The authors have no acknowledgments.

This protocol was approved by the ethics review board of the Saitama Municipal Hospital. The patient, whose case study is presented, provided written informed consent for the publication of his case and accompanying case images.
1. Set-up for PCLeB
<ol>
	<li>When starting coronary angiography (CAG), prepare the manifold for PCLeB. Connect one inlet line of the manifold to a contrast medium bottle and another inlet line to a 500 mL bottle of lactated Ringer&#8217;s solution mixed with 500 U of heparin sodium (Figure 2).</li>
	<li>Perform CAG and pinpoint the occluded lesion. After finishing CAG, introduce a guiding catheter, which is connected to the manifold via a Y-connector, into the aorta and engage it in the ostium of the culprit coronary artery as usual.</li>
	<li>Start wiring procedures with a balloon catheter placed inside the guiding catheter close to its outlet.</li>
	<li>If the coronary flow is incidentally restarted with the passage of the wire through the occluded lesion, move the balloon catheter quickly toward the culprit lesion to re-occlude the lesion site. After re-occluding the lesion site, resume and finish the wiring procedures. 
	NOTE: Such incidental coronary reflow sometimes happens; therefore, the balloon catheter is placed inside the guiding catheter during the wiring procedures.</li>
	<li>After finishing the wiring procedures, move the balloon catheter forward and place the balloon at the occluded lesion. Check the position of the balloon by contrast medium injection, to see if the balloon is appropriately placed at the occluded lesion. 
	NOTE: After finishing the checking of the balloon position, naturally, the contrast medium fills all the lumen from the manifold to the tip of the guiding catheter.</li>
	<li>Disconnect the syringe injector from the manifold and connect a 30 mL syringe to the manifold instead, which is used for lactated Ringer&#8217;s solution injection. 
	NOTE: No special syringe is needed for replacing the syringe, but an ordinary syringe for normal usage is fine as long as it has a &#8805;30 mL volume and a lock connector, so it will not be disconnected from the manifold during rapid injections of lactated Ringer&#8217;s solution.</li>
	<li>Fill the 30 mL syringe with 20&#8211;30 mL of lactated Ringer&#8217;s solution by sucking it from the bottle of lactated Ringer&#8217;s solution through the line connected to the inlet of the manifold.</li>
	<li>Remove air bubbles inside the 30 mL syringe if present. Disconnect the syringe and push them out and re-connect it to the manifold. Suck lactated Ringer&#8217;s solution from the bottle to make up the amount used for pushing air bubbles out. 
	NOTE: Here, the default set-up of the injection system for PCLeB has been completed; that is, the contrast medium (approximately 4 mL) fills all the lumen from the manifold to the tip of the guiding catheter, and 20&#8211;30 mL of lactated Ringer&#8217;s solution fills the 30 mL syringe connected to the manifold.</li>
</ol>
2. Details of PCLeB Procedures
<ol>
	<li>Once the balloon catheter is placed at the occluded lesion and the default set-up of the injection system is completed, inflate the balloon for 20&#8211;30 s at low pressure.</li>
	<li>Deflate the balloon and start the initial 10 s brief reperfusion. Immediately push out all the contrast medium (approximately 4 mL) prefilled in the lumen from the manifold to the tip of the guiding catheter, by injecting &#8805;4 mL of 20&#8211;30 mL of the lactated Ringer&#8217;s solution prefilled in the 30 mL syringe into the guiding catheter, to see if the coronary flow is recovered. 
	NOTE: Approximately 4 mL of contrast medium is usually enough to confirm coronary flow recovery.</li>
	<li>If the coronary flow is recovered, replenish the syringe with the same amount of lactated Ringer&#8217;s solution used for pushing the contrast medium out by sucking the solution from the bottle connected to the manifold through an inlet line, to prepare for lactated Ringer&#8217;s solution injection at the end of reperfusion. Jump to step 2.7. 
	NOTE: If coronary flow recovery is not observed, the true occluded lesion may be located distal to the current location of the balloon. Go to step 2.4.</li>
	<li>Empty the syringe by injecting all the lactated Ringer&#8217;s solution into the coronary artery.</li>
	<li>Fill the syringe with 4 mL of the contrast medium by sucking it from the bottle connected to the manifold through an inlet line and inject it all to the guiding catheter to fill the lumen. Then, fill up the syringe with 20&#8211;30 mL of lactated Ringer&#8217;s solution by sucking it from the bottle of the lactated Ringer&#8217;s solution connected to the manifold through an inlet line. 
