This work was supported by Interdisciplinary Centre for Clinical Research IZKF Aachen (junior research group to E.A.L.) within the faculty of Medicine at RWTH Aachen University. We are grateful Dr. Rusu and Ashley Christina Vourakis for critical review of the manuscript and Mrs. Roya Soltan for the professional help with immunohistochemistry staining.

NOTE: Experiments presented in this paper are performed accordingly to the German low and to the European animal care guidelines. The animals are bred in the Animal facility of Institute for Laboratory Animal Science, Universiy hospital Aachen, Germany, under supervision of Prof. Dr. R. Tolba and Dr. A. Teubner (animal welfare officer).
1. Animal Care
<ol>
	<li>Keep the mice in a specialized care unit, assuring proper access to food and specialized veterinary control and treatment. If the animals are moved or purchased from outside, please assure one-week accommodation before undergoing the procedure.</li>
</ol>
2. Intubation
<ol>
	<li>Anesthetize 8-10 weeks old male C57Bl/6 wild type mice, 25-27 g using intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Monitor the level of anesthesia by toe pinch reflexes. Place vet ointment on the eyes to prevent dryness during the procedure.</li>
	<li>Manage pain therapy with buprenophine 0.1 mg/kg body weight subcutaneously before starting the surgical procedure, following the animal care guidelines of your institution.</li>
	<li>Assure the maintenance of sterile conditions to avoid infections during surgery by using sterile materials and instruments.</li>
	<li>Place the anesthetized mouse in a supine position on a heated surgery table. Remove the hair from both ventral neck area and left half of the thorax using a small razor and disinfect with 70% alcohol prior to incision.
	<ol>
		<li>Perform a small median incision of 0.5 cm using surgery scissors in the center of the neck. Under the skin, go through the 2 fatty bodies with sterile curved forceps and visualize the trachea under stereomicroscope through the transparence of the covering muscle.</li>
	</ol>
	</li>
	<li>Introduce orally the intubation cannula into the trachea under view by using the stereomicroscope (Figure 1A). Distinguish the metal cannula through the transparent tissue. And check, the position and location during the operation at any moment (Figure 1B).</li>
	<li>Connect the cannula to the small animal ventilator and adjust ventilation settings according manufactures guidelines (tidal volume between 100-150 &#181;l and a respiration rate between 100-150 per min).</li>
</ol>
3. Myocardial Infarction Induction
<ol>
	<li>Perform a skin incision less than 0.5 cm in the middle of a line between xyphoid and left axila. Use forceps to separate the muscle layer from the underlying ribs.</li>
	<li>Perform a small incision between ribs by using a small scissor until the thoracic cavity is opened17. For chronic infarction, perform the incision in the 5th intercostal space (Figure 1C) and/or for the ischemia/reperfusion model, in the 4th intercostal space (Figure 1D): for an easier approach number from below the 2th and the 3th intercostal space, respectively).</li>
	<li>Place the retractors into the incision to open thoracic cavity and to visualize the heart.</li>
	<li>Carefully remove the pericardium to prevent excessive fibrotic processes.</li>
	<li>Visualize the left descending coronary artery (LAD) as a deep positioned light red vessel. If the LAD cannot be visualized, consider some reference points to increase the reproducibility.
	<ol>
		<li>For chronic infarction model, place the ligature in the middle of the ventral side of the heart (between the auricle and apex), having as reference the vein as shown in Figure 1C. Bind both branches of the artery using 0/7 silk suture to obtain a transmural anterior and posterior infarction. The gray color indicates the position of the ligature and can be repeated if needed (Figure 1C).</li>
		<li>For ischemia/reperfusion model, place the ligature under the auricle, over the main body of LAD (Figure 1D). The ligature is located over a silicon tube to protect the integrity of the vessel. The gray color indicates the infarcted area and should appear in the entire heart (Figure 1D). Place temporal sutures on the ribs during the ischemia period and moisten using a compress to avoid tissue drying. After ischemia, remove the silicon tube and cut the suture with small scissors to visualize the reperfusion.</li>
	</ol>
	</li>
