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 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.
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 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.
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's Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited animal care facility. Strict protocols were followed to minimize the mice's discomfort, stress, pain, and injury. Mice were euthanized using CO2 gas, which is acceptable for small rodents according to the American Veterinary Medical Association Guidelines on Euthanasia. The studies on mice in this manuscript were carried out with an approved IACUC protocol at the University of Pittsburgh.
1. Fetal echocardiogram
NOTE: An echocardiogram is a powerful tool for identifying cardiovascular malformation and extracardiac defects in mice. Due to the small size of the mouse embryos (about 1-2 mm at midgestation, 3.5 mm at birth), ultrahigh-frequency echocardiographic equipment with ultrasound biomicroscopy (UBM) is required. UBM provides different high-frequency (30-50 MHz) probes with a small imaging window (15 mm x 14 mm) that provides the resolution (30 µm axial x 68 µm lateral) to visualize one mouse fetus at a time. A 40 MHz transducer provides high-resolution images to identify cardiovascular phenotypes7.
2. Necropsy
NOTE: Once abnormal cardiac phenotypes are suspected using fetal echocardiography, fetuses are collected and fixed via full-body submersion in the fixative solution: either 10% buffered formalin phosphate or 4% paraformaldehyde (PFA). Inspect the sample's external and internal morphology, looking for macroscopic anatomical abnormalities or malformations.
3. Embedding
4. Episcopic confocal microscopy (ECM)
NOTE: After appropriate embedding, embryos undergo image collection serially via ECM for histopathology analysis. Individual slides can be recovered from the microtome for further studies.
5. Three-dimensional (3D) reconstruction
NOTE: The purpose of 3D reconstruction is to process a 2D image stack from ECM imaging into 3D videos in the coronal, sagittal, and transverse orientations and to use the 3D videos for diagnosis of the structural and anatomical abnormalities in the samples.
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 visualized using 2D images and color flow images. Blood flow across the ventricles or between the atria and the ventricles can be easily identified (Figure 3). ASD is difficult to distinguish with patent foramen ovale in a fetus.
Outflow tract anomalies: As shown in Figure 3, the flow in the main pulmonary artery is across the ascending aorta flow in normal embryos. In embryos with a double outlet of the right ventricle (DORV), both great arteries can be seen arising from the right ventricle. DORV is also associated with VSD (Figure 3D) and can be identified using color flow. However, given the small size of the embryos, DORV sometimes cannot be reliably distinguished from the overriding of the aorta, pulmonary atresia, or persistent truncus arteriosus (PTA). In PTA, only one outflow tract flow can be seen in the fetal echocardiogram (Figure 3E). The detailed arch structure and main pulmonary artery can be observed using necropsy (Figure 2E,F).
Necropsy can quickly diagnose the situs status in the chest and abdomen and the cardiac position relative to the chest (either levocardia (Figure 2C–E) or dextrocardia). Outflow tract structures and the relative size of both atria and ventricles can be easily visualized (Figure 2E,F).
ECM histopathology is the gold standard technique for assessing any structural cardiac anomaly8,11,12. It provides an unparalleled resolution and detail to the structures of embryos. Three-dimensional reconstruction using different planes and views can easily identify the relationship between the great arteries and ventricles (Figure 3) and the defect between the ventricles and atria.
Figure 1: The locations for measuring the crown-rump length in 2D echocardiogram images of embryos to determine the gestational age. 2D echocardiogram images of embryos at (A) E11.5, (B) E12.5, (C,D) E13.5-E14.5. (D) The diseased embryo is smaller than its (C) sibling and is noted to have a "mushy" appearance with hydrops (arrow). (A–C) Live embryos demonstrate distinct organs. Representative VB-mode with color images of E14.5 heart in a coronal 4-chamber view tilted anteriorly to image the (E) outflow tracts demonstrate intact ventricular septum with the normal relationship of great arteries, which is confirmed with ECM in the (E') coronal view. (F) Representative sagittal view of E14.5 heart demonstrates the left ventricular and right ventricular outflow tracts with the ascending aorta (AO) pointed cranially and the pulmonary artery pointed posteriorly (toward the spine), which is confirmed with ECM in the (F') sagittal view. (G) Representative transverse view of the left ventricle (LV) and right ventricle (RV), which is confirmed with ECM in the (G') transverse view. (H) A transverse view at the base of the heart in ECM demonstrates a distinct and separated aortic valve (AV) and pulmonary valve (PV). PV is anterior to AV. (H') Representative ECM transverse view image of persistent truncus arteriosus (PTA) demonstrates the left pulmonary artery and right pulmonary artery arising posteriorly from the undivided persistent truncal artery, with the left carotid artery arising anteriorly and cranially from the persistent truncal artery. Abbreviations: A: anterior, AO: aorta, AV: aortic valve, Cd: caudal, Cr: cranial, DAO: descending aorta, L: left, LA: left atrium, LCA: left carotid artery, LPA: left pulmonary artery, LV: left ventricle, P: posterior, PA: pulmonary artery, PTA: persistent truncus arteriosus, R: right, RA: right atrium, RPA: right pulmonary artery, RV: right ventricle. Scale bar: 0.5 mm. Please click here to view a larger version of this figure.
