This work was supported by The Danish Council for Independent Research [11e108410], the Danish Heart Foundation [12e04-R90-A3852 and 12e04-R90-A3907], and The Novo Nordisk Foundation [NNF16OC0023244].

All rats were treated according to Danish national guidelines&#160;described in the Danish law on animal experiments and Ministerial order on animal experiments. All experiments were approved by the Institutional Ethics Review Board and conducted in accordance with the Danish law for animal research (authorization number 2012-15-2934-00384, Danish Ministry of Justice).
1. Adjustment of the Ligating Clip Applier
NOTE: The banding of the pulmonary trunk is performed with a modified open ligating clip applier with an angled jaw. The applier is modified with an adjustable stop mechanism to stop the compression when the jaws reach an exact distance from each other. When a small titanium ligating clip is compressed with the modified applier, a lumen persists between the legs of the clip with a specific diameter according to the adjustment of the stop mechanism (Figure 1).
<ol>
	<li>Choose the diameter of the desired banding, e.g., 0.6 mm.</li>
	<li>Adjust the ligating clip applier until the distance between the jaws is 1.0 mm when fully compressed. This leaves a lumen of 0.6 mm as the two clip legs have a thickness of 0.2 mm each.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58050/58050fig1.jpg" /> 
Figure 1: The PTB procedure.&#160;(A) The surgical instruments used for the PTB procedure including the ligating clip applier (blue arrow). (B) The adjustable stop mechanism of the ligating clip applier. Turning the cogwheel (blue arrow) will adjust the position of the pin (yellow arrow), which stops the closing of the applier when the jaws reach a certain distance from each other. The distance corresponds to twice the thickness of the legs of the clip plus the inner diameter of the clip, when the clip is compressed, and can be calibrated by using for example a needle with a known outer diameter. (C) The applier compresses a titanium clip to an exact inner diameter pre-specified by adjustment of the applier. (D) The inner diameter of the compressed clip can be adjusted in order to induce different severities of RV hypertrophy and failure. For the data presented, an inner diameter of 1.0 mm was used to induce mild RV hypertrophy, an inner diameter of 0.6 mm was used to induce moderate RV failure, and an inner diameter of 0.5 mm was used to induce severe RV failure. (E) The clip after application around the pulmonary trunk. <a href="https://www.jove.com/files/ftp_upload/58050/58050fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Preparation of the Rat
NOTE: Other regimens of analgesics may be applied.
<ol>
	<li>Use Wistar rat weanlings weighing approximately 100&#8211;120 g. In order to maintain body temperature during the surgery, use a covered heating pad.</li>
	<li>For surgery, use a mechanical ventilator set to a tidal volume of approximately 1.75 mL and respiratory rate of 75 per min.</li>
	<li>Anaesthetize the rat with sevoflurane (7% mix in 1.5 L of O2) in an induction chamber for 5 minutes. Intubate the rat using a 17 G IV cannula, where the distal 2 mm of the needle have been cut off in order for the soft catheter to cover the tip. Remove the needle and connect the cannula to the ventilator.</li>
	<li>Place the rat on its back on the heating pad. Make sure that the intubation is correct by observing the movements of the thorax. These should be without side differences and in pace with the ventilator. 
	NOTE: Absence of movements of the thorax, abdominal contractions and inflation of the stomach in the upper left abdomen are signs of a misplaced tube. Remove the cannula, put the rat back in the induction chamber and re-intubate.</li>
	<li>After correct intubation, reduce the sevoflurane to maintenance concentration (3.5% mix in O2, 1.5 L/min) and fix the paws of the rat to the heating pad.</li>
	<li>Confirm prober anesthetization by checking withdrawal reflexes of the extremities using a forceps to squeeze the paws of the rat.</li>
	<li>Inject the rats with buprenorphine (0.1 mg/kg subcutaneously (s.c.)) and carprofene (5 mg/kg s.c.) to relieve post-operative pain.</li>
	<li>Shave the chest and disinfect with chlorhexidine.</li>
