1. Preparation
1.1. Anesthesia of the rats
<ol>
	<li>Place the rat into a chamber prefilled with isoflurane (depending on the size of the chamber about 3 minutes of 5%) to anesthetize the rat.</li>
	<li>Once anesthetized, remove the rat and weigh it.</li>
	<li>Place the nose of the rat into the anesthesia mask (isoflurane 2-3% in oxygen 1 L/min) to maintain anesthesia. During anesthesia with isoflurane, the heart rates of rats decrease to 300-340 bpm (380-420 bpm in mice).</li>
</ol>
1.2. Insert a tail vein cannula
<ol>
	<li>Wash the tail of the rat with warm water (38 &deg;C) and soap to allow the blood vessels to be more easily seen.</li>
	<li>Cannulate either vein, which can be easily seen on the lateral aspect of the tail. Using a 22-24 G i.v. catheter, begin attempts approximately 1/3 of the way from the tip.</li>
	<li>If the initial efforts are unsuccessful, retry more proximally.</li>
	<li>Take blood sample for analysis of hematocrit (necessary to calculate extracellular volume fraction= ECV).</li>
	<li>Secure the i.v. catheter with skin glue and tape.</li>
</ol>
1.3. Positioning of the electrodes
<ol>
	<li>Clean all footpads with alcohol saturated swab or with soap and water.</li>
	<li>Four electrodes are needed, one for each foot.</li>
	<li>Attach the electrodes to the middle of the footpad and secure them with adhesive tape.</li>
	<li>Twist or plait the ECG cables around each other to minimize the formation of loops that would cause interference with the ECG signal.</li>
</ol>
2. Scanning
2.1. Positioning of the animal and monitoring
<ol>
	<li>Place the rat in a supine or prone position onto the imaging tube and position the heart in the center of the coil.</li>
	<li>Arrange the rat so that its nose rests in the cone which delivers isoflurane anesthesia (2-3% in oxygen 1 L/min). The rat is allowed to breath freely throughout the entire imaging procedure.</li>
	<li>Place the sensor of the thermometer at the anal orifice and fix it on the table with tape.</li>
	<li>Body temperature is maintained at 38&plusmn;1 &deg;C by a warm water heating system. The warming mat is placed under the lower aspect of the animal which extends outside the coil. Please see the schematic representation of the MRI experimental setup (Figure 2) for more details.</li>
	<li>Connect the ECG electrodes, once the ECG signal is stable begin imaging.</li>
</ol>
2.2. Scan protocol/ Imaging parameters
<ol>
	<li>In this study a Philips Ingenia 3.0T clinical MRI system with a solenoid coil with 70 mm inner diameter was used [MR system requirements are a small bore (e.g. 16 cm) or large bore (human size, e.g. 65 cm) with dedicated small animal coil, ECG gating capabilities for heart rates up to 500/min, oxygen supply and a removal system for anesthetic gases].</li>
	<li>Start with a survey to locate the heart in the center of the MRI field of view.</li>
	<li>Once the position is correct, continue with two-chamber and four-chamber cine MRI views to determine the geometry of the stack of short axis images.</li>
	<li>For SALLI, position mid-cavity short axis slice on the two- and four-chamber images (PreSALLI). A combination of 4,000 msec acquisition duration and 4,000 msec relaxation duration allows for sufficient recovery of magnetization along the z-axis and prevents overheating of gradients. To enable functional analysis, phases should be set to at least 12, with temporal undersampling of factor 2 to accelerate acquisition.</li>
	<li>Inject the gadolinium-based contrast agent into the indwelling catheter, without moving the rat. Wait five minutes.</li>
	<li>Start planning SALLI as multislice short axis stack, consisting of at least 7 slices (slice thickness 2.4 mm). Position the slices so that the most distal is below the apex of the heart and the top level is just distal to the cardiac valves. Try to ensure that the short axes are perpendicular to the septum. Almost every 30 minutes (around three SALLIs) inject another dose of contrast agent.</li>
	<li>When image acquisition is finished remove the rat from the scanner and allow it to recover from anesthesia.</li>
</ol>
3. Image Reconstruction
Image reconstruction may be performed either online using predefined reconstruction parameters, or offline using a dedicated image reconstruction tool:
3.1. Online Reconstruction
<ol>
	<li>Various Parameters can be defined including:</li>
</ol>
<ul>
	<li>For T1 mapping: location and width of the reconstruction window within the cardiac cycle (e.g. from 50 to 100 msec to achieve systolic T1 map at heart rate 300/min)</li>
	<li>For IR-prepared (LGE) images: location and width of the inversion time (TI) window and number of images to be reconstructed within that window (e.g. TI 100-300 msec, 5 images, to generate 5 LGE images with TIs 100-150-200-250-300 msec).</li>
</ul>
3.2. Offline Reconstruction
<ol>
	<li>When using a dedicated software package (Gyrotools, Z&uuml;rich, Switzerland) for image reconstruction, extract the three sets of data (cine MR, inversion recovery and T1 maps) in a more interactive way.</li>
	<li>Adjust the definition of end-systole or end-diastole for the T1 by selecting the corresponding cine image.</li>
	<li>Visualize T1 maps as color maps. Find exact values by passing the mouse over an area of interest. Images can be stored in various image formats for further analysis (e.g. calculation of ECV from pre- and post-contrast T1 values of myocardium and blood pool) 12.</li>
</ol>