Figure 2: Representative images of a necropsy to observe cardiovascular abnormalities. (A) An embryo pinned to a black-background dissecting dish with no obvious phenotypes. The image shows the gross anatomy of the head, digitals, chest cavity, and abdomen. (B) The dermis is removed from the pup, revealing submandibular glands, ribcage, and abdomen. (C) The ribcage is lifted, revealing the gross anatomy of the chest cavity, including the thymus, heart, lungs, and diaphragm. (D) A zoomed view of the chest with the ribcage removed. (E) The thymus is removed, revealing great vessels and trachea. (F) A zoomed view of great vessels. Abbreviations: AAO: ascending aorta, D: diaphragm, LA: left atrium, LCA: left carotid artery, LSVC: left superior vena cava, LV: left ventricle, P: pericardium, RA: right atrium, Rb: ribs, RCA: right carotid artery, RL: right lung, RSVC: right superior vena cava, RV: right ventricle, S: sternum, SCA: right subclavian artery, SMG: submandibular glands, T: trachea, Th: thymus. Scale bar: 1 mm. Please click here to view a larger version of this figure.
Figure 3: Representative fetal echocardiogram (Echo) and images from episcopic confocal microscope (ECM). (A) Echo demonstrates an intact ventricular septum without an interventricular shunt and normal related great arteries in the normal control, confirmed by (A') ECM at E14.5-15.5 stage. (B) Ultrasound detection of an atrioventricular septal defect (AVSD). Echo imaging in the 4-chamber view demonstrates the communication between LA, RA, LV, and RV, confirmed by (B') ECM at the E14.5 stage. (C) Ultrasound diagnosis of the ventricular septal defect (VSD) with color flow demonstrates the flow across LV and RV, confirmed by (C') ECM at the E16.5 stage. (D) Echo and (D') ECM demonstrate DORV with a VSD between LV and RV with side-by-side great arteries (aorta is right to the pulmonary artery) at the E14.5 stage. (E) Ultrasound diagnosis of persistent truncus arteriosus (PTA) demonstrates communication between LV and RV with a single outflow tract overriding both ventricles (PTA). (E') This pathology is confirmed by ECM at the E14.5 stage. Scale bar: 0.5 mm. AO: aorta, AVSD: atrioventricular septal defect, Cd: caudal, Cr: cranial, L: left, LA: left atrium, LV: left ventricle, PA: pulmonary artery, PTA: persistent truncus arteriosus, R: right, RA: right atrium, RV: right ventricle, VSD: ventricular septal defect. Scale bar: 0.5 mm. Please click here to view a larger version of this figure.
Stage | Crown-to-Rump Length (mm) | Fetus Area (mm2) | Heart Area (mm2) | Heart Area/Fetus Area |
E12.5 | 7.9 ± 0.8 (n = 77) | 23.2 ± 5 (n = 77) | ||
E13.5 | 10.5 ± 0.9 (n = 92) | 40.9 ± 7 (n = 92) | ||
E14.5 | 12.5 ± 0.9 (n = 101) | 57.6 ± 8 (n = 101) | 2.9 ± 0.5 (n = 70) | 0.050 ± 0.004 (n = 70) |
E15.5 | 14.1 ± 0.5 (n = 134) | 71.4 ± 6 (n = 134) | 3.8 ± 0.4 (n = 87) | 0.053 ± 0.004 (n = 87) |
E16.5 | 15.4 ± 0.6 (n = 112) | 82.7 ± 6 (n = 112) | 4.9 ± 0.5 (n = 87) | 0.058 ± 0.007 (n = 87) |
E17.5 | 16.6 ± 0.4 (n = 211) | 96.9 ± 7 (n = 211) | 6.1 ± 0.6 (n = 146) | 0.063 ± 0.004 (n = 146) |
E18.5 | 17.7 ± 0.6 (n = 139) | 112.1 ± 8 (n = 139) | 7.1 ± 0.8 (n = 93) | 0.063 ± 0.005 (n = 93) |
E19.5 | 18.7 ± 0.7 (n = 57) | 126.7 ± 8 (n = 57) | 7.7 ± 0.7 (n = 36) | 0.062 ± 0.005 (n = 36) |
Table 1: Developmental profile of Fetal Growth.
E14.5 | E16.5 | E18.5 | Newborn | |
70% Ethanol | 1 h | 1.5 h | 3 h | 4 h |
95% Ethanol | 35 min | 45 min | 1 h | 1 h |
95% Ethanol | 35 min | 45 min | 1 h | 1 h |
100% Ethanol | 15 min | 15 min | 30 min | 6 min |
Xylene 1 | 20 min | 30 min | 40 min | 30 min |
Xylene 2 | 20 min | 30 min | 40 min | 30 min |
Wax 1 | 20 min | 20 min | 20 min | 30 min |
Wax 2 | 20 min | 20 min | 20 min | 30 min |
Wax 3 | Overnight | Overnight | Overnight | Overnight |
Table 2: Protocol for embryo embedding based on embryonic days.
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 cardiovascular pathology. A 2D image allows for the identification of abnormal anatomy and cardiac function, with the assistance of color Doppler to examine the blood flow and detect any communications between the chambers of the heart or abnormal outflow and inflow tracts. To determine the CHD diagnosis, fetuses are scanned from embryonic day E14.5 when the outflow tract septation and cardiac chamber formation are completed. Scanning at earlier stages might reflect the developmental delay.
Histopathology is the standard to characterize CHDs8 by using a microtome followed by optical microscope visualization. The major disadvantage of the standard method is the lack of an intuitive 3D display of the cardiovascular structures for diagnosis and the limitation in the lack of providing different views of the samples. An ECM histology is the most time-efficient method to characterize cardiac defects. If ECM is unavailable, a microtome may be used to section the embryo and determine cardiac phenotypes. Other options for obtaining cardiac imaging are performing high-throughput, high-resolution magnetic resonance imaging (MRI) or computed tomography (CT). One key aspect of MRI or CT is that multiple embryos can be imaged simultaneously; however, even after an ultrasound, CT, or MRI phenotyping, histopathology is required to confirm any CHD diagnosis.
Using ECM imaging for histopathology, the embryo is serially imaged after each cut using a laser scanning confocal microscope mounted over the automatic sliding microtome. The individual images collected from ECM allow for subsequent 3D reconstructions and allow the samples to be digitally resliced in any imaging plane without re-taking the images8,11. Such an operation enables a comprehensive assessment of the cardiac anatomy, which can be used in different developmental stages. Furthermore, individual slides could be recovered from the microtome while gathering the data from the ECM equipment. Although ECM histopathology is the gold standard to assess any structural cardiac anomaly, there are limitations with the availability of the equipment, software, and running time per embryo. The running time to section an embryonic heart can vary from 1-3 h per sample, depending on its size. Due to the complexity of the equipment, researchers must regularly check on the computer to make sure that the region of interest inside the field is being recorded. Researchers must also check the sample to ensure no paraffin wax shavings obstruct the sample from the laser scanner.
ECM and subsequent 3D reconstruction provide a complete high-resolution assessment of any structural cardiac defect regardless of the embedding plan of the specimen. These protocols have helped to successfully diagnose a range of CHDs and allowed us to understand the cardiac embryogenesis in murine models and pathologies related to genetic mutations.
The authors have nothing to disclose.
None.
1x phosphate-buffered saline solution (PBS), PH7.4 | Sigma Aldrich | P3813 | |
1.5 mL Eppendorf tubes (or preferred vial for tissue storage) | SealRite | 1615-5599 | |
10% buffered formalin phosphate solution | Fisher Chemical | SF100-4 | |
100% Ethanol | Decon Laboratories | 2701 | |
16% paraformaldehyde (PFA) fixative | ThermoScientific | 28908 | 4% working concentration freshly prepared in 1x PBS at 4 °C |
50 mL tubes | Falcon | 352070 | |
6–12 Well plate or 20 mL vial for embryo storage | Falcon | 353046 | |
Dissecting microscope | Leica | MDG36 | |
Dissecting Pins (A1 or A2 grade) | F.S.T | 26002-15 | |
Dissecting Plate | F.S.T | FB0875713 | Petri dish with paraffin base |
Embedding molds | Sakura | 4133 | |
Extra narrow scissors (10.5 cm) | F.S.T | 14088-10 | 1–2 pairs |
Fiji application/Image J | NIH | Fiji.sc | |
Fine tip (0.05 mm x 0.01 mm) Dissecting Forceps (11 cm) | F.S.T | 11252-00 | 2 Pairs |
Hot forceps | F.S.T | 11252-00 | For orientation of embryos |
Industrial Marker for Wax Blocks | Sharpie | 2003898 | Formatted for labratory use |
Jenoptik ProgRes C14plus Microscope Camera | Jenoptik | 017953-650-26 | |
Jenoptik ProgRess CapturePro acquisition software | Jenoptik | jenoptik.com | |
Large glass beaker | Fisher Scientific | S111053 | For melting paraffin |
Leica M165 FC binocular microscope (16.5:1 zoom optics) | Leica | M165 FC | |
OsiriX MD Version 12.0 | OsiriX | osirix-viewer.com | |
Paraplast embedding paraffin wax | Millipore Sigma | 1003230215 | |
Small glass beaker | Fisher Scientific | S111045 | |
Small, perforated spoon (14.5 cm) | F.S.T | 10370-17 | |
Straight Vannas Scissors (4–8 mm) | F.S.T | 15018-10 | A pair |
Vevo2100 ultrahigh-frequency ultrasound biomicroscope | FUJIFILM VisualSonics Inc. | Vevo2100 | |
Xylene | Fisher Chemical | UN1307 |