This study was supported by the Netherlands Cardiovascular Research Initiative, the Dutch Heart Foundation, the Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences (CVON nr. 2012-08, PHAEDRA, The Sebald fund, Stichting Hartekind).

Procedures involving animal subjects have been approved by the Dutch Central Committee for Animal Experiments and the Animal Care Committee at University Medical Center Groningen (NL). Both Wistar and Lewis rats with weights between 180 and 300 g were used.
1. Housing and Acclimatization
<ol>
	<li>After arrival at the central animal facility, house rats in groups of 5 per cage. During a 7-day acclimatization period, accustom the rats to human handling, but do not perform any experimental procedures.</li>
</ol>
2. Preparation and Injection of Sterile Monocrotalin
<ol>
	<li>For 1 mL of 60 mg/mL monocrotalin (MCT) solution, weigh 60 mg of monocrotalin in a 2-mL tube. Add 700 µL of 0.9% NaCl. Add 200 µL of 1 M HCl. Warm the solution in the tube under hot running tap water and vortex it. Use 6 N NaOH to bring the pH towards 7.0. Use sterile technique for preparation of MCT for injection into rodents.</li>
	<li>Inject 1 mL of sterile 60 mg/mL MCT solution per kg subcutaneously in the neck (0.3 mL of 60 mg/mL MCT for a 300-g rat). NOTE: We prefer not to use smaller volumes due to the greater chance that the injected dose will not be appropriate.</li>
</ol>
3. Aorta-caval Shunt Surgery
<ol>
	<li>Anesthesia.
	<ol>
		<li>Fill the induction chamber with 5% isoflurane/100% O2 (flow: 1 L/min) and place the rat in the chamber. Check for adequate depth of the anesthesia by performing a hind toe pinch. Weigh the rat.</li>
		<li>Shave and clean the abdomen over an area that is approximately 8 cm long and 3 cm wide. Place the rat on its back on a heat mat (37 °C) covered by a sterile mat.</li>
		<li>Place the snout in a ventilation mask/hood with 2 - 3% isoflurane/100% O2 (flow: 1 L/min). Check the depth of anesthesia by performing a hind toe pinch. Apply eye ointment to prevent dryness while under anesthesia.</li>
	</ol>
	</li>
	<li>Shunt Surgery.
	<ol>
		<li>Scrub the skin with chloride-hexidine for disinfection. Inject 0.01 mg/kg buprenorphine subcutaneously for post-operative analgesia.</li>
		<li>Use sterile instruments for surgery. Make an incision with a #10 scalpel blade in the abdomen on the midline, starting 1 cm below the diaphragm an extending down to just above the genitalia.</li>
		<li>Lift up the intestine with a cotton swab, cover the intestines in a sterile, wet gauze (0.9% NaCl), and place them to the left side of the animal.</li>
		<li>Use cotton swabs to separate the membranes that attach the abdominal aorta and the vena cava inferior to the surrounding tissues. 
		NOTE: Do not dissect the membranes between the aorta and the vena cava.</li>
		<li>Using splinter forceps, remove the perivascular aortic fat just above the bifurcation, only on the right side of the aorta and only on the site where the needle will be inserted.</li>
