A Novel Right Ventricular Volume and Pressure Loaded Piglet Heart Model for the Study of Tricuspid Valve Function.

Summary

A novel recovery piglet heart model with combined pressure and volume overload on the right ventricle is described for the study of tricuspid valve function.

Abstract

Heart conditions in which the tricuspid valve (TV) faces either increased volume or pressure stressors are associated with premature valve failure. Mechanistic studies to improve our understanding of the underlying pathophysiology responsible for the development of premature TV failure are lacking. Due to the inability to conduct these studies in humans, an animal model is required. In this manuscript, we describe the protocols for a novel chronic recovery infant piglet heart model for the study of changes in the TV when placed under combined volume and pressure stress. In this model, volume loading of the right ventricle and the TV is achieved through the disruption of the pulmonary valve. Then pressure loading is accomplished through the placement of a pulmonary artery band. The success of this model is assessed at four weeks post intervention surgery through echocardiography, intracardiac pressure measurement, and pathologic examination of the heart specimens.

Introduction

The normal TV functions in a low volume and pressure stress environment. However, there are pediatric and adult heart conditions where the TV is either congenitally malformed or the cardiac physiology is such that the right ventricle and TV are challenged by increased volume (preload) and/or pressure (afterload) stress, such as Tetralogy of Fallot, Ebstein’s anomaly, congenitally corrected transposition of the great arteries, patients with transposition of the great arteries following an atrial switch procedure, idiopathic pulmonary hypertension and hypoplastic left heart syndrome. In these cardiac conditions, the TV is prone to premature valve failure, which increases morbidity and mortality^1,2,3,4,5. Although one may hypothesize that premature TV failure in these cardiac lesions may be related to the TV being subjected to increased volume and/or pressure stressors, the exact etiology is unknown. Research over the past decade has demonstrated that the mitral valve, the other atrioventricular valve, is capable of eliciting structural changes in response to stressors^6,7,8. However, the current literature lacks mechanistic studies that assess TV adaptation to stressors. This aspect may be in part due to a lack of an adequate animal heart model that will allow for such studies.

In the literature, there are models that individually volume or pressure loaded the right ventricle. However, the combination of chronic pressure and volume loading of the right ventricle has been more challenging to achieve. There are animal models in the literature that use placement of a pulmonary artery band to pressure-load the right ventricle as well as creating an atrial septal defect to volume-load the right ventricle⁹. This technique was not able achieve the goal of chronic simultaneous pressure and volume loading of the right ventricle as the presence of a tight pulmonary artery band may result in a right to left shunt across the atrial septal defect. This results in the atrial septal defect no longer providing a volume load to the right ventricle. An atrial septal right to left shunt will result in a cyanotic animal¹⁰. To overcome this complication, the model requires the exclusion of animals with naturally existing atrial septal defects.

Other models have utilized the hybrid stage I palliation surgery for hypoplastic left heart syndrome in piglets¹¹. This is a recovery model that allows for combined pressure and volume loading of the right ventricle. However, the procedure requires expensive balloon-expandable stents that can be financially prohibitive. Studies by Zeltser et al.¹² and Lambert et al.¹³ involve cutting through the right ventricular outflow tract and pulmonary valve and then sewing a polytetrafluorethylene patch over top, mimicking the transannular patch Tetralogy of Fallot repair technique, to volume load the right ventricle. Then pressure loading of the right ventricle is achieved through placement of a pulmonary artery band. This model can be technically challenging and is disadvantaged by leaving a ventriculotomy scar on the right ventricular outflow tract, which may influence RV function and hence TV function.

This study describes an innovative chronic recovery piglet heart model that elicits combined increased pressure and volume stress on the right ventricle without a ventriculotomy. This model will enable mechanistic studies of TV adaptive changes to simultaneous chronic increase in pressure and volume stressors.

Protocol

The protocol and procedures in this manuscript were developed under the supervision of a veterinarian and performed in compliance with the guidelines of the Canadian Council on Animal Care and the guide for the care and use of laboratory animals. The protocol was approved by the institutional animal care committee at the University of Alberta. All individuals involved in the manuscript procedures received appropriate biosafety training.

