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.
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.
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 PH2,3,4. 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 ligation6,7 and transverse aortic banding8,9,10 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.
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
2. Anesthesia and Endotracheal Intubation
3. Preparation of the Surgical Site
4. Left Atrial Stenosis Surgery
5. Post-operative Care
6. Confirmation of the Success of the Left Atrial Stenosis with Echocardiography
7. Sham Operation
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 left atrial enlargement (Figure 2B), pulmonary congestion (Figure 2E), right ventricular (RV) pressure overload (Figure 2F), and an increased pulmonary venous flow (Figure 2F,G) compared to rats in the SOC group (Figure 2A–E). There is also an increased RV systolic pressure in the LAS group versus the SOC group (Figure 3). A histologic examination of a lung cross section stained with elastic-Van Gieson (EVG) shows increased pulmonary artery (PA) and pulmonary vein (PV) medial thickness, and an increased PV dimension in the LAS group versus the SOC group (Figure 4A–D). Furthermore, alpha-smooth muscle actin (αSMA) immunostaining shows an increased number of smooth muscle cells in the PA and the PV of the LAS group versus control rats (Figure 4E,F). Thus, the LAS model increases muscularization in both the PA and the PV of the LAS rat.
Table 1 summarizes the operative parameters, comparing the SOC group to the LAS group. Specifically, the RV-to-body-weight ratio and lung-to-body-weight ratio are significantly increased in the LAS group versus the SOC group. Hemodynamic parameters, including RV systolic pressure, RV end-diastolic pressure, and estimated LA pressure, are significantly increased in the LAS group compared to the SOC group (Table 1).
Figure 1: Representative echocardiogram comparing the left ventricular inflow velocity of sham-operated control (SOC) versus left atrial stenosis (LAS) rats. (A) Four-chamber view and corresponding color Doppler echo of an SOC rat. (B) Four-chamber inflow velocity and corresponding color Doppler echo of an LAS rat. (C) Peak left ventricular inflow velocity of an SOC rat (0.94 m/s) vs. (D) an LAS rat (2.12 m/s). Please click here to view a larger version of this figure.
Figure 2: Representative macroscopic and echocardiographic findings in sham-operated control (SOC) versus left atrial stenosis (LAS) rats 10 weeks after surgery. (A) Macroscopic findings of the heart from an SOC rat versus (B) the heart from an LAS rat, which show left atrial dilatation. The black scale bar represents 1 cm. (C) Macroscopic findings of the lung from an SOC rat versus (D) the lung from an LAS rat, which show pulmonary congestion. (E) Echocardiographic short-axis view of an SOC rat versus (F) an LAS rat, which shows interventricular septum flattening with increased right ventricular free wall thickness. (G) Pulmonary venous flow of the SOC rat versus (H) the LAS rat, which showed increased PV inflow. This figure is reproduced and modified from Fujimoto et al.11 with permission. Please click here to view a larger version of this figure.
Figure 3: Representative hemodynamic recording of a sham-operated control (SOC) rat versus a left atrial stenosis (LAS) rat, showing no difference in left ventricular (LV) pressure but an increase in right ventricular (RV) pressure in the LAS rat. The figure is reproduced and modified from Fujimoto et al.11 with permission. Please click here to view a larger version of this figure.
Figure 4: Representative histological changes in sham-operated control (SOC) versus left atrial stenosis (LAS) rats, 10 weeks after surgery. Lung cross section stained with elastic-Van Gieson (EVA) shows (A – B) increased pulmonary artery (PA) and (C – D) pulmonary vein (PV) thickness and an increased dimension of the PV in the LAS group. Alpha-smooth muscle actin (αSMA) immunostaining shows an increased number of positively stained cells in the vessel walls of the (E – F) PA and the (G – H) PV in the LAS group. The scale bars represent 100 µm. This figure is reproduced and modified from Fujimoto et al.11 with permission Please click here to view a larger version of this figure.