		<li>Use cotton swabs to separate the aorta and vena cava from 2 mm superior to the site where the needle will be inserted in order to create space for a Biemer clamp.</li>
		<li>At this area, first place a loose ligature (5-0 suture) around the aorta. Create tension on the ligature by placing a Kocher clamp on it, and then place the Kocher superior to the incision (Figure 1A). Place the Biemer clamp just superior to the ligature (Figure 1A).</li>
		<li>Using a cotton swab, compress the vena cava as distally as possible to obstruct the flow (Figure 1A). Bend a needle (18 G in this protocol) into a 45-degree angle, with the orifice pointing towards the outside (Figure 1A).</li>
		<li>At an angle of 90 degrees, insert the needle in the aorta, just above the bifurcation, with the orifice of the needle pointing to the left (Figure 1A). Manipulate the tip of the needle to the left and insert it into the vena cava. 
		NOTE: The needle tip should now be visible in the vena cava (Figure 1B).</li>
		<li>Use a second cotton swab to push the remaining blood in the aorta out of the insertion site to prevent thrombosis. Dry the area around the shunt with a sterile gauze in order for the glue to adequately stick.</li>
		<li>Pull the entire needle out of the aorta and immediately apply a drop of tissue glue onto the puncture site in the aorta. Make sure not to glue the cotton swab to the tissue. Unclamp the aorta.</li>
		<li>Verify the shunt manually by pulling on and releasing the ligature on the aorta proximal to the shunt. Loosening should color the vena cava distal to the shunt in bright red and create turbulence at the shunt site. 
		NOTE: Tightening will turn the blood in the vena cava back to dark red.</li>
		<li>Place the intestines back in the animal. Close the muscle layer and skin with resorbable 4-0 sutures. Ventilate the animal with 100% O2 to recover from anesthesia. 
		NOTE: Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	</ol>
	</li>
	<li>Sham Surgery.
	<ol>
		<li>Perform all of the above procedures except for the insertion of the needle into the aorta.</li>
	</ol>
	</li>
	<li>Post-surgical care.
	<ol>
		<li>Place the rat in a single cage and into an incubator at 37 °C until the next morning.</li>
		<li>Around 6 h after surgery, inject 0.01 mg/kg buprenorphine subcutaneously for post-operative analgesia. Repeat the next morning if the rat shows signs of discomfort. 
		NOTE: The first 3 days after surgery, rats tend to eat and drink less (this is particularly important when chow or drinking water are mixed with drugs). Most rats show normal behavior 3 days after surgery. If not, monitor closely. Weight loss exceeding 15% in 1 week is considered abnormal, and such rats should be euthanized by the extraction of the circulating blood volume while under anesthesia.</li>
	</ol>
	</li>
</ol>
4. Development of PAH
NOTE: In this protocol, the animal is euthanized by the extraction of the circulating blood volume while under anesthesia.