</ol>
3. Isolation of the Pulmonary Trunk
<ol>
	<li>With a pair of scissors, make a 2 cm incision in the skin along the middle part of the sternum. Identify the major pectoral muscle and cut its sternal attachment. Identify the 2nd, 3rd, and 4th costa below.</li>
	<li>Optionally, grab the 2nd costa with a fixation forceps, put a suture (4-0, multifilament, absorbable) around the 2nd costa from the 1st intercostal space to the lower medial part of the 2nd intercostal space. Tie a firm knot in order to ligate the anterior thoracic artery. 
	NOTE:&#160;This can be useful if bleeding from the anterior thoracic artery is a recurrent issue.</li>
	<li>Cut the 4th, 3rd, and 2nd costa close to the sternum with a pair of scissors and carefully dissect the intercostal muscles until a complete left thoracotomy has been performed. If any bleeding from the anterior thoracic artery occurs, compress with a pean and ligate the artery.</li>
	<li>Insert a retractor between the sternum and the costae and open it to get a good operating field. At the top of the field is the thymus covering the aorta and the pulmonary trunk. Carefully lift the thymus using a pean and flip it upwards in order to expose the aorta and the pulmonary trunk below.</li>
	<li>Guide the tip of a small surgical hooklet with a 85&#176; angle through the transverse pericardial sinus located behind the left atrial appendage. Pull it halfway back through the sinus and guide the tip of the ear hook upwards until it appears between the ascending aorta and the pulmonary trunk.
	<ol>
		<li>Remove any connective tissue covering the tip with an iris scissor in order to separate the pulmonary trunk from the ascending aorta.</li>
		<li>Repeat the step with a larger hook (optional).</li>
	</ol>
	</li>
	<li>Guide an angled muscle forceps around the pulmonary trunk through the passage made with the hook(s). Grab the end of an approximately 10 cm ligature (4-0, multifilament) and pull half of the ligature back through the passage. Now the pulmonary trunk is separated from the ascending aorta and can be controlled by the ligature around it.</li>
</ol>
4. Application of the Clip
<ol>
	<li>Load the adjusted ligating clip applier with a clip. Carefully guide one of the jaws and one leg of the clip though the passage around the pulmonary trunk. Use the ligature to gently pull the pulmonary trunk upwards and into the fork of the clip.</li>
	<li>When the pulmonary trunk is in the fork of the clip and the two tips of the clip legs are free of any connective tissue, compress the clip with the applier to apply the banding.</li>
	<li>Observe how the RV immediately dilates in response to the banding and remove the ligature.</li>
</ol>
5. Closing of the Thorax
<ol>
	<li>Remove the pean from the thymus and reposition the thymus&#160;to its natural position. Remove the retractor.</li>
	<li>Close the thorax in three layers: the intercostal layer, the major pectoral muscle, and the skin with suture (4-0, multifilament, absorbable). Inject 2 mL of saline s.c. to replace fluid lost during surgery.</li>
	<li>Turn the sevoflurane off and and keep the rat on the ventilator (1.5 L of O2) until it starts breathing spontaneously. Then, extubate the rat.</li>
	<li>Treat the rats with buprenorphine in the drinking water for the following three days14 or apply a similar analgesic protocol. After three days, the rats have recovered and are without discomfort.</li>
	<li>In the following weeks, the well-being of the rats and possible adverse effects should be evaluated on a daily basis. The healing of the wound from the thoracotomy should receive special attention during the first week in order to detect any signs of infection or insufficiency of the cicatrices. If the rats show signs of failure to thrive including bristly fur, impaired mobility, respiratory problems, and weight loss, they should be monitored closely and euthanized if they lose more than 20% of their body weight or develop fulminant respiratory insufficiency.&#160;</li>
</ol>
6. Sham Surgery
<ol>
	<li>Perform a sham surgery by following all of the steps above except for the application of the clip (step 4).</li>
</ol>

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The authors have nothing to disclose

