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

Summary

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.

Abstract

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.

Introduction

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 β2-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 exist^9,10. 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 diseases^{12,13,14,15,16,17,18,19}. 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° 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° from the gauge needle. Padding needles with different diameters (0.6-0.8 mm) are used, depending on the mice'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.

Protocol

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 the surgical instruments and fabrication of the needles

1. Prepare the sterile surgical instruments (Figure 1A), a 6-0 braided silk suture (Figure 1B[a]) for ligation, and a 5-0 nylon suture for incision closure (Figure 1B[b]).
2. Pass the 6-0 braided silk suture (Figure 1B[a]) through a 25 G needle disassembled from a 1 mL injection syringe. Then, curve the needle 90° with hemostatic forceps to make a latch needle (Figure 1C[a]). Curve the 22 G needle 120° (Figure 1C (b)) to make a padding needle.

2. Preparation for surgery

1. Autoclave all surgical instruments before surgery. Adjust the heating pad to 37 °C. Anesthetize the mice by intraperitoneal injection with a mixture of xylazine (5 mg/kg) and ketamine (100 mg/kg) for pain relief. Place the mice in individual boxes to wait for narcotic onset.
   NOTE: It is also recommended to use 1.5% isoflurane for inhalant anesthesia.
2. Monitor the adequacy of anesthesia by the disappearance of the pedal withdrawal reflex. Keep the mice in the supine position on the heating pad by fixing the incisors with a suture and fixing the legs with adhesive tape. Check the reflex again to ensure the depth of anesthesia.
3. Apply the depilatory paste on the skin from the neck to the xiphoid process. Disinfect the area with iodine followed by 75% alcohol.
4. Perform endotracheal intubation.
   1. Adjust the animal miniventilator's (Figure 1D) parameters and set the respiratory rate to 150/min and the tidal volume to 300 µL.
   2. Pull out the tongue slightly by using pointed pliers, elevate the mandible with a lab made spatula Figure 1C[c]) to expose the glottis, and softly insert a lab made trachea cannula (Figure 1C[d]) through the glottis while a cold light source is directed on the larynx.
   3. Connect the tube and the ventilator to verify whether the cannula has been inserted into the trachea. Fix the trachea using adhesive tape if the cannula has been inserted correctly.

3. Surgery

1. Open the chest.
   1. Make an incision in the skin parallel to the second rib, about 10 mm in length, with ophthalmic scissors. Ensure that the incision starts from the sternal angle and ends on the left anterior axillary line. Identify the second intercostal space by counting the ribs from the sternal angle.
   2. Separate and cut the pectoralis major and pectoralis minor muscles with scissors and elbow tweezers above the second intercostal space to expose this space.
      NOTE: It is also recommended to bluntly separate, mobilize, and move the pectoralis muscles to the right and cranially.
   3. Bluntly penetrate the second intercostal space with elbow tweezers and open this space. Then, bluntly separate the parenchyma and thymus until the pulmonary trunk is visible.
2. Constrict the pulmonary artery.
   1. Bluntly separate the pulmonary trunk and the ascending aorta with elbow tweezers. Pass the suture through the connective tissue between the pulmonary trunk and the ascending aorta with a latch needle.
   2. Place the padding needle (see step 1.2) on the pulmonary trunk and, then, ligate the pulmonary trunk together with the padding needle, using the 6-0 braided silk suture. Remove the padding needle immediately after the filling of the pulmonary conus is observed and cut the ends of the suture.
   3. Observe the filling of the pulmonary conus to evaluate whether there is a constriction present. Evaluate the animal's reflex again to ensure the success of the ligation.
      NOTE: Perform a sham surgery by following all of the above steps except for the constriction.
3. Close the chest and the skin with the 5-0 nylon suture. Disinfect the skin again with 75% alcohol.
4. Inject 0.5 mL of saline subcutaneously to replace any fluid lost during the surgery. Place the mouse in the cage separately with heating pad to promote recovery. Return the mice to their cages in a 12/12 h light/dark cycle room when consciousness returns. Treat the mice with buprenorphine via their drinking water for the following 3 days.
5. Pay special attention to the healing of the thoracotomy wound by monitoring the mice 2x a day during the first week to detect any signs of insufficient healing, impaired mobility, or weight loss.