<ol>
	<li>Sacrifice 1 day after the surgery (MF8) for the early cellular and functional responses to increased pulmonary blood flow (e.g., gene up-regulation or early transcription factors).</li>
	<li>Sacrifice 1 week after the surgery (MF14) for an early-stage PAH vascular phenotype (medial hypertrophy without neointimal lesions).</li>
	<li>Sacrifice 2 weeks after the surgery (MF21) for an advanced-stage PAH vascular phenotype (marked medial hypertrophy and neointimal formation) with mild elevation in RVP and mPAP.</li>
	<li>Sacrifice 3 weeks after the surgery (MF28) for an end-stage PAH vascular phenotype (marked neointimal occlusion) and strong elevation in RVP and mPAP. Clinical signs of right ventricular failure are common in this stage.</li>
	<li>Sacrifice after day 28 (MF-RVF) for PAH-associated right ventricular failure (RVF), clinically defined as dyspnea, severe lethargy, and weight loss (&lt; 10% in 1 week). Terminate rats when one of these signs is present. Frequently, rats develop these symptoms between days 28 and 35 and, if left unguarded, die spontaneously during this time interval.</li>
</ol>
5. Right Heart Catheterization
<ol>
	<li>Anesthesia.
	<ol>
		<li>Fill the induction chamber with 5% isoflurane/100% O2 (flow: 1 L/min) and place the rat in the box. Check for adequate depth of the anesthesia by performing a hind toe pinch. Weigh the rat.</li>
		<li>Shave and clean the neck at the right-ventral side of the rat and, for the echocardiography protocol, the thorax and upper abdomen.</li>
		<li>Place the rat on its back on a heat mat (37 °C) and place the snout in a ventilation mask/hood with 2 - 3% isoflurane/100% O2 (flow: 1 L/min). The snout should be faced towards the researcher.</li>
		<li>Check depth of anesthesia. Be careful with rats with severe PH. If the heart rate decreases, reduce the depth of anesthesia. Preferable, perform all measurements within 20 min. Apply eye ointment to prevent dryness while under anesthesia.</li>
	</ol>
	</li>
	<li>Echocardiography protocol.
	<ol>
		<li>Perform the echocardiography according to the protocol described by Brittain et al. in JoVE13.</li>
	</ol>
	</li>
	<li>Catheterization protocol. 
	NOTE: This protocol uses a rigid cannula with a preformed tip bent 20 degrees to guide the 15-cm silicon catheter with a ball 2 mm from the tip. A 20-G needle with its orifice slightly bent to the inside is used to insert the cannula into the right jugular vein (see the list of materials). Rats in any phase of PAH progression and control can be used in this protocol.
	<ol>
		<li>Disinfect the neck with chloride-hexidine. Make a 1.5-cm incision with a #10 scalpel blade in the right-ventral side of the neck, from the right collar bone to the mandibular bone.</li>
		<li>Spread the tissue using scissors. Using tweezers, gently pull the tissue apart until the jugular vein appears. Dissect the membranes around the jugular vein using splinter forceps.</li>
		<li>Put tension on the jugular vein by placing a loose ligature (5-0 suture) around the vessel. Increase the tension and tape the ligature onto the ventilation mask (Figure 2A).</li>
		<li>Downstream of the insertion site, place a loose ligature around the vessel to tighten after the cannula is in situ in order to prevent leakage and loss of pressure.</li>
		<li>Using the handles of a forceps, slightly bend the tip of a 20-G needle with the orifice to the inside to conduct the cannula with the catheter.</li>
		<li>Introduce the tip of the 20-G needle into the vein and quickly place the cannula containing the catheter inside the vessel. Pull out the needle, and then close the ligature that was prepared in step 5.3.4.</li>
		<li>Conduct the cannula containing the catheter into the jugular vein. The tip of the cannula is at a 20-degree curve (see step 5.3.5). Maneuver the cannula under the collar bone and advance a little to enter the right atrium (Figure 2C).</li>
		<li>To enter the right ventricle, point the tip of the cannula to the left, towards the heart (Figure 2D). On the bedside monitor, an RV pressure curve should appear, matching Figure 2D.</li>
		<li>When the RV pressure curve is constant, write down the systolic and diastolic right ventricular pressure 1 (sRVP1/dRVP1).</li>
		<li>Manipulate the tip of the cannula to the left and upwards. Advance the catheter within the cannula (Figure 2E).</li>
		<li>Advance the catheter into the main pulmonary artery (PA). No resistance should be felt when passing the pulmonary valve. 
		NOTE: When the catheter enters the main pulmonary artery, the diastolic pressure will rise. On the bedside monitor, a PA pressure curve should appear, matching Figure 2E.</li>
		<li>When the PA pressure curve is constant, write down the systolic, diastolic, and mean PA pressure 1 (sPAP1, dPAP1, mPAP1).</li>
		<li>Further advance the catheter within the cannula until the ball at the tip of the catheter gets wedged in a pulmonary artery. Observe the pressure curve on the bedside monitor drop and match the wedge pressure curve in Figure 2F.</li>
		<li>When the wedge pressure curve is constant, write down the systolic, diastolic, and mean wedge pressure.</li>
		<li>Pull back the catheter slowly and subsequently measure and write down the values for sPAP2, dPAP2, mPAP2, sRVP2, and dRVP2, as displayed on the bedside monitor.</li>
		<li>When in the RV, slightly pull back the cannula and catheter to measure the mean right atrial pressure (RAP). The curve should match the RAP curve in Figure 2A. 
		NOTE: In this protocol, the rats are euthanized after the catheterization protocol by the extraction of the circulating blood volume while under anesthesia.</li>
	</ol>
	</li>
</ol>
6. Morphology Assessment and Morphometry

