Effect of Left Atrial Ligation on Avian Embryonic Hearts and HLHS Implications (Video) | JoVE

Bortecine Sevgin; Merve Nur Coban; Faruk Karatas; Kerem Pekkan

doi:10.3791/65330

Developmental Biology

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

Published: June 16, 2023 doi: 10.3791/65330

Bortecine Sevgin*¹, Merve Nur Coban*¹, Faruk Karatas², Kerem Pekkan^1,2

¹Department of Biomedical Sciences and Engineering, Koc University, ²Department of Mechanical Engineering, Koc University

* These authors contributed equally

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 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.

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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

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.
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.
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.
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)

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

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).
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.
Next, use the micro scissors to cut the excess ends of the suture as close as possible to the bud (Supplementary Video 5).
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.
Using the tweezers, remove the excess suture pieces cut away in step 3.3.
Finally, using closed tweezers, return the embryo to its original position, as in step 2.5 (Supplementary Video 6).
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.
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.

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Representative 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 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 removed from the embryo at HH25-26. Then, the samples were fixed in 4% paraformaldehyde (PFA) at 4 °C⁶^,¹⁵. The removed heart samples were then dehydrated in ethanol solutions of increasing concentrations (70%, 96%, and 100%) for 1 h each. Finally, the samples were kept in xylene at RT for 0.5 h, and paraffin embedding was performed before sectioning at a 10 µm thickness. The samples transferred to the glass slide were stained with Elastica van Gieson stain. The sections were examined under a stereomicroscope, where the transverse diameter of the ventricle was measured. We also performed a three-dimensional non-invasive method, optical coherence tomography scanning (OCT), in some samples¹¹^,²⁹. Both control and LAL hearts at HH25-26 were excised, and their cross-lumen diameters were measured under OCT.

The results showed that for LAL, a more compact myocardial structure is achieved with significant morphological changes compared to normal development (Figure 7). In addition, the deposition of extracellular matrix components, such as collagen, is observed around the cardiac interstitium, resembling myocardial fibrosis similar to the HLHS³⁰. To better understand myocardial thickening and trabecular compaction, morphometric porosity measurements were performed in both control and LAL specimens. As expected, the LAL intervention resulted in a smaller left ventricular cavity and trabecular compaction by tightening the inter-trabecular recesses. These findings confirm the hypothesis that LAL alters the healthy ventricular architecture and reorientates the trabecular aspect²². Likewise, the LAL model at HH29 resulted in an enlarged right ventricular cavity, altered trabecular architecture and myocardial volume²⁴.

To support the results obtained in this study, OCT imaging was used to measure the ventricular lumen cross area and axial length (Figure 8). LAL showed a significant reduction in left ventricular size and diameter compared to the control. While only the ventricles are focused here, the influence of LAL on aortic arch development is also reported³¹. A recent study we contributed to reported in 3D that the mid-wall myocardial strains were increased in both ventricles after LAL at HH25³². In addition, LAL groups exhibited an increase in wall thickness compared to control groups at HH25. This study is consistent with the previous study by Tobita et al.²⁵, which demonstrated a significant increase in peak systolic epicardial circumferential strains in the LAL LV at HH27.

Figure 1: Incubation system for embryonic growth. Fertilized white Leghorn chicken eggs (Gallus gallus) were incubated in an incubator at a constant humidity (60%) and temperature (37.5 °C). Please click here to view a larger version of this figure.

Figure 2: Preparation of the LAL knot. Multiple knots ~0.5-1 mm in diameter and 1-2 cm long pieces were prepared using 10-0 nylon surgical sutures. Please click here to view a larger version of this figure.

Figure 3: Replica of the embryonic left atrium made from putty. A replica of the left atrial section is created to train and practice knot orientation and knot closure steps using two tweezers. This allowed us to make many trials and perfect these steps before implementing this skill in the actual embryo. Please click here to view a larger version of this figure.

Figure 4: Station preparation. (A,B) All materials and solutions for microsurgery were placed in the clean area for Station 1 and Station 2. Please click here to view a larger version of this figure.

Figure 5: Opening a window from the blunt end of the egg and removal of both the outer and inner membranes. (A,B) A small window was opened using microsurgical instruments on the eggshell and including the whole embryo in HH20-21. (C) Fragments of the eggshell were removed in the first cracking step. (D,E) Extraembryonic membranes also were dissected under the microscope. (F) After the LAL process was completed, the egg was covered with a double layer of parafilm. Please click here to view a larger version of this figure.

