In Utero Fetal Lamb Model of Mitral Inflow Obstruction To Induce Hypoplastic Left Heart Syndrome-like Physiology

Summary

This protocol describes an in utero. fetal lamb model of mitral inflow obstruction using balloon-based restriction of left atrial inflow to induce hypoplastic left heart syndrome-like physiology, with continuous hemodynamic monitoring and refined left atrial balloon implantation techniques.

Abstract

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect characterized by underdevelopment of the left ventricle (LV), mitral valve, aortic valve, and ascending aorta. Based on the “no flow, no grow” theory, this protocol describes a reproducible fetal sheep model that restricts mitral inflow to impair LV growth in utero. At approximately 120 days of gestation, maternal laparotomy and hysterotomy are performed, followed by fetal left thoracotomy. A balloon catheter is introduced into the left atrium (LA), left deflated at surgery, and subsequently inflated beginning at approximately postoperative day 3 to restrict mitral inflow until antegrade flow in the ascending aorta is markedly reduced or abolished, as verified by a flow probe after maternal recovery. Hemodynamic changes are continuously monitored via fetal carotid arterial pressure and an ascending aortic flow probe. Fetuses are maintained for up to three weeks and undergo necropsy. Critical steps include atraumatic placement and securement of the aortic flow probe and the LA balloon in the fragile fetus, as well as meticulous prevention of amniotic fluid loss before and after uterine closure. This model provides a platform for investigating HLHS mechanisms, validating imaging biomarkers, evaluating prenatal or perinatal therapeutic interventions, and guiding the development of preventive strategies.

Introduction

Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease characterized by underdevelopment of the left heart structures and the aorta¹. Because the small left ventricle (LV) cannot support systemic perfusion, circulation becomes dependent on the right ventricle, resulting in single-ventricle physiology. Consequently, HLHS typically requires a series of staged open-heart surgeries.

The etiology of HLHS is multifactorial, with both genetic factors^2,3 and alterations in embryonic mechanical blood flow^3,4,5,6,7,8 recognized as major contributors to disease development. Recently, fetal interventions aimed at modifying blood flow to promote LV growth have received increasing attention^9,10, highlighting a critical need for large-animal HLHS models to improve mechanistic understanding and support the development of therapeutic strategies.

Fetal sheep are frequently used to model human congenital heart disease because they tolerate chronic instrumentation and surgical procedures and have a body size at birth similar to that of human neonates¹¹. Although swine models are widely used in cardiovascular research and more closely resemble the human heart in certain genetic and anatomical aspects, they are difficult to manage when extracorporeal cables are required, as they may pull on or become entangled in externalized lines. Therefore, for studies requiring chronic exteriorized electrical cables and/or pressure monitoring catheters, sheep or cattle are often preferred. These ruminants are generally calmer than pigs and can be more readily managed in restricted environments.

Currently, large-animal attempts to replicate the HLHS phenotype have primarily used in utero fetal lamb models. Sheep hearts closely resemble human hearts, with a four-chamber configuration, atrioventricular and semilunar valve structures, and similar coronary artery patterns. The first fetal lamb model of HLHS was reported by Fishman et al.¹² in the 1970s, with additional models developed more than four decades later. Several approaches have been used to induce mitral inflow obstruction and create LV hypoplasia in fetal lambs^7,8,13, including in utero. occlusion of the fetal foramen ovale using a stent⁷, occlusion of LV inflow using platinum coils placed above the mitral valve⁸, and insertion and inflation of a balloon catheter within the left atrium (LA) to obstruct mitral inflow¹³. However, survival rates (to a defined postoperative endpoint) remain relatively low, ranging from 42%^8,13 to 58%⁷, underscoring the need for safer and less invasive approaches.

