Name: Ligadura auricular izquierda en embriones de pollo
Uploaded: 2023-06-16T15:00:00
Description: Watch this Scientific Journal Video about Left Atrial Ligation in Chick Embryos at JoVE.com

left atrial ligation in chick embryos

Modelo de ligadura auricular izquierda embrionaria aviar para HLHS e investigación cardiovascular

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.

Autor destacado: Efecto de la ligadura auricular izquierda en los corazones embrionarios aviares y las implicaciones de la HLHS 

Ligadura auricular izquierda en embrión aviar como modelo de carga hemodinámica alterada durante el desarrollo vascular temprano

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

Watch this Scientific Journal Video about Left Atrial Ligation in Chick Embryos at JoVE.com

Ligadura auricular izquierda en embriones de pollo

Comience limpiando suavemente las cáscaras de huevo de los huevos de gallina leghorn blancos fertilizados con toallitas empapadas en etanol sin pelusa. Incubar los huevos hasta el final romo hasta la etapa deseada, generalmente HH20 a 21. Antes de comenzar el procedimiento, prepare el número requerido de nudos atando un nudo simple suelto en una sutura de 1,5 centímetros de largo y 10 ceros.Asegúrese de que el nudo no esté apretado y que sea lo suficientemente grande como para caber sobre las aurículas. Abra la cáscara del huevo suavemente con el extremo inverso de las pinzas, sosteniendo el huevo firmemente para evitar grietas no deseadas. Abre una ventana desde el extremo romo del huevo y retira las membranas externa e interna.Use microtijeras para quitar solo la membrana vitelina necesaria. Una vez que el embrión esté libre de la membrana vitelina, coloque la pinza de punta cerrada debajo del segmento dorsal del embrión y gírela suavemente para exponer el lado izquierdo. Retire todas las membranas inmediatamente alrededor de la yema auricular quitando primero las membranas gruesas y luego usando pinzas más finas para eliminar la membrana fina.Coloque el nudo preparado cerca del embrión. Orientar correctamente el nudo abierto sobre la yema auricular izquierda para ejecutar el apriete del nudo. Con unas microtijeras, corta los extremos de la sutura sobrante lo más cerca posible del cogollo y retira los trozos de sutura sobrantes con unas pinzas.Finalmente, utilizando unas pinzas cerradas, devuelve el embrión a su posición original. Después de completar la microcirugía, cubra el huevo con una doble capa de param y devuélvalo a la incubadora. Los modelos de ligadura auricular izquierda, o LAL, mostraron que se logró una estructura miocárdica más compacta con cambios morfológicos significativos en comparación con el desarrollo normal.Se observó depósito de matriz extracelular alrededor del intersticio cardíaco, parecido a la fibrosis miocárdica. Las mediciones morfométricas de porosidad revelaron una cavidad ventricular izquierda más pequeña y compactación trabecular en los modelos LAL. En la HH 25 del modelo LAL se observó un aumento de tamaño de la cavidad ventricular derecha, alteración de la arquitectura trabecular y del volumen miocárdico.Las imágenes de tomografía de coherencia óptica de los modelos LAL mostraron una reducción significativa en el tamaño y el diámetro del ventrículo izquierdo en comparación con el control. Los grupos LAL también mostraron un aumento en el espesor de la pared en comparación con los grupos de control en HH 25.

Research

JoVE Journal

Developmental Biology

Left Atrial Ligation in Chick Embryos

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 ...