Name: using chicken embryo as a powerful tool in assessment of developmental cardiotoxicities
Uploaded: 2021-03-21T15:00:00
Description: Watch this Scientific Journal Video about Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities at JoVE.com

This work was supported by National Natural Science Foundation of China (Grant No. 91643203, 91543208, 81502835).

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 &#176;C, and gradually decreased to 37.1 &#176;C as incubation proceeded; the humidity started at 50% and gradually increased to 70%.
1. Exposure methods...

<ol>
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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 success5, 7,8,9,10,11<sup...

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 redundant in quantity, relatively accurate exposure-dose controls, an independent, closed system within the shell and easy manipulation of the developing embryo, all of which guarantees its potential to be used as a powerful toxicological assessment model. 
In cardiotoxicity, the chicken embryo features a four chambered heart, similar to mammalian hearts but with thicker walls, allowing easier morphological assessments. Additionally, the chicken embryo allows for developmental inhalation exposure, which is not possible in mammalian models: during the later stage of development, the chicken embryo will transition from internal respiration to external respiration (getting oxygen via the lung); the latter requires that the embryo penetrates the air cell membrane with the beak, and starts to breathe air3, making the air cell a mini-inhalation chamber. Utilizing this phenomenon, the toxicological effects of gas contaminants on the heart (and other organs) may be assessed without the need of dedicated inhalation chamber instruments. 
In this manuscript, several exposure/endpoint assessment methods are described, all of which serve to make the chicken embryo a powerful tool in the assessment of development cardiotoxicity following exposure to environmental contaminants.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% phosphate buffered formaldehydefixative</td>
			<td>Biosharp, Hefei, China</td>
			<td>REF: BL539A</td>
			<td></td>
		</tr>
		<tr>
			<td>75% ethanol</td>
			<td>Guoyao,Shanghai,China</td>
			<td>CAS:64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosignaling monitor BL-420E+</td>
			<td>Taimeng, Chengdu, China</td>
			<td>BL-420E+</td>
			<td></td>
		</tr>
		<tr>
			<td>Candling lamp</td>
			<td>Zhenwei, Dezhou, China</td>
			<td>WZ-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable syringe</td>
			<td>Zhiyu, Jiangsu, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Egg incubator</td>
			<td>Keyu,Dezhou, China</td>
			<td>KFX</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical balance</td>
			<td>OHAUS, Shanghai, China</td>
			<td>AR 224CN</td>
			<td></td>
		</tr>
		<tr>
			<td>Electro-thermal incubator</td>
			<td>Shenxian, Shanghai, China</td>
			<td>DHP-9022</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Guoyao,Shanghai,China</td>
			<td>CAS:64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fertile chicken egg</td>
			<td>Jianuo, Jining, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin Staining Kit</td>
			<td>Beyotime, Bejing, China</td>
			<td>C0105</td>
			<td></td>
		</tr>
		<tr>
			<td>Histology paraffin</td>
			<td>Aladdin, Shanghai, China</td>
			<td>P100928-500g</td>
			<td>Melt point 52~54&#176;C</td>
		</tr>
		<tr>
			<td>Histology paraffin</td>
			<td>Aladdin, Shanghai, China</td>
			<td>P100936-500g</td>
			<td>Melt point 62~64&#176;C</td>
		</tr>
		<tr>
			<td>IV catheter</td>
			<td>KDL, Zhejiang, China</td>
			<td></td>
			<td>The catheters have to be soft, plastic ones.</td>
		</tr>
		<tr>
			<td>Lentivirus</td>
			<td>Genechem, Shanghai, China</td>
			<td></td>
			<td>The lentivirus were individually designed/synthesized by Genechem.</td>
		</tr>
		<tr>
			<td>Masson&#39;s trichrome staining kit</td>
			<td>Solarbio, Beijing, China</td>
			<td>G1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal probe</td>
			<td>Jinuotai, Beijing, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector (5 uL)</td>
			<td>Anting,Shanghai, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>CAIKON, Shanghai, China</td>
			<td>XSP-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica, Germany</td>
			<td>HistoCore BIOCUT</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome blade</td>
			<td>Leica,Germany</td>
			<td>Leica 819</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbitual sodium</td>
			<td>Yitai Technology Co. Ltd.,&#160; Wuhan, China</td>
			<td>CAS: 57-33-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetter(10ul)</td>
			<td>Sartorius, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone iodide</td>
			<td>Longyuquan, Taian, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Anqisheng,Suzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Kelun,Chengdu, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sunflower oil</td>
			<td>Mighty Jiage, Jiangsu, China</td>
			<td></td>
			<td>Any commerical sunflower oil for human consumption should work</td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>M&#38;G, Shanghai, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tedlar PVF Bag (5L)</td>
			<td>Delin, Dalian, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>SCILOGEX, Rocky Hill, CT, US</td>
			<td>MX-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Guoyao,Shanghai,China</td>
			<td>CAS:1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

