Cardiac Catheterization for Assessment of Pulmonary Hypertension in Preterm Lambs

Summary

Here, we present a protocol to assess pulmonary hypertension by cardiac catheterization in a preterm lamb model of evolving bronchopulmonary dysplasia-associated pulmonary hypertension. The described method enables direct measurement of mean pulmonary artery and pulmonary capillary wedge pressures and calculation of pulmonary vascular resistance.

Abstract

Bronchopulmonary dysplasia with pulmonary hypertension (BPD-PH) is a devastating complication of preterm birth that is associated with increased morbidity and mortality. Preterm birth disrupts normal lung and vascular development, and postnatal exposures such as mechanical ventilation and oxygen therapy can further impair lung and pulmonary vascular growth, contributing to the development of BPD-PH. In the preterm lamb model of evolving BPD, lambs are delivered preterm and mechanically ventilated for up to 21 days, incorporating interrupted lung development along with injury from mechanical ventilation and oxidative stress to recapitulate key features of evolving BPD-PH.

The goal of the cardiac catheterization protocol is to directly assess pulmonary hemodynamics in preterm and term neonatal lambs. Lambs are intubated, mechanically ventilated, and sedated throughout the study. At the time of cardiac catheterization, a Swan-Ganz catheter is advanced through the external jugular vein to the superior vena cava and then through the right atrium, right ventricle, and main pulmonary artery. Cather position is confirmed by identification of characteristic pressure waveforms obtained during advancement through the right-sided cardiac chambers. Mean pulmonary artery pressure and pulmonary capillary wedge pressure are measured via pressure transduction, and cardiac output is determined by thermodilution to enable calculation of pulmonary vascular resistance.

The protocol provides reproducible measurements of pulmonary hemodynamics that parallel those obtained during clinical cardiac catheterization in human neonates. By enabling direct assessment of pulmonary vascular pressures and resistance, this method enhances the translational utility of the preterm lamb model for mechanistic evaluation of potential therapies for BPD-PH.

Introduction

Bronchopulmonary dysplasia (BPD) occurs in up to 45% of preterm infants born before 28 weeks of gestation, is increasing in the United States, and can be complicated by pulmonary hypertension (PH, BPD-PH)^1,2,3. The chronic cardiopulmonary disease process begins when normal lung maturation is interrupted by preterm birth and is exacerbated by postnatal injury from mechanical ventilation and oxidative stress. Ongoing lung injury leads to pulmonary vascular remodeling and the development of PH^4,5.

Pediatric PH is defined as mean pulmonary artery pressure (mPAP) ≥20 mmHg in children >3 months of age. If the pulmonary vascular resistance (PVR) index is also >3 Wood units (WU) x m² and pulmonary capillary wedge pressure (PCWP) < 15 mmHg, then patients are defined as having pre-capillary PH, presumed secondary to pulmonary vascular disease in the context of BPD-PH⁶. Pulmonary vascular resistance is calculated from hemodynamic measurements obtained during cardiac catheterization using the equation

The incidence of BPD-PH increases with BPD severity, occurring in 25–41% of infants with severe BPD^7,8. BPD-PH is associated with substantially increased morbidity and mortality. Meta-analyses report an odds ratio for mortality of 4.4–6.4 compared to BPD without PH, and mortality rates of 40–47% within two years for neonates with BPD and severe, persistent PH⁹. BPD-PH also leads to longer hospitalizations, greater need for home oxygen, increased risk of tracheostomy, and higher rates of neurodevelopmental impairment^7,10,11.

Given the high morbidity and mortality associated with BPD-PH, accurate assessments of PH are essential to inform risk stratification, monitoring, and therapeutic management, as well as to facilitate optimal selection of candidates for clinical trials to improve outcomes. Although echocardiography is often used for initial screening for PH, right-heart catheterization is the gold standard for diagnosing PH across all age groups, including neonates, because it enables direct measurement of mPAP, PCWP, and CO¹². Cardiac catheterization accurately and precisely determines PVR and is therefore essential to incorporate into clinical and translational research, especially in large animal models that test potential therapeutic agents for PH^12,13.

