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.

Creating a Murine Model of RV Hypertrophy and Failure by Pulmonary Trunk Banding

<ol>
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The authors have nothing to disclose.

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&#160;and 3&#160;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&#160;to 8&#160;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.

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&#160;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.

<table><tbody><tr><td>Biosyn 6-0, monofilament, absorbable suture</td><td>Covidien</td><td>UM-986</td><td></td></tr><tr><td>Blunt cannula, 27G 0.4x0.25,&amp;nbsp;</td><td>Sterican</td><td>292832</td><td></td></tr><tr><td>Bupaq Multidose vet 0,3 mg/ml (Buprenorphinum)</td><td>Salfarm Danmark</td><td>VNR 472318</td><td></td></tr><tr><td>C57BL/6NTac mice</td><td>Taconic Biosciences</td><td>C57BL/6NTac</td><td></td></tr><tr><td>Dagrofil 1, braided, non-absorbable suture</td><td>B Braun</td><td>C0842273</td><td></td></tr><tr><td>Depilatory cream&amp;nbsp;</td><td>Veet&amp;nbsp;</td><td>3132000</td><td></td></tr><tr><td>Disinfection Swabs (82% Ethanol + 0.5% Chlorhexidine)</td><td>Mediq</td><td>3340122</td><td></td></tr><tr><td>Disposable scalpels, size 11</td><td>Swann-Morton</td><td>11708353</td><td></td></tr><tr><td>Dr&amp;auml;ger Vapor 2000 Sevoflurane</td><td>Dr&amp;auml;ger</td><td>M35054</td><td></td></tr><tr><td>Eye oinment neutral, &quot;Ophta&quot;</td><td>Actavis</td><td>MTnr.: 07586 Vnr: 53 96 68</td><td></td></tr><tr><td>Horizon ligating clips</td><td>Teleflex Medical</td><td>5200 (IPN914931)</td><td></td></tr><tr><td>Horizon Open Ligating Clips applier, curved, 6&quot; (15 cm)</td><td>Teleflex Medical</td><td>537061</td><td></td></tr><tr><td>Kitchen roll holder</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>Metal wire of different thickness</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>Microsurgical instruments set</td><td>Thompson</td><td>n.a.</td><td></td></tr><tr><td>MiniVent Ventilator</td><td>Hugo Sachs</td><td>Type 845</td><td></td></tr><tr><td>MS505S transducer&amp;nbsp;</td><td>Visual sonics</td><td>n.a.</td><td></td></tr><tr><td>Rimadyl Bovis vet. 50 mg/ml (Carprofen)</td><td>Zoetis</td><td>MTnr: 34547, Vnr: 10 27 99,</td><td></td></tr><tr><td>Sevoflurane Baxter 100 %</td><td>Baxter Medical</td><td>MTnr: 35015</td><td></td></tr><tr><td>Silicone tubing</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>Soft plastic sheet</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>Stereomicroscope, &quot;Opmi Pico&quot;</td><td>Carl Zeiss Surgicals GmbH</td><td>n.a.</td><td></td></tr><tr><td>Ultrasonic probe holder/rail</td><td>Visual Sonics</td><td>11277</td><td></td></tr><tr><td>Varming plate&amp;nbsp;</td><td>Visual sonics</td><td>11437</td><td></td></tr><tr><td>Venflon ProSafety, 22G, 0,9 x 25mm</td><td>Becton Dickinson</td><td>393222</td><td></td></tr></tbody></table>

a murine model of pressure overload-induced right ventricular hypertrophy and failure by pulmonary trunk banding

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 &#181;m and severe: 250 &#181;m). 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.

