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

7.7K 次观看.  University of Louisville. 该方法的总体目标是利用超声心动图计算冠状动脉储备，对大鼠左前降冠动脉功能进行可视化。该方法为诊断患病心脏的微血管功能障碍和评估整个研究中的潜在治疗方法提供了重要的评估工具。虽然再灌注疗法已被证明可以挽救危及的心肌，并改善左心室功能，但小冠状动脉重新硬化后，可能会发生功能异常。该方法可深入了解大鼠冠状动脉微循环，以及其他模型，?...