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 frequently exacerbates the structural and functional damage to ischemic myocardium, giving rise to a cascade of adverse outcomes. These include cardiomyocyte apoptosis and necrosis, arrhythmias, and systolic dysfunction, ultimately culminating in myocardial ischemia-reperfusion injury (MI/RI)^1,2. The incidence of MI/RI injury is as high as 25%-30%, and its mechanism has not been fully elucidated³. Therefore, exploring effective prevention and treatment strategies has become a major issue that needs to be tackled in the cardiovascular field.

Animal models are of paramount importance in cardiovascular disease research. They serve as indispensable tools for elucidating the pathological mechanisms of MI/RI and for developing novel therapeutic strategies⁴. The reliability and stability of the myocardial ischemia-reperfusion injury animal model directly affect the successful translation and application of treatment strategies in clinical practice. The left anterior descending coronary artery (LAD) is a common vessel affected by myocardial ischemia⁵. The MI/RI model constructed by ligating the LAD is highly similar to the pathogenesis and pathological process of human myocardial ischemia-reperfusion injury⁶. Among these models, the traditional polyethylene-tube-assisted coronary artery ligation method has been extensively employed in prior studies. Its widespread use can be attributed to advantages such as uniform distribution of ligation force and a degree of protection for the vascular endothelium⁷. However, despite its many advantages, the operational details remain a focus of debate among many researchers.

For the traditional polyethylene-tube-assisted coronary artery ligation method, the chest needs to be reopened to untie the ligature during reperfusion. Currently, there are two main operation methods: the classic thoracotomy ligation method and the modified minimally invasive ligation method. The classic thoracotomy ligation method requires a large-scale incision of the chest to fully expose the heart for ligation⁶. The modified minimally invasive ligation method uses special instruments to complete the ligation through a smaller incision⁸. In the reperfusion stage, the classic thoracotomy ligation method requires reopening the chest to untie the ligature, which is not only time-consuming but also causes severe trauma to the experimental animals and is likely to lead to their death. In addition, repeated thoracotomy may trigger complications such as infection, interfering with the accuracy of experimental results^7,9. Moreover, since the degree of each thoracotomy operation is difficult to be completely consistent, it will increase the variability of experimental data. Regarding the application of the modified minimally invasive ligation method, despite its smaller incision, the procedure poses significant technical challenges. It demands that experimenters possess highly advanced technical proficiency and rely on high-end precision instruments, such as a stereomicroscope. When attempted in a confined space without the aid of such instruments, there is a heightened risk of inadvertently damaging surrounding tissues. This, in turn, can undermine the stability and reliability of experimental results.

Consequently, there is an urgent requirement for an enhanced model - preparation approach to tackle these challenges. This study presents an improved method for modeling MI/RI. The method involves using a double live - knot coil to ligate the LAD and enables in vitro release of the live-knot to achieve reperfusion, thereby obviating the need for a second thoracotomy. Characterized by its simplicity of operation and ease of mastery, this method can consistently induce myocardial ischemia-reperfusion injury, offering valuable reference and inspiration to researchers in the field of MI/RI.

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

1. 1.1.Perform fasting: Subject the rats to an 8-h fasting period with free access to water
2. Calibrate and weigh: Precisely measure the body weight using an electronic balance (0.1 g accuracy) to calculate the anesthetic dose.
3. Induce anesthesia: Administer 20% urethane solution via intraperitoneal injection at a dose of 0.4 mL per 100 g body weight using a sterile 2.5 mL syringe; inject 1 cm lateral to the ventral midline.
4. Confirm anesthesia: Clamp the hind paw with sterile forceps; absence of limb withdrawal reflex confirms deep anesthesia.

2. Electrocardiogram (ECG) monitoring (Figure 1, Step 1)

1. Immobilize the rat in a supine position: Secure the torso and limbs in a "limbs-extended posture" using medical adhesive tape on the surgical table.
2. Connect electrodes: Subcutaneously insert sterilized electrode needles in the following sequence: the right forelimb (connected to the white electrode), the right hindlimb (connected to the black electrode), and the left hindlimb (connected to the red electrode). Subsequently, attach these electrodes to a Lead II ECG monitor. Ensure that a stable baseline is established and that QRS complexes are clearly visible on the monitor.
   NOTE: Maintain continuous ECG monitoring throughout the procedure. Assess anesthesia depth and rat status in real-time via waveform abnormalities.