	NOTE: Here, the default set-up of the injection system for PCLeB has been re-established.</li>
	<li>Move the balloon distally to the true lesion, and repeat the same procedures described in 2.1&#8211;2.6.</li>
	<li>Once coronary flow recovery is observed, and the balloon is confirmed to be placed at the culprit lesion, keep the balloon there throughout the PCLeB procedures. 
	NOTE: The desirable balloon size may be the same as the lesion diameter. By selecting such balloon size, instead of a smaller-sized balloon, the coronary flow may not be hampered by the deflated balloon left at the lesion site during each brief repetitive reperfusion.</li>
	<li>Start lactated Ringer&#8217;s solution injection (20 mL for the right coronary artery and 30 mL for the left coronary artery) directly into the culprit coronary artery through the guiding catheter 4&#8211;5 s before the end of each brief reperfusion. 
	NOTE: Usually, it takes 5&#8211;7 s to inject 20&#8211;30 mL of lactated Ringer&#8217;s solution, which may overlap with the subsequent brief ischemic period; this is an intentional delay. The aim of this intentional delay is to ensure that the lactate is trapped inside the ischemic myocardium.</li>
	<li>In the middle of lactated Ringer&#8217;s solution injection, let the secondary operator start balloon inflation so that the balloon inflation is completed a little before completion of lactated Ringer&#8217;s solution injection. 
	NOTE: This maneuver is intended so that the final small amount of lactated Ringer&#8217;s solution (ideally 2&#8211;3 mL) injected into the culprit coronary artery is bounced back from the inflated balloon. Through this approach, the lactated Ringer&#8217;s solution can be injected into the ischemic myocardium until the last moment of the balloon inflation process. This procedure requires cooperation between the primary operator who injects the lactated Ringer&#8217;s solution and the secondary operator who inflates the balloon.</li>
	<li>Reestablish the default set-up of the injection system, as described in 2.5., during 60 s ischemia for the next brief reperfusion.</li>
	<li>Deflate the balloon and start the next 10 s longer reperfusion after 60 s ischemia. Push out all the contrast medium prefilled in the lumen from the manifold to the tip of the guiding catheter, to reconfirm coronary flow.</li>
	<li>If the balloon is found not to be placed at the center of the lesion site, adjust the balloon position. 
	NOTE: This might be done during the first 10 s reperfusion, but it can be postponed until the next 20 s reperfusion because the 10 s duration may be slightly very short to do it.</li>
	<li>Start lactated Ringer&#8217;s solution injection into the culprit coronary artery through the guiding catheter 4&#8211;5 s before the end of each brief reperfusion</li>
	<li>In the middle of lactated Ringer&#8217;s solution injection, let the secondary operator start balloon inflation so that the balloon inflation is completed a little before completion of lactated Ringer&#8217;s solution injection.</li>
	<li>Reestablish the default set-up of the injection system during 60 s ischemia.</li>
	<li>Repeat these brief ischemia and reperfusion sequences seven times until 60 s reperfusion is performed twice (Figure 1). 
	NOTE: These procedures can be performed without disconnecting the 30 mL syringe from the manifold. The balloon inflation pressure can be increased gradually throughout the procedures, but high pressure is not recommended.</li>
</ol>
3. Alternative Default Set-up
NOTE: There may be an alternative to the default set-up, where replacing the syringe injector with a 30 mL syringe is not needed.
<ol>
	<li>After finishing the wiring procedures, disconnect the inlet line connected to the lactated Ringer&#8217;s solution bottle from the manifold and connect the 30 mL syringe filled with lactated Ringer&#8217;s solution directly with the inlet of the manifold instead. 
	NOTE: This is the alternative default set-up.</li>
	<li>During each brief ischemia, disconnect the 30 mL syringe, which was emptied at the end of the preceding brief reperfusion, from the manifold. Refill the syringe with lactated Ringer&#8217;s solution by sucking it directly from the bottle. Reconnect the refilled 30 mL syringe to the inlet of the manifold to reestablish the alternative default set-up. 
	NOTE: With this set-up, it is possible to obtain more clear images of the culprit coronary artery by making a larger volume of contrast medium available. However, the aforementioned set-up may be more convenient because disconnecting the 30 mL syringe from the manifold is not necessary for each refilling of the syringe with lactated Ringer&#8217;s solution.</li>
</ol>