	<li>Beside the anesthetics and analgesics used at the beginning of procedure (steps 2.1 and 2.2), use 0.5% isoflurane during surgery to ensure the proper comfort of the animal, or follow the animal care guidelines of your institution.</li>
</ol>
4. Suture and Recovery
<ol>
	<li>Eliminate the residual air from thorax by filling with warm isotonic salt solution.</li>
	<li>Close the thorax with 3 sutures 0/6 (as shown in Figure 2A and 2B). Position the medial sutures at an angle of 90&#176;, to assure a sealed closure of the ribs, as shown in Figure 2 (Figure 2A, B).</li>
	<li>Close the muscle layer with 2 sutures (Figure 2C) and the skin with 3-4 sutures 0/6 (Figure 2D). Perform these sutures separately to obtain a proper window for further echocardiographic measurement.</li>
	<li>Disconnect the intubation cannula from the ventilator and allow spontaneous breath. For later identification, mark the mouse using the local system (ask the animal welfare officer from your institution).</li>
	<li>Lay down the mouse on the left side under the red lamp until it wakes up. Do not leave an animal unattended until it has regained sufficient consciousness. Do not allow an animal that has undergone surgery to be in the company of other animals until fully recovered.</li>
	<li>Manage pain therapy with buprenophine 0.1 mg/kg body weight, subcutaneously for the next 3 days, following the animal care guidelines of your institution.</li>
</ol>
5. Analysis of the Myocardial Infarction
<ol>
	<li>Regularly monitor the heart function by means of echocardiography (Figure 3A): the ejection fraction, fractional shortening, cardiac output and heart dimensions.</li>
	<li>Anesthetize the animals using intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Confirm proper anesthetization prior to surgery by the lack of reflexes.</li>
	<li>Open the thoracic cavity and excise the heart, placing it in sterile PBS solution washing extensively the remaining blood.</li>
	<li>If needed, collect the blood directly from the heart by avoiding the injury of the infracted regions, or after removal of the heart, from the thoracic cavity.</li>
	<li>After washing, stop the heart in diastola in saturated KCl solution (steril filtered 3M KCl in PBS). For histological analysis fix the heart in 10% formalin and proceed with Step 5.7.</li>
	<li>If necessary, measure the viability of the cardiac cells by Evans-Blue/ Triphenyl tetrazolium chloride (TTC) staining. After rebuilding the ligature at the initial place, perfuse the heart with 200 &#181;l 1% Evans Blue Solution using an aortic cannula and freeze the heart in a small plastic bag at -20 &#176;C, without washing.
	<ol>
		<li>After 2 hr, perform 5 transversally slides using a sharp scalpel and incubate them for 10-15 min in TTC solution at 37 &#176;C, as described by manufactured. Fix the slides for 10 min in 10% formalin and put them between the microscopic slides for further analysis.</li>
		<li>Embed the heart tissue in paraffin, by positioning the heart on the tip, to perform transversal sectioning. Perform serial section of 5 &#181;m. Collect the first 20 sections and discard the next 300 &#181;m. Continue the section protocol until the mitral valve level has been reached (Figure 3A, B). Serial sections, 400 &#181;m apart along the entire heart are collected and can be stained for the qualitative and quantitative analysis.</li>
	</ol>
	</li>
	<li>Measure the infarction size using Gomori&#39;s one-step staining6-8.</li>
	<li>Analyze angiogenesis, collagen content or inflammatory cells recruitment in serial section using usual immunohistological staining.</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ccr1+deficiency+reduces+inflammatory+remodelling+and+preserves+left+ventricular+function+after+myocardial+infarction.">Ccr1 deficiency reduces inflammatory remodelling and preserves left ventricular function after myocardial infarction.</a> J Cell Mol Med. 12 (2), 496-506 (2008).</li><li>Liehn, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+monocyte+chemotactic+protein-1/chemokine+CC+motif+ligand-2+competitor+limiting+neointima+formation+and+myocardial+ischemia/reperfusion+injury+in+mice.">A new monocyte chemotactic protein-1/chemokine CC motif ligand-2 competitor limiting neointima formation and myocardial ischemia/reperfusion injury in mice.</a> J Am Coll Cardiol. 56 (22), 1847-1857 (2010).</li><li>Liehn, E. 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The authors have nothing to disclose.

