This study was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) to Q.F. (grant #MOP-119600).

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
<ol>
	<li>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.</li>
</ol>
2. Surgery
<ol>
	<li>Induce anesthesia by placing the pup in a sealed isoflurane chamber (approximately 500 &#181;l of 100% v/v isoflurane allowed to dissipate over 1,300 cm3 chamber). Keep the mouse in the chamber until cessation of movement (about 30 sec).</li>
	<li>Remove mouse from isoflurane chamber and induce hypothermic anesthesia by placing mouse on wet ice. To avoid frostbite, place ice inside a sterile surgical glove, wrap the mouse within the glove and cover the glove with ice OR wrap the mouse in sterile wet gauze and place it on ice. 
	NOTE: Mice cool faster when contact with ice is over a larger surface area, and as such melted ice can shorten hypothermia induction time.</li>
	<li>Confirm anesthesia by lack of movement response to toe and tail pinch. 
	NOTE: Cessation of provoked movement occurs at a body temperature between 15 &#176;C and 8 &#176;C. (Cooling time to this temperature approximately 1 min). Complete apnea and asystole is NOT required for LAD ligation.</li>
	<li>Move the ice bed to the surgical area equipped with an operating microscope. Ensure that the mouse pup remains on ice during the whole surgical procedure. 
	NOTE: The surgical area, gauze and surgical instruments should be sterilized. Maintain sterile field throughout the procedure, and wear single use, sterile surgical gloves. Ophthalmic ointment is not required as mice are born with their eyes closed 17.</li>
	<li>Place the mouse pup in the right lateral decubitus position. Disinfect chest by gently wiping with povidone-iodine solution followed by an ethanol swab.</li>
	<li>Perform skin incision on the left chest along the mid-axillary line by cutting skin between the xyphoid and left axilla a few millimeters below the left foreleg. Also use scissors to cut through the underlying pectoral muscle layer.</li>
	<li>Perform a left thoracotomy in the 4th intercostal space by separating ribs and intercostal muscles with forceps. 
	NOTE: Neonatal ribs are very fragile and can be easily broken. To avoid this, gently separate ribs by opening forceps along the intercostal muscle (rather than grasping ribs).</li>
	<li>Visualize the left anterior descending coronary artery (LAD) emerging from the left auricle behind the pulmonary vein and descending beyond the great cardiac vein. 
	NOTE: In this early neonatal window, the thymus is often visualized and may cover part of the cardiac base. The LAD can be seen emerging beside the position of the thymus, depending on the angle of thoracic incision.</li>
	<li>Ligate the LAD by passing an 11-0 nylon suture (0.007 mm diameter needle) below the artery through the mid ventricle below&#160;the left auricle (Figure 1C). Ischemia is confirmed with blanching of the myocardium below the suture site (Figure 1G).</li>
	<li>Close the thoracic incision using 8-0 nylon sutures (0.15 mm diameter needle). Use two sutures to close the ribs. Place both rib sutures before ligating to ensure the needle does not puncture the lungs when passing through the body cavity.</li>
	<li>After closing the ribs, remove the mouse from the ice bed. Keep the mouse within the surgical area and place it directly on the sterile surgical towel over a warm heating pad at 37 &#176;C to begin warming.</li>
	<li>Once on the sterile heated area, close the muscle and skin layers using 8-0 nylon sutures (0.15 mm diameter needle). Use one suture for the muscle layer and use two sutures for the skin incision. 
	NOTE: Muscle and skin layers may be closed on a heating pad to reduce hypothermia exposure time. Mouse temperature begins to rise once placed on the heating pad, but remains sufficiently low enough for anesthesia maintenance during muscle and skin closure.</li>
</ol>
3. Surgical Recovery
<ol>
	<li>Continue rapid warming on a warm heating pad until return of spontaneous movement. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Remove povidone-iodine and blood by gently wiping injury site with ethanol swab.</li>
	<li>After sufficient recovery from anesthesia, remove it from sterile surgical area, smear it with bedding from foster mother&#39;s cage, and return pup to foster mother. This helps prevent maternal rejection or cannibalization. 
	NOTE: Do not return pups to a cage with other animals until fully recovered. If an entire litter has not been used, and littermates remain with foster mother, place recovered pups in the middle of the litter. If performing more than one surgery, complete all surgeries required for an individual litter before returning mice to their foster mother.</li>
