Name: Author Spotlight: Investigating the Underlying Mechanisms of Right Ventricular Failure in Pulmonary Hypertension
Uploaded: 2024-06-14T14:40:00
Description: Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary ...

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.

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 ...

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

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+6-mercaptopurine+in+pressure+overload+induced+right+heart+failure.">Effects of 6-mercaptopurine in pressure overload induced right heart failure.</a> PLoS One. 14 (11), e0225122 (2019).</li><li>Egemnazarov, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+overload+creates+right+ventricular+diastolic+dysfunction+in+a+mouse+model:+Assessment+by+echocardiography.">Pressure overload creates right ventricular diastolic dysfunction in a mouse model: Assessment by echocardiography.</a> J Am Soc Echocardiogr. 28 (7), 828-843 (2015).</li><li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+right+ventricular+failure+by+pulmonary+artery+constriction+and+evaluation+of+right+ventricular+function+in+mice.">Induction of right ventricular failure by pulmonary artery constriction and evaluation of right ventricular function in mice.</a> J Vis Exp. (147), e59431 (2019).</li><li>Kojonazarov, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+peroxisome+proliferator-activated+receptor+&#946;/&#948;+agonist+gw0742+has+direct+protective+effects+on+right+heart+hypertrophy.">The peroxisome proliferator-activated receptor &#946;/&#948; agonist gw0742 has direct protective effects on right heart hypertrophy.</a> Pulm Circ. 3 (4), 926-935 (2013).</li><li>Kojonazarov, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P38+MAPK+inhibition+improves+heart+function+in+pressure-loaded+right+ventricular+hypertrophy.">P38 MAPK inhibition improves heart function in pressure-loaded right ventricular hypertrophy.</a> Am J Respir Cell Mol Biol. 57 (5), 603-614 (2017).</li><li>Rai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Riociguat+and+Sildenafil+on+right+heart+remodeling+and+function+in+pressure+overload+induced+model+of+pulmonary+arterial+banding.">Effect of Riociguat and Sildenafil on right heart remodeling and function in pressure overload induced model of pulmonary arterial banding.</a> Biomed Res Int. 2018, 3293584 (2018).</li><li>Sydykov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+deficiency+and+pharmacological+stabilization+of+mast+cells+ameliorate+pressure+overload-induced+maladaptive+right+ventricular+remodeling+in+mice.">Genetic deficiency and pharmacological stabilization of mast cells ameliorate pressure overload-induced maladaptive right ventricular remodeling in mice.</a> Int J Mol Sci. 21 (23), 9099 (2020).</li><li>Andersen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+combined+angiotensin+ii+receptor+antagonism+and+neprilysin+inhibition+in+experimental+pulmonary+hypertension+and+right+ventricular+failure.">Effects of combined angiotensin ii receptor antagonism and neprilysin inhibition in experimental pulmonary hypertension and right ventricular failure.</a> Int J Cardiol. 293, 203-210 (2019).</li><li>Andersen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+overload+induced+right+ventricular+remodeling+is+not+attenuated+by+the+anti-fibrotic+agent+pirfenidone.">Pressure overload induced right ventricular remodeling is not attenuated by the anti-fibrotic agent pirfenidone.</a> Pulm Circ. 9 (2), 2045894019848659 (2019).</li><li>Labazi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-dependent+changes+in+right+ventricular+gene+expression+in+response+to+pressure+overload+in+a+rat+model+of+pulmonary+trunk+banding.">Sex-dependent changes in right ventricular gene expression in response to pressure overload in a rat model of pulmonary trunk banding.</a> Biomedicines. 8 (10), 430 (2020).</li><li>Sun, X. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+mao-a+activity+promotes+progression+of+pulmonary+arterial+hypertension.">Increased mao-a activity promotes progression of pulmonary arterial hypertension.</a> Am J Respir Cell Mol Biol. 64 (3), 331-343 (2021).</li><li>Axelsen, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Empagliflozin+on+right+ventricular+adaptation+to+pressure+overload.">Effects of Empagliflozin on right ventricular adaptation to pressure overload.</a> Front Cardiovasc Med. 10, 1302265 (2023).</li><li>Mamazhakypov, A., Veith, C., Schermuly, R. T., Sydykov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+protocol+for+pulmonary+artery+banding+in+mice+to+generate+a+model+of+pressure-overload-induced+right+ventricular+failure.">Surgical protocol for pulmonary artery banding in mice to generate a model of pressure-overload-induced right ventricular failure.</a> STAR Protoc. 4 (4), 102660 (2023).</li><li>Boehm, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delineating+the+molecular+and+histological+events+that+govern+right+ventricular+recovery+using+a+novel+mouse+model+of+pulmonary+artery+de-banding.">Delineating the molecular and histological events that govern right ventricular recovery using a novel mouse model of pulmonary artery de-banding.</a> Cardiovasc Res. 116 (10), 1700-1709 (2020).</li><li>Andersen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+bisoprolol+and+losartan+treatment+in+the+hypertrophic+and+failing+right+heart.">Effects of bisoprolol and losartan treatment in the hypertrophic and failing right heart.</a> J Card Fail. 20 (11), 864-873 (2014).</li><li>Hirata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+model+of+pulmonary+artery+banding+leading+to+right+heart+failure+in+rats.">Novel model of pulmonary artery banding leading to right heart failure in rats.</a> Biomed Res Int. 2015, 753210 (2015).</li><li>Vildbrad, M. 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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...

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.

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	<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,&#160;</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&#160;</td>
			<td>Veet&#160;</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&#228;ger Vapor 2000 Sevoflurane</td>
			<td>Dr&#228;ger</td>
			<td>M35054</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye oinment neutral, &#34;Ophta&#34;</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&#34; (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&#160;</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>
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		<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, &#34;Opmi Pico&#34;</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>
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		<tr>
			<td>Varming plate&#160;</td>
			<td>Visual sonics</td>
			<td>11437</td>
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			<td>Venflon ProSafety, 22G, 0,9 x 25mm</td>
			<td>Becton Dickinson</td>
			<td>393222</td>
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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.

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

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

This method can be used to study the underlying mechanisms of right ventricular failure, identify new drug targets, and investigate the effect of potential therapeutic agents for the treatment of right ventricular failure. A major challenge in models of pulmonary trunk bending is to induce different stages of right ventricular pathology, especially the early stages of right ventricular remodeling with mild hypertrophy have been overlooked in published protocols. The underlying mechanisms of right ventricular failure in pulmonary hypertension remain inadequately understood.We investigate the initial molecular changes in mild right ventricular pathology, which might offer greater reversibility than later stages. The method enables us to evaluate right ventricular function independently from the pulmonary vasculature. Thus, we can investigate the direct effects of new therapeutic compounds on the right ventricle.This may increase our understanding of the underlying molecular pathways in the development of right ventricular failure, and thereby lead to the discovery of new therapeutic strategies for treating RV failure in patients with pulmonary hypertension.

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...

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 ...

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

Research

JoVE Journal

Medicine

Surgery for Pulmonary Trunk Banding to Create a Murine Model of Pressure Overload-Induced Right Ventricular Hypertrophy and Failure

Begin by intubating the anesthetized mouse with a 22-gauge IV catheter. Perform the intubation under visual guidance using a surgical microscope and an intubation stand, allowing proper alignment for visualization of the vocal cords. Ensure continuous delivery of inhalant anesthetics on a nasal tube until the endotracheal tube has been connected.Ventilate the mouse at 175 strokes per minute and a tidal volume of 300 microliter per stroke. Before beginning the surgery, subcutaneously administer analgesics for peri and postoperative analgesia and saline to compensate for perioperative fluid loss. Confirm proper placement of the endotracheal tube by observing symmetrical movement of the chest.To perform the surgery, use a scalpel to make a 10-millimeter incision in the skin above the second intercostal space from the sternal angle to the left anterior axillary line. Then, using forceps, bluntly dissect the major and minor pectoral muscles and identify the second and third costae. Place the custom-made thoracic retractor under the pectoral muscles.Cut the intercostal muscles in the second intercostal space. Reposition the thoracic retractor to the intercostal space to keep the operating field accessible. Rotate the mouse's lower body to improve the exposure of the pulmonary trunk.Bluntly dissect the thymus to expose the heart, pulmonary trunk, and aorta Using microscopic forceps, carefully remove the connective tissue between the vessels to separate the pulmonary trunk from the ascending aorta. Pass the guidance cannula through the transverse pericardial sinus posteriorly of the pulmonary trunk. Use forceps to grasp the knot on the tip of the guidance cannula and pull the suture through the cannula.Carefully remove the guidance cannula while the suture stays in place around the pulmonary trunk. Load the ligating clip applier. Use the suture to guide the pulmonary trunk into the jaws of the ligating clip and compress the clip.Release the suture immediately after placing the clip and observe the filling of the pulmonary trunk. Ensure that the clip is correctly placed. Place a 6-0 monofilament absorbable suture around the second and third costae and close the intercostal space.Evacuate as much air as possible from the thoracic cavity by applying gentle pressure to the chest while tightening the suture. Lastly, suture the pectoral muscles and skin with a 6-0 monofilament absorbable suture. To ensure adequate postoperative analgesia, add analgesic to the drinking water as well as administer it by subcutaneous injections for the first three days after surgery.