This paper describes ischemia-reperfusion (I/R) modeling in a 3-day chick embryo using a spinal needle customized hook to better understand I/R development and treatment. This model is simple, quick, and inexpensive.
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Method Article
Generation of Hook Ischemia-Reperfusion Model using a Three-Day Developing Chick Embryo
* These authors contributed equally
In This Article
Summary
Abstract
Ischemia and reperfusion (I/R) disorders, such as myocardial infarction, stroke, and peripheral vascular disease, are a few of the leading causes of illness and death. Many in vitro and in vivo models are currently available for studying the I/R mechanism in disease or damaged tissues. However, to date, no in ovo I/R model has been reported, which would allow for a better understanding of I/R mechanisms and faster drug screening. This paper describes I/R modeling using a spinal needle customized hook in a 3-day chick embryo to understand I/R development and treatment mechanisms. Our model can be used to investigate anomalies at the DNA, RNA, and protein levels. This method is simple, quick, and inexpensive. The current model can be used independently or in conjunction with existing in vitro and in vivo I/R models.
Introduction
Ischemia-reperfusion tissue injury has been linked to a number of pathologies, including heart attacks, ischemic stroke, trauma, and peripheral vascular disease1,2,3,4,5. This is primarily due to a lack of a comprehensive understanding of the disease progression and the lack of an effective research model. Ischemic injury occurs when the blood supply to a specific area of the tissue is cut off. As a result, ischemic tissue eventually necrotizes, though the rate varies depending on the tissue. Hence, restoring the blood supply may help to mitigate the damage. However, it has been observed, in some cases, that reperfusion causes more tissue damage than ischemia alone does6,7,8. Therefore, understanding the molecular and cellular mechanisms of ischemia-reperfusion is required to develop an effective therapeutic intervention. Currently, no effective treatment for I/R injuries is known. This disparity has prompted the creation of new experimental models, ranging from in vitro to in vivo models, to address the existing problem9,10,11,12,13.
Chick embryos (Gallus gallus domesticus) are widely used in research because of their ease of access, ethical acceptability, relatively large size (compared to other embryos), low cost, and rapid growth14. We used a chick embryo at 72 h of development to create an in ovo I/R by occluding and releasing the right vitelline artery with the assistance of a spinal needle. We named it the Hook-I/R ischemia-reperfusion model (Figure 1). The model utilized in this study is capable of accurately simulating all downstream processes, including oxidative and inflammatory pathways, which are frequently associated with I/R damage15,16,17.
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Protocol
The Institutional Animal Ethical Committee at Era's Lucknow Medical College and Hospital issued a written waiver stating that no formal approval was required to conduct these experiments in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). However, Standard Operating Procedures were followed to minimize any potential for embryonic distress.
1. Buffer preparation (Table 1)
- Prepare Ringer's solution
- To prepare Ringer's solution, dissolve 0.72 g of NaCl (123 mM), 0.017 g of CaCl2 (1.53 mM), 0.037 g of KCl (4.96 mM) in 70 mL of sterile distilled H2O, with a final volume of 100 mL. Adjust the pH to 7.4. Let it dissolve completely and autoclave. Then, filter through a 0.22 µm filter, aliquot into single-use amounts (about 10 mL) and store at room temperature.
- Prepare normal saline (0.9% Sodium Chloride, NaCl).
- In 70 mL of sterile distilled H2O, dissolve 0.9 g of NaCl (154 mM). Make up the volume to 100 mL. Autoclave for 15 min at 121 °C. Adjust the pH to 7.4 with 0.1 N HCl or 0.1 N NaOH if necessary. Make 10 mL aliquots in a 15 mL sterile centrifuge tube and store at room temperature.
- Prepare 70% ethanol (v/v).
- Mix 70 mL of pure ethanol (Mol. wt. 46.07 g/L) to 30 mL of sterile H2O. Prepare as needed or store at room temperature. There is no need for sterilizing.
- Prepare 1x Phosphate Buffer Saline (1x PBS).
- Prepare 100 mL of 1x PBS by adding 0.144 g of Na2HPO4·7H2O (5.37 mM), 0.8 g of NaCl (136.8 mM), 0.2 g of KCl (26.8 mM), 0.2 g of KH2PO4 (14. 6 mM) to 70 mL of distilled water. Dissolve and make up the volume to 100 mL and autoclave for 15 min at 121 °C. Bring the pH to 7.4, adding a couple of drops of 0.1 N HCl or 0.1 N NaOH, if needed. Make aliquots of 10 mL in a 15 mL sterile centrifuge tube and store at room temperature.
