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Bioengineering

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

doi: 10.3791/62694 Published: July 15, 2021
Rei Ukita1, John W. Stokes1, W. Kelly Wu1, Jennifer Talackine1, Nancy Cardwell1, Yatrik Patel1, Clayne Benson5, Caitlin T. Demarest1, Erika B. Rosenzweig3, Keith Cook2, Emily J. Tsai4, Matthew Bacchetta1,6

Abstract

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'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 ± 5 and 57 ± 7 mmHg at week 1, respectively, to 44 ± 7 and 93 ± 18 mmHg (mean ± 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 < 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.

Introduction

Decompensated right ventricular (RV) failure is the predominant cause of morbidity and mortality for patients with pulmonary hypertension (PH). RV failure is responsible for over 50% of hospitalizations in patients with PH and is a common cause of death in this patient population1,2. Although current medical treatments for PH can provide temporizing measures, they do not reverse the progression of the disease. As such, the only long-term treatment is lung transplantation. To explore and test novel medical treatments and interventions for PH and RVF, a clinically relevant animal model is needed to recapitulate the disease's complex pathophysiology. In particular, there is a great clinical need to develop RV-targeted therapeutics for PH patients to improve RV function. To date, most published animal studies of PH and RV dysfunction have relied on small mammals such as mice and rats3. On the other hand, there have only been a handful of large animal models to study the disease and RV pathophysiology from abnormal afterload4,5,6,7. In addition, none of the previously published large animal models include descriptions of experimental procedures for controlled titration of disease severity that differentially leads to compensated versus decompensated RV failure phenotypes. An animal model of PH that can be titrated to induce acute and chronic RV failure with varying degrees of compensation is needed to study disease mechanisms and to develop, test, and translate novel diagnostics and therapeutics for PH and RVF into clinical practice. Such a model in a large animal is especially valuable for the development of mechanical circulatory support devices8.

Here, a chronic, large animal PH-RVF model using left pulmonary artery (PA) ligation and progressive main PA banding in adult sheep is presented9,10. The ligation of the left PA (LPA) increases the pulmonary vascular resistance and decreases PA capacitance11,12. The progressive PA banding approach allows for precise titration of disease severity and RV adaptation. This platform can also be readily utilized for longitudinal investigation of disease progression toward RV decompensation. The procedures and processes required to execute this model are presented as a resource for investigators interested in a large animal platform to develop novel treatments for PH and RVF.

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Protocol

The Institutional Animal Care and Use Committee at Vanderbilt University Medical Center approved the protocol. The described procedures were conducted in accordance with the US National Research Council's Guide for the Care and Use of Laboratory Animals, 8th edition. The overview and the timeline of the experimental procedure are provided in Figure 1.

1. One day before surgery, preparation of the animal

  1. Withhold food for 24-40 h prior to the surgical procedure to decompress the animal's rumen.
  2. Apply a 50 µg/h fentanyl patch to a sheared area on the sheep's dorsum 12 h before the procedure. Clean the area with chlorhexidine to remove lanolin oil residues prior to patch application. Cover and protect the patch with an elastic tubular dressing.

2. Day of the surgery, pre-operative steps in the preparation room

  1. Administer tiletamine/zolazepam intramuscularly (2.2-5 mg/kg) and deliver 1%-3% of isoflurane mixed with 80%-100% of oxygen via a face mask to induce anesthesia.
  2. Position the sheep supine on the preparation table and secure its legs.
  3. Intubate with a 10 mm endotracheal tube and start mechanical ventilation under volume-control mode (tidal volume, TV = 10 mL/kg, respiratory rate, RR = 15 breaths per minute).
  4. Shave the surgical field from the sheep's neck to its upper abdomen, as detailed below.
    1. Shave the sheep's anterior neck to expose the skin overlying the jugular veins for central venous catheterization (see step 3.7).
    2. Shave the anterolateral thorax bilaterally in preparation for thoracotomy (see step 4.1).
    3. Shave the left side of the torso from the chest to the back (i.e., as dorsally as the table will allow with the subject in the supine position), and from breast to rear flank caudally, in preparation for implantation of subcutaneous ports (see steps 4.12-4.15).
  5. Insert a 20 G angiocatheter in the auricular artery for arterial pressure monitoring and blood gas sampling.
  6. Place a silicone tube with an inner diameter of 3/8"-1/2" for rumen decompression. The orogastric tube will remain in the rumen throughout the entire procedure.
  7. Transport the animal from the pre-operative prep room to the surgical suite.

