Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat

Summary

Left atrial stenosis (LAS) is a novel surgical technique used for studying group 2 pulmonary hypertension (PH) and mechanisms underlying pulmonary venous arterialization. Here, we present a protocol to constrict the left atrium using a titanium clip to cause pulmonary venous arterialization and moderate PH in a rat.

Abstract

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of group 2 PH, due to mitral stenosis (MS), to facilitate the investigation of disease mechanisms and potential therapeutic strategies. We present a novel rat model of pulmonary venous congestion-induced pulmonary venous arterialization and group 2 PH caused by left atrial stenosis (LAS). LAS is achieved by constricting the left atrium using a half-closed titanium clip. After the LAS surgery, a rat model with a transmitral inflow velocity greater than or equal to 2.0 m/s on echocardiography gradually develops pulmonary venous arterialization and group 2 PH over an 8- to 10-week period. In this protocol, we provide the step-by-step procedure of how to perform the LAS surgery. The presented LAS rat model mimics MS in humans and is useful for studying the underlying molecular mechanism of pulmonary venous arterialization and for the preclinical evaluation of therapies for group 2 PH.

Introduction

The purpose of this article is to demonstrate the step-by-step procedure of how to perform the LAS surgery in rats. Surgically induced LAS closely mimics MS and cor triatriatum in humans, which involve the creation of a mechanical obstruction in the left atrium¹. Obstruction of the left ventricular (LV) inflow often causes a congestion of the pulmonary venous circulation, and patients gradually develop PH. The World Health Organization classifies PH due to left heart diseases as group 2, which is the most prevalent group of PH^2,3,4. The diagnosis of PH in patients with left heart diseases is associated with a greater than a sevenfold increase in the 1-year standardized mortality⁴. Currently, there is no approved therapy for group 2 PH apart from treating the underlying left heart diseases (e.g., surgically replacing the stenotic mitral valve). However, even effective mitral valve replacement does not resolve PH fully in up to half of the patients with MS⁵. This persistent PH is due to adverse pulmonary vascular remodeling, which is poorly understood. Hence, animal models are important to enhancing our understanding of the underlying molecular mechanisms of adverse pulmonary vascular remodeling in group 2 PH.

There are a few animal models of group 2 PH. Coronary artery ligation^6,7 and transverse aortic banding^8,9,10 in rodents are the most commonly used group 2 PH animal models. The major disadvantage of these models is the involvement of LV, which makes the outcome of group 2 PH studies difficult to interpret. In contrast, the LV remains intact in the LAS model. Furthermore, the LAS model is clinically relevant because it results in the slow and progressive development of PH over a 10-week period¹¹. In humans, MS is considered significant if the transmitral Doppler flow velocity is greater than 2.0 m/s¹¹, and we also use this number as a cut-off to determine whether the LAS surgery has produced significant stenosis. Furthermore, although the LAS model generates mild or moderate PH, it demonstrates characteristic histologic changes, similar to those in human patients, namely the development of intrapulmonary venous arterialization¹¹. The LAS rat model is a novel and clinically relevant group 2 PH model with preserved LV function. It is suitable for studying the pathophysiology of persistent pulmonary vascular remodeling, identifying molecular targets, and testing novel therapies for group 2 PH.

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Protocol

The LAS experimental protocol has been approved by the Jikei University School of Medicine Animal Care Committee and the University Research and Ethics Committee (protocol #2015-118).

1. Pre-operative Preparation

1. After arriving at the animal facility, provide 5-week-old male Sprague Dawley rats between 150 to 200 g with 1 week to acclimate to their new home prior to the operation.
2. Prepare the following equipment before the surgery by autoclaving: 1) a small animal respirator, 2) an anesthetic machine, 3) an intubation kit (composed of a pair of hemostat forceps, a tongue depressor, and an 18 G angiocatheter), 4) surgical instruments (which include a pair of curved forceps, a pair of straight forceps, a needle driver, a chest retractor, a pair of scissors, a 5-0 monofilament suture, a clip applicator, medium-large clips, and a 23 G chest tube).
3. Have sterile Q-tips and gauze ready to deal with bleeding.
4. Use a heating pad to maintain the animal body temperature around 37 °C during the surgery.

2. Anesthesia and Endotracheal Intubation

1. Anesthetize the rat in an induction chamber with 5% isoflurane mixed with 2 L/min room air.
2. Prior to intubation, shave the rat's chest hair with a hair shaver and apply hair removal cream to remove fine hair.
3. Check the pedal reflex to confirm successful anesthesia prior to intubation.
4. Hook the front teeth with a string and secure the string with two pins.
5. Open the rat's mouth with the hemostat forceps and insert the tongue depressor into the mouth.
6. Lift the tongue depressor to visualize the vocal cord.
   NOTE: It is helpful to shine a strong light over the head region of the rat to help visualize the vocal cord.
7. Insert the 18 G angiocatheter as an endotracheal tube into the trachea, and then, quickly, connect the catheter to the respirator.
8. Set the tidal volume to 10 µL per gram with a respiratory rate of 100 breaths/minute.
9. Maintain anesthesia with 2% isoflurane mixed with 2 L/min room air.

3. Preparation of the Surgical Site

1. Prepare the surgical site with alternating scrubs of chlorhexidine and alcohol x3.
2. Give buprenorphine 0.01 mg/kg subcutaneously.
3. Cover the rat with a sterile drape.
4. Check the pedal reflex to confirm a successful intubation and maintenance of anesthesia.