2. Day 1
- Arrange all the tools necessary for egg sterilization (70% ethanol, cleaning wipes, an egg rack, and an OHP marker).
- Clean the 0-day eggs with 70% ethanol using tissue paper wipes. Use only a 0-day egg, as older eggs may not give rise to an embryo.
- Write the current date on eggs with an OHP marker.
- Place the eggs in an egg incubator set to a temperature of 36-37 °C and a humidity level of 60%-65%. Incubate the eggs for the next 24 h.
3. Day 2
- Arrange the necessary equipment for the withdrawal of 5-6 mL of albumin (sharp edge scissors, 5 mL syringe, 18 G needle, syringe discarder, and adhesive tape).
- Wipe the surgical scissors with 70% ethanol or sterilize using an autoclave after wiping them with 70% ethanol.
- Now take the egg from the 37 °C egg incubator for layering.
- Place the egg on a clean egg rack.
- Attach a small piece of adhesive tape (size: about 1-inch length x width) to the egg's edge.
- Make a small hole in the edge of the eggshell using sharp-pointed edge scissors. Insert a 5 mL syringe at an approximate angle of 75°.
NOTE: The 5 mL syringe comes with a 24 G x 1 needle (sterile), but it is good to replace the 24 G x 1 needle with an 18 G x 1.5 needle (sterile). The 18 G x 1.5 needle is 1.25 x 38 mm in width. Therefore, it will facilitate the removal of albumin. - After inserting the needle into the yolk sac, slowly withdraw 5-6 mL of albumin.
NOTE: This provides the embryo with a bed on which it can grow. Withdrawing albumin prevents overspill of albumin while establishing a window. Finally, the risk of the embryo being damaged during windowing is mitigated by eliminating 5-6 mL of albumin. - After removing the albumin, reseal the opening with adhesive tape and leave the eggs to incubate at 37 °C for 48 h.
4. Day 4
- Prepare the Ringer's solution, 0.9% normal saline, and 1x PBS as described in section 1 of the protocol. Then, autoclave the three solutions. Following autoclaving, place the respective solution at room temperature.
- Take out the egg from the 37 °C egg incubator and cut the shell into a circular shape. Before cutting the eggshell, cover the area to be cut with adhesive tape.
NOTE: Covering the window area with adhesive tape prevents the breaking of the eggshell into undesired places. However, if you break into an unwanted place, seal the area with the adhesive tape. Covering the places to be cut with adhesive tape prevents shell pieces from falling onto the yolk sac. - Create a small hole in the eggshell with a sharply pointed edge scissor at the place where the windowing is desired and begin cutting a circular opening. This process is known as windowing.
NOTE: Ensure that the circular cut is large enough to allow easy access to the embryo from any direction. If needed, alter the egg position to accommodate the embryo's position. - Next, using a stereo zoom surgical microscope, locate the right vitelline artery (RVA).
NOTE: Chicken embryos normally undergo thoracic torsion (along with cervical flexure, etc.) as they develop, such that the left side of the head is against the yolk at the 72 h stage. More caudally, where the vitelline arteries exit the body, the embryo is not much twisted, and this part of the body lies ventral side down toward the yolk. So, viewing directly, the right of the embryo is to the right of the researcher. - Once the RVA is located, create two small holes on the left and right sides of the RVA using a 26 G needle (Figure 2).
- Place the Doppler blood flow imaging probe above the RVA. Ensure that the Doppler blood flow imaging probe is placed 5 ± 1 mm from the site of ischemia and toward the distal end of the RVA. Take a flux reading for 2 min and 30 s (or longer if desired). This will be the normoxic phase reading.
- In the meantime, using a nose plier and toothed forceps, manually mold the spinal needle's edge into the shape of a hook (Figure 3). Do this by bending the edge of the spinal needle for approximately 1 mm. A larger size will make it more difficult to insert and remove the spinal needle during the I/R procedure.
- Insert the spinal needle directly beneath the right vitelline artery using a micromanipulator.