4. Echocardiographic assessment of the RV function after surgery

NOTE: Echocardiographic changes can be detected 3 days after surgery.

1. Anesthetize the mice with 3% isoflurane through inhalation and maintain the depth of anesthesia with 1.5% isoflurane. Fix a mouse on the platform, tape its claws to the electrode, and maintain the animal in a supine position. Maintain the mouse's heart rate between 450-550 beats/min by adjusting the concentration of isoflurane between 1.5% and 2.5%.
2. Remove the hair on the mouse's chest with depilatory cream and apply ultrasound gel to the skin of the chest.
3. Assess the pulmonary trunk constriction with a 30 MHz probe.
   1. Keep the probe at 30° counterclockwise relative to the left parasternal line, while orienting the notch in the caudal direction. Regulate the y-axis and x-axis under the B-mode until the mitral valve, aorta, and LV chamber are clearly visible.
   2. Rotate the probe 30°-40° on its y-axis toward the chest. Regulate the y-axis and x-axis until the pulmonary conus is clearly visible.
   3. Place the cursor at the tip of the pulmonary valve leaflets to measure the peak flow velocity. Use the Color Doppler mode by pressing Color, followed by PW, and then moving the cursor to place the PW-dashed line parallel to the direction of the blood flow.
   4. Measure the pulmonary artery peak velocity. Save the data and image with Cine Store and Frame Store.
4. Assess the RV parameters with a 30 MHz probe.
   1. Adjust the left side of the pad so that it is lower than the right side. Keep the probe oriented at 30° to the horizon along the right anterior axillary line with the notch pointed in the caudal direction. Regulate the y-axis and x-axis until the RV is clearly shown.
   2. Press M-Mode 2x to show the indicator line. Measure the RV chamber dimension, fractional shortening, and RV wall thickness. Save the data and image with Cine Store and Frame Store.
5. Stop isoflurane inhalation to allow the mice to recover consciousness and then return the animals to their cages in a 12 h light/dark cycle room.

5. Right heart catheterization for assessing the RV function

NOTE: Right heart catheterization was performed 8 weeks after surgery to assess the RV function, using a 1.0 F catheter and a monitoring system.

1. Autoclave all surgical instruments. Anesthetize the animal via intraperitoneal injection with a mixture of xylazine (5 mg/kg) and ketamine (100 mg/kg).
2. After the pedal withdrawal reflex disappears, fix the mouse on the platform, tape its claws to the electrode, and maintain the mouse in supine position. Remove the hair in the surgical area with depilatory cream.
3. Disinfect the skin of the surgical area with 75% alcohol. Using pointed pliers,pull the tongue out slightly, elevate the mandible with a spatula made inhouse to expose the glottis, and softly insert the trachea cannula made inhouse through the glottis while a cold light source is directed on the larynx. Use a ventilator (Figure 1E) to assist with ventilation.
4. Open the chest cavity by means of a 1.5 cm bilateral incision below the xiphoid process through the diaphragm with ophthalmic scissors and forceps. Cut through the diaphragm and ribs with ophthalmic scissors to expose the heart. Penetrate the RV free wall with a 23 G needle and remove the needle; press the puncture point gently with a cotton swab to stop any bleeding. Puncture the ventricle with the catheter tip through the wound.
   NOTE: It is also recommended to perform right heart catheterization via the right jugular vein⁶. When the catheter tip is in the ventricle, the monitor will display the RV pressure curve.
5. Record the RV systolic blood pressure, the RV end-diastolic pressure, the RV dP/dt, the mouse's heart rate, and the RV exponential time constant of relaxation (Tau) for 10 min when the curve is stable. Using the software, click Select and then click Analyze.
6. Regulate the tip of the catheter toward the RV outflow tract. Pull the catheter out after the recording is complete. Place the catheter in saline when the measurements are finished.
7. Euthanize the mice by intraperitoneal injections of pentobarbital sodium 150 mg/kg, followed by cervical dislocation. Then, harvest the heart, lungs, and tibia for histomorphological and molecular biological analyses.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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