The authors have nothing to disclose.

NOTE: This study was authorized by Ethics committee number CEEA34.PB.103.12.
1. Use of Piglets as an Animal Model
<ol>
	<li>Perform the in two groups (n = 6 in each group), matched in terms of sex, age (15 &#177; 3 months) and weight (30 &#177; 10 kg) (control group and chronic hypoxia (CH) group). House the CH group for 3 weeks in a hypobaric chamber (0.4 atm), and house the control group in regular normobaric (1 atm) conditions.</li>
	<li>Use a hypobaric chamber consisting of a Plexiglas box with a 2 square meter footprint and a height of 1.6 meters, as shown in Figure 1.</li>
	<li>Continuously monitor and maintain a temperature of 18 &#176;C and a pressure of 0.4 atm. Ensure adequate ventilation by a vacuum pump, enabling an air renewal rate of 8 m3 per hr.</li>
	<li>Place two animals in the chamber of the appropriate litter. Every 48 hr, return the pressure in the box to normobaric conditions for half an hour to clean the box, in the presence of the two animals, with the box kept closedto prevent them escaping. This method of inducing pulmonary hypertension has been widely validated 13.</li>
	<li>Anesthetizing of the animals
	<ol>
		<li>Anesthetize the piglets with an initial intravenous injection of sodium thiopental (10 mg/kg) and maintain anesthesia by continuous inhalation of isoflurane (1.5 to 3.5%). Apply two drops of Carbopol gel into the piglets&#8217; eyes to prevent corneal dryness.</li>
		<li>Place the animals in the left lateral decubitus position with their forelegs tied in a flexed position to expose the chest. Clean the skin with soap and water and then shave using an electric shaver to remove any hair that could impede penetration of the ultrasound through the chest.</li>
	</ol>
	</li>
	<li>Echocardiogram</li>
	<li>Monitor the development of PAH longitudinally and non-invasively by echocardiography. Perform an echo every week using a 3 MHz transducer. Record an electrocardiogram by placing 3 electrodes on the right and left paws and on the right side of the chest.</li>
	<li>Record two-dimensional and M-mode imaging data in three different incidences (longitudinal, minor axis and apical) using a-Doppler probe. Hold the probe in the right hand and place between the fourth and fifth left intercostal spaces. Move the probe slowly up and down, rotating it right and left until a good image resolution is obtained for the different cardiac structures (i.e. the septum). The acoustic window differs slightly from one animal to another depending on the position of the heart in the chest.</li>
	<li>Record the ECG at the same time for each of these incidences and for at least 10 cardiac cycles to enable off-line analysis. End diastole is defined as the point in the cardiac cycle coinciding with the onset of the Q wave on the ECG. End systole coincides with the onset of the T wave.</li>
	<li>Measure mitral and tricuspid valve blood flow by apical view Doppler ultrasound. Place the-Doppler probe at the tip of the manubrium sterni; the sampling zone was located just above the valve to record the blood flow crossing the valve.</li>
	<li>Record the blood velocity during the cardiac cycle by Doppler flowmetry to obtain the velocity-time integral of the blood flow through the mitral and tricuspid valves and the aortic and pulmonary valves. Use the video graphics array of the echograph to store images at a frame rate of 25 per sec in order to get fixed images or sequence images provided by the echograph.</li>
	<li>Measure the dimensions and surface area of the heart cavities using measuring instruments and the contours of the surface areas proposed by the software integrated into the echograph in accordance with international recommendations 14.</li>
	<li>Measure the free wall thickness of the right ventricle during diastole and systole by TM (time-motion) recording in minor axis incidence 14.</li>
	<li>Measure the pulmonary artery root diameter at the tip of the pulmonary valves on the minor axis view 14.</li>
	<li>Measure the septum and the posterior wall of the left ventricle during diastole in TM motion on the longitudinal view at the beginning of the Q waves on the ECG and in systole at the tip of the T wave on the ECG 14.</li>