	<li>Observe behavior of foster mother toward the pup every 10 - 15 min for 2 - 3 hr to ensure acceptance of the pup. If the mother displays aggression towards the injured pup, remove the pup and euthanize by isoflurane overdose (&#62; 5%; until cessation of breathing), followed by decapitation.&#160;NOTE: Perioperative analgesia medications are not required as the centralized pain reflexes are not fully developed at this early age15.</li>
</ol>
4. Measurement of Myocardial Infarct Size 4 - 6 hr Post-MI
<ol>
	<li>Allow pups to recover in the care of a CD-1 foster mother for a period of 4 - 6 hr. Remove the pup from cage with mother and euthanize it by isoflurane overdose followed by decapitation with large scissors.</li>
	<li>Excise the heart under dissecting microscope, careful not to rip the myocardium.
	<ol>
		<li>Cut the skin from the xyphoid process to the top of the thorax. Open abdominal wall below the ribcage. Grasp the lower rib cage and cut through the ribs and musculature longitudinally along the left mid-axillary line from the diaphragm to the axilla.</li>
		<li>Hold scissors in transverse plane and carefully cut through diaphragm from left to right side. Make sure to place scissors below the level of the heart so as to avoid any damage to the apex.</li>
		<li>Grasp rib cage and cut right side of ribs and musculature along the right mid-axillary line. Remove all vascular connections to the heart with scissors. Remove the heart from chest cavity by grasping the base.</li>
	</ol>
	</li>
	<li>Section the heart into three pieces using a surgical carbon steel razor blade. Make the first cut along the short-axis of the heart at the midpoint between the suture and the cardiac apex. Make the second cut at the level of the suture (Figure 3A). 
	NOTE: This leaves an apex section approximately 0.75 mm thick weighing 0.0018 g; a mid-base section approximately 1 mm thick, weighing 0.0065 g; and the base of the heart.</li>
	<li>Place the heart sections in 1% 2,3,5-triphenyltetrazolium chloride (TTC) at room temperature for 10 - 15 min. Carefully watch specimen to avoid over-staining.</li>
	<li>To increase contrast, fix stained heart slices with 4% paraformaldehyde overnight at 4 &#176;C. To calculate % infarct area, photograph heart sections and measure infarct area on imaging software. The viable myocardium stains red while the infarct area is demarcated as white18.</li>
</ol>
5. Measurement of Cardiac Function by Echocardiography Post-MI
<ol>
	<li>Allow pups to recover in the care of a CD-1 foster mother for a period of 24 - 48 hr. Induce anesthesia by placing the pup in a sealed isoflurane chamber (5% isoflurane). Keep the mouse in the chamber until cessation of movement (about 30 sec).</li>
	<li>Secure the pup in the supine position on a heated dock (temperature 37 &#176;C) with its nose in a cone to deliver 0.5 - 1% isoflurane (for anesthesia maintenance). Place pre-warmed echo gel on the left thoracic area.</li>
	<li>Obtain a parasternal long-axis view of the left ventricle (LV). Ensure the images are obtained below the level of the suture in the LV. After acquiring the position, turn the ultrasound probe (40 MHz) 90&#176; to obtain a parasternal short-axis view, and record M-mode echocardiographic images.</li>
	<li>Measure the end diastolic and end systolic left ventricular internal diameters from the short-axis M-mode images. Calculate ejection fraction and fractional shortening.</li>
</ol>
6. Measurement of Myocardial Infarct Size 24 hr Post-MI
<ol>
	<li>Allow pups to recover in the care of a CD-1 foster mother for a period of 24 hr. Remove the pup from cage with mother and euthanize it by isoflurane overdose followed by decapitation with large scissors.</li>
	<li>Open the chest cavity as in step&#160;4.2. Before excising the heart, carefully grasp the thoracic aorta just above the diaphragm with fine forceps. Hold fine scissors in the coronal plane, flat against the thoracic wall, and cut the thoracic aorta off the posterior thoracic wall moving scissors in the cranial direction.</li>
	<li>Cut the aorta free from the thoracic wall and any other connections until it reaches the heart. Excise the heart by grasping the superior vena cava and cutting all other vascular connections to the body.</li>
	<li>Rinse the heart (with aorta attached) in saline. Carefully cannulate the thoracic aorta with a 30 gauge needle and tie aorta to cannula with 8-0 nylon suture thread.</li>
	<li>Perfuse the heart with 1 ml of saline through the aorta via a 30 gauge needle at a rate of 1 ml/min. Perfuse the heart with 150 &#181;l of 2% Evans blue solution at a rate of 1 ml/min. Remove the heart from the cannula, section it into three pieces and stain it with TTC as described in section 4.</li>
	<li>To calculate infarct size, photograph heart sections and measure infarct area on imaging software. The viable myocardium stains blue, the area at risk stains red, and the infarct area is demarcated as white.</li>
</ol>