C57BL/6N mice (male, 5-week old, 17-20 g)&#160;were randomized to either severe PTB (sPTB, 250 &#181;m, n = 12), mild PTB (mPTB, 450 &#181;m, 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&#160;week but no signs of RV dysfunction as assessed by echocardiography (Figure 6).
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66851/66851fig06.jpg" /> 
Figure 6: Echocardiographic measurements 1 week after surgery. (A) Cardiac output (CO).&#160;(B) Tricuspid annular plane systolic excursion (TAPSE).&#160;(C) Left ventricular eccentricity index (LVEI). Abbreviations: mPTB: mild pulmonary trunk banding (PTB); sPTB: severe PTB. Data are presented by scatterplots with means &#177; SD. *p &#60; 0.05, **p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66851/66851fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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&#160;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.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66851/66851fig07.jpg" /> 
Figure 7: Echocardiographic, invasive pressure volume, and anatomical measurements 3 weeks after surgery. (A) Cardiac output (CO).&#160;(B) Tricuspid annular plane systolic excursion (TAPSE).&#160;(C) Right ventricular systolic pressure (RVSP).&#160;(D) End-diastolic elastance (Eed).&#160;(E) End-systolic elastance (Ees).&#160;(F) Ratio of right ventricular weight and body weight (RV/BW).&#160;(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 &#60; 0.001. Abbreviations: mPTB: mild pulmonary trunk banding (PTB); sPTB: severe PTB. Normally distributed data are presented by scatterplots with means &#177; 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 &#60; 0.05, **p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66851/66851fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/66851/66851fig08.jpg" /> 
Figure 8: Histological analyses of RV tissue. (A) RV cross-sectional area.&#160;(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.&#160;(F-H) Picrosirius stained RV tissue from (F) sham, (G) mPTB, and (H) sPTB. Scalebars: (B-D) = 50 &#181;m, (F-H) = 250 &#181;m. Data are presented by boxplots with medians, IQR, and whiskers representing values within 1.5 x IQR from Q1 and Q3, respectively. *p &#60; 0.05, **p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66851/66851fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/66851/66851fig09.jpg" /> 
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.&#160;(B) 1: Light speckles without discoloration.&#160;(C) 2: moderate speckles and light discoloration.&#160;(D) 3: Severe speckles and discoloration (nutmeg liver). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66851/66851fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Investigating the Underlying Mechanisms of Right Ventricular Failure in Pulmonary Hypertension

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.

A Murine Model of Pressure Overload-Induced Right Ventricular Hypertrophy and Failure by Pulmonary Trunk Banding

Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary ...

a-murine-model-pressure-overload-induced-right-ventricular

Nanyang Technological University

Research

JoVE Journal

Medicine

578 Views.  Aarhus University. 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. 

Video: Author Spotlight: Investigating the Underlying Mechanisms of Right Ventricular Failure in Pulmonary Hypertension

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript discusses the latest development of the previously published ovine PH-RVF model that utilizes left pulmonary artery (PA) ligation and main PA occlusion. This model of PH-RVF is a versatile platform to control not only the disease severity but also the RV&#39;s phenotypic response.
Adult sheep (60-80 kg) underwent left PA (LPA) ligation, placement of main PA cuff, and insertion of RV pressure monitor. PA cuff and RV pressure monitor were connected to subcutaneous ports. Subjects underwent progressive PA banding twice per week for 9 weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). At the initiation and endpoint of this model, ventricular function and dimensions were assessed using echocardiography. In a representative group of 12 animal subjects, RV mean and systolic pressure increased from 28 &#177; 5 and 57 &#177; 7 mmHg at week 1, respectively, to 44 &#177; 7 and 93 &#177; 18 mmHg (mean &#177; standard deviation) by week 9. Echocardiography demonstrated characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The longitudinal trend of SvO2 and PA cuff pressure demonstrates that the rate of PA banding can be titrated to elicit varying RV phenotypes. A faster PA banding strategy led to a precipitous decline in SvO2 &#60; 65%, indicating RV decompensation, whereas a slower, more paced strategy led to the maintenance of physiologic SvO2 at 70%-80%. One animal that experienced the accelerated strategy developed several liters of pleural effusion and ascites by week 9. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and enabling therapeutic innovation to manage RV adaptation and maladaptation from PH.

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing ...