3. Surgical site depilation and disinfection (Figure 1, Step 2)

1. Mechanical hair removal: Use an electric clipper to remove fur from the neck and left thorax (residual length ≤0.5 mm).
   NOTE: Hold the blade at a 30° angle and move against hair growth direction.
2. Chemical depilation: Apply depilatory cream evenly over exposed skin for 2 min. Remove thoroughly with a sterile scraper, followed by pulsed flushing using a 2.5 mL syringe with 37 °C saline. Repeat 3 times until no residue remains.
3. Graded disinfection: Disinfect the skin three times in concentric circles (inner to outer) with a 75% ethanol-iodophor mixture (1:1) using sterile cotton balls.

4. Ventilator parameter setup

1. Preoperative equipment check: Connect the ventilator circuit to the Y-shaped tracheal tube adapter.
2. Adjust parameters ( Figure 1, Step 3): Set species-specific parameters: tidal volume 3.0 ml, respiratory rate 80 breaths/min, and inspiratory-expiratory ratio (I:E) 2:1.
3. Verify circuit patency: Activate the ventilator and immerse the circuit end in saline. Observe rhythmic bubble formation. If absent, systematically check for leaks and troubleshoot.

5. Tracheal intubation

1. Incision positioning: Drape the surgical field with sterile cloth. Lift the subthyroid cartilage skin with sharp forceps and make a 2.5 cm longitudinal incision along the trachea using straight scissors.
2. Layer-by-layer dissection: Retract the skin with sterile forceps (left hand) and perform superficial progressive dissection with microscissors (right hand).
   NOTE: Limit the first layer to epidermis and dermis; avoid muscle layer damage.
3. Tracheal exposure (Figure 1, Step 4): Use hemostatic forceps for blunt dissection of platysma and strap muscles to expose 4-5 tracheal cartilages. Mobilize the trachea with forceps and place a silicone tube beneath it for stabilization.
   NOTE: Carotid artery and vagus nerve can be seen on both sides of the trachea, so avoid damaging these structures during operation.
4. Tracheal intubation ( Figure 1, Step 5): Create a transverse Ω-shaped incision (approximately 1/3 circumference) between the 3rd and 4th tracheal cartilage rings using microscissors. Insert a 14 G silicone endotracheal tube rapidly, aligning the depth marker with the incision edge.
   NOTE: Post-intubation, synchronize thoracic movement with ventilator rhythm. Monitor airway pressure and respiratory frequency for abnormalities. Adjust ventilator settings or check tube patency if deviations occur.

6. Intercostal incision preparation

1. Mark the incision site: Use a sterile surgical marker to delineate the left sternal border at the 3rd-4th intercostal space (identified by palpating the point of maximal apical impulse). Draw a 3.0 cm longitudinal line.
2. Layer-by-layer dissection: Elevate the skin with sterile forceps and incise along the marked line using sterile scissors. Insert hemostatic forceps subcutaneously at a 30° angle and perform blunt dissection parallel to the intercostal space to separate the muscle layer.
3. Rib transection: Transect the 3rd and 4th ribs and intercostal muscles using microscissors.Note: If intercostal artery hemorrhage occurs, immediately apply a sterile cotton swab with firm pressure for 60 s to achieve hemostasis¹⁰.

7. Left anterior descending (LAD) artery localization and ligation

1. Confirm ligation site (Figure 1, Step 6): Expand the intercostal space to 4 cm using a sternal retractor to expose the heart. Gently dissect the pericardium (identify thymic tissue as the white structure above the heart). Locate the LAD 2 mm inferior to the midpoint of the "V" formed by the pulmonary conus (right auricular conical protrusion) and left auricle (triangular valvular structure).
2. Needle calibration(Figure 1, Step 7-1): Clamp a 6-0 polypropylene suture (with needle) using micro-forceps. Enter the myocardium 1 mm lateral to the mid-distal LAD junction at a 15° angle relative to the myocardial surface (depth: 1 mm; width: 2 mm), advancing from right to left.
3. Double slipknot technique (Figure 1, Step 7-1 - 7-5): After the ligature thread was passed through the myocardium, roll one end of the ligature thread with needle tip into a coil with microscopic forceps, and thread the other end into the coil and gently pull it to tighten the fixation. Knot the other end of the ligature thread again by repeating the above operation to form a double-knot structure, and making sure that the two ends of the ligature thread are left parallel to each other outside of the thoracic cavity.
   NOTE: During the tightening process, microtweezers were used to tighten gradually with gentle and even force, while myocardial colour changes were observed to determine the degree of ischemia, so as to avoid over-exertion leading to vessel rupture or insecure ligation. The successful establishment of the model was confirmed by observing the ischemic pallor of the anterior wall/apical region of the left ventricle and the sustained ST-segment elevation ≥ 0.2 mV on the electrocardiogram, and the 30-min ischemic timer was started immediately after confirming the successful ligation.

