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.
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.
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.
The study was approved by the Danish Animal Experiments Inspectorate (authorization number: 2021-15-0201-00928) and was performed in accordance with the national laboratory animal legislation. This study used 5-week-old male C57BL/6N mice.
1. Customization of instruments for intubation and surgery (Figure 1)
NOTE: This section details the most important steps in the preparation of custom-made instruments for intubation and PTB surgery from inexpensive and readily available materials.
Figure 1: Instruments for intubation and PTB surgery. (A) Endotracheal tube made from an IV catheter. (B) Thoracic retractor. (C) Intubation stand and mouse placed in intubation stand receiving anesthesia on a nasal tube. (D) Surgical instruments and modified ligating clip applier used for PTB surgery. (E) Guidance cannula. (F) Custom-made adjustable stop-mechanism. Please click here to view a larger version of this figure.
2. Adjustment of the ligating clip applier
3. Preparations for surgery
4. PTB surgery
5. Echocardiography
Figure 2: Parasternal long axis view (PLAX). (A–D) Positioning of the ultrasonic probe. (E, F) The normal murine heart in PLAX. (G, H) RV dilation and hypertrophy after PTB. Abbreviations: LV: left ventricle, RV: right ventricle, PV: pulmonary valve, PT: pulmonary trunk, Ao: aorta. Please click here to view a larger version of this figure.
Figure 3: Parasternal short axis view (PSAX). (A–D) Positioning of the ultrasonic probe. (E, F) The normal murine heart in PSAX. (G, H) PSAX after PTB. Abbreviations: LV: left ventricle, RV: right ventricle, PM: papillary muscle. Please click here to view a larger version of this figure.
Figure 4: Apical 4-chamber view (A4CH). (A–D) Positioning of the ultrasonic probe. (E, F) The normal murine heart in the A4CH view. (G, H) RV and RA dilatation after PTB. Abbreviations: LV: left ventricle, RV: right ventricle, RA: right atrium, LA: left atrium. Please click here to view a larger version of this figure.
Figure 5: Tricuspid regurgitation visualized by color Doppler in the A4CH-view. (A) In diastole, flow from the RA to the RV is observed (arrow). (B) During systole, a thin jet of flow from the RV to the RA is visible (arrow). Abbreviations: LV: left ventricle, RV: right ventricle, RA: right atrium, LA: left atrium. Please click here to view a larger version of this figure.
6. Data analyses
7. Right heart catheterization
C57BL/6N mice (male, 5-week old, 17-20 g) were randomized to either severe PTB (sPTB, 250 µm, n = 12), mild PTB (mPTB, 450 µm, n = 9), or sham surgery (sham, n = 15). Evaluation of cardiac function was performed by echocardiography 1 week and 3 weeks after surgery. Right heart catheterization with subsequent euthanasia was performed 3 weeks post-surgery. Organs were weighed, and cardiac tissue was prepared for histological analyses.
Echocardiography 1 week after surgery revealed signs of elevated RV pressure and RV dysfunction in sPTB compared with mPTB and sham groups. RV dysfunction was evident by decreased cardiac output (CO) and TAPSE (Figure 6A,B). Decreased left ventricular eccentricity index (LVEI) indicated increased RV pressure (Figure 6C). Therefore, reversal interventions may be investigated already after 1 week in severe PTB models. The mPTB group showed echocardiographic signs of elevated RV pressure compared with sham (decreased LVEI) after 1 week but no signs of RV dysfunction as assessed by echocardiography (Figure 6).
Figure 6: Echocardiographic measurements 1 week after surgery. (A) Cardiac output (CO). (B) Tricuspid annular plane systolic excursion (TAPSE). (C) Left ventricular eccentricity index (LVEI). Abbreviations: mPTB: mild pulmonary trunk banding (PTB); sPTB: severe PTB. Data are presented by scatterplots with means ± SD. *p < 0.05, **p < 0.001. Please click here to view a larger version of this figure.
After 3 weeks, the effects of the PTB surgery were accentuated in sPTB with a further reduction in cardiac output and a consistent decrease in TAPSE compared with sham (Figure 7A,B). No difference in echocardiographic parameters was observed between mPTB and sham groups after 3 weeks (Figure 7A,B).
Right heart catheterization revealed increased RV systolic pressure (RVSP) in both PTB groups compared with sham 3 weeks after surgery (Figure 7C). RV diastolic function was affected in the sPTB group, indicated by an increase in RV end-diastolic elastance (Eed), a load-independent measure of RV stiffness (Figure 7D). RV contractility, as assessed by end-systolic elastance (Ees), was also increased in the sPTB group compared with the sham, whereas the mPTB group showed a non-significant increase (Figure 7E).
