We thank Christiane Pahrmann for her technical assistance. D.W. was supported by the Max Kade Foundation. T.D. received grants from the Else Kro&#776;ner Fondation (2012_EKES.04) and the Deutsche Forschungsgemeinschaft (DE2133/2-1_. S. S. received research grants from the Deutsche Forschungsgemeinschaft (DFG; SCHR992/3- 1, SCHR992/4-1).

Animals received humane care in compliance with the Guide for the Principles of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, and published by the National Institutes of Health. All animal protocols were approved by the responsible local authority (the University of California San Francisco (UCSF) Institutional Animal Care and Use Committee).
1. Animal care
<ol>
	<li>Obtain mice at the age of 14 weeks weighing approximately 27 g (e.g., from the Institute of Laboratory Animals). 
	NOTE: BALB/c mice are used for this article.</li>
	<li>Keep mice under conventional conditions in ventilated cabinets, feeding them standard mice chow and autoclaved water ad libitum.</li>
</ol>
2. Mouse preparation
<ol>
	<li>Use an induction chamber to anaesthetize mouse with isoflurane (3.5%).</li>
	<li>Remove the hair over the chest and neck using a hair trimmer.</li>
	<li>Place mouse in supine position on a heated pad and maintain anesthesia with a facemask covering mouth and nose of the mouse.</li>
	<li>Check for sufficient depth of anesthesia by pinching the hind feet and tail to verify an absence of reflexes.</li>
	<li>Inject subcutaneous buprenorphine (0.03 mg/kg) for analgesia.</li>
	<li>Spread the hind and fore limbs and fix their position using tape.</li>
	<li>With povidone iodine, disinfect the shaved area, followed by scrubbing with 80% ethanol. Repeat this step twice.</li>
	<li>Use a small scissor to make a midline skin incision from the lower third of the sternum to the chin.</li>
	<li>Use curved forceps and carefully separate the muscles around the neck to expose the trachea.</li>
	<li>Use a micro-scissor to perform a tracheotomy between the second and third cartilage rings.</li>
	<li>Set the ventilator to a ventilation frequency of 110/min with a tidal volume of 0.5 mL.</li>
	<li>Remove the facemask and insert a plastic cannula (20 G), connected to the ventilator, into the trachea. Ventilate the animal. 
	NOTE: Ensure that the ventilation cannula is not inserted too deep by confirming bilateral lung ventilation.</li>
	<li>Use cautery to detach the right pectoralis muscle from its sternal origin between the third and seventh ribs.</li>
	<li>Use side angled spring scissors to cut the fourth to sixth ribs as close as possible to the sternum.</li>
	<li>Cauterize the mammary artery, if bleeding is visible.</li>
	<li>Decrease isoflurane to 2.5%.</li>
	<li>Dissect underlying connective tissue to obtain a clear view into the chest cavity.</li>
	<li>Use blunt forceps to open the pericardium and expose the heart.</li>
	<li>Use a mini Goldstein retractor to spread the ribs and keep the chest cavity open.</li>
	<li>Lift the heart from the thoracic cavity with a blunt rod.</li>
	<li>Decrease the tension of the retractor to reduce chest opening and to keep the heart from falling back.</li>
	<li>Precool the cryoprobe (3 mm diameter) for 10 s.</li>
	<li>Apply the cryoprobe on the anterior left ventricle wall and freeze for 10 s to generate a left ventricular cryo-injury infarct. 
	NOTE: The cryoprobe can be applied to different heart walls depending upon the scientific question and need.&#160;</li>
	<li>Irrigate the cryoprobe with room temperature saline to detach the probe from the left ventricular wall.</li>
	<li>Use the retractor to enlarge the chest opening.</li>
	<li>Gently return the heart to the thoracic cavity with a blunt rod.</li>
	<li>Remove the retractor and connect the sternotomy with a single knot using 6-0 suture.</li>
	<li>Close the chest cavity using 6-0 running suture. Use a 10 mL syringe to evacuate any remaining air from the chest before tying the knot.</li>
	<li>Adapt the skin at the caudal edge and suture it to the point of the tracheal opening with running suture (5-0).</li>
	<li>Set isoflurane to 1.5% and wait until the animal gains spontaneous breathing.</li>
	<li>Remove the tracheal catheter and reapply the facemask onto the animal mouth and nose to maintain anesthesia.</li>
	<li>Close the tracheal incision with one 8-0 suture.</li>
	<li>Reposition the ventral neck muscles back to their position to cover the trachea.</li>
	<li>Complete the skin suture.</li>
	<li>Add metamizole to the drinking water (50 mg metamizole per 100 mL) for pain analgesia for 3 days and monitor the animal daily. 
	NOTE: The observation period for this model is 8 weeks. Be sure to follow your institution&#39;s guidelines regarding analgesia regimen.&#160;</li>
</ol>

