Name: generation and characterization of right ventricular myocardial infarction induced by permanent ligation of the right coronary artery in mice
Uploaded: 2022-02-01T14:50:00.000+00:00
Duration: 10 min 18 s
Description: Watch this Scientific Journal Video about Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice at JoVE.com

This work was supported by grants from the National Natural Science Foundation of China (82073851 to Sun) and the National China Postdoctoral Science Foundation (2021M690074 to Lin).

All procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised in 1996) and were approved by the Animal Ethics Committee of Nanfang Hospital, Southern Medical University (Guangzhou, China). Healthy male C57BL/6J mice (8-10 weeks old; body weight, 25-30 g) were obtained from the Animal Center of Southern Medical University. Female mice can also be used, but mixing both genders is not recommended due to the potential influences of sex differences. After arrival, the mice were housed under a 12-h/12-h dark/light cycle (3-4 mice per cage), with ad libitum food and water.
1. Preparation for surgery
<ol>
	<li>Sterilize surgical instruments by autoclaving&#160;before the surgery. Adjust the heating pad to 37 &#176;C.</li>
	<li>Anesthetize the mice by an intraperitoneal injection of 50 mg/kg of pentobarbital (see Table of Materials) to relieve surgical pain. Place the mice in separate boxes for anesthesia induction. Ensure the depth of anesthesia by the absence of a toe-withdrawal response. 
	NOTE: It is also recommended to use 1.5% isoflurane for inhalation anesthesia because it is better for analgesia.</li>
	<li>Place the mice supine on the pad by fixing their incisors with a suture and immobilizing their limbs with adhesive tape. Ensure the depth of anesthesia again by checking the reflex.</li>
	<li>Remove hair from the neck to xiphoid with a depilatory cream. Disinfect the surgical area 3 times with alternating antiseptic scrub and 75% alcohol and&#160;then drape the surgical field.</li>
	<li>Perform intubation following the steps below.
	<ol>
		<li>Adjust the breathing frequency of the animal with a mini ventilator (see Table of Materials) to 150/min and the tidal volume to 300 &#181;L. 
		&#8203;NOTE: It is unnecessary to use positive end-expiratory pressure mode.</li>
		<li>Pull out the tongue slightly with tweezers, lift the mandible with a tongue depressor to expose the glottis, and perform intra-tracheal intubation by inserting a 22 G cannula into the glottis.</li>
		<li>Switch on the mini ventilator and connect the tracheal cannula to the ventilator. The phenomenon of thoracic undulation becoming equal to the ventilator frequency indicates successful intubation. Fix the cannula with tape to prevent slipping during the operation.</li>
	</ol>
	</li>
</ol>
2. Permanent ligation of the right coronary artery
<ol>
	<li>Connect the electrocardiography (ECG) electrodes (see Table of Materials) to the mouse limbs correctly and record the ECG. 
	NOTE: One of the II, III, or AVF lead is selected as a monitoring lead; Lead III is more appropriate.</li>
	<li>Open the chest.
	<ol>
		<li>Make a 1 cm long incision in the skin parallel to the third right rib with ophthalmic scissors. Determine the third intercostal again and ensure adequate space according to the sternum angle. 
		NOTE: The direction of the skin incision is made from the sternum angle to the right anterior axillary line.</li>
		<li>Separate and cut the pectoralis major and pectoralis minor muscles with scissors and micro forceps above the third intercostal space. After that, bluntly separate the intercostal muscle with elbow forceps to expose the surgical field. 
		NOTE: Only a small part of pectoral muscles needs to be cut, and then a blunt separation is recommended to expose the heart.</li>
		<li>Incise the pericardium. Lift the right atrium with sterile cotton and ligate the RCA with a sterile 8-0&#160;nylon thread with a ligation range of 3-5 mm. After ligating the RCA, the monitoring ECG (lead III) shows ST-segment elevation.&#160; 