NOTE: In this protocol, the animal is euthanized by the extraction of the circulating blood volume while under anesthesia. Rats in any phase of PAH progression and control can be used in this protocol.
<ol>
	<li>After sacrifice, take out the lungs by cutting the trachea about 5 mm above the bronchial bifurcation and the vessels that connect the lungs to the heart. Put the lungs in cold saline. Dissect the left lung. Cut the left main bronchus at the bifurcation.</li>
	<li>Fill a 50-mL syringe with 4% paraformaldehyde, attach a tube with a cannula to the syringe, and hang the syringe about a meter above the work table. Fit the cannula in the left main bronchus to passively fill the lung with paraformaldehyde. Handle paraformaldehyde with caution.</li>
	<li>Incubate the left lung in paraformaldehyde for 48 h.</li>
	<li>Dehydrate the left lung by incubating it consecutively in 70% ethanol (1 h), 80% ethanol (1 h), 90% ethanol (1 h), 100% ethanol (3 h), xylol (2 h), and paraffin (2 h).</li>
	<li>Embed the left lung in paraffin, with the hilum of the lung facing the cassette.</li>
	<li>Stain the paraffin-embedded, 4-µm lung sections using a Verhoeff or Elastica-van Gieson staining, as per manufacturer's instructions29. Make sure the elastic laminae are well differentiated (as in Figure 3). Scan the stained sections at 40X magnification.</li>
	<li>Divide the lung into 4 quadrants. In each quadrant, find 10 vessels with an outer diameter &lt; 50 µm (intra-acinar) and 10 vessels with an outer diameter &gt; 50 µm (pre-acinar). Take a picture (2 x 40 pictures per lung). Zoom in randomly up to 20x magnification and photograph every vessel in this field of view to minimize selection bias.</li>
	<li>Exclude vessels that have a longest/shortest diameter ratio of &gt; 2, an incomplete circular shape, or a collapse of more than one quarter of the vessel wall. 
	NOTE: An example of an excluded vessel is shown in Figure 3b Make each picture on the same magnification (40X) and include a scale bar.</li>
	<li>Open ImageJ and the first picture. Draw a straight line on the scale bar in the picture to set the scale via "Analyse" and "Set scale." For "known distance," use the value on the picture's scale bar. Use micrometers (µm) as the unit of length. Set the scale to global.</li>
	<li>Using "freehand selections," draw a line on the inner border of the luminal area (Figure 3), and use "measure" (crtl m) to measure this area. Then, draw a line around the outer elastic lamina (Figure 3) to measure the total vessel area.</li>
	<li>Calculate the luminal and outer diameter (<img alt="Empty set symbol (∅), mathematical notation, set theory concept." src="/files/ftp_upload/55065/55065eq1.jpg" data-altupdatedat="1747911167000" data-alt="Equation">) using <img alt="Formula: Expression for diameter related to area; math equation in educational context." src="/files/ftp_upload/55065/55065eq2.jpg" data-altupdatedat="1747911167000" data-alt="Equation">.</li>
	<li>Calculate wall thickness using <img alt="Static equilibrium equation: (outer Ø − lumen Ø)/2, diagram, educational use in physics." src="/files/ftp_upload/55065/55065eq3.jpg" data-altupdatedat="1747911167000" data-alt="Equation">.</li>
	<li>Calculate the wall/lumen ratio using <img alt="Wall thickness to lumen diameter ratio formula, diagram illustrating measurement concept." src="/files/ftp_upload/55065/55065eq4.jpg" data-altupdatedat="1747911167000" data-alt="Equation">.</li>
	<li>Calculate the occlusion score using <img alt="Static equilibrium equation, ΣFx=0, calculation formula with outer and lumen diameter variables" src="/files/ftp_upload/55065/55065eq5.jpg" data-altupdatedat="1747911167000" data-alt="Equation">.</li>
	<li>Score the vessel on muscularization (no, partial, or total muscularization) (Figure 3B). 
	NOTE: Vessels with a double elastic lamina for more than half the circumference are defined as totally muscularized. Vessels with a double elastic lamina less than half the circumference are defined as partially muscularized.</li>
	<li>Score the vessel on the presence of a neointima (yes or no) (Figure 3C). 
	NOTE: Vessels without a clearly-defined internal elastic lamina combined with (often eccentric) luminal occlusion are defined as neointimal lesions.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumonectomy+combined+with+SU5416+induces+severe+pulmonary+hypertension+in+rats.">Pneumonectomy combined with SU5416 induces severe pulmonary hypertension in rats.</a> Am J Physiol Lung Cell Mol Physiol. 310 (11), L1088-L1097 (2016).</li><li>Ranchoux, B., Antigny, F., Rucker-Martin, C., 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=Endothelial-to-mesenchymal+transition+in+pulmonary+hypertension.">Endothelial-to-mesenchymal transition in pulmonary hypertension.</a> Circulation. 131 (11), 1006-1018 (2015).</li><li>de Raaf, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SuHx+rat+model:+Partly+reversible+pulmonary+hypertension+and+progressive+intima+obstruction.">SuHx rat model: Partly reversible pulmonary hypertension and progressive intima obstruction.</a> Eur Respy J. 44 (1), 160-168 (2014).</li></ol>

