Name: Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods (Video) | JoVE Journal
Uploaded: 2020-03-10T15:00:00.000+00:00
Duration: 8 min 28 s
Description: Learn standardized tissue analysis techniques for studying congenital heart defects in mouse embryos using qualitative and quantitative histological methods. This protocol enhances reproducibility and supports improved cardiology diagnostics.

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The Aguirre Lab is supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under award number K01HL135464 and by the American Heart Association under award number 19IPLOI34660342.

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The authors have no disclosures to report.

This protocol explores the techniques involved in the analysis of cardiac development in embryonic hearts. Some limitations of this method are the required physical dexterity for the preparatory techniques, which may require practice, and skill with microscope imaging. If the slices obtained at the cryostat are messy, the hematoxylin and eosin staining will not be clear, or if the images taken at the microscope have poor lighting, then the method used with ImageJ will not work. A limitation of the threshold feature of Im...

CHDs are the most common type of birth defect in humans and are the leading cause of birth defect-related deaths1,2,3,4,5,6. Although about 90% of newborn children survive CHD, it is frequently associated with significant morbidity and medical interventions over the years, imposing a heavy burden on the patients&#39; lives and the healthcare system7,8,9,10. Outside of purely genetic factors, the causes of CHD are poorly understood4. Unidentified causes account for ~56-66% of all CHD cases according to the American Heart Association and other sources2,3,4,11. Well-known factors include genetic mutations, CNVs, de novo single nucleotide variants, and aneuploidy. It is suspected that environmental and dietary factors are also important sources contributing to CHD, as suggested by epidemiological studies linking maternal lifestyle2,12, economic deprivation, and race13, and by research into dietary factors such as folic acid11,14 and the bioactive lipid retinoic acid15,16. Investigating the mechanisms and causes of CHD and other cardiovascular defects is important to develop preventive strategies and novel therapeutic options1,4,17,18,19.
The developing mouse is a cornerstone model for studying CHD in mammals. However, some of the methods and analyses employed, such as dissections preserving heart morphology, analysis of developmental stages, and identification of CHD-associated defects, can be daunting for scientists that are new to the analysis of murine hearts. The goal of the methods described in this protocol is to offer qualitative and quantitative guidelines for these processes. Thus, in this protocol we explain how to perform timed matings to produce embryos of the desired gestational stage, dissect pregnant females for intact heart recovery (including associated tissues such as the outflow tract), heart fixation and preparation for cryostat sectioning, basic histology methods, quantitative analyses of common heart defects, and qualitative analysis of heart muscle compaction, a common precursor phenotype to some types of CHD.

<table><tbody><tr><td>15 mL Conical Tube(s)</td><td>Fisher Scientific</td><td># 1495970C</td><td></td></tr><tr><td>C57BL/6J Mice</td><td>Jackson Labs</td><td>C57BL/6J - stock 000664</td><td></td></tr><tr><td>Coplin Staining Jars (x6)</td><td>VWR Scientific</td><td># 25457-006</td><td></td></tr><tr><td>Coverslips 24X50MM #1.5</td><td>VWR Scientific</td><td># 48393-241</td><td></td></tr><tr><td>Cryostat - Leica CM3050S</td><td>Leica</td><td>N/A</td><td></td></tr><tr><td>Dissecting Dish(s)</td><td>Fisher Scientific</td><td># 50930381</td><td></td></tr><tr><td>Dumont #5 - Fine Forceps (x2)</td><td>Fine Science Tools</td><td># 11254-20</td><td></td></tr><tr><td>Eosin Y Solution</td><td>Millipore Sigma</td><td># HT110116-500ML</td><td></td></tr><tr><td>Ethyl Alcohol (Pure, 200 proof)</td><td>Fisher Scientific</td><td># BP2818-500</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Millipore Sigma</td><td># E9884-100G</td><td></td></tr><tr><td>Eukitt</td><td>Millipore Sigma</td><td># 03989-100ML</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td># 14060-10</td><td></td></tr><tr><td>Fluorescent Stereo Microscope Leica M165 FC</td><td>Leica</td><td>N/A</td><td></td></tr><tr><td>Glycine</td><td>Millipore Sigma</td><td># 410225-250G</td><td></td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td># 11052-10</td><td></td></tr><tr><td>Graphpad Prism 8 Software</td><td>Graphpad</td><td></td><td></td></tr><tr><td>ImageJ Software</td><td>ImageJ</td><td></td><td></td></tr><tr><td>Kimwipes</td><td>Fisher Scientific</td><td># 06666A</td><td></td></tr><tr><td>Mayer&#039;s hematoxylin solution</td><td>Millipore Sigma</td><td># MHS16-500ML</td><td></td></tr><tr><td>Micropipette tip(s) - p200</td><td>Fisher Scientific</td><td># 02707448</td><td></td></tr><tr><td>Microsoft Excel Software</td><td>Microsoft</td><td></td><td></td></tr><tr><td>OCT Compound</td><td>VWR Scientific</td><td># 102094-106</td><td></td></tr><tr><td>Olympus CkX53 Microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Paint Brushes (at least 2)</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>VWR Scientific</td><td># 0215014601</td><td>Make into 4% solution (dissolved in PBS)</td></tr><tr><td>Pasteur pipette(s)</td><td>Fisher Scientific</td><td># 13-711-7M</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>ThermoFisher Scientific</td><td># 15140122</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>ThermoFisher Scientific</td><td># 70011044</td><td>Dilute from 10x to 1x before using</td></tr><tr><td>Scale</td><td>Mettler Toledo</td><td># MS1602TS</td><td></td></tr><tr><td>Scale</td><td>Mettler Toledo</td><td># MS105</td><td></td></tr><tr><td>Scalpel Handle #3</td><td>VWR Scientific</td><td># 10161-918</td><td></td></tr><tr><td>Scalpel Blades</td><td>VWR Scientific</td><td># 21909-612</td><td></td></tr><tr><td>Square Mold</td><td>VWR Scientific</td><td># 100500-224</td><td>For OCT molds</td></tr><tr><td>Sucrose</td><td>Millipore Sigma</td><td># S9378-500G</td><td></td></tr><tr><td>Superfrost Plus Slides</td><td>Fisher Scientific</td><td># 1255015</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td># 14002-14</td><td></td></tr><tr><td>Tissue-Tek Accu-Edge Disposable Microtome Blades</td><td>VWR Scientific</td><td># 25608-964</td><td></td></tr><tr><td>Travel Scale</td><td>Acculab</td><td>VIC 5101</td><td></td></tr><tr><td>Xylene</td><td>Millipore Sigma</td><td>214736-1L</td><td></td></tr></tbody></table>

analysis of congenital heart defects in mouse embryos using qualitative and quantitative histological methods

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

The muscle compaction index was compared between hearts developing under two different environments, a control and an experimental group. These protocols were used to analyze the compaction of muscle tissue quantitatively, which permitted statistical analysis. Muscle compaction was shown to be significantly reduced in the experimental hearts relative to the embryos that developed in nonexperimental conditions.
Embryos were disca...

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

analysis-congenital-heart-defects-mouse-embryos-using-qualitative

Research

JoVE Journal

Developmental Biology

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In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

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Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

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Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Learn standardized tissue analysis techniques for studying congenital heart defects in mouse embryos using qualitative and quantitative histological methods. This protocol enhances reproducibility and supports improved cardiology diagnostics.

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

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