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>
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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 a...

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 border="0" cellpadding="0" cellspacing="0" style="width:595px;" width="593">
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	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">10 ml Syringe</td>
			<td style="width:155px;">Thermo Scientific</td>
			<td style="width:145px;">03-377-23</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">5-0 prolene suture</td>
			<td style="width:155px;">Ethicon</td>
			<td>EH7229H</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">6-0 prolene suture</td>
			<td style="width:155px;">Ethicon</td>
			<td>8706H</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">8-0 Ethilon suture</td>
			<td style="width:155px;">Ethicon</td>
			<td>2808G</td>
			<td style="width:132px;"></td>
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			<td height="17" style="height:17px;width:163px;">Absorption Spears</td>
			<td style="width:155px;">Fine Science Tools</td>
			<td>18105-01</td>
			<td style="width:132px;"></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:163px;">BALB/c</td>
			<td style="width:155px;">The Jackson Laboratory</td>
			<td>Stock number 000651</td>
			<td style="width:132px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:163px;">Bepanthen Eye and Nose ointment</td>
			<td style="width:155px;">Bayer</td>
			<td>1578675</td>
			<td style="width:132px;">Eye ointment</td>
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		<tr height="34">
			<td height="34" style="height:34px;width:163px;">Betadine Solution</td>
			<td style="width:155px;">Betadine Purdue Pharma</td>
			<td>NDC:67618-152</td>
			<td style="width:132px;"></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Blunt Forceps</td>
			<td style="width:155px;">Fine Science Tools</td>
			<td>18025-10</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:163px;">Buprenex</td>
			<td style="width:155px;">Reckitt Benckiser</td>
			<td style="width:145px;">NDC Codes:&#160;12496-0757-1, 12496-0757-5</td>
			<td style="width:132px;">Buprenorphine</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:163px;">Cryoprobe 3mm</td>
			<td style="width:155px;">Brymill Cryogenic Systems</td>
			<td>Cry-AC-3 B-800</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Ethanol 70%</td>
			<td style="width:155px;">Th. Geyer</td>
			<td>2270</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Forceps curved</td>
			<td style="width:155px;">S&#38;T</td>
			<td>00284</td>
			<td style="width:132px;"></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Forceps fine</td>
			<td style="width:155px;">Fine Science Tools</td>
			<td>11251-20</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Forceps standard</td>
			<td style="width:155px;">Fine Science Tools</td>
			<td>11023-10</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Gross Anatomy Probe</td>
			<td style="width:155px;">Fine Science Tools</td>
			<td>10088-15</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Hair clipper</td>
			<td style="width:155px;">WAHL</td>
			<td>8786-451A ARCO SE</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:163px;">High temperature cautery kit</td>
			<td style="width:155px;">Bovie</td>
			<td>18010-00</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:163px;">ISOFLURANE</td>
			<td style="width:155px;">Henry Schein Animal Health</td>
			<td>029405</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">IV Catheter 20G</td>
			<td style="width:155px;">B. Braun</td>
			<td>603028</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Mini-Goldstein Retractor</td>
			<td style="width:155px;">Fine Science Tools</td>
			<td>17002-02</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:163px;">NaCl 0.9%</td>
			<td style="width:155px;">B.Braun</td>
			<td style="width:145px;">PZN 06063042&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Art. Nr.: 3570160</td>
			<td style="width:132px;">saline</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Needle holder</td>
			<td style="width:155px;">Fine Science Tools</td>
			<td>12075-14</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Needle Holder, Curved</td>
			<td style="width:155px;">Harvard Apparatus</td>
			<td>72-0146</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Novaminsulfon</td>
			<td style="width:155px;">Ratiopharm</td>
			<td>PZN 03530402</td>
			<td style="width:132px;">Metamizole</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Operating Board&#160;</td>
			<td style="width:155px;">Braintree Scientific</td>
			<td>39OP</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Replaceable Fine Tip</td>
			<td style="width:155px;">Bovie</td>
			<td>H101</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Scissors</td>
			<td style="width:155px;">Fine Science Tools</td>
			<td>14028-10</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Small Animal Ventilator</td>
			<td style="width:155px;">Kent Scientific</td>
			<td>RV-01</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:163px;">Spring Scissors - Angled to Side</td>
			<td style="width:155px;">Fine Science Tools</td>
			<td>15006-09</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Surgical microscope</td>
			<td style="width:155px;">Leica&#160;</td>
			<td>M651</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Transpore Surgical Tape</td>
			<td style="width:155px;">3M</td>
			<td>1527-1</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:163px;">Vannas Spring Scissors</td>
			<td style="width:155px;">Fine Science Tools</td>
			<td>15400-12</td>
			<td style="width:132px;"></td>
		</tr>
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			<td height="17" style="height:17px;width:163px;">Vaporizer&#160;</td>
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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 (<strong class="xfi...

Watch this Scientific Journal Video about A Cryoinjury Model to Study Myocardial Infarction in the Mouse at JoVE.com

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 ...

a-cryoinjury-model-to-study-myocardial-infarction-in-the-mouse

Research

JoVE Journal

Medicine

9.1K 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

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

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

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.

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

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 transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

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