<ol><li>Writing Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+Disease+and+Stroke+Statistics-2016+Update%3A+A+Report+From+the+American+Heart+Association%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association.</a> Circulation. 133 (4), 38-360 (2016).</li>
<li>de Alencar Neto, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphine%2C+Oxygen%2C+Nitrates%2C+and+Mortality+Reducing+Pharmacological+Treatment+for+Acute+Coronary+Syndrome%3A+An+Evidence-based+Review%5BTitle%5D)+AND+%22Cureus%22%5BJournal%5D)">Morphine, Oxygen, Nitrates, and Mortality Reducing Pharmacological Treatment for Acute Coronary Syndrome: An Evidence-based Review.</a> Cureus. 10 (1), 2114(2018).</li>
<li>Detry, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pathophysiology+of+myocardial+ischaemia%5BTitle%5D)+AND+%22European+Heart+Journal%22%5BJournal%5D)">The pathophysiology of myocardial ischaemia.</a> European Heart Journal. 17, Suppl G 48-52 (1996).</li>
<li>Ertl, G., Frantz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Healing+after+myocardial+infarction%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">Healing after myocardial infarction.</a> Cardiovascular Research. 66 (1), 22-32 (2005).</li>
<li>Jugdutt, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ventricular+remodeling+after+infarction+and+the+extracellular+collagen+matrix%3A+when+is+enough+enough%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough.</a> Circulation. 108 (11), 1395-1403 (2003).</li>
<li>Velagaleti, R. S., Vasan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+failure+in+the+twenty-first+century%3A+is+it+a+coronary+artery+disease+or+hypertension+problem%5BTitle%5D)+AND+%22Cardiology+Clinics%22%5BJournal%5D)">Heart failure in the twenty-first century: is it a coronary artery disease or hypertension problem.</a> Cardiology Clinics. 25 (4), 487-495 (2007).</li>
<li>Benjamin, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+Disease+and+Stroke+Statistics-2017+Update%3A+A+Report+From+the+American+Heart+Association%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association.</a> Circulation. 135 (10), 146-603 (2017).</li>
<li>Morrissey, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+method+of+standardized+myocardial+infarction+in+aged+rabbits%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Heart+and+Circulatory+Physiology%22%5BJournal%5D)">A novel method of standardized myocardial infarction in aged rabbits.</a> American Journal of Physiology-Heart and Circulatory Physiology. 312 (5), 959-967 (2017).</li>
<li>Degabriele, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Critical+appraisal+of+the+mouse+model+of+myocardial+infarction%5BTitle%5D)+AND+%22Experimental+Physiology%22%5BJournal%5D)">Critical appraisal of the mouse model of myocardial infarction.</a> Experimental Physiology. 89 (4), 497-505 (2004).</li>
<li>Chen, J., Ceholski, D. K., Liang, L., Fish, K., Hajjar, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Variability+in+coronary+artery+anatomy+affects+consistency+of+cardiac+damage+after+myocardial+infarction+in+mice%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Variability in coronary artery anatomy affects consistency of cardiac damage after myocardial infarction in mice.</a> American Journal of Physiology-Heart and Circulatory Physiology. 313 (2), 275-282 (2017).</li>
<li>Reichert, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Murine+Left+Anterior+Descending+%28LAD%29+Coronary+Artery+Ligation%3A+An+Improved+and+Simplified+Model+for+Myocardial+Infarction%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments+%3A+JoVE%22%5BJournal%5D)">Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction.</a> Journal of Visualized Experiments : JoVE. (122), e55353(2017).</li>
