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 at 5% with 1.5&#8211;2.0 L/min O2 flow followed by 1.5&#8211;2.0% with 1.5&#8211;2.0 L/min O2 flow. This anesthesia is maintained throughout the experiment, during the resting and stress stages.</li>
	<li>Place the animal in a supine position and shave the thorax. Maintain the body temperature at 37-38 &#176;C using the built-in warming platform. Monitor the heart rate using the software. 
	NOTE: Proper anesthesia is crucial for maintaining the heart rate at normal physiological rates (between 295-350 beats/min).</li>
	<li>Apply ophthalmic vet ointment to prevent the dryness of eyes prior to the imaging.</li>
	<li>Perform echocardiography using 13&#8211;24 MHz linear probe (e.g., MS250) (Figure 1A).</li>
	<li>Place the animal in the supine position on the heated platform. Ensure that the anesthesia is controlled via the nose cone. Then position the probe to obtain the parasternal short axis view (PSAX) using the rail system (Figure 1B).</li>
	<li>Moving the probe in the rostral direction from the PSAX, locate the pulmonary artery (Figure 1B).</li>
	<li>Move the probe slightly in the direction caudal from the pulmonary artery to view the LAD and store the image. 
	NOTE: The LAD is difficult to find without Color Doppler, so B-Mode images of the LAD are not always possible.</li>
	<li>When individual differences make it difficult to locate the LAD coronary artery, follow the technique mentioned below:
	<ol>
		<li>Move the probe lateral to the pulmonary artery.</li>
		<li>Angle the platform so that the animal is inclined, inverted, or slightly towards the right side so that the left ventricle is more readily visible with the probe.</li>
	</ol>
	</li>
	<li>Once the image in B Mode is captured or cine-stored, click the Color Doppler on the touch screen (Figure 1C). Visualize the coronary artery (white arrow indicates LAD) in the short axis (Figure 1C). The red color, as seen in real time, is indicative of the direction of the flow (i.e., blood flow is towards the probe).</li>
	<li>After visualizing LAD on a color-Doppler mode, change the mode to the pulse wave (PW)-mode. Look for the presence of a yellow-indicator line on the coronary artery (Figure 2A).</li>
	<li>Place the yellow PW-line in the middle of the coronary artery. Ensure that the angle is parallel to the direction of the flow. 
	NOTE: Velocity of the flow is highly dependent on the angle of the PW line, so be sure to match the angle of the on-screen probe with the angle of the LAD. Use the touch screen to adjust the angle; PW angle should be less than 60&#176;.</li>
	<li>Use cine store to capture the velocity of the resting LAD coronary flow at the peak diastole as wave forms (Figure 2B).</li>
	<li>After obtaining of resting LAD flow velocity, measure the maximum flow velocity of LAD during the stress to calculate CFR. To measure the maximum flow velocity during the stress, infuse Dobutamine at a dose of 20 &#181;g/kg/min via tail vein8 (Figure 2C). 
	NOTE: Dobutamine infusion should not be more than 8 min. Use an infusion pump and set the diameter of the infusion pump to 14.43 for BD 10 mL syringe.
	<ol>
		<li>Use 25-G infusion butterfly set for the tail vein cannulation. To place the infusion needle, place a small strip of gauze around the base of the rat&#8217;s tail, then grab with hemostats and twist the tourniquet to apply pressure and cause the vein to enlarge.</li>
		<li>Place the needle while directly connected to Dobutamine syringe. 
		NOTE: Take extra care not to accidentally introduce drug when drawing up blood to ensure proper placement of the needle in the tail vein. Also, be sure to avoid introducing an air bubble into the vein, as an embolism may be fatal to the animal.</li>
		<li>Once the needle is placed, stabilize it with a glue and a piece of surgical tape, securing the infusion line to the tail.</li>
		<li>Remove the hemostats and tourniquet to recover the flow.</li>
		<li>Place Dobutamine syringe in the infusion pump and set it to inject 20 &#181;g/kg/min.</li>
		<li>During Dobutamine infusion, carefully monitor the LAD peak and heart rate. Periodically record LAD PW peaks in the Doppler mode, especially whenever it increases in response to Dobutamine. 
		NOTE: The stress challenge induced by Dobutamine causes the heart to work harder; this often results in the movement of the heart and the LAD. Be prepared to move the animal, the probe, or both in order to keep the LAD in view.</li>
		<li>After LAD peaks and heart rate have plateaued during the challenge, stop the Dobutamine infusion, remove the tail vein infusion set and remove the animal from the platform. Allow the animal to recover in its home cage.</li>
	</ol>
	</li>
	<li>Select Peak Vel tool to obtain the peak diastolic velocities from the images shown in Figure 2B and C.</li>
	<li>Calculate the CFR index as the ratio of the LAD stress (Dobutamine) peak diastolic flow velocity to the resting LAD peak diastolic flow velocity (Figure 2A).</li>
</ol>
3. Ischemia Reperfusion Injury
<ol>
	<li>Anesthetize rat with isoflurane - induction chamber at 5% with 1.5&#8211;2.0 L/min O2 flow followed by 1.5&#8211;2.0% with 1.5&#8211;2.0 L/min O2 flow.</li>
	<li>Confirm the depth of anesthesia by the lack of the pedal withdrawal reflex. Use ophthalmic vet ointment on the eyes to prevent dryness. Maintain the proper body temperature of 37-38 &#176;C using a heating pad or an animal temperature controller.</li>
	<li>Administer pre-surgical Meloxicam, 5 mg/kg intramuscularly, 15 min before the surgery, followed by 2 mL subcutaneous bolus of 0.9% saline to prevent dehydration during the surgery. Sterile gloves and instruments were used, and aseptic techniques were employed. Using the fiber-optic light source, intubate rat using 18-gauge IV catheter and connect to the ventilator.</li>
	<li>Ligate LAD using 8-0 monofilament suture through 15 mm opening at the 5th intercostal space. Tie a plain knot and leave for 30 min. Visually confirm ischemia in all rats via discoloration of the heart surface12. Release the ligature after 30 min and verify reperfusion by the reddening of the previously discolored area of heart muscle for 1-2 min12.</li>
	<li>Close the rib cage using 4-0 absorbable suture with an interrupted suture pattern, then close the skin using 5-0 non-absorbable silk suture with continuous suture pattern.</li>
	<li>Remove the animal from anesthesia. Allow the animal to recover in the home cage.&#160;5mg/kg Meloxicam was injected intramuscular in every 24 hours before euthanizing the animal (72hours after surgery).</li>
</ol>
4. Ultrasound imaging after IR surgery
<ol>
	<li>72 h after IR surgery, measure the coronary flow and CFR again. Compare the measurements to before IR measurements. Repeat steps 2.1 to 2.15 as described above.</li>
</ol>