		NOTE: Because the mouse RCA is invisible, its anatomic location must be carefully confirmed. The myocardium of the RV is much thinner than that of the LV. Therefore, it is difficult to grasp the depth of the inserted needle. It is easy to induce sinus bradycardia and atrioventricular block if the depth of the inserted needle is too deep and the ligation range is too large.</li>
	</ol>
	</li>
	<li>Remove the sterile cotton and&#160;suture muscles and skin with a sterile 5-0 nylon thread to close the intercostal incision.&#160;Disinfect the skin again with 75% alcohol and&#160;single-house the mouse&#160;after surgery. 
	NOTE: The well-sutured muscle is important for avoiding aerothorax. A sterile drainage tube is placed in the chest cavity until the completion of chest closing, and then the chest cavity is evacuated by an injection syringe connecting the drainage tube. 
	NOTE: After surgery, mice are placed on a heating&#160;pad. Analgesics such as buprenorphine (0.1 mg/kg body weight, subcutaneously injection) are required to reduce the animals&#39; pain after the surgery. The expected complications are sinus bradycardia and atrioventricular block, and the mortality rate post-surgery is 10-20%.</li>
</ol>
3. Echocardiographic assessment of the RV function after surgery
NOTE: For echocardiography, use an MS400D probe with a center frequency of 30 MHz, connected to a high-resolution ultrasound imaging system (see Table of Materials). The echocardiography examination is performed 4 weeks after surgery.
<ol>
	<li>Anesthetize the mouse with 3% isoflurane via inhalation.</li>
	<li>Place the mouse in the supine position on an ultrasonic platform for animal fixation and ultrasonic operation. Tape its claws to the electrode to obtain an ECG recording through a system attached to the ultrasonic machine.</li>
	<li>Monitor heart rate through ECG and maintain it between 450-550 beats/min by adjusting the anesthetic concentration between 1.5% and 3%.</li>
	<li>Remove the hair from the mouse&#39;s chest with a depilatory cream and apply ultrasound gel to the skin of the chest.</li>
	<li>Set the platform to the horizontal position. Orient the transducer parallel to the left leg and obtain the left ventricular long-axis image. Rotate the probe 90&#176; clockwise to obtain the LV short-axis view. Press the Cine store button to save the images. 
	NOTE: The upper-left of the platform is tilted at the lowest point. The LV short-axis rotation angle of the transducer is maintained while the transducer is oriented toward the right shoulder of the mouse.</li>
	<li>Move down the transducer vertically, maintaining its position over the upper abdomen and below the mouse&#39;s diaphragm under B-mode. Adjust the platform position slightly by rotating its x- and y-axes until the RV, right atrium (RA), left atrium (LA), and LV are clearly seen on the screen. Save apical four-chamber images by pressing the Cine store or Frame store button. 
	NOTE: B-mode is used to show the two-dimension (2D) view of the heart.</li>
	<li>Press M-mode; after the 2x indicator line appears, locate the indicator line at the tricuspid valve orifice to obtain the movement of the tricuspid annular plane. Press the Cine store or Frame store button to save data and images. 
	NOTE: M-mode means motion mode, which reveals the motion of the heart or vessel in a curve form.</li>
	<li>Press Measure button to enter measurement mode. Click on Area measurement button to zone into RV and LV. Calculate the area of RV and LV to obtain the area ratio of RV to LV.
	<ol>
		<li>Click on Timeline button and make two baselines to define the movement range of the tricuspid annular plane during the systolic and diastolic periods. Click on Distance button and measure the distance between two baselines to obtain tricuspid annular plane systolic excursion (TAPSE).</li>
	</ol>
	</li>
	<li>Tilt the left side of the platform at the lowest point. Keep the probe at a 30&#176; angle to the horizontal axis along the right anterior axillary line. Rotate the x- and y-axes of the platform to display the RV.
	<ol>
		<li>Press M-mode button and locate the indicator line at the septum&#39;s hyperechoic point to obtain the M-mode image of the RV interface. Press Cine store button to save the picture.</li>