<ol>
	<li>Rosamond, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics--2008+update:+a+report+from+the+American+Heart+Association+Statistics+Committee+and+Stroke+Statistics+Subcommittee.">Heart disease and stroke statistics--2008 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.</a> Circulation. 117 (4), 25-146 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-analysis+Global+Group+in+Chronic+Heart+Failure.+The+survival+of+patients+with+heart+failure+with+preserved+or+reduced+left+ventricular+ejection+fraction:+an+individual+patient+data+meta-analysis.">Meta-analysis Global Group in Chronic Heart Failure. The survival of patients with heart failure with preserved or reduced left ventricular ejection fraction: an individual patient data meta-analysis.</a> Eur Heart J. 33 (14), 1750-1757 (2012).</li><li>Soonpaa, M. H., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cardiomyocyte+DNA+synthesis+in+normal+and+injured+adult+mouse+hearts.">Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts.</a> Am J Physiol. 272, 220-226 (1997).</li><li>D'Uva, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERBB2+triggers+mammalian+heart+regeneration+by+promoting+cardiomyocyte+dedifferentiation+and+proliferation.">ERBB2 triggers mammalian heart regeneration by promoting cardiomyocyte dedifferentiation and proliferation.</a> Nat Cell Biol. 17 (5), 627-638 (2015).</li><li>Oberpriller, J. O., Oberpriller, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+the+adult+newt+ventricle+to+injury.">Response of the adult newt ventricle to injury.</a> J Exp Zool. 187 (2), 249-253 (1974).</li><li>Poss, K. D., Wilson, L. G., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+regeneration+in+zebrafish.">Heart regeneration in zebrafish.</a> Science. 298 (5601), 2188-2190 (2002).</li><li>Jopling, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+heart+regeneration+occurs+by+cardiomyocyte+dedifferentiation+and+proliferation.">Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation.</a> Nature. 464 (7288), 606-609 (2010).</li><li>Porrello, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+regenerative+potential+of+the+neonatal+mouse+heart.">Transient regenerative potential of the neonatal mouse heart.</a> Science. 331 (6020), 1078-1080 (2011).</li><li>Haubner, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+cardiac+regeneration+in+a+mouse+model+of+myocardial+infarction.">Complete cardiac regeneration in a mouse model of myocardial infarction.</a> Aging. 4 (12), 966-977 (2012).</li><li>Soonpaa, M. H., Kim, K. K., Pajak, L., Franklin, M., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+DNA+synthesis+and+binucleation+during+murine+development.">Cardiomyocyte DNA synthesis and binucleation during murine development.</a> Am J Physiol. 271, 2183-2189 (1996).</li><li>Li, F., Wang, X., Capasso, J. M., Gerdes, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+transition+of+cardiac+myocytes+from+hyperplasia+to+hypertrophy+during+postnatal+development.">Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development.</a> J Mol Cell Cardiol. 28 (8), 1737-1746 (1996).