A Murine Model of Pressure Overload-Induced Right Ventricular Hypertrophy and Failure by Pulmonary Trunk Banding
Summary
We describe a murine model of right ventricular pressure overload-induced by pulmonary trunk banding. Detailed protocols for intubation, surgery, and phenotyping by echocardiography are included in the paper. Custom-made instruments are used for intubation and surgery, allowing for fast and inexpensive reproduction of the model.
Abstract
Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary diseases. The pathogenesis of RV failure is complex and remains inadequately understood. To identify new therapeutic strategies for the treatment of RV failure, robust and reproducible animal models are essential. Models of pulmonary trunk banding (PTB) have gained popularity, as RV function can be assessed independently of changes in the pulmonary vasculature.
In this paper, we present a murine model of RV pressure overload induced by PTB in 5-week-old mice. The model can be used to induce different degrees of RV pathology, ranging from mild RV hypertrophy to decompensated RV failure. Detailed protocols for intubation, PTB surgery, and phenotyping by echocardiography are included in the paper. Furthermore, instructions for customizing instruments for intubation and PTB surgery are given, enabling fast and inexpensive reproduction of the PTB model.
Titanium ligating clips were used to constrict the pulmonary trunk, ensuring a highly reproducible and operator-independent degree of pulmonary trunk constriction. The severity of PTB was graded by using different inner ligating clip diameters (mild: 450 µm and severe: 250 µm). This resulted in RV pathology ranging from hypertrophy with preserved RV function to decompensated RV failure with reduced cardiac output and extracardiac manifestations. RV function was assessed by echocardiography at 1 week and 3 weeks after surgery. Examples of echocardiographic images and results are presented here. Furthermore, results from right heart catheterization and histological analyses of cardiac tissue are shown.
Introduction
Right ventricular (RV) failure is a clinical syndrome with symptoms of heart failure and signs of systemic congestion resulting from RV dysfunction1. RV dysfunction is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary diseases2. The etiology of RV dysfunction is complex, and its underlying signaling pathways and regulation remain inadequately elucidated.
Observations from current therapies show that improved RV function correlates closely to afterload reduction, suggesting pulmonary vasculature as the primary treatment target3. This indicates that current therapies only have a minimal direct effect on RV function, which can deteriorate even after the improvement of pulmonary vascular resistance3. Further research into improving RV function independently of afterload reduction is thus highly needed.
Robust and reproducible animal models are essential in the search for new therapeutic agents. In most models of chronic RV failure, the underlying cause is pulmonary hypertension induced by structural alteration of the pulmonary vasculature4,5,6. Well-characterized models include the chronic hypoxia model7,8, the Sugen-hypoxia model9,10,11, and the monocrotaline model12,13. Because the RV failure is secondary to pulmonary hypertension in these models, it is impossible to differentiate the effects of interventions on the pulmonary vasculature from the direct effects on the RV6.
To study the RV independently from the pulmonary vasculature, the pulmonary trunk banding (PTB) model has gained popularity and has been described in several animal species, including mice, rats, rabbits, dogs, sheep, and pigs6,14,15,16,17,18,19,20,21,22,23,24,25,26,27. In PTB models, constriction of the pulmonary trunk is achieved surgically, causing an increase in RV pressure6. Different approaches to the application of PTB exist, including constriction of the vessel with a ligature or with a metal ligating clip18,28. In models using ligatures, the pulmonary trunk is tied to a needle, and the needle is retracted, leaving the ligature in place. This results in a constriction of the vessel that depends on the needle size and the tension of the knot18,29. In models employing metal ligating clips, the degree of pulmonary trunk constriction may be more reproducible. Modified ligating clip appliers are used to close the ligating clips to a predefined and constant diameter. This makes the method operator-independent and reduces PTB-related variability in the disease phenotype15,27,28.
