Name: 병아리 배아의 좌심방 결찰
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

HLHS 및 심혈관 연구를 위한 조류 배아 좌심방 결찰 모델

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.

저자 스포트라이트: 좌심방 결찰술이 조류 배아 심장에 미치는 영향과 HLHS 시사점 

초기 혈관 발달 중 변경된 혈류역학적 하중에 대한 모델로서의 조류 배아의 좌심방 결찰

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

병아리 배아의 좌심방 결찰

보푸라기가 없는 에탄올을 적신 물티슈로 수정된 흰 레그혼 닭고기 달걀의 달걀 껍질을 부드럽게 청소하는 것으로 시작하십시오. 알을 무딘 것으로 부화시키고 원하는 단계, 보통 HH20에서 21까지 끝난다. 절차를 시작하기 전에 느슨한 오버핸드 매듭을 1.5cm 길이의 10-0 봉합사에 묶어 필요한 매듭 수를 준비하십시오.매듭이 빡빡하지 않고 심방에 맞을 만큼 충분히 큰지 확인하십시오. 핀셋의 반대쪽 끝으로 달걀 껍질을 부드럽게 깨서 열고, 원치 않는 균열을 방지하기 위해 달걀을 단단히 지지합니다. 달걀의 뭉툭한 끝에서 창문을 열고 외막과 내막을 모두 제거합니다.마이크로 가위를 사용하여 필요한 vitelline 멤브레인만 제거합니다. 배아가 유리체막에서 분리되면 끝이 닫힌 핀셋을 배아의 등쪽 부분 아래에 놓고 부드럽게 뒤집어 왼쪽을 노출시킵니다. 굵은 막을 먼저 제거한 다음 미세한 핀셋을 사용하여 미세한 막 제거를 통해 심방 새싹 주변의 모든 막을 즉시 제거합니다.미리 준비된 매듭을 배아 가까이에 놓습니다. 열린 매듭의 방향을 왼쪽 심방 새싹 위로 올바르게 지정하여 매듭 조임을 실행합니다. 마이크로 가위를 사용하여 여분의 봉합사 끝을 새싹에 최대한 가깝게 자르고 핀셋으로 여분의 봉합사 조각을 제거합니다.마지막으로 닫힌 핀셋을 사용하여 배아를 원래 위치로 되돌립니다. 미세 수술이 끝나면 난자를 파라믹의 이중층으로 덮고 인큐베이터로 되돌립니다. 좌심방결찰술(left atrial ligation, LAL model)은 정상 발달에 비해 상당한 형태학적 변화와 함께 더 조밀한 심근 구조가 달성되었음을 보여주었습니다.심근 섬유증과 유사한 심장 간질 주위의 세포외 기질 침착이 관찰되었습니다. 형태학적 다공성 측정은 LAL 모델에서 더 작은 좌심실강과 섬유주 압축을 보여주었습니다. LAL 모델의 HH 25에서, 확대된 우심실강, 변화된 섬유주 구조 및 심근 용적이 관찰되었다.LAL 모델의 광간섭 단층촬영 영상은 대조군에 비해 좌심실의 크기와 직경이 유의하게 감소한 것으로 나타났습니다. LAL 그룹은 또한 HH 25에서 대조군에 비해 벽 두께의 증가를 나타냈다.

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 ...