</li><li>Feng, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevation+of+an+endogenous+inhibitor+of+nitric+oxide+synthesis+in+experimental+congestive+heart+failure.">Elevation of an endogenous inhibitor of nitric oxide synthesis in experimental congestive heart failure.</a> Cardiovasc Res. 37 (3), 667-675 (1998).</li><li>Xiang, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte-specific+overexpression+of+human+stem+cell+factor+improves+cardiac+function+and+survival+after+myocardial+infarction+in+mice.">Cardiomyocyte-specific overexpression of human stem cell factor improves cardiac function and survival after myocardial infarction in mice.</a> Circulation. 120 (12), 1065-1074 (2009).</li><li>van Kats, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-converting+enzyme+inhibition+and+angiotensin+II+type+1+receptor+blockade+prevent+cardiac+remodeling+in+pigs+after+myocardial+infarction:+role+of+tissue+angiotensin+II.">Angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade prevent cardiac remodeling in pigs after myocardial infarction: role of tissue angiotensin II.</a> Circulation. 102 (13), 1556-1563 (2000).</li><li>Mahmoud, A. I., Porrello, E. R., Kimura, W., Olson, E. N., Sadek, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+models+for+cardiac+regeneration+in+neonatal+mice.">Surgical models for cardiac regeneration in neonatal mice.</a> Nat Protoc. 9 (2), 305-311 (2014).</li><li>Ahn, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+myocardial+infarcts+of+a+predictable+size+and+location+by+branch+pattern+probability-assisted+coronary+ligation+in+C57BL/6+mice.">Induction of myocardial infarcts of a predictable size and location by branch pattern probability-assisted coronary ligation in C57BL/6 mice.</a> Am J Physiol Heart Circ Physiol. 286 (3), 1201-1207 (2004).</li><li>Kao, W. W., Xia, Y., Liu, C. Y., Saika, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+in+morphogenesis+of+cornea+and+eyelid.">Signaling pathways in morphogenesis of cornea and eyelid.</a> Ocul Surf. 6 (1), 9-23 (2008).</li><li>Redfors, B., Shao, Y. Z., Omerovic, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarct+size+and+area+at+risk+assessment+in+mice.">Myocardial infarct size and area at risk assessment in mice.</a> Experimental &amp; Clinical Cardiology. 17 (4), 268-272 (2012).</li><li>Phifer, C. B., Terry, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+hypothermia+for+general+anesthesia+in+preweanling+rodents.">Use of hypothermia for general anesthesia in preweanling rodents.</a> Physiol Behav. 38 (6), 887-890 (1986).</li><li>Jesty, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-kit++precursors+support+postinfarction+myogenesis+in+the+neonatal,+but+not+adult,+heart.">c-kit+ precursors support postinfarction myogenesis in the neonatal, but not adult, heart.</a> Proc Natl Acad Sci U S A. 109 (33), 13380-13385 (2012).</li><li>Mahmoud, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meis1+regulates+postnatal+cardiomyocyte+cell+cycle+arrest.">Meis1 regulates postnatal cardiomyocyte cell cycle arrest.</a> Nature. 497 (7448), 249-253 (2013).</li></ol>

