Generation of Hook Ischemia-Reperfusion Model using a Three-Day Developing Chick Embryo

Hi, I am Dr.Syed Shadab Raza, the principal investigator for the Laboratory for Stem Cells and Restorative Neurology, located at the Department of Biotechnology at Era University Campus, Lucknow, India. In my laboratory, we strive to understand the pathophysiological mechanism underlying ischemia-reperfusion disorders and the interventions that can reverse those processes. In this context, we frequently employ the in vitro and the in vivo model of ischemic stroke.

Recently, we have created a ischemic-reperfusion injury model in a three day developing chick embryo. So now my students are going to show you how to replicate the ischemia-reperfusion injury in a three day developing chick embryo. Now we are going to show you how to create ischemia-reperfusion in a three days chick embryo.

Our model is cost effective, time effective and highly reproducible. So let's get started. Day one.

Requirements for first day.Procedure. Clean the zero day egg with 70%ethanol using tissue paper wipes. Next, write the current day, date, on eggs with OHP marker and then place the eggs in an egg incubator with a temperature of 36 to 37 degrees Centigrade and 60 to 65%humidity.

Incubate the eggs for next 24 hours. Day two. Requirements for second day.Procedure.

Wipe the surgical scissor with 70%ethanol or followed by an autoclave. After 24 hours of incubation, remove the egg out of the incubator for layering. Next, attach a small piece of Sellotape approximately one inch length and width to the egg's edge and then make a small hole in the edge of the eggshell using the sharp-pointed edge scissor followed by insertion of a five mL syringe at approximately 75 degrees angle.

After inserting the needle into the yolk sack, slowly withdraw five to six mL of albumin. After removing the albumin, reseal the hole with Sellotape and put back the egg into the 37 degrees Centigrade egg incubator for next 48 hours. Day four.

Requirements for fourth day.Procedure. Prepare the Ringer's solution, 0.9%normal saline and one X phosphate buffer saline as per the tables provided in this manuscript. Then autoclave the three solutions.

After autoclave, the respective solutions may be placed at room temperature. Next, take out the egg from the 37 degree egg incubator. Now, before cutting the shell, cover the area of windowing with Sellotape.

And now make a small hole onto the eggshell and begin cutting a circular opening. This process is known as windowing. Make sure that the circular cut is big enough so that the embryo can easily be accessible from any direction, as the positioning of the egg may have to be adjusted as per the position of the embryo.

Now, using a stereo zoom surgical microscope, locate the right vitalline artery or RVA. Here, the arrow indicates the embryo's three day developmental stage. Once the RVA is located, with the use of a 26 G needle make two small holes on the left and right sides of the RVA.

Next, adjust the Doppler blood flow imaging probe onto the RVA. The Doppler blood flow imaging probe should be placed five plus minus one millimeter from the site of ischemia and two thirds the distal end of the RVA. Take flux reading for 30 seconds to two minutes, as desired.

This will make the normoxia phase reading. Now manually mold the edge of the spinal needle to give the edge a shape of hook with the help of a nose plier followed by teeth forceps. Now, using a micro manipulator, insert the spinal needle just beneath the right vitalline artery.

You are ready to lift the spinal needle. Now with the assistance of the micro manipulator, slowly lift the artery until the Doppler blood flow flux shows a minimum decrease of 80%in arterial flow. Once a dropdown of 80%or more in Doppler flux is achieved, leave the spinal needle lifted upward, pulling the artery for five minutes.

This will be the period of ischemia. After this ischemic timing of five minutes, slowly release the artery with the help of micro manipulator to restore normal blood flow levels. Now the Doppler blood flow meter should show readings similar to those observed during normoxia.

This will be the period of reperfusion in the RVA. After the I/R process, apply two, three drops of 1X PBS to the embryo. Finally, reseal the window with Sellotape and then place the egg back in the egg incubator for five hours and 55 minutes.

After five hours and 55 minutes, take out the egg from the egg incubator and place onto the egg tray and reopen the window. Treatment requirements.Procedure. For the treatment of the arteries with the drugs, activators or inhibitors, the RVA should be excised after one hour of I/R process.

Remove the embryo out of the shell by taking it out of the shell onto a sterile 100 mm Petri dish. Once the embryo is released in the Petri dish, excise the RVA using ocular iris in the guidance of stereo zoom surgical microscope. The excision dimension of RVA should be up to 15 plus minus one millimeter distal from the trunk and two plus minus one millimeter towards the trunk.