1. Pre-procedure preparation, anesthesia and access

1. Animal inclusion and exclusion criteria.
   1. Inclusion criteria: Use both female and male Duroc crossbred piglets between four to five weeks of age for this model. Piglets of this age have a human maturity equivalent to infants between four to six months of age.
   2. Exclusion criteria: Exclude piglets with atrial septal defect and/or patent ductus arteriosus (assessed by echocardiography), cardiac or extracardiac malformation, or where infectious disease was suspected.
2. Transfer piglets to the housing facility at least five to seven days prior to surgery for acclimatization.
3. A day prior to surgery, examine the piglet to ensure fitness for surgery and absence of signs of illness, such as coughing, emesis, diarrhea, paleness, weakness, lethargy, or skin lesions.
4. Fast piglets from solid feed for at least three hours prior to surgery, allowing access to water up to the time of surgery.
5. Transfer piglet to the surgical procedure room.
6. Administer procedural premedication of atropine (0.04 mg/kg), midazolam (0.2 mg/kg) and ketamine (2 mg/kg) through an intramuscular injection. Also, administer a slow-release preparation of buprenorphine (0.01 mg/kg) subcutaneously for perioperative analgesia.
7. On the operating table, place a recirculating water blanket and set the water temperature initially at approximately 40 °C. Adjust the water blanket temperature to maintain core body temperature between 38.0 to 39.5 °C in the piglet.
8. Once the jaw is fully relaxed, spray the larynx with 1% lidocaine. Perform standard endotracheal intubation with portable pulse oximeter monitoring.
9. Check, attach and ensure proper function of monitoring equipment: pulse oximeter, capnograph, ECG telemetry leads and temperature probe.
10. Give the piglet inhaled isoflurane (2-5%) for anesthetic during the surgical procedure, adjusted depending on the depth of anesthesia. Monitor the depth of anesthesia through assessment of heart rate, respiratory rate and breathing pattern, oxygen saturation, eyelid and withdrawal reflex.
11. Use the following initial ventilator settings: PEEP of 4 cmH₂O, tidal volume between 8-10 mL/kg and an inspiratory to expiratory ratio of 1:1. The ventilator rate ranges between 26-36/min, adjusted to achieve pCO₂ between 35-42 mmHg on an arterial blood gas analyzed using a point-of-care blood gas analyzer.
12. Place the piglet in a supine position. Apply sterile ophthalmic ointment to both eyes for lubrication during procedure.
13. Shave hair over the surgical site. Using sterile gauze and disinfectant scrub (povidone-iodine 7.5%), disinfect the chest in a circular movement from inside to outside three times. Wipe away the excess scrub solution with sterile gauze. Using aseptic technique, drape the surgical sites (neck and left side of chest) and surrounding area.
14. Using a modified Seldinger technique, place central catheters in both the carotid artery (3.5 French single lumen catheter or 24 gauge IV catheter) and internal jugular vein (five or six French double or triple lumen catheter) for pressure monitoring, blood sampling and venous access during the procedure for medication delivery.
15. Administer an intravenous dose of cefazolin (30 mg/kg) for perioperative sepsis prophylaxis and ranitidine (1 mg/kg) for stress ulcer prophylaxis. Initiate intravenous maintenance fluid (D5WNS) through the central venous catheter.

2. Bioptome disruption of pulmonary valve cusps

1. After confirmation of surgical plane, perform a left thoracotomy at the third intercostal space to ensure adequate exposure to visualize main pulmonary artery.
2. Using sterile gel and a sleeve, perform epicardial echocardiographic study through the left thoracotomy to assess for presence of atrial septal defect, patent ductus arteriosus, and then sweep through the pulmonary and tricuspid valves to assess for congenital abnormalities.
3. Through the left thoracotomy incision, place purse-string sutures on the main pulmonary artery (MPA) and attach to a snare.
4. Using a needle introducer, puncture the MPA in the middle of the purse-string sutures and advance a wire. Over the wire, advance a custom-designed flanged seven-French catheter sheath through the MPA incision. Securely anchor the catheter on the surface of the MPA by wrapping purse-string sutures around the catheter flange, and snare can be tightened down (Figure 1).
5. Introduce a bioptome into the sheath and advance under epicardial echocardiographic guidance to the pulmonary valve.
6. Under direct epicardial echocardiographic visualization, use the bioptome to secure bites in the pulmonary valve cusps. Withdraw the bioptome result in cusp tears and disrupt the pulmonary valve.
   1. Repeat this process as needed to achieve moderate to severe pulmonary regurgitation. This is assessed using pulse-wave Doppler interrogation in the branch pulmonary arteries. We aim to achieve a reverse to forward velocity time integral (VTI) ratio between 0.6 to 0.7 at the time of the procedure. Once satisfied, withdraw the bioptome.
7. During procedure, administer intravenous infusions of epinephrine (0.05 - 0.15 µg/kg/min) and/or norepinephrine (0.05 - 0.15 µg/kg/min) as required to maintain normal blood pressure during procedure (mean systemic arterial blood pressure between 60 – 80 mmHg).