We describe an accessible and highly reproducible method of pulmonary trunk banding using a modified ligating clip applier to compress a titanium clip around the pulmonary trunk. By adjusting the applier to compress the clip to different inner diameters, distinct phenotypes of RV hypertrophy and failure can be induced including severe RV failure with extra-cardiac manifestation of decompensation.
Although simple, the protocol contains a few critical steps. Importantly, the rats cannot be too big when they undergo the PTB procedure. In our experience, Wistar rat weanlings weighing 100&#8211;120 g are suitable for the procedure. In larger rats, application of a severe banding may lead to acute RV failure and death. Other studies6,7,8,9,10,16&#160;have mainly used larger rats (160&#8211;260 g) but also larger diameters of their respective bandings (1.27&#8211;1.65 mm).
The application of banding of less severity may also explain the relative modest increases in RV pressures reported by other groups. Banding with an 18 G needle (1.27 mm) leads to RV systolic pressures in the range of 70&#8211;90 mmHg6,7,8,9. In one study6, this was not sufficient to cause RV fibrosis or reduce cardiac output. Here, we report RV pressures of approximately 90 mmHg for a moderate banding and 110 mmHg for a severe banding. With a severe banding, we have also been able to create a phenotype of decompensated RV failure with extra-cardiac manifestations including hepatic congestion and ascites12. Pulmonary trunk banding by ligation using a 20G needle (0.902 mm) caused liver fibrosis, nutmeg-like liver congestion and ascites in Sprague Dawley rats16&#160;despite the relatively mild constriction compared to our study. This may be explained by different rat strains responding differently to the banding. There are significant differences with regards to metabolism17, adrenergic tone and heart rate18&#160;between rat strains. Even within the same rat strain, various characteristics including growth rate may vary with different vendors19. This should always be taken into account. For the specific rat strain used, it is therefore critical that well-designed pilot studies are performed in order to determine the banding diameter and the follow-up time needed for the desired RV failure phenotype to develop. The clip model can potentially be used in rat neonates as opposed to the ligating technique previously used20, but we have no experience with this and the same considerations as mentioned above applies before initiating a study.
The PTB model has a few limitations. Firstly, the very proximal occlusion by the clip around the pulmonary trunk represents the conditions of pulmonic stenosis or CTEPH more than the distal narrowing of the smaller pulmonary arteries seen in PAH. The adaptation of the RV to the increased afterload may vary depending on the location of the obstruction(s)21. Secondly, the application of the clip during surgery causes a very sudden increase in RV afterload different from the gradual increase in pulmonary vascular resistance in PAH. The procedure is, however, performed in rat weanlings (100&#8211;120 g) giving rise to a progressively increased RV afterload relative to body weight with growth of the animals. During the seven weeks period after surgery, the body weight of the rats increases approximately fourfold and hence relative RV afterload increases proportionally inducing a progressive disease development6.
Using a modified ligating clip applier and a titanium clip for banding of the pulmonary trunk, we were able to induce RV failure. The method has several advantages including high reproducibility and the possibility of creating different disease severities from mild RV hypertrophy to decompensated RV failure by adjusting the diameter of the banding clip. Changing the diameter by 0.1 mm resulted in distinct RV failure phenotypes ranging from moderate and compensated RV failure to severe and decompensated RV failure demonstrating accuracy of this pulmonary trunk banding method.

