The use of mice for these studies is necessary as mice have four-chambered hearts that can mimic human CHDs. Mice were provided veterinary care and housed in the institution&#39;s Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited animal care facility. Strict protocols were followed to minimize the mice&#39;s discomfort, stress, pain, and injury. Mice were euthanized using CO2 gas, which is acceptable for small rodents according to the American Veterinary Medical Associa...

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	<li>Wu, W., He, J., Shao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+and+mortality+trend+of+congenital+heart+disease+at+the+global,+regional,+and+national+level,+1990-2017.">Incidence and mortality trend of congenital heart disease at the global, regional, and national level, 1990-2017.</a> Medicine. 99 (23), e20593 (2020).</li><li>vander Linde, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+prevalence+of+congenital+heart+disease+worldwide:+a+systematic+review+and+meta-analysis).">Birth prevalence of congenital heart disease worldwide: a systematic review and meta-analysis).</a> Journal of the American College of Cardiology. 58 (21), 2241-2247 (2011).</li><li>Yang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Racial+differences+in+infant+mortality+attributable+to+birth+defects+in+the+United+States.">Racial differences in infant mortality attributable to birth defects in the United States.</a> Birth Defects Research. 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Cardiovascular Imaging. 7 (1), 31-42 (2014).</li><li>Liu, X., Tobita, K., Francis, R. J., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+techniques+for+visualizing+and+phenotyping+congenital+heart+defects+in+murine+models.">Imaging techniques for visualizing and phenotyping congenital heart defects in murine models.</a> Birth Defects Research. Part C, Embryo Today: Review. 99 (2), 93-105 (2013).</li><li>Tsuchiya, M., Yamada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+histological+3D-imaging:+episcopic+fluorescence+image+capture+is+widely+applied+for+experimental+animals.">High-resolution histological 3D-imaging: episcopic fluorescence image capture is widely applied for experimental animals.</a> Congenital Anomalies (Kyoto. 54 (4), 250-251 (2014).</li><li>Yu, Q., Tian Leatherbury, ., Lo, X., W, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+assessment+of+fetal+mice+by+in+utero+echocardiography).">Cardiovascular assessment of fetal mice by in utero echocardiography).</a> Ultrasound in Medicine and Biology. 34, 741-752 (2008).</li><li>Rosenthal, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+high+resolution+three-dimensional+reconstruction+of+embryos+with+episcopic+fluorescence+image+capture.">Rapid high resolution three-dimensional reconstruction of embryos with episcopic fluorescence image capture.</a> Birth Defects Research. Part C, Embryo Today: Review. 72 (3), 213-223 (2004).</li><li>Weninger, W. J., Mohun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotyping+transgenic+embryos:+a+rapid+3-D+screening+method+based+on+episcopic+fluorescence+image+capturing.">Phenotyping transgenic embryos: a rapid 3-D screening method based on episcopic fluorescence image capturing.</a> Nature Genetics. 30 (1), 59-65 (2002).</li></ol>

The authors declare no conflicts of interest in this manuscript.

Genetically modified mice have been used to understand the pathomechanisms of congenital heart defects. The protocols we provide in this study attempt to streamline and standardize the process of assessing murine fetal heart defects. However, there are critical steps to note during the protocol. Mouse embryos grow significantly during each day of gestation, and the correct time to harvest a mouse can be determined by performing a fetal echocardiogram accurately. The fetal echocardiogram can be used to screen the fetal ca...