4. Left Atrial Stenosis Surgery

1. Mark the incision site 2 cm below the rat's left armpit with a rule.
2. Make a 2 cm left lateral chest wall incision with a pair of scissors.
3. Separate the intercostal muscles between the fourth and the fifth rib, using the straight and the curved forceps, until entering the chest cavity.
4. Insert the chest retractor into the chest cavity. Continue to use the straight and curved forceps to separate the intercostal muscle to obtain a direct visualization of the thymus and the heart.
5. Lift the thymus with a pair of straight forceps. Remove the thymus covering the heart with a pair of scissors. Avoid cutting or poking into any major blood vessels.
6. Carefully pass a 5-0 monofilament suture through the surface of the left ventricle, right below the left atrial appendage. Avoid passing the needle through the major coronary arteries.
7. After the suture is in place and there is no significant bleeding, tie a loose knot.
8. Pull the suture thread up and forward to lift the heart out of the chest.
9. Once the heart is lifted out of the chest, quickly apply a medium-large clip to the left atrium, just above the mitral valve.
   NOTE: The clip is half-way closed, with the tip of the clip pinching the left atrium, causing left atrial stenosis.
10. Quickly put the heart back into the chest. Ensure the heart is not outside of the chest for longer than 30 s.
11. Remove the stay suture used to lift the heart.
12. Close the chest with a 5-0 monofilament suture, using a simple interrupted pattern.
13. Insert a 23 G chest tube attached to a 10 mL syringe into the chest, and then, proceed with closing the chest wall muscle and skin with simple interrupted sutures.
14. Draw out any air, blood, and pleural effusion via the inserted chest tube, using the attached 10 cc syringe, and then, pull the tube.
15. Close the skin layer with a 5-0 monofilament suture, using a simple interrupted pattern.
16. Give buprenorphine 0.01 mg/kg subcutaneously.
17. Turn off the isoflurane.
18. Disconnect the respirator after spontaneous respiration is observed.
19. Keep the rat intubated and allow it to recover on the heating pad until it wakes up.
20. Safely extubate the rat after one or more of the following signs is/are observed: the rat starts moving its four limbs, it regains its righting reflex, it regains its gag reflex, or it displays spontaneous voiding.

5. Post-operative Care

1. Every 8 - 12 h, give buprenorphine 0.01 mg/kg subcutaneously. Carprofen 5 mg/kg is given subcutaneously on a daily basis for 2 days and, then, as needed, if the rat is not moving around well and looks like it is in pain.
2. Give 5 mL of normal saline subcutaneously right after the surgery, as the rat may have difficulty drinking from the water spigot, immediately postoperative.

6. Confirmation of the Success of the Left Atrial Stenosis with Echocardiography

1. Perform a transthoracic echocardiography 2 weeks after the surgery to determine the LV inflow velocity.
2. Anesthetize the rat following the steps outlined in section 1.
3. After the induction of anesthesia, maintain anesthesia using a nose cone with 2% isoflurane mixed with 2 L/min room air.
4. Shave the rat's chest wall with a hair shaver and use hair removal cream to remove any remaining hair.
5. Place the ultrasound probe at the apex of the heart, which is around the fifth intercostal space on the left side of the chest. Move the probe around in this region until a good four-chamber view is obtained.
6. Measure the LV inflow velocity using the pulsed-wave Doppler mode just above the mitral valve annulus.
7. An LV inflow velocity greater than 2.0 m/s is required for the development of moderate pulmonary hypertension at 8 - 10 weeks post-LAS surgery.

7. Sham Operation

1. Except for applying the clip (step 4.9), perform all the steps above to create age-matched, sham-operated control (SOC) rats.

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Results

The effectiveness of the LAS is confirmed using echocardiography, 2 weeks postoperative. Rats with an LV inflow velocity greater than 2.0 m/s, measured with a four-chamber view, are considered to have developed significant stenosis (Figure 1) and reliably develop moderate PH and pulmonary venous arterialization 8 - 10 weeks post-LAS surgery.

Ten weeks post-LAS surgery, the rats in the LAS group show...

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Discussion

The LAS rat is a novel group 2 PH model that has already received substantial interest from researchers in the field^12,13. Comparing to the two existing group 2 models, namely the pulmonary vein stenosis (PVS) model¹⁴, using piglets, and the supracoronary aortic banding (SAB) rat model^8,9,10, the LAS rat model has several advantages. Compared to t...

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Materials

Name	Company	Catalog Number	Comments
5-0 Prolene Suture	Johnson & Johnson - Ethicon	8725H	Polypropylene suture with HEMO-SEAL Technology
Anaesthesia Machine	Wakenyaku Co., Ltd.	BRTK-100A	Air pump and anaethesia machine
Angiocatheter guidewire	Self-made		10 cm guidewire glued to a 1 cc syringe
Chest retractor	Natsume Seisakusho Co., Ltd.	F-2
Chest tube 23G	Self-made		10 cc syringe attached to a 23G needle plus plastic tube
Curved forceps	Natsume Seisakusho Co., Ltd.	A-14
Heating pad	Vivaria	MP-916-NV	Keep body temperature at 37 degree celsius
Horizon Ligating Clips	Teleflex	REF 003200	Size Medium-Large
Horizon Manual-Load Ligating Clip Applier For Medium-Large Size Horizon	Teleflex	REF 337085	Ligation Clips Angled Jaw, (20cm)
Needle holder	Natsume Seisakusho Co., Ltd.	MC-40
Rodent Respirator	CWE Inc	SAR-830/P	Small animal ventilator
Scissors	Natsume Seisakusho Co., Ltd.	B-12	Straight scissors ideally with round tips
Straight forceps	Natsume Seisakusho Co., Ltd.	A-7
Tongue depressor	Uchida Yoko Co., Ltd.	8-615-2417	Use the wide end