The right ventricle (RV) can adapt to a persistent pressure overload. In time, however, adaptive mechanisms fail to sustain cardiac output, the RV dilates and eventually the RV fails. RV function is the main prognostic factor of several cardiopulmonary disorders including pulmonary arterial hypertension (PAH), thromboembolic pulmonary hypertension (CTEPH), and various forms of congenital heart disease with a pressure (or volume) overload of the RV. Despite intense treatment, RV failure remains a predominant cause of death in these conditions.
As a consequence of the unique properties1,2 and embryological development3 of the RV, knowledge derived from left heart failure cannot simply be extrapolated to right heart failure. Animal models of right heart failure are therefore needed in order to investigate the mechanisms of RV failure and potential pharmacological treatment strategies.
There are experimental models of pulmonary hypertension induced by SU5416 combined with hypoxia (SuHx)4 or monocrotaline (MCT)5, which induce RV failure secondary to disease in the pulmonary vasculature. These models are used to evaluate therapeutic effects of drugs that target the pulmonary vasculature. Both the SuHx and the MCT model are non-fixed afterload models of RV failure. Consequently, it is not possible to conclude if an improvement in RV function after an intervention is secondary to afterload reducing pulmonary vascular effects or if it is caused by direct effects on the RV. In addition, the MCT model has several extra-cardiac effects.
In experimental pulmonary trunk banding models, the afterload of the RV is fixed due to a mechanical constriction of the pulmonary trunk. This allows for the investigation of direct cardiac effects of an intervention on the RV independent from any pulmonary vascular effects6,7,8,9. Usually, the banding is performed by placing a needle along the pulmonary trunk. Then a ligature is placed around the needle and the pulmonary trunk and tied with a knot, and the needle is removed leaving the suture around the pulmonary trunk. Depending on the gauge of the needle, different degrees of constrictions can be applied, but despite this approach being widely used, it has some disadvantages. First, the diameter of the banding is not exactly the same as the outer diameter of the needle as the ligature is tied around both the needle and the pulmonary trunk. Second, there may be significant variation to how tightly the knot is tied making it difficult to reproduce a certain degree of banding. This will lead to a variation in banding diameter and thereby a larger dispersion. Finally, the knot may come loose over time.
One study applies a half-closed tantalum clip around the pulmonary trunk10. They compressed the clip around the pulmonary trunk to an inner area of 1.10 mm2 and compared it to rats subjected to banding with a suture using an 18 G needle. Overall, banding with the clip was associated with less peri-surgical complications and data variance.
Based on the principles described by Schou et al.11, we further developed and characterized the pulmonary trunk banding (PTB) model of RV hypertrophy and failure. Here, we present our experience using this model based on results from previous studies12,13. For this model, a titanium clip is compressed around the pulmonary trunk to an exact preset inner diameter, which may be adjusted in order to induce distinct RV failure phenotypes.