Congenital heart diseases (CHDs) are the most common neonatal birth defect1,2, affecting about 0.8%-1.7% of neonates and resulting in significant neonatal mortality and morbidity3. A genetic etiology is strongly indicated with CHDs4,5. Genetically modified mouse models have been used widely to understand the complexity of CHDs and the mechanisms that cause them due to the mice having four-chamber hearts and comparable cardiac developmental DNA sequences in mouse and human fetuses6. Identifying the phenotype of the mouse mutants is the fundamental first step in characterizing the function of the targeted gene. Mouse models expressing gene dosage effects, in which a single genetic mutation can result in a spectrum of cardiac defects that mimic human CHDs, are important for understanding the complexity of CHDs and the mechanisms that cause them.
This article outlines a pipeline to characterize cardiac phenotypes in mouse models. The applied methods utilize fetal echocardiogram7, followed by necropsy and ECM histopathology7,8, which can display the detailed anatomy of developing murine cardiac phenotypes. A fetal echocardiogram is a noninvasive modality that allows direct visualization of multiple embryos with reasonable imaging resolution. In addition, a fetal echocardiogram provides a quick determination of the total number of embryos in a litter, their developing stages, and the relative orientation and location in the uterine horn. Using a spectral Doppler/color flow, abnormal embryos can be identified based on the structure, the hemodynamic disturbance, the growth restriction, or the development of&#160;hydrops. Since a fetal echocardiogram study is a noninvasive technique, it can be used to scan on multiple days and to observe the changes in hemodynamics or cardiac morphology. Obtaining high-quality imaging of fetal echocardiograms requires practice and skill, as specific heart defects may be missed due to a lack of experience and knowledge. Because of this, a more definitive analysis of cardiac morphology may be obtained through a combination of necropsy and ECM histopathology. Necropsy provides direct visualization of the arch structure, the relative relationships of the aorta and pulmonary artery, the size of the ventricles and atria, the position of the heart relative to the chest, and the bronchopulmonary structures. However, interior features such as the heart valves and wall thickness may be difficult to assess through necropsy alone. Thus, ECM histopathology is recommended for a conclusive diagnosis. ECM histopathology is a high-resolution visualization technique that allows for both 2D and 3D reconstruction of the image stack9. These images are obtained through serial Episcopic fluorescent imaging of a paraffin-embedded sample as it is thinly sectioned at a consistent interval by an automatic microtome. Unlike classical histology, images are captured as a section before it is cut from the block such that all images are captured within the same reference frame. Because of this, the 2D image stack produced by ECM histopathology may easily and reliably be reconstructed in three dimensions. This is done using a DICOM viewer, which allows 3D visualization of the images in the three anatomical planes: coronal, sagittal, and transverse. From these high-resolution 3D reconstructions, a definitive cardiac diagnosis may be made. The application of these three different visualization modalities, either individually or in combination, can provide accurate characterizations of structural heart defects in mouse embryos.

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			<td>1x phosphate-buffered saline solution (PBS), PH7.4</td>
			<td>Sigma Aldrich</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes (or preferred vial for tissue storage)</td>
			<td>SealRite</td>
			<td>1615-5599</td>
			<td></td>
		</tr>
		<tr>
			<td>10% buffered formalin phosphate solution</td>
			<td>Fisher Chemical</td>
			<td>SF100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>16% paraformaldehyde (PFA) fixative&#160;</td>
			<td>ThermoScientific</td>
			<td>28908</td>
			<td>4% working concentration freshly prepared in 1x PBS at 4 &#176;C</td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>6&#8211;12 Well plate or 20 mL vial&#160; for embryo storage</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope&#160;</td>
			<td>Leica</td>
			<td>MDG36</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Pins (A1 or A2 grade)</td>
			<td>F.S.T</td>
			<td>26002-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Plate&#160;</td>
			<td>F.S.T</td>
			<td>FB0875713</td>
			<td>Petri dish with paraffin base</td>
		</tr>
		<tr>
			<td>Embedding molds</td>
			<td>Sakura</td>
			<td>4133</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra narrow scissors (10.5 cm)</td>
			<td>F.S.T</td>
			<td>14088-10</td>
			<td>1&#8211;2 pairs&#160;</td>
		</tr>
		<tr>
			<td>Fiji application/Image J</td>
			<td>NIH</td>
			<td></td>
			<td>Fiji.sc</td>
		</tr>
		<tr>
			<td>Fine tip (0.05 mm x 0.01 mm) Dissecting Forceps (11 cm)</td>
			<td>F.S.T</td>
			<td>11252-00</td>
			<td>2 Pairs</td>
		</tr>
		<tr>
			<td>Hot forceps&#160;</td>
			<td>F.S.T</td>
			<td>11252-00</td>
			<td>For orientation of embryos</td>
		</tr>
		<tr>
			<td>Industrial Marker for Wax Blocks&#160;</td>
			<td>Sharpie</td>
			<td>2003898</td>
			<td>Formatted for labratory use</td>
		</tr>
		<tr>
			<td>Jenoptik ProgRes C14plus Microscope Camera&#160;</td>
			<td>Jenoptik</td>
			<td>017953-650-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Jenoptik ProgRess CapturePro acquisition software</td>
			<td>Jenoptik</td>
			<td></td>
			<td>jenoptik.com</td>
		</tr>
		<tr>
			<td>Large glass beaker&#160;</td>
			<td>Fisher Scientific</td>
			<td>S111053</td>
			<td>For melting paraffin</td>
		</tr>
		<tr>
			<td>Leica M165 FC binocular microscope (16.5:1 zoom optics)</td>
			<td>Leica</td>
			<td>M165 FC</td>
			<td></td>
		</tr>
		<tr>
			<td>OsiriX MD Version 12.0</td>
			<td>OsiriX</td>
			<td></td>
			<td>osirix-viewer.com&#160;</td>
		</tr>
		<tr>
			<td>Paraplast embedding paraffin wax</td>
			<td>Millipore Sigma</td>
			<td>1003230215</td>
			<td></td>
		</tr>
		<tr>
			<td>Small glass beaker</td>
			<td>Fisher Scientific</td>
			<td>S111045</td>
			<td></td>
		</tr>
		<tr>
			<td>Small, perforated spoon (14.5 cm)</td>
			<td>F.S.T</td>
			<td>10370-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Vannas Scissors (4&#8211;8 mm)</td>
			<td>F.S.T</td>
			<td>15018-10</td>
			<td>A pair</td>
		</tr>
		<tr>
			<td>Vevo2100 ultrahigh-frequency ultrasound biomicroscope</td>
			<td>FUJIFILM VisualSonics Inc.</td>
			<td>Vevo2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Chemical</td>
			<td>UN1307</td>
			<td></td>
		</tr>
	</tbody>
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a pipeline to characterize structural heart defects in the fetal mouse