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	<li>Messroghli, D. R., Nordmeyer, S., Buehrer, M., 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=Small+animal+Look-Locker+inversion+recovery+(SALLI)+for+simultaneous+generation+of+cardiac+T1+maps+and+cine+and+inversion+recovery-prepared+images+at+high+heart+rates:+initial+experience.">Small animal Look-Locker inversion recovery (SALLI) for simultaneous generation of cardiac T1 maps and cine and inversion recovery-prepared images at high heart rates: initial experience.</a> Radiology. 261 (1), 258-2565 (2011).</li><li>Messroghli, D. R., Nordmeyer, S., Dietrich, T., 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=Assessment+of+diffuse+myocardial+fibrosis+in+rats+using+small-animal+Look-Locker+inversion+recovery+T1+mapping.">Assessment of diffuse myocardial fibrosis in rats using small-animal Look-Locker inversion recovery T1 mapping.</a> Circ. Cardiovasc. Imaging. 4 (6), 636-640 (2011).</li><li>Flett, A. S., Hayward, M. P., Ashworth, M. T., 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=Equilibrium+contrast+cardiovascular+magnetic+resonance+for+the+measurement+of+diffuse+myocardial+fibrosis:+preliminary+validation+in+humans.">Equilibrium contrast cardiovascular magnetic resonance for the measurement of diffuse myocardial fibrosis: preliminary validation in humans.</a> Circulation. 122 (2), 138-144 (2010).</li><li>Arheden, H., Saed, M., Higgins, C. 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Med. 58 (1), 34-40 (2007).</li><li>O H-Ici, D., Ridgway, J., Kuehne, T., 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=Cardiovascular+magnetic+resonance+of+myocardial+edema+using+a+short+inversion+time+inversion+recovery+(STIR)+black-blood+technique:+Diagnostic+accuracy+of+visual+and+semi-quantitative.">Cardiovascular magnetic resonance of myocardial edema using a short inversion time inversion recovery (STIR) black-blood technique: Diagnostic accuracy of visual and semi-quantitative.</a> 14 (1), (2012).</li><li>Sagar, S., Liu, P. P., Cooper, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocarditis.">Myocarditis.</a> Lancet. 379, 738-747 (2012).</li><li>Skouri, H. N., Dec, G. W., Friedrich, M. G., Cooper, L. 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G., Sechtem, U., Schulz-Menger, J., 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=Cardiovascular+magnetic+resonance+in+myocarditis:+a+JACC+White+Paper.">Cardiovascular magnetic resonance in myocarditis: a JACC White Paper.</a> J. Am. Coll. Cardiol. 54, 1475-1487 (2009).</li><li>Korkusuz, H., Esters, P., Naguib, N., 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=Acute+myocarditis+in+a+rat+model:+late+gadolinium+enhancement+with+histopathological+correlation.">Acute myocarditis in a rat model: late gadolinium enhancement with histopathological correlation.</a> Eur. Radiol. 19 (11), 2672-2678 (2009).</li><li>Ugander, M., Oki, A. J., Hsu, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+volume+imaging+by+magnetic+resonance+imaging+provides+insights+into+overt+and+sub-clinical+myocardial+pathology.">Extracellular volume imaging by magnetic resonance imaging provides insights into overt and sub-clinical myocardial pathology.</a> Eur. Heart J. 33 (10), 1268-1278 (2012).</li><li>Gilson, W. D., Kraitchman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+magnetic+resonance+imaging+in+small+rodents+using+clinical+1.5.">Cardiac magnetic resonance imaging in small rodents using clinical 1.5.</a> T and. 3 (1), 35-45 (2007).</li><li>Messroghli, D. R., Radjenovic, A., Kozerke, S., Higgins, D. M., Sivananthan, M. U., Ridgway, J. 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All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals, and approved by the local animal care authorities. No conflicts of interest declared.

