NHLBI R01 HL073087 to CKS.

Experimental Protocols
The following different protocols were used to establish feasibility of measurements aimed at investigating the IR murine heart non-invasively. Baseline echocardiographic imaging was followed by surgical induction of ischemia-reperfusion (IR, protocol 1) which was followed by echocardiography at multiple time-points during recovery from IR.
Protocol 1. Left Anterior Descending (LAD) artery IR: Surgical Procedure
Male C57BL/6 adult mice (8-week old; 24.5&plusmn;1.5g, mean&plusmn;SD; Harlan Technologies, IN) were maintained under standard laboratory housing conditions with access to chow and drinking water ad libitum. Mice were anesthetized using intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (10mg/kg), laid on a warm surgical table, and intubated endotracheally. Proper anesthetic plane for rodents is deduced by a cessation of toe pinch reflexes and a slowing of respiration. Instruments were sterilized via 70% ethanol washes and autoclaving. Any instruments that left the sterile field were inserted into a hot-bead instrument sterilizer for 1 minute before continuing use. The mice were ventilated (Harvard Apparatus, Boston, MA) using air-isoflurane mixture at an appropriate rate and tidal volume. Cardiac electrophysiology was monitored throughout the surgery using a three-lead ECG setup, and changes were recorded using PC Powerlab software (AD Instruments).
The heart was accessed via left thoracotomy. The left lung was retracted to allow access to the pericardium. The left auricle was elevated to expose the coronary LAD artery, which was isolated using 7-0 Prolene suture mounted on a tapered needle. The suture was tightened over a piece of PE-10 tubing to induce reversible ischemia. LAD was occluded for 60 minutes. After 60 min, the suture was released to allow for reperfusion of the injured myocardium. Upon successful reperfusion, the thorax was closed with interrupted 7-0 Prolene sutures as well as the skin incision with 5-0 Prolene sutures. 
Throughout the surgery, body temperature was maintained at 36.7 &plusmn; 0.5&deg;C using a heated surgical table and monitored with a rectal thermal probe. The ischemic-episode causes pale coloration of LV myocardium that can be visually appreciated. Thorax was closed, sutured, and tracheal tube was disconnected enabling the mouse to breathe on its own. The animal was then returned to its cage and placed on a heating block set to 37&deg;C. Once the recovery was complete, the animal was returned to the rodent vivarium unit prior to echocardiographic imaging. All procedures were approved by the Institutional Laboratory Animal Care and Use Committee (ILACUC) of the Ohio State University.
Protocol 2. M-mode imaging protocol for the determination of cardiac function and detection of wall motion abnormality
To assess radial and longitudinal changes in cardiac function and wall motion abnormalities, we performed pre-IR imaging using high frequency, high resolution ultrasound scanner (Visual Sonics Inc., Toronto, Canada). Standard M-mode echocardiography parameters such as wall dimensions, volumes at systole, diastole and stroke volume, ejection fractions (EF), fractional shortening (FS), cardiac output (CO), and internal diameters at systole and diastole, were assessed using in-built M-mode protocol at each time point (pre-IR, day 3 and day 7).
Protocol 3. VevoStrain Speckle tracking algorithm protocol
B-mode movies were acquired and subjected to process using in-built VevoStrain algorithm to perform strain analyses. This allows assessment of velocity, displacement, strain, and strain rate through separate radio buttons provided in the interface giving interactive plots and M-mode images along with the data values which was done by clicking desired icons in the control panel.
Speckle Tracking Assessment of Strains
2-D Grayscale cine loops were acquired in the LV short-axis view at a frame rate of &gt;275 frames/s. For each experiment, at least three consecutive cardiac cycles were recorded and digitally stored on a hard disk for offline analysis on a workstation. We used a speckle-tracking algorithm incorporated into Vevo2100. The strain analysis was performed by the same trained observer. The region of interest was overlaid across a cross section of the ventricle at the image corresponding to the minimal endocardial area. The software algorithm then automatically divided the LV short-axis view into six segments for speckle tracking throughout the cardiac cycle. The tracking quality was then visually inspected, and, if it was satisfactory for at least five segments, the tracing was accepted.
Protocol 4. Doppler flow measurement protocol
To study the mitral valve regurgitation and to calculate the rate of change of LV systolic blood pressure (dP/dt), Doppler flow detection protocol was used. For this purpose, power Doppler blood flow velocity profiles were acquired by placing the probe on the mitral valve regurgitant jet and aortic jet. Imaging of blood flow velocity was followed by measurement to obtain values of the parameters such as peak and mean velocity, peak and mean pressure gradients, velocity time integral, and time. For the computation of dP/dt, mitral valve regurgitant velocity profile was used to measure velocity at early systole (steepest slope of the velocity profile to minimize load dependency) and corresponding time of slope. Finally, Bernoulli s equation provided the velocity-pressure conversion to determine the rate of increase of systolic pressure.