3. Day of surgery, pre-operative steps in the operating suite

  1. Reconnect the sheep to the ventilator in the surgical suite, and continue ventilation at the same setting in step 2.3 (isoflurane 1%-3%, TV = 10 mL/kg, RR = 15 breaths per minute)
  2. Connect the pulse oximetry (SpO2), arterial blood pressure, temperature, end-tidal capnograph, and electrocardiogram (ECG) sensors to the anesthetic monitor.
  3. Connect the sensors for vital signs to the animal.
    1. Place the pulse oximeter on the tongue of the animal.
    2. Place the temperature probe into the rectum.
    3. Connect 3-lead electrocardiogram probes: Place the red lead on the left rear leg, the white lead on the right front leg, and the black lead on the left anterior leg.
    4. Connect the three-way stopcock's male luer end to the auricular artery angiocatheter and connect the opposite female luer end to the pressure transducer for arterial line monitoring using an appropriately sized pressure tubing.
      1. Align the transducer to the level of the operating table.
      2. Open the three-way stopcock on the transducer.
      3. Scroll the main knob of the vitals monitor to highlight the arterial blood pressure channel, and then press the knob to select the channel.
      4. Select ZERO IBP to zero the transducer.
    5. Connect the male luer connection of the capnography monitor line to the female luer connection on the ventilator tube to monitor end-tidal CO2.
  4. Set up the IV pumps for continuous fluid administration and inotropic or vasopressor support.
    1. Perforate the septum on the saline bag with the IV administration set. Make sure that the IV tubing is clamped prior to perforating the bag to prevent spillage.
    2. Align and fit the IV administration set tubing into the IV roller pump, and check whether the direction specified on the pump matches the direction of fluid administration.
      NOTE: Ensure that the IV administration set is compatible with the IV pump.
    3. Turn on the pump and specify PRIME to remove all the air in the line.
  5. Position the sheep for the operative procedure.
    1. From the supine position, rotate the sheep to a partial right lateral decubitus position.
    2. Secure the right front foot downward and secure the left front foot while retracting it cephalad and lateral with rope or atraumatic straps.
    3. Perform transthoracic echocardiography for baseline assessment of the ventricular anatomy and function. Ultrasonography is also useful to determine the optimal intercostal space that facilitates surgical access to both the main pulmonary artery and the left pulmonary artery.
  6. Clean the surgical field free of dirt and other contaminants using soap or scrub brush. Prep the neck and the chest with chlorhexidine or betadine solution and drape the surgical field in a sterile fashion.
  7. Using ultrasound guidance or anatomic landmarks, access the left or right internal jugular vein using a finder needle or angiocath. Using Seldinger technique, insert a 7-French triple-lumen central venous catheter into the internal jugular vein for intravenous access and central venous pressure monitoring.
    1. Use the proximal port for pressure monitoring and distal port for fluid and drug administration.
  8. Administer 20 mg/kg of cefazolin and 5 mg/kg of enrofloxacin intravenously. Repeat the dosing of cefazolin every 2-4 h during the procedure.
  9. Administer a 500 mL bolus of normal saline solution to augment the preload before surgery. Begin a maintenance intravenous fluid rate of 15 mL/kg/h.

