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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

None.

....

Materials

NameCompanyCatalog NumberComments
1x phosphate-buffered saline solution (PBS), PH7.4Sigma AldrichP3813
1.5 mL Eppendorf tubes (or preferred vial for tissue storage)SealRite1615-5599
10% buffered formalin phosphate solutionFisher ChemicalSF100-4
100% EthanolDecon Laboratories2701
16% paraformaldehyde (PFA) fixative ThermoScientific289084% working concentration freshly prepared in 1x PBS at 4 °C
50 mL tubesFalcon352070
6–12 Well plate or 20 mL vial  for embryo storageFalcon353046
Dissecting microscope LeicaMDG36
Dissecting Pins (A1 or A2 grade)F.S.T26002-15
Dissecting Plate F.S.TFB0875713Petri dish with paraffin base
Embedding moldsSakura4133
Extra narrow scissors (10.5 cm)F.S.T14088-101–2 pairs 
Fiji application/Image JNIHFiji.sc
Fine tip (0.05 mm x 0.01 mm) Dissecting Forceps (11 cm)F.S.T11252-002 Pairs
Hot forceps F.S.T11252-00For orientation of embryos
Industrial Marker for Wax Blocks Sharpie2003898Formatted for labratory use
Jenoptik ProgRes C14plus Microscope Camera Jenoptik017953-650-26
Jenoptik ProgRess CapturePro acquisition softwareJenoptikjenoptik.com
Large glass beaker Fisher ScientificS111053For melting paraffin
Leica M165 FC binocular microscope (16.5:1 zoom optics)LeicaM165 FC
OsiriX MD Version 12.0OsiriXosirix-viewer.com 
Paraplast embedding paraffin waxMillipore Sigma1003230215
Small glass beakerFisher ScientificS111045
Small, perforated spoon (14.5 cm)F.S.T10370-17
Straight Vannas Scissors (4–8 mm)F.S.T15018-10A pair
Vevo2100 ultrahigh-frequency ultrasound biomicroscopeFUJIFILM VisualSonics Inc.Vevo2100
XyleneFisher ChemicalUN1307

References

  1. Wu, W., He, J., Shao, X. Incidence and mortality trend of congenital heart disease at the global, regional, and national level, 1990-2017. Medicine. 99 (23), e20593 (2020).
  2. vander Linde, D., et al.

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