Operative Parameters | SOC group (n=5) | Column1 | LAS group (n=5) | Column2 | Column3 |
Median | IQR | Median | IQR | P-value | |
BW operation (g) | 195 | 190-205 | 194 | 190-208 | 0.98 |
BW sacrifice (g) | 416 | 410-420 | 452 | 390-505 | 0.65 |
RV weight/BW | 0.39 | 0.38-0.43 | 0.54 | 0.50-0.59 | <0.01 |
LV weight/BW | 1.91 | 1.85-1.95 | 1.98 | 1.78-2.20 | 0.69 |
RV weight/LV weight | 0.2 | 0.19-0.22 | 0.27 | 0.27-0.28 | <0.01 |
Lung weight/BW | 0.37 | 0.36-0.41 | 0.47 | 0.42-0.51 | <0.01 |
Cardiac catheterization | |||||
RVSP (mmHg) | 18 | 16-20 | 40.6 | 30-50 | <0.01 |
RVEDP (mmHg) | 1.6 | 1.0-2.0 | 3.4 | 3.0-4.0 | <0.01 |
LVSP (mmHg) | 84 | 60-80 | 77.6 | 70-80 | 0.72 |
LVEDP (mmHg) | 2.8 | 2.0-3.0 | 7.6 | 7.0-8.0 | 0.013 |
RVSP/LVSP | 0.22 | 0.15-0.27 | 0.52 | 0.54-0.60 | 0.021 |
Estimated LA pressure (mmHg) | 7.9 | 6.8-8.4 | 28.1 | 22.8-27.0 | <0.01 |
Table 1: Operative and cardiac catheterization parameters and estimated left atrial pressure in the sham-operated control and left atrial stenosis groups. Abbreviations: SOC = sham-operated control; LAS = left atrial stenosis; IQR = interquartile range; BW = body weight; RVSP = right ventricular systolic pressure; RVEDP = right ventricular end diastolic pressure; LVSP = left ventricular systolic pressure; LVEDP = left ventricular end diastolic pressure; LA = left atrium. This table is reproduced and modified from Fujimoto et al.11 with permission.
Supplemental Figure 1: Landmarks for clip placement and tightness of clip closure. (A) One end of the clip is placed next to the base of the pulmonary artery. (B) Another end of the clip is placed just above the coronary sinus, halfway across the left ventricle. (C) The clip should be halfway closed, so the ends just touch each other. Please click here to download this figure.
The LAS rat is a novel group 2 PH model that has already received substantial interest from researchers in the field12,13. Comparing to the two existing group 2 models, namely the pulmonary vein stenosis (PVS) model14, using piglets, and the supracoronary aortic banding (SAB) rat model8,9,10, the LAS rat model has several advantages. Compared to the PVS piglet model, the LAS rat model costs less to generate and the surgical procedure in rat is less complicated than in piglet. Compared to the SAB rat model, which is the most commonly used group 2 PH animal model, the pathophysiology of group 2 PH in the LAS model is less complicated than in the SAB model, as aortic banding first causes left ventricular failure before developing pulmonary congestion and PH. It is likely that the LAS and SAB models in rodents will be complementary tools to better understand the etiology of group 2 PH.
Two most critical steps in LAS surgery are the placement of the stay suture and the application of the metal clip. Regarding the placement of the stay suture, the choice suture is crucial. Avoid suturing with a cutting needle. Use a monofilament suture, as it produces less drag and friction when passing through the left ventricle. Regarding the application of the metal clip, it is important to identify the surface landmarks. One end of the clip is ideally placed next to the base of the pulmonary trunk and the other end placed just above the coronary sinus, halfway across the LV (Supplementary Figure 1). The clip should be halfway closed, so the ends just touch each other (Supplementary Figure 1C).
The LAS rat model has several limitations. First, the LAS model is only able to generate moderate PH with PASP around 40 mmHg11. We have explored the use of tighter atrial clips, but the operative mortality increased significantly as a consequence. Second, the fast-beating heart made it difficult to accurately place the clip at the desired landmarks. As a result, the success rate is around 50%, due to either a loose band or incorrect clip placement. A modified clip applier with a stopper would improve the consistency of the clip tightness. Third, with the currently available technology, it is still difficult to obtain direct pulmonary arterial pressure and pulmonary capillary wedge pressure measurements in a rat model. Finally, the fidelity of molecular mechanisms in rat PH models to human PH remains questionable, and it remains an area of active investigation.
Despite these limitations, the LAS rat is a clinically relevant, economical, and reproducible small animal model that is suitable for studying the pathophysiology and molecular mechanism of group 2 PH and pulmonary venous arterialization. It can also serve as a workhorse for the preclinical testing of novel therapies developed to treat group 2 PH.
The authors have nothing to disclose.
The authors acknowledge the Mitacs-Japan Society for the Promotion of Science (JSPS) Summer Program. Ping Yu Xiong was supported by funding from the Mitacs-JSPS Summer Program to visit the Jikei University School of Medicine. Dr. Minamisawa is supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (S.M.), the MEXT-Supported Program for the Strategic Research Foundation at Private Universities (S.M.), the Vehicle Racing Commemorative Foundation (S.M.), and The Jikei University Graduate Research Fund (S.M.) with financial support for this project. Dr. Archer is supported in part by U.S. National Institutes of Health (NIH) grants NIH 1R01HL113003-01A1 (S.L.A.) and NIH 2R01HL071115-08 (S.L.A.), the Canada Foundation for Innovation, Tier 1 Canada Research Chair in Mitochondrial Dynamics and Translational Medicine (S.L.A.), the William J. Henderson Foundation, the Canadian Vascular Network, and the Queen's Cardiopulmonary Unit (QCPU).
The authors acknowledge Mr. Tadashi Kokubo, Chief of Photographic Services of the Academic Information Center at the Jikei University School of Medicine, for filming the video.
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 |