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S., 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=Long-term+trends+in+the+incidence+of+heart+failure+after+myocardial+infarction.">Long-term trends in the incidence of heart failure after myocardial infarction.</a> Circulation. 118 (20), 2057-2062 (2008).</li><li>Ezekowitz, J. A., Kaul, P., Bakal, J. A., Armstrong, P. W., Welsh, R. C., McAlister, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Declining+in-hospital+mortality+and+increasing+heart+failure+incidence+in+elderly+patients+with+first+myocardial+infarction.">Declining in-hospital mortality and increasing heart failure incidence in elderly patients with first myocardial infarction.</a> Journal of American College of Cardiology. 53 (1), 13-20 (2009).</li><li>Piper, H. M., Garcia-Dorado, D., Ovize, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fresh+look+at+reperfusion+injury.">A fresh look at reperfusion injury.</a> Cardiovascular Research. 38 (2), 291-300 (1998).</li><li>Yellon, D. M., Hausenloy, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+reperfusion+injury.">Myocardial reperfusion injury.</a> New England Journal of Medicine. 357 (11), 1121-1135 (2007).</li><li>Koyama, T., Shibata, M., Moritani, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+postconditioning+with+lactate-enriched+blood+in+patients+with+acute+myocardial+infarction.">Ischemic postconditioning with lactate-enriched blood in patients with acute myocardial infarction.</a> Cardiology. 125, 92-93 (2013).</li><li>Koyama, 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=Impact+of+ischemic+postconditioning+with+lactate-enriched+blood+on+early+inflammation+after+myocardial+infarction.">Impact of ischemic postconditioning with lactate-enriched blood on early inflammation after myocardial infarction.</a> IJC Metabolic &amp; Endocrine. 2, 30-34 (2014).</li><li>Koyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactated+Ringer&#8217;s+solution+for+preventing+myocardial+reperfusion+injury.">Lactated Ringer&#8217;s solution for preventing myocardial reperfusion injury.</a> IJC Heart &amp; Vasculature. 15, 1-8 (2017).</li><li>Staat, P., 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=Postconditioning+the+human+heart.">Postconditioning the human heart.</a> Circulation. 112 (14), 2143-2148 (2005).</li><li>Hahn, J. Y., 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=Ischemic+postconditioning+during+primary+percutaneous+coronary+intervention:+the+effects+of+postconditioning+on+myocardial+reperfusion+in+patients+with+ST-segment+elevation+myocardial+infarction+(POST)+randomized+trial.">Ischemic postconditioning during primary percutaneous coronary intervention: the effects of postconditioning on myocardial reperfusion in patients with ST-segment elevation myocardial infarction (POST) randomized trial.</a> Circulation. 128 (17), 1889-1896 (2013).</li><li>Limalanathan, S., Andersen, G. &. #. 2. 1. 6. ;., Kl&#248;w, N. E., Abdelnoor, M., Hoffmann, P., Eritsland, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ischemic+postconditioning+on+infarct+size+in+patients+with+ST-elevation+myocardial+infarction+treated+by+primary+PCI+results+of+the+POSTEMI+(POstconditioning+in+ST-Elevation+Myocardial+Infarction)+randomized+trial.">Effect of ischemic postconditioning on infarct size in patients with ST-elevation myocardial infarction treated by primary PCI results of the POSTEMI (POstconditioning in ST-Elevation Myocardial Infarction) randomized trial.</a> Journal of American Heart Association. 3 (2), e000679 (2014).</li><li>Engstr&#248;m, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ischemic+postconditioning+during+primary+percutaneous+coronary+intervention+for+patients+with+ST-segment+elevation+myocardial+infarction:+a+randomized+clinical+trial.">Effect of ischemic postconditioning during primary percutaneous coronary intervention for patients with ST-segment elevation myocardial infarction: a randomized clinical trial.</a> JAMA Cardiology. 2 (5), 490-497 (2017).</li><li>Inserte, J., Barba, I., Hernando, V., Garcia-Dorado, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+recovery+of+intracellular+acidosis+during+reperfusion+prevents+calpain+activation+and+determines+protection+in+postconditioned+myocardium.">Delayed recovery of intracellular acidosis during reperfusion prevents calpain activation and determines protection in postconditioned myocardium.</a> Cardiovascular Research. 81 (1), 116-122 (2009).</li><li>Koyama, 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=Impact+of+postconditioning+with+lactate-enriched+blood+on+in-hospital+outcomes+of+patients+with+ST-segment+elevation+myocardial+infarction.">Impact of postconditioning with lactate-enriched blood on in-hospital outcomes of patients with ST-segment elevation myocardial infarction.</a> International Journal of Cardiology. 220, 146-148 (2016).</li><li>Koyama, T., Munakata, M., Akima, T., Miyamoto, K., Kanki, H., Ishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+squeezing+immediately+after+coronary+reperfusion+therapy+using+postconditioning+with+lactate-enriched+blood.">Muscle squeezing immediately after coronary reperfusion therapy using postconditioning with lactate-enriched blood.</a> International Journal of Cardiology. 275, 36-38 (2019).</li><li>Hale, S. L., Mehra, A., Leeka, J., Kloner, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postconditioning+fails+to+improve+no+reflow+or+alter+infarct+size+in+an+open-chest+rabbit+model+of+myocardial+ischemia-reperfusion.">Postconditioning fails to improve no reflow or alter infarct size in an open-chest rabbit model of myocardial ischemia-reperfusion.</a> American Journal of Physiology-Heart and Circulatory Physiology. 294 (1), H421-H425 (2008).</li><li>Hasdai, D., Holmes, D. R., Garratt, K. N., Edwards, W. D., Lerman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+pressure+and+stretch+release+endothelin-1+from+human+atherosclerotic+coronary+arteries+in+vivo.">Mechanical pressure and stretch release endothelin-1 from human atherosclerotic coronary arteries in vivo.</a> Circulation. 95 (2), 357-362 (1997).</li><li>Kohmoto, O., Ikenouchi, H., Hirata, Y., Momomura, S., Serizawa, T., Barry, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+effects+of+endothelin-1+on+[Ca2+]i+transients,+pHi+and+contraction+in+ventricular+myocytes.">Variable effects of endothelin-1 on [Ca2+]i transients, pHi and contraction in ventricular myocytes.</a> American Journal of Physiology-Heart and Circulatory Physiology. 265, H793-H800 (1993).</li><li>Koyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postconditioning+with+lactate-enriched+blood+in+patients+with+ST-segment+elevation+myocardial+infarction.">Postconditioning with lactate-enriched blood in patients with ST-segment elevation myocardial infarction.</a> Cardiology. 142, 79-80 (2016).</li></ol>

