We are grateful for funding from the British Heart Foundation to TK and to the UK Medical Research Council for a postgraduate research studentship to GB.

All components of this study were carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986, and with the approval of the University of Cambridge Ethical Review Panel.
1. Animal Preparation
<ol>
	<li>Induce infarcts with a surgical intervention7,8. For measuring at the acute stage, perform imaging 24 hr after intervention.</li>
	<li>Prepare a thin syringe tubing for drug delivery with a 25 G needle at one extremity and a syringe with a 25 G needle at the other end. The length of the line should be enough to allow the injection from outside the magnet&#8217;s bore (approximately&#160;1.5 m).</li>
	<li>Prepare a solution diluting Gadovist in saline. Load a syringe with the right amount of solution to achieve 3 mmol/kg adjusted for mouse bodyweight. Keep the injection volume between 50-100 &#181;l.</li>
	<li>Prefill the line with the Gadovist solution. To avoid blood clotting, fill the extremity of the line close to the cannula with heparin saline.</li>
	<li>Place the mouse in the induction box for anesthesia with 3% isoflurane in O2&#160;1 L/min for 3-5 min as necessary. Correct anesthesia depth can be identified with the slowing breath rate, which should decrease to less than 70 bpm.</li>
	<li>Cannulate the tail vein using the line previously prepared, use surgical tape to keep the needle in place during the procedure.</li>
</ol>
2. Animal Positioning and Monitoring
<ol>
	<li>Place the mouse on the MRI bed, delivering 1-2% isoflurane in 1 L/min O2 for anesthesia maintenance as required. Maintain respiration rates between 20-70 bpm.</li>
	<li>Ensure the center of the MRI coil is located over the mouse heart position.</li>
	<li>Monitor respiration by placing a respiratory pillow sensor slightly below the diaphragm. Use a rectal thermometer with a prelubricated cover slip to monitor core temperature. Ensure that temperature is kept constant during the scans at 37&#177;2 &#176;C.</li>
	<li>For ECG monitoring, place three electrodes on the anterior paws and on the left rear paw, making sure that the palm of the toe is completely open. Choose the pair of electrodes achieving the best signal quality to derive the ECG. Twist the ECG cables together, to insure that they do not form resonant circuits at the MRI resonant frequency, which would severely corrupt the ECG signal when running pulse sequences.</li>
	<li>Ensure the electrodes are firmly attached to the bed using tape. The electrodes and the nose cone will hold the animal in place during the whole procedure.</li>
	<li>Position a water-heated blanket over the mouse, encapsulating the monitoring and coil leads to maintain body temperature.</li>
	<li>Align the laser of the bed with the heart position; use the anterior toes line as a landmark. Position this in the magnet isocenter using an automatic bed.</li>
	<li>Run the standard adjustments for the MRI system.</li>
	<li>Set the monitoring equipment to detect the R-wave in the ECG. Adjust the thresholds for each mouse and within imaging sessions so that there is reliable triggering.</li>
</ol>
3. MRI
<ol>
	<li>Acquire a pilot image to plan the multiplanar pilot images.</li>
	<li>Identify the heart in the image, most easily by its flow artifacts. If the images show the mouse is not well centered in the coil or the isocenter, retract the bed and repeat positioning.</li>
	<li>Acquire a fast gradient echo (GE) image with five slices per orientation and a 3 cm field of view (FOV)&#160;with ECG gating turned on.</li>
	<li>Run an ungated 3D scan centered on the heart for PET coregistration (see Table 1 for scan parameters). While this scan is running, plan the successive scans.</li>
	<li>Plan a four chamber (4 ch) view scan (see Table 1 for scan parameters). This should cut through the apex and the tricuspid and mitral valves, showing all 4 chambers.</li>
	<li>Plan a two chamber (2 ch) view (see Table 1 for scan parameters). It should cut through the apex and the tricuspid valve, showing the left atrium and ventricle.</li>
	<li>Crosscheck the geometry of the two long-axis views. If the slice planning is sub-optimal, repeat the scan.</li>
	<li>Plan a stack of short axis slices (see Table 1 for scan parameters) orthogonal to both the 4 ch and to the 2 ch view. Cover the whole heart, starting from the first apical slice without blood pool until the first basal slice without any RV. Slices should be equally spaced with no gaps.</li>
</ol>
