Chronic Ovine Model of Right Ventricular Failure and Functional Tricuspid Regurgitation

Summary

Right ventricular failure and functional tricuspid regurgitation are associated with left-sided heart disease and pulmonary hypertension, which contribute significantly to morbidity and mortality in patients. Establishing a chronic ovine model to study right ventricular failure and functional tricuspid regurgitation will help in understanding their mechanisms, progression, and possible treatments.

Abstract

The pathophysiology of severe functional tricuspid regurgitation (FTR) associated with right ventricular dysfunction is poorly understood, leading to suboptimal clinical results. We set out to establish a chronic ovine model of FTR and right heart failure to investigate the mechanisms of FTR. Twenty adult male sheep (6-12 months old, 62 ± 7 kg) underwent a left thoracotomy and baseline echocardiography. A pulmonary artery band (PAB) was placed and cinched around the main pulmonary artery (PA) to at least double the systolic pulmonary artery pressure (SPAP), inducing right ventricular (RV) pressure overload and signs of RV dilatation. PAB acutely increased the SPAP from 21 ± 2 mmHg to 62 ± 2 mmHg. The animals were followed for 8 weeks, symptoms of heart failure were treated with diuretics, and surveillance echocardiography was used to assess for pleural and abdominal fluid collection. Three animals died during the follow-up period due to stroke, hemorrhage, and acute heart failure. After 2 months, a median sternotomy and epicardial echocardiography were performed. Of the surviving 17 animals, 3 developed mild tricuspid regurgitation, 3 developed moderate tricuspid regurgitation, and 11 developed severe tricuspid regurgitation. Eight weeks of pulmonary artery banding resulted in a stable chronic ovine model of right ventricular dysfunction and significant FTR. This large animal platform can be used to further investigate the structural and molecular basis of RV failure and functional tricuspid regurgitation.

Introduction

Right ventricular failure (RVF) is recognized as an important factor contributing to the morbidity and mortality of cardiac patients. The most common causes of RVF are left-sided heart disease and pulmonary hypertension¹. During the progression of RVF, functional tricuspid regurgitation (FTR) may arise as a consequence of right ventricular (RV) dysfunction, annular dilatation, and subvalvular remodeling. Moderate to severe FTR is an independent predictor of mortality^2,3, and it is estimated that 80%-90% of tricuspid regurgitation cases are functional in nature⁴. FTR itself may promote adverse ventricular remodeling by influencing either afterload or preload⁵. The tricuspid valve has historically been considered the forgotten valve⁶, and it was believed that the treatment of left-sided heart disease would resolve the associated RV pathology and FTR⁷. Recent data have shown this to be a faulty strategy, and current clinical guidelines advocate a much more aggressive approach to FTR⁴. However, the pathophysiology of severe FTR associated with right ventricular dysfunction is still poorly understood, leading to suboptimal clinical results⁸. The currently available large animal models of RVF are based on pressure, volume, or mixed overload. We have previously described a large animal model of RVF and TR but only in an acute setting⁹.

The current study focuses on a chronic ovine model of pulmonary artery banding (PAB) to increase RV afterload (pressure overload) and induce RV dysfunction and FTR. The afterload model is reliable and reproducible compared to pulmonary hypertension models, in which changes in microvasculature are less predictable and more likely¹⁰. The goal of the study was to develop a chronic large animal model of RVF and FTR that would most accurately mimic RV pressure overload in patients with left-sided heart disease and pulmonary hypertension. The establishment of such a model would permit in-depth studies on the pathophysiology of the ventricular and valvular remodeling associated with RV dysfunction and tricuspid insufficiency. The ovine model was chosen based on our prior work on the mitral valve and the published literature supporting the anatomical and physiological similarities between human and ovine hearts^11,12,13.

For this study, 20 adult sheep (62 ± 7 kg) underwent a left thoracotomy and main pulmonary artery banding (PAB) to at least double the systolic pulmonary artery pressure (SPAP), thus inducing RV pressure overload. The animals were followed for 8 weeks, and the symptoms of heart failure were treated with diuretics when clinically apparent. Surveillance echocardiography was performed periodically to assess RV function and valvular competence. Following the completion of the experimental protocol for model development (8 weeks), the animals were taken back to the operating room for median sternotomy and the implantation of sonomicrometry crystals on the epicardial and intra-cardiac structures. This procedure was performed using cardiopulmonary bypass with the heart beating and with bicaval control. There were no problems in weaning the animals from the cardiopulmonary bypass or acquiring the sonomicrometry data in a stable steady-state hemodynamic environment without the need for inotropes for right heart support. We anticipate performing tricuspid ring annuloplasty and other right heart procedures in the near future using a right thoracotomy approach in both terminal and survival experiments. The current experience leads us to believe that it will be possible to wean the animals from the cardiopulmonary bypass without difficulty and that long-term survival is feasible. As such, we believe the model will permit the performance of clinically pertinent cardiac procedures. Below is a description of the steps (perioperative and operative) performed for carrying out the ovine experimental protocol.