using chicken embryo as a powerful tool in assessment of developmental cardiotoxicities

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.

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 ef...

Watch this Scientific Journal Video about Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities at JoVE.com

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

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.

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

Hello everyone. I am Qixiao Jiang, Associate Professor at Department of Toxicology, School of Public Health at Qingdao University. On behalf of my research group, I am going to present the assets of using the chicken embryo in development of our cardio toxicity assessment.Chicken embryo is a classical developmental model that has multiple advantages like easy manipulation, high accessibility, closed independent system, et cetera. Working with multiple environmental pollutants, such as phenolic tannic acid. Diesel exhaust, particularly the matter on the carbon nano tubes.We have a multiple exposure methods established in the lab, but for larger cause, the functional assessments are also routinely performed. In this video, I'm going to show you the details, of exposure methods and the morphological functional assessment methods to you. Feel free to contact us if you are interested.Thank you. Here are our exposure methods that I can introduce to you. Now the first method that I am going to introduce is a air cell injection, where you'll directly inject into the air cell.Clean the surface of the eggs with poly-iodide solution. This is commercially available poly-iodide solution, diluted with deionized water, 1/5. And then, I dry the eggshell with tissue paper, without scrubbing.It's important because scrubbing will break their protective layer coating on the outside of the shell. Cleaned all the eggs in the dark room and the mark the air cell with a pencil. Exclude eggs with cracks on the shell.Also exclude eggs with air cells on the side. If that has a blunt tip, those are highly unlikely to hatch normally. Sanitize the air cell area, as well as the metal probe, with 75%alcohol.Wipe them dry, and then drill a small hole at the center of the air cell area with the probe. Do not stick the probe deep into the air cell. Instead, only break the shell with the tip of the probe.If the hole is still not large enough, break the shell again at the vicinity of the existing hole, until it's large enough to allow you insertion of the tip. Insert pipette tip into the hole with the tip touching the inner membrane. Slowly, inject the mixture and hold it there for at least 10 seconds.This is to allow the dispensed oil to be fully dispensed. And then remove the tip. Now, seal the hole with a brush and a drop of melted paraffin.Be careful not to drip melted paraffin onto the inner memory. Once sealed, place eggs in the incubator until they reach desired embryonic stage. The second, the method I'm going to introduce is the Micro injection.First of all, the eggs needs to be cleaned and handled as described in the air injection section. This procedure needs to be performed in sterilized hood. Sterilize the air cell area, and instruments with acetone.Drill a small hole at the center of air cell area with the metal probe. Do not stick the probe deeply into the air cell, or the inner memory may be damaged. Then, use the same process to carefully enlarge the hole until the diameter is approximately two millimeter.This can allow the ratio confirmation of the inner memory. Then load the solution in micro injector and carefully insert the needle through the hole into the inner membrane for approximately total three millimeters. Gently dispense the solution and then remove the needle.Keep the needle as perpendicular as possible. Thin seal the hole with a small piece of tape. Completely cover the hole to prevent embryo dehydration and and death during subsequent incubation.The third method I'm going to introduce is the air cell infusion. where you instill a gas contaminant into the air cell. Clean and handle the eggs as described in the air cell injection section.And then you can read eggs until embryonic day 17. Handle the eggs at embryonic day 17 to mark the air cell area. At the embryonic day 18, take eggs out from the incubator, sterilize the air cell area and the instruments, with acetone.And then carefully drill two small holes on two sides of the air cell. One is for injection of gas or aerosol. The other is for expanding our air.Carefully control the size of the holes, so that the side of the injection hole is just enough for the catheter needle to be inserted, while the diameter of expanding hole should be a bit larger. Approximately one millimeter in diameter. Now this is the sample bag we use to hold the target gas or aerosol.You can substitute with whatever device you use. Gently