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

Summary

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.

Abstract

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 °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.

Introduction

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 loading^2,3. Hypoplastic left heart syndrome (HLHS), a congenital heart disease, results in an underdeveloped ventricle/aorta at birth⁴ with a high rate of mortality^5,6. 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 migration^2,8,9,10

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 culture^11,12,13,14. On the other hand, advanced imaging access of zebrafish embryos and transgenic/knockout mice provide distinct advantages^11,12. 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 prognosis^11,13. 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 LA^19,20. In some studies, a surgical nylon thread is applied to the atrioventricular node^19,21. 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 septate^6,14,22,23. 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 observed^14,24.

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.

Protocol

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 "live animals" 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 10^th 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

1. 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.
2. Embryo inclusion/exclusion criteria
   1. Do not incubate eggs that were cracked or damaged during transport.
   2. If bleeding is noted during the LAL procedure or following re-incubation, do not use the embryo.
   3. Do not use embryos developing in the left side-up position, as hemodynamic blood flow may differ from normal orientation.
   4. Dıscard embryos developing with congenital defects in both pre- and post-surgical procedures.
   5. Include embryos that reach the targeted stage developing in their original location, mimicking HLHS as a LAL model.
3. Incubate the fertilized white-Leghorn chicken eggs (Gallus gallus domesticus L.), blunt end-up, to the desired stage typically at HH20-21¹⁵ (37.5 °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 "operable" embryos. Shaking can also work with incubators with this capacity and increase productivity further.
4. 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).

2. Operations at Station 1 (Figure 4A)

1. Open a window from the blunt end of the egg and remove both the outer and inner membranes¹⁵ (Figure 5A-D).
2. 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.
3. 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'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.
4. Remove only the necessary vitelline membrane using micro scissors (Supplementary Video 1).
5. 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).
6. 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.
7. 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).
8. 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.
   ​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.

3. Operations at Station 2 (Figure 4B)

1. Under the stereomicroscope, position the pre-prepared knot from step 1.4 close to the embryo at an accessible location (Figure 6B). The atrial bud is now ready to be tied off (Supplementary Video 4).
2. 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.
   1. Correctly orient the knot to execute the tightening process without damaging the embryos.
   2. Clear the fine membranes optimally in step 2.7 to reduce the effect of a beating heart.
   3. 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.
3. Next, use the micro scissors to cut the excess ends of the suture as close as possible to the bud (Supplementary Video 5).
4. 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.
5. Using the tweezers, remove the excess suture pieces cut away in step 3.3.
6. Finally, using closed tweezers, return the embryo to its original position, as in step 2.5 (Supplementary Video 6).
7. 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.
8. 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.

Results

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 methods^19,28. 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...

Discussion

In HLHS, blood flow is altered due to structural defects, leading to abnormal morphology on the left side^4,6. 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 ...

Materials

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