Bioengineering

A Pulmonary Trunk Banding Model of Pressure Overload Induced Right Ventricular Hypertrophy and Failure

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary disorders. Reliable and reproducible animal models of RV failure are therefore warranted in order to investigate disease mechanisms and effects of potential therapeutic strategies. Banding of the pulmonary trunk is a common method to induce isolated RV hypertrophy but in general, previously described models have not succeeded in creating a stable model of RV hypertrophy and failure.
We present a rat model of pressure overload induced RV hypertrophy caused by pulmonary trunk banding (PTB) that enables different phenotypes of RV hypertrophy with and without RV failure. We use a modified ligating clip applier to compress a titanium clip around the pulmonary trunk to a pre-set inner diameter. We use different clip diameters to induce different stages of disease progression from mild RV hypertrophy to decompensated RV failure.
RV hypertrophy develops consistently in rats subjected to the PTB procedure and depending on the diameter of the applied banding clip, we can accurately reproduce different disease severities ranging from compensated hypertrophy to severe decompensated RV failure with extra-cardiac manifestations.
The presented PTB model is a valid and robust model of pressure overload induced RV hypertrophy and failure that has several advantages to other banding models including high reproducibility and the possibility of inducing severe and decompensated RV failure.

We present a surgical method to induce right ventricular hypertrophy and failure in rats.

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary ...

Quantification of Biventricular Function and Morphology by Cardiac Magnetic Resonance Imaging in Mice with Pulmonary Artery Banding

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. Assessment of RV function and morphology is complex, partly due to the complex shape of the RV. Currently, cardiac magnetic resonance (CMR) imaging is the golden standard for noninvasive assessment of RV function and morphology. The current protocol describes CMR imaging in a mouse model of RV pressure load induced by pulmonary artery banding (PAB). PAB is performed by placing a 6-0 suture around the pulmonary artery over a 23 G needle. The PAB gradient is determined using echocardiography at 2 and 6 weeks. At 6 weeks, the right and left ventricular morphology and function is assessed by measuring both end-systolic and end-diastolic volumes and mass by ten to eleven cine slices 1 mm thick using a 9.4 T magnetic resonance imaging scanner equipped with a 1,500 mT/m gradient. Representative results show that PAB induces a significant increase in RV pressure load, with significant effects on biventricular morphology and RV function. It is also shown that at 6 weeks of RV pressure load, cardiac output is maintained. Presented here is a reproducible protocol for the quantification of biventricular morphology and function in a mouse model of RV pressure load and may serve as a method for experiments exploring determinants of RV remodeling and dysfunction.

To understand the pathophysiology of right ventricular (RV) adaptation to abnormal loading, experimental models are crucial. However, assessment of RV dimensions and function is complex and challenging. This protocol provides a method to perform cardiac magnetic resonance imaging (CMR) as a noninvasive benchmark procedure in mice subjected to RV pressure load.

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. ...

O-Ring Aortic Banding Versus Traditional Transverse Aortic Constriction for Modeling Pressure Overload-Induced Cardiac Hypertrophy

Aortic banding in mice is one of the most commonly used experimental models for cardiac pressure overload-induced cardiac hypertrophy and the induction of heart failure. The previously used technique is based on a threaded suture around the aortic arch tied over a blunted 27 G needle to create stenosis. This method depends on the surgeon manually tightening the thread and, thus, leads to high variance in the diameter size. A newly refined method described by Melleby et al. promises less variance and more reproducibility after surgery. The new technique, o-ring- aortic banding (ORAB), uses a non-slip rubber ring instead of a suture with a thread, resulting in reduced variation in pressure overload and reproducible phenotypes of cardiac hypertrophy. During surgery, the o-ring is placed between the brachiocephalic and left carotid arteries. Successful constriction is confirmed by echocardiography. After 1 day, correct placement of the ring results in an increased flow velocity in the transverse aorta over the o-ring-induced stenosis. After 2 weeks, impaired cardiac function is proven by decreased ejection fraction and increased wall thickness. Importantly, besides less variance in the diameter size, ORAB is associated with lower intra- and post-operative mortality rates compared with transverse aortic constriction (TAC). Thus, ORAB represents a superior method to the commonly used TAC surgery, resulting in more reproducible results and a possible reduction in the number of animals needed.

The present protocol describes a new technique of aortic banding in mice to induce pressure overload cardiac hypertrophy. For banding, a rubber ring with a fixed inner diameter is used. This new technique promises less variance and more reproducible data for future experiments.

