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>

<ol>
	<li>Malenfant, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+in+the+development+of+pulmonary+arterial+hypertension.">Signal transduction in the development of pulmonary arterial hypertension.</a> Pulm Circ. 3 (2), 278-293 (2013).</li><li>Paulin, R., 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=Signal+transducers+and+activators+of+transcription-3/pim1+axis+plays+a+critical+role+in+the+pathogenesis+of+human+pulmonary+arterial+hypertension.">Signal transducers and activators of transcription-3/pim1 axis plays a critical role in the pathogenesis of human pulmonary arterial hypertension.</a> Circulation. 123 (11), 1205-1215 (2011).</li><li>Courboulin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+miR-204+in+human+pulmonary+arterial+hypertension.">Role for miR-204 in human pulmonary arterial hypertension.</a> J Exp Med. 208 (3), 535-548 (2011).</li><li>Bonnet, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+factor+of+activated+T+cells+in+pulmonary+arterial+hypertension+can+be+therapeutically+targeted.">The nuclear factor of activated T cells in pulmonary arterial hypertension can be therapeutically targeted.</a> Proc Natl Acad Sci U S A. 104 (27), 11418-11423 (2007).</li><li>Meloche, J., 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=Role+for+DNA+damage+signaling+in+pulmonary+arterial+hypertension.">Role for DNA damage signaling in pulmonary arterial hypertension.</a> Circulation. 129 (7), 786-797 (2014).</li><li>Humbert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+pathobiology+of+pulmonary+arterial+hypertension.">Cellular and molecular pathobiology of pulmonary arterial hypertension.</a> J Am Coll Cardiol. 43 (12 Suppl S), 13S-24S (2014).</li><li>Archer, S. L., Michelakis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evidence-based+approach+to+the+management+of+pulmonary+arterial+hypertension.">An evidence-based approach to the management of pulmonary arterial hypertension.</a> Curr Opin Cardiol. 21 (4), 385-392 (2006).</li><li>Li, M., Scott, D. E., Shandas, R., Stenmark, K. R., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+pulsatility+flow+induces+adhesion+molecule+and+cytokine+mRNA+expression+in+distal+pulmonary+artery+endothelial+cells.">High pulsatility flow induces adhesion molecule and cytokine mRNA expression in distal pulmonary artery endothelial cells.</a> Ann Biomed Eng. 37 (6), 1082-1092 (2009).</li><li>Li, M., Stenmark, K. R., Shandas, R., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+pathological+flow+on+pulmonary+artery+endothelial+production+of+vasoactive+mediators+and+growth+factors.">Effects of pathological flow on pulmonary artery endothelial production of vasoactive mediators and growth factors.</a> J Vasc Res. 46 (6), 561-571 (2009).</li><li>Nour, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrapulmonary+shear+stress+enhancement:+a+new+therapeutic+approach+in+pulmonary+arterial+hypertension.">Intrapulmonary shear stress enhancement: a new therapeutic approach in pulmonary arterial hypertension.</a> Pediatr Cardiol. 33 (8), 1332-1342 (2012).</li><li>Barst, R. J., 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=Diagnosis+and+differential+assessment+of+pulmonary+arterial+hypertension.">Diagnosis and differential assessment of pulmonary arterial hypertension.</a> J Am Coll Cardiol. 43 (12 Suppl S), 40S-47S (2004).</li><li>Galie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+on+diagnosis+and+treatment+of+pulmonary+arterial+hypertension.+The+Task+Force+on+Diagnosis+and+Treatment+of+Pulmonary+Arterial+Hypertension+of+the+European+Society+of+Cardiology.">Guidelines on diagnosis and treatment of pulmonary arterial hypertension. The Task Force on Diagnosis and Treatment of Pulmonary Arterial Hypertension of the European Society of Cardiology.