Name: Venstre atriel ligering i kyllingembryoner
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

Aviær embryonal venstre atriel ligeringsmodel til HLHS og kardiovaskulær forskning

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.

Forfatter Spotlight: Effekt af venstre atriel ligation på fugleembryonale hjerter og HLHS-implikationer 

Venstre atriel ligering i fugleembryoet som model for ændret hæmodynamisk belastning under tidlig vaskulær udvikling

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

Venstre atriel ligering i kyllingembryoner

Start med forsigtigt at rengøre æggeskallerne af befrugtede hvide leghorn kyllingæg med fnugfri ethanol gennemblødte servietter. Inkuber æggene stump ende indtil det ønskede stadium, normalt HH20 til 21. Før du starter proceduren, skal du forberede det krævede antal knuder ved at binde en løs overhåndsknude i en 1,5 centimeter lang 10-nul sutur.Sørg for, at knuden ikke er stram og er stor nok til at passe over atrierne. Knæk æggeskallen forsigtigt med den omvendte ende af pincetten, og støt ægget fast for at forhindre uønskede revner. Åbn et vindue fra den stumpe ende af ægget og fjern både de ydre og indre membraner.Brug mikrosaks til kun at fjerne den nødvendige vitellinmembran. Når embryoet er fri for vitellinmembranen, skal du placere pincetten med lukket spids under embryoets dorsale segment og forsigtigt vende den for at udsætte venstre side. Fjern alle membraner umiddelbart omkring atrieknoppen ved først at fjerne de grove membraner og derefter bruge finere pincet til fjernelse af fine membraner.Placer den forberedte knude tæt på embryoet. Orienter den åbne knude korrekt over venstre atrieknopp for at udføre knudestramningen. Brug mikrosaks til at skære de overskydende suturender så tæt som muligt på knoppen og fjern de overskydende suturstykker med pincet.Endelig skal du bruge lukket pincet til at returnere embryoet til sin oprindelige position. Efter afslutningen af mikrokirurgien skal du dække ægget med et dobbeltlag param og returnere det til inkubatoren. Den venstre atriale ligation, eller LAL-modeller, viste, at en mere kompakt myokardiestruktur blev opnået med signifikante morfologiske ændringer sammenlignet med normal udvikling.Ekstracellulær matrixaflejring blev observeret omkring hjerteinterstitium, der lignede myokardiefibrose. Morfometriske porøsitetsmålinger afslørede et mindre venstre ventrikelhulrum og trabekulær komprimering i LAL-modellerne. Ved HH 25 i LAL-modellen blev der observeret et forstørret højre ventrikelhulrum, ændret trabekulær arkitektur og myokardievolumen.Optisk kohærenstomografibilleddannelse af LAL-modeller viste en signifikant reduktion i venstre ventrikels størrelse og diameter sammenlignet med kontrollen. LAL-grupper udviste også en stigning i vægtykkelse sammenlignet med kontrolgrupper ved 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 ...