Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Summary

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Abstract

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

Introduction

The burden of cardiovascular disease that results from fetal intrauterine growth restriction (IUGR) cannot be overstated. It is the leading cause of stillbirth after congenital abnormalities.¹ IUGR refers to a fetus that fails to reach its growth potential and is commonly a result of placental insufficiency.² In survivors, cardiovascular ill health is evident across the life span with myocardial dysfunction apparent in infancy and childhood^3,4 and subsequent hypertension⁵, diabetes⁶, and obesity developing in adult life - all cumulative cardiac risk factors from birth towards premature death from ischemic heart di....

Protocol

The following experimental protocol is approved by the Animal Ethics Committee, Katholieke Universiteit Leuven, Leuven, Belgium. We followed previously described surgical procedure¹³ including some methodological changes, especially in anesthesia procedure.

1. Inducing Intrauterine Growth Restriction (IUGR)

1. Time dated pregnant rabbits (hybrid Dendermonde and New Zealand White) are housed in individual cages on a 12/12 hr light schedule with access to water and standard rabbit chow ad libitum from gestational age (GA) 18 days. At GA 25 days (full term is 31 days approximately), rabbits are transferred to the o....

Results

An asymmetrical growth restricted fetus and placenta from uteroplacental vascular ligation is compared to a normal control fetus and placenta in Figure 1F. Asymmetrical growth is confirmed by reduced neonatal birth weight and increased head circumference:abdominal circumference ratio to controls. Representative results from fetoplacental Doppler studies are shown in Figure 2. A normal low resistance pattern of positive end-diastolic flow in a control fetus is shown. With progressive incr.......

Discussion

We have used a previously validated approach of surgically reducing uteroplacental blood flow in a rabbit to produce IUGR^13-16 and later examining fetal cardiac function¹⁴ to describe microultrasound technology and cardiac function analysis available on the VisualSonics VEVO 2100. The ability to reproduce fetoplacental Doppler changes of human IUGR fetuses in a small animal model and to then allow examination of cardiac function with recently described echocardiography is likely to progress fetal ca.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ketamine	Ceva Sante Animale		http://www.ceva.com/en
Xylazine	Ceva Sante Animale		http://www.ceva.com/en
Depot Provera	Pharmacia Upjohn
Penicillin G	Kela Pharma		http://www.kela.be
Lidocaine	B Braun Medical		http://www.bbraun.com/
Temgesic	Schering-Plough		http://www.merck-animal-health-usa.com/
Isolurane	Isoba Vet; Abbott Laboratories Ltd		http://www.abbottanimalhealth.com/index.html
Ethicon	Johnson and Johnson		http://www.ethiconproducts.co.uk/products/sutures
Ethicon	Johnson and Johnson		http://www.ethiconproducts.co.uk/products/sutures
Ethicon	Johnson and Johnson		http://www.ethiconproducts.co.uk/products/sutures
VEVO 2100	VisualSonics	SN100-0032	http://www.visualsonics.com/
Aquasonic Gel	Parker Laboratories	01 02	http://www.parkerlabs.com/ultrasound_products.html
Nellcor N-20PA Pulse oximeter	Covidien		http://www.nellcor.com/prod/PRODUCT.ASPX?S1=POX&S2=MON&id=282&V