Echocardiographic Data Acquisition
Mice were imaged in a conscious state at a room temperature of 37&deg;C with decreased ambient lighting while held by an experienced handler in a prone left decubitus position. The heart was imaged in the 2-D mode in the parasternal long- and short-axis views with a depth setting of 1.0 cm and at a frame rate of &ge; 270 frames/s. An M-mode image was obtained at a sweep speed of 200 mm/s. All measurements were done according to guidelines provided by the American Society of Echocardiography.
Imaging was performed in conscious mice along with tandem registration of the electrocardiogram. End systole was defined as the minimum LV area. Segmental Scirc and Srad curves were then constructed by integrating appropriate signals starting from the end-diastolic points and then averaged to obtain average segmental strain curves. End-systolic strains were then obtained from the average strain curve.
Representative Results
Two-dimensional movies acquired at high frame rate (8 kHz) were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles as a function of time helped appreciate the functional consequence of IR.&nbsp; The above-mentioned approach led to the reliable detection of the following parameters:
Standard IR Outcomes (commonly reported in the literature)
<ul>
 <li>left-ventricular (LV) contraction
 <ul>
 <li>injury impairs the contraction and relaxation in the heart resulting in functional loss </li>
 <li>changes in LV chamber area and mass (hypertrophy)</li>
 </ul>
 </li>
 <li>LV relaxation of isovolumetric phases of both pre-IR and post-IR of beating hearts </li>
 <li>compromised sector-wise wall motion </li>
 <li>turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles</li>
 <li>LV shape changes towards sphericity</li>
 <li>compromised ejection fraction</li>
 <li>attenuated fractional shortening</li>
</ul>
Unique post-IR Outcomes
(never before reported in a murine IR setting but known to be of clinical significance in humans)
<ul>
 <li>effective regurgitant orifice (ERO)
<ul>
 <li>a fundamental measure of valvular incompetence </li>
 </ul>
 </li>
 <li>decreased myocardial strains (circumferential as well as radial)
 <ul>
 <li>a fundamental measure of myocardial strength and anatomical deformation </li>
 </ul>
 </li>
 <li>asynchrony in LV sectors
 <ul>
 <li>a fundamental measure of inherent periodic pumping characteristics of the heart </li>
</ul>
</li>
 <li>wall motion and symmetry
<ul>
 <li>changes in magnitude and direction of vectors demonstrating magnitude, direction and&nbsp; symmetry at the affected site</li>
</ul>
</li>
 <li>dimension of mitral valve and aorta
<ul>
 <li>changes in internal diameters of mitral valve orifice and aorta</li>
 </ul>
</li>
 <li>mitral regurgitation
<ul>
 <li>back flow of blood from LV to left atrium due to incomplete closure of mitral leaflets</li>
</ul>
</li>
</ul>
<ul>
 <li>sector-wise changes in magnitude of the radial (Srad) and circumferential (Scir) strain </li>
 <li>changes in strain rates along with the marked variation in their changing patterns relative to pre-IR data </li>
 <li>synchronicity of LV sectors as divided according to the American Society of Echocardiography</li>
 <li>association of ventricular mechanical synchronicity with the myocardial stiffness and decreased myocardial strength as assessed by tissue Doppler and strain imaging</li>
 <li>changes in magnitude and direction of vectors shown in movies depicting compromised movement (slow and asymmetric) of the IR-affected site</li>
 <li>internal diameters of mitral valve orifice and aorta</li>
</ul>
<img src="/files/ftp_upload/1781/1781fig1.1.jpg" alt="Figure 1 I" /> Fig. 1i. myocardial strain measurement: end-diastole full relaxation shown by shortest vectors (M1)
<img src="/files/ftp_upload/1781/1781fig1.2.jpg" alt="Figure 1 II" /> Fig. 1ii. myocardial strain measurement: LV is contracting shown by length and direction of vectors, the injury site (green point) has limited contraction (M2)
<img src="/files/ftp_upload/1781/1781fig1.3.jpg" alt="Figure 1 III" /> Fig. 1iii. myocardial strain measurement: end-systole, full contraction shown by shortest vectors (M3)
<img src="/files/ftp_upload/1781/1781fig1.4.jpg" alt="Figure 1 IV" /> Fig. 1iv. myocardial strain measurement: long axis at diastole (M4)
<img src="/files/ftp_upload/1781/1781fig1.5.jpg" alt="Figure 1 V" /> Fig. 1v. myocardial radial strain (Srad) measurement: the color coded curves represent the strain at respective colored points in images of Fig1.i-iv (M5)
<img src="/files/ftp_upload/1781/1781fig1.6.jpg" alt="Figure 1 VI" /> Fig. 1vi. myocardial circumferential strain (Scir) measurement: the color coded curves represent the strain at respective colored points in images of Fig1.i-iv (M6)
Figure 1. B-mode movies visualizing end-diastole (ED) and end-systole (ES) and changes in LV chamber size post-IR. B-mode images to determine left ventricular (LV) circumferential strain (Scirc) and radial strain (Srad) by two-dimensional speckle tracking echocardiography.