The model described here uses left atrial balloon inflation to obstruct mitral inflow and represents a promising experimental approach for inducing HLHS-like physiology and testing interventions aimed at reversing LV unloading. However, as with other fetal lamb models, relatively low survival rates remain a significant limitation. Further surgical refinement toward less invasive techniques is therefore necessary. This study describes methodologies to improve the safety and feasibility of LA balloon implantation in chronic fetal lambs. The primary focus is on surgical feasibility and safety; although long-term outcome data remain limited, this model may be useful for investigating mechanisms of impaired LV development and for developing therapeutic strategies.

Protocol

This experimental protocol was approved by the Institutional Animal Care and Use Committee of the Abigail Wexner Research Institute at Nationwide Children’s Hospital (AR24-00184). All procedures followed the guidelines in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This study adhered to the ARRIVE guidelines (Animal Research: Reporting of In Vivo. Experiments). Dorset pregnant sheep (65–79 kg, 117–120 days of gestation) were housed in a specific pathogen-free environment with free access to food and water for at least 1 week prior to surgery. The equipment used in this study is listed in the Table of Materials.

Humane Endpoint Criteria:

Complications from line maintenance.

The catheters and flow probe cable inserted into the fetus are externalized through the ewe’s uterus and flank and secured outside the body. These externalized lines may become occluded due to thrombosis or kinking, or may be displaced by animal movement. Resulting complications may include infection or major bleeding. Signs of complications include lack of blood return during sampling, persistent monitoring errors, inability to inject through the line, line dislodgement, and major bleeding. Occluded lines may remain in place if there are no signs of infection or bleeding. In cases of uncontrolled bleeding following line removal, or clear signs of infection related to an occluded line, the ewe and fetuses will be euthanized.

Preterm labor.

Preterm labor may occur at any stage of pregnancy and is more likely following fetal cardiac intervention. Early signs include vulvar swelling and relaxation. The appearance of the amniotic sac indicates imminent delivery, which typically occurs within 30 min after rupture. If signs of preterm labor are observed, the ewe and fetuses will be euthanized to terminate the experiment and proceed to necropsy.

Fetal demise.

Fetal demise may occur at any stage of pregnancy, particularly following fetal cardiac intervention. Although fetal demise often leads to preterm labor, it may also result in systemic infection in the ewe. If the ewe shows clear signs of labor progression or if a fetus is determined to be nonviable, a terminal procedure will be performed, and the ewe and fetuses will be euthanized.

Abortion with dystocia following fetal demise.

If one fetus expires while a co-twin survives, retention of the deceased fetus may increase the risk of abortion with or without dystocia. This may present as changes in vital signs or agitation in the ewe. If dystocia occurs, the ewe and fetuses will be euthanized to terminate the experiment and proceed to necropsy.

Massive maternal bleeding.

A pregnant ewe may experience hemorrhage as a complication of uterine or abdominal incision. Signs include vaginal bleeding, lethargy, and persistent abnormalities in vital signs. Severe hemorrhage may result in hematoma formation or shock. If blood loss prevents maintenance of physiological mean arterial pressure, or if tachycardia persists despite intervention, and signs of impending cardiovascular collapse are observed, euthanasia will be performed.

Balloon damage or migration.

Balloon damage or displacement may result in ineffective occlusion of LV inflow. Simple balloon deflation due to damage may not produce clinical signs if fetal hemodynamics remain stable. If dislodgement occurs in the inside-LA configuration, intrathoracic bleeding and fetal demise may result. In cases of significant bleeding leading to fetal demise, the ewe and fetuses will be euthanized.

Surgical site dehiscence and/or wound infection.

Surgical site dehiscence typically occurs 5–8 days after surgery and may present with infection, tissue necrosis, or herniation of internal organs. Wound infections may occur within several weeks postoperatively. If intervention is feasible, treatment will be provided; if the condition becomes life-threatening, euthanasia will be considered.