</a> Eur Heart J. 25 (24), 2243-2278 (2004).</li><li>Naeije, R., Dewachter, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+pulmonary+arterial+hypertension.">Animal models of pulmonary arterial hypertension.</a> Rev Mal Respir. 24 (4 pt 1), 481-496 (2007).</li><li>Via, G., 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=International+evidence-based+recommendations+for+focused+cardiac+ultrasound.">International evidence-based recommendations for focused cardiac ultrasound.</a> J Am Soc Echocardiogr. 27 (7), e681-e683 (2014).</li><li>Folland, E. D., 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=Assessment+of+left+ventricular+ejection+fraction+and+volumes+by+real-time,+two-dimensional+echocardiography.+A+comparison+of+cineangiographic+and+radionuclide+techniques.">Assessment of left ventricular ejection fraction and volumes by real-time, two-dimensional echocardiography. A comparison of cineangiographic and radionuclide techniques.</a> Circulation. 60 (4), 760-766 (1979).</li><li>Meloche, J., 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=Critical+role+for+the+advanced+glycation+end-products+receptor+in+pulmonary+arterial+hypertension+etiology.">Critical role for the advanced glycation end-products receptor in pulmonary arterial hypertension etiology.</a> J Am Heart Assoc. 2 (1), e005157 (2013).</li><li>Nour, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrapulmonary+shear+stress+enhancement:+a+new+therapeutic+approach+in+acute+myocardial+ischemia.">Intrapulmonary shear stress enhancement: a new therapeutic approach in acute myocardial ischemia.</a> Int J Cardiol. 168, 4199-4208 (2013).</li><li>Barrier, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Today's+and+tomorrow's+imaging+and+circulating+biomarkers+for+pulmonary+arterial+hypertension.">Today's and tomorrow's imaging and circulating biomarkers for pulmonary arterial hypertension.</a> Cell Mol Life Sci. 69 (17), 2805-2831 (2012).</li><li>Budev, M. M., Arroliga, A. C., Jennings, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+evaluation+of+pulmonary+hypertension.">Diagnosis and evaluation of pulmonary hypertension.</a> Cleve Clin J Med. 70, S9-S17 (2003).</li><li>Barst, R. J., Channick, R., Ivy, D., Goldstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+perspectives+with+long-term+pulsed+inhaled+nitric+oxide+for+the+treatment+of+pulmonary+arterial+hypertension.">Clinical perspectives with long-term pulsed inhaled nitric oxide for the treatment of pulmonary arterial hypertension.</a> Pulm Circ. 2 (2), 139-147 (2012).</li><li>Pepke-Zaba, J., Higenbottam, T. W., Dinh-Xuan, A. T., Stone, D., Wallwork, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhaled+nitric+oxide+as+a+cause+of+selective+pulmonary+vasodilatation+in+pulmonary+hypertension.">Inhaled nitric oxide as a cause of selective pulmonary vasodilatation in pulmonary hypertension.</a> Lancet. 338 (8776), 1173-1174 (1991).</li><li>Zapol, W. M., Rimar, S., Gillis, N., Marletta, M., Bosken, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+the+lung.">Nitric oxide and the lung.</a> Am J Respir Crit Care Med. 149 (5), 1375-1380 (1994).</li><li>Stenmark, K. R., Meyrick, B., Galie, N., Mooi, W. J., McMurtry, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+pulmonary+arterial+hypertension:+the+hope+for+etiological+discovery+and+pharmacological+cure.">Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure.</a> Am J Physiol Lung Cell Mol Physiol. 297 (6), L1013-L1032 (2009).</li><li>Torre-Amione, G., 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=Reversal+of+secondary+pulmonary+hypertension+by+axial+and+pulsatile+mechanical+circulatory+support.">Reversal of secondary pulmonary hypertension by axial and pulsatile mechanical circulatory support.</a> J Heart Lung Transplant. 29 (2), 195-200 (2010).</li></ol>