	<li>Measure the left ventricular end-diastolic diameters (LVEDD) and left ventricular end-systolic diameters (LVESD). Calculate fractional shortening (FS) using the formula FS (%) = (LVEDD-LVESD)/LVEDD. Measure the diameter of the aorta and the surface area of the right and left atrium.</li>
	<li>From the velocity-time integral of the mitral and tricuspid blood flows, measure the following: maximum amplitude of the E and A wave, deceleration half-time of the E wave, flow duration, velocity-time index 14.</li>
	<li>From Doppler measurements of the pulmonary artery, measure the following: maximum velocity, ascension half-time, flow duration and its integral 14.</li>
	<li>Assess the left ventricular volume using the Simpson 15 method.</li>
	<li>Store the data for each piglet in a database for subsequent statistical analysis on the whole group.</li>
</ol>
2. Right Heart Catheterization
<ol>
	<li>Animal preparation
	<ol>
		<li>Before pulsatile catheter placement, pig is placed in a hypobaric chamber during 3 weeks in order to induce pulmonary hypertension.</li>
		<li>Fast the animals for 24 hr before surgery (24 hr for solid food, 8 to 12 hr for water).</li>
		<li>24 hr prior to RHC, have a well-trained vet perform a pre-anesthesia clinical exam to assess mucous membrane color, capillary refill time, global lung and heart functions using a stethoscope and body temperature using a rectal thermometer.</li>
		<li>Administer injections of Midazolam (intramuscular, 0.5 mg/kg) and morphine hydrochloride (intramuscular, 0.1 mg/kg) 15 to 30 min&#160;before the induction of anesthesia. Repeat injection of morphine hydrochloride (0.05 to 0.5 mg/kg, intramuscular) during induction every 4 to 6 hr.</li>
		<li>Administer sodium thiopental (10 mg/kg, intravenous) via an initial bolus injection of 5 mg/kg then by partial injection until it was effective. Follow this by bag and mask ventilation until endotracheal intubation.</li>
		<li>Piglet intubation
		<ol>
			<li>Lubricate the tube with pramocaine gel. Insert a metal stylus into the tube to stiffen it and facilitate the intubation process.</li>
			<li>Touch the eyelid to ensure deep anesthesia. Perform intubation by direct visualization of the larynx, using a laryngoscope to raise the tongue and avoid injury to the vocal cord. Inflate the tube balloon to prevent regurgitation-related issues.</li>
		</ol>
		</li>
		<li>Control the respiratory rate at 10-12 breaths per minute; current volume of 7-10 ml/kg, insufflation pressure of 25 to 30 cm H2O and an inspiratory phase of 2 sec with a positive end-expiratory pressure of 5 cm H2O.</li>
		<li>Anesthetize the animal with isoflurane in 100% oxygen (induction 3-5% with oxygen flow of 2 to 3 L/min, maintenance of 1.5-2.5% oxygen flow at 1 L/min). Apply carbopol gel to the cornea as in step 1.5.1.</li>
		<li>Insert a heparinized catheter into the caudal auricular artery (5 ml of 0.9% saline spiked with 5,000 IU/ml heparin) via subcutaneous infusion with green cannula fixed in place with a suture stitch.</li>
		<li>Infuse lactated Ringer&#8217;s solution (10-20 ml/kg/hr).</li>
		<li>Place the animal on a slightly tilted examination table. Keep the head inclined slightly downwards to promote salivary flow.</li>
	</ol>
	</li>
	<li>Monitoring
	<ol>
		<li>Every 5 min, check and record the following values on the individual anesthesia case report: mucous membrane color and capillary refill time, mandibular muscle tone and eyeball position, miosis/mydriasis, palpebral reflex.</li>
		<li>Continuously monitor the heart and respiratory rate, pulse oximetry, body temperature and electrocardiograms. Insert an arterial introducer into the femoral artery. Insert a large-caliber 10 cm long catheter, into the femoral artery and monitor the systemic blood pressure.</li>
	</ol>
	</li>
	<li>Setting up of the pulsatile catheter 