<table><tbody><tr><td>17 G IV Venflon Cannula</td><td>Becton Dickinson, US</td><td>393228</td><td>Distal 2 mm of the needle have been cut off</td></tr><tr><td>1 mL syringe + 26 G needle</td><td>Becton Dickinson, US</td><td>303172 &amp;amp; 303800</td><td></td></tr><tr><td>4-0 absorbable multifilament suture</td><td>Covidien, US</td><td>GL-46-MG</td><td>Polysorb, violet, 5x18&quot;</td></tr><tr><td>4-0 multifilament ligature</td><td>Covidien, US</td><td>LL-221</td><td>Polysorb, violet, 98&quot;</td></tr><tr><td>Buprenorphine</td><td>Indivior UK Limited</td><td></td><td>Local procurement, Temgesic 0.3 mg/mL</td></tr><tr><td>Carprofene</td><td>ScanVet, DK</td><td>27693</td><td>Norodyl 50 mg/mL</td></tr><tr><td>Chlorhexidine</td><td>Faaborg Pharma, DK</td><td></td><td>Local procurement</td></tr><tr><td>Contractor</td><td>Aesculap, Germany</td><td>BV010R</td><td>Blunt, self retaining, 70 mm</td></tr><tr><td>Ear Hooklet</td><td>Lawton, Germany</td><td>66-0261</td><td>Small, 14 cm, tip modified to an angle of 85&amp;deg;</td></tr><tr><td>Eye gel</td><td>Decra, UK</td><td></td><td>Lubrithal, Local procurement</td></tr><tr><td>Forceps, Delicate Tissue</td><td>Lawton, Germany</td><td>09-0020</td><td></td></tr><tr><td>Forceps, Dissecting</td><td>Lawton, Germany</td><td>09-0013</td><td>1 regular, 1 with tip modified to an angle of 100&amp;deg;</td></tr><tr><td>Gas Anesthesia System</td><td>Penlon Limited, UK</td><td>SD0217SL</td><td>Sigma Delta Vaporizer</td></tr><tr><td>Hair trimmer</td><td>Oster</td><td>76998-320-051</td><td></td></tr><tr><td>Horizon Open Ligating Clip Applier</td><td>Teleflex, US</td><td>137085</td><td>Modified with adjustable stop mechanism</td></tr><tr><td>Horizon Titanium Clips</td><td>Teleflex, US</td><td>001200</td><td>Small</td></tr><tr><td>Induction chamber</td><td>N/A</td><td></td><td></td></tr><tr><td>Iris Scissor</td><td>Lawton, Germany</td><td>05-1450</td><td></td></tr><tr><td>Iris Scissor</td><td>Aesculap, Germany</td><td>BC060R</td><td></td></tr><tr><td>Mechanical ventilator</td><td>Ugo Basile, Italy</td><td>7025</td><td></td></tr><tr><td>Microscissor</td><td>Lawton, Germany</td><td>63-1406</td><td></td></tr><tr><td>Microscope</td><td>Carl Zeiss, Germany</td><td>303294-9903</td><td></td></tr><tr><td>Needle Holder</td><td>Lawton, Germany</td><td>08-0011</td><td>TITEGRIP</td></tr><tr><td>Pean</td><td>Lawton, Germany</td><td>06-0100</td><td>Halsted-Mosquito, straight</td></tr><tr><td>Pro-Optha</td><td>Lohmann &amp;amp; Rauscher, Germany</td><td>16515</td><td>Tampon</td></tr><tr><td>Saline 9 mg/mL</td><td>Fresenius Kabi, DK</td><td>209319</td><td></td></tr><tr><td>Sevoflurane</td><td>AbbVie, US</td><td></td><td>Sevorane, Local procurement</td></tr><tr><td>Surgical hook</td><td>Lawton, Germany</td><td>51-0665</td><td>Cushing, 19 cm, tip modified to an angle of 90&amp;deg;</td></tr><tr><td>Surgical Tape</td><td>3M, US</td><td>1530-0</td><td>Micropore</td></tr><tr><td>Temperature Controller</td><td>CMA Microdialysis; Sweden</td><td>8003760</td><td>CMA 450</td></tr><tr><td>Weighing machine</td><td>VWR, US</td><td></td><td></td></tr><tr><td>Wistar rat weanlings</td><td>Janvier Labs, France</td><td></td><td>RjHan:WI, 100-120 g</td></tr></tbody></table>

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.