4. Operative procedure

  1. Perform a muscle-sparing mini-thoracotomy (length < 8 cm) at the left fourth intercostal space to obtain mediastinal exposure. Choose mini-thoracotomy to expedite postoperative recovery.
    1. After dividing the skin, split the underlying muscle (pectoralis major) longitudinally along its fibers, which run slightly oblique to the intercostal space. Place a self-retaining retractor to spread the muscle layer and expose the chest wall.
    2. Divide the serratus anterior and the underlying intercostal muscle in the selected intercostal space, taking care to stay immediately cephalad to the rib.
    3. Enter the pleural space and then continue to fully release the intercostal muscles posteriorly toward the spine and anteromedially toward the sternum to prevent inadvertent rib fracture or dislocation at the sternum. Avoid injury to the mammary vessels medially.
    4. Place the self-retaining retractors to open the rib space and the overlying soft tissue. Use a small or medium Finochietto retractor to separate the ribs and a Tuffier retractor (5 cm retractor blade) to sit perpendicular to the Finochietto within the intercostal space, which retracts the soft tissue within the intercostal space to improve exposure.
  2. Incise the pericardium anterior to the phrenic nerve without injuring it and create a pericardial well with 2-0 silk sutures to expose the main PA and RV. Identify the left atrial appendage within the exposure as a landmark for the level of the PA bifurcation.
    1. Assess the exposure and ensure whether the appropriate intercostal space has been entered. Ideally, the proximal PA and the left atrial appendage are readily visible directly below the incision, suggesting the optimal intercostal space has been selected to provide exposure to both the main PA and LPA.
    2. If exposure is deemed inadequate to safely reach both the main PA and LPA, do not hesitate to open an additional intercostal space to accomplish all the necessary steps of the operation; however, this will not be necessary with appropriate incision selection.
  3. Dissect around the main PA and isolate it with an umbilical tape. Ensure adequate posterior dissection for the eventual occluder placement and PA flow probe as distal as possible on the main PA.
    1. Place a sterile flow probe into a bowl of water or saline on the sterile field to calibrate the data acquisition software. Handoff the electrical plug on the other end to a non-sterile designee to connect the probe to the meter.
      1. Refer to the supplementary documents for details of connecting and calibrating PA flow probe and meter.
    2. Apply a generous amount of sterile ultrasound gel in the groove of the PA flow probe.
    3. Fit the silicone liner into the groove of the PA flow probe and apply an additional layer of ultrasound gel onto the liner.
    4. Place the PA flow probe onto the PA and acquire PA flow readings on the flow meter and the data acquisition interface.
      1. Placement of the PA flow probe may cause partial occlusion of the PA that can decrease left ventricular preload and mean arterial pressure. Pay careful attention to the hemodynamics during PA flow acquisition.
      2. Check on the flow meter screen to ensure that the PA flow signal strength is 5 bars. If the meter displays fewer than 5 bars, ensure adequate contact between the flow probe and the main PA. Apply additional ultrasound gel if needed.
  4. Complete intra-pericardial dissection of LPA and encircle it with an umbilical tape.
    1. Use a small sponge stick or thin malleable retractor for caudal retraction of the left atrial appendage.
      NOTE: Exposure to the LPA is facilitated by caudal retraction of the left atrial appendage, cephalad retraction of the main PA, and lateral retraction of the pericardium just anterior to where the LPA exits the pericardium.
  5. Place a heavy-duty silicone vascular occluder around the main PA (Figure 2A,B, circle). Occluder size can be adjusted based on PA diameter; ensure the fit is snug. Use a 0 silk suture on a Keith needle to secure the ends of the vascular occluder together with a U stitch. Once secured around the main PA, slide the occluder distally along the main PA.
  6. Encircle the proximal main PA with a ½" Penrose drain to facilitate dissection and reserve space to place a flow probe at subsequent re-operative surgery. Trim the Penrose drain to fit loosely around the PA and secure the Penrose to itself with a running 4-0 Prolene suture (Figure 2B).
  7. Establish an RV pressure line for monitoring of RV pressures (Figure 2B, white arrow).
    1. Select a location for the RV pressure line in the RV outflow tract-free wall. Place a 5-0 monofilament, nonabsorbable polypropylene purse-string suture with pledgets surrounding the selected location and seat a vascular snare. Make the pledgets from a sterile surgical glove.
    2. Prepare the RV pressure line: cut off the male end of sterile 36'' pressure tubing at a 30° angle to facilitate insertion through the myocardium. Use a 2-0 silk tie to mark the pressure line at an optimal depth for placement within the RV.
    3. Using an 11-blade scalpel, make a small cardiotomy in the RVOT free wall within the previously placed purse-string suture. Control bleeding with manual pressure or by tightening the snare on the purse-string suture.
      NOTE: Obtain a baseline biopsy of the RV free wall at this step by sampling RV tissue within the purse-string suture. This biopsy site can then serve as the entry point for the RV pressure line.
    4. Insert and secure the cut end of the pressure tubing into the RV outflow tract (RVOT). Tie down the purse-string and then secure the purse-string to the pressure tubing to secure the pressure line.
  8. Extend the RVOT tubing by connecting an additional pressure tubing to the RVOT pressure line.
  9. Hand off the additional pressure tubing to a non-sterile designee to connect the tubing to a pressure transducer and monitor for the measurement of the baseline RV pressure. Set up the pressure transducer as follows.
    1. Connect IV administration set's male luer end to transducer's female luer end.
    2. Connect pressure tubing's female luer end to transducer's male luer end.
    3. Spike the IV administration set into a heparinized saline bag (2 IU/mL).
    4. Fit the saline bag into a pressure bag and pump the pressure bag to 250-300 mmHg as indicated on the gauge.
    5. Fully prime the line by releasing the valve on the transducer, ensuring proper de-airing.
    6. Follow Supplementary Methods for transducer calibration.
  10. After carefully dissecting around the LPA, encircle it with an umbilical tape. Ligate the LPA by tying down the umbilical tape. Note the animal's hemodynamic response to ligation if relevant to the study. Increase the minute ventilation to compensate for the increased dead-space ventilation created upon LPA ligation. These ventilator adjustments mitigate respiratory acidosis.
  11. Slowly inject up to 3 mL of saline into the main PA occluder to ensure there is no leakage while monitoring RV pressure from the RVOT pressure line. Once the RV response is confirmed, withdraw the instilled saline.
  12. Bring the RVOT pressure line and PA occluder tubing out of the chest one intercostal space below the thoracotomy incision.
  13. Form two subdermal pockets along the fascial layer on the left dorsum of the sheep as far posteriorly toward the spine as feasible within the sterile field. These serve as the sites for indwelling ports (Figure 2C).
  14. Using a chest tube puller, tunnel the RVOT pressure line and occluder tubing from the chest incision out to the left dorsum port sites.
  15. Secure both the occluder tubing and RV pressure line to the port's barb connections. Anchor the occluder and pressure tubing around the port connectors with additional ties. Use the provided barbed connector fitting to protect the connection (Figure 1C). Seat the ports within the pre-formed subdermal pockets.
  16. Anchor the ports in three locations around its rim to the underlying fascia with 3-0 polypropylene sutures to prevent port migration. Reapproximate the subcutaneous tissue, dermis, and skin in layers with polyglactin 910 sutures. Reconfirm the pressure readings through percutaneous access of the ports. Flush the RVOT port with 5 mL (1000 IU/mL, 5000 units) of heparin sodium.
  17. Place a 16-French chest tube in the left pleural cavity through a separate incision, secure it to the skin, and then connect to a closed chest tube drainage unit at a pressure of -20 cm·H2O. Place an untied U-stitch around the tube to facilitate closure after chest tube removal.
  18. Administer an intercostal nerve block (0.5-1 mg/kg bupivacaine) for postoperative analgesia.
  19. Close the thoracotomy with figure-of-eight, #2 polyglactin 910 sutures. Close the pectoralis muscle layer with running #0 polyglactin 910. Close the subcutaneous tissue in layers of running #2-0 polyglactin 910 sutures and staple the skin.
  20. Reposition the animal to dorsal recumbency, remove the orogastric tube, and then discontinue isoflurane.
  21. Continue mechanical ventilation and supportive care until arterial blood pH > 7.35 and pCO2 < 55 mmHg.
  22. Extubate once the animal is breathing spontaneously, lifting its head, and chewing on the endotracheal tube. Remove the chest tube prior to full anesthetic recovery. Tie the U-stitch to close the chest tube incision.
  23. Transfer the animal to its cage while monitoring its anesthesia recovery. Ensure supplemental oxygen (3-5 L/min by facemask) is available at all times while the sheep remains immobile. Monitor vital signs every hour for the first 4 h, every 8 h for the next 24 h, and once daily after that.

5. Postoperative recovery

  1. Monitor the thoracotomy and port implantation sites daily for signs of infection. Administer long-acting antibiotic (ceftiofur, 5 mg/kg intramuscularly) within 24 h after the procedure and every 3-4 days after that for 1 week.
  2. Continue the fentanyl patch postoperatively for a total of 72 h. After that, provide additional analgesia (e.g., meloxicam, 1 mg/kg once daily intramuscularly) if the animal continues to show signs of pain (i.e., teeth grinding, elevated heart rate).
  3. Remove the external sutures and skin staples 10-14 days after the surgery or as recommended by veterinary staff.
  4. Ensure port site protection from the animal rubbing or scraping the port sites against surrounding structures using a tubular dressing (Figure 2D).