pour 10 mL of gas or aerosol into a syringe. And then, carefully attach the syringe to our catheter needle with the inner needle remote so that it's soft.Gently insert this catheter needle into the infusion hole. Once it's in there, apply pressure against the catheter needle. This is to minimize the leakage from the injection site.Another finger can be put around with the expel hole for your infusion confirmation. If you do the infusion gently, the air flow from the expel hole should be easily felt. That's original air in the air cell being expelled.When the infusion is done, seal both holes immediately. These tapes and the return the eggs to your incubator. I can introduce our endpoint assessment process.Fully anesthetize the animals with pentobarbital 33 mg/kg. IP injection tested by the opens. Then place chickens on the operation table.Sterilize the abdomen area and the electrodes with ethanol. Then, insert two needle electrodes from two sides of the abdomen sub continuously. Make sure that the needles do not enter the abdomen cavity by lifting the skin a little bit.And insert the needle from there. Once inserted, carefully push the needle forward until it reaches the side of the chest cavity. Make sure that the needle does not go deep into the body or stick out from the skin.Then make the measurements with an electrocardiographic instrument. Other seminary instruments capable of EKG can be used as well. This is our second end point assessment method is to map for Motrin.This is the cross section of the heart in the way of HME. This is the right ventricle we are going to be measuring with. To do this, we use two rulers.Ruler 1, and ruler 2. Now we just copy ruler one to the screen and we resize it to the appropriate size and then rotate that. And we move it so that the two ends of the horizontal line rest on the 2 ends of the 3 right ventricle walls.And now we have seven measurement lines that go across our three right ventricle wall. Now we copy the secondary ruler to the picture as well. We also resize it a little bit and rotate and replace that so that it will be placed alike.This horizontal line go parallel with the outer wall of the right ventricle. And the perpendicular one would go across the point to where the ruler's white matter lines meet the inner wall or the right ventricle. And then we make measurement dots so that it can be measured later on with image J.We repeat this process 7 times per sample, so that our representative measurement of right ventricle.I'll show you a summary of present results. This is the PFOA injection into the egg before incubation, and you can see that with increasing those of the injection the serum concentration LPF in serum increased effectively. Per the demonstration, the effectiveness, of the micro injection.You can see that A is control, and the B, is where the antivirus express in great fluorescence q protein has been injected. You can see a strong fluorescence. And this is the demonstration for the effectiveness of the epithelium infusion.You can see that with the infusion, dramatic fiber-optic changes has been observed in the myocardium tissue.Okay. And you also can see that the R-R Intervals of the hatching chickens 0, 1, and 2 weeks. It also significantly changed after the infusion with diesel exhaust.Indicating that you factor in this, this method. Here, you can also see that the effectiveness of the epithelium infusion in this example. you can see that the right ventricles of the heart effectively increased after being aerosol infused with Diesel exhaust.And also of course, here we are using the histomorphometric methods. We just show to you. This is quite effective.Okay.This are all the methods I'm going to present to you. I hope this can facilitate other people's work with chicken embryo as a powerful research tool in the Developmental Toxicology. Have a nice day.

Research

JoVE Journal

Developmental Biology

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

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Microbead Implantation in the Zebrafish Embryo

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Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

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In Ovo Electroporation in the Chicken Auditory Brainstem

In Ovo Electroporation in the Chicken Auditory Brainstem

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Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

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Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease

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Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

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The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. ...

In Ovo Intravascular Injection in Chicken Embryos

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...