<li>Kim, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+murine+closed-chest+model+of+myocardial+ischemia+and+reperfusion%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments+%3A+JoVE%22%5BJournal%5D)">A murine closed-chest model of myocardial ischemia and reperfusion.</a> Journal of Visualized Experiments : JoVE. (65), e3896(2012).</li>
<li>Antonio, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Left+ventricle+radio-frequency+ablation+in+the+rat%3A+a+new+model+of+heart+failure+due+to+myocardial+infarction+homogeneous+in+size+and+low+in+mortality%5BTitle%5D)+AND+%22J+Card+Fail%22%5BJournal%5D)">Left ventricle radio-frequency ablation in the rat: a new model of heart failure due to myocardial infarction homogeneous in size and low in mortality.</a> J Card Fail. 15 (6), 540-548 (2009).</li>
<li>Ovsepyan, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+myocardial+infarction+in+mice%3A+methodology%2C+monitoring%2C+pathomorphology%5BTitle%5D)+AND+%22Acta+Naturae%22%5BJournal%5D)">Modeling myocardial infarction in mice: methodology, monitoring, pathomorphology.</a> Acta Naturae. 3 (1), 107-115 (2011).</li>
<li>Ciulla, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Left+ventricular+remodeling+after+experimental+myocardial+cryoinjury+in+rats%5BTitle%5D)+AND+%22Journal+of+Surgical+Research%22%5BJournal%5D)">Left ventricular remodeling after experimental myocardial cryoinjury in rats.</a> Journal of Surgical Research. 116 (1), 91-97 (2004).</li>
<li>Grisel, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+MRL+mouse+repairs+both+cryogenic+and+ischemic+myocardial+infarcts+with+scar%5BTitle%5D)+AND+%22Cardiovascular+Pathology%22%5BJournal%5D)">The MRL mouse repairs both cryogenic and ischemic myocardial infarcts with scar.</a> Cardiovascular Pathology. 17 (1), 14-22 (2008).</li>
<li>Duerr, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+myocardial+remodeling+between+cryoinfarction+and+reperfused+infarction+in+mice%5BTitle%5D)+AND+%22Journal+of+Biomedicine+and+Biotechnology%22%5BJournal%5D)">Comparison of myocardial remodeling between cryoinfarction and reperfused infarction in mice.</a> Journal of Biomedicine and Biotechnology. 2011, 961298(2011).</li>
<li>Ma, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intramyocardial+delivery+of+human+CD133%2B+cells+in+a+SCID+mouse+cryoinjury+model%3A+Bone+marrow+vs.+cord+blood-derived+cells%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">Intramyocardial delivery of human CD133+ cells in a SCID mouse cryoinjury model: Bone marrow vs. cord blood-derived cells.</a> Cardiovascular Research. 71 (1), 158-169 (2006).</li>
<li>Takagawa, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myocardial+infarct+size+measurement+in+the+mouse+chronic+infarction+model%3A+comparison+of+area-+and+length-based+approaches%5BTitle%5D)+AND+%22Journal+of+Applied+Physiology+%281985%29%22%5BJournal%5D)">Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches.</a> Journal of Applied Physiology (1985). 102 (6), 2104-2111 (2007).</li>
<li>van den Bos, E. J., Mees, B. M., de Waard, M. C., de Crom, R., Duncker, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+model+of+cryoinjury-induced+myocardial+infarction+in+the+mouse%3A+a+comparison+with+coronary+artery+ligation%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Heart+and+Circulatory+Physiology%22%5BJournal%5D)">A novel model of cryoinjury-induced myocardial infarction in the mouse: a comparison with coronary artery ligation.</a> American Journal of Physiology-Heart and Circulatory Physiology. 289 (3), 1291-1300 (2005).</li>
</ol>