The authors have nothing to disclose.

A detailed description of the PCLeB procedures was provided with a representative case treated using PCLeB. PCLeB consists of intermittent reperfusion and timely coronary injections of lactated Ringer&#39;s solution to achieve controlled reperfusion with tissue oxygenation and minimal lactate washout7,8,9. Not only intermittent reperfusion but also supplementary lactate, administered as lactated Ringer&#39;s solution, may increase the delay in recovery from tissue acidosis produced during ischemia; in this supposed mechanism, PCLeB may potentiate the beneficial effects of the original postconditioning protocol that consists of intermittent reperfusion only10.
Several critical steps need to be pointed out to successfully perform PCLeB. First, before starting PCLeB, incidental coronary flow recovery should be prevented as much as possible because uncontrolled reperfusion before PCLeB ruins the subsequent&#160;controlled reperfusion achieved by PCLeB in terms of tissue oxygenation with minimal lactate washout. Spontaneous reperfusion before CAG cannot help. However, during the initial wiring procedures, coronary flow is sometimes unintentionally re-started before balloon delivery to the occluded coronary artery. This is an unwanted phenomenon. To minimize the effects of this phenomenon, the balloon catheter is recommended to be placed inside the guiding catheter beforehand, close to its outlet during the wiring procedures, so that the balloon can be quickly moved forward to the culprit lesion to re-occlude the lesion site once the coronary flow is unintentionally re-started.
Second, once PCLeB is started, confirming the restoration of coronary flow during each brief reperfusion is important because the failure to restore the coronary flow during PCLeB procedures makes PCLeB meaningless. Therefore, during each brief reperfusion, contrast medium injection into the culprit coronary artery should be performed to check if the coronary flow was restored. This can be achieved by injecting &#8805;4 mL of 20&#8211;30 mL of the lactated Ringer&#39;s solution prefilled in the 30 mL syringe into the guiding catheter, which pushes out approximately 4 mL of the contrast medium prefilled within the lumen from the manifold to the tip of the guiding catheter in the default set-up. Injections of both the contrast medium and 20&#8211;30 mL of lactated Ringer&#39; solution need to be performed even during the first 10 s reperfusion; thus, the busiest time during the entire procedures of PCLeB occurs at the very start of this protocol. Even if the initial brief reperfusion took 12&#8211;13 s instead of 10 s, it might be still acceptable. Since the reason for starting brief reperfusion with 10 s duration is to achieve minimal lactate washout during the very early phase of reperfusion, the initial brief reperfusion of 12&#8211;13 s duration can still achieve this goal.
Third, the size of the balloon used for PCLeB is important. Since the balloon is left at the lesion site throughout the procedures of PCLeB, if a small-sized balloon is selected, the lumen area gained after balloon dilation may be small and coronary flow may be hampered by the deflated balloon left at the lesion site during each brief reperfusion. Therefore, the balloon should ideally have the same size as the lumen diameter of the target lesion. However, one size smaller balloon may still be acceptable. Selection of balloons of such sizes is also beneficial in imposing adequate stretch stimuli on the vessel wall before stenting because of the reason mentioned later.
Fourth, the speed of lactated Ringer&#39;s solution injection and the timing of the balloon inflation are vital. The core objective of PCLeB is to keep tissue lactate concentrations high during the early reperfusion period. To achieve this objective, a larger amount of lactated Ringer&#39;s solution should be trapped inside the ischemic myocardium in a less diluted form. To make it possible, fast and continued injection of lactated Ringer&#39;s solution until the last moment of the balloon inflation process is needed. Therefore, 20&#8211;30 mL of lactated Ringer&#39;s solution needs to be injected within several seconds and the balloon inflation should be completed a little before completion of lactated Ringer&#39;s solution injection. To trap a larger amount of lactated Ringer&#39;s solution inside the ischemic myocardium, a larger amount of the solution, instead of 20&#8211;30 mL, can be used for each injection, but care should be taken to avoid volume overload.