Figure 6: A snapshot of the in ovo left atrial ligation (LAL) surgery. (A) Dorsal view of the chick embryo in its normal orientation. The embryonic ventricle reaching HH20-21 has a primitive common atrium (a), ventricle (v), and the outflow tract (ot). (B) A close-up dorsal view of the flipped, left side-up embryo and the knot location is displayed. (C) LA with the suture knot. Abbreviations: LA = left atrium; AA = aortic arch. Please click here to view a larger version of this figure.

Figure 7: Removal and examination of the control and LAL hearts. Both (A) control and (B) LAL hearts that reached the HH25-26 stage were removed from the embryo and examined under a stereomicroscope. The bar graph shows the differences in transverse heart diameter between the LAL group and the control group, and the mean ± standard deviation (SD) of at least four replicates. Scale bar = 100 µM. Histological examination of heart tissues in (C) control and (D) LAL samples using the Elastica van Gieson staining technique. The bar graph shows the differences in porosity (%) to show myocardial compression between the LAL and the control group, and the mean ± SD of at least two replicates. **p < 0.01. GraphPad Prism version 9.5.1 was used for statistical analysis. Scale bar = 50 µM. Abbreviations: LAL = left atrial ligation; LA = left atrium; RA = right atrium; RV = right ventricle; LV = left ventricle. Please click here to view a larger version of this figure.

Figure 8: Optical coherence tomography. Both (A) control and (B) LAL hearts reaching HH25-26 were examined by OCT. (C,D) The size and length of the lumen cross of the ventricles are indicated in panels (C) and (D), respectively. The histograms represent the mean ± SD with at least three repeats. *p < 0.05; **p < 0.01; GraphPad Prism version 9.5.1 was used for statistical analysis. Scale bar = 100 µM. Abbreviations: LAL = left atrial ligation; RV = right ventricle; LV = left ventricle. Please click here to view a larger version of this figure.

Table 1: A review of research studies that employ the left atrial ligation (LAL) model in avian embryos⁶^,¹⁰^,¹²^,¹⁴^,²²^,²⁴^,²⁷^,³⁰^,³²^,³³.
In almost all papers, the LA is tied at HH21. The effect of LAL is investigated at later HH stages (assessment stage). Abbreviations: AVC = atrioventricular cushion; LAV = left atrioventricular canal; RAV = right atrioventricular canal; RA = right atria; LA = left atria; RV = right ventricle; LV = left ventricle; SEN = subendocardium; IVS = interventricular septum; micro-CT = microcomputed tomography. Please click here to download this Table.

Table 2: Typical effectiveness of the left atrial ligation (LAL) model created at HH21 and incubated until HH25. Please click here to download this Table.

Supplementary Video 1: Both the outer and inner membranes of the avian embryo are removed. Then, only the vitelline membrane is removed, as shown using micro scissors. Please click here to download this File.

Supplementary Video 2: Tweezers are placed under the dorsal segment of the right side-up embryo and carefully flipped to expose its left atrial bud. Please click here to download this File.

Supplementary Video 3: The membranes around the left atrial bud are gradually cleared. The atrium slightly expands, which makes knot placement and tying possible. Please click here to download this File.

Supplementary Video 4: An approximately 0.1-0.3 mm long suture is positioned near the embryo and tightened around it using two tweezers. Please click here to download this File.

Supplementary Video 5: The excess suture ends of the knot are carefully cut using micro scissors. Please click here to download this File.

Supplementary Video 6: The embryo is gently returned to its normal orientation. LAL is completed. Please click here to download this File.

