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...

<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 b...

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 border="0" cellpadding="0" cellspacing="0" width="804">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:217px;">17G IV Venflon Cannula</td>
			<td style="width:189px;">Becton Dickinson, US</td>
			<td style="width:108px;">393228</td>
			<td style="width:291px;">Distal 2 mm of the needle have been cut off</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">1 mL syringe + 26G needle&#160;</td>
			<td>Becton Dickinson, US</td>
			<td>303172 &#38; 303800</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">4-0 absorbable multifilament suture</td>
			<td>Covidien, US</td>
			<td>GL-46-MG</td>
			<td>Polysorb, violet, 5x18&#34;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">4-0 multifilament ligature</td>
			<td>Covidien, US</td>
			<td>LL-221</td>
			<td>Polysorb, violet, 98&#34;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Buprenorphine</td>
			<td>Indivior UK Limited</td>
			<td></td>
			<td>Local procurement, Temgesic 0.3 mg/mL</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Carprofene</td>
			<td>ScanVet, DK</td>
			<td>27693</td>
			<td>Norodyl 50 mg/mL</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chlorhexidine</td>
			<td>Faaborg Pharma, DK</td>
			<td></td>
			<td>Local procurement</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Contractor</td>
			<td>Aesculap, Germany</td>
			<td>BV010R</td>
			<td>Blunt, self retaining, 70 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ear Hooklet</td>
			<td>Lawton, Germany</td>
			<td>66-0261</td>
			<td>Small, 14 cm, tip modified to an angle of 85&#176;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Eye gel</td>
			<td>Decra, UK</td>
			<td></td>
			<td>Lubrithal, Local procurement</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Forceps, Delicate Tissue&#160;</td>
			<td>Lawton, Germany</td>
			<td>09-0020</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Forceps, Dissecting&#160;</td>
			<td>Lawton, Germany</td>
			<td>09-0013</td>
			<td>1 regular, 1 with tip modified to an angle of 100&#176;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gas Anesthesia System</td>
			<td>Penlon Limited, UK</td>
			<td>SD0217SL</td>
			<td>Sigma Delta Vaporizer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hair trimmer</td>
			<td>Oster</td>
			<td>76998-320-051</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Horizon Open Ligating Clip Applier</td>
			<td>Teleflex, US</td>
			<td>137085</td>
			<td>Modified with adjustable stop mechanism</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Horizon Titanium Clips</td>
			<td>Teleflex, US</td>
			<td>001200</td>
			<td>Small</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Induction chamber</td>
			<td>N/A</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Iris Scissor</td>
			<td>Lawton, Germany</td>
			<td>05-1450</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Iris Scissor&#160;</td>
			<td>Aesculap, Germany</td>
			<td>BC060R</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mechanical ventilator</td>
			<td>Ugo Basile, Italy</td>
			<td>7025</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscissor</td>
			<td>Lawton, Germany</td>
			<td>63-1406</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope</td>
			<td>Carl Zeiss, Germany</td>
			<td>303294-9903</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Needle Holder</td>
			<td>Lawton, Germany</td>
			<td>08-0011</td>
			<td>&#160;TITEGRIP</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pean</td>
			<td>Lawton, Germany</td>
			<td>06-0100</td>
			<td>Halsted-Mosquito, straight</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pro-Optha</td>
			<td>Lohmann &#38; Rauscher, Germany</td>
			<td>16515</td>
			<td>Tampon</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Saline 9 mg/mL</td>
			<td>Fresenius Kabi, DK</td>
			<td>209319</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sevoflurane</td>
			<td>AbbVie, US</td>
			<td></td>
			<td>Sevorane, Local procurement</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical hook</td>
			<td>Lawton, Germany</td>
			<td>51-0665</td>
			<td>Cushing, 19 cm, tip modified to an angle of 90&#176;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical Tape</td>
			<td>3M, US</td>
			<td>1530-0</td>
			<td>Micropore</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Temperature Controller</td>
			<td>CMA Microdialysis; Sweden</td>
			<td>8003760</td>
			<td>CMA 450&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Weighing machine</td>
			<td>VWR, US</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:217px;">Wistar rat weanlings</td>
			<td>Janvier Labs, France</td>
			<td style="width:108px;"></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 (<stro...

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 ...