<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 authors have nothing to disclose

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. CFR is a noninvasive method to assess both coronary artery stenosis and coronary microvascular circulation and is an indicator of myocardial blood supply, explicitly the ability of the coronaries to increase blood flow under stress conditions7. A normal CFR (&#62;2.0) often reflects a good prognosis, while CFR less than 1.90 provides incremental diagnostic information for the identification of high-risk coronary artery disease14,15,16.
Our results show that, even though the systolic function was preserved after ischemia reperfusion (Figure 3C), CFR was significantly lower (Figure 3B). Thus, recanalization of stenotic coronary arteries does not improve microvascular perfusion. Decreased CFR enables detection of impaired microvascular vasodilation after ischemia reperfusion.
This study demonstrates the serial CFR evaluations to explore the effect of various pharmacological therapies using noninvasive transthoracic Doppler echocardiography. This method of coronary functional assessment can be used in small animal research as a feasible and viable clinical diagnostic tool. This will lead to minimizing the requirement of animal use, euthanasia, or necropsy in the small animal models. Critical steps in this protocol are visualizing the coronary artery and obtaining the PW velocity images of good quality. Another critical step is to maintain LAD visualization during the stress state. During Dobutamine challenge, the heart rate increases and the LAD may move from the field of view; researchers should be prepared to move the field in order to follow the coronary artery. Limitations in the current study include the relatively small sample size, the lack of correlation between CFR and the coronary artery lumen diameter in vivo in rats, due to the difficulty in obtaining accurate visualization for the size measurement of the coronary artery. However, the methods described here are reliable, reproducible, and offer insightful information on the damage inflicted on the cardiac microvasculature following ischemia reperfusion.

