This work is supported by a Hamilton-Fairley NHMRC Fellowship (RH, AL); the Victorian Government's Operational Infrastructure Support Program (RH, EW) and the Marie Curie Industria-Academia Partnership and Pathways grant sponsored by the European Commission (ME, PD). The authors would like to thank Dr. Andre Miyague, Dr. Francesca Russo, Ms. Rosita Kinnart and Mr. Ivan Laermans for their technical expertise in producing this video.

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)
<ol>
	<li>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 operating theatre for surgical creation of IUGR. Prior to surgery, randomly allocate a "case" (IUGR) and "control" uterine horn in the bicornuate rabbit uterus by using randomization software (GraphPad Software, USA)19. Sample size should be determined by power calculations by individual researchers for their question of interest.</li>
	<li>Anesthesia Procedure. 
	Induction anesthesia is achieved by administering ketamine 35 mg/kg (Ceva Sante Animale, Brussels, Belgium) and xylazine 5 mg/kg (Ceva Sante Animale, Brussels, Belgium) intramuscularly in the hind leg. Anesthesia is then maintained with an inhalational mixture of 1-4% isoflurane (Isoba Vet; Abbott Laboratories Ltd., Queenborough, Kent, UK) in O2 at 2 L/min. Place a pulse oximeter (Nellcor N-20, Covidien, Ireland) on the maternal second and third toe to continuously monitor heart rate and arterial O2 saturation.</li>
	<li>Surgical Procedure.
	<ol>
		<li>Premedication. Prior to commencing surgery, administer antibiotic prophylaxis Penicillin G, 300,000 IU (Kela Pharma, Hoogstraten, Belgium), uterine tocolytic medroxyprogesterone acetate 0.9 mg/kg (Depo-Provera, Pharmacia Upjohn, Puurs, Belgium) and analgesic buprenorphine 0.03 mg/kg (Temgesic; Schering-Plough) subcutaneously in a single 2.5 ml syringe.</li>
		<li>Rabbit preparation. Place the rabbit on a warming pad for the duration of the surgery. Remove hair from the abdominal surgical field and apply antiseptic Povidone-iodine 7.5% (Betadine, Purdue, Connecticut, USA). A complete aseptic protocol is then employed.</li>
		<li>Abdominal laparotomy. Infiltrate the incision site with local anesthetic (2 ml 1% lidocaine, B Braun Medical, Belgium) subcutaneously. The incision site should be approximately 5 cm in length in the midline, in the lower 2/3 of the abdomen. Once the skin is opened, use a mixture of sharp and blunt dissection to expose the rectus sheath. Care must be taken to avoid the vascular maternal mammary glands located either side of the midline on entry (Figure 1A). Grasp the rectus sheath and peritoneum, tent to prevent inadvertent inclusion of abdominal contents, and carefully enter using sharp dissection (Figure 1B).</li>
		<li>Uterine exteriorization. Surround the surgical field with gauze drapes soaked in warmed saline. Carefully grasp and exteriorize both sides of the bicornuate uterus. Care must be taken not to place either the vaginal or ovarian end of each uterine horn under tension, as this may result in unwelcome bleeding, pain and uterine contractility. The assistant continuously irrigates the exposed uterus with warmed saline to prevent uterine contractions. Palpate and record the number of fetuses present in each horn. By convention, the fetus at the ovarian end is designated the first fetus. One uterine horn is the case horn and the other is the control horn. Identify the previously randomly allocated case horn for IUGR induction and then replace the control horn back into the abdomen (internal control fetuses).</li>
		<li>Surgically induced IUGR. Identify the uteroplacental vessels to each gestational sac in the designated case horn (Figure 1C). Surgically ligate 40-50% of these vessels using a 5-0 polyglactin suture (Ethicon Vicryl, Johnson and Johnson Medical, USA) (Figures 1D-1E). In practice, there are often three vascular bundles supplying each gestational sac. In this case, the largest vascular bundle is ligated. Once complete, the uterus is again irrigated with warmed saline and carefully returned to the abdomen.</li>
		<li>Abdominal closure. Close the abdomen in two layers using a continuous running 2-0 polyglactin suture (Ethicon Vicryl, Johnson and Johnson Medical, USA). Close the skin with subcuticular 3-0 polyglactin suture (Ethicon Vicryl, Johnson and Johnson Medical, USA). Spray aluminum aerosol onto the wound to prevent the rabbit from later interfering with the sutures.</li>
		<li>Recovery. Cease anesthesia and closely observe recovery of respiration. The rabbit is then housed as described above with access to food and water ad libitum and observed daily.</li>
	</ol>
	</li>
</ol>
2. Performing Fetal Echocardiography and Pulsed-wave Doppler Ultrasound
<ol>
	<li>The same rabbit is then transferred at GA 30 days for echocardiographic and ultrasound evaluation. The rabbit is placed on a warming pad for the duration of the examination.</li>
	<li>Anesthesia Procedure.
	<ol>