The authors have nothing to disclose.

This method describes the surgical procedure of an aorto-caval shunt in rats pre-treated with MCT to create flow-induced PAH and the techniques to assess the principle hemodynamic and histopathological end points that characterize PAH and this model.
Critical Steps within the Protocol and Troubleshooting
Surgery and post-surgery. During the aorto-caval shunt surgery, the most critical step is the dissection of the aorta and vena cava. The membranes that enclose the aor...

The goal of this method is to create a reproducible model for severe, flow-induced pulmonary arterial hypertension in rats and to measure its principle hemodynamic and histopathological end points.
Pulmonary arterial hypertension (PAH) is a clinical syndrome that encompasses a progressive increase in pulmonary vascular resistance leading to right ventricular failure and death. Within the superordinate disease spectrum of pulmonary hypertensive diseases (PH), PAH is the most severe form and one that remains without a cure1. The underlying arteriopathy in PAH is characterized by a typical form of vascular remodeling that occludes the vessel lumen. Muscularization of normal non-muscularized vessels and hypertrophy of the medial vessel layer are regarded as early disease phenomena in PAH, are also seen in other forms of PH2, and are thought to be reversible3. As PAH advances, the intimal layer begins to remodel, eventually forming characteristic neointimal lesions2. Neointimal-type pulmonary vascular remodeling is exclusive to PAH and is currently regarded to be irreversible4.
As PAH is a rare disease, advances in its pathobiological comprehension and development of novel therapies have relied heavily on animal models. The monocrotalin (MCT) model in rats is a simple single hit model that has been, and still is, used frequently. MCT is a toxin that causes injury to the pulmonary arterioles and regional inflammation5. 60 mg/kg MCT leads to an increase in the mean pulmonary artery pressure (mPAP), pulmonary vascular resistance (PVR), and right ventricular hypertrophy (RVH) after 3 - 4 weeks6. The histomorphology is characterized by isolated medial hypertrophy without neointimal lesions5. The MCT rat model thus represents a moderate form of PH, and not PAH, although it is commonly presented as the latter.
In children with PAH associated with a congenital left-to-right shunt (PAH-CHD), increased pulmonary blood flow is regarded as the essential trigger for the development of neointimal lesions7,8,9. In rats, increased pulmonary blood flow can be induced by the creation of a shunt between the abdominal aorta and the vena cava, a technique first described in 199010. Alternatives to create increased pulmonary flow are by unilateral pneumonectomy or by subclavian to pulmonary artery anastomosis11. Conceptual disadvantages of these models consist of potential compensatory growth of the remaining lung and adaptive pathway activation induced by the pneumonectomy, or of iatrogenic injury of the pulmonary vasculature due to pulmonary artery anastomosis, both confounding the effects of increased pulmonary blood flow.
When an aorto-caval shunt is created and increased pulmonary blood flow is induced as a second hit in MCT-treated rats, characteristic neointimal lesions occur, and a severe form of PAH and associated right ventricular failure (RVF) develop 3 weeks after the increased flow12. The hemodynamic progression of PAH in this model can be assessed in vivo by echocardiography and right heart catheterization. The vascular histomorphology, vessel wall thickness, degree of arteriolar occlusion, and parameters for right ventricular failure form the pillars of the ex vivo characterization of PAH.
