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.