VevoStrain speckle tracking algorithm to observe vectors depicting the magnitude and direction of the motion of myocardium, axial and parasternal long axis views demonstrating the vectors at the injury site of the impaired myocardium.
Radial and longitudinal strains on a baseline mouse and a mouse post IR day7. The color coded strain profiles showing different color coded points in sites on the myocardium of baseline (pre-IR) and treated (post-IR, day7) hearts. LV-left ventricle, RV-right ventricle, Srad-radial strain, Scir-circumferential strain.
<img src="/files/ftp_upload/1781/1781fig2.jpg" alt="Figure 2" /> Figure 2. Myocardial sectoral synchronicity. Analyses based on the strain data pre- and post-IR. LV divided into six sectors (1 = posterior basal, 4 = anterior basal, 2 = posterior mid, 5 = anterior mid, 3 = posterior apex, 6 = anterior apex) according to American Society of Echocardiography. Validation of ventricular mechanical synchrony to see its association with myocardial stiffness as assessed by tissue Doppler and strain imaging.
Color coded image panel showing the sectors from 1-6 with values for time to peak on the corresponding sectors in milliseconds. Radial and longitudinal synchronicities provided for pre-IR and post-IR in terms of graphs. The colors on the graphs correspond to colors of the corresponding sectors.
<img src="/files/ftp_upload/1781/1781fig3.1.jpg" alt="Figure 3 I" /> Fig. 3i. anatomical measurements: mitral regurgitation orifice diameter in mice heart (blue lines with repeated measurements), sample volume in aorta (yellow lines) (M8)
<img src="/files/ftp_upload/1781/1781fig3.2.jpg" alt="Figure 3 II" /> Fig. 3ii. regurgitant flow profiles in mouse heart, upper forward and lower backward flow at mitral valve (M9)
<img src="/files/ftp_upload/1781/1781fig3.3.jpg" alt="Figure 3 III" /> Fig. 3iii. aortic out flow velocity profiles in mouse heart (M10)
<img src="/files/ftp_upload/1781/1781fig3.4.jpg" alt="Figure 3 IV" /> Fig. 3iv. regurgitant orifice at mitral Valve, Ao = aorta, LA = left atrium, LV = left ventricle, r = radius (M11)
Figure 3. Mitral valve V regurgitant fraction (RF). 
Echocardiographic image panel for the measurement of regurgitant fraction (RF). Measurement of aortic diameter (AoD) and mitral valve orifice diameter (MVD) showing placement of the sample volume in the regurgitant jet and aorta. These measurements provide the regurgitant velocity profiles and ascending aortic flow in which velocity profiles above and below baseline indicate forward and backward blood flow to the LV and back to left atrium. RF (in %) computation using the formula, RF = MV CSA*VTI of MV jet at diastole - Ao CSA*VTI of Ao jet at systole)*100 / MV CSA*VTI of MV.
Quantification of regurgitant fraction showing the regurgitant fraction in post-IR mice indicator to regurgitated mitral valve. Changes in mitral valve effective regurgitant orifice (ERO) post-IR due to sphericity of LV. Representative color Doppler images depicting the blood flowing from left atrium to the LV. Enlarged blood flow orifice indicates turbulent flow and increased abnormal flow velocity. ERO indicating valvular incompetence. The flow velocity aliased. The pulsed wave is utilized as to benefit in determining transitions from laminar to turbulent flow. ERO computed using the formula, ERO = Flow/Vmax = 2 &pi; r2 Va /Vmax, Va = Aliasing velocity, r = orifice radius, Vmax = maximum velocity.
<img src="/files/ftp_upload/1781/1781fig4.1.jpg" alt="Figure 4 I" /> Fig. 4i. measurement of functional parameters in pre-IR mouse. Soft ware calculates parameters by tracing, red vertical line = diastole, green vertical line systole (M12)
<img src="/files/ftp_upload/1781/1781fig4.2.jpg" alt="Figure 4 II" /> Fig. 4ii. measurement of functional parameters in post-IR mouse. Soft ware calculates parameters by tracing, red vertical line = diastole, green vertical line systole (M13)
Figure 4. The rate of change of ventricular systolic pressure (dP/dt): pre- and post-IR. Correlation of ejection fraction (EF) with dP/dt. Image panel showing an M-mode from which the basic functional measurements are taken.
The discrimination of heart contractility over time; bar graphs showing dP/dt post-IR. Indicating measurement of one parameter gives clinical information to the other.
<img src="/files/ftp_upload/1781/1781fig5.jpg" alt="Figure 5" /> Figure 5. Short axis movie demonstrating pre-IR strain measurement. 
Echocardiographic movie obtained from the parasternal view of heart to visualize the morphology in the mouse heart pre-IR.
<img src="/files/ftp_upload/1781/1781fig6.jpg" alt="Figure 6" /> Figure 6. Short axis movie demonstrating post-IR LV strain measurement 
Representative axial movie of a mouse LV traced to endocardium demonstrating the tissue strain vectors with five specific points in measurement considered.
<img src="/files/ftp_upload/1781/1781fig7.jpg" alt="Figure 7" /> Figure 7. Parasternal long-axis color Doppler movie 
Color Doppler flow movie demonstrating the blood pumped in from left atrium through mitral valve into the LV during diastole and the blood is pumped out through the aorta during systole phase of the heart with the direction of flow blue color away and red color towards (BART) the ultrasound probe.