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 NO production can be stimulated mechanically and safely within the pulmonary circulation without affecting systemic functions. The study design complies with the latest preclinical research recommendations in pulmonary hypertension, which have recently recommended tests on large animals before new methods are adopted in clinics. The translational potential of these findings is very high.
However the pathophysiological mechanism of PAH is not unique. Classification of PAH differentiates between five different groups. Group three is representative of medial hypertrophy secondary to chronic hypoxia. Although it can be included in this group, the model does not perfectly represent human PAH. Human PAH secondary to chronic hypoxia is mainly linked to chronic bronchitis in which lesions of the bronchi and pulmonary parenchyma induce peripheral pulmonary-to-systemic shunts. These shunts induce hypoxemia via a mechanism different to that used in the model. Here, the model is closer to life at high altitude than lung disease. However the cardiac hemodynamics of the model are close to those in humans: same heart rate, same cardiac output and same pulmonary pressure.
A previous study by Nour et al. demonstrated coronary 17 and pulmonary circulation 10 vasodilation secondary to pulsation. Their model of PAH was an aortopulmonary shunt. Aortopulmonary shunts increase blood flow in the pulmonary artery. It is our view that placing a balloon in the pulmonary artery creates an obstacle to right ventricular ejection that could interfere with the decrease in pulmonary pressure observed in their experiment. It is for that reason that a model of PAH was chosen that does not affect cardiac output. Instead the cardiac blood flow remains constant throughout the experiment.
Moreover, their experimental conditions are quite different since the chest of the animal is opened surgically during application of pulsatility, which modifies normal ventilation and pulmonary circulation. It is therefore difficult to compare the two experiments.
The mechanism via which pulsatility decreases PAH has not been demonstrated. The study conducted by Nour showed an increase in e-NOS, while we demonstrated an increase in exhaled NO. These two conclusions are strongly suggestive of a role played by NO in the PAH reduction mechanism. However other mediators may be involved 18. It would be useful to perform further studies to examine the biomarkers involved in pulsation-induced vasodilation.
NO delivery has been reported as being beneficial to PAH patients 19-21. However, NO has a very short half-life and its use to treat patients therefore involves a complicated procedure. In addition, the release of NO secondary to pulsatile flow needs to be more clearly demonstrated. The increase in eNOS and the systemic vasodilation observed in the study conducted by Nour but not in the study suggest that it is necessary to conduct further studies examining NO release induced by pulsatility and its role in vasodilation. This is another reason why NO therapy is not yet used in clinical practice.
Right heart catheterization is a standard procedure used to assess the severity of the condition and to monitor patient treatment 16,18. It would be entirely feasible to use pulsatile catheters instead of regular catheters. This would not require any additional procedures and could be used to assess the severity of PAH as well as to treat patients and improve their condition. Another advantage of this technique is that it has few, if any, toxic effects. Since these devices induce NO generation via a physiological process, any detrimental interaction with other therapies is unlikely. Consequently, pulsatile catheters could be rapidly and safely used in patients requiring ongoing treatment.
Although novel, the findings of this study confirm previous observations made in left heart failure patients who developed secondary pulmonary hypertension. Indeed, patients with pulmonary hypertension associated with chronic congestive heart failure have a significant risk of morbidity and death following heart transplants. Torre-Amione et al. 22 observed that patients who received a pulsatile left ventricular assist device had a significant decrease in pulmonary pressure, enabling them to qualify for heart transplants. Although not explored in the study, NO release could probably explain the improvement in pulmonary pressure, as in this study. This study not only confirms previous findings but suggests that the use of pulsatile catheters should also be explored in left heart failure patients who develop secondary pulmonary hypertension of high clinical significance.
Despite being very promising, this study has several limitations. Firstly, the efficacy of this device has only been tested with one model of pulmonary hypertension. Currently there are very few large animal pulmonary hypertension models 23. It is essential to develop new models. In the light of the findings by Torre-Amione et al. 22 concerning secondary pulmonary hypertension, a pulmonary hypertension model associated with left heart failure would be of great therapeutic interest. The effect of pulsatile catheters on NO release was estimated by measuring exhaled NO and not directly via the NO circulating in the PA. Ozkan et al. 24 have shown that exhaled NO is a good indicator of the amount of NO within the PA circulation. Indeed in their study, they demonstrated that exhaled NO decreased in PAH patients vs. controls and that stimulating NO generation in the PA using epoprostenol was reflected in the exhaled NO. This means that exhaled NO measurement is a valid method of estimating NO levels circulating in the PA. This study also measured PA pressure over 30 min, but the long-term effects (weeks, months and years) have not been studied. Sharing this technique will allow other teams to test different protocols (i.e. combinations with other treatments), analyze the benefits and perhaps discover adverse effects. Above all, it is hoped that this study may lead to rapid and safe use of the device in human PAH patients.