1. Animal preparation

1. Collect blood from the ewe’s jugular vein to confirm Q fever negativity upon arrival.
2. Acclimate the sheep to a stanchion for postoperative continuous monitoring.
3. Evaluate the sheep by the veterinary team 1 week prior to surgery. Fast healthy animals overnight before anesthesia.
4. Administer diazepam (0.5 mg/kg) and ketamine (4 mg/kg) via the internal jugular vein for sedation.
5. Intubate the ewe with an 8–9 mm single-lumen endotracheal tube and initiate anesthesia with isoflurane (1%–5%). Maintain anesthesia with isoflurane (1%–5% in oxygen) or a continuous propofol infusion (starting at 30 mg/kg/h; range: 20–45 mg/kg/h).
6. Insert an orogastric tube to decompress the stomach and rumen.
7. Insert a single-lumen venous catheter (16–18G) into the cephalic or jugular vein for fluid administration, propofol infusion, and drug delivery as needed.
8. Place an arterial line (22–24G) in the auricular, femoral, or radial artery for continuous blood pressure monitoring and blood sampling.
9. Shave the entire abdominal area, extending to the dorsal and inguinal regions. Prepare the surgical site using chlorhexidine, povidone-iodine, or alcohol.
10. Transfer the sheep to the surgical suite. Place the animal in a supine position on a surgical table and position a warming device beneath the ewe to prevent hypothermia. Secure all limbs.
11. Apply noninvasive monitors, including a blood pressure cuff on the right forelimb, a pulse oximeter on the ear or tongue, and electrocardiogram leads to monitor maternal blood pressure, oxygen saturation, and heart rate.
12. Administer cefazolin (50 mg/kg) for antibiotic prophylaxis before incision. Repeat every 4 h during the procedure as needed.

2. Laparotomy and hysterotomy of the ewe and thoracotomy of the fetus

1. Perform a midline laparotomy to expose the uterus. Palpate the uterus and count the number of fetuses.
2. Gently exteriorize the uterus. Select a hysterotomy site measuring approximately 10–15 cm, depending on fetal size.
3. Place a purse-string suture (1 absorbable braided suture) before performing the hysterotomy to control amniotic fluid leakage. Open the uterine wall layer by layer using electrocautery.
4. Place four Babcock clamps along the uterine incision to secure all layers and prevent amniotic fluid loss. Ensure that the amniotic membrane (innermost layer) is included.
5. Place a continuous over-and-over suture along the incision edge using 2-0 silk to maintain apposition of the amniotic membrane to the uterine wall.
6. Gently externalize the fetal head and secure the uterine purse-string suture with a hemostat (Figure 1A). Cover the fetal face with a custom plastic cover with an opening for the intracardiac echocardiography (ICE) probe to maintain moisture.
7. Stabilize the fetal head using gauze to maintain a supine position.
8. Insert an ICE probe into the fetal esophagus via the mouth and perform transesophageal echocardiography (TEE) (Figure 1A).
9. Acquire baseline echocardiographic measurements.
   NOTE: Baseline echocardiographic assessment includes qualitative evaluation of cardiac anatomy and ventricular contractility, as well as quantitative measurements of ventricular chamber size and systolic function when image quality is sufficient.
10. Administer an intramuscular drug combination containing an analgesic, an anticholinergic, and a neuromuscular blocking agent (fentanyl 20 µg/kg, atropine 20 µg/kg, and vecuronium 20 µg/kg) into the fetal thigh to reduce movement during surgery.
11. Make a neck incision and expose the jugular vein and carotid artery.
12. Insert a fluid-filled catheter into the carotid artery for arterial pressure monitoring and another into the jugular vein for central venous pressure monitoring (Figure 1B).
    NOTE: Ligate distal vessel ends and secure catheters with two 2-0 silk sutures.
13. Close the neck incision using 2-0 silk sutures while externalizing the catheters.
14. Align the two pressure lines with an additional amniotic pressure line and secure them along the fetal neck using 3-0 silk sutures.
15. Expose the upper fetal body by releasing the uterine purse-string suture. Place stay sutures at the xiphoid and dorsal aspect to secure positioning.
16. Secure the fetal head and forelimbs using gauze to position the fetus in the right lateral decubitus position.
17. Perform a fetal thoracotomy at the fourth intercostal space and open the rib cage using a rib spreader.
18. Open the pericardium and suspend it with 4-0 silk stay sutures to expose the left atrium (LA) and pulmonary artery (PA).
19. Dissect the pulmonary artery and ascending aorta and encircle each with vessel loops.
20. Pass the cable of a 6 mm flow probe through the second intercostal space and position it around the ascending aorta.
21. Confirm adequate signal quality by connecting the probe to the flow measurement unit.

