We would like to thank the Helmsley foundation for providing ultrasound equipment to perform our experiments. This work was supported by NIA R01 053585 grant.

All procedures were performed in accordance with protocols approved by the University of Louisville Institutional Animal Care and Use Committee (IACUC-approved protocol 18223) and the NIH Guide for the Care and Use of Laboratory Animals11.
1. Animals
<ol>
	<li>Use 4-month-old female Fisher 344 rats (BW~150-180 g) for the study.</li>
</ol>
2. Ultrasound Imaging before IR surgery
<ol>
	<li>Anesthetize the rat with isoflurane - induction chamber...

<ol>
	<li>Eltzschig, H. K., Eckle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia+and+reperfusion--from+mechanism+to+translation.">Ischemia and reperfusion--from mechanism to translation.</a> Nature medicine. 17 (11), 1391-1401 (2011).</li><li>French, C. J., Zaman, A. K., Sobel, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+of+erythropoietin+to+render+jeopardized+ischemic+myocardium+amenable+to+incremental+salvage+by+early+reperfusion.">Failure of erythropoietin to render jeopardized ischemic myocardium amenable to incremental salvage by early reperfusion.</a> Coronary Artery Disease. 20 (4), 295-299 (2009).</li><li>Marzilli, M., Mariani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+and+microvascular+dysfunction:+implications+for+salvage+of+jeopardized+myocardium+and+reduction+of+infarct+size.">Ischemia-reperfusion and microvascular dysfunction: implications for salvage of jeopardized myocardium and reduction of infarct size.</a> Italian Heart Journal. 2 Suppl 3, (2001).</li><li>Prasad, A., Gersh, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+microvascular+dysfunction+and+reperfusion+injury.">Management of microvascular dysfunction and reperfusion injury.</a> Heart (British Cardiac Society). 91 (12), 1530-1532 (2005).</li><li>Ishihara, 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=Impaired+coronary+flow+reserve+immediately+after+coronary+angioplasty+in+patients+with+acute+myocardial+infarction.">Impaired coronary flow reserve immediately after coronary angioplasty in patients with acute myocardial infarction.</a> British heart journal. 69 (4), 288-292 (1993).</li><li>Camici, P. G., d&#39;Amati, G., Rimoldi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+microvascular+dysfunction:+mechanisms+and+functional+assessment.">Coronary microvascular dysfunction: mechanisms and functional assessment.</a> Nature Reviews Cardiology. 12 (1), 48-62 (2015).</li><li>Chung, K. S., Nguyen, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+measures+of+coronary+microcirculation:+Taking+the+long+road+to+the+clinic.">Non-invasive measures of coronary microcirculation: Taking the long road to the clinic.</a> Journal of Nuclear Cardiology. , (2017).</li><li>Kelm, N. Q., 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=Adipose-derived+cells+improve+left+ventricular+diastolic+function+and+increase+microvascular+perfusion+in+advanced+age.">Adipose-derived cells improve left ventricular diastolic function and increase microvascular perfusion in advanced age.</a> PLoS One. 13 (8), e0202934 (2018).</li><li>Pan, 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=Late+recovery+of+coronary+flow+reserve+in+patients+successfully+treated+with+a+percutaneous+procedure.">Late recovery of coronary flow reserve in patients successfully treated with a percutaneous procedure.</a> Revista Espa&#241;ola de Cardiolog&#237;a. 56 (5), 459-464 (2003).</li><li>Samim, A., Nugent, L., Mehta, P. K., Shufelt, C., Bairey Merz, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+angina+and+microvascular+coronary+dysfunction.">Treatment of angina and microvascular coronary dysfunction.</a> Current treatment options in cardiovascular medicine. 12 (4), 355-364 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , (2011).</li><li>Ciuffreda, M. C., 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=Rat+experimental+model+of+myocardial+ischemia/reperfusion+injury:+an+ethical+approach+to+set+up+the+analgesic+management+of+acute+post-surgical+pain.">Rat experimental model of myocardial ischemia/reperfusion injury: an ethical approach to set up the analgesic management of acute post-surgical pain.</a> PloS one. 9 (4), (2014).</li><li>van de Hoef, T. P., Siebes, M., Spaan, J. A. E., Piek, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+in+clinical+coronary+physiology:+why+coronary+flow+is+more+important+than+coronary+pressure.">Fundamentals in clinical coronary physiology: why coronary flow is more important than coronary pressure.</a> European Heart Journal. 36 (47), 3312-3319 (2015).</li><li>Naya, 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=Preserved+coronary+flow+reserve+effectively+excludes+high-risk+coronary+artery+disease+on+angiography.">Preserved coronary flow reserve effectively excludes high-risk coronary artery disease on angiography.</a> Journal of nuclear medicine : official publication, Society of Nuclear Medicine. 55 (2), 248-255 (2014).</li><li>Cortigiani, L., 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=Diagnostic+and+prognostic+value+of+Doppler+echocardiographic+coronary+flow+reserve+in+the+left+anterior+descending+artery.">Diagnostic and prognostic value of Doppler echocardiographic coronary flow reserve in the left anterior descending artery.</a> Heart (British Cardiac Society). 97 (21), 1758 (2011).</li><li>Kawata, 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=Prognostic+value+of+coronary+flow+reserve+assessed+by+transthoracic+Doppler+echocardiography+on+long-term+outcome+in+asymptomatic+patients+with+type+2+diabetes+without+overt+coronary+artery+disease.">Prognostic value of coronary flow reserve assessed by transthoracic Doppler echocardiography on long-term outcome in asymptomatic patients with type 2 diabetes without overt coronary artery disease.</a> Cardiovascular diabetology. 12, 121-121 (2013).</li></ol>

