Name: induction of right ventricular failure by pulmonary artery constriction and evaluation of right ventricular function in mice
Uploaded: 2019-05-13T13:30:00
Description: Watch this Scientific Journal Video about Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice at JoVE.com

This work was supported by grants from the National Natural Science Foundation of China (81570464, 81770271; to Dr. Liao) and the Municipal Planning Projects of Scientific Technology of Guangzhou (201804020083) (to Dr. Liao).

All procedures were performed in accordance with institutional guidelines for animal research, which conform 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). C57BL/6 male mice (8-10 weeks old, weighing 20-25 g) were provided by the Animal Center of South Medical University. After arrival, the mice were housed under a 12/12 h dark/light cycle, with sufficient food and water.
1. Preparation of th...

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Pathological increases in RV filling pressures result in a leftward shift of the septum, which can alter the LV geometry21. These changes contribute to reduced cardiac output and LV ejection fraction (LVEF), which can cause a hemodynamic disorder of the circulatory system22. Therefore, an efficient, stable, and economical model for studying the mechanism of RVF is valuable.
We developed a more effective and highly reproducible approach to PAC usi...

RV dysfunction (RVD), defined here as evidence of an abnormal RV structure or function, is associated with poor clinical outcomes. RVF, as the end stage of RV function, is a clinical syndrome with signs and symptoms of heart failure that result from progressive RVD1. With differences in structure and physiological function, left ventricular (LV) failure and RVF have different pathophysiological mechanisms. A few independent pathophysiological mechanisms in RVF have been reported, including overexpression of &#946;2-adrenergic receptor signaling2, inflammation3, transverse tubule remodeling, and Ca2+ handling dysfunction4.
RVF can be caused by volume or pressure overload of the RV. Previous animal models have used SU5416 (a potent and selective inhibitor of the vascular endothelial growth factor receptor) combined with hypoxia (SuHx)5,6 or monocrotaline7 to induce pulmonary hypertension, which results in RVF secondary to pulmonary vascular disease2. The researchers conducting these studies focused on the vasculature instead of the pathological progression of RVF. Moreover, monocrotaline has extra-cardiac effects that cannot precisely represent cardiogenic disease. Other models have used arteriovenous shunts to induce volume overload and RVF8. However, this surgery is difficult to perform and inappropriate for mice, who require long induction periods for the production of RVF.
Rat PAC models using banding clips also exist9,10. Compared with rats, mice have many advantages as animal models of cardiac diseases, such as easier reproduction, more widespread use, reduced costs, and access to gene modification11. However, the diameters of the banding clips usually range from 0.5 mm to 1.0 mm, which are too large for mice9. In addition, the banding clip is hard to produce, imitate, and popularize in other labs.
We provide a protocol to develop a modified reproductive RVF mouse model based on reported studies, which uses PAC to mimic the tetralogy of Fallot and Noonan syndrome or other pulmonary arterial hypertensive diseases12,13,14,15,16,17,18,19. This PAC approach is created by ligating the pulmonary trunk of mice using a latch and padding needle made inhouse to control the degree of constriction. The latch needle is made of a 90&#176; curved injection syringe with a braided silk suture passed through the syringe. The needle is made from common materials using a process that is easy to master. The padding needle is curved 120&#176; from the gauge needle. Padding needles with different diameters (0.6-0.8 mm) are used, depending on the mice&#39;s weight (20-35 g). Additionally, we establish an evaluation criterion to determine the stability and quality of the RVF model by echocardiography and right heart catheterization. We use mice as the model animal because of their widespread use in other experiments. The needles made in the lab are easy to reproduce and can be widely used in other labs. This study provides a good approach for researchers to investigate the mechanism of RVF.