		<li>Induction anesthesia is achieved by administering ketamine 35 mg/kg (Ceva Sante Animale, Brussels, Belgium) and xylazine 5 mg/kg (Ceva Sante Animale, Brussels, Belgium) intramuscularly. Once sedate, obtain intravenous access using a 24 Gauge cannula (BD Insyte-W, Becton Dickinson Infusion Therapy Systems, Utah, USA) inserted into a peripheral auricular vein.</li>
		<li>Maintain anesthesia with a continuous infusion of ketamine and xylazine: 2 ml ketamine (100 mg/ml) and 1.5 ml xylazine (20 mg/ml) to 46.5 ml of normal saline, then infused via a syringe pump at 40 ml/hr. Secure a three-way-tap and attach a 5 ml syringe of rescue anesthesia: 2 ml ketamine (100 mg/ml) and 1.5 ml xylazine (20 mg/ml). Boluses of 0.5 ml rescue anesthesia are rarely required. Administer analgesic buprenorphine 0.03 mg/kg (Temgesic; Schering-Plough) subcutaneouslyprior to surgery. Oxygen 2 L/min is applied by mask. Place a pulse oximeter (Nellcor N-20, Covidien, Ireland) on the maternal second and third toe to continuously monitor heart rate and arterial O2 saturation.</li>
	</ol>
	</li>
	<li>Abdominal laparotomy and uterine exteriorization. Refer to 1.3.3 and 1.3.4.</li>
	<li>Fetal positioning for echocardiographic and ultrasound examination.
	<ol>
		<li>Place the rabbit in a lateral position facing towards the researcher. This facilitates easier retrieval of a single gestational sac for examination and prevents excessive traction on uteroplacental vessels. As much as possible, try to exteriorize only one gestational sac at a time to minimize exposure. Ensure the exposed gestational sac is placed on gauze irrigated with warmed saline. To ensure standardized results between different study groups, generally only the fetus at the ovarian and vaginal ends of each uterine horn are used in the ultrasound examination.</li>
		<li>Fetal cardiac function is very susceptible to the effect of uterine exteriorization, change in temperature and pressure from the ultrasound transducer over the placental vessels. To minimize these environmental and iatrogenic confounding effects on cardiac function, the ultrasound examination must begin immediately after exteriorization of the gestational sac and should be systematic and efficient. In our experience, changes in fetal cardiac function seldom occur in the first 5 min of exposure. If fetal bradycardia is encountered, remove the ultrasound probe, reposition the fetus to prevent kinking of fetoplacental vessels, irrigate with warmed saline or briefly return the gestational sac back into the maternal abdomen. If fetal bradycardia is persistent, the examination for this fetus should be abandoned in favor of examining the next fetus.</li>
	</ol>
	</li>
	<li>Fetal echocardiography.
	<ol>
		<li>The Visualsonics VEVO 2100 (Toronto, Canada) high-resolution microultrasound platform with cardiac and strain analysis software installed is required. A VisualSonics MS-250 transducer (VisualSonics, Toronto, Canada) is used for data acquisition. This has a center frequency of 21 MHz with a bandwidth 13-24 MHz. The geometric focus is 15 mm, the maximum image width is 23 mm and the maximum image depth is 30 mm. The footprint is 28 x 5.75 mm. Data is acquired in accordance with the American Society of Echocardiography guidelines and standards for performance of the fetal echocardiogram.20</li>
		<li>Data acquisition 1: lateral view. At the level of the lateral four-chamber-view, record a B-mode cine loop of at least five cardiac cycles to determine strain, strain rate, velocity and displacement. This is analyzed offline using the VevoStrain (Visualsonics, Toronto, Canada) algorithm as previously described in the journal in detail.21 M-mode echocardiography indices can then be obtained in this standard view. Offline measurements of both cardiac structure and function can then be analyzed: internal ventricular and septal diameters in systole and diastole, volumes, stroke volume, fractional shortening, ejection fraction and cardiac output.21</li>
		<li>Data acquisition 2: apical view. At the level of the apical four-chamber-view, B-mode cine loop can be repeated for VevoStrain analyses as described above. M-mode echocardiography is used to assess tricuspid (TAPSE) and mitral annular plane systolic excursion (MAPSE) and longitudinal strain and strain rate.22,23 This apical view also permits assessment of valvular flow velocity waveforms and the modified myocardial performance index as previously described.24</li>
	</ol>
	</li>
	<li>Fetoplacental pulse-waved Doppler ultrasound.</li>
</ol>
Flow velocity waveforms (FVW) of the umbilical artery are obtained by locating the umbilical vessels using color Doppler and then placing the pulsed Doppler sample gate over the umbilical artery on a free loop of umbilical cord.25 The middle cerebral artery (MCA) FVW is located by placing the pulsed Doppler sample gate just beyond the origin of MCA once the Circle of Willis is located by color Doppler.26 The ductus venosus (DV) FVW is obtained by placing the pulsed Doppler sample gate at the proximal portion of the DV seen with color Doppler where it originates from the intrahepatic umbilical vein either in a sagittal or transverse view of the fetus.26 The pulsatility index (PI) is calculated offline using the VisualSonics cardiovascular analysis software.

