A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

Summary

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

Abstract

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

Introduction

Abnormally formed outflow valves are the most common type of congenital heart defects ¹. However, defective cardiac valve structure and function, even though present at birth, may become symptomatic only in adulthood. In fact, several adult valve diseases can be attributed to a congenital origin. Treatment of such patients often involves replacing defective valves, and, importantly, replaced aortic valves have been shown to have congenital anomalies ². Given the fact that critical processes involved in valve development begin early during embryogenesis, the importance of better understanding the mechanisms that regulate these events is highlighte....

Protocol

Avian embryos are not considered vertebrate animals under IUCAC regulations.

1. Obtaining Embryos for Surgery

1. Incubate 80 - 90 fertilized Bovan chicken eggs (blunt end up) in a humidified (60%) rocker incubator at 40 °C for approximately 72 hr to obtain embryos at Hamilton and Hamburger (HH) stage 17. Determine exact number of eggs based on power analysis and survival rate of embryos ¹⁷. Use plastic egg trays for incubation to allow adequate air circulation.
2. Once this desired developmental stage is attained, pour approximately 20 ml of warm (37 °C) Tyrode's buffer (supplemented with sodium bica....

Results

Shown in Figure 1 are recommended instruments needed for OFT banding. The petri dish (Figure 1A) containing the embryo ex ovo should be deep enough so as to not destroy the embryo when covered with the lid. Deep petri dishes (Figure 1C) should also be used for ultrasound imaging to allow an adequate volume of Tyrode's buffer to be poured atop the yolk.

A single 1 cm thr.......

Discussion

This technique is relatively quick and easy to perform, however certain key points need to be kept in mind so as to get accurate downstream results. Embryos should be maintained ex ovo in a petri dish that contains Tyrode's buffer to provide adequate rehydration. It is also important to hydrate the yolk post-surgery with Tyrode's buffer and to make sure the incubation chamber is adequately hydrated. Surgeries should not be performed on an embryo if any bleeding is visible or the yolk is even slightly bro.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fertilized Bovan chicken eggs	Clemson University, Clemson, SC
11 / 0 Nylon suture	Ashaway	S30001	UV sterilize knots before surgery
100 x 26 mm petri dish	VWR	25387-030
Transfer pipettes	Thermo Scientific	232-20S
Scalpel handle #3	Fine Science Tools	91003-12
Straight scissor	Roboz	RS-6702
Dumont #5 fine forceps	Fine Science Tools	11254-20
Tyrodes buffer	Sigma-Aldrich	2145-10L	Filter sterlize before use
Sodium bicarbonate	Fisher Scientific	S233-500
Vevo 770 Ultrasound Imaging system	VisualSonics, Inc.	VS-11392
708 Ultrasound transducer	VisualSonics, Inc.	VS-11171