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><tbody><tr><td>Drugs for anesthesia</td><td></td><td></td><td></td></tr><tr><td>sodium thiopental, THIOPENTAL SODIUM</td><td>Abbott, France</td><td>0000071-73-8</td><td>powder&lt;br/&gt; 3 place Gustave Eiffe 94518 RUNGIS CEDEX.</td></tr><tr><td>&amp;nbsp;isoflurane, FORANE</td><td>Abbott, France</td><td>05260-05</td><td>glass bottle 250 ml&lt;br/&gt; 3 place Gustave Eiffe 94518 RUNGIS CEDEX.</td></tr><tr><td>midazolam, Hypnovel</td><td>Accord Healthcare&amp;nbsp;</td><td>Vidal</td><td>injectable ampoules 1mg/ml&lt;br/&gt; 45 Rue du Faubourg de Roubaix 59000 Lille France</td></tr><tr><td>pramocaine,TRONOTHANE 1%&amp;nbsp;</td><td>Laboratoires LISAPHARM</td><td>Vidal</td><td>Gel appl locale T/30g&lt;br/&gt; 3, rue Scheffer. 75016 Paris.</td></tr><tr><td>morphine chlohydrate Lavoisier</td><td>CMD Lavoisier Laboratoires CHAIX et DU MARAIS</td><td>Vidal</td><td>injectable ampoules&amp;nbsp;&lt;br/&gt; 7, rue Labie -75017 Paris - France</td></tr><tr><td>Acrylates Copolymer-Carbopol&amp;reg; Aqua SF-1 Polymer</td><td>Lubrizol</td><td></td><td>gel appl local&lt;br/&gt; Elys&amp;eacute;es La D&amp;eacute;fense 19 le Parvis 92073 Paris la d&amp;eacute;fense</td></tr><tr><td>Material&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Ventilateur Harvard 683</td><td>Harvard apparatus</td><td>Harvard apparatus</td><td>DRIM 75 rue des Anglais - 78700 Conflans Ste Honorine &amp;nbsp;&amp;nbsp;</td></tr><tr><td>Echographe Voluson E8 with a 3.5 MHz probe</td><td>General Electric</td><td>GEHealthcare</td><td>DRIM 75 rue des Anglais - 78700 Conflans Ste Honorine &amp;nbsp;&amp;nbsp;</td></tr><tr><td>Pulsatil Catheter</td><td>Cardio inovating system</td><td></td><td>Cardio innovative systems, 33 rue Vivienne, Paris, France 75002</td></tr><tr><td>NO breath Analyseur</td><td>Respur</td><td>Respur</td><td>26 rue Felix Rouget 95490 Vaureal France</td></tr></tbody></table>