The major findings from the present study are that IR increases the resting LAD coronary artery velocity and impairs CFR, even in the absence of any residual angiographic stenosis.
Understanding the coronary physiology is an essential part of the clinical decision-making for cardiologists to treat coronary artery disease. CFR is one of the important functional parameters in understanding the pathophysiology of coronary microcirculation7,13</...

Myocardial ischemia reperfusion (IR) is a condition where blood supply is restricted to the heart followed by the restoration of perfusion and simultaneous reoxygenation1. Occlusion of coronary arteries can be caused by an embolus or cholesterol plaque rupture, which results in a severe imbalance of metabolic supply and demand, causing tissue hypoxia. Salvage of jeopardized myocardium, improve left ventricular function, and enhance survival in patients with acute myocardial infarction have been observed by the reperfusion therapy. However, after recanalization of the coronary artery, functional abnormalities of small coronary vessels may occur2,3,4,5. A significant proportion of patients, perhaps as many as 40%, do not regain microvascular and myocardial perfusion despite the restoration of coronary flow. Visualization and evaluation of the coronary microvasculature can be difficult in vivo, but there are a number of invasive and noninvasive techniques that can be used to assess parameters directly depending on the coronary microvascular function6,7. Also, the endothelial function can be assessed at the level of the coronary circulation via the CFR5.
Transthoracic Doppler echocardiography is a noninvasive tool which allows us to study coronary artery flow velocity and CFR5. CFR represents the ratio of maximal coronary blood flow to the resting coronary blood flow8. During the stress challenge, a normal heart increases coronary blood flow up to four times above the resting value. Cardiovascular risk increases when CFR is diminished9. Ishihara et al showed that the CFR was severely impaired immediately after the coronary angioplasty5. In the absence of coronary artery stenosis, CFR decreases during the coronary microvascular dysfunction and is present in about half of the patients with stable coronary artery disease10.
The overall goal of this method is noninvasive visualization of left anterior descending coronary artery (LAD) function in rats via echocardiography, which may be used to calculate CFR. This offers an important assessment tool for diagnosing microvascular dysfunction and evaluating potential therapeutic treatments.