Protocol

The protocol was approved by the Michigan State University Institutional Animal Care and Use Committee (IACUC) (Protocol 2020-035, approved on 7/27/2020). For this study, 20 adult male sheep weighing 62 ± 7 kg were used.

1. Preoperative steps

1. Fast the animal 12 h prior to surgery (overnight).
2. Place the animal in a sheep chair (Figure 1), and prepare for the right jugular vein cannulation using a long 11 Fr introducer sheath (sheath length = 10 cm).
3. Shave with clippers along the IV placement site-the right anterior aspect of the neck around 10-15 cm lateral from the midline for the right jugular vein.
4. Turn the animal's head to the left so that the right anterior and lateral aspects of the neck are exposed. Localize the jugular vein course. To facilitate this, compress the bottom of the neck to distend the vein.
5. Clean with chlorhexidine and alcohol-based scrub, and anesthetize locally with 1% lidocaine.
6. Canulate the jugular vein in the mid to upper third of the neck, as described.
   1. Cut the skin above the vein with a number 11 blade perpendicular to the vein.
   2. Cannulate with a 14 G angiocatheter; when in place (blood is coming out of the needle or blood is spotted), remove the needle, leave the catheter, pass the guidewire, remove the catheter, place the 11 Fr sheath, and secure it.
   3. Ensure the patency and proper placement of the cannula by withdrawing dark-red blood and performing a saline flush to confirm the flow and absence of swelling at the insertion site.
7. Start the induction of propofol at 1.0-1.5 mg/kg intravenous (IV).
8. Intubate with a number 9 endotracheal (ET) tube using a laryngoscope with a number 5 blade. For this, one person should secure the jaws and tongue, while the other person identifies the trachea, inserts the ET tube, and inflates the sealing cuff. Confirm proper placement by the bilateral breath sounds and condensation on the ET tube.
9. Administer intravenously the analgesic buprenorphine at 0.01 mg/kg and use 240 mg of gentamicin and 1 g of cefazolin for antibiotic prophylaxis.
10. Transfer the animal from the sheep chair onto a surgical table, and position it on its right side.