Using the described PTB procedure in previous studies from our group12,13, we induced RV hypertrophy (PTB mild) by banding with a 1.0 mm clip, a moderate degree of RV failure (PTB moderate) by banding with a 0.6 mm clip and a severe degree of RV failure (PTB severe) by banding with a 0.5 mm clip. The rats subjected to the severe banding developed extra-cardiac manifestations of RV failure including liver failure and ascites (Figure 5E). All rats were evaluated seven weeks after the PTB and sham rats underwent the same procedure only without the application of the clip. Perioperative mortality was less than 1 in 6. Seven weeks survival rate was 80% for rats subjected to severe banding and close to 100% in rats subjected to mild or moderate banding or sham surgery.
For evaluation of the effects of the PTB procedure, we used echocardiography together with cardiac magnetic resonance imaging (MRI) to assess RV volumes and cardiac output. Tricuspid annular plane systolic excursion (TAPSE) was measured as the distance of the tricuspid annular plane with the RV contraction in the apical four-chamber view. An average of three cycles outside respiration was used as a representative value. RV end-diastolic volume (EDV) and end-systolic volume (ESV) were assessed by drawing of the endocardium in a series of short axis images through the RV obtained by MRI for each rat, and RV ejection fraction (EF) calculated as EF = (EDV-ESV)/EDV. Cardiac output was measured between the pulmonary valves and the clip using a phase-contrast MRI sequence. Digital recordings of RV pressures were obtained by a micro tip catheter installed in the RV before euthanasia. Further details of the methods have been described previously12. RV hypertrophy was evaluated as the ratio of the RV weight divided by the weight of the left ventricle (LV) plus septum and as the weight of the RV divided by the length of the tibia to correct for the size of the rat. All methods were applied as described previously12.
In one week, elevated RV pressures and RV dysfunction evident by a decrease in cardiac output and TAPSE had developed in the PTB rats compared to sham operated rats. Accordingly, interventions or pharmacological treatments can be initiated already at this time point if one aims to investigate effects on established RV failure. After additional six weeks, RV pressure had further increased. The differences of the moderate vs the severe RV failure phenotype were even more pronounced shown by a stepwise decrease in both cardiac output and TAPSE with increased severity of the banding (Figure 2 and Figure 3). Detailed hemodynamic differences between PTB mild rats and PTB severe rats 4 weeks after surgery have been published by our group previousely15.
The PTB procedure also caused RV dilatation evident by an increase in both RV EDV and RV ESV in the moderate PTB rats compared to sham operated rats and in the severe PTB rats compared to both moderate PTB and sham. A stepwise decrease in RV EF was also seen (Figure 4).
The development of RV hypertrophy was associated to the magnitude of the pressure overload applied by the clip. The ratio of the RV over the LV plus septum weight increased stepwise from rats with a mild banding with a clip of 1.0 mm over rats banded with a moderate 0.6 mm clip to rats banded with a severe 0.5 mm clip. Similar results were seen for the RV weight corrected for the size of the rat by dividing with the length of the tibia. The hypertrophy was also seen as an increase in cardiomyocyte cross sectional area in the PTB rats compared to sham operated rats. Apart from hypertrophy of the cardiomyocytes, the pressure overload also induced other morphological changes of the RV associated with RV failure including RV fibrosis. In rats subjected to severe banding, decompensated RV failure was induced. This phenotype was characterized by signs of backward failure including hepatic congestion seen as a dark discoloration of the liver. Hepatic congestion was usually accompanied by ascites (Figure 5).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58050/58050fig2.jpg" /> 
Figure 2: Effects of PTB one week and seven weeks after the procedure.&#160;(A) Right ventricular (RV) systolic pressure (B) cardiac output and (C) tricuspid annular plane systolic excursion (TAPSE) measured one week after sham or PTB operation with a moderate or a severe banding respectively. (D, E&#160;and F) The same measures seven weeks after the procedures and further development of RV failure12. Data presented as mean &#177; SEM. One-way ANOVA with post hoc Bonferroni analysis. **p &#60; 0.01, ***p &#60; 0.001, and ****p &#60; 0.0001 PTB vs sham and PTB severe vs PTB moderate. <a href="https://www.jove.com/files/ftp_upload/58050/58050fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58050/58050fig3.jpg" /> 
Figure 3: Effects of the PTB procedure assessed by echocardiography.&#160;(A) A representative four chamber view and (B) measurements of velocity time integral (VTI) in the pulmonary trunk (upper panel) and tricuspid annular plane systolic excursion (TAPSE) (lower panel) in a sham operated rat. (C and D) Similar images for a PTB rat subjected to moderate banding. All images are seven weeks after sham operation or banding. <a href="https://www.jove.com/files/ftp_upload/58050/58050fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58050/58050fig4.jpg" /> 
Figure 4: Magnetic resonance imaging of PTB rats.&#160;Cardiac magnetic resonance imaging (MRI) of PTB and sham operated rats. (A) Representative four chamber images (upper panel) and short axis views (lower panel) of sham rats (left) and PTB rats with a moderate degree of RV failure (right). In the PTB rat, the high RV pressures caused septum bulging (blue asterisk). (B) The PTB procedure induced RV dilatation evident by an increase in both RV end diastolic volume (EDV) and RV end systolic volume (ESV). (C) RV ejection fraction (EF) decreased12. Data presented as mean &#177; SEM. One-way ANOVA with post hoc Bonferroni analysis. *p &#60; 0.05, **p &#60; 0.01, and ****p &#60; 0.0001 PTB vs sham and PTB severe vs PTB moderate. <a href="https://www.jove.com/files/ftp_upload/58050/58050fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58050/58050fig5.jpg" /> 
Figure 5: Anatomical data and histology.&#160;RV hypertrophy measured as (A) RV divided by the LV plus septum and (B) RV weight divided by tibia length from rats with mild RV hypertrophy, moderate RV failure, and severe RV failure seven weeks after the PTB procedure. (C) Representative images of histological sections stained with hematoxyline eosine for measurement of cardiomyocyte cross sectional area and (D) picrosirius red analyzed under polarized light for fibrosis from sham rats (left) and PTB rats with moderate RV failure (right). (E) A healthy liver (left) and a discolored liver with congestion (right) from a PTB rat with severe RV failure12,13. Data presented as mean &#177; SEM. One-way ANOVA with post hoc Bonferroni analysis. **p &#60; 0.01 and ****p &#60; 0.0001 PTB vs sham, PTB moderate vs PTB mild and PTB severe vs PTB moderate. <a href="https://www.jove.com/files/ftp_upload/58050/58050fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Pulmonary Trunk Banding Model of Pressure Overload Induced Right Ventricular Hypertrophy and Failure at JoVE.com

