 2.5&#160;(Supplementary Video 6).</li>
	<li>After completing the LAL process, cover the egg with a double layer of parafilm and incubate it again. A tight and sterile closing of the eggs is paramount for survival, especially past 24 h of incubation. If visual access is also desired, use paraffin wax with glass slides. 
	NOTE: As the early embryonic period is studied, the eggs are typically incubated for 24-48 h until they reach approximately HH25 or HH27. However, there is no limit, and later stages can be studied, as attempted by other researchers. For fast operation speeds, at least a two-person team is recommended. One person should be trained for and is responsible for egg opening, initial membrane cleanup, rotation, and membrane cleanup around the left atrial bud. The other person is responsible only for initial knot preparation, knot placement, and tightening. The final embryo rotation can be performed by Person 1. The surgical operation for a single embryo takes about 4-5 min.</li>
	<li>Before/after surgical operations, clean the bench-top surfaces and instruments with ethanol. Ensure to apply fresh chick ringer solution (NaCl, KCl, CaCl2, and NaHCO3)16,27 to the metal instruments touching the embryonic tissues.</li>
</ol>

Avian Embryonic Left Atrial Ligation Model for HLHS and Cardiovascular Research

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Y., 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=Induction+of+left+ventricular+hypoplasia+by+occluding+the+foramen+ovale+in+the+fetal+lamb.">Induction of left ventricular hypoplasia by occluding the foramen ovale in the fetal lamb.</a> Scientific Reports. 10 (1), 880 (2020).</li><li>Nie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+frogs+as+a+model+to+study+the+etiology+of+HLHS.">Use of frogs as a model to study the etiology of HLHS.</a> Journal of Cardiovascular Development and Disease. 10 (2), 51 (2023).</li><li>Vilches-Moure, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+chicken+(Gallus+gallus+domesticus)+as+a+model+of+cardiac+biology+and+development.">Embryonic chicken (Gallus gallus domesticus) as a model of cardiac biology and development.</a> Comparative Medicine. 69 (3), 184-203 (2019).</li><li>Kain, K. 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=The+chick+embryo+as+an+expanding+experimental+model+for+cancer+and+cardiovascular+research.">The chick embryo as an expanding experimental model for cancer and cardiovascular research.</a> Developmental Dynamics. 243 (2), 216-228 (2014).</li><li>Sukparangsi, W., Thongphakdee, A., Intarapat, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+embryonic+culture:+A+perspective+of+in+ovo+to+ex+ovo+and+in+vitro+studies.">Avian embryonic culture: A perspective of in ovo to ex ovo and in vitro studies.</a> Frontiers in Physiology. 13, 903491 (2022).</li></ol>

The authors have nothing to disclose.

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><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&amp;nbsp;</td><td>39602012</td><td>Tissue embedding medium</td></tr><tr><td>Stereo Microscope</td><td>Zeiss Stemi 508</td><td>&amp;nbsp;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 &amp;amp; Fils, Switzerland</td><td></td><td>Used to return the embryo</td></tr><tr><td>Tweezer (Super Fine Dumont #5SF)&amp;nbsp;</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.

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

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.

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 ...

left-atrial-ligation-avian-embryo-as-model-for-altered-hemodynamic

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

1.0K Views.  Koc University. 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. 

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

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.

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).

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

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Medicine

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Bioengineering

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

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Evaluation of Hydrodynamic Properties in Mounted Porcine Aortic Valves

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a method for mounting a porcine aortic valve in a pulse duplicator to evaluate its hydrodynamic properties. These properties can then be evaluated before and after the procedure under investigation is performed and/or the investigative medical device is applied. Securing the inflow segment presents some difficulty owing to the lack of circumferential myocardium in the left ventricular outflow tract. This method addresses that issue by securing the inflow segment using the anterior leaflet of the mitral valve and then suturing the left ventricular free wall around the inflow fixture. The outflow segment is secured simply by inserting the fixture into an incision in the superior aspect of the aortic arch. We found that specimens had significantly different hydrodynamic properties before and after tissue fixation. This finding induced us to use fresh specimens in our testing and should be considered when using this method. In our work, we used this method to test novel intracardiac patch materials for use in the valvular position by performing an aortic valve neocuspidization procedure (Ozaki procedure) on the mounted porcine aortic valves. These valves were tested before and after the procedure to assess the change in hydrodynamic properties in comparison to the native valve. Herein, we report a platform for hydrodynamic testing of experimental aortic valve procedures that enables comparison with the native valve and between different devices and techniques used for the procedure under investigation.

Author Spotlight: Advancing Cardiac Procedure Testing Prior to Embarking on Large Animal Studies 

We present a method for mounting a porcine aortic valve on a pulse duplicator to test its hydrodynamic properties. This method can be used to determine the change in hydrodynamics after the application of an experimental procedure or novel medical device prior to use in a large animal model.

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Generating Murine Model of Myocardial Infarction: A Surgical Procedure for Permanent Ligation of Left Anterior Descending Coronary Artery in Mouse Model

Source: Lugrin, J. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/59591" target="_blank">Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery</a>. J. Vis. Exp. (2019)
In this video, we describe a surgical procedure to generate a mouse model of myocardial infarction by permanently ligating the left anterior descending coronary artery.

Source: Lugrin, J. et al. Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery. J. Vis. Exp. (2019)
In ...

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

Summary

Explore More Videos

Chapters in this video

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

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

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

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Generating Murine Model of Myocardial Infarction: A Surgical Procedure for Permanent Ligation of Left Anterior Descending Coronary Artery in Mouse Model