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&amp;ndash;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&quot;,</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 the resting LAD coronary artery velocity before the IR surgery (743 &#177; 40mm/s vs 423 &#177; 59 mm/s) (Figure 3A). Stress response to Dobutamine test after the IR surgery was significantly reduced compared to the before IR surgery responses (937 &#177; 67ms/s vs 1005 &#177; 77mm/s) (Figure 3A).
CFR is calculated as the ratio of the peak flow velocity during stress (Dobutamine) to the resting flow velocity (measured prior to Dobutamine infusion)8.&#160; CFR was 2.1&#160;&#177; 0.35 in young rats before the IR surgery (Figure 3B) but significantly reduced (1.1 &#177; 0.25) after 72 h of the IR surgery, even though the resting LAD coronary artery velocity was higher in these rats compared to data obtained before IR surgery (Figure 3C). In addition, there were no significant changes in the systolic function of the left ventricle in rats after 72 h of the IR surgery (Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59406/59406fig1.jpg" /> 
Figure 1: Coronary artery location. A. Probe position on the rat while obtaining LAD coronary artery velocity. B. Short axis anatomical representation of the pulmonary artery, aorta, and LAD coronary artery. C. Anatomical visualization of LAD coronary artery on echocardiography. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59406/59406fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59406/59406fig2.jpg" /> 
Figure 2: The Pulse Wave Velocity imaging of the LAD coronary artery. A. Representation of the pulse wave velocity sensor placement on LAD coronary artery. B. LAD coronary artery pulse wave image during the rest condition. C. LAD coronary artery pulse wave image during the stress (Dobutamine) condition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59406/59406fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59406/59406fig3v2.jpg" /> 
Figure 3. Measurement of the coronary flow using Doppler echocardiography. A. Pulse wave velocity measured in Control during the rest (Cont B) and during Dobutamine infusion (Cont D) and in animals after 72 h of IR surgery during the rest (IR B) and during Dobutamine challenge (IR D), p &#60; 0.05 Cont B vs. ischemia reperfusion B (*). B. &#160;CFR was calculated from pulse wave in the experimental animals (n=9), p &#60; 0.05 Cont vs. IR (*). C. Fractional shortening assessment from the experimental groups (n=9). Data are presented as mean&#8201;&#177;&#8201;SD, analyzed with one-way ANOVA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59406/59406fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

7.8K Views.  University of Louisville. 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.

Video: Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

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 ...

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 ...

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 ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

A New Model of Heart Failure Post-Myocardial Infarction in the Rat

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial infarction (MI). However, the disadvantages of this model include high mortality rate after ligation and larger variations both in the infarct size and the degree of impaired cardiac function. In addition, a ventilator or exteriorization of the heart is indispensable for the previous models, which complicates the procedure during the ligation. In this study, we developed a reliable and reproducible model without the ventilator or exteriorization of the heart by ligating the LAD coronary artery. Four weeks after the procedure, we found that the serum concentrations of CK-MB, NT-proBNP, and Renin, which were used to assist diagnoses of MI and HF, were significantly higher in the MI group compared to the sham group. In contrast, the value of left ventricle ejection fraction (LVEF) in the MI group was obviously less than in the sham group. Furthermore, the infarct size and cardiac fibrosis area were individually confirmed and quantitatively analyzed by TTC staining and Masson's trichrome staining. Smaller variations were found in either infarct size or fibrosis area in the MI group, which helped to develop a reliable and reproducible model of HF post-MI. This new model of HF post-MI in the rat is vital for studying the potential mechanisms of MI and HF. This new method can be used to develop the new drug for treatment of MI and HF in rats by using pharmacological strategies.