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe CHD died before adulthood, with the maximum mortality during the first week of life. Remarkable advances in surgical techniques, diagnostic approaches, and medical management have led to marked improvements in outcomes. To address the critical research needs of understanding congenital heart defects, murine models have provided an ideal research platform, as they have very similar heart anatomy to humans and short gestation rates. The combination of genetic engineering with high-throughput phenotyping tools has allowed for the replication and diagnosis of structural heart defects to further elucidate the molecular pathways behind CHDs. The use of noninvasive fetal echocardiography to screen the cardiac phenotypes in mouse models coupled with the high fidelity of Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) histopathology with three-dimensional (3D) reconstructions enables a detailed view into the anatomy of various congenital heart defects. This protocol outlines a complete workflow of these methods to obtain an accurate diagnosis of murine congenital heart defects. Applying this phenotyping protocol to model organisms will allow for accurate CHD diagnosis, yielding insights into the mechanisms of CHD. Identifying the underlying mechanisms of CHD provide opportunities for potential therapies and interventions.

The mouse embryos with significant hemodynamic defects were noted to be embryonic lethal. A wide variety of CHDs can be identified through the high output, noninvasive fetal echocardiogram using different views (Figure 1).
Septal defects: The most common CHDs are septal defects such as a ventricular septal defect (VSD), an atrioventricular septal defect (AVSD), and an atrial septal defect (ASD)1. VSD or AVSD can be easily v...

Watch this Scientific Journal Video about A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse at JoVE.com

A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse

This article details murine congenital heart disease (CHD) diagnostic methods using fetal echocardiography, necropsy, and Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) followed by three-dimensional (3D) reconstruction.

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe ...

