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

Summary

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

Abstract

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.

Introduction

CHDs are the most common type of birth defect in humans and are the leading cause of birth defect-related deaths^1,2,3,4,5,6. 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' lives and the healthcare system^7,8,9,

Protocol

All animals used in the experiments referenced in this paper were treated using the animal care guidelines of the Michigan State University Institutional Animal Care and Use Committee (IACUC).

1. Timed mating of C57BL6/J mice for embryo production

1. Once mice have reached breeding age (6-8 weeks), put them together in harem breeding format (i.e., two females per one male). Set them up for breeding sometime in the afternoon or evening.
   NOTE: Mice often breed within 1 h after lights have been turned off within their housing facility. Females should be retired from breeding at about 6-8 months and males should be used no later....

Results

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.......

Discussion

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.......

Materials

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