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Discussion

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 have attempted to replicate the morphological, hemodynamic, and pathophysiological features of HLHS prognosis. In mice embryos, mechanical loading is altered via an embolizing agent injected into the LA through a fetal intervention at embryonic day (ED) 14.5 (approximately HH40-41 in chick embryos)³⁴. A total of 48% of fetuses that were positively embolized survived to gestation with small LVs and retrograde aortic flow. The long gestation period, further challenges in interventions at early time points, and challenging fetal surgery can limit the feasibility of this model. LV hypoplasia was also mimicked in the fetal lamb models by filling the LA with silicone rubber through a balloon catheter³⁵. In this large animal model, the LV volume is reduced sufficiently, but the survival time is not long, resulting in low disease penetration. An alternative intervention approach is to occlude the foramen ovale via percutaneous transhepatic catheterization, as attempted in fetal lamb³⁶, even though directing the occlusive stent to the foramen ovale is very challenging. Sheep models in general are very challenging due to their pre-existing vascular morphologies and adverse pulmonary physiology. As such, the need for seasonal breeding, a later gestational age, and small sample size further limit this model. Finally, the Xenopus embryo is also introduced as another convenient model for mimicking human heart disease³⁷, in spite of its three-chamber heart with a single ventricle and differences in the neural crest cell patterns. The availability of full genome-established microinjection and microsurgical interventions and the long survival time also make this model attractive.

In previous studies, the disease penetration of six out of 39 operated avian embryos was presented as 15% in the LAL model³⁴. However, the average HLHS penetration of the chick embryo model that we examined at the early stage (reaching HH25 and HH27) was 66% (12 of 18 operated avian embryos). Prof. Sedmera reported that the average survival time of embryos could be HH40 (ED14)¹⁹. Other groups regularly performing this ligation have reported high success rates at early time points (e.g., 75% until HH29, 50% until HH34, and 20% until HH38)³⁰. However, in later stages, a distinct phenotype emerges and mortality increases. Although disease penetration can be relatively low in chick embryos, the goal of this model is to better examine the etiology and pathology of prenatal HLHS, especially at the early stages.

Due to the very small size of the early-stage chick embryo, pre- and postoperative LAL intervention can lead to several complications. As a remedy to prevent contamination, eggshells and benches are cleaned with 70% ethanol, and gloves can be used throughout the procedure. A critical step in the LAL protocol is the removal of the entire pericardial membrane to allow a good fit of the knot around the left atrial bud. In addition, we have found the two-person team to be extremely useful, particularly in training and specializing in a specific skill set needed during the protocol steps. This approach speeds up the procedure and its learning curve. As such, the risk of bleeding and contamination is secondary and outweighs the benefits of the proposed buddy system. The use of a pre-prepared suture stored in a sterile chick ringer solution is recommended. In addition, maintaining a low tightening strength of the suture is also critical for higher embryo yield. Tighter knots on the atrial bud applied in HH21 may lead to premature failure of the LV that is unable to elaborate the clinically critical engagement of conduction, coronary, and secondary myoarchitecture remodeling defects, which have been well described in previous studies²². Specifically, using the finest and unused tweezers and scissors at certain steps and relatively blunt ones at others can reduce accidental bleeding. Finally, immediately after completion of the knot, the embryo should be turned to its original position rapidly, and the eggshell window should be closed with a double layer of parafilm to preserve its temperature and humidity. The typical yield for the LAL model reaching HH25 is presented in Table 2. In addition to the decreased ventricular size, valvular malformations may also develop, such as mitral/aortic atresia. The severity of these lesions increases morbidity at the later stages, as in HLHS. Due to the challenges discussed here, compared to other mechanical interventions performed in chick embryos, such as conotruncal banding, the LAL model results in much lower yield levels. We believe, through the precautions presented here, a 50% yield is achievable.

The avian embryo is an ideal vertebrate animal model for researching cardiovascular development due to its morphological structure, size, low cost, ease of culture, and manipulation³⁸^,³⁹. This model also provides natural protection against pathogens⁴⁰. Instrumented embryos can be utilized in advanced in vivo imaging and local siRNA manipulation. As such, conserved gene regions of the human and chicken are available through Sanger shotgun sequencing and physical-based mapping³⁹, leading to advanced mechanosensitive studies¹⁹. Moreover, the microarray approach applied by Krejčí et al. has been used to measure the success regarding the potential reversibility of hemodynamic changes in myocardial structure. Thus, the identification of genes differentially expressed between the left and right ventricles can be used as a criterion for the ideal period of intervention when irreversible changes begin³³.

In conclusion, potential future directions for microsurgical applications in the chick embryo model include the use of cardiovascular gene editing techniques focusing on specific matrix genes and molecular signaling pathways, supporting advances in tissue-cell culture and imaging technologies³².

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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

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

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