	NOTE: This medical device consists of two catheters placed side-by-side and welded together. The distal part of the first one is connected to a standard balloon with a diameter of 20 mm and a maximum volume capacity of 5 ml. The second catheter enables insertion of a wire to facilitate positioning in the pulmonary artery. The device is 750 mm long with an internal gauge of 0.035 and an external diameter of 12 Fr.
	<ol>
		<li>For the experiments, perform pulsation with a small Harvard 683 animal ventilator, which applies an active vacuum to the balloon during deflation and positive pressure during inflation, with a volume of 2.5 ml for each pulse. Use helium as the propellant gas for the balloon pump in order to prevent gas embolism.</li>
		<li>Record the surface electrocardiographic tracings continuously to document any heart rhythm disturbances during the protocol. In the left femoral artery, connect a sensor device to a 20 gauge catheter, connected to a hemodynamic monitoring system.</li>
	</ol>
	</li>
	<li>Right heart catheterization
	<ol>
		<li>Wash and shave the neck of the animal. Clean the skin with a cutaneous antiseptic solution (Betadine scrub) using a gauze compress. To demarcate the surgical site, place sterile drapes around the right jugular between the right shoulder and the manubrium sterni.</li>
		<li>Make a 4 cm longitudinal incision with sterile scissors half-way between the right shoulder and the manubrium sterni.</li>
		<li>Carefully remove the skin and muscle layers with forceps. Then gently remove the connective tissue surrounding the vein over a length of approximately 5 cm. Clip the distal side to prevent bleeding. Place a binder wire around the proximal side to be able to control the vein opening after hemi-section.</li>
		<li>Using a specific small very sharp chisel, cut the vein in half transversely. Ensure that the edges of the incision are neat. Using thin foam, raise one edge of the incision and gently push the catheter into the proximal side of the vein. Control bleeding with binder wire.</li>
		<li>Introduce the catheter into the jugular vein and successively push through the superior vena cava, right atrium, right ventricle and, finally, the pulmonary artery.</li>
		<li>Record the pressures over 10 stable cardiac cycles, on each cardiac cavity and the pulmonary artery (T0). Measure cardiac blood flow three times at 1 min intervals.</li>
		<li>Position the balloon catheter in the pulmonary artery under radioscopic control. Inflate and deflate the balloon (pulsation) with 1 cm3 of helium. Continue pulsation for 10 min. Record pressures over 10 stable cardiac cycles, in each cardiac cavity and the pulmonary artery and measure the cardiac blood flow after 10 min (T1).</li>
		<li>Measure the cardiac blood flow again 30 min after the pulsatility protocol (T2).</li>
	</ol>
	</li>
</ol>
3. NO Measurement
<ol>
	<li>Connect a Douglas bag to the exhaled respiratory gas outlet pipe until it is completely filled. Place it over the in-pipe of a NO breath analyzer.</li>
	<li>Slowly and constantly force the exhaled air into the analyzer by compressing the elastic Douglas bag. Measure the bag outflow by the flowmeter of the analyzer to maintain a constant flow.</li>
</ol>
4. Histology Measurements
<ol>
	<li>Under anesthesia, inject 30 ml of Dolethal (injectable solution for euthanasia) into the pulsatile catheter placed in the heart.</li>
	<li>Immediately after euthanasia, open the chest by sawing the manubrium longitudinally, slightly move the heart and hold the right lung. Then using dissecting scissors, cut two 2 or 3 cm3 samples from the middle lobe of the lung.</li>
	<li>Snap freeze one sample in liquid nitrogen and stored at -80 &#176;C. Fix the second sample in 3.7% paraformaldehyde for 24 h and then embed in paraffin for subsequent histological analysis.</li>
	<li>Perform histology measurements as previously described 16. Measure the PA wall thickness as follows: 2 measurements/artery in 10 arteries/piglet and in 6 piglets/group.</li>
	<li>Perform statistical analysis. Values were expressed as fold change &#177; SEM.
	<ol>
		<li>To compare two means, use an unpaired Student&#8217;s t test. To compare more than two means, use a one-way ANOVA followed by a Dunn&#8217;s test. A p &#60;0.05 was considered statistically significant (*).</li>
	</ol>
	</li>
</ol>