The strain data based on the measurement using speckle tracking algorithm is relatively less observer dependent as the most of the analysis is performed by the software. However the observer should be careful during tracing the boarders of epicardium and endocardium in the B-mode movies which depends more on the experience. Moreover, M-mode analysis and measurement of velocity profiles is also minimal observer dependent. However, in the measurement of pressure rise and measurement of times, observer requires very careful attention during measurements because the pressure depends directly with the square of the peak velocity as expressed by Bernoulli s formula in which even a small amount of error in the measurement can produce squared effect on the total error in the measurement of dP/dt. Furthermore, the regurgitant orifice is not necessarily constant throughout systole, and this can potentially affect the estimation of regurgitant severity. The higher doses of anesthesia may result in the dramatic reduction of the fractional shortening affecting other functional parameters. Therefore, standard use of sedation is important for better outcome. Most parameters can be measured using other modalities, for example, MRI tagging can measure 2D and 3D strains. However, echocardiography is more user friendly.

<table border="1">
<tbody>
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
</tr>
<tr>
<td>Vevo2100</td>
<td>&nbsp;</td>
<td>VisualSonics, Inc., Canada</td>
<td>SN2100-0032</td>
</tr>
<tr>
<td>Ventilation device</td>
<td>&nbsp;</td>
<td>Harvard Apparatus, MA</td>
<td>n/a</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">PC Powerlab software</a></td>
<td>&nbsp;</td>
<td><a href="http://www.adinstruments.com/" target="_blank">AD Instruments</a></td>
<td>n/a</td>
</tr>
<tr>
<td>Isoflurane</td>
<td>&nbsp;</td>
<td>VEDCO, Inc., MO</td>
<td>5260-04-12</td>
</tr>
<tr>
<td>Aquasonic gel 100</td>
<td>&nbsp;</td>
<td>Parker Lab. Inc., NJ</td>
<td>01-02</td>
</tr>
</tbody>
</table>