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			<td height="21" style="height:21px;width:395px;">ALC-V8S ventilator</td>
			<td style="width:310px;">SHANGHAI&#160; ALCOTT&#160; BIOTECH&#160; CO</td>
			<td style="width:123px;">ALC-V8S</td>
			<td style="width:327px;">Assist ventilation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Animal Mini Ventilator</td>
			<td style="width:310px;">Haverd</td>
			<td style="width:123px;">Type 845</td>
			<td style="width:327px;">Assist ventilation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Animal ultrasound system VEVO2100</td>
			<td style="width:310px;">Visual Sonic</td>
			<td style="width:123px;">&#160;VEVO2100</td>
			<td style="width:327px;">Echocardiography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Cold light illuminator</td>
			<td style="width:310px;">Olympus</td>
			<td style="width:123px;">ILD-2</td>
			<td style="width:327px;">Light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Heat pad- thermostatic surgical system (ALC-HTP-S1)</td>
			<td style="width:310px;">SHANGHAI&#160; ALCOTT&#160; BIOTECH&#160; CO</td>
			<td style="width:123px;">ALC-HTP-S1</td>
			<td style="width:327px;">Heating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Isoflurane</td>
			<td style="width:310px;">RWD life science</td>
			<td style="width:123px;">R510-22</td>
			<td style="width:327px;">Inhalant anaesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Matrx VIP 3000 Isofurane Vaporizer</td>
			<td style="width:310px;">Midmark Corporation</td>
			<td style="width:123px;">VIP 3000</td>
			<td style="width:327px;">Anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Medical braided silk suture (6-0)</td>
			<td style="width:310px;">Shanghai Pudong Jinhuan Medical Supplies Co.</td>
			<td style="width:123px;">6-0</td>
			<td style="width:327px;">Ligation</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Medical nylon suture (5-0)</td>
			<td style="width:310px;">Ningbo Medical Needle Co.</td>
			<td style="width:123px;">5-0</td>
			<td style="width:327px;">Suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Millar Catheter (1.0 F)</td>
			<td style="width:310px;">AD instruments</td>
			<td style="width:123px;">1.0F</td>
			<td style="width:327px;">For right heart catheterization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Pentobarbital sodium salt</td>
			<td style="width:310px;">Merck</td>
			<td style="width:123px;">25MG</td>
			<td style="width:327px;">Anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">PowerLab multi-Directional physiological Recording System</td>
			<td style="width:310px;">AD instruments</td>
			<td style="width:123px;">4/35</td>
			<td style="width:327px;">Record the result of right heart catheterization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Precision electronic balance</td>
			<td style="width:310px;">Denver Instrument</td>
			<td style="width:123px;">TB-114</td>
			<td style="width:327px;">Weighing sensor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Self-made latch needle</td>
			<td style="width:310px;"></td>
			<td style="width:123px;"></td>
			<td style="width:327px;">Separate the aorta and pulmonary trunk</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Self-made padding needle&#160;</td>
			<td style="width:310px;"></td>
			<td style="width:123px;"></td>
			<td style="width:327px;">Constriction</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Self-made tracheal intubation</td>
			<td style="width:310px;"></td>
			<td style="width:123px;"></td>
			<td style="width:327px;">Tracheal intubation&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Small animal microsurgery equipment</td>
			<td style="width:310px;">Napox</td>
			<td style="width:123px;">MA-65</td>
			<td style="width:327px;">Surgical instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Transmission Gel</td>
			<td style="width:310px;">Guang Gong pai</td>
			<td style="width:123px;">250ML</td>
			<td style="width:327px;">Echocardiography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Veet hair removal cream</td>
			<td style="width:310px;">Reckitt Benchiser</td>
			<td style="width:123px;">RQ/B 33 Type 2</td>
			<td style="width:327px;">Remove hair of mice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Vertical automatic electrothermal pressure steam sterilizer</td>
			<td style="width:310px;">Hefei Huatai Medical Equipment Co.</td>
			<td style="width:123px;">LX-B50L</td>
			<td style="width:327px;">Auto clean the surgical instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Vertical small animal surgery microscope</td>
			<td style="width:310px;">Yihua Optical Instrument</td>
			<td style="width:123px;">Y-HX-4A</td>
			<td style="width:327px;">For right heart catheterization</td>
		</tr>
	</tbody>
</table>

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.

In this study, mice were randomly assigned to the PAC group (n = 9) or the sham operation group (n = 10). Echocardiography was performed at 1, 4, and 8 weeks after the surgery. Eight weeks after surgery, following the last echocardiography and catheterization assessments, the mice were euthanized, and their hearts were harvested for a morphological and histological assessment.
Pulmonary trunk constriction cause...

Watch this Scientific Journal Video about Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice at JoVE.com

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

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 ...

