Here, we present a detailed visual protocol for executing the left atrial ligation (LAL) model in the avian embryo. The LAL model alters the intracardiac flow, which changes wall shear stress loading, mimicking hypoplastic left heart syndrome. An approach to overcome the challenges of this difficult microsurgery model is presented.
Due to its four-chambered mature ventricular configuration, ease of culture, imaging access, and efficiency, the avian embryo is a preferred vertebrate 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.
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.
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 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
2. Operations at Station 1 (Figure 4A)
3. Operations at Station 2 (Figure 4B)
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.
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.
The authors have nothing to disclose.
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.
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 |