Anatomical measurements revealed increasing RV hypertrophy with increased severity of PTB expressed by a stepwise increase in RV weight to body weight (RV/BW) ratio (Figure 7F). RVSP was linearly proportional to the degree of RV hypertrophy (Figure 7G). RV cardiomyocyte cross-sectional area (CSA) confirmed a 40% increase in RV hypertrophy in sPTB compared with sham, while there was a non-significant increase in mPTB compared with sham (Figure 8A–D). The degree of fibrosis in the RV and right atrium (RA) was seven-fold higher in sPTB compared with sham. No increase in RV fibrosis was observed in mPTB (Figure 8E–H).
Tricuspid regurgitation (TR) was observed in 64% in the sPTB group after 1 week and in 91% at 3 weeks, whereas no TR was found in the sham and mPTB groups (data not shown). Varying degrees of liver discoloration, a sign of extracardiac decompensation, were seen in the sPTB group (Figure 9). No discoloration was seen in the mPTB and sham groups.
In summary, we show a correlation between banding diameter and disease severity. The mPTB group mimics an early disease stage with mild RV hypertrophy without RV dysfunction, whereas the sPTB group shows signs of RV dysfunction and failure.
Figure 7: Echocardiographic, invasive pressure volume, and anatomical measurements 3 weeks after surgery. (A) Cardiac output (CO). (B) Tricuspid annular plane systolic excursion (TAPSE). (C) Right ventricular systolic pressure (RVSP). (D) End-diastolic elastance (Eed). (E) End-systolic elastance (Ees). (F) Ratio of right ventricular weight and body weight (RV/BW). (G) Correlation between right ventricular systolic pressure (RVSP) and the weight of the RV/BW at 3 weeks post-surgery. A linear regression analysis was performed and is presented in the plot. Adjusted R2 = 0.75, p < 0.001. Abbreviations: mPTB: mild pulmonary trunk banding (PTB); sPTB: severe PTB. Normally distributed data are presented by scatterplots with means ± SD. Non-normally distributed data are presented by boxplots with medians with IQR and with whiskers representing values within 1.5 x IQR from Q1 and Q3, respectively *p < 0.05, **p < 0.001. Please click here to view a larger version of this figure.
Figure 8: Histological analyses of RV tissue. (A) RV cross-sectional area. (B–D) HE stained RV tissue from (B) sham, (C) mPTB, and (D) sPTB. (E) Percentage of interstitial fibrosis relative to healthy tissue in RV. (F–H) Picrosirius stained RV tissue from (F) sham, (G) mPTB, and (H) sPTB. Scalebars: (B–D) = 50 µm, (F–H) = 250 µm. Data are presented by boxplots with medians, IQR, and whiskers representing values within 1.5 x IQR from Q1 and Q3, respectively. *p < 0.05, **p < 0.001. Please click here to view a larger version of this figure.
Figure 9: Different degrees of liver discoloration. Discoloration was rated 0-3. The rating was based on the degree of speckled appearance and color of liver tissue. Pictures were taken through a microscope, making the tissue appear lighter than in ambient lighting. (A) 0: normal liver. (B) 1: Light speckles without discoloration. (C) 2: moderate speckles and light discoloration. (D) 3: Severe speckles and discoloration (nutmeg liver). Please click here to view a larger version of this figure.
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 resulting increase in RV pressure. This model may help elucidate the pathogenesis of RV failure secondary to RV pressure overload, as we show that the PTB model is suited to study different stages of RV pathology.
