Name: Legatura atriale sinistra negli embrioni di pulcino
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

Modello di legatura dell'atrio sinistro embrionale aviario per HLHS e ricerca cardiovascolare

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.

Autore in primo piano: Effetto della legatura atriale sinistra sui cuori embrionali aviari e implicazioni HLHS 

Legatura atriale sinistra nell'embrione aviario come modello per l'alterato carico emodinamico durante lo sviluppo vascolare precoce

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

Legatura atriale sinistra negli embrioni di pulcino

Inizia pulendo delicatamente i gusci delle uova di gallina livornese bianca fecondate con salviette imbevute di etanolo senza lanugine. Incubare le uova fino alla fine smussata fino allo stadio desiderato, di solito da 20 a 21 HH. Prima di iniziare la procedura, preparare il numero richiesto di nodi legando un nodo a rovescio sciolto in una sutura a 10 zeri lunga 1,5 centimetri.Assicurati che il nodo non sia stretto e sia abbastanza grande da adattarsi agli atri. Apri delicatamente il guscio dell'uovo con l'estremità posteriore delle pinzette, sostenendo saldamente l'uovo per evitare crepe indesiderate. Apri una finestra dall'estremità smussata dell'uovo e rimuovi sia la membrana esterna che quella interna.Utilizzare le microforbici per rimuovere solo la membrana vitellina necessaria. Una volta che l'embrione è libero dalla membrana vitellina, posizionare la pinzetta a punta chiusa sotto il segmento dorsale dell'embrione e capovolgerla delicatamente per esporre il lato sinistro. Rimuovi tutte le membrane immediatamente intorno alla gemma atriale rimuovendo prima le membrane grossolane e poi usando una pinzetta più fine per rimuovere la membrana fine.Posizionare il nodo pre-preparato vicino all'embrione. Orientare correttamente il nodo aperto sul bocciolo atriale sinistro per eseguire il serraggio del nodo. Usando delle microforbici, taglia le estremità della sutura in eccesso il più vicino possibile al bocciolo e rimuovi i pezzi di sutura in eccesso con una pinzetta.Infine, utilizzando una pinzetta chiusa, riportare l'embrione nella sua posizione originale. Dopo aver completato la microchirurgia, coprire l'ovulo con un doppio strato di param e rimetterlo nell'incubatrice. La legatura atriale sinistra, o modelli LAL, ha mostrato che è stata ottenuta una struttura miocardica più compatta con cambiamenti morfologici significativi rispetto allo sviluppo normale.È stata osservata una deposizione di matrice extracellulare intorno all'interstizio cardiaco, simile alla fibrosi miocardica. Le misurazioni morfometriche della porosità hanno rivelato una cavità ventricolare sinistra più piccola e una compattazione trabecolare nei modelli LAL. A HH 25 del modello LAL, sono stati osservati un allargamento della cavità ventricolare destra, un'architettura trabecolare alterata e un volume miocardico.L'imaging con tomografia a coerenza ottica dei modelli LAL ha mostrato una significativa riduzione delle dimensioni e del diametro del ventricolo sinistro rispetto al controllo. I gruppi LAL hanno anche mostrato un aumento dello spessore della parete rispetto ai gruppi di controllo a 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 ...