	</ol>
	</li>
	<li>Open the M-mode image of the RV interface, press Measure button to enter measurement mode. Measure the RV inner distance at the end of diastole (RVIDd), RV ejection fraction (RVEF), and RV fraction shortening (RVFS) using the in-built measurement tool of the echocardiographic system.</li>
	<li>Stop administering isoflurane and place the mouse on the heating pad for 3-5 min until it regains consciousness. After that, return the mouse to its cage with 12 h light/dark cycle.</li>
</ol>
4. Invasive measurements of RV hemodynamic
NOTE: RV hemodynamic is assessed through right heart catheterization 4 weeks after RVI. A 1.0 F catheter together with a monitoring system is applied.
<ol>
	<li>Anesthetize the mouse with an intraperitoneal injection of 50 mg/kg of sodium pentobarbital (see Table of Materials).</li>
	<li>After confirming the disappearance of the pedal withdrawal reflex, keep the mouse in the supine position and immobilize it with adhesive tape.</li>
	<li>Shave the chest hair from the sternal angle to the xiphoid. Disinfect the operating area with 75% alcohol.</li>
	<li>Perform tracheal intubation and set the parameter of the animal ventilator as described in steps 1.5.2-1.5.3.</li>
	<li>Make a 1 cm bilateral incision on the skin above the xiphoid process and transect the diaphragm and rib with ophthalmic scissors to expose the heart.</li>
	<li>Puncture the right ventricular free wall with a 32 G needle. Remove the needle and press the wound with cotton to stanch bleeding.</li>
	<li>Insert the tip of the catheter into the right ventricle through the puncture site and push the catheter forward slowly. Adjust the position of the tip to obtain a typical RV pressure waveform shown on a monitor and recording system. 
	NOTE: Right jugular vein is also an appropriate route for hemodynamic measurement.</li>
	<li>After 10 min of stabilization, record the data of RV systolic blood pressure (RVSBP), RV end-diastolic pressure (RVEDP), and RV dP/dt. Click on Select button to select cardiac cycles for calculation and then click on Analyze button to calculate the mean values of the selected cycles.</li>
	<li>Remove the catheter after completion of recording and then place it inside normal saline solution.</li>
	<li>Euthanize the mouse with an intraperitoneal injection of overdose pentobarbital sodium (150 mg/kg) and then sacrifice it by cervical dislocation.</li>
	<li>Collect the heart and tibia for histological analysis.</li>
</ol>
5. Coronary vascular cast using a vascular casting agent
<ol>
	<li>Heparinize the mouse with an intraperitoneal injection of 200 IU/mL of heparin sodium at 2000 IU/kg (see Table of Materials).</li>
	<li>Anesthetize the mouse with an intraperitoneal injection of 50 mg/kg of sodium pentobarbital.</li>
	<li>Place the animal supine on the pad and intubate for artificial ventilation following steps 1.5.2-1.5.3.</li>
	<li>Open the chest with surgical scissors as described in step 4.5 and expose the heart.</li>
	<li>Make a 3 mm notch with ophthalmic scissors on the right atria and perfuse the heart with 5 mL of normal saline through the cardiac apex with an injector.</li>
	<li>Block the blood from the aorta with an aortic clamp and perfuse 0.1 mL of nitroglycerin (1 mg/mL) through the cardiac apex with an injector to dilate the coronary artery.</li>
	<li>Prepare the cast reagent by mixing the ingredients in the kit according to the manufacturer&#39;s instructions (see Table of Materials). 
	NOTE: It is recommended to prepare the cast reagent and perfusion with normal saline and nitroglycerin simultaneously to prevent microvascular closure.</li>
	<li>Perfuse the heart with 1 mL of cast reagent through the cardiac apex and wait for 2-3 h.</li>
	<li>Erode the heart with 50% sodium hydroxide for 2-3 days and remove the muscle tissue or connective tissue by rinsing with normal saline.</li>
	<li>Take pictures under a camera. 
	CAUTION: The cast reagent is harmful to the eyes, skin, and respiratory tract. Sodium hydroxide is corrosive. Wearing protective gloves, goggles, and a lab coat is required. The cast reagent must be prepared in a fume hood.</li>
</ol>

