Left Atrial Ligation in the Avian Embryo as a Model for Altered Hemodynamic Loading During Early Vascular Development

Summary

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.

Abstract

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.

Introduction

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 loading^2,3. Hypoplastic left heart syndrome (HLHS), a congenital heart disease, results in an underdeveloped ventricle/aorta at birth⁴ with a high rate of mortality^5,6. Despite the recent advances in its clinical management, the vascular growth and development dynamics of HLHS are still unclear^7....

Protocol

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 t.......

Discussion

In HLHS, blood flow is altered due to structural defects, leading to abnormal morphology on the left side^4,6. 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 .......

Materials

Name	Company	Catalog Number	Comments
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