Some modification of the PCLeB protocol might be possible if the modification adheres to the two critical components of PCLeB, i.e., starting with a very short period of reperfusion (i.e., 10&#8211;15 s) and trapping lactated Ringer&#39;s solution inside the ischemic myocardium in each brief repetitive ischemia. Reduction of the number of intermittent reperfusions and modification of the period of brief ischemia/reperfusion may be allowed. However, whether such modifications will reduce the beneficial effects of PCLeB is unknown.
Coronary flow recovery is generally very good after reperfusion therapy with PCLeB8,9,15. No-reflow phenomenon may not be experienced with this approach; this cannot be prevented by the original postconditioning protocol in animal experiments, reportedly17. However, at the end of the reperfusion therapy with PCLeB, if good coronary flow recovery cannot be achieved (i.e., TIMI grade flow II instead of III), there might be two possible explanations. First, insufficient stretch stimuli to the culprit lesion before stenting might reduce the beneficial effects of PCLeB. Stretch stimuli to the culprit lesion by balloon dilation or stenting procedures induces endothelin release from the endothelium of the lesion site18 and causes intracellular alkalization in myocardial cells19 distal to the lesion, which is an opposite effect of PCLeB20 and may reduce the beneficial effects of PCLeB. Therefore, it is recommended that supposed endothelin storage in the culprit lesion should be released as much as possible during the PCLeB procedures when tissue acidosis is maintained. If a smaller-sized balloon relative to the vessel size was used during the PCLeB procedures, stretch stimuli to the culprit lesion may be suboptimal and the endothelin inside the culprit lesion will be spared. Subsequent larger stimuli imposed by stenting procedures may induce an intense release of spared endothelin from the lesion site, possibly causing abrupt intracellular alkalization; this may attenuate the beneficial effects of PCLeB. Second, if a fairly longer stent, relative to the balloon used for PCLeB, is implanted, the similar phenomenon will ensue even if a sufficiently large balloon is used for the PCLeB procedures because no stretch stimuli is imposed on the coronary vessel wall excessively covered by the stent. Therefore, if possible, spot stenting using a shorter stent is preferable after the PCLeB procedures.
There might be no limitation in the application of PCLeB in patients with STEMI as long as reperfusion therapy is indicated. Cardiogenic shock is not a contraindication at all but rather a good indication for PCLeB. Increase in aortic pressure can be expected during PCI using PCLeB, as shown in the representative results. Simultaneous use of IABP may reduce the beneficial effects of PCLeB because IABP may enhance the washout of lactate via a mechanically driven force and may promote recovery from tissue acidosis produced during ischemia. Therefore, simultaneous use of IABP is not recommended even in severe cases, such as in patients with cardiogenic shock. Spontaneous reperfusion before CAG may reduce or remove the beneficial effects of PCLeB. However, spontaneous reperfusion before CAG does not necessarily preclude the application of PCLeB because coronary flow may still be insufficient and low-flow ischemia may still exist in the reperfused myocardium in such cases. Therefore, PCLeB may be worth trying as long as TIMI flow grade III is not achieved before PCI. Conversely, cases with spontaneous reperfusion with TIMI flow grade III achieved before PCI may be a clear limitation of the application of PCLeB.
The protocol of PCLeB appears complicated at a glance. However, once the initial busiest part of the protocol has finished, it can be realized that the patient&#39;s condition is becoming more stabilized as the procedures proceed to the less busy, later stages of the protocol. Before finishing the entire protocol, secondary operators often start to prepare for the next procedures after PCLeB, such as intravascular ultrasonography, because they know that nothing worse will happen, thereafter. Currently, myocardial reperfusion injury is generally left untreated during reperfusion therapy for STEMI. Despite the absence of firm evidence for the beneficial effects of PCLeB, considering its safety aspect and the current lack of alternative effective approaches, PCLeB is worth trying instead of leaving myocardial reperfusion injury as it occurs. Once the effectiveness of PCLeB is generally confirmed in the future, this technique might, hopefully, be applied to other arterial occlusive disorders, such as acute limb ischemia.