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The authors have nothing to disclose.

Sicard and colleagues from France first reported a mouse model of RVI in 2019, which described the surgical process and focused on the interaction between LV and RV after RVI9. However, to date, no study has reported using this model for further studies. A more detailed procedure would be helpful for researchers to use the mouse model of RVI for investigation. In contrast to the report by Sicard et al.9, we provided step-by-step information for model generation and strategy for quality control and further evaluated the anatomical distribution of RCA, RV hemodynamics, and the survival of cardiomyocytes in the infarct area. A recent report demonstrated that cardiomyocytes in the infarct area play an essential role in myocardial regeneration23. The RV function in patients with RVI would recover spontaneously within 3-12 months, even without reperfusion16,24. These findings suggest that the mouse RVI model would help search for potential therapeutic targets for right heart failure or cardiac regeneration. Therefore, it is necessary to popularize the model.
Due to the invisibility of RCA and the variation of RCA distribution, it would be difficult for the junior operators to generate RVI models with stable infarct sizes. To overcome this limitation, controlling the ligation level and range and ensuring sufficient elevation of ST-segment in II or III lead of ECG is recommended. The most critical step for successfully generating a mouse RVI model is to locate the anatomical structure of RCA. As shown in Figure 1A, mouse RCA may contain a primary or several parallel arteries; thus, the infarct size depends on how many arteries are blocked. Therefore, intraoperatively, the position of the RCA can be confirmed according to the anatomical characteristics of neighboring the right atrium and the visible vein. RVI mice usually exhibit myocardial infarction in the free wall of RV. Still, the septum can also be seldomly involved if the septal artery originates from the RCA, as shown in Figure 4D. The septum can be irrigated in mice by its own septal coronary artery branch25 or a branch of RCA or LCA26,27. After ligating the RCA, the classical ECG change of ST-segment elevation in EEG leads II or III leads is the gold standard to judge the success of RVI.
Since RV dilation induced by RCA ligation would increase intrapericardial pressure and then restrain cardiac filling, which would result in aggravation of hemodynamic disorder9,10, the pericardium should be torn apart during the operation. In contrast to the high incidence of cardiac rupture in mice with LCA ligation, no cardiac rupture was observed in the RVI mice. However, surgical mortality due to bleeding and atrioventricular block could be as high as 50% for beginners, which can be avoided by decreasing the piercing depth of needle stitch and myocardial range of suture ligation, lowering the ligation position, and gentle manipulation. Experienced technicians in our laboratory can complete the generation of an RVI mouse model in about 30 min with an 80%-90% success rate calculated by the survival proportion of mice with significant infarct size. Operation success was judged by instant elevation of ST-segment in Lead II or III of ECG after RCA ligation, TTC negative staining of myocardium in the 1st 24 h after surgery, and RV dilation measured by echocardiography at 3 days or 1 week after surgery. ST elevation in ECG leads of inferior wall and echocardiographic dilation of RV at 3 days after surgery may be used as the inclusion criteria for studies using mouse RVI model. 
During the 4-week follow-up period, quite a few surviving cardiomyocytes were observed in the infarct area of RVI mice, which may be a reasonable basis for regenerative research. RV remodeling and dysfunction recovery at 4 weeks were not noted after RVI in this model, suggesting that this model is also feasible for basic research on right heart remodeling and failure.

The right ventricle (RV), long thought to be a simple tube connected to the pulmonary artery, has been wrongfully neglected for many years1. However, there has been an increasing interest in RV function recently since it plays an essential role in hemodynamic disorders2,3 and may serve as an independent risk predictor of cardiovascular disease4,5,6,7. RV diseases include RV infarction (RVI), pulmonary artery hypertension, and valvular disease8. In contrast to the immense interest in pulmonary artery hypertension, RVI has remained neglected7,9.
RVI, usually accompanied by inferior-posterior myocardial infarction10,11, is caused by right coronary artery (RCA) occlusion. According to clinical investigations, severe RVI likely induces hemodynamic disturbances and arrhythmias, such as hypotension, bradycardia, and atrioventricular block, associated with higher hospital morbidity and mortality12,13,14. RV function could recover spontaneously to a certain extent even in the absence of reperfusion15,16. Several morphological and functional differences exist between the left ventricle (LV) and RV17. RV is believed to be more resistant to ischemia than LV8, partially due to the more extensive collateral circulation formation after RVI. Clarifying the differences between LV infarction (LVI) and RVI and identifying the underlying mechanisms would provide new therapeutic targets for cardiac regeneration and ischemic heart failure. However, owing to the difficulty associated with&#160;RVI mouse model generation, basic research on RVI is mainly limited.
A large animal model of RVI has been generated by ligating RCA in swine18, which is easier to operate because of the visible RCA. Compared with the large animal model, the mouse model has the following advantages: more accessibility in gene manipulation, lower economic cost, and shorter experimental period19,20. Although a mouse RVI model focusing on the influence of RVI on LV function was reported previously, the detailed steps of the procedure, the difficulties and key points of operation, and the model characteristics such as hemodynamic changes were not fully introduced9,21.
This article provides detailed surgical procedures for generating a mouse model of RVI. Moreover, this model was characterized by echocardiographic measurement, invasive hemodynamic evaluation, and histological analysis. Furthermore, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. The technique introduced in this paper would help beginners to quickly grasp the generation of the mouse RVI model with acceptable operation mortality and reliable evaluation approaches. The mouse model of RVI would help research the mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