4. Late Gadolinium Enhancement
<ol>
	<li>Increase blank time for gating to acquire every other heart beat (TR = 550-750 msec). A delay must be applied such that acquisition is performed at the end-diastolic phase of the ECG.</li>
	<li>Perform a low-resolution version of the LGE sequence (see Table 1 for scan parameters) before the injection in order to check that the gating is correct and no flow artifacts are present. If flow artifacts are present in the diastolic phase, increasing the delay slightly (i.e. imaging at the very early stage of systole) will get rid of them.</li>
	<li>Perform the injection of the Gadovist solution slowly and steadily over 15 sec.</li>
	<li>Initiate LGE imaging (see Table 1 for scan parameters) after the injection. Good contrast is achieved when imaging within the first 15 min.</li>
</ol>
5. PET Imaging
<ol>
	<li>Transfer the MRI bed to the PET scanner leaving the receiver in place and firmly anchor it to the PET bed support. Connect the anesthetic and monitoring equipment.</li>
	<li>Position the heart in the center of the PET field of view moving the bed only axially.</li>
	<li>Acquire a single-pass transmission scan with a germanium source.</li>
	<li>Extract 10-30 Mbq of radioactively labeled FDG from its container with a syringe. Take necessary care with radioactive material. Measure the activity with a well counter. A good injection volume for mice ranges between 50-100 &#181;l, if the volume is too small, dilute the solution with saline.</li>
	<li>Start the emission scan, set to acquire list-mode gated PET for 45 min. Contemporaneously, inject the tracer slowly and steadily over 15 sec. Flush the line with enough saline to deliver the whole of the radioactive tracer.</li>
	<li>For animal health, do not to exceed 300 &#181;l with the three injections (i.e. Gadovist, FDG, flush).</li>
	<li>Place the syringe back in the well counter to measure the residual activity.</li>
	<li>Follow local rules throughout for handling radioactive material.</li>
</ol>
6. MRI Segmentation
<ol>
	<li>Load the Cine-MRI images in Segment v1.9&#160;9.</li>
	<li>Run the automatic tools for segmentation. Starting from a point inside the ventricle, these will inflate the estimated ventricle walls until the best match is found.</li>
	<li>Delineate the LV and the RV manually at end systole (ES) and end diastole (ED), defined respectively as the frames with the maximal and with the minimal global LV volume. Exclude the papillary muscles and trabeculations throughout. Do not adjust image contrast, as inconsistency in the contrast leads to a different interpretation of partial volume effects.</li>
	<li>Delineate manually the epicardium at ES and ED for LV mass calculations, as shown in Figure 6. The cardiac muscle is incompressible, so the LV mass must be consistent in ES and ED. If the difference is more than 5% operator error is likely and these should be reviewed.</li>
	<li>At the base of the ventricles, use a straight line to discriminate ventricles from atria as shown in Figure 6, identifying the angle of the slice with the help of the long-axis views.</li>
	<li>Cross-check the segmentation on the long-axis views.</li>
	<li>Delineate the scar area on the LGE images. Divide the scar volume by the LV mass derived in the cine imaging to obtain the infarct as a percentage of the LV.</li>
</ol>
7. PET Analysis and Coregistration with MRI
<ol>
	<li>Reconstruct the PET images using a 3D filtered backprojection algorithm and export data to Nifti format (http://nifti.nimh.nih.gov/) showing standard uptake values.</li>
	<li>Transform all the PET images using the fixed transformation matrix between the PET and the MRI system. If the matrix is not known, fill a geometrical phantom with radioactive material and perform an MRI, then move the bed to the PET and perform an emission scan. The matrix obtained to transform the PET to the MRI image is the standard matrix to go from the PET to the MRI system.</li>
	<li>Register the average PET image to the 3D MRI using SPM-mouse bulk coregistration tool10.</li>
	<li>Stack the slices from Cine-MRI to obtain a 3D reconstruction, interpolate to obtain 0.3 mm isotropic resolution. Register the end-diastolic PET volume to the end-diastolic MRI volume using SPM-Mouse coregistration tools. Use the same matrix for all the other heart phases.</li>
	<li>To evaluate PET infarct size, choose 50% of the maximum intensity in the heart to discriminate between infarcted and noninfarcted voxels. Express infarcted voxels as a percentage of the LV.</li>
</ol>