The widespread use of coronary reperfusion therapy has markedly improved the survival of patients with ST-segment elevation myocardial infarction (STEMI) over the past decades1,2. On the contrary, the proportion of patients with post-myocardial-infarction (post-MI) heart failure has increased, paradoxically3,4. To reduce the incidence of post-MI heart failure, further reduction of the infarct size is of paramount importance.
Timely restoration of the coronary blood flow is crucial for salvaging myocardial cells from the ischemic cell death. However, restoration of the coronary blood flow to the ischemic myocardium induces another type of cell death, which is myocardial reperfusion injury and reduces myocardial-salvaging effects of reperfusion therapy5,6. Therefore, to further reduce the infarct size and improve the outcomes of patients with STEMI, prevention of myocardial reperfusion injury is vital. However, despite notable efforts over the decades, no approach has been proven successful in preventing this injury in the clinical setting to date. Recently, a new approach for cardioprotection in patients with STEMI, i.e., postconditioning with lactate-enriched blood (PCLeB), has been reported7,8,9. PCLeB is a modification of the original protocol of postconditioning reported by Staat et al10. In the original postconditioning protocol, the culprit coronary artery is reopened and immediate application of four brief cycles of intermittent reperfusion is performed. Despite its initial success in a small pilot study10, the original postconditioning protocol has failed to improve the outcomes of patients with STEMI in large-scale clinical trials11,12,13. In&#160;the&#160;PCLeB protocol, the original postconditioning protocol&#160;was modified to increase the delay in recovery from tissue acidosis produced during ischemia because the delay in recovery from the tissue acidosis was thought to be responsible for the cardioprotective effects of postconditioning14. For this purpose, in addition to intermittent reperfusion, timely coronary injections of the lactated Ringer&#39;s solution were incorporated into the postconditioning protocol, aimed at achieving controlled reperfusion with tissue oxygenation and minimal lactate washout. Through this approach, the speed of the recovery from tissue acidosis relative to that of tissue re-oxygenation can be substantially reduced, thus making the delay in recovery from tissue acidosis definitive. The duration of each brief reperfusion, in this modified postconditioning protocol (Figure 1), is gradually increased in a stepwise manner from 10 to 60 s. This approach prevents the abrupt and rapid washout of lactate during the very early phase of reperfusion. Each brief ischemic period lasts for 60 s. Injection of lactated Ringer&#39;s solution is performed to supply lactate directly into the culprit coronary artery at the end of each brief reperfusion. 20 mL of lactated Ringer&#39;s solution is injected for the right coronary artery and 30 mL is injected for the left coronary artery. This is done immediately before the balloon inflation. During each brief repetitive ischemic period, to trap the lactate inside the ischemic myocardium, the balloon is quickly inflated at the lesion site. Full reperfusion is performed after seven cycles of balloon inflation and deflation. Stenting is performed thereafter, and the percutaneous coronary intervention (PCI) is completed. Excellent in-hospital15 and 6 month16 outcomes in a limited number of patients with STEMI treatment using PCLeB have already been reported. This method article provides a detailed description of each step of the PCLeB procedures, which is developed for preventing myocardial reperfusion injury in patients with STEMI.