The preterm lamb model is a unique large-animal model of evolving BPD-PH that can be used to advance understanding of disease mechanisms and test novel therapies¹⁴. Preterm lambs are delivered during the late canalicular to early saccular stage of lung development (equivalent to approximately 24 to 28 weeks of gestation) by cesarean section after exposure to antenatal steroids. Lambs are intubated and resuscitated, using a standardized technique based on neonatal guidelines. They receive exogenous surfactant, caffeine, and early enteral nutrition (in addition to intravenous (IV) dextrose) as the standard of care in the neonatal intensive care unit¹⁵. Previous studies using this model demonstrated that, after 21 days of mechanical ventilation with physiologic targets similar to those used in preterm infants, measurements of pulmonary vascular pressures and postmortem lung histology revealed elevated PVR, alveolar simplification, and other histologic changes consistent with evolving BPD-PH^15,16. However, earlier approaches made thoracotomies to directly insert a thermistor wire and catheter into the pulmonary trunk, which in and of themselves may have contributed to the development of BPD-PH.

Here, a less invasive protocol for right-heart catheterization via the external jugular vein to directly measure pulmonary hemodynamics and calculate PVR in preterm and term neonatal lambs is described. The technique provides reproducible measurements of pulmonary vascular pressures and CO that parallel those obtained during clinical cardiac catheterization in human neonates. The protocol improves the translational utility of the preterm lamb model for mechanistic studies and preclinical therapeutic testing in BPD-PH.

Protocol

All experimental procedures were prospectively approved and done in compliance with the animal handling and training standards of the University of Utah Institutional Animal Care and Use Committee.

1. Position setup

1. Position the lamb prone in a sling and ensure that previously placed vascular access is functioning (umbilical or jugular central venous catheter).
2. Maintain dexmedetomidine infusion at 0.2 to 1.0 mcg/kg/h for sedation and adjust the infusion rate to achieve the desired sedation depth.
3. Provide additional faster-acting sedation and analgesia with pentobarbital (1 mg/kg/dose IV push) and buprenorphine (5 µg/kg/dose IV push) as needed.
4. Shave the right or left neck over the external jugular vein and inject 0.5 mL of 1% lidocaine subcutaneously over the site. Sterilize the site with three alternating betadine and alcohol scrubs.
5. Don sterile gloves and appropriate personal protective equipment. Dispose of all needles and other sharps immediately after use in an approved sharps container.
6. Prime a Swan-Ganz catheter with 2% heparinized saline and connect the distal port to a pressure transducer for continuous recording in LabChart.
7. Zero the pressure transducer.
8. Monitor oxygen saturation (SpO2), arterial blood pressure, and heart rate throughout the procedure. Halt the procedure to allow vitals to stabilize or abort the procedure if SpO2 <90% for 2 min, heart rate <60 beats per minute, or respiratory rate >100 breaths per minute despite ventilator and inhaled oxygen fraction adjustments or if arterial blood pressure decreased >15% from pre-procedure baseline.

2. Catheter introduction and advancement

1. Use an 11 blade to make a 1 cm incision over the external jugular vein and expose the vessel.
2. Dissect the external jugular vein, then isolate it by passing a 3.0 silk suture beneath the vein. Use this suture to maintain proximal control of the vessel in the event of bleeding as the catheter is introduced.
3. Introduce an 18 G, 2.5 inch needle into the vessel.
4. Advance a guide wire through the needle using the Seldinger technique.
5. Advance a 6 Fr introducer (filled with heparinized saline) into the vessel over the guide wire.
6. Remove the guidewire while the introducer remains in place.
7. Suture the introducer to secure its position in the vessel at a depth of approximately 3.0 cm to 3.5 cm, using the previously placed silk tie.
8. Confirm blood return from the introducer by aspiration and then flush the introducer with heparinized saline.
9. Pass the previously prepared Swan-Ganz catheter through the introducer and gradually advance while monitoring the transduced pressure waveform.
   NOTE: Check and recheck transducer leveling and zeroing prior to introducing the catheter. Small errors in transducer leveling can alter pulmonary artery pressure, PCWP, mPAP, and PVR. Even a 1–2 cm leveling error can meaningfully alter results.