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 increase in RV hypertrophy (M-mode). During RHC, invasive measurements of both right ventricular systolic pressure (RVSP) and mean PA pressure confirmed the presence of pulmonary hypertension in chronic hypoxic piglets vs. control piglets (values T0) (Figure 3). Diastolic pressure in the right ventricle was of -3 &#177; 1 mmHg in the control group and -2 &#177; 1 in the PAH group and the atrial pressure was -4 &#177; 2 mmHg in each group. The systemic pressure was 106 &#177; 13 vs 95 &#177; 18 mmHg in the control group vs the PAH group. Pulmonary hypertension was also confirmed by quantification of vascular remodeling on histological slices. To assess the probable therapeutic effect of pulsatile flow on pulmonary hypertension, a pulsatile catheter was used to generate an artificial pulsatile pulmonary flow for 10 min. As shown in Figure 3 an increase in the pulsatile flow for 10 min induced a significant reduction in both RVSP and mean PA pressure (T1 values) compared to baseline (T0 values). Since the cardiac output was not changed, pulsatility reduced the vascular pulmonary artery resistance by 26 &#177; 3% in the control group and by 41 &#177; 4% in the PAH group. To ensure that the decrease in PA pressure was sustained over time, animals in which the right catheter did not generate further pulsatile flow stayed in place for a further 30 min. As shown (T2 values), both RVSP and mean PA pressure continued to decrease compared to baseline (T0 values). It should be noted that systemic vascular parameters were not affected by the generation of pulsatile flow in the pulmonary artery and that neither systemic pressure nor cardiac output changed significantly.
Exhaled NO was measured for 6 chronically hypoxic piglets (T0) and 40 min after the generation of the artificial pulsatile flow (T2). This preliminary result, which needs to be confirmed - demonstrated a significant (p &#60;0.001) increase in exhaled NO from 2 &#177; 1 ppm to 22 &#177; 8 ppm.
Finally, the pulsatile flow showed no significant effects on circulating ET-1 and 5-HT levels, suggesting that the decrease in PA pressure was mainly due to an increase in NO generation.
<img alt="Figure 1" src="/files/ftp_upload/52571/52571fig1.jpg" /> 
Figure 1: Diagram of the Protocol. (A) Diagram of the hypoxic box with the equipment required. (B) Timeline of the experiment. Echocardiographs were performed every week for 3 weeks before, during and after hypoxia. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52571/52571fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52571/52571fig2.jpg" /> 
Figure 2: Echocardiography Results. Typical analysis measured non-invasively. (A) Over the 3 weeks (W1, W2, W3) of chronic hypoxia exposure (0.4 atmospheres), modifications in heart rate were observed as right ventricular hypertrophy progressively increased (B). In addition, pulmonary artery acceleration time (PAAT) measurements (C) demonstrated that PAAT decreased significantly in PAH piglets as PAH progressed, whereas no modifications occurred in the control group. An ANOVA statistical analysis adapted to the small number of animals concerned was performed (* p &#60;0.05; ** p &#60;0.005; *** p &#60;0.001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52571/52571fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 3" src="/files/ftp_upload/52571/52571fig3.jpg" /> 
Figure 3: Pulmonary Pressure was Reversed Following Pulsatile Catheter Exposure. (A) Piglets were exposed to pulsatile catheterization for 10 min. Mean pulmonary artery pressure (PAPcm H2O) was evaluated before (T0), immediately after (T1), and 30 min after the pulsation (T2). Each time, the pressure was measured over 10 stable cardiac cycles in order to obtain a representative value for the results. (B) Vascular remodeling was quantified and was increased in PAH piglets compared to control piglets. An ANOVA statistical analysis adapted to the small number of animals concerned was performed (* p &#60;0.05; ** p &#60;0.005; *** p &#60;0.001).
<img alt="Figure 4" src="/files/ftp_upload/52571/52571fig4.jpg" /> 
Figure 4: Pulsatile Catheters did not Modify Hemodynamic Parameters. The systemic systolic pressure (A) and the cardiac output (B) were evaluated in PAH and control piglets before, during and after the pulsatile protocol. No significant difference was observed between the groups and the different times. (C) Systolic pulmonary pressures were measured (mmHg) by right catheterization at T0, T1 and T2. An ANOVA statistical analysis adapted to the small number of animals concerned was performed (* p &#60;0.05; ** p &#60;0.005; *** p &#60;0.001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52571/52571fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 | Medicine | Video