<table>
	<tbody>
		<tr>
			<td>Heparin Na (5000 U/5 mL)</td>
			<td>Mochida Pharmaceutical Company</td>
			<td></td>
			<td>Heparin sodium</td>
		</tr>
		<tr>
			<td>Lactec Injection (500 mL)</td>
			<td>Otsuka Pharmaceutical Factory</td>
			<td></td>
			<td>Lactated Ringer&#39;s solution</td>
		</tr>
		<tr>
			<td>NAMIC CONVENIENCE KIT AKK-3435</td>
			<td>NIPRO</td>
			<td>6069410</td>
			<td>a manifold-plus-syringe-injector kit</td>
		</tr>
		<tr>
			<td>Y Connector</td>
			<td>GOODMAN CO.,LTD.</td>
			<td>YOL9A</td>
			<td>Y-connector is connceted between a guiding catheter and a manifold, and enables both pressure momitoring and introduction of a balloon catheter into a guiding catheter simultaneously.</td>
		</tr>
	</tbody>
</table>

postconditioning with lactate-enriched blood for cardioprotection in st-segment elevation myocardial infarction

The beneficial effects of reperfusion therapy for ST-segment elevation myocardial infarction (STEMI) is attenuated by reperfusion injury. No approach has been proven successful in preventing this injury in the clinical setting to date. Meanwhile, a novel approach for cardioprotection in patients with STEMI, i.e., postconditioning with lactate-enriched blood (PCLeB), has recently been reported. PCLeB is a modification of the original protocol of postconditioning, aimed at increasing the delay in the recovery from tissue acidosis produced during ischemia. This was sought to achieve controlled reperfusion with tissue oxygenation and minimal lactate washout. In this modified postconditioning protocol, the duration of each brief reperfusion is gradually increased in a stepwise manner from 10 to 60 s. Each brief ischemic period lasts for 60 s. At the end of each brief reperfusion, injection of lactated Ringer&#39;s solution (20&#8211;30 mL) is performed directly into the culprit coronary artery immediately before the balloon inflation and the balloon is quickly inflated at the lesion site, so that the lactate is trapped inside the ischemic myocardium during each brief repetitive ischemic period. After seven cycles of balloon inflation and deflation, full reperfusion is performed. Stenting is performed thereafter, and the percutaneous coronary intervention is completed. Excellent in-hospital and 6 month outcomes in a limited number of patients with STEMI treated using PCLeB have already been reported. This method article provides a detailed description of each step of the PCLeB procedures.