<table>
	<tbody>
		<tr>
			<td>10 mL syringe</td>
			<td>BD Syringe</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>250S 13&#8211;24 MHz linear probe</td>
			<td>FUJIFILM VisualSonics Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dobutamine hydrochloride</td>
			<td>Sigma</td>
			<td>D0676-10mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RRC</td>
			<td>27376</td>
			<td></td>
		</tr>
		<tr>
			<td>Legato 100 Syringe pump</td>
			<td>KD Scientific</td>
			<td>788100</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo 3100</td>
			<td>FUJIFILM VisualSonics Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Winged infusion set, 27G x 1/2&#34;,</td>
			<td>Medline.com</td>
			<td>TMOSV27ELZ</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of coronary flow reserve after myocardial ischemia reperfusion in rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

For this study, we used 12 female Fisher 344 rats. We performed a stress test with Dobutamine and measured LAD coronary artery velocity before and 72 hours after the IR surgery. Before the IR surgery, resting LAD coronary artery velocity was measured as 423 &#177; 59 mm/s, which was increased after Dobutamine infusion (1005 &#177; 77mm/s) (Figure 3A). After 72 h of ischemia reperfusion, resting LAD coronary artery velocity was significantly higher compared to...

Watch this Scientific Journal Video about Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats at JoVE.com

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

The overall goal of this method is the visualization of the left anterior descending coronary artery function in rats using echocardiography to calculate coronary flow reserve. This method offers an important assessment tool for a diagnosis of microvascular dysfunction in diseased hearts and for evaluating for potential treatments throughout the study. Although reperfusion therapy has been shown to salvage jeopardized myocardium and to improve left ventricular function, functional abnormalities of small coronary vessels may occur after recanalization of the coronary artery.This method can provide insight into the coronary microcirculation of rats, as well as into other models, including myocardial infarction, and pressure volume overload of the left ventricle. Before pre-ischemia reperfusion surgery ultrasound imaging, confirm a lack of response to toe pinch in an anesthetized Fischer 344 rat. Shave the hair from the chest.Apply ointment to the animal's eyes and place the animal in the supine position on a 37 to 38 degree Celsius built-in warming platform. Connect the 13 to 24-megahertz linear probe to the ultrasound machine. Set the application preset to cardiac imaging setting and use the rail system to position the probe to obtain the parasternal short axis view.Use the XY axis micromanipulator to move the probe in the rostral direction to locate the pulmonary artery. Move the probe, making the left ventricle more readily visible. Once the B-mode image has been captured and Cine Stored, click color Doppler and visualize the coronary artery in the short axis.The red color, as observed in real time, is indicative of the direction of the flow. After visualizing the lower anterior descending coronary artery in color Doppler mode, change the mode to the pulse wave and look for the presence of a yellow indicator line on the coronary artery. Place the yellow pulse wave line in the middle of the coronary artery, taking care that the angle is parallel to the direction of the flow and use Cine Store to capture the velocity of the resting left anterior descending coronary flow and the peak diastole of the wave forms.After obtaining the resting left anterior descending flow velocity, place a small strip of gauze around the base of the tail and use hemostats to grab and twist the tourniquet to apply pressure, causing the vein to enlarge. Carefully place a 25-gauge infusion butterfly needle attached to a 10-milliliter syringe loaded with dobutamine into the dilated tail vein and stabilize the needle with glue and a piece of surgical tape. When the infusion line has been secured, remove the hemostats and tourniquet to recover the flow and place the syringe into an infusion pump set to inject 20 micrograms per kilogram per minute.During the infusion, carefully monitor the left anterior descending artery peak and heart rate, periodically recording the left anterior descending artery pulse wave peaks in the Doppler mode under the dobutamine-induced stress conditions. After the anterior descending coronary artery peaks and heart rate have plateaued during the challenge, stop the infusion, remove the tail vein infusion set, and remove the animal from the platform. Then, use the peak velocity tool to obtain the peak diastolic velocities from the images and calculate the coronary flow reserve index as the ratio of the left anterior descending artery stress peak diastolic flow velocity to the resting left anterior descending artery peak diastolic flow velocity.Using a fiber optic light source, intubate rat with an 18-gauge IV catheter and connect to the catheter to a ventilator. Next, use an 8-0 monofilament suture to ligate the left anterior descending artery through a 15-millimeter opening at the fifth intercostal space with a plain knot for 30 minutes. At the end of the ischemia period, visually confirm the ischemia via discoloration of the heart surface and release the ligature.Reperfusion can be verified by the reddening of the previously-discolored area of heart muscle. 72 hours after the surgery, measure the coronary flow and the coronary flow reserve as demonstrated, and compare the measurements to those obtained before the injury. In this representative experiment, before the ischemia reperfusion surgery, the resting left anterior descending coronary artery velocity was measured as approximately 423 millimeters per second to more than double the velocity after dobutamine infusion.72 hours after ischemia reperfusion, the resting left anterior descending coronary artery velocity was significantly higher compared to the resting left anterior descending coronary artery velocity before the surgery. The stress response to a dobutamine test after the injury was significantly reduced compared to the response before the surgery responses. The coronary flow reserve is calculated as the ratio of the peak flow velocity during stress to the resting flow velocity and was approximately 2.1 in young rats before surgery, but significantly reduced 72 hours after the injury.In addition, there were no significant changes in the systolic function of the left ventricle 72 hours after the injury. After its development, this technique will allow researchers to explore and use ultrasound imaging of coronary arteries in small animal models that accurately mimic human disease conditions. After watching this video, you should have a very good understanding of how to measure coronary blood flow and evaluate coronary microcirculation in a small animal cardiac disease model.

