Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Summary

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.

Abstract

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.

Introduction

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’ 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 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.

Protocol

NOTE: This study was authorized by Ethics committee number CEEA34.PB.103.12.

1. Use of Piglets as an Animal Model

1. Perform the in two groups (n = 6 in each group), matched in terms of sex, age (15 ± 3 months) and weight (30 ± 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.
2. 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.
3. Continuously monitor and maintain a temperature of 18 °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.
4. 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.
5. Anesthetizing of the animals
   1. 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’ eyes to prevent corneal dryness.
   2. 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.
6. Echocardiogram
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.
12. 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.
13. Measure the free wall thickness of the right ventricle during diastole and systole by TM (time-motion) recording in minor axis incidence ¹⁴.
14. Measure the pulmonary artery root diameter at the tip of the pulmonary valves on the minor axis view ¹⁴.
15. 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.
16. 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.
17. 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.
18. From Doppler measurements of the pulmonary artery, measure the following: maximum velocity, ascension half-time, flow duration and its integral ¹⁴.
19. Assess the left ventricular volume using the Simpson ¹⁵ method.
20. Store the data for each piglet in a database for subsequent statistical analysis on the whole group.

2. Right Heart Catheterization

1. Animal preparation
   1. Before pulsatile catheter placement, pig is placed in a hypobaric chamber during 3 weeks in order to induce pulmonary hypertension.
   2. Fast the animals for 24 hr before surgery (24 hr for solid food, 8 to 12 hr for water).
   3. 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.
   4. Administer injections of Midazolam (intramuscular, 0.5 mg/kg) and morphine hydrochloride (intramuscular, 0.1 mg/kg) 15 to 30 min 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.
   5. 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.
   6. Piglet intubation
      1. Lubricate the tube with pramocaine gel. Insert a metal stylus into the tube to stiffen it and facilitate the intubation process.
      2. 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.
   7. 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.
   8. 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.
   9. 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.
   10. Infuse lactated Ringer’s solution (10-20 ml/kg/hr).
   11. Place the animal on a slightly tilted examination table. Keep the head inclined slightly downwards to promote salivary flow.
2. Monitoring
   1. 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.
   2. 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.
3. 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.
   1. 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.
   2. 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.
4. Right heart catheterization
   1. 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.
   2. Make a 4 cm longitudinal incision with sterile scissors half-way between the right shoulder and the manubrium sterni.
   3. 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.
   4. 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.
   5. Introduce the catheter into the jugular vein and successively push through the superior vena cava, right atrium, right ventricle and, finally, the pulmonary artery.
   6. 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.
   7. 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).
   8. Measure the cardiac blood flow again 30 min after the pulsatility protocol (T2).

3. NO Measurement

1. 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.
2. 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.

4. Histology Measurements

1. Under anesthesia, inject 30 ml of Dolethal (injectable solution for euthanasia) into the pulsatile catheter placed in the heart.
2. 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.
3. Snap freeze one sample in liquid nitrogen and stored at -80 °C. Fix the second sample in 3.7% paraformaldehyde for 24 h and then embed in paraffin for subsequent histological analysis.
4. 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.
5. Perform statistical analysis. Values were expressed as fold change ± SEM.
   1. To compare two means, use an unpaired Student’s t test. To compare more than two means, use a one-way ANOVA followed by a Dunn’s test. A p <0.05 was considered statistically significant (*).

Results

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...

Discussion

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...

Materials

List of materials used in this article

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