The authors have nothing to disclose.

This article describes a mouse cryoinjury model to investigate ACS and related pharmacological and therapeutic options.
The most crucial step is the application of the cryoprobe on the cardiac tissue. Contact duration must be tightly controlled in order to obtain the optimal infarct size and to guarantee reproducible results. Prolonged cooling of the myocardium will lead to oversized infarcts or ventricular perforation. In contrast, shortened cooling time generates limited epicardial lesions and does not eliminate all resident cells. Hence, this can be confounding when studying regenerative cell transplantation.
Compared to other cryoinfarction methods20, the open chest approach described in this article has the advantage that the infarct can be induced freely on different positions of the heart. Moreover, this approach facilitates therapeutic cell injection or patch applications, as the infarct border is visible, and the site of cell transplantation can be chosen accordingly.&#160;
A drawback of this model is the etiology of myocardial injury. Cryoinjury results in cell death due to the generation of ice crystals disrupting the cell membrane rather than a direct ischemia. In addition, the direction of injury is usually from epicardium inwards, whereas ischemic infarcts tend to propagate outwards from the endocardial to the epicardial layer. Therefore, this model is limited to study the pathophysiological mechanisms of myocardial ischemia or to imitate the ischemia-reperfusion setting.
In conclusion, the model described here is inexpensive, easy to perform, can be established fast and reliably. Cardiomyocyte necrosis and subsequent scar formation develop over time, resulting in progressive impaired pump function and electrical conductance. Well-controllable infarct size, shape and location make this model ideal to evaluate experimental interventions aiming to restore cardiac function or cardiac regeneration. Successfully tested treatment options should be further confirmed in large animal studies.&#160;

Acute coronary syndrome (ACS) is the leading causes of death in the Western world1,2. Acute occlusion of the coronary arteries leads to activation of ischemic cascade and necrosis of the affected cardiac tissue3. Damaged myocardium is gradually replaced by non-contractile scar tissue, which manifestz clinically as a heart failure4,5. Despite recent advances in the treatment of ACS, the prevalence of ACS and ACS-related heart failure is rising, and therapeutic options are limited6,7. Therefore, developing animal models to study ACS and its complications are of immense interest.
To date, the most widely used animal model to study ACS and ACS-induced myocardial remodeling is the ligation of the left descending coronary artery (LAD). Ligation of the LAD leads to acute ischemia of the myocardium, similar to human myocardial tissue during ACS.&#160; However, inconsistent infarct sizes remain the Achilles&#39;&#160;heel of LAD ligation. Surgical variation and anatomical variability of the LAD lead to inconsistent infarct sizes and hinder the reproducibility and replicability of this procedure8,9,10. In addition, LAD ligation has a high intra- and postsurgical mortality. Despite recent endeavors to improve reproducibility and reduce mortality11,12, large numbers of animals are still needed to properly evaluate anti-remodeling therapies.
Alternative models of ACS have been proposed and studied over the recent years, including radio-frequency13, thermal14 or cryogenic injuries15,16,17,18. Current cryoinjury methods apply a metal rod pre-cooled in liquid nitrogen to damage the subject&#39;s cardiac tissue15,16. However, this procedure needs to be repeated several times to generate a sufficient infarct size. Due to the high conductivity and low heat capacity of the rod compared to the tissue, the probe warms quickly, and the tissue is cooled (and thus infarcted) heterogeneously. To overcome these limitations, we describe herein a cryoinfarction model utilizing a hand-held liquid nitrogen delivery system. This model is reproducible, easy to perform and can be established fast and reliably. A reproducible transmural infarct lesion independent of coronary anatomy is generated, which eventually leads to cardiac failure. This method is especially suitable to study the remodeling process for the evaluation of novel therapeutic pharmacological and tissue engineering-based strategies.