evaluation-coronary-flow-reserve-after-myocardial-ischemia

Research

JoVE Journal

Medicine

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 ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture of an atherosclerotic plaque; hence, treatment is aimed at plaque stabilization in order to prevent cellular death. However, there is considerable debate among clinicians, about which treatment pathway is best: early invasive using percutaneous coronary intervention (PCI/stent) when indicated or a conservative approach (i.e., medication only with PCI/stent if recurrent symptoms occur).
There are three types of ACS: ST elevation myocardial infarction (STEMI), non-ST elevation MI (NSTEMI), and unstable angina (UA). Among the three types, NSTEMI/UA is nearly four times as common as STEMI. Treatment decisions for NSTEMI/UA are based largely on symptoms and resting or exercise electrocardiograms (ECG). However, because of the dynamic and unpredictable nature of the atherosclerotic plaque, these methods often under detect myocardial ischemia because symptoms are unreliable, and/or continuous ECG monitoring was not utilized.
Continuous 12-lead ECG monitoring, which is both inexpensive and non-invasive, can identify transient episodes of myocardial ischemia, a precursor to MI, even when asymptomatic. However, continuous 12-lead ECG monitoring is not usual hospital practice; rather, only two leads are typically monitored. Information obtained with 12-lead ECG monitoring might provide useful information for deciding the best ACS treatment.
Purpose. Therefore, using 12-lead ECG monitoring, the COMPARE Study (electroCardiographic evaluatiOn of ischeMia comParing invAsive to phaRmacological trEatment) was designed to assess the frequency and clinical consequences of transient myocardial ischemia, in patients with NSTEMI/UA treated with either early invasive PCI/stent or those managed conservatively (medications or PCI/stent following recurrent symptoms). The purpose of this manuscript is to describe the methodology used in the COMPARE Study.
Method. Permission to proceed with this study was obtained from the Institutional Review Board of the hospital and the university. Research nurses identify hospitalized patients from the emergency department and telemetry unit with suspected ACS. Once consented, a 12-lead ECG Holter monitor is applied, and remains in place during the patient's entire hospital stay. Patients are also maintained on the routine bedside ECG monitoring system per hospital protocol. Off-line ECG analysis is done using sophisticated software and careful human oversight.

Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture ...

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 ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in G&#246;ttingen Minipigs and Landrace Pigs

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an unmet clinical need for prevention and therapy of post-myocardial infarction heart failure (post-MI HF). Clinically relevant pig models of post-MI HF are prerequisites for final proof-of-concept studies before entering into clinical trials in drug and medical device development.
Here we aimed to characterize a closed-chest porcine model of post-MI HF in adult G&#246;ttingen minipigs with long-term follow-up including serial cardiac magnetic resonance imaging (CMRI) and to compare it with the commonly used Landrace pig model.
MI was induced by intraluminal balloon occlusion of the left anterior descending coronary artery for 120 min in G&#246;ttingen minipigs and for 90 min in Landrace pigs, followed by reperfusion. CMRI was performed to assess cardiac morphology and function at baseline in both breeds and at 3 and 6 months in G&#246;ttingen minipigs and at 2 months in Landrace pigs, respectively.
Scar sizes were comparable in the two breeds, but MI resulted in a significant decrease of left ventricular ejection fraction (LVEF) only in G&#246;ttingen minipigs, while Landrace pigs did not show a reduction of LVEF. Right ventricular (RV) ejection fraction increased in both breeds despite the negligible RV scar sizes. In contrast to the significant increase of left ventricular end-diastolic (LVED) mass in Landrace pigs at 2 months, G&#246;ttingen minipigs showed a slight increase in LVED mass only at 6 months.
In summary, this is the first characterization of post-MI HF in G&#246;ttingen minipigs in comparison to Landrace pigs, showing that the G&#246;ttingen minipig model reflects post-MI HF parameters comparable to the human pathology. We conclude that the G&#246;ttingen minipig model is superior to the Landrace pig model to study the development of post-MI HF.

Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in G&amp;#246;ttingen Minipigs and Landrace Pigs

The overall goal of the current study is to present the techniques of induction of myocardial infarction (MI) and post-myocardial infarction heart failure (post-MI HF) in closed-chest, adult G&#246;ttingen minipigs and the characterization of post-MI HF model in G&#246;ttingen minipigs as compared to Landrace pigs.

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an ...

Rodent Heart and Brain Tissue Preparation for Digital Macro Photography after Ischemia-reperfusion

Macro photography is applicable for imaging various tissue samples at high magnification to perform qualitative and quantitative analyses. Tissue preparation and subsequent image capture are steps performed immediately after the ischemia-reperfusion (IR) experiment and must be performed in a timely manner and with appropriate care. For the evaluation of IR-induced damage in the heart and brain, this paper describes 2,3,5-triphenyl-2H-tetrazolium chloride (TTC)-based staining followed by macro photography. Scientific macro photography requires controlled lighting and an appropriate imaging setup. The standardized methodology ensures high-quality, detailed digital images even if a combination of an inexpensive up-to-date digital camera and macro lens is used. Proper techniques and potential mistakes in sample preparation and image acquisition are discussed, and examples of the influence of correct and incorrect setups on image quality are provided. Specific tips are provided on how to avoid common mistakes, such as overstaining, improper sample storage, and suboptimal lighting conditions. This paper shows the appropriate methodology for rat heart and brain tissue slicing and staining and provides guidelines for establishing lighting and camera setups and photography techniques for high-resolution image acquisition.

Presented here is a protocol for the standardized methodology of rodent tissue preparation after the ischemia-reperfusion experiment and guidelines for establishing lighting and camera setups for high-resolution image acquisition. This method is applicable to all experimental small-animal organ photography.

Macro photography is applicable for imaging various tissue samples at high magnification to perform qualitative and quantitative analyses. Tissue ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...