Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Summary

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

Abstract

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.

Introduction

The chicken embryo is a classic developmental model, which has been used for over two hundred years¹. The chicken embryo model has various advantages compared to traditional models. First of all, as early as over 70 years ago, the normal development of the chicken embryo had been illustrated very clearly in the Hamburger-Hamilton staging guide², in which a total of 46 stages during chicken embryo development were defined with precise time and morphological characteristics, facilitating detections of abnormal development. Additionally, the chicken embryo model has other features such as being relatively low-cost and redun....

Protocol

All procedures described were approved by the Institutional Animal Care and Use Committee (IACUC) of Qingdao University. In our lab, the eggs were incubated in two incubators. Eggs were held upright in the incubator and randomly placed on the shelves. The incubation conditions for the eggs were as follows: incubation temperature started at 37.9 °C, and gradually decreased to 37.1 °C as incubation proceeded; the humidity started at 50% and gradually increased to 70%.

1. Exposure methods

NOTE: Exposure of environmental contaminants to chicken embryos may be achieved in several ways. In this section, three r....

Results

Exposure results
Air cell injection
Air cell injection can effectively expose developing chicken embryos to various agents, which may be subsequently detected in the collected samples (serum, tissue, etc.) of embryos/hatchling chickens. Here is an example, in which perfluorooctanoic acid (PFOA) was air-cell injected, and serum PFOA concentrations were then determined with Ultra-performance liquid chromatography-mass spectrometry. The serum concentrations corresponded with the injected doses, indicating the effect.......

Discussion

The chicken embryo has been a classical model in developmental studies for 200 years¹. Our methods presented in this manuscript have been used in the assessment of several environmental contaminants, including perfluorooctanoic acid, particulate matter, and diesel exhaust with success^{5, 7,8,9,10,11}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% phosphate buffered formaldehydefixative	Biosharp, Hefei, China	REF: BL539A
75% ethanol	Guoyao,Shanghai,China	CAS:64-17-5
Biosignaling monitor BL-420E+	Taimeng, Chengdu, China	BL-420E+
Candling lamp	Zhenwei, Dezhou, China	WZ-001
Disposable syringe	Zhiyu, Jiangsu, China
Egg incubator	Keyu,Dezhou, China	KFX
Electrical balance	OHAUS, Shanghai, China	AR 224CN
Electro-thermal incubator	Shenxian, Shanghai, China	DHP-9022
Ethanol absolute	Guoyao,Shanghai,China	CAS:64-17-5
Fertile chicken egg	Jianuo, Jining, China
Hematoxylin and Eosin Staining Kit	Beyotime, Bejing, China	C0105
Histology paraffin	Aladdin, Shanghai, China	P100928-500g	Melt point 52~54°C
Histology paraffin	Aladdin, Shanghai, China	P100936-500g	Melt point 62~64°C
IV catheter	KDL, Zhejiang, China		The catheters have to be soft, plastic ones.
Lentivirus	Genechem, Shanghai, China		The lentivirus were individually designed/synthesized by Genechem.
Masson's trichrome staining kit	Solarbio, Beijing, China	G1340
Metal probe	Jinuotai, Beijing, China
Microinjector (5 uL)	Anting,Shanghai, China
Microscope	CAIKON, Shanghai, China	XSP-500
Microtome	Leica, Germany	HistoCore BIOCUT
Microtome blade	Leica,Germany	Leica 819
Pentobarbitual sodium	Yitai Technology Co. Ltd.,  Wuhan, China	CAS: 57-33-0
Pipetter(10ul)	Sartorius, Germany
Povidone iodide	Longyuquan, Taian, China
Scissor	Anqisheng,Suzhou, China
Sterile saline	Kelun,Chengdu, China
Sunflower oil	Mighty Jiage, Jiangsu, China		Any commerical sunflower oil for human consumption should work
Tape	M&G, Shanghai, China
Tedlar PVF Bag (5L)	Delin, Dalian, China
Vortex mixer	SCILOGEX, Rocky Hill, CT, US	MX-F
Xylene	Guoyao,Shanghai,China	CAS:1330-20-7