NOTE: Insert the spinal needle with extreme caution to avoid damaging the RVA or any adjacent arteries. The optimal technique is to adjust the spinal needle's custom-designed hook exactly above the right side of the RVA hole, followed by gradually inserting the spinal needle's custom-designed edge into the yolk sac with the assistance of a micromanipulator under the guidance of a stereo zoom surgical microscope through the right hole. Once the spinal needle's hook is in the yolk sac, gradually adjust the hook beneath the RVA so that its edge is exactly placed beneath the left hole. Now is the time to lift the spinal needle. - Now, with the assistance of the micromanipulator, gradually lift the artery until the Doppler blood flow flux indicates 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 (pulling the artery upward) for 5 min. This will be the period of ischemia in the RVA.
NOTE: It is critical to monitor the Doppler flux during the duration of ischemia. If a significant amount of fluctuation is found, terminate the tests. - After the 5 min ischemia period, gradually release the artery to restore normal blood flow levels. Ensure a Doppler blood flowmeter reading displays values comparable to those obtained during normoxia. This will be the period of reperfusion in the RVA (Figure 4).
- After the I/R procedure, apply a few drops (2-3) of 1x PBS to the embryo and watch it for 2-3 min.
NOTE: The use of 1x PBS helps prevent the embryo from drying out. - Finally, reseal the window with adhesive tape and place the egg back in the egg incubator for 5 h and 55 min.
- After 5 h and 55 min, take the egg from the egg incubator, place it on the egg rack, reopen the window, and follow the downstream treatment protocol.
5. Treatments
- For the treatment of the arteries with drugs, activators, or inhibitors, excise the RVA after 1 h of the I/R process.
- For the downstream studies, first remove the embryo from the eggshell by placing it on a sterile 90 mm Petri dish.
- Once the embryo is released into the Petri dish, excise the RVA using the ocular iris under the guidance of a stereo zoom surgical microscope.
- Ensure that the excision dimension of RVA is up to 15 ± 1 mm (distal from the trunk), 5 ± 1 mm each on the left and right side of the artery, and 2 ± 1 mm toward the trunk.
NOTE: A ruler can be utilized to measure the area to be excised (optional). - After excising the RVA, wash it with 1x PBS in a sterile Petri dish containing 1x PBS.
- For the desired treatments, place the artery in a 1.5 mL centrifuge tube (sterilized) filled with 500 µL of Ringer's solution. Place the RVA in the centrifuge tube and place it in a 37 °C incubator for 5 h and 55 min.
NOTE: Depending on the treatments, either use the Ringer's solution without any treatment, or treatment with the desired volume and concentration of drug, activator, or inhibitor. - After 5 h and 55 min of incubation, take out the RVA from the 37 °C laboratory incubator, and proceed with the desired treatments.
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Results
The Doppler Blood Flow Imaging technique was used to evaluate the effectiveness of our model. In short, we compared the data from the control group with the data from the RVA group to determine the success of our creation. Figure 4A depicts a typical flux associated with the control animal, while Figure 4B depicts the results obtained from an RVA. The numeric 1-8 represents the various events associated with I/R phases. In brief, numeric 1-3 correspond to the ph...
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Discussion
The goal of ischemia-reperfusion research is to create therapeutic strategies that prevent cell death and promote recovery29,30. To overcome current constraints in I/R research, we designed a Hook I/R chick embryo model to produce a reliable and reproducible I/R model. To our knowledge, ours is the first I/R model ever created in a 3-day chick embryo for routine I/R experiments, besides studying stress signals (e.g., oxidative and inflammatory stress). Given...
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Disclosures
The authors declare no competing interests.
