Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Summary

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Abstract

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

Introduction

Myocardial infarction (MI) is a leading cause of death worldwide, and remains responsible for about one third of heart failure cases¹. While the advent of percutaneous intervention and continuous optimization of the use of thrombolytics has increased reperfusion following MI, cardiomyocyte death and loss of contractile myocardium nevertheless occurs. There also remain large numbers of "no-option" patients who are not candidates for or do not see benefit from these interventions. These patients continue to experience disabling ischemia leading to scar formation and deleterious ventricular remodeling as a mechanism of infarct healing. This process ult....

Protocol

Breeding pairs of C57BL/6 and CD-1 IG-S mice were purchased from Charles River. Animals used in this study were handled in accordance with the guidelines of the Canadian Council on Animal Care, and study protocols were approved by the Animal Use Subcommittee at Western University, London, Canada.

1. Animal Care

1. After birthing is complete and pups have been initially breast-fed by their mother for a few hr, place them in a different cage with a CD-1 foster mother. CD-1 mothers display a calmer phenotype with a strong fostering instinct, and have a lower tendency to cannibalize injured pups¹⁵.

Results

The myocardial infarction procedure at P1 can be completed in 10 - 15 min and has a mortality rate of 7.8% (5 out of 64 pups). After surgery, mice recover from hypothermic anesthesia within the next 5 - 20 min (time of recovery depends on body temperature reached during anesthesia and speed of surgeon). When using P7 pups (for comparison with a non-regenerative myocardium), a longer period of cooling is required to reach torpor. P7 pups are much larger and have more difficulty recovering .......

Discussion

The surgical LAD ligation demonstrated herein is a reliable method to produce MI in neonatal mice. This model provides researchers with a reproducible model with which to study mammalian heart regeneration. Visualization of the coronary vasculature is a key component of this method, ensuring correct suture placement and thus guaranteeing reproducibility. While adult mice do not possess poikilothermic capabilities, the body temperature and metabolic rate of neonatal mice is closely associated with ambient temperature. Fur.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
8-0 Nylon Suture	Microsurgery Instruments	8-0 Nylon
11-0 Nylon Suture	Shanghai Pudong Medical Products Co Ltd	H1101
Fine Scissors	Fine Science Tools	14058-09
Small forceps	Fine Science Tools	11063-07
Micro Needle Holder	Fine Science Tools	12060-02
Zeiss Opmi 6s/S3 Microscope	Zeiss	300002
Isoflurane	Baxter	CA2L9100
Isoflurane Chamber	Made in Feng laboratory
Bead Sterilizer	Fine Science Tools	18000-45
2,3,5-Triphenyltetraolium chloride (TTC)	Sigma	T8877
Stereomicroscope SteREO Discovery. V8	Zeiss	435400
AxioVision 8.0	Zeiss
Axiocam Icc5	Zeiss	426554
Heat pad	Sunbeam	731A0-CN
Sterile Gloves	VWR	414004-430
Gauze Sponges	Ducare	90212
Ice
Ultrasound imaging system, Vevo2100	Visual Sonics	VEVO2100
Ultrasound transducer, 40 MHz	Visual Sonics	MS400