During the procedure, there are some critical points to be noted: the intubation, the opening the thoracic cavity and the LAD ligature. The first critical step is the intubation of the animal before experiements. Many groups are using a vertical support for fixing the mouse and a source of light to insert the cannula directly into the trachea. This method has uncertainty concerning the correct insertion of the cannula into the trachea and is the most prone to failure by the novices. Making a small incision, the position of the cannula can be controlled during the entire maneuver, thus decreasing the default rate. Moreover, the tracheotomy is surpassed, thus decreasing complications and reducing the time of operation.
The next critical step is the opening of the thoracic cavity. The median sternotomy represents a high-risk maneuver delaying the recovery of the animals. The lateral left incision implying the cutting of 2-3 ribs15, leads to deficient recovery and increased mortality. We used in the model small, discrete incision between the ribs offering minimal burden. The animals recover very quickly after the surgery and do not present defects or disturbed healing. The lower inter-costal space is taken as a reference point. Considering this, the proper and differentiated access to the ligature place for chronic and ischemia/reperfusion model, does not raise serious problems.
The ligature itself represents the most critical step. The left descending coronary artery is hard to be visualized, and often needs to be bound without view. Therefore, some anatomic reference points are pointed out to help the surgeon to perform the correct ligation. For the chronic infarction model, the ligature is placed in the middle of the ventral side of the heart, between the auricle and the apex, above the ending of the major anterior vein (Figure 2B). The efficiency can be controlled by visualizing the appearance of the grey color in the affected areas. If the infarcted area appears anterior and does not include the posterior wall, a new suture can be placed to the left of the first suture. The main root of LAD is always visible under the auricle18, and therefore does not present serious problems in detecting this part. However, the auricle presents the major risk of bleeding and needs to be handled carefully.
The procedure is limited by existence of appropriate equipment. A ventilator and appropriate anesthesia system for the small animals are expensive and require connections to gas and ventilation system of the room. Further, a close supervision of the animals is necessary in the first week after procedure to detect the possible clinical. To examine the heart function during the experiment, high-resolution ultrasound, complex Langendorf perfusion-system, or small intraventricular catheter measurements are required, involving high costs and additional expertise.
Considering the myocardial infarction, there is no alternative methods available to reproduce the complexity of the events in vitro. Depending on the point of interest, ex vivo perfusion of an isolated heart in Langendorff system provides information about the contractility, heart function and myocardial viability in response to different stimuli or drugs. However, it excludes all interferences of blood components and immune system, and it is not indicated for long-studies of remodeling and healing after myocardial infarction.
After performing the myocardial infarction procedure, all other functional analysis can be carried out, like intraventricular pressure measurements, ultrasound (small animal ultrasound systems) or isolated heart Langendorff-perfusion. Moreover, all biological and molecular analysis can be performed to identify cells, proteins, mRNAs, microRNAs, genes or other biomarkers, which can be used as therapeutic targets to develop new treatment strategies for myocardial infarction.

