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

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 Ta....

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 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