3. Placement of pulmonary artery band

1. Insert a single lumen five-French umbilical catheter through the previously placed sheath and advance it into the right ventricle (RV) for RV pressure monitoring during the placement of a pulmonary artery band.
2. Weave either silk or a synthetic, braided non-absorbable suture through the middle of a silastic band for reinforcement and strength. Wrap this silastic band around the MPA in between the pulmonary valve and proximal to the catheter puncture site.
3. Through direct RV pressure measurement, tighten the pulmonary artery band and adjust using a vascular clip to achieve ≥60% systemic RV systolic pressure. Once the desired pressure is reached, tie sutures to secure the pulmonary artery band.
4. Advance the umbilical catheter across the pulmonary artery band and into the distal MPA for a pull-back pulmonary artery band gradient. Remove the umbilical catheter and sheath.
5. Tie off the purse-string suture to close the MPA puncture site (Figure 2).
6. Close the left thoracotomy incision in three layers using nonabsorbable sutures for the first layer and then absorbable sutures to close the muscle layers. Apply skin staples to close the skin layer.
7. Infiltrate around the skin incision with Bupivacaine (0.5%, max dose 2 mg/kg) for local analgesia. Administer a single intravenous dose of meloxicam (0.2 mg/kg) for postoperative analgesia in addition to the previously given subcutaneous slow-release buprenorphine.
8. Remove both the arterial and venous central lines and tie off the vessels to ensure hemostasis.
9. Apply Hibitane cream (1% chlorhexidine) to the incisions and secure a bandage dressing to cover the incision.
10. Turn off inhaled isoflurane. Once piglet is breathing on its own with adequate airway protection mechanisms, extubate with postoperative recovery monitoring and care.
11. For prophylaxis of incisional infection, give an empiric five-day course of oral cephalexin 30 mg/kg BID to the piglet.

4. Echocardiographic assessment

1. Qualitatively assess pulmonary regurgitation by color Doppler and grade from 0 to 4 [0 = none, 1 = trivial (single narrow jet), 2 = mild (single slightly broader jet with jet length < 10 mm, proximal jet width to RV outflow tract ratio < 0.25), 3 = moderate (single or multiple jets with the combined proximal jet width to RV outflow tract ratio between 0.25 to 0.65), and 4 = severe (wide jet with proximal jet width to RV outflow tract ratio > 0.65)].
   1. In the intervention group, semi-quantify the pulmonary regurgitation by measuring a reverse to forward velocity time integral (VTI) ratio using pulse-wave Doppler in the branch pulmonary artery (Figure 3).
   2. Qualitatively grade the severity of tricuspid regurgitation from 0 to 4 through color Doppler assessment [0 = none, 1 = trivial (single narrow jet), 2 = mild (multiple narrow jets), 3 = moderate (wide jet reaching the midportion of the right atrium), and 4 = severe (wide jet reaching the back wall of the right atrium)]. Both the pulmonary and tricuspid valve regurgitation grading systems are in accordance with current guidelines^{14,15,16,17,18,19}.
2. Assess right ventricular systolic function using right ventricular fractional area change (RV FAC), which was measured by tracing the area bound by endocardial borders of the right ventricle at end-diastole and end-systole in the apical four-chamber view. Calculate the RV FAC as the difference between the two areas, expressed as a percentage of the end-diastolic RV area. An RVFAC value < 35% represents abnormal RV systolic function²⁰.

Results

For model validation, ten piglets (5 male and 5 female) that underwent left thoracotomy with pulmonary valve disruption and placement of pulmonary artery band (intervention group, IP) were compared with ten age and gender-match control piglets that underwent left thoracotomy (control group, CP). At baseline, prior to intervention, all the piglets had either none or trivial pulmonary regurgitation with normal right ventricular geometry and function. There was one piglet in the control group with mild tricuspid regurgitati...

Discussion

During the development of this novel heart model, several considerations have impacted the final model design.

Piglet age and surgical exposure
Prior to formulation of the current piglet model, the team has worked on piglet cadavers ranging from one to six weeks of age with the goal to determine the age range where procedural instrumentation and exposure is adequate. As the surgical procedure required direct echocardiographic guidance, the piglet had to be of the appropr...