high-frequency high-resolution echocardiography: first evidence on non-invasive repeated measure of myocardial strain, contractility, and mitral regurgitation in the ischemia-reperfused murine heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

Watch this Scientific Journal Video about High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

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24.0K Views.  The Ohio State University. High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

Video: High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

Murine Echocardiography and Ultrasound Imaging

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1 Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal.2 However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals.3 Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound.4,5 Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function.6 This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment.

This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1  ...

Magnetic Resonance Derived Myocardial Strain Assessment Using Feature Tracking

Purpose: An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that strain is a more sensitive and earlier marker for contractile dysfunction than the frequently used parameter EF. Current technologies for CMR are time consuming and difficult to implement in clinical practice. Feature tracking is a technology that can lead to more automization and robustness of quantitative analysis of medical images with less time consumption than comparable methods.
Methods: An automatic or manual input in a single phase serves as an initialization from which the system starts to track the displacement of individual patterns representing anatomical structures over time. The specialty of this method is that the images do not need to be manipulated in any way beforehand like e.g. tagging of CMR images.
Results: The method is very well suited for tracking muscular tissue and with this allowing quantitative elaboration of myocardium and also blood flow.
Conclusions: This new method offers a robust and time saving procedure to quantify myocardial tissue and blood with displacement, velocity and deformation parameters on regular sequences of CMR imaging. It therefore can be implemented in clinical practice.

An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that strain is a more sensitive and earlier marker for contractile dysfunction than the frequently used parameter EF.

Purpose: An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

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

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

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

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

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

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

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

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.

The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

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 ...

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the risks of uncorrected MR and its poor prognosis are known, the longitudinal changes in cardiac function, structure and remodeling are incompletely understood. This knowledge gap has limited our understanding of the optimal timing for MR correction, and the benefit that early versus late MR correction may have on the left ventricle. To investigate the molecular mechanisms that underlie left ventricular remodeling in the setting of MR, animal models are necessary. Traditionally, the aorto-caval fistula model has been used to induce volume overload, which differs from clinically relevant lesions such as MR. MR represents a low-pressure volume overload hemodynamic stressor, which requires animal models that mimic this condition. Herein, we describe a rodent model of severe MR in which the anterior leaflet of the rat mitral valve is perforated with a 23G needle, in a beating heart, with echocardiographic image guidance. The severity of MR is assessed and confirmed with echocardiography, and the reproducibility of the model is reported.&#160;&#160;

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance.&#160;

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the ...

Morphological and Functional Assessment of the Right Ventricle Using 3D Echocardiography

Traditionally, it was believed that the right side of the heart has a minor role in circulation; however, more and more data suggest that right ventricular (RV) function has strong diagnostic and prognostic power in various cardiovascular disorders. Due to its complex morphology and function, assessment of the RV by conventional two-dimensional echocardiography is limited: the everyday clinical practice usually relies on simple linear dimensions and functional measures. Three-dimensional (3D) echocardiography overcame these limitations by providing volumetric quantification of the RV free of geometrical assumptions. Here, we offer a step-by-step guide to obtain and analyze 3D echocardiographic data of the RV using the leading commercially available software. We will quantify 3D RV volumes and ejection fraction. Several technical aspects may help to improve the quality of RV acquisition and analysis as well, which we present in a practical manner. We review the current opportunities and the limiting factors of this method and also highlight the potential applications of 3D RV assessment in current clinical practice.

Here, we provide a step-by-step acquisition and analysis protocol for the 3D volumetric assessment of the right ventricle, mainly focusing on the practical aspects that maximize the feasibility of this technique.