A representative case treated using PCLeB is shown. A 48-year-old man presented at the emergency department of the Saitama Municipal Hospital for prolonged chest pain. Electrocardiography (ECG) revealed marked ST-segment elevation in precordial leads (Figure 3), and he was then diagnosed with anterior STEMI. Blood levels of glucose, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were 111, 179, and 36 mg/dL, respectively. Glycated hemoglobin level was 5.9%. Creatine kinase (CK) and creatine kinase-MB (CK-MB) levels were 106 and 3 IU/L, respectively. The catheterization team was mobilized, and he was transferred to the catheterization laboratory immediately. He experienced frequent ventricular fibrillation before reperfusion therapy (Figure 4). DC cardioversion was performed three times at the emergency department and three times at the catheterization laboratory before CAG was performed.
CAG revealed proximal occlusion of the left anterior descending artery with thrombolysis-in-myocardial-infarction (TIMI) flow grade I (Figure 5A). No collateral circulation was observed. At this time, the aortic systolic pressure was approximately 70 mmHg (Figure 6A). However, intra-aortic balloon counterpulsation (IABP) was withheld because it enhances lactate washout via a mechanically driven force, which is opposite to what PCLeB does.
Reperfusion using PCLeB was started 60 min after symptom onset. No ventricular tachycardia/fibrillation was observed during and after PCLeB. The aortic systolic pressure increased to approximately 75 mmHg at the end of PCLeB (Figure 6B) and to 80 mmHg after the PCI (Figure 6C). Not only the systolic pressure but also the shape of the aortic pressure curve had improved; the apparent increase in the area under the curve of the aortic pressure suggested an increase in cardiac output. TIMI flow grade III was achieved after PCLeB. Stenting was performed, thereafter. TIMI flow grade III was still observed after stenting and the PCI was completed (Figure 5B). ECG recorded upon admission to the intensive care unit revealed complete ST resolution (Figure 7). The peak plasma CK and CK-MB levels were 4830 and 172 IU/L, respectively. The higher level of CK relative to that of CK-MB might be caused by a series of DC cardioversion before reperfusion therapy. No complication or adverse event was observed throughout his admission period. Resting thallium scintigraphy before discharge revealed well-preserved myocardial viability (Figure 8).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59672/59672fig1.jpg" /> 
Figure 1: Overview of the protocol for postconditioning with lactate-enriched blood. The duration of each brief reperfusion is gradually increased from 10 to 60 s in a stepwise manner. Each brief ischemic period lasts for 60 s. At the end of each brief reperfusion, lactate is supplied by injecting lactated Ringer&#8217;s solution directly into the culprit coronary artery immediately before balloon inflation and the balloon is quickly inflated at the lesion site so that the lactate is trapped inside the ischemic myocardium. After seven cycles of balloon inflation and deflation, full reperfusion is performed, followed by stenting. LCA = left coronary artery; RCA = right coronary artery. This figure has been modified from reference8. <a href="https://www.jove.com/files/ftp_upload/59672/59672fig1large.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/59672/59672fig2.jpg" /> 
Figure 2: Schematic representation of the manifold system for PCLeB. One inlet line of the manifold is connected to a bottle of contrast medium and another inlet line to a 500 mL bottle of lactated Ringer&#8217;s solution. The syringe injector is to be replaced by a 30 mL syringe equipped with a lock connector before starting PCLeB. PCLeB = postconditioning with lactate-enriched blood. <a href="https://www.jove.com/files/ftp_upload/59672/59672fig2large.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/59672/59672fig3.jpg" /> 
Figure 3: Electrocardiography in the emergency department. Marked elevation of ST-segment in precordial leads was observed. <a href="https://www.jove.com/files/ftp_upload/59672/59672fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59672/59672fig4.jpg" /> 
Figure 4: An electrocardiography recording of ventricular fibrillation observed at the catheterization laboratory <a href="https://www.jove.com/files/ftp_upload/59672/59672fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59672/59672fig5.jpg" /> 
Figure 5: Coronary angiography view before and after reperfusion therapy with PCLeB. (A) Coronary angiography view before reperfusion therapy. Subtotal occlusion of the proximal left anterior descending artery with thrombolysis-in-myocardial-infarction flow grade I was observed. (B) Final coronary angiography view after reperfusion therapy with PCLeB. Coronary flow in the left anterior descending artery was resumed. PCLeB, postconditioning with lactate-enriched blood. <a href="https://www.jove.com/files/ftp_upload/59672/59672fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59672/59672fig6.jpg" /> 
Figure 6: Aortic pressure before and after reperfusion with PCLeB. (A) Aortic pressure before reperfusion. The systolic aortic pressure was around 70 mmHg. (B) The aortic pressure immediately after PCLeB. The systolic aortic pressure increased to around 75 mmHg and the shape of the aortic pressure curve had improved in view of an increase in the area under the curve. (C) Aortic pressure after PCI completion. The systolic aortic pressure increased to 80 mmHg. PCI = percutaneous coronary intervention; PCLeB = postconditioning with lactate-enriched blood. <a href="https://www.jove.com/files/ftp_upload/59672/59672fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59672/59672fig7.jpg" /> 
Figure 7: Electrocardiography at the intensive care unit immediately after reperfusion therapy <a href="https://www.jove.com/files/ftp_upload/59672/59672fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Complete ST resolution was observed.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59672/59672fig8.jpg" /> 
Figure 8: Resting thallium scintigraphy before discharge. A bull&#8217;s eye imaging is shown. Myocardial viability was well preserved in the left anterior descending artery area. <a href="https://www.jove.com/files/ftp_upload/59672/59672fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Postconditioning with Lactate-enriched Blood for Cardioprotection in ST-segment Elevation Myocardial Infarction at JoVE.com

Postconditioning with Lactate-enriched Blood for Cardioprotection in ST-segment Elevation Myocardial Infarction

Herein, we present a protocol for postconditioning with lactate-enriched blood, which is a novel approach for cardioprotection. This protocol comprises&#160;intermittent reperfusion and timely coronary injections of lactated Ringer&#39;s solution. This can be applied to ST-segment elevation myocardial infarction patients who undergo primary percutaneous coronary intervention.

The beneficial effects of reperfusion therapy for ST-segment elevation myocardial infarction (STEMI) is attenuated by reperfusion injury. No approach has ...

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9.2K Views.  Saitama Municipal Hospital. Herein, we present a protocol for postconditioning with lactate-enriched blood, which is a novel approach for cardioprotection. This protocol comprises intermittent reperfusion and timely coronary injections of lactated Ringer's solution. This can be applied to ST-segment elevation myocardial infarction patients who undergo primary percutaneous coronary intervention.

Video: Postconditioning with Lactate-enriched Blood for Cardioprotection in ST-segment Elevation Myocardial Infarction

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 ...

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

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 ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

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 ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

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 ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...