A Minimally Invasive Double Live-Knot Technique for Myocardial Ischemia-Reperfusion Injury Modeling in Rats

Summary

Here, we propose a scheme to induce myocardial ischemia-reperfusion injury by left anterior descending coronary artery (LAD)-targeted double active knot coil ligation, which can be released in vitro for reperfusion on demand without secondary thoracotomy and is minimally invasive.

Abstract

The myocardial ischemia-reperfusion injury (MI/RI) model is of great significance in exploring the pathophysiological mechanisms of myocardial infarction and facilitating the development of relevant drugs. However, among the commonly used modeling methods, the traditional polyethylene-tube-assisted coronary artery ligation method has significant drawbacks. It requires reopening the chest to untie the ligature, which is not only time-consuming but also causes substantial damage to the experimental animals and can easily lead to their death. The aim of this study is to introduce an improved model-preparation method. In this method, the Left Anterior Descending Coronary Artery (LAD) is ligated using a double live-knot coil, and the live-knot can be opened in vitro to achieve reperfusion, thus avoiding a second invasive procedure. Evaluation methods such as electrocardiogram (ECG) monitoring, M-mode echocardiography (ME) determination, Evans Blue and 2,3,5-triphenyte-trazoliumchloride staining (EB-TTC) showed that this method could induce stable MI/RI. This improved MI/RI model scheme is characterized by its simplicity and speed, and the modeling success rate is highly satisfactory. Overall, it offers practical and valuable guidance for researchers engaged in the field of MI/RI research.

Introduction

According to the World Health Organization, coronary heart disease holds a significant position in the global burden of cardiovascular diseases, characterized by high incidence, high mortality, and a high rate of complications. Moreover, the disease shows an increasingly younger trend. Statistics from some developed countries indicate that the incidence of coronary heart disease in the 35 to 40 year-old age group has increased by approximately 15%-20% over the past decade. Reperfusion methods such as drug thrombolysis, interventional therapy, and coronary artery bypass grafting are safe, effective, and widely used. However, the abrupt restoration of blood flow frequen....

Protocol

The experimental protocol was conducted in compliance with the Use of Laboratory Animals and Institutional Animal Care and Use Committee guidelines at ShaanXi University of Traditional Chinese Medicine (Record number: SUCML20240820006). All animal research data have been documented following the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. Male Sprague Dawley (SD) rats weighing 250 g ± 20 g and aged 6-8 weeks were utilized for this study. The specifics regarding the animals, reagents, and equipment employed are listed in the Table of Materials.

1. Preoperative preparation<....

Results

ECG
The electrocardiogram was recorded throughout the process from before the ligation of the rat heart to 120 min after ischemia-reperfusion. The electrocardiogram changes are shown in Figure 2A. During ischemia, the ST-segment showed an upward-arched elevation, and the T-wave was tall and peaked. After reperfusion, the elevation of the ST-segment gradually dropped back.

