Name: myocardial infarction in neonatal mice, a model of cardiac regeneration
Uploaded: 2016-05-24T15:00:00
Description: Watch this Scientific Journal Video about Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration at JoVE.com

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
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	<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 displ...

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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.

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			<td height="21" style="height:21px;width:261px;">8-0 Nylon Suture</td>
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			<td height="21" style="height:21px;width:261px;">Isoflurane</td>
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			<td height="21" style="height:21px;width:261px;">Isoflurane Chamber</td>
			<td style="width:244px;">Made in Feng laboratory</td>
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			<td height="21" style="height:21px;width:261px;">Bead Sterilizer</td>
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			<td height="42" style="height:42px;width:261px;">2,3,5-Triphenyltetraolium chloride (TTC)</td>
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			<td height="22" style="height:23px;width:261px;">Stereomicroscope SteREO Discovery. V8</td>
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			<td height="21" style="height:21px;width:261px;">AxioVision 8.0</td>
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			<td height="21" style="height:21px;width:261px;">Axiocam Icc5</td>
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			<td height="21" style="height:21px;width:261px;">Heat pad</td>
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			<td height="21" style="height:21px;width:261px;">Sterile Gloves</td>
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			<td height="21" style="height:21px;width:261px;">Ice</td>
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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 ...

The overall goal of this surgical procedure is to reproducibly induce myocardial infarction in neonatal mice. This method can help answer key questions in cardiac regeneration such as what mechanisms support regeneration in neonates and how can adult heart regeneration be promoted after myocardial infarction. The main advantage of this technique is that the left anterior descending coronary artery, or LAD, can be visualized to ensure highly reproducible LAD ligation in the neonatal mouse heart.The changes in cardiac function after LAD ligation can be assessed by echocardiography. Before beginning the procedure place an anesthetized pup in the right lateral decubitus position on ice under the operating microscope and confirm the appropriate level of sedation by a lack of response to toe and tail pinch. Then gently disinfect the chest with povidone iodine followed by an ethanol swab.Next use a pair of forceps to carefully lift the skin between the xiphoid and left axilla and make an incision a few millimeters below the left fore leg. Using scissors cut through the underlying pectoral muscle layer, then open a pair of forceps along the muscles and ribs in the fourth intercostal space to perform a gentle thoracotamy. Visualize the left anterior descending coronary artery emerging from the left auricle behind the pulmonary vein and descending beyond the great cardiac vein.Location of the coronary artery is critical for the reproducibility of the technique. To ensure visualization of the artery maintain the mouse body temperature between eight and 15 degrees Celsius during the procedure. Then use a 007 mL diameter needle to pass an 11-0 nylon suture under the artery through the mid ventricle below the left auricle and ligate the artery.The ischemia is confirmed by the blanching of the myocardium below the suture site. Next use 0.15 mL diameter needles to place two 8-0 nylon rib sutures without tying to ensure both sutures are placed without injury to internal organs such as the lungs. Then tie the rib sutures to close the thoracic incision and move the mouse onto a sterile surgical towel over a 37 degrees Celsius heating pad.Close the muscle and skin with more 8-0 nylon sutures. Then use an ethanol swab to gently remove the povidone iodine and blood. When the animal has fully recovered from the anesthesia wipe it with bedding from the foster mother's cage to reduce the chance of maternal rejection and cannibalization and return the pup to the foster mother.Observe the behavior of the foster mother toward the pup every 10 to 15 minutes for two to three hours to confirm the acceptance of the pup. To measure the myocardial infarct size after four to six hours with the foster mother, the pup is euthanized by isofluorane overdose followed by decapitation. Next, 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.Holding the scissors at a transverse angle carefully cut through the diaphragm from the left to the right side. Then grasp the rib cage and cut the right side of the ribs and musculature along the right mid axillary line. Use the scissors to remove all of the vascular connections to the heart.Then grasp the base of the heart and remove it from the chest cavity. Using a surgical carbon steel razor blade section the heart along the short axis at the midpoint between the suture and the cardiac apex followed by a second cut at the suture. Then place the sections in 1%TTC at room temperature for 10 to 15 minutes, carefully watching the specimens to avoid overstaining.24 to 48 hours after LAD surgery cardiac function is assessed by echocardiography. Secure the pup in the supine position on a heated dock with its nose in an anesthesia cone and apply pre warmed echo gel to the left thoracic area. Next obtain a peristernal long axis view of the left ventricle below the suture with a 40 Mhz ultrasound probe.Then turn the probe 90 degrees and acquire a parasternal short axis view recording the end mode echocardiographic images. The end diastolic and end systolic left ventricular internal diameters can the be measured from the short axis end mode images. Upon histological examination at three days post myocardial infarction the typical evidence of an infarction including the infiltration of inflammatory cells is observed.At day seven the left ventricular tissue appears normal and by 21 the cardiac tissue has completely regenerated. In a total of 13 left anterior descending coronary artery ligation surgeries, 100%of the hearts were infarcted with an average infarct size of 36%To further confirm the infarct induction echocardiography can be performed 24 and 48 hours after the procedure. In this representative experiment at 24 hours post myocardial infarction the ejection fraction and the fractional shortening were significantly reduced.By 48 hours the ejection fraction and the fractional shortening were further reduced compared to the control animals which demonstrated no reduction at all. Once mastered this technique can be completed in 10 to 15 minutes if it is performed properly. While implementing the myocardial infarction it is important to remember to operate quickly and accurately to ensure the procedure is completed within the time window during which the LAD is visible.Following this procedure other methods like histological analysis can be performed to answer additional questions about how different treatments affect cardiac regeneration. The development of this technique paves the way for researchers in the field of cardiac biology to explore cardiac regeneration in mice. After watching this video you should have a good understanding of how to reproducibly induce myocardial infarction in neonatal mice by visualizing and ligating the LAD.

Research

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Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

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Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

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Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...