To ensure success and reproducibility of the murine PTB model, great attention should be paid to the following steps. (i) Choosing the appropriate ligating clip diameter. The right clip diameter depends on several factors, such as the desired degree of RV pressure overload and follow-up time. The age and body weight of the mice are also important factors to consider. Accordingly, the protocol can be adjusted depending on the current scientific question. For this reason, we recommend performing pilot surgeries and echocardiography at relevant time points after surgery to evaluate the suitability of the selected clip diameters. (ii) Precise adjustment of the clip applier is essential to ensure reproducible pathology. We recommend using metal wire or cannulas with a well-defined outer diameter as a guide to adjust the ligating clip applier. (iii) Blood loss was the most frequent severe complication during the PTB surgery and the most common cause of peri- and postoperative mortality. To limit blood loss, use blunt microsurgical instruments and blunt dissection whenever possible. Only use surgical scissors when indicated in the protocol. (iv) Another cause of perioperative mortality is cardiac arrythmias. Short episodes of bradycardia can occur during the dissection of the pulmonary trunk and application of the ligating clip. To reduce the risk of cardiac arrest, the time of manipulation of the pulmonary trunk should be limited. When bradycardia is observed, pause manipulation of the pulmonary trunk until the heart rate has normalized. (v) Reproducible echocardiographic assessment can be challenging and requires training before reproducible results are achieved. Due to the small size of the RV, it is especially challenging to achieve good visualization of the RV in sham-operated mice in PLAX and A4CH view. (vi) Heart rate may vary significantly during echocardiographic assessment. To reduce variability, monitor the heart rate closely and use low doses of anesthesia while ensuring sufficient anesthesia.
A limitation of the murine PTB model is the need for surgical equipment and training. Pilot surgeries should always be performed for training, and the methods should be adjusted to locally available equipment. We used titanium ligating clips, which have been found to be a highly reproducible method for the induction of RV pressure overload15. Additionally, the use of ligating clips has been shown to result in lower peri-surgical mortality and better post-surgical recovery compared with surgical ligatures31.
Echocardiography for assessment of RV function has some limitations, as it is operator-dependent, and inter- and intraoperator variability may influence the results of echocardiography. In the present study, echocardiography was performed by two operators. To avoid bias from interobserver variation, both operators performed echocardiography on an equal number of mice from all groups. Furthermore, echocardiography in mice has its challenges. This is especially true in small and relatively healthy mice, as evidenced by the larger CO and TAPSE measurements variation at 1 week (Figure 5) compared with 3 weeks (Figure 6). To achieve a more accurate measurement of CO, VTI in the pulmonary trunk was measured in three cardiac cycles in three different locations (in the center of the pulmonary trunk and near the walls of the vessel). Further, echocardiography has been shown to overestimate CO compared with MRI in rats subjected to PTB32. MRI is the gold standard for measuring CO in mice and may therefore be considered when using the present model32. Another challenge in mice with little or no elevation of RV pressure, is the small size of the RV, complicating a good A4CH-view to measure, e.g., TAPSE. This may result in an underestimation of TAPSE in some mice in the sham and mPTB groups. Despite these challenges, statistically significant results were achieved for CO and TAPSE in sPTB compared with sham and mPTB. This shows that CO and TAPSE are useful markers of RV function in the present model, but also highlights the need for extensive training of investigators prior to echocardiographic assessment of mice.
An inherent limitation to all animal models is that results cannot be directly extrapolated to humans due to genetic and physiologic differences.
To our knowledge, this is the only PTB model in juvenile mice. This approach has been used in rats, where a gradual and slow increase in afterload develops as the rats grow. This slower disease induction better mimics the development of chronic RV failure15.
The current protocol includes two distinct groups of mice with different degrees of RV dysfunction. To our knowledge, no other published murine PTB models include a group with only mild RV hypertrophy without signs of RV dysfunction, hemodynamic impairment, or fibrosis. This early stage of the disease is thus largely overlooked. Studying this early stage of disease development may increase our understanding of the gradual progression from RV hypertrophy to RV failure seen in patients with chronic PAH17.
In this model, the pathology of chronic RV failure develops within 3 weeks after surgery, making it one of the most time-efficient models of chronic RV failure6. Other murine PTB models report study durations of 1 to 8 weeks17,18,33,34,35. We observed signs of RV dysfunction already 1 week post-surgery in sPTB. This time-efficient approach with 3 weeks follow-up, however, comes with limitations. Mice are not fully grown at 8 weeks and a longer follow-up might result in an increasingly severe pulmonary trunk stenosis and an even more severe RV failure.
Genetically modified mice are readily available, and new genetically modified strains can be produced in mice relatively fast and time-efficiently36. This constitutes a major advantage of murine PTB models in contrast to models using rats or larger mammals.
In conclusion, we present a reproducible murine model of pressure overload-induced RV hypertrophy and failure in juvenile mice. 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. The model may be used to identify mechanisms that govern the RV adaptation to pressure overload, as well as processes underlying RV pathology ultimately resulting in RV failure. Finally, the model can be used to test new therapeutic targets for the treatment of RV failure.
The authors have nothing to disclose.
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.
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 | |
Disinfection Swabs (82% Ethanol + 0.5% Chlorhexidine) | Mediq | 3340122 | |
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 |