Acknowledgements
We want to express our gratitude to Hari Shankar for his critical inputs during videography and editing, Mr. Baqer Hussain for voice-over, Mr. Asghar Rizvi for video editing, Mr. Mohammad Haider for video shoots, Mr. Mohammad Danish Siddiqui for assistance during the experiments.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| (-80°C) freezer | Haier, China | - | |
| 1.5mL Centrifuge tube | TARSONS, India | 500010X | |
| 100mm Petri dish (sterile) | Tarsons, India | 460050 | |
| 18G Needle (18G×1.5 (1.25×38mm) | Ramsons, India | 13990 | |
| 1mL Syringe | DISPO VAN | - | |
| 26G Needle (26G×1/2 (10.45x13mm) | DISPO VAN, india | 30722D | |
| 37°C egg incubator with adjustable percentage humidity | Gentek, India | GL-100 | |
| 37°C laboratory incubator | SCIENCE TECH, India | CB 101-14 | |
| 3-Methyladenine (3-MA) | Sigma Aldrich, USA | M9281 | |
| 3mL Pasture Pipette | TARSONS, India | 940050 | |
| 50mL Beaker | TARSONS, India | - | |
| 5mL Syringe | DISPO VAN, India | IP53 | |
| 70% ethanol | Merck Millipore, United States | 64-17-5 | |
| Adhesive tape/Cello tape | Sunrise, India | - | |
| Ambra1 primers | Applied Biosystems, Foster city, USA | Hs00387943_m1 | |
| Anti-mouse IgG | Cell Signaling Technology, USA | 7076S | |
| Anti-Rabbit IgG | Jackson Immuno Research Laboratories, USA | 711-035-152 | |
| Atg7 | R&D Systems, USA | MAB6608 | |
| Atg7 primers | Applied Biosystems, Foster city, USA | Hs00893766_m1 | |
| Autoclave Bag | Tarsons, India | 550022 | |
| Autoclave Machine | Local made, Lucknow, India | - | |
| Beclin-1 | Proteintech, USA | 66665-1-Ig | |
| Beta Actin | ImmunoTag, USA | ITT07018 | |
| Bovine Serum Albumin | Himedia, Mumbai, India | TC194 | |
| Calcium Chloride | Himedia, Mumbai, India | GRM534 | |
| Catalase | ImmunoTag, USA | ITT5155 | |
| Cleaning wipes | Kimberly-Clark, India | 370080 | |
| Cleaved Caspase3 | ImmunoTag, USA | ITT07022 | |
| di-Sodium hydrogen phosphate heptahydrate | Himedia, Mumbai, India | GRM39611 | |
| Doppler blood flowmeter | Moors instrument, United Kingdom | moorVMS-LDF1 | |
| Egg rack | - | - | |
| Egg rack | - | - | |
| GAPDH | ImmunoTag, USA | M1000110 | |
| GAPDH primers | Applied Biosystems, Foster city, USA | Hs02758991_g1 | |
| Glycine | Himedia, Mumbai, India | MB013 | |
| Kidney tray | HOSPITO | - | |
| LC3A/B | Cell Signaling Technology, USA | 4108S | |
| Methanol | Rankem laboratories, Mumbai, India | M0252 | |
| Micromanipulator | Narishige, Japan | M-152 | |
| N-acetyl-L-cysteine (NAC) | Sigma Aldrich, USA | A7250 | |
| Naringenin | Sigma Aldrich, USA | 67604-48-2 | |
| NF-kβ | Thermo Fisher Scientific, USA | 51-0500 | |
| NLRP3 | ImmunoTag, USA | ITT07438 | |
| Nose plier | Local made, Lucknow, India | - | |
| Ocular forceps | Stoelting, Germany | 52106-40 | |
| Ocular iris | Tufft Surgical Instruments, Jaipur, India | Hard Age Vannas Micro Scissors Angled 8CM / 3 1/8" | |
| OHP marker pen | Camlin, India | - | |
| ORP-150 | ImmunoTag, USA | ITT08329 | |
| Pointed sharp edge scissor | Stoelting, Germany | 52132-11 | |
| Potassium Chloride | Himedia, Mumbai, India | MB043 | |
| Potassium phosphate monobasic anhydrous | Himedia, Mumbai, India | MB050 | |
| Protease Inhibitor | Abcam, United States | Ab65621 | |
| SOD-1 | ImmunoTag, USA | ITT4364 | |
| Sodium Chloride | Fisher Scientific, Mumbai, India | 27605 | |
| Sodium dodecyl sulphate | Himedia, Mumbai, India | GRM886 | |
| Spinal needle 25GA; 3.50 IN (90.51 X 90mm) | Ramson, India | GS-2029 | |
| Stereo Zoom surgical microscope | Olympus, Japan | SZ2-STU3 | |
| Syringe discarder | BIOHAZARD | 882210 | |
| Toothed forceps | Stoelting, Germany | 52102-30 | |
| Tris Base | G Biosciences, United States | RC1217 | |
| Tris Hydrochloric Acid | Himedia, Mumbai, India | MB030 | |
| Tween 20 | G Biosciences, United States | RC1227 | |
| White Leghorn Chicken 0-day eggs | - | - | |
| Z-Val-Ala-Asp(OMe)-FMK | MP Biomedicals, LLC, USA | FK009 |
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