Myocardial infarction is one of the main causes of death in industrialized countries. Despite undeniable progress of diagnostic and therapeutic approaches, cardiovascular diseases are still the major cause of mortality. Given the improved life expectancy and life-related risks, a continuous increase in the incidence of cardiovascular diseases is expected in the future. Therefore, there is a strong need to establish and validate novel approaches for the treatment of cardiovascular disease. The information of human studies suffer from its limitations, these studies generally are insufficient to explain and understand the mechanisms at the molecular level, being unable to provide solutions to these major health problems.
Moreover, basic research has been limited due to complexity and difficulty to reproduce the mechanisms of cardiovascular disease in the laboratory. Therefore, to increase our knowledge about the pathophysiology of cardiovascular diseases, it is essential to validate animal models1,2. However, to identify all cascades of molecular events involved in the healing after myocardial infarction, analysis at different time points is necessary, causing a large number of animals experiments.
Myocardial infarction experiments are often performed by using animal models. Inducing myocardial infarction in small animals3-11 is the most suitable and efficient model employed to investigate cellular and molecular events than large animal models. Moreover, no other species presents the availability of transgenic or knockout strains as mice12. These mouse models are highly useful in other diseases, including cardiovascular pathologies (such as atherosclerosis, in stent restenosis)13,14. In addition, the low pregnancy period and the high number of progenies qualify mouse models as most attractive system to study molecular mechanisms of myocardial infarction12.
Nevertheless, the size of the heart in mice expects high precision of manipulation during microsurgery. Teaching such qualified and skilled surgery personnel is a time-consuming and work-intensive process. Therefore, we herein present a detailed microsurgery procedure, including tips and tricks to guide collaborators even with average qualifications, such as students or technicians to perform the complex myocardial infarction model in mice.
Initially, intubation is performed by means of a short cannula without using the tracheotomy. The thoracic incision is located in the intercostal area, avoiding injury of ribs or/and surrounding tissue. This sub-step is highly relevant to assure fast recovery and healing15. The ligature is made differential for chronic ischemia and ischemia/reperfusion models, for a high survival rate while still maintaining a significant infarction size. Our experience shows that using silk suture assures a higher reproducibility compared to cryo-injuries16.
In conclusion, the method described here is applicable in both chronic ischemia and ischemia/reperfusion models in small animals. The tips and tricks presented in this procedure are meant to enable personnel with even low or average qualification to apply it in small animal models.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereomicroscope</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">SZ/X9</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mouse ventilator</td>
			<td style="width:71px;">Harvard Apparatus</td>
			<td style="width:119px;">730043</td>
			<td style="width:167px;">Model Minient 845</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Dual Anesthesia System (Tabletop Version)</td>
			<td style="width:71px;">Harvard Apparatus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Selfcontained isofluranebased anesthesia unit for use on lab tables, with a compact 8&#34; x 11&#34; footprint.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Intubation cannula</td>
			<td style="width:71px;">Harvard Apparatus</td>
			<td style="width:119px;">732737</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Forceps</td>
			<td style="width:71px;">FST, Germany</td>
			<td style="width:119px;">9119700</td>
			<td style="width:167px;">standard tip curved 0.17 mm x 0.1 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scissors</td>
			<td style="width:71px;">FST, Germany</td>
			<td style="width:119px;">9146011</td>
			<td style="width:167px;">straight</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vannas scissor</td>
			<td style="width:71px;">Aesculap, Germany</td>
			<td style="width:119px;">OC 498 R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Retractors</td>
			<td style="width:71px;">FST, Germany</td>
			<td>1820010</td>
			<td>2.5mm wide</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Retractors</td>
			<td style="width:71px;">FST, Germany</td>
			<td>1820011</td>
			<td>5mm wide</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wire handles</td>
			<td style="width:71px;">FST, Germany</td>
			<td>1820005</td>
			<td>&#160;10cm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wire handles</td>
			<td style="width:71px;">FST, Germany</td>
			<td>1820006</td>
			<td>&#160;14cm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ketamine 10%</td>
			<td style="width:71px;">CEVA, Germany</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Xylazine 2%</td>
			<td style="width:71px;">Medistar, Germany</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bepanthene eye and nose cream</td>
			<td style="width:71px;">Bayer, Germany</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="105" rowspan="3" style="height:105px;width:216px;">Silicon tube</td>
			<td rowspan="3" style="width:71px;">IFK Isofluor, Germany</td>
			<td rowspan="3" style="width:119px;">custommade&#160; product</td>
			<td style="width:167px;">diameter 500&#181;m</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">section thickness 100 &#181;m</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">polytetrafluorethylene catheter</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">PROLENE Suture 6/0&#160;</td>
			<td style="width:71px;">ETHICON</td>
			<td style="width:119px;">8707H</td>
			<td style="width:167px;">polypropylene monofilament suture, unresorbable, needle CC1, 13mm, 3/8 Circle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">7/0 Silk</td>
			<td style="width:71px;">Seraflex</td>
			<td style="width:119px;">IC 1005171Z</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultrasound&#160;</td>
			<td style="width:71px;">Vevo, Canada</td>
			<td style="width:119px;">770 Vevo</td>
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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.