This method provides a useful technique for studying the mechanisms of right ventricular failure. The main advantages of this technique are saving time and increasing the success rate for surgery. This technique can help us imitate right heart hypertrophy and failure in humans by constriction of the pulmonary artery to create a pulmonate pressure overload.This method can be applied to the transverse aortic constriction technique and can provide insight into post heart transplantation complications, and congenital heart disease studies. The procedure will be demonstrated by Xiaoxia Huang, a technician from my laboratory. Before beginning the procedure, pass a piece of 6-0 braided silk suture through a 25 gauge needle disassembled from a one milliliter syringe.Use hemostatic forceps to bend the needle 90 degrees. Then bend a 22 gauge needle 120 degrees. After confirming a lack of response to toe pinch, place an anesthetized mouse in the supine position on a 37 degrees Celsius heating pad, and secure the incisors with a suture.Fix the limbs with tape, and use depilatory cream to remove the hair from the neck to the xiphoid process. Disinfect the exposed skin with sequential iodine and 75%ethanol wipes, and use pointed pliers to extract the tongue slightly. Elevate the mandible with a custom made spatula to expose the glottis, and use a cold light source to direct the careful insertion of a custom made trachea cannula through the glottis.Adjust the mini ventilator parameters and set the respiratory rate to 150 breaths per minute, and a tidal volume of 300 microliters. Then connect the tube and the ventilator to confirm that the cannula has been correctly inserted before fixing the cannula with tape. To open the chest, make an approximately 10 millimeter skin incision parallel to the second rib.Use scissors and elbow tweezers to separate the pectoralis major and minor muscles above the second intercostal space. Count the ribs from the sternal angle to identify the second intercostal space and use scissors to open this space. Then bluntly separate the parenchyma and thymus until the pulmonary trunk is visible.To constrict the pulmonary artery, use the elbow tweezers to bluntly separate the pulmonary trunk and the ascending aorta. Use the 90 degree latch needle to pass the suture through the connective tissue between the pulmonary trunk and the ascending aorta. Place the 120 degree padding needle on the pulmonary trunk.Use the 6-0 braided silk suture to ligate the padding needle to the pulmonary trunk, removing the padding needle immediately after filling of the pulmonary conus is observed. Cut both ends of the sutures and continue to observe the filling of the pulmonary conus to evaluate whether a constriction is present. Then evaluate the animal's pedal reflex again to ensure the success of the ligation and close the chest and skin with a 5-0 nylon suture.At least three days after the surgery, apply ultrasound gel to the chest of the pulmonary artery constricted animal. Place a 30 megahertz probe on the chest at a 30 degree counter clockwise position relative the the left parasternal line, while orienting the notch in the caudal direction. Regulate the Y and X axes under the B-mode until the mitral valve, aorta, and left ventricle chamber are clearly visible.Rotate the probe 30 to 40 degrees on its Y axis toward the chest. Regulate the Y and X axes until the pulmonary conus is clearly visible and place the cursor at the tip of the pulmonary valve leaflets to measure the peak flow velocity. Select color and PW and place the pulsed wave dashed line parallel to the direction of the blood flow.Adjust the left side of the pad so that it is higher than the right side, keeping the probe oriented 30 degrees to the horizon along the right interior axillary line, with the notch pointed in the caudal direction. Regulate the X and Y axes until the right ventricle is clearly visible. Then click M-mode two times to show the indicator line.Eight weeks after the surgery, intubate the mouse as demonstrated, and make a 1.5 centimeter bilateral incision below the xiphoid process through the diaphragm to open the chest cavity. Cut through the ribs to expose the heart, and use a 23 gauge needle to penetrate the right ventricle free wall. Press the puncture point gently with a cotton swab to stop any bleeding, and use the catheter tip to puncture the ventricle through the wound.When the curve is stable, regulate the tip of the catheter toward the right ventricle outflow tract. Click select and analyze to record the right ventricle's systolic blood pressure and diastolic pressure, maximal rate of rise, exponential time constant of relaxation, and heart rate for 10 minutes. Then transfer the catheter from the heart into a container of saline.Compared to the Sham group, a higher peak velocity, greater pressure gradient, and greater right ventricle wall thickness are obtained from the parasternal long axis view at eight weeks after surgery in the Pulmonary Artery Constriction group. Additionally, the systolic function of the right ventricle is significantly reduced in the Pulmonary Artery Constriction group, compared to the Sham group, eight weeks after the surgery. The Pulmonary Artery Constriction group has a higher right ventricle pressure in the systole and diastole, and the contractility index is reduced compared to that of the Sham group.The right ventricle Tau and the maximal rate of rise are also greater in the Pulmonary Artery Constriction group than that in the Sham group. Demonstrating that right ventricle dysfunction is induced in mice after eight weeks of pulmonary artery banding. Invasive hemodynamic testing in the right ventricle, reveals that the heart rate remains stable before and after the catheter monitoring.Compared to the Sham group, the right ventricle dimensions are significantly enlarged, and the right ventricle weight is higher. Moreover, histological examination shows that cardiac fibrosis in the area covered by cardiomyocytes are greater in the Pulmonary Artery Constriction group than in the Sham group. The mostly important part of the procedure is ensuring that the pulmonary artery is separated correctly and completely.To avoid an incorrect separation, the separation point should be near the branch of the pulmonary artery, where the connective tissue between the arteries will be looser. With this method, we have provided a more convenient and efficient approach to inducing right ventricular hypertrophy. Generally, the surgical procedure and evaluation approaches are very safe.

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