2. Surgery steps

1. Ventilate at 15 mL/kg (12-18 breaths/min), with oxygen flow at 4 L/min and isoflurane at 2.5%-4.0%. Confirm proper anesthesia to ensure the subject is at the surgical level (stage 3) by checking the jaw tone and eye rotation.
2. Lubricate both eyes by applying ophthalmic oinment and insert a gastric tube to ensure gas and food evacuation. Connect the electrocardiogram (EKG), pulse oximeter (SpO₂), capnograph (ETCO₂), and body temperature monitors. Attach the EKG limb leads (I, II, III) to the skin via alligator clips, the SpO₂ sensor to the animal's cheek, and the ETCO₂ tube to the endotracheal tube, and pass the temperature probe through the nostril into the nasopharynx.
3. Prepare the operative field. Shave the left anterior thorax, clean with chlorhexidine and alcohol-based scrub, and cover with sterile drapes.
4. Make a 10 cm long skin and subcutaneous incision at the level of the fourth intercostal space.
5. Confirm the correct intercostal space by identifying the thoracic inlet and counting the intercostal spaces downward. Subsequently, continue the incision in the center and along the fourth intercostal space.
6. Divide the intercostal muscles, open the chest cavity, and spread the ribs with a mini-thoracotomy Finochietto-style retractor. While performing the thoracotomy, take care not to injure the left internal mammary artery (LIMA) at the sternal border of the incision and the lung at the superior border.
7. Perform baseline epicardial echocardiography to assess the biventricular function and valvular competence. The occurrence of non-standard views may be due to the mini-thoracotomy focused on the tricuspid valve (TV), right and left ventricular function, and pulmonary artery flow.
8. Identify the LIMA at the sternal border of the incision, remove the adjacent tissues around it, and prepare to establish an arterial line for pressure monitoring.
9. Place two 4-0 silk sutures around the artery, with one proximal and one distal to the cannulation site (used to secure the arterial catheter).
10. Use titanium clips with a clip applier to clip the LIMA distal to the planned cannulation site to prevent backflow bleeding during cannulation.
11. Make a perpendicular incision that is half of the circumference of the catheter in the LIMA with a number 11 blade.
12. Insert an 18 G angiocatheter, and attach it to the arterial line module. When a pressure of around 120/80mmHg is reached, secure the catheter in place using the two 4-0 silk sutures placed earlier.
13. Perform pericardiotomy starting at the level of pulmonary artery sinuses and going 4-5 cm laterally along the main pulmonary artery (MPA), taking care not to injure the left phrenic nerve.
14. Apply four to five retraction stitches to the opened pericardium to create a pericardial well, as this facilitates exposure and dissection between the pulmonary trunk and aorta.
15. Dissect the MPA from ascending aorta (AA) around 2-3 cm from its origin using blunt right-angle forceps, starting at the level of the left atrial appendage and working toward the AA. To fully separate the MPA from the AA, use electrocautery or scissors to remove the connective tissue between the two structures.
16. Pass an umbilical tape around the MPA with a blunt right-angle clamp. Establish an MPA pressure line by placing a 5-0 monofilament purse-string suture 1 cm distal from the MPA sinuses.
17. Insert a 20 G angiocatheter, and connect it to a monitoring line. Ensure correct MPA and arterial line readings are achieved prior to cinching the umbilical tape; the arterial and pulmonary pressures may vary but should be comparable to human patient values.
18. Hold both ends of the umbilical tape, and clip them together to reduce the lumen of the MPA.
19. Progressively tighten the band with the successive application of a clip applier, with each clip placed below the previous clip until the systemic blood pressure begins to steadily decline (Figure 2). At this point, remove the last placed clip to stabilize the systemic blood pressure.
20. When maximal cinching and stable hemodynamic conditions are achieved, secure the umbilical tape to the adventitia of the MPA using a 5-0 monofilament suture to avoid distal migration.
21. Perform post-banding echocardiography to assess the biventricular function and valvular competence, as in step 2.7. Remove the MPA pressure line and arterial line, and ensure good hemostasis by checking for any bleeding coming from the area where the band and arterial lines were placed.
22. Place a chest tube into the left thorax, with the entry site one intercostal space below the initial incision. Close the ribs with two Vicryl size 2 sutures, and close the wound with three-layer continuous sutures: Vicryl 2-0 for the muscle and subcutaneous tissues and Prolene 3-0 for the skin.
23. When no signs of bleeding are seen, remove the chest tube before weaning the animal from the ventilator.
24. Wean the animal from the ventilator, extubate, move it to a single cage, and closely follow it for at least 1 h. Leave the central IV line in place, and secure it using a loosely applied bandage around the neck.
    NOTE: Postoperative intravenous analgesia was maintained with buprenorphine (0,05 mg/kg) and Flunixin (1.2 mg/kg) for 3 days post-surgery. 

Results

Following the completion of the experimental protocol for model development (nearly 8 weeks), the animals were taken back to the operating room for median sternotomy and the implantation of sonomicrometry crystals on the epicardial and intra-cardiac structures. This procedure was performed using cardiopulmonary bypass with the heart beating and with bicaval control, as described by our group in detail previously⁹. There were no problems in weaning the animals from the cardiopulmonary bypass or acq...

Discussion

In this model, 8 weeks of pulmonary artery banding resulted in a stable chronic ovine model of right ventricular dysfunction and, in most cases, significant FTR. The strengths of the presented chronic PAB model include the precise afterload adjustment during the procedure, although its influence on RV responses may differ. The model is suitable for evaluating varying degrees of RV failure or FTR, with the severity modulated by the degree of pulmonary artery constriction. Moreover, the application of fixed and stable resi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anesthesia Machine Drager Narkomed MRI-2	Drager	4116091-001
angiocatheter	BD	BD382268	14GAx8.25cm
BD ChloraPrep Scrub Teal 26 ml applicator with a sterile solution
Blade #11	Bard-Parker	371111
Buprenorphine	HIKMA
cefazolin 1.0g	Hikma	0143-9924-90
Diprivan 200mg/20ml	63323-0269-29	FRESENIUS KABI
Electrosurgical generator Valleylab Force FX	Valleylab	CF5L44233A
Gentamicin Sulfate 40 mg / mL	Fresenius	406365
i-Stat Blood analyzer MN 300	Abbott
Lidocaine HCl 1%	Pfizer	243243
Open ligating clip appliers Horizon Medium	Teleflex	237061
PERMAHAND Silk Suture	PERMA HAND	SA 63H
Pinnacle Introducer sheath	Terrumo	RSS102	sheath length 10cm
Prolene 3-0	ETHICON	8684H
Titanium Clips Medium	Teleflex	2200
Umbilical tape	Ethicon	EFA 1165
VICRYL 2 coated undyed 1X54" TP-1	ETHICON	J 880T
Vicryl 2-0	ETHICON	J269H