After excising the RVA, wash it with 1X PBS in a new, sterile Petri dish. For desired treatments, put the artery in a sterilized 1.5 milliliter centrifuge tube filled with 500 microliters of Ringer's solution. And after placing the artery in the centrifuge tube, put into the 37 degree Centigrade laboratory incubator for five hours.

After five hours of incubation, take out the RVA from the 37 degrees Centigrade laboratory incubator and proceed for desired treatments. And now, to present the results. To verify the utility of our model in ischemic-reperfusion research in ischemic arteries, first we looked at the activities of oxygen regulatory proteins 150 or ORP150, cytoplasmic superoxide dismutase one or SOD1 and catalase.

The I/R-treated RVAs showed an increased activity of ORP150, cytoplasmic SOD1 and catalase when compared to the control group, however, supplementing with N-acetyl-L-cysteine, or NAC, a ROS quencher, reduced oxidative stress in the I/R group, as can be evident in the current figure. To establish the utility of this model for inflammatory investigations, we looked at the expression of I/R-1 beta and TNF-alpha in I/R-treated RVAs versus control RVAs. Both interleukins were found to be over-expressed in the Hook-I/R group compared to the control group.

The supplementation of Naringenin, an anti-inflammatory drug, significantly reduced the levels of IL1-beta as well as TNF-alpha. Next, we examined the expression of NLRP3 and NF-kappa beta through Western blotting. This analysis discovered evidence of activation of the NLRP3 inflamasome, as well as NF-kappa beta in response to I/R produced in the RVA.

Similar to previous results, Naringenin reduced the expression of NLRP3 and NF-kappa beta. These results demonstrated that our model could be used to investigate inflammatory alterations. Next, we examined the effect of I/R on apoptosis and autophagy pathways.

First, in figure A, we accessed the expression of cleaved caspase-3. We observed an enhanced expression of cleaved caspase-3 in the I/R group compared to the control group, while the I/R group receiving ZVADFMK showed a decrease in the expression of caspase-3. Similarly for autophagy, the I/R group exhibited high protein levels of LC3, autophagosome marker Beclin-1, a key regulator of autophagy in mammalian cells, and ATG-7, a protein required for basal autophagy, than the control group, while the groups receiving 3-ma, an autophagy inhibitor, showed a decrease in the expression of the above three autophagy proteins, as evident from figures B to D.The figure E demonstrates the effect of Hook-mediated I/R on ambra-1 and ATG-7 mRNA levels.

These results were in line with the previous results, that is, enhanced mRNA expression was observed for ambra-1 and ATG-7 in I/R-treated RVAs compared to their respective controls. However, the outcomes were reversed when 3-ma was added to the I/R group. The current figure represents the effectiveness of our model to study the alterations in the DNA.

We observed an intense DNA smear in the I/R-treated group compared to the control group. However, the supplementation of NAC to the I/R RVA group reversed the outcome. To examine how effective our model is in contrast to other models, we compared the data generated by our Hook-I/R model and the MCAO model in the present experiment.

To brief, the expression of the apoptotic protein, cleaved caspase-3, the autophagy proteins, Beclin-1 and ATG-7, and the inflammatory interleukins, TNF and IFN, was bound to be higher in I/R-treated RVAs than in control RVAs. Interestingly, the Hook-I/R model gave results that were very similar to those obtained by the MCAO model, whether it was the analysis for inflammatory stress or cellular death pathways. Our model can be used for routine ischemia-reperfusion experiments, either alone or parallel to the existing models.

However, during mimicking the ischemia-reperfusion, a highest precaution should be taken during making holes, right and left side of the RVA, and also while occluding or releasing the RVA so that it should not damage any arteries or veins. In routine molecular biology such as Western blotting, QRT-PCR analyzer, chemical acids and imaging techniques could be improvised to associate pathophysiology associated with the ischemia-reperfusion in a three days developing chick embryo. This model could efficiently be used to study molecular mechanics at DNA, RNA and protein levels.

As we have shown you, the I/R modeling in a three day developing chick embryo, we believe that our model will open new avenues in the field of I/R research. With this work, I wish you all the best for your experiments. Thank you.