This method describes detailed protocols for the aorto-caval shunt (AC-shunt) surgery, right heart catheterization, and qualitative and quantitative assessment of vascular histomorphology.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Shunt Surgery&lt;/strong&gt;</td></tr><tr><td>Sterile surgical gloves</td><td></td><td></td><td></td></tr><tr><td>Duratears Eye ointment</td><td>Alcon</td><td>10380</td><td></td></tr><tr><td>Chloride-Hexidine</td><td></td><td></td><td></td></tr><tr><td>Cotton swabs</td><td></td><td></td><td></td></tr><tr><td>Histoacryllic tissue glue</td><td>B. Braun Medical</td><td>1050052</td><td></td></tr><tr><td>Silkam 5-0 sutures black non-resorbable</td><td>B. Braun Medical</td><td>F1134027</td><td></td></tr><tr><td>Safil 4-0 sutures violet resorbable</td><td>B. Braun Medical</td><td></td><td></td></tr><tr><td>18 G needle</td><td>Luer</td><td>NN1838R BD</td><td>tip bent in 45 degrees orifice to the outside</td></tr><tr><td>Gauzes 10 x 10 cm</td><td>Paul Hartmann</td><td>407825</td><td></td></tr><tr><td>Temgesic Buprenorphine</td><td>RB Pharmaceuticals</td><td>5429</td><td>subcutaneous injection</td></tr><tr><td>Sodium Chloride 0.9%</td><td></td><td></td><td></td></tr><tr><td>Ventilation mask Rat</td><td></td><td></td><td></td></tr><tr><td>Scalple blade</td><td></td><td></td><td></td></tr><tr><td>Biemer clamp 18 mm, 5 mm opening&amp;nbsp;</td><td>&lt;a href=&quot;https://www.google.nl/url?sa=i&amp;amp;rct=j&amp;amp;q=&amp;amp;esrc=s&amp;amp;source=images&amp;amp;cd=&amp;amp;cad=rja&amp;amp;uact=8&amp;amp;ved=0ahUKEwitqICpkITNAhWiL8AKHe6iCC8QjhwIBQ&amp;amp;url=http%3A%2F%2Fwww.agnthos.se%2Fquote%2Fquote%2Fcaturl%2Fid%2F242%2FBiemer&amp;amp;psig=AFQjCNEnfRXuMQ6Doi4_-2KMYu3qeRovMA&amp;amp;ust=1464777707579746&quot;&gt;AgnTho&lt;/a&gt;</td><td>64-562</td><td></td></tr><tr><td>Heat mat</td><td></td><td></td><td></td></tr><tr><td>Kocher Clamp</td><td></td><td></td><td></td></tr><tr><td>Shaving machine</td><td></td><td></td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Right Heart Catheterization&lt;/strong&gt;</td></tr><tr><td>Sterile surgical gloves</td><td></td><td></td><td></td></tr><tr><td>Eye ointment</td><td>Duratears</td><td></td><td></td></tr><tr><td>Chloride-Hexidine</td><td></td><td></td><td></td></tr><tr><td>Cotton swabs</td><td></td><td></td><td></td></tr><tr><td>Gauzes 10 x 10 cm</td><td>Paul Hartmann</td><td>407825</td><td></td></tr><tr><td>Silkam 5-0 sutures black non-resorbable</td><td>B. Braun Medical</td><td>F1134027</td><td></td></tr><tr><td>Needle 20 G</td><td>Luer</td><td></td><td>Tip slightly bent to the inside</td></tr><tr><td>Cannula 20 G</td><td>Luer</td><td></td><td>to introduce catheter, tip pre-formed in 20 degrees</td></tr><tr><td>Silastic Catheter 15 cm long</td><td></td><td></td><td>0.5 mm ball 2 mm from tip</td></tr><tr><td>Pressure transducer</td><td>Ailtech</td><td></td><td></td></tr><tr><td>Bedside monitor Cardiocap/5</td><td>Datex-Ohmeda</td><td></td><td></td></tr><tr><td>Shaving machine</td><td></td><td></td><td></td></tr><tr><td>10 mL Syringe</td><td></td><td></td><td></td></tr><tr><td>Sodium Chloride 0.9%</td><td></td><td></td><td>for flushing</td></tr><tr><td colspan="4">&lt;strong&gt;Vascular Morphology&lt;/strong&gt;</td></tr><tr><td>50 mL Syringe</td><td></td><td></td><td></td></tr><tr><td>4% Formaldehyde</td><td></td><td></td><td></td></tr><tr><td>18 G cannula with tube</td><td></td><td></td><td></td></tr><tr><td>Verhoef staining kit</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/ht254?lang=en&amp;amp;region=NL&quot;&gt;HT254&lt;/a&gt;</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/ht254?lang=en&amp;amp;region=US</td></tr><tr><td>Digital slide scanner</td><td>Hamamatsu</td><td>C9600</td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td></td></tr><tr><td>Elastic (Connective Tissue Stain)&amp;nbsp;</td><td>Abcam</td><td>ab150667</td><td>http://www.abcam.com/elastic-connective-tissue-stain-ab150667.html&lt;br /&gt;
http://www.abcam.com/ps/products/150/ab150667/documents/ab150667-Elastic%20Stain%20Kit%20(website).pdf</td></tr></tbody></table>