<table><tbody><tr><td>10 ml Syringe</td><td>Thermo Scientific</td><td>03-377-23</td><td></td></tr><tr><td>5-0 prolene suture</td><td>Ethicon</td><td>EH7229H</td><td></td></tr><tr><td>6-0 prolene suture</td><td>Ethicon</td><td>8706H</td><td></td></tr><tr><td>8-0 Ethilon suture</td><td>Ethicon</td><td>2808G</td><td></td></tr><tr><td>Absorption Spears</td><td>Fine Science Tools</td><td>18105-01</td><td></td></tr><tr><td>BALB/c</td><td>The Jackson Laboratory</td><td>Stock number 000651</td><td></td></tr><tr><td>Bepanthen Eye and Nose ointment</td><td>Bayer</td><td>1578675</td><td>Eye ointment</td></tr><tr><td>Betadine Solution</td><td>Betadine Purdue Pharma</td><td>NDC:67618-152</td><td></td></tr><tr><td>Blunt Forceps</td><td>Fine Science Tools</td><td>18025-10</td><td></td></tr><tr><td>Buprenex</td><td>Reckitt Benckiser</td><td>NDC Codes:&amp;nbsp;12496-0757-1, 12496-0757-5</td><td>Buprenorphine</td></tr><tr><td>Cryoprobe 3mm</td><td>Brymill Cryogenic Systems</td><td>Cry-AC-3 B-800</td><td></td></tr><tr><td>Ethanol 70%</td><td>Th. Geyer</td><td>2270</td><td></td></tr><tr><td>Forceps curved</td><td>S&amp;amp;T</td><td>00284</td><td></td></tr><tr><td>Forceps fine</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Forceps standard</td><td>Fine Science Tools</td><td>11023-10</td><td></td></tr><tr><td>Gross Anatomy Probe</td><td>Fine Science Tools</td><td>10088-15</td><td></td></tr><tr><td>Hair clipper</td><td>WAHL</td><td>8786-451A ARCO SE</td><td></td></tr><tr><td>High temperature cautery kit</td><td>Bovie</td><td>18010-00</td><td></td></tr><tr><td>ISOFLURANE</td><td>Henry Schein Animal Health</td><td>029405</td><td></td></tr><tr><td>IV Catheter 20G</td><td>B. Braun</td><td>603028</td><td></td></tr><tr><td>Mini-Goldstein Retractor</td><td>Fine Science Tools</td><td>17002-02</td><td></td></tr><tr><td>NaCl 0.9%</td><td>B.Braun</td><td>PZN 06063042&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Art. Nr.: 3570160</td><td>saline</td></tr><tr><td>Needle holder</td><td>Fine Science Tools</td><td>12075-14</td><td></td></tr><tr><td>Needle Holder, Curved</td><td>Harvard Apparatus</td><td>72-0146</td><td></td></tr><tr><td>Novaminsulfon</td><td>Ratiopharm</td><td>PZN 03530402</td><td>Metamizole</td></tr><tr><td>Operating Board&amp;nbsp;</td><td>Braintree Scientific</td><td>39OP</td><td></td></tr><tr><td>Replaceable Fine Tip</td><td>Bovie</td><td>H101</td><td></td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Small Animal Ventilator</td><td>Kent Scientific</td><td>RV-01</td><td></td></tr><tr><td>Spring Scissors - Angled to Side</td><td>Fine Science Tools</td><td>15006-09</td><td></td></tr><tr><td>Surgical microscope</td><td>Leica&amp;nbsp;</td><td>M651</td><td></td></tr><tr><td>Transpore Surgical Tape</td><td>3M</td><td>1527-1</td><td></td></tr><tr><td>Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15400-12</td><td></td></tr><tr><td>Vaporizer&amp;nbsp;</td><td>Kent Scientific</td><td>VetFlo-1205S</td><td></td></tr></tbody></table>

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.