Aortic banding in mice is one of the most commonly used experimental models for cardiac pressure overload-induced cardiac hypertrophy and the induction of ...

Chronic Ovine Model of Right Ventricular Failure and Functional Tricuspid Regurgitation

The pathophysiology of severe functional tricuspid regurgitation (FTR) associated with right ventricular dysfunction is poorly understood, leading to suboptimal clinical results. We set out to establish a chronic ovine model of FTR and right heart failure to investigate the mechanisms of FTR. Twenty adult male sheep (6-12 months old, 62 &#177; 7 kg) underwent a left thoracotomy and baseline echocardiography. A pulmonary artery band (PAB) was placed and cinched around the main pulmonary artery (PA) to at least double the systolic pulmonary artery pressure (SPAP), inducing right ventricular (RV) pressure overload and signs of RV dilatation. PAB acutely increased the SPAP from 21 &#177; 2 mmHg to 62 &#177; 2 mmHg. The animals were followed for 8 weeks, symptoms of heart failure were treated with diuretics, and surveillance echocardiography was used to assess for pleural and abdominal fluid collection. Three animals died during the follow-up period due to stroke, hemorrhage, and acute heart failure. After 2 months, a median sternotomy and epicardial echocardiography were performed. Of the surviving 17 animals, 3 developed mild tricuspid regurgitation, 3 developed moderate tricuspid regurgitation, and 11 developed severe tricuspid regurgitation. Eight weeks of pulmonary artery banding resulted in a stable chronic ovine model of right ventricular dysfunction and significant FTR. This large animal platform can be used to further investigate the structural and molecular basis of RV failure and functional tricuspid regurgitation.

Right ventricular failure and functional tricuspid regurgitation are associated with left-sided heart disease and pulmonary hypertension, which contribute significantly to morbidity and mortality in patients. Establishing a chronic ovine model to study right ventricular failure and functional tricuspid regurgitation will help in understanding their mechanisms, progression, and possible treatments.

The pathophysiology of severe functional tricuspid regurgitation (FTR) associated with right ventricular dysfunction is poorly understood, leading to ...

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

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.

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.

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Biology

Optimizing Pulmonary Artery Banding for Consistent Right Heart Failure Induction

Rat Model of Right-Sided Cardiac Remodeling and Arrhythmia Using Pulmonary Artery Banding

Clinical conditions, including chronic obstructive pulmonary disease or pulmonary arterial hypertension (PAH), can lead to chronic right ventricle pressure overload and progressive right heart failure (RHF). RHF can be identified by right-sided cardiac hypertrophy and dilation associated with abnormal myocardial function affecting the RV and the right atrium (RA). We recently demonstrated that severe RHF is accompanied by an increased risk of atrial inflammation, atrial fibrosis, and atrial fibrillation (AF), the most common type of cardiac arrhythmia (CA). Recent studies have shown that RV and RA inflammation plays an important role in the arrhythmogenesis of CA, including AF. However, the impact of inflammation in the development of CA and AF in RHF is poorly described.
Experimental models of RHF are required to better understand the association between right-sided myocardial inflammation and CA. The rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH) is well-established to provoke RHF. However, MCT triggers severe pneumo-toxicity and pulmonary inflammation. Hence, MCT-induced RHF does not help to distinguish whether the subsequent myocardial inflammation originates from the RHF per se or circulating inflammatory signals secreted by the injured lung.
In this article, a mechanical method involving pulmonary artery trunk banding (PAB) was used to provoke right-sided cardiac arrhythmogenesis. The PAB consists of performing a permanent suture of the pulmonary artery trunk for 3 weeks. Such an approach generates increased right-sided pressure overload. At D21 post-PAB, the suture results in hypertrophied, dilated, and inflamed RV and RA. The PAB-induced RHF is also accompanied by vulnerability to ventricular and atrial arrhythmias, including AF.

Right heart failure (RHF) is characterized by right-sided cardiac dilation and hypertrophy, leading to ventricular and atrial malfunction. Cardiopulmonary conditions associated with RHF are accompanied by an increased risk for cardiac arrhythmias. This article describes a standardized model of pulmonary artery banding-induced RHF associated with enhanced ventricular and atrial arrhythmogenesis.