shunt surgery, right heart catheterization, and vascular morphometry in a rat model for flow-induced pulmonary arterial hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

Representative results are presented in Figure 4. The presented results show characteristics of MCT+FLOW in Lewis rats in the following groups: Control (n = 3), MF8 (n = 5), MF14 (n = 5), MF28 (n = 5), and MF-RVF (n = 10). Statistical analyses were performed using the one-way ANOVA with Bonferroni correction.
60 mg/kg MCT and increased pulmonary blood flow lead to a mean rise in systolic right ventricular pressu...

Watch this Scientific Journal Video about Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension at JoVE.com

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

shunt-surgery-right-heart-catheterization-vascular-morphometry-rat

Research

JoVE Journal

Medicine

16.7K Views.  University Medical Center Groningen, University of Groningen. This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

Video: Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease. 
Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.

Pulmonary arterial hypertension (PAH) is a disease of pulmonary arterioles that leads to their obliteration and the development of right ventricular failure. Rodent models of PAH are critical in understanding the pathophysiology of PAH. Here we demonstrate hemodynamic characterization, with right heart catheterization and echocardiography, in the mouse and rat.

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of group 2 PH, due to mitral stenosis (MS), to facilitate the investigation of disease mechanisms and potential therapeutic strategies. We present a novel rat model of pulmonary venous congestion-induced pulmonary venous arterialization and group 2 PH caused by left atrial stenosis (LAS). LAS is achieved by constricting the left atrium using a half-closed titanium clip. After the LAS surgery, a rat model with a transmitral inflow velocity greater than or equal to 2.0 m/s on echocardiography gradually develops pulmonary venous arterialization and group 2 PH over an 8- to 10-week period. In this protocol, we provide the step-by-step procedure of how to perform the LAS surgery. The presented LAS rat model mimics MS in humans and is useful for studying the underlying molecular mechanism of pulmonary venous arterialization and for the preclinical evaluation of therapies for group 2 PH.

Left atrial stenosis (LAS) is a novel surgical technique used for studying group 2 pulmonary hypertension (PH) and mechanisms underlying pulmonary venous arterialization. Here, we present a protocol to constrict the left atrium using a titanium clip to cause pulmonary venous arterialization and moderate PH in a rat.

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of ...

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

A Model of Reverse Vascular Remodeling in Pulmonary Hypertension Due to Left Heart Disease by Aortic Debanding in Rats

Pulmonary hypertension due to left heart disease (PH-LHD) is the most common form of PH, yet its pathophysiology is poorly characterized than pulmonary arterial hypertension (PAH). As a result, approved therapeutic interventions for the treatment or prevention of PH-LHD are missing. Medications used to treat PH in PAH patients are not recommended for treatment of PH-LHD, as reduced pulmonary vascular resistance (PVR) and increased pulmonary blood flow in the presence of increased left-sided filling pressures may cause left heart decompensation and pulmonary edema. New strategies need to be developed to reverse PH in LHD patients. In contrast to PAH, PH-LHD develops due to increased mechanical load caused by congestion of blood into the lung circulation during left heart failure. Clinically, mechanical unloading of the left ventricle (LV) by aortic valve replacement in aortic stenosis patients or by implantation of LV assist devices in end-stage heart failure patients normalizes not only pulmonary arterial and right ventricular (RV) pressures but also PVR, thus providing indirect evidence for reverse remodeling in the pulmonary vasculature. Using an established rat model of PH-LHD due to left heart failure triggered by pressure overload with subsequent development of PH, a model is developed to study the molecular and cellular mechanisms of this physiological reverse remodeling process. Specifically, an aortic debanding surgery was performed, which resulted in reverse remodeling of the LV myocardium and its unloading. In parallel, complete normalization of RV systolic pressure and significant but incomplete reversal of RV hypertrophy was detectable. This model may present a valuable tool to study the mechanisms of physiological reverse remodeling in the pulmonary circulation and the RV, aiming to develop therapeutic strategies for treating PH-LHD and other forms of PH.

The present protocol describes a surgical procedure to remove ascending-aortic banding in a rat model of pulmonary hypertension due to left heart disease. This technique studies endogenous mechanisms of reverse remodeling in the pulmonary circulation and the right heart, thus informing strategies to reverse pulmonary hypertension and/or right ventricular dysfunction.

Pulmonary hypertension due to left heart disease (PH-LHD) is the most common form of PH, yet its pathophysiology is poorly characterized than pulmonary ...

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

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

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

Biology

Optimizing Pulmonary Artery Banding for Consistent Right Heart Failure Induction

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

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

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

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

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