Murine PTB models have been shown to induce RV hypertrophy and failure18,28. One major challenge when using the PTB model is choosing the appropriate PTB diameter to achieve the desired degree of RV pathology. This is especially challenging when attempting to model decompensated RV failure. For this, the constriction needs to be tight enough to induce chronic RV failure without leading to acute RV failure and death shortly after surgery6. One approach to solving this challenge is to use weanlings or juvenile animals6,15. A PTB model has been used successfully to study different stages of RV failure using Wistar rat weanlings15,30. To achieve this, juvenile rats with remaining growth potential underwent PTB surgery with the application of titanium ligating clips. When the rats grew, the pulmonary stenosis gradually became more severe and resulted in RV hypertrophy or chronic RV failure, depending on the severity of PTB15,30. Inspired by this model, we hypothesized that different stages of RV pathology could be produced in a murine PTB model using juvenile mice. Studying a broad spectrum of RV pathology from mild to severe disease may help elucidate our understanding of disease progression and the transition from RV hypertrophy to RV failure.
Here, we present a murine model of RV pressure overload induced by PTB in juvenile mice. With this model, different degrees of RV pathology can be produced, ranging from RV hypertrophy to decompensated RV failure. This study includes detailed protocols for intubation, PTB surgery, and phenotyping by echocardiography.
Protocol
Representative Results
Discussion
In this paper, we present a murine model of pressure overload-induced RV hypertrophy and failure. We demonstrate that: (i) PTB in juvenile mice can induce varying degrees of RV pathology, ranging from mild RV hypertrophy to RV failure with extracardiac signs of decompensation and histologically confirmed RV fibrosis. (ii) Signs of RV dysfunction can be observed and quantified by echocardiography at 1 and 3 weeks after PTB surgery. (iii) The degree of RV hypertrophy is proportional to the severity of PTB and the…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by Snedkermester Sophus Jacobsen og Hustru Astrid Jacobsens Fond, Helge Peetz og Verner Peetz og hustru Vilma Peetz Legat, Grosserer A.V. Lykfeldt og Hustrus Legat. Furthermore, the authors would like to acknowledge the staff of the animal facilities at the Department of Clinical Medicine, Aarhus University, for their support during the execution of the experimental work.
Materials
| Biosyn 6-0, monofilament, absorbable suture | Covidien | UM-986 | |
| Blunt cannula, 27G 0.4×0.25, | Sterican | 292832 | |
| Bupaq Multidose vet 0,3 mg/ml (Buprenorphinum) | Salfarm Danmark | VNR 472318 | |
| C57BL/6NTac mice | Taconic Biosciences | C57BL/6NTac | |
| Dagrofil 1, braided, non-absorbable suture | B Braun | C0842273 | |
| Depilatory cream | Veet | 3132000 | |
| Disposable scalpels, size 11 | Swann-Morton | 11708353 | |
| Dräger Vapor 2000 Sevoflurane | Dräger | M35054 | |
| Eye oinment neutral, "Ophta" | Actavis | MTnr.: 07586 Vnr: 53 96 68 | |
| Horizon ligating clips | Teleflex Medical | 5200 (IPN914931) | |
| Horizon Open Ligating Clips applier, curved, 6" (15 cm) | Teleflex Medical | 537061 | |
| Kitchen roll holder | n.a. | n.a. | |
| Metal wire of different thickness | n.a. | n.a. | |
| Microsurgical instruments set | Thompson | n.a. | |
| MiniVent Ventilator | Hugo Sachs | Type 845 | |
| MS505S transducer | Visual sonics | n.a. | |
| Rimadyl Bovis vet. 50 mg/ml (Carprofen) | Zoetis | MTnr: 34547, Vnr: 10 27 99, | |
| Sevoflurane Baxter 100 % | Baxter Medical | MTnr: 35015 | |
| Silicone tubing | n.a. | n.a. | |
| Soft plastic sheet | n.a. | n.a. | |
| Stereomicroscope, "Opmi Pico" | Carl Zeiss Surgicals GmbH | n.a. | |
| Ultrasonic probe holder/rail | Visual Sonics | 11277 | |
| Varming plate | Visual sonics | 11437 | |
| Venflon ProSafety, 22G, 0,9 x 25mm | Becton Dickinson | 393222 |
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