Here we present a method to generate multi-modality (cine MR, inversion-recovery and T1 maps) MR images in small animals. Small animal research plays an increasingly important role in cardiovascular disease research, and CMR is a powerful tool allowing us to study the function, structure and tissue composition of the myocardium. However, small animal CMR has a number of unique challenges owing to the high heart rates and small heart size. Improved methods of imaging, including more time efficient methods will play a further role in the development of this technique.
The initial set-up of the animal is a critical part of this protocol. It is important not only to allow for generation of optimal images, but also to allow for the animal to remain stable throughout the often prolonged examination (complete protocol including reconstruction takes about 2 hours). As the images are gated with the ECG, interference from poor lead contact, or from loops formed from the ECG cables, can lead to prolonged scan times or failure of the sequences. A poor ECG signal due to high heart rates or arrhythmia is one of the main challenges of the small animal CMR. In addition, temperature and anesthesia must be carefully controlled to obtain accurate, reproducible measurements and allow the animals undergo prolonged examination. In a similar manner to a standard human CMR examination, the heart is located, followed by two long-axis cine scans that then allow the short-axis SALLI images (T1 maps) to be performed. Depending on the given application, there are a number of variations regarding the SALLI images: 1) One single short-axis pre-contrast SALLI and multiple short-axis post-contrast SALLI (as presented here). This approach allows functional information, infarct size, and basic information on ECV to be obtained. 2) Multiple short-axis pre-contrast and multiple short-axis post-contrast SALLI. This protocol adds information on regional myocardial edema and distribution of ECV (e.g. in acute myocardial infarction) at the cost of prolonged scan time. 3) Single-slice pre- and post-contrast SALLI, multi-slice conventional cine. This quick approach yields functional information and global ECV, and might be sufficient in models of diffuse myocardial disease.
Existing CMR sequences can obtain excellent cine MR images in both humans and small animals. LGE imaging in small animals remains a challenge. Due to the high heart rates, acquisition time for an inversion recovery-prepared sequence is increased to 6-10 minutes per image 13, which causes serious problems with adjusting the inversion time to null signal from healthy myocardium.
T1 quantification provides information about tissue characteristics and gadolinium kinetics. Typical cardiac T1 methods require the acquisition of multiple images with different inversion times to allow for an accurate fitting of the underlying T1 curve 14. T1 mapping techniques for human applications typically acquire non-segmented raw images with durations of 150-200 msec each. This method is not suitable for small animals, where heart rates from 200-600 beats per minute are expected (i.e. cardiac cycles of 100 - 300 msec). SALLI solves this problem by using a segmented approach, thereby allowing myocardial T1 to be quantified in rats.
T1 mapping provides a quantitative approach to characterization of myocardium. It therefore allows diffuse processes to be studied. T1 values can be compared between individual studies and individual subjects, allowing the study of chronic changes in the myocardium. The ability to simultaneously assess function and myocardial properties will allow study of the chronologic formation of myocardial injury. In the future, multimodal imaging will allow the combination of functional information with qualitative and quantitative assessment of the myocardium to provide a more complete picture of cardiovascular disease.