The cryoinjury infarct model is suitable to study ACS and its complications. Low mortality rates and efficient postsurgical recovery is seen in this model. Cryoinjury induced myocardial damage leads to reduced cardiac function, electrical uncoupling, and transmural remodeling.
Echocardiography can be used to monitor cardiac function noninvasively in vivo. In cryo-injured hearts, echocardiography demonstrates significantly reduced ejection fraction and fractional area change (Figure 1a-c). Functional impairment continues from day 7 post-surgery until the observational endpoint of 56 days. &#160;
Detailed cardiac function can be assessed invasively through pressure volume loop (PV-loop) analysis. A 1.2 Fr conductance catheter is introduced into the left ventricle, and the left ventricular pressure is plotted against the left ventricular volume. Hemodynamic parameters such as stroke volume, stroke work, cardiac output, and preload-adjusted maximal power can be calculated. As shown in Figure 1d-h, cryoinfarction leads to impaired left ventricle (LV0 function, which is reflected as a decrease in stroke volume, stroke work, cardiac output and preload-adjusted maximal power.
To study cardiac electrophysiology, optical mapping can be performed ex vivo. Hearts are removed, perfused with Langendorff perfusion technique, and stained with a fluorescent voltage sensitive dye. Cryoinjured hearts demonstrate blockage of electrical conduction at the border of injury, indicating local electrical uncoupling (Figure 1i).
Histological staining with Masson&#39;s trichrome demonstrates transmural fibrotic tissue formation at the site of injury (Figure 2a). Infarct size can be calculated by measuring infarct scar area or midline scar length19 (Figure 2b). Immunofluorescence staining against alpha- sarcomeric actinin (cardiomyocyte marker) and collagen-I confirm fibrotic remodeling and loss of cardiomyocytes at the site of injury (Figure 2c).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59958/59958fig1.jpg" /> 
Figure 1: Functional and electrophysiological analysis of cryoinjured heart. Representative two-dimensional echocardiography images taken pre-operatively (D0) and at post-operative day 7 (D7), 28 (D28), and 56 (D56). (a) The top panel shows the parasternal long-axis view at end-diastole and the bottom panel at end-systole. (b, c) Ejection Fraction (EF) and Fractional Area Change (FAC) decrease after cryo-infarction and remained diminished over time Cardiac function was assessed invasively by pressure volume curve analysis. (d-g) Day 56 post injury stroke volume (SV), stroke work (SW), cardiac output (CO), and preload-adjusted maximal power (PAMP) were significantly lower than in pre-operative native animals. (h) Representative PV-loops from native and 56 days post-surgery animals showed characteristic right shift and decline in amplitude of the pressure signal following thoracic vena cava (TVC) occlusion. (i) Isochrone map of cardiac optical mapping from native and cryoinjured hearts 14 days post-surgery. Top and bottom panels show hearts paced from the apex and base, respectively. Infarct area is marked by dashed white line. Intergroup differences were assessed by one-way analysis of variance (ANOVA) with Bonferroni&#39;s post-Hoc test or Student&#8217;s t-test. N = 3 animals. * indicates p &#60; 0.05. The error bars represent the standard deviation (SD). ESPVR = end-systolic pressure volume relationship; EDPVR = end-diastolic pressure volume relationship. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59958/59958fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59958/59958fig2.jpg" /> 
Figure 2: Histologic assessment of native and cryo-injured hearts. (a) Masson&#8217;s trichrome staining shows collagen deposition (green) in the infarcted area. The infarcted percentage of the left ventricle was measured as (b) area and (c) midline infarct length. (d) Immunofluorescence staining demonstrates increased collagen-I deposition with concomitant loss of cardiomyocytes in infarcted area. LV = left ventricle; RV = right ventricle; endo = endocardial; epi = epicardial.&#160; N = 3 animals. Error bars show SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59958/59958fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A Cryoinjury Model to Study Myocardial Infarction in the Mouse

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 ...

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Nanyang Technological University

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9.2K Views.  University Heart Center. This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

Video: A Cryoinjury Model to Study Myocardial Infarction in the Mouse

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

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

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

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 Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

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 ...

Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In order to explore the pathophysiological changes after myocardial infarction and design future treatments, research models of MI are required. Permanent ligation of the left coronary artery (LCA) in mice is a popular model to investigate cardiac function and ventricular remodeling post MI. Here we describe a less invasive, reliable, and reproducible surgical murine MI model by permanent ligation of the LCA. Our surgical model comprises of an easily reversible general anesthesia, endotracheal intubation that does not require a tracheotomy, and a thoracotomy. Electrocardiography and troponin measurement should be performed to ensure MI. Echocardiography at day 28 after MI will discern heart function and heart failure parameters. The degree of cardiac fibrosis can be evaluated by Masson's trichrome staining and cardiac MRI. This MI model is useful for studying the pathophysiological and immunological alterations after MI.

Presented here is a surgical procedure for permanent ligation of the left coronary artery in mice. This model can be used to investigate the pathophysiology and associated inflammatory response after myocardial infarction.

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In ...