The author Professor Jan D'hooge has a research agreement with VisualSonics. The remaining authors have no disclosures.

We have used a previously validated approach of surgically reducing uteroplacental blood flow in a rabbit to produce IUGR13-16 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...

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 childhood3,4 and subsequent hypertension5, diabetes6, and obesity developing in adult life - all cumulative cardiac risk factors from birth towards premature death from ischemic heart disease.7
Developing animal models to characterize the maternal-fetal communication that establishes IUGR and the subsequent fetal response to reduced substrate availability is necessary if we are to both better understand the pathophysiology of IUGR-related cardiac impairment and to develop cardio-protective strategies to improve fetal and postnatal health. In this regard, many different models across different species have been described.8 IUGR is commonly induced by maternal under nutrition with a low protein diet in rodents,9 surgical ablation or ligation of uterine blood flow in rodents and guinea pigs10 or occlusion of the umbilical artery in sheep.11 However, it is apparent that no model fully recapitulates the human IUGR.12
In this current methodological study, we used a well validated approach of selective uteroplacental vascular interruption in a rabbit13-16 that not only produces fetal cardiovascular responses observed with ultrasound clinically14, but also allows interrogation of fetal cardiac function with novel echocardiography using microultrasound technology of the VisualSonics VEVO 2100. While Doppler ultrasound of fetoplacental vessels remains the cornerstone of current antenatal surveillance of IUGR fetuses17, functional echocardiography is increasingly being utilized to provide new insights into disease pathophysiology and to assess fetal wellbeing.18 Accordingly, here we take these advances from clinical research and describe an animal model that harbors not only this imaging sophistication but also provides the experimental platform to investigate mechanistic pathways and novel therapeutics.