Myocarditis is a major cause of acute heart failure, sudden death, and chronic dilated cardiomyopathy 7. CMR has been established as the gold standard technique for measurement of function and for in vivo tissue analysis. Novel imaging techniques and improvements in imaging could not only improve the diagnosis of myocarditis, but also aid the study of the pathophysiology and speed identification of therapeutic targets 8-10. Small animal imaging is an important tool for the study of cardiovascular diseases. Techniques routinely used in clinical CMR, such as late gadolinium enhancement (LGE) cannot easily be transferred to small animal CMR due to the animals' high heart rates; but could already be shown 11. The small animal Look-Locker inversion recovery (SALLI) approach generates multi-modal image data sets allowing for the comprehensive assessment of both cardiac function and morphology (Figures 1 and 3). Here we show, in detail, the procedure and setup for small animal imaging with a typical SALLI protocol. In particular we show the reconstruction and analysis of the T1 imaging data sets.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Adhesive Tape (Silkafix)</td>
			<td>Lohmann und Rauscher</td>
			<td>34327</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gadopentetat-Dimeglumin (Magnevist)</td>
			<td>Bayer</td>
			<td>G-00012163</td>
			<td>2mmol/Kg</td>
		 </tr>
		 <tr>
			<td>Introcan Safety-W (G24)</td>
			<td>B. Braun</td>
			<td>4254503-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Red Dot, Neonatal Monitoring Electrode with Pre-Attached Lead Wire</td>
			<td>3M</td>
			<td>2269T</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Skin glue (Histoacryl)</td>
			<td>B. Braun</td>
			<td>1050052</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scales (Typ 440)</td>
			<td>Kern</td>
			<td>95088</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Skin desinfection (Softasept N)</td>
			<td>B. Braun Petzold</td>
			<td>360250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Thermometer </td>
			<td>LumaSense Technologies</td>
			<td>Luxtron 812</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