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

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Universidad Científica del Sur

Research

JoVE Journal

Medicine

9.6K Views.  Aarhus University Hospital. We present a surgical method to induce right ventricular hypertrophy and failure in rats.

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

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 ...

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

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. ...

A Novel Right Ventricular Volume and Pressure Loaded Piglet Heart Model for the Study of Tricuspid Valve Function.

Heart conditions in which the tricuspid valve (TV) faces either increased volume or pressure stressors are associated with premature valve failure. Mechanistic studies to improve our&#160;understanding of the underlying pathophysiology responsible for the development of premature TV failure are lacking. Due to the inability to conduct these studies in humans, an animal model is required. In this manuscript, we describe the protocols for a novel chronic recovery infant piglet heart model for the study of changes in the TV when placed under combined volume and pressure stress. In this model, volume loading of the right ventricle&#160;and the TV is achieved through the disruption of the pulmonary valve. Then pressure loading is accomplished through the placement of a pulmonary artery band. The success of this model is assessed at four weeks post intervention surgery through echocardiography, intracardiac pressure measurement, and pathologic examination of the heart specimens.

A novel recovery piglet heart model with combined pressure and volume overload on the right ventricle is described for the study of tricuspid valve function.

Heart conditions in which the tricuspid valve (TV) faces either increased volume or pressure stressors are associated with premature valve failure. ...

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 ...

Ascending Aortic Banding for Studying Pressure-Overload Induced Heart Failure

A Minimally Invasive Model of Aortic Stenosis in Swine

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological similarities to humans. Efforts have been dedicated to creating a model of pressure-overload induced heart failure, and ascending aortic banding while still supra-coronary and not a perfect mimic of aortic stenosis in humans, closely resembling the human condition.
The purpose of this study is to demonstrate a minimally invasive approach to induce left ventricular pressure overload by placing an aortic band, precisely calibrated with percutaneously introduced high-fidelity pressure sensors. This method represents a refinement of the surgical procedure (3Rs), resulting in homogenous trans-stenotic gradients and reduced intragroup variability. Additionally, it enables swift and uneventful animal recovery, leading to minimal mortality rates. Throughout the study, animals were followed for up to 2 months after surgery, employing transthoracic echocardiography and pressure-volume loop analysis. However, longer follow-up periods can be achieved if desired. This large animal model proves valuable for testing new drugs, particularly those targeting hypertrophy and the structural and functional alterations associated with left ventricular pressure overload.

Author Spotlight: Development of a Minimally Invasive Large-Animal Model for Reliable and Reproducible Cardiovascular Research 

This protocol describes a minimally invasive surgical procedure for ascending aortic banding in swine.

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological ...

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

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

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

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 ...

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.