3. Measurement of pressures and cardiac output (CO)

1. Advance the Swan-Ganz catheter until the typical venous pressure tracing is replaced by a typical right atrial pressure tracing (Figure 1). Observe that right atrial pressure does not typically reach zero (although this may occur in the setting of severe hypovolemia). Record transduced pressures.
2. Advance the Swan-Ganz catheter further until a typical right ventricle (ventricular) pressure tracing is seen (Figure 1). Observe that right ventricular pressure reaches zero. Record transduced pressures.
   NOTE: Catheter whip artifact caused by movement of the catheter tip with blood flow can result in spuriously high systolic spikes.
3. Slowly advance the Swan-Ganz catheter until a pulmonary arterial tracing is obtained. Observe that pulmonary artery (arterial) pressure does not reach zero. Record transduced pressures.
   NOTE: If there is difficulty advancing the catheter into the pulmonary artery, the balloon may be briefly inflated to encourage the catheter tip to move towards the pulmonary valve. Another troubleshooting technique is repositioning the animal. Over-dampening of the waveform can occur from kinks in the tubing and produce a falsely low pressure reading.
4. Further advance the catheter and inflate its balloon to create a wedge against the vessel’s wall, allowing for measurement of PCWP. Limit balloon inflation to a maximum of 10 s once the waveform is confirmed.
   NOTE: Balloon inflation should be accompanied by a feeling of resistance. If no resistance is appreciated, consider the possibility that the balloon has ruptured and do not instill any additional air.
5. Measure cardiac output by thermodilution, referencing instructions on cardiac output machine (exact steps may differ slightly based on model used).
   1. Connect the cardiac output machine to the port on the Swan-Ganz catheter.
   2. Place the thermistor cable in a container of ice water.
   3. Input the appropriate computational constant based on the catheter model and injectate temperature to compute cardiac output.
   4. Fill eight 5 mL syringes with 0.9% saline and place in ice water.
   5. Instill a 3–5 mL (dependent upon model of Swan-Ganz catheter used) cold injectate (0–5°C) via the catheter.
   6. Record the cardiac output from the machine.
   7. Follow the prompts from the cardiac output machine to repeat the cold injectate 5–6 times.
6. Deflate the balloon and withdraw the Swan-Ganz catheter. If the cardiac catheterization is not done on the final day of the study, remove the introducer and ligate the external jugular vein using 3.0 silk sutures. Monitor the animal for complications.

Results

Expected pressure tracings from the right atrium, right ventricle, pulmonary artery and pulmonary capillary wedge locations are illustrated in Figure 1. Upon entry into a central vein, the pressure tracing should show a very low-pressure undulating waveform (not pictured). Respiratory variation may be present. No sharp upstrokes should be present (characteristic of arterial tracing). Upon entry to the right atrium, the waveform becomes organized and rhythmic with two small peaks per cycle (a...

Discussion

Cardiac catheterization provides direct measurement of mPAP and calculation of CO in the preterm lamb model of evolving BPD-PH. The procedure is technical and requires specialized equipment, but it provides accurate and reproducible measurements, which is why it is the preferred diagnostic procedure in humans and is considered a superior clinical trial endpoint. When cardiac catheterization is done in the preterm lamb model, the critical steps of the procedure are successful advancement of the catheter to the right heart...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.45% Sodium Chloride Injection USP	Baxter	2B1313
3-Way Stopcock w/ Swivel Male Luer Lock	Medex	MX5311L
48 in (121.9 cm) Extension Set	Medex	MX048
5 mL Syringe	Terumo	SS-05L
Bridge Amp	ADInstruments	FE221/CS
CONTINU-FLO Solution Set with CONTROL-A-FLOW Regulator	Baxter	2C6895
DB9M - DB9F Cable (400 mm)	ADInstruments	MLAC02
DIN(8) to MLT0699 or MLT0670 Cable (3.9 m)	ADInstruments	MLAC06
Disposable BP Transducer (Stopcock)	ADInstruments	MLT0670
Injectate Temperature Probe	Edwards Lifesciences	9850A
LabChart 8.1.30 for Windows	ADInstruments		Pro Upgrade
Patient CCO Cable	Edwards Lifesciences	70CC2
Percutaneous Sheath Introducer Kit with Integral Hemostasis Valve/Side Port for use with 4 - 6 Fr. Catheters	Arrow	AK-09601
Power Lab C	ADInstruments	PLC01
Swan-Ganz True Size Thermodilution Catheter	Edwards Lifesciences	132F5	6 Fr, 10 cm, Ballon Volume 0.7 mL
Vigilance II Monitor	Edwards Lifesciences	VIG2