3. Balloon implantation method 1: inside the left atrium (LA) (fetuses older than GA110)

1. Clamp the left atrium (LA) slightly above the atrioventricular groove using a small angled vessel clamp (Figure 2A).
2. Place a purse-string suture (6-0 polypropylene suture) on the LA appendage and secure it with a tourniquet.
3. Incise the LA wall within the purse-string suture using fine scissors. Confirm that the opening is sufficient for balloon insertion.
4. Insert the custom-made balloon catheter into the LA. Secure the catheter using the previously placed purse-string suture and reinforce fixation with two additional 4-0 silk sutures (Figure 2B).
   NOTE: The balloon diameter is approximately 1.5 cm at 5 mL and 2.7 cm at 10 mL of 30% glycerin. Although the balloon was bench-tested to tolerate inflation up to 17 mL, approximately 10 mL was used as the practical upper limit for in vivo. inflation because larger volumes may indicate malposition or leakage and may increase the risk of excessive compression or tissue injury.
5. Perform transesophageal echocardiography (TEE) to confirm proper balloon positioning. Confirm that the balloon lies on the mitral plane and does not remain within the appendage. Temporarily inflate the balloon to confirm functional obstruction by reduction in mitral inflow on TEE and reduction or disappearance of antegrade ascending aortic flow on the flow probe, then deflate it.
6. Close the rib cage loosely using three 2-0 silk sutures. Avoid compressing the balloon catheter.
7. Externalize the balloon catheter from the thoracic cavity into the same subcutaneous plane as the flow probe cable and route both to the dorsal skin.
8. Secure both cables to the dorsal skin at a minimum of two points using 2-0 silk sutures (anchor the skin, tie one cable, tie the second cable, and re-anchor the skin).

4. Balloon implantation method 2: outside the left atrium (LA) (fetuses younger than GA110)

1. Release the pericardial suspension around the LA and assess the relationship between the pericardium and the LA wall (Figure 3).
2. Place two 6-0 polypropylene sutures on the balloon catheter (one at the tip and one at the base). Anchor these sutures to the pericardium at the corresponding positions.
   NOTE: Position the balloon at the superior aspect of the LA when the pericardium is closed.
3. Close the pericardium loosely using 2-0 silk sutures.
4. Adjust balloon position under TEE guidance to achieve adequate LA compression. Ensure that the inflated balloon compresses the LA from the caudal aspect rather than the posterior aspect. Temporarily inflate the balloon to confirm the hemodynamic effect, then deflate it.
   NOTE: Adequate compression is defined qualitatively as visible reduction of LA size and mitral inflow on TEE without compression of adjacent structures, and functionally as reduction or disappearance of antegrade ascending aortic flow on the flow probe.
5. Close the rib cage loosely using three 2-0 silk sutures. Avoid compressing the balloon catheter.
6. Externalize the balloon catheter from the thoracic cavity into the same subcutaneous plane as the flow probe cable and route both to the dorsal skin.
7. Secure both cables to the dorsal skin at a minimum of two points using 2-0 silk sutures (anchor the skin, tie one cable, tie the second cable, and re-anchor the skin).