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S., 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=The+isolated+working+mouse+heart:+methodological+considerations.">The isolated working mouse heart: methodological considerations.</a> Pfl&#252;gers Arch. 437, 979-985 (1999).</li><li>Epstein, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+in+mouse+models+of+cardiac+disease.">MR in mouse models of cardiac disease.</a> NMR Biomed. 20, 238-255 (2007).</li><li>Schneider, J. E., Wiesmann, F., Lygate, C. 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There is nothing to disclose.

PET/MRI is a comprehensive measurement method for the noninvasive and longitudinal evaluation of systolic function, tissue viability and specific metabolic markers in mouse models of myocardial infarction. Here we have described a protocol for performing MRI and PET sequentially, where coregistration is simplified by the transfer of the same bed between systems. This strategy, also adopted by manufacturers of clinical systems11, does not require a combined PET/MRI scanner and can be performed with standard equ...

In order to measure the efficacy of new treatment strategies for myocardial infarction (MI) in preclinical studies, the assessment of the acute stage as well as long-term outcome is required1. Methods such as histopathology, intracardiac catheters2, and ex vivo heart models3 are commonly used for preclinical studies in mice. It is impossible, however, to follow up treatment with ex vivo&#160;or highly invasive methods. Noninvasive measurement techniques as in vivo&#160;magnetic resonance imaging (MRI) and positron emission tomography (PET) allow longitudinal experimental designs for disease staging in single subjects. Cine-MRI is used to derive global functional parameters such as left ventricular mass (LVM), ejection fraction (EF) and cardiac output (CO). In addition, after the injection of a gadolinium contrast agent, due to impaired perfusion and washout in the infarction, tissue viability can be assessed with late gadolinium enhancement (LGE) MRI. Complementarily, PET offers a sensitive measure of radiolabeled molecules in order to assess tissue metabolism. The high accuracy of these techniques permits significant reductions in the number of animals required for testing new drugs targeting MI.
The PET and MRI procedures are involved, and without a carefully designed protocol reproducibility is hard to maintain. Procedures established in the clinic for patients require substantial modification for use in mice, due to their considerably faster heart rate and smaller dimensions of the heart4. There is wide variation between individual subjects, both at baseline and in response to induced injury, so a considerable number of mice are needed to establish treatment efficacy.
In this report, we describe our method for sequential PET/MRI imaging of the mouse heart. Both modalities use intravenous contrast agents, which are delivered through the tail vein. MRI consists of standard assessment with Cine-MRI5&#160;with an optimal protocol for LGE as described in our previous work6. The entire MRI procedure lasts 30 min. We have obtained consistent fittings for the beds of our instruments so it is possible to transfer the animal on a platform with the same monitoring and anesthetic delivery apparatus between the machines. The PET scan lasts 45 min with in situ injection once the scan is started. The final step is to measure the parameters from both MRI and PET images following coregistration.

<table border="0" cellpadding="0" cellspacing="0" width="516">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:111px;">Material name</td>
			<td style="width:91px;">Company</td>
			<td style="width:136px;">Catalogue number</td>
			<td style="width:179px;">Comments</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:111px;">Gadovist</td>
			<td style="width:91px;">Bayer Schering Pharma</td>
			<td style="width:136px;">PL 00010/0535</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:111px;">FDG</td>
			<td style="width:91px;">IBA Molecular</td>
			<td style="width:136px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:111px;">Material name</td>
			<td style="width:91px;">Company</td>
			<td style="width:136px;">Catalog number</td>
			<td style="width:179px;">Comments</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:111px;">Bruker BioSpec 47/40</td>
			<td style="width:91px;">Bruker</td>
			<td style="width:136px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:111px;">Bruker mouse bed</td>
			<td style="width:91px;">Bruker</td>
			<td style="width:136px;"></td>
			<td style="width:179px;">This bed includes tubing for anesthesia delivery and scavenging.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:111px;">12 cm diameter birdcage transmitter</td>
			<td style="width:91px;">Bruker</td>
			<td style="width:136px;">T5346</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="74">
			<td height="74" style="height:74px;width:111px;">2 cm diameter surface coil receiver</td>
			<td style="width:91px;">Bruker</td>
			<td style="width:136px;">T7027</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:111px;">Red Dot Neonatal monitoring electrodes</td>
			<td style="width:91px;">3M</td>
			<td style="width:136px;">P/N: 2330</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="98">
			<td height="98" style="height:98px;width:111px;">Monitoring equipment</td>
			<td style="width:91px;">SA Systems</td>
			<td style="width:136px;"></td>
			<td style="width:179px;">The monitoring kit includes respiratory pillow and rectal probe for temperature measurements.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:111px;">Anesthesia equipment</td>
			<td style="width:91px;">General Anesthetic Services</td>
			<td style="width:136px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:111px;">Induction box</td>
			<td style="width:91px;">Vet Tech Solutions LTD</td>
			<td style="width:136px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:111px;">Cambridge split-magnet PET/MRI scanner</td>
			<td style="width:91px;">University of Cambridge</td>
			<td style="width:136px;"></td>
			<td style="width:179px;">A custom built PET/MRI scanner22 was used to perform the PET, its PET performance similar to an F120 micropet scanner23</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:111px;">Segment software</td>
			<td style="width:91px;">Medviso, Lund University</td>
			<td style="width:136px;"></td>
			<td style="width:179px;">Freely available</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:111px;">SPM-mouse</td>
			<td style="width:91px;">University of Cambridge</td>
			<td style="width:136px;"></td>
			<td style="width:179px;">Freely available</td>
		</tr>
	</tbody>
</table>

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.

One of the advantages of using MRI is that a longitudinal design can be used in order to stage disease. This is especially important when evaluating novel compounds, as the time course of effect may not be known. Variations in heart volumes due to the disease progression in the same animal are shown in Figure 4. The figure clearly shows the effects of remodeling post myocardial infarction.
Correct slice geometry is crucial for the success of a cardiac MRI experiment. Represent...

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MRI and PET in Mouse Models of Myocardial Infarction

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 ...

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11.6K Views.  Unversity of Cambridge. 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.

Video: MRI and PET in Mouse Models of Myocardial Infarction

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 ...

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 ...

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 ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

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 ...

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

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 ...