The authors have nothing to disclose.

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...

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 &#34;no-option&#34; 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 ultimately results in heart failure, for which prognosis remains poor despite optimal pharmacologic management with angiotensin-converting enzyme (ACE) inhibitors and beta blockers. Unfortunately, the one-year mortality rate for patients with severely impaired left ventricular function still remains as high as 26%2. Heart transplant is the final treatment option for patients with heart failure. However, the limited donor pool for heart transplantation does not make this a viable option for most patients. Thus, the discovery of novel therapeutic agents to restore the damaged myocardium remains paramount to solving the cardiac disease problem. Reliable animal models of cardiac injury are therefore required as a critical component of this process.
Traditional dogma has dictated that adult cardiomyocytes are post-mitotic, terminally differentiated cells, incapable of dividing or de-differentiating to replace the damaged myocardium3. As such, an adult mammalian heart could never completely recover from injury, and lost cardiomyocytes would be replaced with fibrous tissue. Thus, research has focused primarily on therapeutic agents to minimize infarct expansion and reduce scar formation. More recently however, a paradigm shift has occurred in the thinking surrounding cardiac healing and many research efforts have been redirected to focus on the potential for cardiac regeneration4.
Until recently, in vivo study of cardiac regeneration was restricted to non-vertebrate models, such as those in urodele amphibians and teleost fish5-7. However, the discovery of the capacity for cardiac regeneration in the neonatal mouse has led to the development of two surgical models of mammalian cardiac regeneration: resection of the cardiac apex and coronary artery occlusion to induce myocardial infarction8,9. In 2011, a mouse apex resection model was used to demonstrate that complete cardiac regeneration is possible at postnatal day 1 (P1). However, this capacity declines rapidly after the initial neonatal period. The mammalian heart loses its regenerative potential shortly after birth at P7 as progenitor cell numbers decline, and cardiomyocytes become binucleated, lose their proliferative competency, and permanently exit the cell cycle10,11. Understanding the fundamental differences between the neonatal and adult mammalian heart may lead to novel insights into cardiac regeneration.
While apex resection indeed offers insight into re-growth of contractile tissue, the model does not simulate typical human cardiac injury, and thus does not lend itself as well to the development of therapeutics. The coronary artery occlusion model, however, more directly simulates the pathophysiologic aspects of MI pathology, and thus may provide more useful insights into mechanisms that may be applicable to therapeutic advancement for human use.
Surgical coronary ligation has been used as a useful experimental technique in many animal models12-14. In the adult coronary artery ligation model, animals are anesthetized and intubated to allow opening of the chest cavity while maintaining respiration. The heart continues to beat regularly, permitting visualization of the coronary vasculature and allowing for accurate suture placement. Furthermore, the heart remains pink as perfusion continues, and after ligation the ischemic myocardium appears pale, indicating successful coronary artery ligation. The protocol described for neonatal mice, however, is less reliable as the coronary artery is not visualized and the surgeon must estimate where to place the suture15. Although the general anatomy of the coronary vasculature is the same, individual animal variability in the direction and branching of the LAD exists16. Thus, when &#34;going in blind,&#34; the artery could be easily missed. Other techniques such as echocardiography are then required to confirm successful induction of MI, and to ensure all surgeries result in a similar infarct size. Described here is an improvement on a recently published method15, where the position of the LAD can be established and thus LAD may be ligated to reproducibly induce MI.
This technique does not require endotracheal intubation or mechanical ventilation, as thoracotomy in a hypothermic state in the neonatal mouse does not result in lung collapse. However, in the previously described method, severe hypothermia must be induced to the point of both complete apnea and cessation of the cardiac rhythm15. The major limitation of this approach is that the coronary artery is no longer perfused and the heart appears pale even before LAD ligation. In the approach described herein, coronary artery visualization is possible at a point of torpor before deep hypothermia and cardiac rhythm cessation, with full recovery of the neonatal mouse after the surgery. This method offers a major advantage of 100% reproducibility.

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

myocardial infarction in neonatal mice, a model of cardiac regeneration

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.

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 ...

Watch this Scientific Journal Video about Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration at JoVE.com

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

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.

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 ...

myocardial-infarction-in-neonatal-mice-a-model-of-cardiac-regeneration

Research

JoVE Journal

Medicine

18.4K Views.  Western University. 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.

Video: Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Biology

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study reveals that transplantation of neonatal cardiac macrophages recruited by injury promotes adult heart regeneration after myocardial infarction with improvement of cardiac function and cardiomyocyte proliferation. The results indicate that neonatal cardiac macrophage transplantation could be a promising strategy for cardiac injury treatment. Here, we provide the technical details, including the isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages into myocardial-infarcted adult mice, and the estimation of heart regeneration after a macrophage graft.

We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study ...

Biochemistry

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

Developmental Biology

Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling

By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting bona fide heart regeneration limits the validation of potential regenerative factors. One method to detect new cardiomyocytes is multicolor lineage tracing with clonal analysis. Clonal analysis experiments can be difficult to undertake, because labeling conditions that are too sparse lack sensitivity for rare events such as cardiomyocyte proliferation, and diffuse labeling limits the ability to resolve clones. Presented here is a protocol to undertake clonal analysis of the neonatal mouse heart by using statistical modeling of nearest neighbor distributions to resolve cardiomyocyte clones. This approach enables resolution of clones over a range of labeling conditions and provides a robust analytical approach for quantifying cardiomyocyte proliferation and regeneration. This protocol can be adapted to other tissues and can be broadly used to study tissue regeneration.

Presented here is a protocol for using multicolor lineage tracing and nearest-neighbor modeling to identify clonally derived cardiomyocytes during growth and regeneration in mice. This approach is objective, works across different labeling conditions, and can be adapted to incorporate a variety of image analysis pipelines.

By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting ...

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Bioengineering

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI. 
The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson's trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected. 
In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson's trichrome.

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video