A reproducible protocol for neonatal ischemic inju

Authors: Bernhard J Haubner Thomas Schuetz Josef M Penninger

Publish Date: 2016/09/24

Volume: 111, Issue: 6, Pages: 64-

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Abstract

Cardiac regeneration is one of the prime visions in cardiovascular research The mouse neonatal apical resection and left anterior descending artery LAD ligation model introduced novel in vivo mammalian assays to study cardiac regeneration However recent reports and editorials discussed and critically questioned the value and technical reproducibility of the mouse neonatal myocardial infarction approach making it paramount to develop and use a reproducible model system We established a mouse neonatal myocardial infarction model by visually confirmed ligation of the LAD using microsurgery TdTmediated dUTP nickend labeling TUNEL proved reproducible massive myocardial infarctions in a defined region of the apex and anterior wall of neonatal and 7dayold mice Whereas hearts ligated on postnatal day 7 displayed chronic injury cardiac samples ligated immediately after birth always showed complete structural regeneration after longterm followup Cardiac regeneration was observed in all mouse stains C57BL/6J ICR and mixed background C57BL/6JxSv129 tested so far We present a detailed in vivo protocol to study complex mechanisms of complete cardiac repair following ischemic cardiac damage Neonatal LAD ligation surgery is feasible and results in reproducible myocardial infarctions 24 h after ligation and no structural myocardial defects are detectable following longterm followup We encourage the cardiovascular community to use our protocol and teaching video to answer key scientific questions in the field of cardiac regenerationIschemic heart disease is the most common cause of death worldwide 22 Yet cardiovascular mortality has significantly declined in the Western world due to improved primary prevention reperfusion therapies and secondary prevention following myocardial infarction 12 Despite these major advances within the last few decades our society still faces a high burden of cardiac mortality and morbidity due to inefficient regeneration of the heart following cardiac injury 13 Accumulating experimental evidence has uncovered a minor cardiomyocyte turnover in the adult mammalian heart that is however not sufficient to replace the millions of lost heart muscle cells upon injury 3 It is therefore paramount to find novel strategies to regenerate the heart using for instance stem cells transdifferentiation of cardiac cells or approaches to boost the endogenous regenerative potential of the heart 6 7 21Newts and zebrafish are wellknown in vivo animal models for cardiac regeneration but the evolutionary distance to humans limits their translational character 14 19 Porrello et al reported cardiac regeneration following apical resection in the neonatal mouse 17 Our group and Olson and Sadek subsequently and independently established a neonatal model of left anterior descending artery LAD ligation which for the first time showed that murine neonatal hearts can be repaired following a clinically relevant complex myocardial infarction 8 18 However recent studies challenged the scientific value of the apex resection and LAD ligation model in the neonatal mouse 1 2 10 resulting in a controversy on the usefulness and technical reproducibility of this model system to study mechanisms of cardiac regeneration Of note we recently reported a human case of complete cardiac regeneration in a baby following a massive neonatal myocardial infarction thus newborn humans also have the intrinsic capacity to repair myocardial damage and completely recover cardiac function 9To resolve recent controversies and to address the issue of reproducibility we here present a detailed method and learning videos for LAD ligation in neonatal mouse hearts We show data on complete and reproducible structural cardiac repair following LAD ligation and critically highlight the possible pitfalls of the neonatal mouse heart attack model A reproducible protocol is in our opinion an absolute prerequisite to exploit the great potential of the neonatal mouse heart as a model system to uncover fundamental principles of mammalian cardiac regenerationLeft anterior descending artery LAD ligation was induced in neonatal mice on the day of birth Approximately 10–12 h after birth we performed the LAD or sham surgery Moreover we subjected mice to LAD ligation on postnatal day 7 To assess whether regeneration might be influenced by the genetic mouse background we analyzed three different mouse strains C57BL/6J ICR and mixed background C57BL/6JxSv129 Surgery was performed on hypothermic mice in cardiac arrest to induce hypothermic anesthesia we put the neonates as well as 7dayold animals into an isoflurane induction box oxygen 1 l/min with 4  isoflurane and then placed the mice into shallow ice water in a Petri dish for approximately 4 min This induces cardiac arrest and apnea Next the mice were tapped onto an ice pack in the right decubitus position using Transpore 15271 3 M St Paul Minnesota USA The surface of the ice pack needs to be slightly prewarmed to prevent freezing damage of the skin The thoracic cavity was opened at the fourth intercostal space beginning from the edge of the lung to the left internal mammary artery Following spreading of the ribs with curved forceps the LAD was visualized and ligated via microsurgery using 100 Ethilon EH7467G Ethicon Somerville New Jersey USA The rib cage including the Mm pectorales was closed with a single 80 Coated Vicryl suture V542G Ethicon Somerville New Jersey USA Finally we closed the skin using two sutures with 80 coated Vicryl All surgery was performed using a M80 Leica microscope ocular 10× objective achromat 063× WD = 148 Leica Solms Germany including vertical illumination LED3000 NVI Leica Solms Germany a microneedle holder 1207514 Fine Science Tools Heidelberg Germany roundhandled Vannas spring scissors 1540012 Fine Science Tools Heidelberg Germany and two curved finetip forceps 1129700 Fine Science Tools Heidelberg GermanyFollowing microsurgery mice were placed onto a 38 °C warm plate and left for spontaneous recovery Of note we routinely limit the time of surgery to about 10 min To allow mothers to accept their pups after surgery we always removed half of the pups for surgery while keeping the other half with the mothers for nursing After recovery from anesthesia the mice received a dose of subcutaneous buprenorphine 005 mg/kg for pain treatment following ethical requirements Sham surgery was identical to the LAD ligation surgery without tying the LAD Mice were euthanized by cervical dislocation or decapitation depending on the size and age of the rodents All animal experiments were performed in accordance with the institutional guidelines and approved by the Austrian Animal Ethical Board BMWF66015/002511/3b/2011 and BMWF66015/0024WF/V//3b/2014 The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health

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