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The authors would like to thank the INRA institute, which housed the piglets, and Cardio Innovating systems for the funding.

For the first time, it has been shown that changes in pulmonary pulsatile flow are causally related to the development of PAH secondary to chronic hypoxic exposure. This translational approach provides evidence that inducing an artificial increase in pulmonary pulsatile flow using a specifically designed catheter improves pulmonary hypertension, probably by increasing NO generation.
These findings are not only original, they are also of great therapeutic interest, demonstrating that endogenous...

Pulmonary arterial hypertension is a life-threatening disease affecting the pulmonary vasculature. There is agreement in the field that an imbalance between an increase in vasoconstrictors (endothelin, serotonin) and a decrease in vasodilators (NO, prostacyclin) contributes to the development of PAH. Over time, this pro-constrictive phenotype evolves into a complex pro-proliferative and anti-apoptotic phenotype, contributing to the development of vascular lesions 1.
Prolonged exposure to vasoconstrictors leads to a significant and sustained increase in [Ca2+]i in pulmonary artery smooth muscle cells, allowing the activation of several calcium-regulated transcription factors, such as NFAT 2-4, promoting PASMC proliferation and resistance to an apoptosis phenotype 5. This phenotype leads to pulmonary vascular lesions, contributing to an increase in both PA pressure and pulmonary resistance, which ultimately leads to fatal right heart failure 6.
Currently, there is no treatment available that reverses PAH although there are several that improve patients&#8217; quality of life 7. Among these treatments, the effectiveness of inhaled NO treatment has been demonstrated but because of its short half-life it is difficult to use in clinical practice. For this reason, more stable and durable treatments have been preferred, such as prostacyclin analogs, or endothelin receptor blockers 7. To develop better treatments, it is essential to improve and extend knowledge of the pathophysiology of PAH.
Pulsatility is a well-known stimulus activating shear stress-induced vasodilation, protecting the non-elastic distal artery from high-pressure flow injuries 8,9. In a model of PAH secondary to aortopulmonary surgical shunting, Nour et al. demonstrated intrapulmonary shear stress-mediated endothelial function enhancement 10. Several studies have demonstrated that NO, prostacyclin and ET-1 expression are closely regulated by changes in pulsatile flow. Indeed, a moderate increase in pulsatile flow increases eNOS activity and prostacyclin levels, both of which are reduced in PAH. Pulsatile flow modulation is probably implicated in the etiology of PAH and artificially increasing it is an attractive and novel way of increasing NO and prostacyclin production within the pulmonary circulation.
The present study aims to assess the effects of a 10&#160;min pulsatile flow using a newly developed pulsatile catheter on hemodynamic measurements in a pulmonary hypertension (PH) model in piglets in whom hypoxia has been induced. It has been hypothesized that increasing pulmonary artery pulsatility induces vasorelaxation of the pulmonary arteries, thereby decreasing pulmonary artery pressure.
Right heart catheterization (RHC) is a critical clinical intervention for the diagnosis and follow-up of PAH patients. Indeed, it is the most reliable way of diagnosing PAH and allows physicians to assess vascular reactivity 11,12 as well as disease progression. In fact every PAH patient undergoes RHC several times. The present study in large animals aims to demonstrate the efficacy and safety of pulsatile catheters in assessing and treating PAH during a regular RHC procedure. Because pulsatile catheters are already available and RHC is routinely performed in PAH patients, this study provides all the information required to be able to conduct clinical trials rapidly.

<table border="0" cellpadding="0" cellspacing="0" width="1421">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:311px;">Drugs for anesthesia</td>
			<td style="width:336px;"></td>
			<td style="width:141px;"></td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium thiopental, THIOPENTAL SODIUM</td>
			<td>Abbott, France</td>
			<td>0000071-73-8</td>
			<td>powder 
			3 place Gustave Eiffe 94518 RUNGIS CEDEX.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;isoflurane, FORANE</td>
			<td>Abbott, France</td>
			<td style="width:141px;">05260-05</td>
			<td>glass bottle 250 ml 
			3 place Gustave Eiffe 94518 RUNGIS CEDEX.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">midazolam, Hypnovel</td>
			<td>Accord Healthcare&#160;</td>
			<td>Vidal</td>
			<td>injectable ampoules 1mg/ml 
			45 Rue du Faubourg de Roubaix 59000 Lille France</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">pramocaine,TRONOTHANE 1 %&#160;</td>
			<td>Laboratoires LISAPHARM</td>
			<td>Vidal</td>
			<td>Gel appl locale T/30g 
			3, rue Scheffer. 75016 Paris.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">morphine chlohydrate Lavoisier</td>
			<td>CMD Lavoisier Laboratoires CHAIX et DU MARAIS</td>
			<td>Vidal</td>
			<td>injectable ampoules&#160; 
			7, rue Labie -75017 Paris - France</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Acrylates Copolymer-Carbopol&#174; Aqua SF-1 Polymer</td>
			<td>Lubrizol</td>
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			<td height="21" style="height:21px;">Ventilateur Harvard 683</td>
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			<td height="17" style="height:17px;">Pulsatil Catheter</td>
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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.

Increasing Pulmonary Artery Pulsatile Flow Improves Induced Chronic Hypoxic Pulmonary Hypertension in Piglets
Prior to exposing animals to an increase in pulsatile flow, ultrasound was used non-invasively to check that the piglets had developed pulmonary hypertension. As shown in Figure 2, three weeks of chronic hypoxia induced the development of pulmonary hypertension in piglets, characterized by a significant reduction in pulmonary artery acceleration time (Doppler) and an i...

Watch this Scientific Journal Video about Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets at JoVE.com

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

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 ...

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14.0K Views.  Laval University. 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.

Video: Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

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

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

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

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

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 ...

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.

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 ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

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 ...

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart&#160;catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to&#160;treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right&#160;ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...