This method can help answer key questions about underlying mechanisms and treatment of right ventricular failure. The main advantage of this technique is that it allows a precise and reproducible afterload increase that induces right ventricular hypertrophy and failure. The implications of this technique extend toward the the therapy of right ventricular failure, because it eliminates effects secondary to pulmonary vasodilation.Before beginning the trunk isolation procedure select an appropriate diameter for the banding and adjust the ligating clip applier until the distance between the jaws is one millimeter when the clip is fully compressed leaving a lumen of 6 millimeters. Next, confirm a lack of response in toe pinch in an anesthetized 100 to 120-gram Wistar rat weanling and make a two-centimeter skin incision along the midline of the sternum. Under a dissecting microscope cut the major pectoral muscle at its sternal attachment and identify the second, third, and fourth costae.Use a pair of scissors to cut the fourth, third, and second costae close to the sternum and carefully dissect the intercostal muscles until a complete left thoracotomy has been performed. When performing the left lateral thoracotomy you should be very careful as the left internal mammorial artery is just bellow the ribcage, so be very careful. Insert a retractor between the sternum and the costae and open the jaws until the thymus is visible at the top of the viewing field.Use the peen forceps to carefully flip the thymus to expose the aorta and the pulmonary trunk and guide the tip of a small surgical hooklet, bent to an 85 degree angle, through the reverse paracardial sinus behind the left atrial appendage. Pull the hooklet halfway back through the sinus and guide the tip of the ear hook upward until it appears between the ascending aorta, and the pulmonary trunk. When placing the ear hook make sure you have the right angle.you shouldn't use any force it might be a bit difficult initially but you can master it with practice. Use iris scissors to remove any connective tissue covering the tip to separate the pulmonary trunk from the ascending aorta. Use angled foreceps to guide a 10 centimeter piece of 4/0 multifilimant ligature around the pulmonary trunk through the passage made by the hook.Seize the end of the ligature with another forceps. The pulmonary trunk should now be separated from the ascending aorta and can be controlled by the ligature around it. When the pulmonary trunk has been secured, use a clip to load the adjusting ligating clip applier.Carefully guide one jaw and one leg of the clip through the passage around the pulmonary trunk using the ligature to gently pull the pulmonary trunk up and into the fork of the clip. When the pulmonary trunk is in the fork, and the two tips of the clip are free of any connective tissue, compress the clip to apply the banding removing the ligature as soon as the right ventricle dilates in response to the banding. When the trunk has been banded remove the peen forceps from the thymus and return the tissue to it's natural position.Remove the retractor, and use absorbable 4/0 multifiliment sutures to close the thorax in three layers. Then subcutaneously inject 2 milliliters of saline to replace any fluid lost during the surgery and monitor the animal until full recumbency. Selecting different diameters of the clip it is possible to induce different stages of disease severity, as evident here by an increasing degree of right ventricular hypertrophy.Only severe banding causes extra cardiac manifestations including liver failure. Differences in moderate versus severe pulminary trunk banding are evident by increasing right ventricular pressure and cause moderate versus severe right ventricular failure as revealed by a stepwise decrease in cardiac output and tricuspid annular plane systolic excursion as the severity of the banding increases. Pulmonary trunk banding procedure induced right ventricle dilation is evident by an increase in both right ventricular end diastolic volume and in systolic volume in the moderate pulmonary trunk banded rats compared to sham-operated animals, and in the severe pulmonary trunk banded rats compared to both the moderate and sham animals.A stepwise decrease in right ventricular ejection fraction is also observed. Right ventricular hypertrophy is further indicated by an increase in cardiomyocyte cross sectional area in pulmonary trunk banded rats, compared to sham-operated animals as well as other morphological changes to the right ventricle associated with right ventricle failure, including right ventricle fibrosis. Once mastered this technique can be performed in 30 minutes if it's performed properly.While attempting this procedure it's important to obtain consistent results with the model before initiating experimental research. After its development this technique paved the way for researchers in the field of right heart failure to explore pharmacologic interventions and basic mechanisms. After watching this video you should have a good understanding on how to perform pulmonary trunk banding.

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Research

JoVE Journal

Medicine

9.4K Görüntüleme.  Aarhus University Hospital. Bu yöntem altta yatan mekanizmalar ve sağ ventrikül yetmezliği tedavisi hakkında anahtar sorulara cevap yardımcı olabilir. Bu tekniğin en büyük avantajı sağ ventrikül hipertrofisi ve yetmezliği neden hassas ve tekrarlanabilir afterload artış sağlar. Bu tekniğin etkileri sağ ventrikül yetmezliği tedavisine doğru uzanır, çünkü pulmoner vazodilatasyona sekonder etkileri ortadan kaldırır.Gövde izolasyon prosedürü başlamadan önce bantlama için uygun bir ça...