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 ...

increasing-pulmonary-artery-pulsatile-flow-improves-hypoxic-pulmonary

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Biology

Medicine

Human Biology

Circulatory and Pulmonary Systems

SCIENTISTS IN ACTION

14.1K 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

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

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 ...

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript discusses the latest development of the previously published ovine PH-RVF model that utilizes left pulmonary artery (PA) ligation and main PA occlusion. This model of PH-RVF is a versatile platform to control not only the disease severity but also the RV&#39;s phenotypic response.
Adult sheep (60-80 kg) underwent left PA (LPA) ligation, placement of main PA cuff, and insertion of RV pressure monitor. PA cuff and RV pressure monitor were connected to subcutaneous ports. Subjects underwent progressive PA banding twice per week for 9 weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). At the initiation and endpoint of this model, ventricular function and dimensions were assessed using echocardiography. In a representative group of 12 animal subjects, RV mean and systolic pressure increased from 28 &#177; 5 and 57 &#177; 7 mmHg at week 1, respectively, to 44 &#177; 7 and 93 &#177; 18 mmHg (mean &#177; standard deviation) by week 9. Echocardiography demonstrated characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The longitudinal trend of SvO2 and PA cuff pressure demonstrates that the rate of PA banding can be titrated to elicit varying RV phenotypes. A faster PA banding strategy led to a precipitous decline in SvO2 &#60; 65%, indicating RV decompensation, whereas a slower, more paced strategy led to the maintenance of physiologic SvO2 at 70%-80%. One animal that experienced the accelerated strategy developed several liters of pleural effusion and ascites by week 9. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and enabling therapeutic innovation to manage RV adaptation and maladaptation from PH.

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing ...

Bioengineering

Invasive Hemodynamic Monitoring of Aortic and Pulmonary Artery Hemodynamics in a Large Animal Model of ARDS

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to pulmonary hypertension. For a better understanding and treatment of this disease, precise hemodynamic monitoring of left and right ventricular parameters is important. For this reason, it is essential to establish experimental pig models of cardiac hemodynamics and measurements for research purpose. 
This article shows the induction of ARDS by using oleic acid (OA) and consequent right ventricular dysfunction, as well as the instrumentation of the pigs and the data acquisition process that is needed to assess hemodynamic parameters. To achieve right ventricular dysfunction, we used oleic acid (OA) to cause ARDS and accompanied this with pulmonary artery hypertension (PAH). With this model of PAH and consecutive right ventricular dysfunction, many hemodynamic parameters can be measured, and right ventricular volume load can be detected. 
All vital parameters, including respiratory rate (RR), heart rate (HR) and body temperature were recorded throughout the whole experiment. Hemodynamic parameters including femoral artery pressure (FAP), aortic pressure (AP), right ventricular pressure (peak systolic, end systolic and end diastolic right ventricular pressure), central venous pressure (CVP), pulmonary artery pressure (PAP) and left arterial pressure (LAP) were measured as well as perfusion parameters including ascending aortic flow (AAF) and pulmonary artery flow (PAF). Hemodynamic measurements were performed using transcardiopulmonary thermodilution to provide cardiac output (CO). Furthermore, the PiCCO2 system (Pulse Contour Cardiac Output System 2) was used to receive parameters such as stroke volume variance (SVV), pulse pressure variance (PPV), as well as extravascular lung water (EVLW) and global end-diastolic volume (GEDV). Our monitoring procedure is suitable for detecting right ventricular dysfunction and monitoring hemodynamic findings before and after volume administration.

We present a protocol of creating right ventricular dysfunction in a pig model by inducing ARDS. We demonstrate invasive monitoring of left and right ventricular cardiac output using flow probes around the aorta and the pulmonary artery, as well as blood pressure measurements in the aorta and pulmonary artery.

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to ...

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 ...

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 ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, there is no cure for PAH. It is important to discover new compounds that can be used to treat PAH. The mouse hypoxia-induced PAH model is a widely used model for PAH research. This model recapitulates human clinical manifestations of PAH Group 3 disease and is an important research tool to evaluate the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds in mice. For a compound that needs to be given directly into the bloodstream, optimizing intravenous (IV) administration is a key part of the experimental procedures. Ideally, the IV injection system should permit multiple injections over a set time course. Although the mouse hypoxia-induced PAH model is very popular in many laboratories, it is technically challenging to perform multiple IV bolus dosing and invasive hemodynamic assessment in this model. In this protocol, we present step-by-step instructions on how to carry out multiple IV bolus dosing via mouse jugular vein and perform arterial and right ventricle catheterization for hemodynamic assessment in mouse hypoxia-induced PAH model.

This protocol provides a step-by-step procedure for executing multiple intravenous bolus dose administration and invasive hemodynamic monitoring in mice. Investigators can use this protocol for future therapeutic compound screening for pulmonary artery hypertension.

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video