Discussion

Coronary artery ligation, a classic method for establishing the myocardial ischemia-reperfusion injury model, holds great significance in cardiovascular disease research¹². Since the 1980s, the polyethylene tube-assisted coronary artery ligation method, pioneered by Yamamoto et al., has been widely adopted in scientific research laboratories worldwide. This method offers several advantages, including controllable ligation force, visualized operation, and precise regulation of ischemia time. These .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 mL insulin syringe	Wuxi Yushou Medical Equipment Co.	https://e.tb.cn/h.6NJ9o4dBdpXt4bT?tk=YobMVflhtgI	Used to absorb EB and inject through the aortic arch
2,3,5-triphenyte-trazoliumchloride	Shanghai Yuanye Biotechnology Co., Ltd.	S19026	For heart staining
2.5 mL Disposable sterile syringe	Robust Ping An Medical Technology Co., Ltd.	20231230	Used to aspirate anaesthetics and anaesthetise rats by intraperitoneal injection; used to extract air from the thoracic cavity of rats during closed-chest operations.
4 % paraformaldehyde	Guangzhou Viggs Biotechnology Co., Ltd.	SW002	Used to fix the heart after staining
4-0 suture	Shandong Haidike Medical Products Co., Ltd.		For stitching ribs, muscles and skin
6-0 suture	Jiangsu Beiyueshen Medical Device Co., Ltd.	https://e.tb.cn/h.6NKQ9XNVD1J56np?tk=R8NKVfPJIUM	For ligation of LAD
Acupuncture needle	Beijing Luoya Shanchuan Medical Equipment Co.	https://e.tb.cn/h.6NQZzrSfBLQ9OYZ?tk=yW5aVfPuXP3	Used to pierce the skin of rat limbs and connect the electrocardiogram electrode line.
Animal hair remover	Jinyun New Concept Household Products Co.	https://e.tb.cn/h.6NJe67IEMgxPfUt?tk=9boIVflb6mm	For hair removal
Depilatory cream	Guangzhou Miyanshe Brand Management Co., Ltd.	https://3.cn/2fP-knZw	For deep hair removal
Ethanol	Tianjin Tianli Chemical Reagent Co., Ltd.	https://e.tb.cn/h.6NnxsK8EAvBV1Zu?tk=ja1XVflVhfM	For disinfection
Evans Blue	Sigma	E2129	For heart staining
Hemostatic forceps	Shanghai Aide Medical Instrument Co.	https://e.tb.cn/h.6NQkHWD25dxnsv4?tk=xA1VVflTXjW	Used to fix the insulin syringe and stop the blood of the aortic arch
Medical micro scissors	Nanjing Ji Xiaobao Trading Co.	https://e.tb.cn/h.6NJu33PgNRJCO1L?tk=smZjVflr730	Used to cut open the trachea and ribs
Medical microscopic tweezers	Nanjing Ji Xiaobao Trading Co.	https://e.tb.cn/h.6Nqn2dyJIUjUN6n?tk=MEMaVflGK0b	For pinching 6-0 suture to ligate LAD
Medical scalpel	Shandong Boryuida Analytical Instrument Co.	https://e.tb.cn/h.6NJyfb9CWH5Sj32?tk=HW0kVflEyOL	For cutting the heart during EB-TTC staining
Medical scissors	Shandong Boryuida Analytical Instrument Co.	https://e.tb.cn/h.6NJCLzckQnXXEPe?tk=CtcTVflFJQw	Used to cut rat skin and suture.
Medical surgical cloth	Henan Meideqi Medical Equipment Co.	https://e.tb.cn/h.6NqItN9yA0xttr9?tk=cCCIVflzFjr	Used to provide sterile surgical area
Medical tape	Zhejiang Aotai Medical Technology Co.	https://e.tb.cn/h.6N93etxLPPlJ9ZQ?tk=DfblVflAo5d	Used to fix rats
Medical tweezers	Jiangsu Videocon Medical Technology Co.	https://e.tb.cn/h.6NreHXQv2YTJkIl?tk=S1imVfO17QV	Used to judge the degree of anesthesia in rats, clamp skin, ribs and other tissues, and peel off subcutaneous muscles and pericardium.
Medical ultrasonic coupling agent	Qingdao Hainuo Biological Engineering Co., Ltd.	https://3.cn/2fPpwB-j	Used to improve the efficiency of ultrasonic transmission
Normal saline	Shandong Qidu Pharmaceutical Co., Ltd.	https://e.tb.cn/h.6NMCUFJIti2EsiU?tk=FXBBVfOV1iN	Used for washing residual depilatory cream
Portable color Doppler ultrasound	VINNO Technology Co., Ltd.	Vinno 6	Used for determination of M-mode echocardiography in rats
Povidone iodine	Shandong Anjie High-tech Disinfection Technology Co., Ltd.	https://e.tb.cn/h.6NqAb4WsT3xjMb9?tk=mhFxVfO4G1G	For disinfection
Rat surgical plate	Shandong Boryuida Analytical Instrument Co.	https://e.tb.cn/h.6NQyjjGNGInF3Eu?tk=qgQwVflHilH	Used to fix rats
Refrigeration refrigerator	Zhejiang Zhixing Ultra-low Temperature Technology Co.	DL-78	For freezing the heart
Silica gel tubes	Shandong Chengwu Sano Medical Equipment Co.	https://e.tb.cn/h.6N9L82XWNGlgDDG?tk=pWzCVfOQIgN	Used to support the trachea
Small animal artificial breathing machine	Anhui Zhenghua Biological Equipment Co., Ltd.	DW-3000H	Used to help rats breathe after thoracotomy
Sterile cotton ball	Qingdao Hainuo Biological Engineering Co., Ltd.	https://e.tb.cn/h.6NJbKVmD0b7QhTf?tk=wMceVfOTrBX	For dipping ethanol and povidone iodine disinfection
sterile cotton swab	Qingdao Hainuo Biological Engineering Co., Ltd.	https://e.tb.cn/h.6NMAQz1252EJHSM?tk=nyfLVfOUSBi	Used to clean the residual blood in the chest cavity
Thermal insulation blanket	Guangdong Zhongke Life Science & Technology Co.	https://e.tb.cn/h.6N9OyBVWuSXTUDy?tk=d6OqVfOSTlI	For postoperative body insulation in rats
Thoracotomy retractor	Guangdong Zhongke Life Science & Technology Co.	https://e.tb.cn/h.6NQra4Z9UG8dnvo?tk=6eFUVfllCFW	Used to expose the surgical field of vision
Type II lead electrocardiogram monitor	Chengdu Taimeng Technology Co., Ltd.	BL-420N	For monitoring electrocardiogram
Urethane	Shandong Keyuan Biochemical Co., Ltd.	U820333	Used in anesthetized rats
Water constant temperature oscillator	Shanghai Boxun Medical Biological Instrument Co., Ltd.	SHZ-B	For heart staining