Materials

Name	Company	Catalog Number	Comments
Drugs
1% lidocaine spray	WDDC	103365	Lidodan 30 mL
atropine sodium injection	WDDC/Rafter 8 Products		0.5 mg/mL
bupivacaine	WDDC/Sterimax		5 mg/mL
buprenorphine HCl slow release injection	Chiron Compounding Pharmacy		1 mg/mL
buprenorphine regular	WDDC/Champion Alstoe	121378	Vetergesic 0.3 mg/mL
cefazolin	WDDC/Fresenius Kabi	102016	1 g/vial
cephalexin capsule	WDDC/Novopharm		Novo-Lexin 250 mg/capsule
epinephrine	WDDC		Adrenalin (1:1000) 1 mg/mL
furosemide tablet	WDDC/Novopharm		10 mg tablet
Iodine Scrub/Spray	Cardinal Health	KF22422	Betadine brand
ketamine hydrochloride injection	WDDC/Vetoquinol	131771	Narketan 100 mg/mL
midazolam	WDDC/Sandoz	101100	5 mg/mL
norepinephrine	WDDC		1 mg/mL
pentobarbital sodium	WDDC/Bimeda-MTC	127189	Euthanyl 240 mg/mL
ranitidine injection	WDDC		25 mg/mL
ranitidine tablet	Sanis		300 mg tablet
Surgical Scrub Sponge	Stevens	333-377479	4% CHG Surgical Soap scrub brush
Equipment
24G peripehral IV catheter	BD		Insyte-N
5Fr double lumen central venous catheter	Arrow	CS-14502	20cm
5Fr single lumen umbilical vessel catheters	Covidien/Kendall	8888160333	Argyle 15 inches
6" Chest retractor	RRSMRI
6Fr triple lumen central venous catheter	Arrow	JR-42063-HPHNM	20cm
7Fr catheter sheath with flange	Dr. Coe custom designed
Adson forceps	RRSMRI
Aestiva/5 Ventilator	GE Datex Ohmeda
Atraumatic forceps	Teleflex	351865
bioptome	Dr. Coe lab		Mansfield Biopsy Forceps
Curved hemostat	RRSMRI
Curved mosquito hemostat	RRSMRI
Debakey Forceps (Long)	Teleflex	351804
Debakey Forceps (Narrow)	Teleflex	351802
Debakey Forceps (Rg)	Teleflex	351800
Echo probe cover	Civco
Endotracheotube	Stevens	180-112082055	Rusch Murphy Eye Low Press. Cuff
Iris Spring scissors	Fisher Scientific	NC0127560
iSTAT 1 blood gas analyzer	Abbott Laboratories		MN:300-G
iSTAT CG4+ cartridges	Abbott Laboratories	03P85-50
Kelly Hemostat	Fine Science Tools	1301914
Kocher forceps	RRSMRI
Large Army/Navy Retractor	RRSMRI
Laryngoscope	MACO CE Miller#4 Blade	LA6226-4	Macolaryngoscope.com
Liga-clip applicator L	Ethicon	LC430
Liga-clip applicator M	Ethicon	LX210
Liga-clip applicator S	Ethicon	LX110
Metal suction tip	RRSMRI
Metzenbaum Scissors (Lg)	RRSMRI
Metzenbaum Scissors (Md)	RRSMRI
Mixter - long/mid wide	RRSMRI
Mixter - long/narrow	RRSMRI
Mixter - long/wide	RRSMRI
Mixter - short/narrow	RRSMRI
Needle Driver 10"	RRSMRI
Needle Driver 6"	RRSMRI
Needle Driver 7"	RRSMRI
Philips iE33 Echocardiography machine	Philips		X7 and S12 probes
pressure line tubing and 3-way stopcock	Dr. Freed lab
Rat tooth forcep	RRSMRI
silastic reinforced sheeting	Bioplexus	SH-21001-040	6" x 8" x .040" Gloss
Small Army/Navy Retractor	RRSMRI
Straight hemostat	RRSMRI
Straight mosquito hemostat	RRSMRI
Sutures: 4-0 and 5-0 synthetic, non-absorbable suture, 2-0 silk	Dr. Freed lab
Towel clamp	RRSMRI
vascular tourniquet	Dr. Freed lab
Weitlaner retractor (Md)	RRSMRI
Weitlaner retractor (Sm)	RRSMRI
Zoll R Series Monitor Defibrillator	Zoll technologies