6. Chronic PA banding (9 - 10 weeks)

  1. Transfer the sheep to a small enclosure. Shear off the excess wool around the implanted ports.
  2. Clean the shaved areas with 70% isopropyl alcohol. Apply topical lidocaine spray for local anesthetic.
  3. Prepare two pressure transducers for monitoring RV and occluder cuff pressures (Figure 3A).
    1. For both transducers: Connect the female luer end of pressure tubing (36 in or longer) to the male luer end of the transducer. Connect the male luer end of the pressure tubing to one of the female luer connections on a three-way stopcock. Finally, connect a 22 G Huber needle to the male luer end of that three-way stopcock.
    2. For RV pressure transducer: Hang a heparinized saline bag (2 IU/mL), puncture the bag with the IV administration set, and connect the IV administration set's male luer connection to the female luer connection of the RV pressure transducer. Then, pressurize the saline bag (e.g., pressure bag).
    3. For the occluder transducer: Prime the transducer and the pressure tubing fully. Put a male luer cap on the female luer end of the pressure transducer to prevent the cuff fluid from leaking out back to the transducer.
    4. Connect both the transducers to the data acquisition hardware using an appropriate cable or adapter.
  4. Calibrate the transducers as specified in Supplementary File 1.
  5. Click on Start on the top right of the software window to start recording the data acquisition software to capture RV and PA cuff pressure waveforms at 400 Hz.
  6. Have an assistant provide mild restraint of the animal prior to port access. Insert Huber needle from the RV pressure transducer to the RV port. Attach a 5 mL syringe to the three-way stopcock and attempt to draw blood back into the syringe from the RV port (Figure 3B).
    1. If it is difficult to pull back on the syringe, first inject 5-10 mL saline into the RV port to dislodge the source of occlusion.
    2. If clogging persists, instill 2 mg of tissue plasminogen activator (tPA) into the port as fibrinolytic agent and leave it overnight. Check the following day to aspirate the tPA.
  7. Once the RV pressure line is established, connect the Huber needle from the PA cuff transducer.
  8. Capture the starting values of RV and PA cuff pressures (Figure 3C). Note any drastic changes from previous readings.
    1. If PA cuff and/or RV pressure dropped substantially from the previous reading, it may be a sign that the PA cuff is leaking.
    2. Observe another obvious sign of PA cuff leak by studying the PA cuff waveform. If the average PA cuff pressure drops at a discernible rate, then there is a high chance that the cuff is leaking.
      NOTE: Re-check that all the luer connections on the pressure transducer, tubing, and stopcock are tightened. The highly pressurized fluid content from the PA cuff can flow back and leak out of loose luer connections.
      1. If the PA cuff is leaking, determine the extent of leakage. If the leakage rate is slow, then a more frequent banding strategy can overcome the leakage to make the disease model still effective.
  9. Slowly inject 3% hypertonic saline into the occluder port while paying attention to RV and cuff pressures.
    1. Adjust the amount of injection based on desired PH disease severity and RV phenotype. A weekly increase of cuff pressure by 100-150 mmHg is a reasonable target to develop an adaptive compensating RV phenotype.
    2. More rapid increases in cuff pressure (>250 mmHg per week) will likely produce a decompensating RV phenotype.
  10. Once the PA cuff is inflated to the desired amount, remove the Huber needle from the cuff port.
  11. Obtain a blood sample from the RV port.
    1. Aspirate 10 mL of blood out of the RV port in a sterile fashion and set aside.
    2. Place a new syringe in place of the aspiration syringe and aspirate as much blood as needed without going over the weekly blood draw limit of 7.5% of the total blood volume.
    3. Reconnect the original syringe with aspirated blood and return it through the RV port.
    4. Pull on the valve lever of the pressure transducer to flush heparinized saline from the saline bag into the RV port. Continue flushing until the entire line becomes clear and colorless.
  12. Flush the RV port with 10 mL of saline. Then, further flush the port with 5 mL of 1000 U/mL of heparin sodium.
  13. Repeat the steps 6.1-6.12 every 1-4 days for 9-10 weeks.

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Representative Results

A representative group of 12 sheep is used to show the efficacy of this model for developing varying degrees of PH-RVF. Among these sheep, the mean PA cuff pressure increased from 32 ± 20 mmHg at week 1 to 1002 ± 429 mmHg at week 9. This resulted in increasing the RV mean and systolic pressures from 28 ± 5 and 57 ± 7 mmHg at week 1, respectively, to 44 ± 7 and 93 ± 18 mmHg by week 9. Furthermore, PA cuff pressure profile was superimposed onto mixed venous oxygen saturation (SvO2) to demonstrate the efficacy of the model at fine-tuning disease phenotype (Figure 4). Specifically, faster PA banding led to a more rapid decline in SvO2. In comparison, those that experienced a more gradual PA banding strategy maintained a physiologic range of SvO2 between 70% and 80%. A representative transthoracic echocardiogram acquired after 9 weeks of progressive PA banding shows RV dilation and septal bowing due to pressure overload (Supplementary Video 1). In a previously published case report10, the model can also be used to induce end-stage RV failure, which leads to pleural effusions and abdominal ascites.

Figure 1
Figure 1: Overview and timeline for the overall experiment. (A) Experimental timeline for the chronic pulmonary hypertension (PH) right ventricular failure (RVF) model and the suggested data acquisition strategy. (B) The schematic diagram for the first survival surgery to establish the foundation for the chronic pulmonary hypertension (PH) right ventricular failure (RVF) model. The main pulmonary artery (PA) occluder is implanted, the left pulmonary artery (LPA) is ligated, and a pressure tubing is placed in the right ventricular outflow tract (RVOT). Finally, both RVOT and PA cuff pressure lines are connected to their respective ports, both of which are implanted subcutaneously for recurrent access and monitoring. (C) Photograph of the PA cuff, the subcutaneous port, and the plastic fitting to protect their barbed connection. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Photographs of key surgical steps to establish the ovine pulmonary hypertension (PH) model. (A) Isolation of the main pulmonary artery (PA) and implantation of the PA cuff (circle). (B) Implanted PA cuff (circle), Penrose tubing (star), and right ventricular outflow tract (RVOT) pressure tubing (white triangle). (C) Subcutaneous implantation of ports for RVOT and PA cuff. (D) Tubular dressing and foam padding fitted around the sheep's body to protect the implanted ports. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Experimental approach for chronic pulmonary artery (PA) banding. (A) Schematic for setting up pressure transducers to measure and adjust right ventricular (RV) and PA cuff pressure values. (B) Photograph depiction of accessing RV outflow tract (RVOT) and PA cuff ports. (C) Representative pressure tracing of RV and PA cuff pressures. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Pulmonary artery (PA) cuff pressure and corresponding mixed venous oxygen saturation (SvO2). Longitudinal trends between pulmonary artery (PA) cuff pressure and corresponding mixed venous oxygen saturation (SvO2) show differentiation in right ventricular phenotype based on the PA banding strategy. The color profile varies considerably between subjects that experienced a more rapid PA banding strategy in comparison to subjects that underwent a more gradual banding strategy. Please click here to view a larger version of this figure.

Supplementary Video 1: Representative transthoracic echocardiograms between healthy baseline state and after the pulmonary hypertension right ventricular failure (PH-RVF) disease model. The PH-RVF model recapitulates key features of the disease, including RV dilation and hypertrophy, and septal bowing. Please click here to download this Video.

Supplementary File 1: Data acquisition setup and calibration steps. Please click here to download this File.

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Discussion

The presented PH-RVF model can reliably induce varying levels of disease severity to match the goals of the investigation. Two different approaches are used in combination to induce this disease model. First, the LPA ligation serves to increase pulmonary vascular resistance and decrease PA capacitance11,12, thereby establishing the starting point of the chronic model at an already increased RV afterload state. Then, the implantation of the PA cuff and its progressive inflation serves to develop a targeted phenotype of PH-RVF. Controlling PA cuff pressure and its rate of change can differentially create compensating or decompensating RVs, demonstrated by either maintenance or decline of SvO2 (Figure 4). By increasing cuff pressure by 250-300 mmHg per week, the sheep will start to display early signs of decompensation around 5-6 weeks. Increasing the cuff pressure by 100-150 mmHg per week, on the other hand, allows for a more adaptive profile over the entire 9-week duration.

Few large animal models of chronic PH and RVF exist in the literature. Pulmonary artery embolization in sheep has been most extensively reported and discussed4,5. However, this approach has a high mortality rate, upward of 86%4 depending on dosage frequency and bead sizes, yet it yields only a marginal change in RV hemodynamics and function. On the other hand, the presented model can induce a much greater range of RV pressure overload with minimal procedurally related deaths. One animal that died due to this PH-RVF model developed several liters of pleural effusion and ascites10, correlating with the clinical and research findings of right heart failure in humans13,14,15 and large animals16. These signs were observed without any evidence of left heart failure. This model can therefore serve as a clinically translatable large animal platform with the ability to produce titratable pathophysiology.

There are several notable challenges to executing this model. First, while using a left mini-thoracotomy facilitates expedient postoperative recovery, simultaneous surgical exposure of both the main PA and the LPA is technically challenging via this minimally invasive incision. Selecting the optimal intercostal space is essential and ultrasonography can be a helpful guide. The PA bifurcation is more distal and posterior compared to human anatomy, making ligation of the LPA the most challenging step of this procedure. While the ligation serves as a critical step to increase pulmonary vascular resistance and decrease PA capacitance, it is feasible that the main PA banding alone might achieve sufficiently high RV pressure.

Infection of indwelling ports and port-site wound dehiscence can be difficult to address and lead to devastating complications. High standards for sterile technique, meticulous skin closure, and port site protection significantly limit the incidence and impact of these occurrences.

Cuff rupture is a specific issue with the model that could lead to decreased RV pressure. Though uncommon, this problem has been observed previously. There are a few preventative and remedial steps for this issue. First, care should be taken to avoid puncturing the cuff while securing it around the PA with suture. Testing the cuff prior to closing the chest ensures its integrity at the conclusion of the initial operation. Next, the PA cuff size should be chosen based on the main PA diameter size. If the cuff leaks, then it will be important to assess the magnitude of leakage. If more frequent inflation of the PA band can overcome the rate of leakage, then the model can still achieve moderate PH-RVF, although it may no longer induce the desired severity of PH-RVF.

Finally, a key scientific limitation of the presented animal model is that it does not convey a key feature of pulmonary arterial hypertension, namely, pulmonary vascular remodeling. Hence, this model is not the ideal platform to develop and test therapeutics that are focused solely on the pulmonary vasculature. Instead, it is an effective platform to study RV dysfunction and failure from abnormal RV afterload. Patient outcomes in PH are largely driven by RV function, and favorable outcomes are associated with the preservation of this RV function17. Although this model does not capture all aspects of PH, it is a valuable model for understanding the molecular pathways leading to RVF and developing RV-targeted therapeutics to ameliorate RVF.

The LPA ligation and main incremental PA banding model can successfully recapitulate the complex pathophysiology of RVF secondary to PH. This model will provide investigators an experimental platform to develop new diagnostic biomarkers that differentiate between adaptive and maladaptive responses to PH on the RV, elucidate critical response pathways in RVF, and enable therapeutic innovations to treat RVF.

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgments

This work was funded by the National Institutes of Health R01HL140231. We thank the Division of Animal Care for their animal husbandry and veterinary care. We thank the SR Light Laboratory and its staff, Jamie Adcock, Susan Fultz, Codi VanRooyen, and José Diaz, for their dedicated technical support with large animal surgeries.