This protocol outlines cutting edge methods for generating high resolution imaging data for diagnosing congenital heart defects. We conduct functional assessments with non-invasive fetal echocardiography combined with necropsy followed by episcopal confocal microscopy histopathology to generate serial 2D image stacks and 3D reconstructions for comprehensive CHD diagnosis. This method can elucidate the detailed anatomical changes not only in congenital heart diseases, but also can be used to investigate structural birth defects in other organs including cranial facial defects, limb and skeletal anomalies, as well as defects of the brain and other visceral organs.Demonstrating these procedures will be Meghan Holbrook, Carla Guzman, Peizhao Zhang, and Benjamin Glennon, all researchers from our laboratory. After preparing the mouse for the procedure, warm up the ultrasound gel to a body temperature and apply it generously over the imaging area. Then place the transducer on the abdomen oriented in a horizontal plane and identify the bladder on the screen.Once the bladder is identified, scan cranially from the bladder, look for the fetus, and determine the gestational age by measuring the crown to rump length. Next, use the color doppler to analyze blood flow from the heart. To allow fixative penetration into the internal organs, make incisions in the lateral thorax and abdomen using forceps or dissecting scissors and allow the sample to fix for at least 24 hours in a cold room.Then set up the software to save the images with a name, including the sample's identification, the microscope magnification, and the content of the picture. Pin the sample through its wrists and ankles. Before cutting the skin along the median axis towards the tail, lift the skin in the middle of the neck using forceps and cut the skin toward both the armpits with the scissors.Then cut the skin from the umbilicus to the legs. After embedding the samples, adjust the laser projection position to the center of the specimen on the screen and adjust the zoom knob to 20x. To optimize the resolution, set the gain to 1, 250V.Maximize the blue area using the focus knob, and then reset the gain to approximately 750V for imaging. Next, switch the cutting method to auto. Set the thickness to around 50 micrometers and run the slides.Stop cutting when the lungs and airway are in view. Choose a cutting thickness between eight to 10 micrometers and stop the live view open. Open Microtome Communicator to start imaging.Ensure the temporary storage folder is empty before collecting images. Stop the image collection when no hard structure is visible. Output the temporary stored files into one TIFF image series.For 3D image reconstruction, drag and drop the image files into the image processing software. Once the pop-up window appears, select the links or the files to copy into the database. Once the file is opened, click on the 2D 3D reconstruction tools menu from the toolbar and select 3D MPR.For pixel X resolution, and pixel Y resolution, input the image resolution by giving the zoom used during ECM imaging. For the slice interval, input the slice thickness used to cut during ECM imaging. Next, use the WWWL tool to adjust the window width and window level.Click and drag the tool on the image upward to decrease image brightness and downward to increase it. Click and drag the tool on the image rightward to decrease image contrast and left word to increase it. Drag the images into the desired positions using the pan tool.Use the zoom tool to enlarge or shrink the image and the rotate tool to rotate the image as desired. Now, click and drag the colored axis of the first panel. Notice how rotating this axis changes the orientation of the other two panels.Rotate the three panels axes until the three panels represent coronal, sagittal, and transverse views of the sample. Once all three panels are appropriately positioned, oriented, and brightened, click on the panel representing the coronal view. Then click on Movie Export on the right side of the menu bar.Click on batch and drag the from and to sliders to encompass the entire region of interest. For interval, select the same as thickness option and save the video indicating the views orientation. The echo shows an intact ventricular septum without an intraventricular shunt and normal related great arteries in the normal control, which was confirmed by ECM.The frontal view demonstrates communication between LA, RA, LV, and RV in both echo and ECM, suggesting atrioventricular septal defect. The color flow demonstrates the flow across LV and RV indicating ventricular septal defect. Echo and ECM demonstrated a double outlet of right ventricle with a ventricular septal defect between LV and RV with side by side great arteries.Ultrasound diagnosis of persistent truncus arteriosus demonstrates communication between LV and RV with a single outflow tract overriding both ventricles, also confirmed by ECM at the E14.5 stage. The sections collected from ECM histology can be further processed for immunostaining, hematoxylin and eosin staining and even nucleic acid extraction for genotyping and transcriptional profiling or protein extraction for proteomic analysis. The images from three-dimensional reconstruction can be further processed for assessments of quantitative perimeters such as area or volume.Accurate phenotyping to identify defects in the cardiovascular anatomy is essential for establishing genetically engineered and mutant mouse models of congenital heart disease to elucidate the pathomechanisms of congenital heart disease. This can lead to potential opportunities that develop therapies and intervention to improve the clinical outcome in patients with congenital heart disease.

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1.7K Views.  University of Pittsburgh. This protocol outlines cutting edge methods for generating high resolution imaging data for diagnosing congenital heart defects. We conduct functional assessments with non-invasive fetal echocardiography combined with necropsy followed by episcopal confocal microscopy histopathology to generate serial 2D image stacks and 3D reconstructions for comprehensive CHD diagnosis. This method can elucidate the detailed anatomical changes not only in congenital heart diseases, but also can be used to i...