assessment of cardiac function and myocardial morphology using small animal look-locker inversion recovery (salli) mri in rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Here we demonstrate the findings from a healthy Sprague Dawley Rat. With a well-prepared animal setup we described before, it is possible to get a stable ECG signal. Similar to human cardiac MRI protocols, we start with a survey to locate the heart. If the position of the rat in the coil is correct, we continue with a two-chamber view and a four-chamber view to detect the geometry for the SALLI stack.
The SALLI technique produces three types of images acquired at the same time. These are, firstly, cine images that can be used to study local and global LV function. LV parameters to be assessed with the help of a CMR software package include end systolic and end diastolic volumes, ejection fraction, and mass. The second type of image is multiple IR images with different nominal TI, from which the images with optimal nulling of normal myocardium can be selected. This ensures maximum enhancement of areas with focal myocardial fibrosis/ scar, as necessary for LGE imaging. Thirdly, T1 maps are generated. These should demonstrate homogenous relaxation times over healthy myocardium as shown in Figure 3 (representative T1 map). Before administration of gadopentetate dimeglumine, myocardium exhibits shorter T1 values than blood within the LV cavity. In the first minutes after administration of contrast agent, T1 of blood is shortened more than that of myocardium. While the presented data from a healthy animal shows homogenous T1 behavior across the entire myocardium, the presence of myocardial injury e.g. in models of myocardial infarction would demonstrate T1 shortening in the affected region. T1 maps can be reconstructed for any phase of the cardiac cycle (Figure 3c).
<img alt="Figure 1 " fo:content-width="6.5in" fo:src="/files/ftp_upload/50397/50397fig1highres.jpg" src="/files/ftp_upload/50397/50397fig1.jpg" /> 
 Figure 1. Pulse sequence scheme for fully sampled data sets and reconstruction schemes of multimodal images. Top: SALLI pulse sequence scheme. After an adiabatic inversion pulse (180&deg;), image data for the first radial segment (segment = colored sectors of circles) are continuously sampled for consecutive cardiac phases (circles of different sizes) and heart cycles (different circle colors) during a predefined AD while magnetization recovers with a time constant T1* (solid line). Temporal resolution (eg, number of cardiac phases) is theoretically limited by minimum repetition time. After a predefined RD, during which magnetization recovers without any readout-induced perturbations with a time constant T1 (dashed line), the process is repeated for the next radial segment and so on. A. Reconstruction of T1 maps. In a first step, raw images for the number of cardiac cycles encompassed by AD are reconstructed from all available image data acquired during a predefined time window within the R-R interval (e.g. during systole). In a second step, pixel-wise nonlinear curve fitting is performed and resulting T1* values are corrected for readout-induced deviation of the magnetization recovery curve to generate T1 map from these raw images. B. Reconstruction of IR-prepared images. For a predefined interval after inversion (e.g. 100-300 msec), a set of IR-prepared images with predefined steps of inversion time (e.g. 25 msec) is reconstructed from all image data available within the interval. C. Reconstruction of cine images. Cine images for a predefined number of cardiac phases are reconstructed from all image data available beyond the time point at which all longitudinal magnetization within the field of view had recovered by at least 90%. (Messroghli et al., Small animal look locker inversion recovery (SALLI) for simultaneous generation of T1 maps and cine and reversion recovery-prepared images at high heart rates: initial experience, Radiology 2011. Reprinted with permission from RSNA). <a href="https://www.jove.com/files/ftp_upload/50397/50397fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/50397/50397fig2.jpg" /> 
 Figure 2. MRI experimental setup. MRI experimental setup, including animal positioning onto the animal holder and the monitoring possibilities while scanning.
<img alt="Figure 3" src="/files/ftp_upload/50397/50397fig3.jpg" /> 
 Figure 3. Representative SALLI stack from a healthy Sprague-Dawley rat: a) cine image diastole, b) cine image systole, c) T1 map before intravenous administration of gadopentetate-dimeglumine (diastole), d) LGE image before intravenous administration of gadopentetate-dimeglumine. Typically SALLI Data: slice thickness (mm) 2.4; FOV (mm) 64 x 64; pixel slice (mm) 0.59 x 0.59; flip angle 10&deg;; AD (msec) 4,000; RD (msec) 4,000, Act. TR/TE (msec) 6.7/2.7; NSA 2, heart phases 12; temporal undersampling factor 2, cardiac frequency (bpm) 320; scan duration 7 minutes.
AD = acquisition duration 
bpm = beats per minute 
CMR = cardiac magnetic resonance 
ECV = extracellular volume fraction 
IR = inversion recovery 
LGE = late gadolinium enhancement 
NSA = Number of Signals averaged 
RD = relaxation duration 
SALLI = small animal Look-Locker inversion recovery 
TR = repetition time 
TE = echo time

Watch this Scientific Journal Video about Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats at JoVE.com

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

12.7K Views.  German Heart Institute Berlin. Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Video: Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

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 ...

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 ...

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

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Chapters in this video

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using ³¹P NMR Spectroscopy

Acute Myocardial Infarction in Rats

MRI and PET in Mouse Models of Myocardial Infarction

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats