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* These authors contributed equally
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 phenoty....
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 Associa.......
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.......
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.......
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....Name | Company | Catalog Number | Comments |
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 |
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