Materials

Name Company Catalog Number Comments
 0.9% Sodium Chloride Irrigation Pour Bottle by Baxter Healthcare, 1000 mL Medline  BHL2F7124 Surgical Disposable
0.25% Bupivacaine Hospira Inc 0409-1160-18 Medication, Intra-Operative
0.9% Normal Saline, 1000 mL Baxter Healthcare Corp 0338-0049-04 Medication, Intra-Operative
0.9% Normal Saline, 500 mL Baxter Healthcare Corp.,  0338-0049-03 Medication, Chronic PH
16 mm Heavy Duty Occluder with actuating tubing Access Technologies  OC-16HD Surgical Disposable
3-mL Skin Prep Applicator Medline  MDF260400 Surgical Disposable
70% isopropyl alcohol prep pads Medline MDS090670 Disposable, Chronic PH
Adhesive bandage tape Patterson Veterinary 07-835-7776 Disposable, Chronic PH
Adson forceps V. Mueller NL1400 Surgical Instrument
Allis tissue forceps V. Mueller CH1560 Surgical Instrument
Aortic clamp, straight (bainbridge forceps) V. Mueller SU6001 Surgical Instrument
Backhaus towel forceps V. Mueller SU2900 Surgical Instrument
Bags, Infusion: Nonsterile Novaplus Infusion Bag, 500 mL Medline TCV4005H Disposable, Chronic PH
Berry sternal needle holder V. Mueller CH2540 Surgical Instrument
Blades, Electrode: Electrode Blade, 6.5", with 0.24 cm Shaft Medline  VALE15516 Surgical Disposable
Blades: Stainless-Steel Sterile Surgical Blade, Size #10 Medline  B-D371210 Surgical Disposable
Blades: Stainless-Steel Sterile Surgical Blade, Size #11 Medline  B-D371211 Surgical Disposable
Blades: Stainless-Steel Sterile Surgical Blade, Size #15 Medline  B-D371215 Surgical Disposable
BNC Male to BNC Male Cable Digi-Key 415-0198-036 Equipment
Castroviejo needle holder V. Mueller CH8589 Surgical Instrument
Cefazolin Apotex Corp 60505-6142-0 Medication, Intra-Operative
Ceftiofur Crystalline Free Acid Zoetis Inc 54771-5223-1 Medication, Post-Operative
Chest Drain, with Dry Suction, Adult-Pediatric Medline  DEKA6000LFH Surgical Disposable
Chest tube passer V. Mueller CH04189 Surgical Instrument
COnfidence Flowprobes for Research (PAU-Series) Transonic 24PAU Equipment, Perivascular Flow Probe
Cooley tangential occlusion clamp V. Mueller CH6572 Surgical Instrument
Data Acquisition Hardware ADInstruments  PowerLab 16/30 Equipment
DeBakey Aorta clamp V. Mueller CH7247 Surgical Instrument
DeBakey multi-purpose clamp V. Mueller CH7276 Surgical Instrument
Debakey tissue forceps, 12’’ V. Mueller CH5906 Surgical Instrument
Debakey vascular tissue forceps 7 3/4’’ V. Mueller CH5902 Surgical Instrument
Debakey vascular tissue forceps, 9’’ V. Mueller CH5904 Surgical Instrument
Electrosurgical Generator Covidien  Force FX-C Equipment
Endotracheal Tube, 10mm Patterson Veterinary 07-882-9008 Surgical Disposable
Enrofloxacin Norbrook Laboratories Limited 55529-152-05 Medication, Intra-Operative
Fentanyl Transdermal Patch Apotex Corp 60505-7007-2 Medication, Pre-Operative
Ferris smith tissue forceps V. Mueller SU2510 Surgical Instrument
Finochietto rib spreaders, large V. Mueller CH1220-1 Surgical Instrument
Finochietto rib spreaders, medium V. Mueller CH1215-1 Surgical Instrument
Flexsteel ribbon retractor, 1” x 13” V. Mueller SU3340 Surgical Instrument
Flexsteel ribbon retractor, 2” x 13” V. Mueller SU3346 Surgical Instrument
Foerster sponge forceps, curved V. Mueller GL660 Surgical Instrument
Gauze Sponges: Sterile X-ray Compatible Gauze Sponges, 16-Ply, 4" x 4" Medline  PRM21430LFH Surgical Disposable
Gerald-DeBakey forceps V. Mueller CH04242 Surgical Instrument
Glassman Allis V. Mueller SU6152 Surgical Instrument
Halsted mosquito forceps V. Mueller SU2702 Surgical Instrument
Harken clamp V. Mueller CH6462 Surgical Instrument
Heat Therapy Pump Gaymar/Stryker  TP-400 Equipment
Heparin Fresenius Kabi,  63323-540-31 Medication, Chronic PH
Hospira Primary IV Sets, 80" Patterson Veterinary 07-835-0123 Surgical Disposable
Hypertonic saline 3% Baxter Healthcare Corp.,  0338-0054-03 Medication, Chronic PH
Hypodermic Needle with Bevel and Regular Wall, 20 G x 1" Medline B-D305175Z Disposable, Chronic PH
Interface Cable, Edwards LifeScience Transducer to ADInstruments  Bridge Amplifier Fogg System 0395-2434 Equipment
Intravenous Infusion Pump Heska  Vet/IV 2.2 Infusion Pump Equipment
Isoflurane Patterson Veterinary 14043-704-06 Medication, Pre-Operative
Kantrowitz thoracic clamp, 9-1/2” V. Mueller CH1722 Surgical Instrument
Kelly hemostats V. Mueller 88-0314 Surgical Instrument
Lidocaine HCl, 2.46% PRN Pharmacal,  49427-434-04 Medication, Chronic PH
Ligaclip Multiple-Clip Appliers by Ethicon Medline  ETHMCS20 Surgical Disposable
Loop, Vessel, Mini, Red, 2/pk, Sterile Medline  DYNJVL12 Surgical Disposable
Lorna non-perforating towel forceps V. Mueller SU2937 Surgical Instrument
Mayo dissecting scissors, curved V. Mueller SU1826 Surgical Instrument
Mayo dissecting scissors, straight V. Mueller SU1821 Surgical Instrument
Medipore Dress-It Pre-Cut Dressing Covers by 3M Medline  MMM2955Z Surgical Disposable
Meloxicam Patterson Veterinary 14043-909-10 Medication, Post-Operative
Mixter thoracic forceps, 9” V. Mueller CH1730-003 Surgical Instrument
Mosquito hemostats V. Mueller 88-0301 Surgical Instrument
Multi-Channel Research Consoles Transonic T402/T403 Equipment, Perivascular Flow Meter
Multi-Lumen Central Venous Catheterization Kits Medline  ARW45703XP1AH Surgical Disposable
Multi-Parameter Vital Signs Monitor Smiths Medical  SurgiVet Advisor 3 Equipment
Needles: Hypodermic Needle with Regular Bevel, Sterile, 18 G x 1.5" Medline  B-D305185Z Surgical Disposable
No. 3 knife handle V. Mueller SU1403-001 Surgical Instrument
No. 7 knife handle V. Mueller SU1407 Surgical Instrument
Non-Vented Male Luer Cap Qosina 13614 Disposable, Chronic PH
Octal Bridge Amplifier ADInstruments  FE228 Equipment
Ophthalmic Ointment Akorn Animal Health 59399-162-35 Medication, Pre-Operative
Penrose Tubing, 6 mm x 46 cm, 11 mm Flat Medline  SWD514604H Surgical Disposable
Perma-Hand Black Braided Silk:  2-0 SH Taperpoint Needle, Control Release, 30" Medline   ETHD8552 Surgical Disposable
Perma-Hand Suture, Black Braided, Size 0, 6 x 30” Medline   ETHA306H Surgical Disposable
Perma-Hand Suture, Black Braided, Size 4-0, 12 x 30" Medline  ETHA303H Surgical Disposable
Phenylephrine West-Ward 0641-6142-25 Medication, Intra-Operative
Polyhesive Cordless Patient Return Electrodes, Adult Medline  SWDE7509 Surgical Disposable
Port-A-Cath Huber Needle, Straight, 22 G x 1-1/2" Medline AAKM21200724 Disposable, Chronic PH
PROLENE Monofilament Suture, Blue, Size 4-0, 36", Double Arm, RB-1 Needle Medline  ETHD7143 Surgical Disposable
PROLENE Polypropylene Monofilament Suture, Blue, Double-Armed, RB-1 Needle, Size 5-0, 24" Medline  ETH8555H Surgical Disposable
Regional Block Needles, 22-gauge Medline  B-D408348Z Surgical Disposable
Schnidt tonsil artery forceps V. Mueller M01700 Surgical Instrument
Skin staple extractor Medline CND3031 Disposable, Chronic PH
Skin stapler 35 wide, with counter Medline  STAPLER35W Surgical Disposable
Sphygmomanometer Patterson Veterinary 07-815-0464 Equipment
Sponge bowl V. Mueller GE-75 Surgical Instrument
Sponge, Lap: X-Ray Detectable Sterile Lap Sponge, 18" x 18", 5/Pack Medline  MDS241518HH Surgical Disposable
Sponge, Peanut: X-Ray Detectable Sterile Peanut Sponge, Small, 3/8" Medline  MDS72038 Surgical Disposable
Sterile Disposable Deluxe OR Towel, Blue, 17'' x 27'', 2/Pack Medline  MDT2168202 Surgical Disposable
Sterile Luer-Lock Syringe, 3 mL Medline SYR103010Z Disposable, Chronic PH
Sterile Luer-Lock Syringe, 5 mL Medline SYR105010Z Disposable, Chronic PH
Sterile Surgical Equipment Probe Covers Medline  DYNJE5930 Surgical Disposable
Stopcock: 3-Way Stopcock with Handle in OFF Position, Rotating Adaptor Male Collar Fitting, 45 PSI Medline  DYNJSC301 Surgical Disposable
Stopcock: 3-Way Stopcock with Handle in OFF Position, Rotating Adaptor Male Collar Fitting, 45 PSI Medline DYNJSC301 Disposable, Chronic PH
Subcutaneous Port with 5-French Connector and Blue Boot Access Technologies CP2AC-5NC Surgical Disposable
Super cut metzenbaum dissecting scissors V. Mueller CH2032-S Surgical Instrument
Super cut nelson-metzenbaum dissecting scissors V. Mueller CH2025-S Surgical Instrument
Syringes: Sterile Luer-Lock Syringe, 10 mL Medline  SYR110010Z Surgical Disposable
Thoracic Catheter, Straight, 28 Fr x 20" Medline SWD570549H Surgical Disposable
Three-quarter surgical drape Medline  DYNJP2414H Surgical Disposable
Tiletamine + Zolazepam Zoetis Inc 54771-9050-1 Medication, Pre-Operative
TourniKwik Tourniquet Set with Four 7.5" Bronze-Colored Tubes and 1 Snare, 12 French Medline  CVR79013 Surgical Disposable
Transducer clip Edwards LifeScience TCLIP05 Equipment
Trigger Aneroid Gauge (Sphygmomanometer) Patterson Veterinary 07-815-0464 Equipment
TruWave Disposable Pressure Transducer Kits by Edwards Lifesciences Medline  VSYPX260 Surgical Disposable and Chronic PH
TS420 Perivascular Flow Module Transonic TS420 Equipment, Perivascular Flow Meter
Tubing, Suction: Sterile Universal Suction Tubing with Straight Ribbed Connectors, 1/4" x 12' Medline  OR612 Surgical Disposable
Tubing: Pressure Monitoring Tubing with Fixed Male Luer Lock and Female Fitting, Low Pressure, 72" L Medline DYNJPMTBG72MF Surgical Disposable
Tubing: Pressure Monitoring Tubing with Fixed Male Luer Lock and Female Fitting, Low Pressure, 72" L Medline DYNJPMTBG72MF Disposable, Chronic PH
Tubular Elastic Dressing Retainer Medline DERGL711 Disposable, Chronic PH
Tuffier rib retractor V. Mueller CD1101 Surgical Instrument
Tygon E-3603 Flexible Tubings Fisher Scientific 14-171-227 Surgical Disposable
U.S.A retractor V. Mueller SU3660 Surgical Instrument
Umbilical Tape, Cotton, 3-Strand, 1/8 x 36" Medline  ETHU12TH Surgical Disposable
Valleylab Button Switch Pencil Medline  VALE2516H Surgical Disposable
Vanderbilt deep vessel forceps V. Mueller CH1687 Surgical Instrument
Veterinary Anesthesia Machine Midmark  Matrx VMC Equipment
Veterinary Anesthesia Ventilator Hallowell EMC  Model 2000 Equipment
Vicryl: Undyed Coated Vicryl 0 CT-1 36" Suture Medline  ETHVCP946H Surgical Disposable
Vicryl: Undyed Coated Vicryl 2 TP-1 Taper 54" Suture Medline  ETHVCP880T Surgical Disposable
Vicryl: Undyed Coated Vicryl 2-0 CT-1 18" Suture Medline  ETHVCP739D Surgical Disposable
Vital crile-wood needle holder, 10-3/8” V. Mueller CH2427 Surgical Instrument
Vital mayo-hegar needle holder, 7-1/4” V. Mueller CH2417 Surgical Instrument
Vital metzenbaum dissecting scissors, 14’’ V. Mueller CH2009 Surgical Instrument
Vital metzenbaum dissecting scissors, 9” V. Mueller CH2006 Surgical Instrument
Vital ryder needle holder, 9” V. Mueller CH2510 Surgical Instrument
Yankauer, Bulb Tip: Sterile Rigid Yankauer with Bulb Tip, No Vent Medline  DYND50130 Surgical Disposable

