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

Summary

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.

Abstract

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.

Introduction

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 disorders^2,3 and may serve as an independent risk predictor of cardiovascular disease^4,5,6,7. 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 neglected^7,9.

RVI, usually accompanied by inferior-posterior myocardial infarction^10,11, 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 mortality^12,13,14. RV function could recover spontaneously to a certain extent even in the absence of reperfusion^15,16. 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 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 period^19,20. 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 introduced^9,21.

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.

Protocol

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

1. Sterilize surgical instruments by autoclaving before the surgery. Adjust the heating pad to 37 °C.
2. 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.
3. 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.
4. 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 then drape the surgical field.
5. Perform intubation following the steps below.
   1. Adjust the breathing frequency of the animal with a mini ventilator (see Table of Materials) to 150/min and the tidal volume to 300 µL.
      ​NOTE: It is unnecessary to use positive end-expiratory pressure mode.
   2. 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.
   3. 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.

2. Permanent ligation of the right coronary artery

1. 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.
2. Open the chest.
   1. 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.
   2. 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.
   3. Incise the pericardium. Lift the right atrium with sterile cotton and ligate the RCA with a sterile 8-0 nylon thread with a ligation range of 3-5 mm. After ligating the RCA, the monitoring ECG (lead III) shows ST-segment elevation. 
      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.
3. Remove the sterile cotton and suture muscles and skin with a sterile 5-0 nylon thread to close the intercostal incision. Disinfect the skin again with 75% alcohol and single-house the mouse 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 pad. Analgesics such as buprenorphine (0.1 mg/kg body weight, subcutaneously injection) are required to reduce the animals' pain after the surgery. The expected complications are sinus bradycardia and atrioventricular block, and the mortality rate post-surgery is 10-20%.

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.

1. Anesthetize the mouse with 3% isoflurane via inhalation.
2. 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.
3. Monitor heart rate through ECG and maintain it between 450-550 beats/min by adjusting the anesthetic concentration between 1.5% and 3%.
4. Remove the hair from the mouse's chest with a depilatory cream and apply ultrasound gel to the skin of the chest.
5. 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° 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.
6. Move down the transducer vertically, maintaining its position over the upper abdomen and below the mouse'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.
7. 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.
8. 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.
   1. 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).
9. Tilt the left side of the platform at the lowest point. Keep the probe at a 30° angle to the horizontal axis along the right anterior axillary line. Rotate the x- and y-axes of the platform to display the RV.
   1. Press M-mode button and locate the indicator line at the septum's hyperechoic point to obtain the M-mode image of the RV interface. Press Cine store button to save the picture.
10. 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.
11. 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.

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.

1. Anesthetize the mouse with an intraperitoneal injection of 50 mg/kg of sodium pentobarbital (see Table of Materials).
2. After confirming the disappearance of the pedal withdrawal reflex, keep the mouse in the supine position and immobilize it with adhesive tape.
3. Shave the chest hair from the sternal angle to the xiphoid. Disinfect the operating area with 75% alcohol.
4. Perform tracheal intubation and set the parameter of the animal ventilator as described in steps 1.5.2-1.5.3.
5. 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.
6. Puncture the right ventricular free wall with a 32 G needle. Remove the needle and press the wound with cotton to stanch bleeding.
7. 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.
8. 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.
9. Remove the catheter after completion of recording and then place it inside normal saline solution.
10. Euthanize the mouse with an intraperitoneal injection of overdose pentobarbital sodium (150 mg/kg) and then sacrifice it by cervical dislocation.
11. Collect the heart and tibia for histological analysis.

5. Coronary vascular cast using a vascular casting agent

1. Heparinize the mouse with an intraperitoneal injection of 200 IU/mL of heparin sodium at 2000 IU/kg (see Table of Materials).
2. Anesthetize the mouse with an intraperitoneal injection of 50 mg/kg of sodium pentobarbital.
3. Place the animal supine on the pad and intubate for artificial ventilation following steps 1.5.2-1.5.3.
4. Open the chest with surgical scissors as described in step 4.5 and expose the heart.
5. 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.
6. 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.
7. Prepare the cast reagent by mixing the ingredients in the kit according to the manufacturer'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.
8. Perfuse the heart with 1 mL of cast reagent through the cardiac apex and wait for 2-3 h.
9. Erode the heart with 50% sodium hydroxide for 2-3 days and remove the muscle tissue or connective tissue by rinsing with normal saline.
10. 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.

Representative Results

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 τ (tau) 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).

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 ± SEM, *P < 0.01 vs. sham group, n = 6 per group (two independent sample t-test). Please click here to view a larger version of this figure.

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 ± SEM. *P < 0.01 vs. sham group, n = 6 per group (two independent sample t-test). LV, left ventricle; RVI, right ventricular infarction. Please click here to view a larger version of this figure.

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 (τ). *P < 0.01 vs. sham group, n = 6 per group (two independent sample t-test). Data are presented as mean ± SEM. RVI, right ventricular infarction; RVP, right ventricular pressure. Please click here to view a larger version of this figure.

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 µm) is an enlargement of the tissue in the left box (scale bar = 1 mm). *P < 0.01 vs. sham group, n = 6 per group (two independent sample t-test). Data are presented as mean ± SEM. RVI, right ventricular infarction. Please click here to view a larger version of this figure.

Discussion

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 reperfusion^16,24. 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 LCA^26,27. 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 disorder^9,10, 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 1^st 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.

Materials

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