<table><tbody><tr><td>2,3,5-triphenyltetrazolium chloride</td><td>Sigma</td><td>T8877</td><td>For TTC staining</td></tr><tr><td>Animal Mini Ventilator</td><td>Havard</td><td>Type 845</td><td>For artificial ventilation</td></tr><tr><td>Animal ultrasound system VEVO2100</td><td>Visual Sonic</td><td>VEVO2100</td><td>Measurement for Doppler flow velocity and AS plaque</td></tr><tr><td>Batson&amp;rsquo;s #17 Anatomical Corrosion Kit</td><td>Polyscience Inc</td><td>7349</td><td>For vasculature casting</td></tr><tr><td>buprenorphine</td><td>Isoreag</td><td>1134630-70-8</td><td>For reduce the pain of mice after surgery</td></tr><tr><td>C57BL/6J mice + D29A1A2:D27</td><td>Animal Center of South Medical University</td><td>-</td><td>For the generation of mouse RVI model</td></tr><tr><td>Camera</td><td>Sangnond</td><td></td><td>For taking photograph</td></tr><tr><td>Cold light illuminator</td><td>Olympus</td><td>ILD-2</td><td>Light for operation</td></tr><tr><td>electrocardiograph</td><td>ADI Instrument</td><td>ADAS1000</td><td>For recording electrocardiogram</td></tr><tr><td>hair removal cream</td><td>Reckitt Benchiser</td><td>RQ/B 33 Type 2</td><td>Remove mouse hair</td></tr><tr><td>Heat pad- thermostatic surgical system (ALC-HTP-S1)</td><td>SHANGHAI ALCOTT BIOTECH CO</td><td>ALC-HTP-S1</td><td>Heating</td></tr><tr><td>Hematoxylin-eosin dye</td><td>Leagene</td><td>DH0003</td><td>Hematoxylin-eosin staining</td></tr><tr><td>Heparin sodium salt</td><td>Macklin</td><td>H837056</td><td>For heparization</td></tr><tr><td>Isoflurane</td><td>RWD life science</td><td>R510-22</td><td>Inhalant anaesthesia</td></tr><tr><td>Lab made spatula</td><td></td><td></td><td>Work as a laryngoscope</td></tr><tr><td>Lab made tracheal cannula</td><td></td><td></td><td>For intubation</td></tr><tr><td>Matrx VIP 3000 Isofurane Vaporizer</td><td>Midmark Corporation</td><td>VIP 3000</td><td>Anesthetization</td></tr><tr><td>Medical nylon suture (5-0)</td><td>Ningbo Medical Needle Co.</td><td>5-0</td><td>For chest close</td></tr><tr><td>Microsurgical elbow tweezers</td><td>RWD life science</td><td>F11021-11</td><td>For surgery</td></tr><tr><td>Microsurgical scissors</td><td>NAPOX</td><td>MB-54-1</td><td>For arteriotomy</td></tr><tr><td>Millar Catheter</td><td>AD Instruments, Shanghai</td><td>1.0F</td><td>Measurement of pressure gradient</td></tr><tr><td>MS400D ultrasonic probe</td><td>Visual Sonic</td><td>MS400D</td><td>Measurement for Doppler flow velocity and AS plaque</td></tr><tr><td>needle forceps</td><td>Visual Sonic</td><td>F31006-12</td><td>For surgery</td></tr><tr><td>nitroglycerin</td><td>BEIJING YIMIN MEDICINE Co</td><td></td><td>For dilating coronary artery</td></tr><tr><td>Ophthalmic scissors</td><td>RWD life science</td><td>S11022-14</td><td>For surgery</td></tr><tr><td>Pentobarbital sodium salt</td><td>Merck</td><td>25MG</td><td>Anesthetization</td></tr><tr><td>PowerLab Multi-Directional Physiological Recording System</td><td>AD Instruments, Shanghai</td><td>4/35</td><td>Pressure recording</td></tr><tr><td>Precision electronic balance</td><td>Denver Instrument</td><td>TB-114</td><td>Weighing scale</td></tr><tr><td>Silk suture (8-0)</td><td>Ningbo Medical Needle Co.</td><td>6-0</td><td>coronary artery ligation</td></tr><tr><td>Small animal microsurgery equipment</td><td>Napox</td><td>MA-65</td><td>Surgical instruments</td></tr><tr><td>tissue forceps</td><td>Visual Sonic</td><td>F-12007-10</td><td>For surgery</td></tr><tr><td>tissue scissor</td><td>Visual Sonic</td><td>S13052-12</td><td>Open chest for hemodynamic measurement</td></tr><tr><td>Transmission Gel</td><td>Guang Gong pai</td><td>250ML</td><td>preparation for Echocardiography measurement</td></tr><tr><td>Vascular Clamps</td><td>Visual Sonic</td><td>R31005-06</td><td>For blocking blood from aorta</td></tr></tbody></table>