<table border="1">
	<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Ceva Sante Animale</td>
 <td></td>
 <td><a href="http://www.ceva.com/en" target="_blank">http://www.ceva.com/en</a></td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Ceva Sante Animale</td>
 <td></td>
 <td><a href="http://www.ceva.com/en" target="_blank">http://www.ceva.com/en</a></td>
 </tr>
 <tr>
 <td>Depot Provera</td>
 <td>Pharmacia Upjohn</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin G</td>
 <td>Kela Pharma</td>
 <td></td>
 <td><a href="http://www.kela.be" target="_blank">http://www.kela.be</a></td>
 </tr>
 <tr>
 <td >Lidocaine</td>
 <td>B Braun Medical</td>
 <td></td>
 <td><a href="http://www.bbraun.com/" target="_blank">http://www.bbraun.com/</a></td>
 </tr>
 <tr>
 <td>Temgesic</td>
 <td>Schering-Plough</td>
 <td></td>
 <td><a href="http://www.merck-animal-health-usa.com/" target="_blank">http://www.merck-animal-health-usa.com/</a></td>
 </tr>
 <tr>
 <td>Isolurane</td>
 <td>Isoba Vet; Abbott Laboratories Ltd</td>
 <td></td>
 <td><a href="http://www.abbottanimalhealth.com/index.html" target="_blank">http://www.abbottanimalhealth.com/index.html</a></td>
 </tr>
 <tr>
 <td>Ethicon</td>
 <td>Johnson and Johnson</td>
 <td></td>
 <td><a href="http://www.ethiconproducts.co.uk/products/sutures" target="_blank">http://www.ethiconproducts.co.uk/products/sutures</a></td>
 </tr>
 <tr>
 <td>Ethicon</td>
 <td>Johnson and Johnson</td>
 <td></td>
 <td><a href="http://www.ethiconproducts.co.uk/products/sutures" target="_blank">http://www.ethiconproducts.co.uk/products/sutures</a></td>
 </tr>
 <tr>
 <td>Ethicon</td>
 <td>Johnson and Johnson</td>
 <td></td>
 <td><a href="http://www.ethiconproducts.co.uk/products/sutures" target="_blank">http://www.ethiconproducts.co.uk/products/sutures</a></td>
 </tr>
 <tr>
 <td>VEVO 2100</td>
 <td>VisualSonics</td>
 <td>SN100-0032</td>
 <td><a href="http://www.visualsonics.com/" target="_blank">http://www.visualsonics.com/</a></td>
 </tr>
 <tr>
 <td>Aquasonic Gel</td>
 <td>Parker Laboratories</td>
 <td>01 02</td>
 <td><a href="http://www.parkerlabs.com/ultrasound_products.html" target="_blank">http://www.parkerlabs.com/ultrasound_products.html</a></td>
 </tr>
 <tr>
 <td>Nellcor N-20PA Pulse oximeter</td>
 <td>Covidien</td>
 <td></td>
 <td><a href="http://www.nellcor.com/prod/PRODUCT.ASPX?S1=POX&amp;S2=MON&amp;id=282&amp;V" target="_blank">http://www.nellcor.com/prod/PRODUCT.ASPX?S1=POX&amp;S2=MON&amp;id=282&amp;V</a></td>
 </tr>
	 </tbody>
 </table>

fetal echocardiography and pulsed-wave doppler ultrasound in a rabbit model of intrauterine growth restriction

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.

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...

Watch this Scientific Journal Video about Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction at JoVE.com

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

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.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

fetal-echocardiography-pulsed-wave-doppler-ultrasound-rabbit-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

28.1K Views.  University Hospitals Leuven. 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.

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

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

Murine Echocardiography and Ultrasound Imaging

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1 Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal.2 However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals.3 Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound.4,5 Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function.6 This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment.

This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1  ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

Model Surgical Training: Skills Acquisition in Fetoscopic Laser Photocoagulation of Monochorionic Diamniotic Twin Placenta Using Realistic Simulators

Fetoscopic laser coagulation of arterio-venous anastomoses (AVA) in a monochorionic placenta is the standard of care for twin-twin transfusion syndrome (TTTS), but is technically challenging and can lead to significant complications. Acquiring and maintaining the necessary surgical skills require consistent practice, a critical caseload, and time. Training on realistic surgical simulators can potentially shorten this steep learning curve and enables several proceduralists to acquire procedure-specific skills simultaneously. Here we describe realistic simulators designed to allow the user familiarity with the equipment and specific steps required in the surgical treatment of TTTS, including fetoscopic handling, approaches to anterior and posterior placenta, recognition of anastomoses, and efficient coagulation of vessels. We describe the skills that are especially important in conducting placental laser coagulation that the surgeon can practice on the model and apply in a clinical case. These models can be adapted easily depending on the availability of materials and require standard fetoscopy equipment. Such training systems are complementary to traditional surgical apprenticeships and can be useful aids for fetal medicine units that provide this clinical service.

Practicing the specific skills required for fetoscopic laser coagulation of monochorionic placental anastomoses on realistic models can aid less experienced surgeons in overcoming the steep learning curve associated with this procedure that is now regarded as the standard of care for twin-twin transfusion syndrome.

Fetoscopic laser coagulation of arterio-venous anastomoses (AVA) in a monochorionic placenta is the standard of care for twin-twin transfusion syndrome ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

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

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Chapters in this video

Transthoracic Echocardiography in Mice

Murine Echocardiography and Ultrasound Imaging

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Murine Fetal Echocardiography

A Mouse Fetal Skin Model of Scarless Wound Repair

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Model Surgical Training: Skills Acquisition in Fetoscopic Laser Photocoagulation of Monochorionic Diamniotic Twin Placenta Using Realistic Simulators

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica