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

Summary

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.

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 disease^1,2,3,4,5. 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, restori....

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)

1. Prepare Ringer's solution
   1. 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 m....

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.......

Discussion

The goal of ischemia-reperfusion research is to create therapeutic strategies that prevent cell death and promote recovery^29,30. 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.......

Materials

List of materials used in this article

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