5. Closure of hysterotomy and laparotomy with externalization of cables and catheters

1. Return the fetus gently to the uterus. Preserve as much amniotic fluid and as many placentas as possible.
2. Remove the purse-string suture and the 2-0 silk over-and-over suture. Remove all residual sutures from the uterine incision.
3. Close the uterus using absorbable braided sutures in three continuous layers: horizontal mattress, over-and-over, and continuous covering suture.
   NOTE: Externalize the three pressure lines, the flow probe cable, and the balloon catheter between the suture layers (Figure 4).
4. Administer antibiotics (ampicillin 500–1000 mg and ciprofloxacin 2 mg) into the amniotic sac via the amniotic pressure catheter to reduce the risk of intra-amniotic infection after hysterotomy, fetal thoracotomy, and chronic catheter/cable externalization.
5. Return the uterus to the abdominal cavity.
6. Place a purse-string suture in the abdominal fascia 2–3 cm to the right of the linea alba and pass the cables through the opening.
7. Adjust the cable length within the abdomen and secure the purse-string suture at the fascia. Allow slight mobility of the cables; do not overtighten.
8. Insert a custom-made tunneling device into the subcutaneous tissue of the ewe’s right flank and advance it toward the dorsal region for approximately 50–60 cm.
9. Identify the exit site. Make a small skin incision at the distal tip of the tunneling device and remove the inner core.
10. Pass two additional PVC tubes through the tunneling device. Connect the three pressure lines and the balloon catheter to a single PVC tube, then pull them through the tunnel.
11. Connect the flow probe cable to the second PVC tube and externalize it through the tunnel.
12. Remove the tunneling device from the flank.
13. Close the laparotomy in three layers: fascia, subcutaneous tissue, and skin using appropriate sutures.
14. Partially close the cable exit site using a 3-0 polyglactin 910 suture.
15. Secure a storage pouch to the ewe’s right flank using 0 braided polyester sutures. Position the pouch 2–3 cm dorsal to the cable exit site to minimize friction. The total operative time is typically 4–4.5 h from skin incision to closure.

6. Recovery

1. Discontinue anesthetics and remove the orogastric tube. Extubate the ewe after confirming spontaneous respiration.
   NOTE: Extubation typically occurs after signs of arousal, including neck movement, blinking, jaw tone, and chewing.
2. Remove the arterial line from the ewe.
3. Transfer the ewe to an isolated recovery area. Assist the animal into sternal recumbency until it can stand and ambulate independently.
4. Administer intramuscular buprenorphine (0.015–0.05 mg/kg) every 8–12 h for postoperative analgesia. Administer meloxicam (0.3–1 mg/kg) as needed, following veterinary guidance.
5. Monitor the ewe closely in an individual enclosure for the first 3 days during the acute recovery period.

7. Chronic housing and evaluation

1. House the ewe in a custom stanchion designed to enable continuous hemodynamic monitoring while minimizing stress.
2. Connect the externalized catheters to monitoring systems. Continuously measure fetal aortic pressure, central venous pressure, amniotic fluid pressure, and aortic flow.
3. Obtain fetal blood gas samples from the arterial catheter when abnormal hemodynamic values are observed.
4. Initiate balloon inflation at approximately postoperative day 3 using sterile 30% glycerin. Increase balloon volume by 1 mL/day. After each 0.5 mL increment, monitor the fetal hemodynamic response for at least 5–10 min to ensure stability, including absence of bradycardia or a mean arterial pressure below 30 mm Hg. Continue inflation until antegrade ascending aortic flow is reduced to zero.
5. If zero antegrade flow is not achieved by 10 mL of balloon inflation, suspect balloon leakage or malposition, rather than continuing inflation toward the bench-tested maximum balloon capacity. Deflate the balloon and evaluate for leakage or displacement using catheter inspection or ultrasound imaging.