generation and characterization of right ventricular myocardial infarction induced by permanent ligation of the right coronary artery in mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

In this study, mice were randomly assigned to the RVI (n = 11) or sham operation (n = 11) group. The coronary cast in 2 normal mouse hearts is shown in Figure 1A. In response to RCA ligation, ST-segment elevation was seen in lead III of the ECG (Figure 1B). Moreover, 2,3,5-triphenyl tetrazolium chloride (TTC) staining showed that the infarct area accounts for 45% of the RV free wall at 24 h postoperatively (Figure 1C,D). The above data indicated the successful generation of the RVI mouse model.
Recordings of the 4-chamber apex view (Figure 2A) and 2-chamber view at LV short axis and the corresponding M-mode echocardiography (Figure 2B) measurements were performed at 4 weeks after the surgery to evaluate the RV remodeling and function. Compared with that in the sham group, the RV internal dimension at the end of diastole (RVIDd) increased in the RVI group (Figure 2C), and it was more than 2 times in the sham group (Figure 2A). RV ejection fraction (RVEF), RV fraction shortening (RVFS), and tricuspid annular plane systolic excursion (TAPSE) were significantly smaller in the RVI group than in the sham group (Figure 2D-F). The RV/LV area ratio increased by approximately 50% relative to the sham group (Figure 2G).
The mice were subjected to RV hemodynamic measurement 4 weeks after surgery. In the RVI group, RVSBP, dp/dt max, dp/dt min, and RV contractility were significantly smaller. At the same time, RVEDP and &#964;&#160;(tau)&#160;index were considerably more significant than those in the sham group (Figure 3A-E).
Four weeks after the surgery, the mice were sacrificed. An RV aneurysm was visible in the infarcted area (Figure 4A). The heart weight to body weight (HW/BW) ratio and heart weight to tibia length (HW/TL) ratio in the RVI group were slightly larger (without statistical significance) than those in the sham group (Figure 4B,C). Masson staining22 indicated significant fibrosis in the RV-free wall, and seldomly fibrosis occurred in the septum in the RVI group (Figure 4D,E). In contrast, a few surviving cardiomyocytes were in the infarct area (Figure 4F).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63508/63508fig01.jpg" /> 
Figure 1: Electrocardiography (ECG) changes and infarct size after ligation of the right coronary artery (RCA). (A) Representative images of mouse coronary vascular cast. Scale bar = 4 mm. (B) Lead III ECG change in response to RCA ligation. (C) Representative pictures of 2,3,5-triphenyl tetrazolium chloride (TTC) staining (white indicates infarct area, red indicates viable tissue). Scale bar = 4 mm. (D) Quantitation of myocardial infarct size of RVI mice. Data are presented as mean &#177; SEM, *P &#60; 0.01 vs. sham group, n = 6 per group (two independent sample t-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63508/63508fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63508/63508fig02.jpg" /> 
Figure 2: Echocardiography assessment of right ventricular (RV) remodeling and function in mice subjected to RCA ligation. (A) Representative B-mode images in four-chamber view 4 weeks after RCA ligation; scale bar = 2 mm. (B) Typical pictures of B-Mode at right ventricle interface (upper) and the corresponding M-Mode (lower) showing both LV and RV at 4 weeks after RCA ligation; scale bar = 2 mm. (C) RV internal dimension at the end of diastole (RVIDd). (D) RV fraction shortening (RVFS). (E) RV ejection fraction (RVEF). (F) Tricuspid annular plane systolic excursion (TAPSE). (G) RV/LV area ratio. Data are presented as mean &#177; SEM. *P &#60; 0.01 vs. sham group, n = 6 per group (two independent sample t-test). LV, left ventricle; RVI, right ventricular infarction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63508/63508fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63508/63508fig03.jpg" /> 
Figure 3: Right ventricular (RV) hemodynamics at 4 weeks after right coronary artery ligation. (A) Representative pressure curves were obtained with a pressure catheter. (B) Right ventricular systolic blood pressure (RVSBP) and right ventricular end-diastolic pressure (RVEDP). (C) The maximum and minimum rising rate of RV pressure (dp/dt max, dp/dt min). (D) RV contractility. (E) The exponential time constant of RV relaxation (&#964;). *P &#60; 0.01 vs. sham group, n = 6 per group (two independent sample t-test). Data are presented as mean &#177; SEM. RVI, right ventricular infarction; RVP, right ventricular pressure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63508/63508fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63508/63508fig04.jpg" /> 
Figure 4: Histological results at 4 weeks after RVI. (A) Pictures of whole representative heart from sham and RVI group (red circle indicates infarct wall; scale bar = 3 mm). (B) Heart weight to body weight ratio (HW/BW), P = 0.0536 between RVI and sham group. (C) HW to tibia length ratio (HW/TL), P = 0.1682 between RVI and sham group. (D) Represent pictures of hematoxylin-eosin staining and Masson staining of heart sections (scale bar = 3 mm). (E) Quantitative results of myocardial fibrosis. (F) Representative Masson staining pictures showing survival cardiomyocytes in the infarct area (the right picture (scale bar = 100 &#181;m) is an enlargement of the tissue in the left box (scale bar = 1 mm). *P &#60; 0.01 vs. sham group, n = 6 per group (two independent sample t-test). Data are presented as mean &#177; SEM. RVI, right ventricular infarction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63508/63508fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice at JoVE.com