The myocardial infarction procedure occurs within 25-30 min and shows a mortality rate of 10%. After surgery, the mice recover from anesthesia within the next 15 min. No physical impairment was observed to the operated mouse. However, there is a higher risk of heart rupture one week after post-chronic myocardial infarction, if the repairing processes are disturbed during the inflammatory phase. Since heart is able to change significantly its dimensions during the pumping, it is important for all the collected hearts to be stopped in the same position, for example in diastola. This can be achieved by perfusing the heart with saturated KCl solution. Increased extracellular K+ concentration blocks the ionic pumps, decreases the membrane resting potential of cardiac cells, resulting in a diastolic arrest of cardiac activity.
The infarction area can be seen in ultrasound analysis (Figure 3A, lower panel). In comparison to the normal myocardium, ischemic regions appear thin and hypokinetic (Figure 3A, upper panel). Depending on the model used, the infarction size will differ. The chronic infarction model induces circular, transmural infarction of the apex (Figure 3B), while the ischemia/reperfusion induces a thin, middle-wall and throughout all heart (Figure 3C). There are many methods to determine infarction size. If the aim is to analyze the direct effect on cardiac viability, an Evans-Blue/TTC staining18 is indicated to be performed at least 2 hours after reperfusion, to be able to see any changes in the myocardium. Sections can be analyzed immediately (Figure 3B, middle panel) after staining or can be kept between glass slides in formalin for 2-3 days (Figure 3C, middle panel). The blue area represents the healthy myocardium, not affected by ischemia. The red area represents the viable myocardium inside the ischemic area (risk myocardium), and the white area represents the dead tissue. Usually, the infarction size is expressed as percent from the risk area.
The mature scar resulting after remodeling processes can be easily measured by immunohistolgy using Gomori&#39;s one-step staining. Blue-stained infarcted and red-stained healthy ventricular areas (Figure 3B and C, right panels) are determined in the first section from each level until the mitral valve. To avoid the variation due to binding of LAD at different levels, the infarction from all section is considered and expressed as a percentage of total left ventricular volume. An infarction volume of 15-20% in chronic infarction model and of 10-15% after ischemia/reperfusion model can be achieved. Further, the chronic infarction model will induce an accentuated dilatation, not observed in the ischemic/reperfusion model (Figure 3B and C right panel).
Conventional staining procedures can be used, such as: CD31 staining used to reveal the angiogenesis (red, Figure 4A) or smooth muscle actin staining to determine myofibroblasts (green, Figure 4B). Double fluorescence staining can also be applied to identify different target molecules in the infarction area, since the absence of cardiomyocytes gives no auto-immunofluorescence (Figure 4C).
<img alt="Figure 1" src="/files/ftp_upload/52197/52197fig1.jpg" /> 
Figure 1: Medial incision and insertion of the intubation&#39;s cannula (A). The stereomicroscopic visualization of the metal cannula through the transparence of the tissue (B). The tracheal rings (blue arrows) and the cannula (black arrow) are pointed out. The intercostal incision for the chronic infarction model and the ligature of LAD (C). The ligature is located at middle of the heart (between the auricle and apex, black in lower panel), taking as reference the end of the vein (schematic in blue, lower panel). Both branches of the artery should be bound (red in lower panel). The gray color indicates infarcted area and it appears in the lower half part of the heart (right lower panel). The ligature for the ischemia/reperfusion model is made under the auricle, binding the main body of LAD (red in lower panel) over a silicon tube (right side) (D). The gray color indicates the infracted area, which is present on the entire heart (right lower panel). <a href="https://www.jove.com/files/ftp_upload/52197/52197fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52197/52197fig2.jpg" /> 
Figure 2: The ribs suture seals the thoracic incision if the medial sutures are positioned at an angle of 90&#176; in both chronic (A) and ischemia/reperfusion model (B, left panel). In vivo imaging of ribs suture (C, left panel), muscle suture (C, middle panel) and skin suture (C, right panel). <a href="https://www.jove.com/files/ftp_upload/52197/52197fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52197/52197fig3.jpg" /> 
Figure 3: Echocardiographic images. Images of normal (A, upper panel) and infarcted areas (A, lower panel), are acquired in the long axis (longitudinal, left panels) or in the short axis (transversal, right panels).. Infarction induced by chronic ligature (B) and by one hr ischemia followed by reperfusion (C). Evans Blue/TTC Staining allows identification of perfused (blue)/non-perfused areas as well as the viable (red)/dead (white) myocardium (B, C, middle panels). Gomori&#39;s one-step staining allows the identification of infarcted areas (blue), and differentiates them from the normal regions (red) (B, C, right panels). <a href="https://www.jove.com/files/ftp_upload/52197/52197fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52197/52197fig4.jpg" /> 
Figure 4: Different stainings can be performed in infracted area, such as CD31 to described neo-angiogenesis (A, red, simple arrows), or smooth muscle actin for myofibroblasts (B, green, simple arrows), as well as double staining (C, CD31-red/smooth muscle actin-green), counterstained with DAPI for nuclei (blue). Myofibroblasts can be differentiated easily from smooth muscle cells from small or big arteries, which are always accompanied by a endothelial layer (C, arrows). Double arrows point the erythrocytes autofluorescence. Scale bars 50 &#181;m.

Watch this Scientific Journal Video about Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice at JoVE.com

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

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 ...

minimal-invasive-surgical-procedure-inducing-myocardial-infarction

Research

JoVE Journal

Medicine

27.4K vues.  RWTH Aachen University, Germany. 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).

Vidéo: Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

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 ...

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 ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

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

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

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

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

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

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

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 ...