8. Necropsy

1. Disconnect the monitoring catheters and flow probe cable and store them in the external pouch.
2. Sedate and intubate the ewe. Maintain anesthesia and insert an orogastric tube and vascular catheters using the same protocol as the initial surgery.
3. Transfer the ewe to the surgical suite. Position the animal supine on a surgical table and place a warming device beneath the ewe. Secure all limbs.
4. Apply noninvasive monitors as described in the initial procedure.
5. Reconnect the pressure catheters and flow probe to the monitoring system.
6. Reopen the previous laparotomy and uterine incision. Exteriorize the experimental fetus.
7. Stabilize the fetal head using gauze to maintain a supine position.
8. Insert an intracardiac echocardiography (ICE) probe into the fetal esophagus via the mouth and perform transesophageal echocardiography (TEE).
9. Acquire balloon position images and collect echocardiographic, flow, and pressure data. Deflate the balloon and repeat measurements.
10. Administer heparin (10,000 U) to the fetus. Ligate the umbilical cord and inject saturated potassium chloride into the umbilical vein to arrest the heart in diastole.
11. Remove the experimental fetus. Reopen the thoracic cavity and assess balloon position, migration, and evidence of infection.
12. Excise the heart, measure heart weight, and collect myocardial tissue samples.
13. Repeat steps 8.6–8.12. for the control fetus.
14. Euthanize the ewe using sodium pentobarbital while under anesthesia.

Results

Surgical outcomes

A total of three ewes carrying twins underwent laparotomy and hysterotomy. Three fetuses (one from each ewe) underwent balloon implantation, while the co-twins served as controls. The procedures were performed at a gestational age of 119 ± 1.4 days, with a maternal body weight of 73 ± 4.2 kg.

All three experimental fetuses survived for at least 1 week without surgical complications during the procedure or acute recovery phase. No p...