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...

generation-characterization-right-ventricular-myocardial-infarction

Nanyang Technological University

Research

JoVE Journal

Medicine

3.0K Views.  The First Clinical College of Dalian Medical University. Rechterventrikel is beter bestand tegen ischemie dan de linker. We introduceerden een techniek om een rechterventrikelinfectiemodel te genereren, dat het pathofysiologische mechanisme kan verduidelijken tijdens de ontwikkeling van een rechterventrikelinfarct. We vonden de juiste kransslagader door middel van vasculaire kostenberekening en definieerden het ligatiegebied dat van cruciaal belang is voor het succesvol genereren van een muis rechterventrikelinfarctmodel.Linhai Ruo, een post...

Video: Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Biology

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In order to explore the pathophysiological changes after myocardial infarction and design future treatments, research models of MI are required. Permanent ligation of the left coronary artery (LCA) in mice is a popular model to investigate cardiac function and ventricular remodeling post MI. Here we describe a less invasive, reliable, and reproducible surgical murine MI model by permanent ligation of the LCA. Our surgical model comprises of an easily reversible general anesthesia, endotracheal intubation that does not require a tracheotomy, and a thoracotomy. Electrocardiography and troponin measurement should be performed to ensure MI. Echocardiography at day 28 after MI will discern heart function and heart failure parameters. The degree of cardiac fibrosis can be evaluated by Masson's trichrome staining and cardiac MRI. This MI model is useful for studying the pathophysiological and immunological alterations after MI.

Presented here is a surgical procedure for permanent ligation of the left coronary artery in mice. This model can be used to investigate the pathophysiology and associated inflammatory response after myocardial infarction.

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In ...

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI. 
The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson's trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected. 
In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson's trichrome.

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

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