We successfully developed a reliable and reproducible model of heart failure post-myocardial infarction in the rat without ventilation or exteriorization of the heart. This simplifies the procedure and benefits the further studies about the potential mechanisms behind the heart failure.

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial ...

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. 
Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the ...

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group.

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence ...

Left Anterior Descending Coronary Artery Ligation for Ischemia-Reperfusion Research: Model Improvement via Technical Modifications and Quality Control

Coronary heart disease is the leading cause of death globally. Complete cessation of blood flow in coronary arteries causes ST-segment elevation myocardial infarction (STEMI), resulting in cardiogenic shock and fatal arrhythmia, which are associated with high mortality. Primary coronary intervention (PCI) for recanalizing the coronary artery significantly improves the outcomes of STEMI, but advancements made in shortening the door-to-balloon time have failed to reduce in-hospital mortality, suggesting that additional therapeutic strategies are required. Left anterior descending coronary artery (LAD) ligation in rats is an animal model for acute myocardial IR research that is comparable to the clinical scenario in which rapid coronary recanalization through PCI is used for STEMI; however, PCI-induced STEMI is a technically challenging and complicated operation associated with high mortality and great variation in infarction size. We identified the ideal position for LAD ligation, created a gadget to control a snare loop, and supported a modified surgical maneuver, thereby reducing tissue damage, to establish a reliable and reproducible acute myocardial ischemia-reperfusion (IR) research protocol for rats. This is a non-survival surgery. We also propose a method for validating the quality of study results, which is a critical step for determining the accuracy of subsequent biochemical analyses.

Herein, we present a protocol focusing on the quality control of the left anterior descending coronary artery ligation by technically modifying the traditional procedure in rats for acute myocardial ischemia-reperfusion research.

Coronary heart disease is the leading cause of death globally. Complete cessation of blood flow in coronary arteries causes ST-segment elevation ...

Model of Ischemia and Reperfusion Injury in Rabbits

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and survival experiments. New Zealand White adult rabbit is sedated with atropine, acepromazine, butorphanol, and isoflurane. The animal is intubated and placed on mechanical ventilation. An intravenous catheter is inserted into the marginal ear vein for the infusion of medications. The animal is pre-medicated with heparin, lidocaine, and lactated Ringer&#39;s solution. A carotid cut-down is performed to obtain arterial line access for blood pressure monitoring. Select physiologic and mechanical parameters are monitored and recorded by continuous real-time analysis.
With the animal sedated and fully anesthetized, either a fourth intercostal space small left thoracotomy (survival) or midline sternotomy (non-survival) is performed. The pericardium is opened, and the left anterior descending (LAD) artery is located.
A polypropylene suture is passed around the second or third diagonal branch of the LAD artery, and the polypropylene filament is threaded through a small vinyl tube, forming a snare. The animal is subjected to 30 min of regional ischemia, achieved by occluding the LAD by tightening the snare. Myocardial ischemia is confirmed visually by regional cyanosis of the epicardium. Following regional ischemia, the ligature is loosened, and the heart is allowed to re-perfuse.
For both survival and non-survival experiments, the myocardial function can be assessed via an echocardiography (ECHO) measurement of the fractional shortening. For non-survival studies, data from sonomicrometry collected using three digital piezoelectric ultrasonic probes implanted within the ischemic area and the left ventricle developed pressure (LVDP) using an apically inserted left ventricle (LV) catheter can be continuously acquired for evaluating the regional and global myocardial function, respectively.
For survival studies, the incision is closed, a left needle thoracentesis is performed for pleural air evacuation, and postoperative pain control is achieved.

The present study demonstrates a highly reproducible animal model of acute regional myocardial ischemia and reperfusion injury in rabbits using a left mini-thoracotomy for survival cases or a midline sternotomy for non-survival cases.

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and ...