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References

  1. Campo, A., et al. Outcomes of hospitalization for right heart failure in pulmonary arterial hypertension. European Respiratory Journal. 38, (2), 359-367 (2011).
  2. Tonelli, A. R., et al. Causes and circumstances of death in pulmonary arterial hypertension. American Journal of Respiratory and Critical Care Medicine. 188, (3), 365-369 (2013).
  3. Urashima, T., et al. Molecular and physiological characterization of RV remodeling in a murine model of pulmonary stenosis. American Journal of Physiology- Heart and Circulatory Physiology. 295, (3), (2008).
  4. Sato, H., et al. Large animal model of chronic pulmonary hypertension. ASAIO Journal. 54, (4), 396-400 (2008).
  5. Pohlmann, J. R., et al. A low mortality model of chronic pulmonary hypertension in sheep. Journal of Surgical Research. 175, (1), 44-48 (2012).
  6. Noly, P. -E., Guihaire, J., Coblence, M., Dorfmuller, P., Fadel, E., Mercier, O. Chronic thromboembolic pulmonary hypertension and assessment of right ventricular function in the piglet. Journal of Visualized Experiments: JoVE. (105), e53133 (2015).
  7. Pereda, D., et al. Swine model of chronic postcapillary pulmonary hypertension with right ventricular remodeling: Long-term characterization by cardiac catheterization, magnetic resonance, and pathology. Journal of Cardiovascular Translational Research. 7, (5), 494-506 (2014).
  8. Silva, K. A. S., Emter, C. A. Large animal models of heart failure: A translational bridge to clinical success. JACC: Basic to Translational Science. 5, (8), 840-856 (2020).
  9. Ukita, R., et al. Left pulmonary artery ligation and chronic pulmonary artery banding model for inducing right ventricular - pulmonary hypertension in sheep. ASAIO Journal (American Society for Artificial Internal Organs: 1992. 67, (1), 44-48 (2020).
  10. Ukita, R., et al. Progression toward decompensated right ventricular failure in the ovine pulmonary hypertension model. ASAIO Journal (American Society for Artificial Internal Organs: 1992. (2021).
  11. Mercier, O., et al. Piglet model of chronic pulmonary hypertension. Pulmonary Circulation. 3, (4), 908-915 (2013).
  12. Guihaire, J., et al. Right ventricular plasticity in a porcine model of chronic pressure overload. Journal of Heart and Lung Transplantation. 33, (2), 194-202 (2014).
  13. Tang, K. J., Robbins, I. M., Light, R. W. Incidence of pleural effusions in idiopathic and familial pulmonary arterial hypertension patients. Chest. 136, (3), 688-693 (2009).
  14. Luo, Y. F., et al. Frequency of pleural effusions in patients with pulmonary arterial hypertension associated with connective tissue diseases. Chest. 140, (1), 42-47 (2011).
  15. Brixey, A. G., Light, R. W. Pleural effusions occurring with right heart failure. Current Opinion in Pulmonary Medicine. 17, (4), 226-231 (2011).
  16. Holt, T. N. Bovine High-mountain Disease. Merck and the Merck Veterinary Manual. Available from: https://www.merckvetmanual.com/circulatory-system/bovine-high-mountain-disease/bovine-high-mountain-disease (2019).
  17. Van De Veerdonk, M. C., et al. Progressive right ventricular dysfunction in patients with pulmonary arterial hypertension responding to therapy. Journal of the American College of Cardiology. 58, (24), 2511-2519 (2011).
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Cite this Article

Ukita, R., Stokes, J. W., Wu, W. K., Talackine, J., Cardwell, N., Patel, Y., Benson, C., Demarest, C. T., Rosenzweig, E. B., Cook, K., Tsai, E. J., Bacchetta, M. A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep. J. Vis. Exp. (173), e62694, doi:10.3791/62694 (2021).More

Ukita, R., Stokes, J. W., Wu, W. K., Talackine, J., Cardwell, N., Patel, Y., Benson, C., Demarest, C. T., Rosenzweig, E. B., Cook, K., Tsai, E. J., Bacchetta, M. A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep. J. Vis. Exp. (173), e62694, doi:10.3791/62694 (2021).

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