8. Suture and thoracic negative pressure restoration

1. Rib closure: Remove the sternal retractor. Close the intercostal muscles around ribs 3-4 using a 4-0 absorbable suture in a continuous locking pattern (2 mm stitch intervals).
   NOTE: Avoid contact between the ligation suture and closure sutures to prevent interference with reperfusion.
2. Muscle layer closure and air evacuation: Approximate the superficial pectoralis major and minor muscles with a 4-0 suture. Prior to final tightening, insert a needle-free 2.5 mL syringe into the last stitch hole to aspirate intrathoracic air while an assistant secures the suture.
   NOTE: Monitor thoracic movement for spontaneous respiration (as indicated by asynchronous rhythm relative to the ventilator).
3. Skin closure and disinfection: Suture the skin continuously with 4-0 suture. Disinfect three times following Protocol 3.3 guidelines.

9. Disengaging the ventilator

1. Extubation: Withdraw the endotracheal tube slowly while observing for spontaneous breathing.
2. Tracheal and muscle closure: Suture tracheal cartilage rings longitudinally using a 4-0 suture to ensure an airtight seal. Reapproximate neck muscles.
3. Skin closure and disinfection (Figure 1, Step 8): Close the neck skin with a 4-0 suture. Disinfect using ethanol- and povidone-iodine-soaked sterile swabs. Remove drapes and transfer the rat to a 38 °C heating pad for reperfusion.
   NOTE: If spontaneous respiration fails, re-insert the endotracheal tube and repeat Steps 8.1-8.3 before reattempting extubation.

10. Ischemia-reperfusion

1. Loosen the ligature (Figure 1, Step 9): After 30 min of ischemia, untie the first knot by pulling horizontally on the ligature wire at one end of the banding needle, then untie the second knot by pulling on the ligature wire at the other end, and then pulling horizontally from side to side to ensure that there is no resistance proving that the double knot had been successfully untied.
   NOTE: If EB-TTC double dyeing is required, the ligature line cannot be pulled out.
2. Reperfusion validation: Confirm ST-segment resolution on ECG as evidence of successful reperfusion¹².

11. M-mode echocardiography (ME) determination ¹⁰

1. Pre-procedure preparation: At 2 h post-reperfusion, assess anesthesia depth and supplement if necessary. Immobilize the rat supine and apply 2 mm of 37 °C ultrasound gel to the precordial region.
2. Data acquisition: Power on the ultrasound system. Select the preset protocol, set the MX250 probe to 18 MHz, and record M-mode images of left ventricular motion. Save images appropriately.
3. Data analysis: Calculate left ventricular ejection fraction (LVEF), fractional shortening (LVFS), end-systolic diameter (LVIDs), end-diastolic diameter (LVIDd), and stroke volume (SV) using system-integrated software.
   NOTE: Capture data from ≥3 consecutive cardiac cycles; report mean values for statistical rigor.

12. Evans Blue and 2,3,5-Triphenyte-trazoliumchloride (EB-TTC) staining

1. Re-intubation: Sequentially remove sutures from the neck skin, muscles, and trachea. Insert an endotracheal tube and connect it to the ventilator to maintain respiration.
2. Re-thoracotomy: Remove sutures from the chest skin, muscles, and ribs. Use a thoracic retractor to widen the intercostal space and fully expose the heart.
3. Religate the LAD artery: Re-ligate the LAD artery; confirm successful ligation by observing apical pallor and persistent ST-segment elevation on ECG.
4. Inject EB: Using a 0.5 mL insulin syringe, aspirate 0.3 mL of 1.5% Evans Blue solution. Then, quickly dissect the aortic arch, insert the syringe needle into the aorta, and secure it with a micro-hemostatic clamp. Next, inject the Evans Blue solution and observe the heart for uniform blue staining.
5. Excise the heart: After 30 s post-injection, excise the heart with sterile scissors. Immediately immerse it in saline and rinse repeatedly to remove residual blood and Evans Blue. Freeze the heart at -80 °C.
6. Section the heart: After 20 min of freezing, remove the heart. Using a surgical blade, cut five 1 mm-thick transverse slices perpendicular to the cardiac long axis from apex to base.
7. Stain with TTC: Allow the slices to equilibrate to room temperature. Transfer them into a 50 mL amber centrifuge tube containing 1.5% TTC solution. Incubate the tube in a preheated thermostatic shaker at 37 °C for 30 min under light-protected conditions.
8. Image acquisition and analysis: After staining, fix the slices in paraformaldehyde for 12 h. Capture high-resolution images and quantify the infarct area (IA), area at risk (AAR), and total left ventricular area (LV) using Image J software. Calculate the myocardial infarction ratio as IA/(AAR+IA) and the ischemia-risk ratio as (AAR+IA)/LV, following the methodology described by Cai et al¹².

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