Discussion

A fetal lamb model of mitral inflow obstruction using a balloon inserted into the left atrium (LA) has previously been described¹³; however, a key limitation has been the short duration of mitral inflow restriction. Longer periods of restricted left ventricular (LV) inflow have been suggested to induce more pronounced HLHS-like changes^8,13. Therefore, implantation of the LA balloon at earlier gestational ages is necessary...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0 Ti-Cron suture	Covidien	88863374-62	To fix the pouch to the ewe's right flank
1 Polysorb suture	Covidien	CL-932	Purse string suture on the uterus and closure of the uterus
16 G intravenous catheter	BD	382259	For fluid and drug administration
2-0 perma-hand silk (no needle)	Ethicon	A185H	For blood vessel ligation and to fix the pressure catheters at the vessels of the fetus
2-0 silk suture	DemeTECH	SK262026B0P	To fix the catheters and cables at the fetal skin and close the fetal rib cage and skin
22 G intravascular catheter	BD	381423	For arterial blood pressure monitoring
3-0 silk suture	Ethicon	K832H	To fix the catheters and cables at the fetal skin
3-0 Vicryl plus	Ethicon	VCP944H	Laparotomy closure of the ewe
4-0 silk suture	Covidien	GS-64-M	For stay sutures on pericardium of the fetus
6 mm flow probe	Transonic System Inc. (ADInstruments)	6PSS	For blood flow measurement
6-0 Prolene suture	Ethicon	8805H	Purse string suture on the fetal left atrium
7-0 Prolene suture	Ethicon	8702H	Purse string suture on the fetal left atrium and to stop bleeding from the fetal tissues
70% isoptopyl alchohol	Aspen Vet	11795782	Topical cleaning solution
Acuson Acunav Ultrasound catheter	Johnson&Johnson	IPX8	Intracardiac echocardiography probe to perform fetal transesophageal echocardiography
Ampicillin	Hospira Pharmaceuticals	Pharmacy	Antibiotic prophylaxis
Atropine	Hospira Pharmaceuticals	Pharmacy	To prevent fetal bradicardia
Babcock tissue forceps	NIFTY medical supplies	1100-FCP-03	To hold the wall of the uterus
Balloon catheter	handmade		To occlude the blood flow to the fetal left ventricle
Blant fill needle 18G	Medline	SYR110021Z	To connect V8 PVC tube to infusion catheter or male-male connecters
Blant needle 25G	SAI Infusion Technologies	B25-50	To connect Gilson PVC tube to infusion catheter or male-male connecters
Blood pressure cuff	Royal Pilips	989803000000	Non-invasive blood pressure monitoring
Buprenorphine	Hospira Pharmaceuticals	Pharmacy	postoperative pain control: concentration 0.3 mg/mL, dose 0.03 mg/kg
Cautery pencil	Medline	ESRK3002L	For cut and dissection using electrocautery
Cefazolin	Hospira Pharmaceuticals	Pharmacy	Antibiotic prophylaxis
Centurion Heplock injection site adapters	Medline	HL50LT100	To cap the pressure lines and for injection or blood drawing
Chloraprep	BD	930825	Topical antiseptic
Ciprofloxacin	Hospira Pharmaceuticals	Pharmacy	Antibiotic prophylaxis
Cooley bulldog vessel clamp	NAZMED SMS SDN BHD	SMS-05-7237	For clamping the fetal left atrium
DELTA-CAL transducer simulator and tester	ADInstruments	MLA6595	Pressure transducer simulater/tester
ECG leads	3M	2570	ECG monitoring
Endotracheal tube, size 8-9	Covidien	86452, 86114, or 86454	To secure airway
Flow measurement unit	Transonic System Inc. (ADInstruments)	TS420	For blood flow measurement
Gilson pump	Gilson. Inc	F155006	For precise continuous infusion of small amount of fluid for fetuses
Heparin	Hospira Pharmaceuticals	Pharmacy	Anticoagulant: 1,000 USP units/mL
Isoflurane	Baxter Healthcare Corporation	Pharmacy	Anesthetic: dose 1-5%
Laparotomy drape with pouches	Medline	AAMI PB70	Sterile drape
M/M adapter	Medline	DYNJADAPMM	Male-male adapter for pressure catheters
Orogastric tube	Jorgensen Lab, Inc.	J0348R	For stomach and rumen decompression
Plastic Occluding Tube Clamp (Hemostat)	Cardinal Health	X0029XO5ET	For tube clamp of the pressure catheters and balloon catheter
Potassium chloride	Hospira Pharmaceuticals	Pharmacy	To arrest the fetal heart in diastole
PowerLab system (instrument interface, quad bridge AMP)	ADInstruments	FE224, PLCF1/4	Recording of the pressures, blood flow, heart rate
Probe cover kit	Medline	DYNJE5930	Sterile cover of the echocardiographic probe
Propofol	Fresenius Kabi	Pharmacy	Anesthetic: concentration 10 mg/mL, dose 20-45 mg/kg/hr
Pulse oximeter lingual clip	Nellcor	PO736	For pulse oxmetry monitoring
PVC tubing for Gilson pump (Orange/Green)	Gilson. Inc	F117933	For Gilson pump tubing
Rib spreader for newborn	Surgical Republic	550-020-030	For fetal thoracotomy
Scalpel #10 blade	Bard-Parker	371610	For skin incisions of the ewe
Sodium pentobarbital	Hospira Pharmaceuticals	Pharmacy	For an ewe's euthanasia
Tunnelling device (metal round tube)	Everbilt	N/A	To exteriorize the cables and catheters to the ewe's flank
Umbilical tape	McKesson	894782	To ligate the umbilical cord at necropsy
V8 PVC tube (micro medical tubing)	Scientific Commodities, Inc.	BB31785-V/8	Fluid-filled catheter for pressure monitoring of the fetus
Vecuronium	Hospira Pharmaceuticals	Pharmacy	To prevent fetal movement during surgery
Vein pick	Fisher Scientific	50-19506098	To insert pressure lines into vessels
Vessel loop	Medline	DYNJVL03	To hold ascending aorta and main pulmonary artery
Warming blanket	Jorgensen Lab, Inc.	J1034B	To maintain animal's body tempreture