The authors acknowledge the following funding sources: Else Kr&#246;ner-Fresenius-Stiftung (Tienush Rassaf); Hans und Gertie Fischer Stiftung (Tienush Rassaf), grant from the medical faculty, University Duisburg-Essen, Germany (Tienush Rassaf, Lars Michel); Ernst- und Berta Grimmke-Stiftung (Christos Rammos).

All experiments were completed in accordance and compliance with all relevant regulations&#160;(&#8216;European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes&#8217; (Directive 2010/63/EU) and animal care was in accordance with institutional guidelines. All experiments have been performed with male C57BL/6JRj mice at the age of 6 months.
1. Preparation
<ol>
	<li>Prepare a surgical microscope and a heating pad as well as a rectal probe ...

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	<li>Sanchis-Gomar, F., Perez-Quilis, C., Leischik, R., Lucia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+coronary+heart+disease+and+acute+coronary+syndrome.">Epidemiology of coronary heart disease and acute coronary syndrome.</a> Annals of Translational Medicine. 4 (13), 256 (2016).</li><li>Anderson, J. L., Morrow, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Myocardial+Infarction.">Acute Myocardial Infarction.</a> New England Journal of Medicine. 376 (21), 2053-2064 (2017).</li><li>McNamara, R. 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=Predicting+in-hospital+mortality+in+patients+with+acute+myocardial+infarction.">Predicting in-hospital mortality in patients with acute myocardial infarction.</a> Journal of the American College of Cardiology. 68 (6), 626-635 (2016).</li><li>Kurian, G. A., Rajagopal, R., Vedantham, S., Rajesh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Oxidative+Stress+in+Myocardial+Ischemia+and+Reperfusion+Injury+and+Remodeling:+Revisited.">The Role of Oxidative Stress in Myocardial Ischemia and Reperfusion Injury and Remodeling: Revisited.</a> Oxidative Medicine and Cellular Longevity. , (2016).</li><li>Turer, A. T., Hill, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+myocardial+ischemia-reperfusion+injury+and+rationale+for+therapy.">Pathogenesis of myocardial ischemia-reperfusion injury and rationale for therapy.</a> American Journal of Cardiology. 106 (3), 360-368 (2010).</li><li>Totzeck, M., Hendgen-Cotta, U. B., French, B. A., Rassaf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+remote+ischemic+preconditioning+and+ischemic+preconditioning+against+myocardial+ischemia/reperfusion+injury.">A practical approach to remote ischemic preconditioning and ischemic preconditioning against myocardial ischemia/reperfusion injury.</a> Journal of Biological Methods. 3 (4), (2016).</li><li>Respress, J. L., Wehrens, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transthoracic+echocardiography+in+mice.">Transthoracic echocardiography in mice.</a> Journal of Visualized Experiments. (39), (2010).</li><li>Pacher, P., Nagayama, T., Mukhopadhyay, P., Batkai, S., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cardiac+function+using+pressure-volume+conductance+catheter+technique+in+mice+and+rats.">Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats.</a> Nature Protocols. 3 (9), 1422-1434 (2008).</li><li>Zhang, B., Davis, J. P., Ziolo, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+catheterization+in+mice+to+measure+the+pressure+volume+relationship:+Investigating+the+Bowditch+effect.">Cardiac catheterization in mice to measure the pressure volume relationship: Investigating the Bowditch effect.</a> Journal of Visualized Experiments. (100), e52618 (2015).</li><li>Rossello, X., Hall, A. R., Bell, R. M., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Langendorff+perfused+isolated+mouse+heart+model+of+global+ischemia-reperfusion+injury:+Impact+of+ischemia+and+reperfusion+length+on+infarct+size+and+LDH+release.">Characterization of the Langendorff perfused isolated mouse heart model of global ischemia-reperfusion injury: Impact of ischemia and reperfusion length on infarct size and LDH release.</a> Journal of Cardiovascular Pharmacology and Therapeutics. 21 (3), 286-295 (2016).</li><li>Task Force on the management of ST-segment elevation acute myocardial infarction of the European Society of Cardiology (ESC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESC+Guidelines+for+the+management+of+acute+myocardial+infarction+in+patients+presenting+with+ST-segment+elevation.">ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation.</a> European Heart Journal. 33 (20), 2569-2619 (2012).</li><li>Shigemitsu, O., 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=Acute+myocardial+infarction+due+to+left+main+coronary+artery+occlusion.+Therapeutic+strategy.">Acute myocardial infarction due to left main coronary artery occlusion. Therapeutic strategy.</a> Japanese Journal of Thoracic and Cardiovascular Surgery. 50 (4), 146-151 (2002).</li></ol>

The authors have nothing to disclose.

PV monitoring of LV hemodynamics in acute myocardial infarction serves as a novel method for real-time in vivo assessment of cardiogenic shock and impaired LV function in I/R injury. PV catheterization can provide a broad spectrum of parameters with regard to LV systolic and diastolic function. In addition to the LV volumetric parameters typically obtained by echocardiography or MRI (chamber volumes, EF, stroke volume, and cardiac output), PV analysis yields a more complete profile of LV function by simultaneous...

Cardiovascular disease is the most common cause of death in western civilization1. Acute myocardial infarction is a critical event, which is associated with high acute and chronic mortality2. Even if revascularization is achieved via emergency percutaneous coronary intervention (PCI), mortality remains high, particularly within the first 48 h after onset of symptoms in patients with acute myocardial infarction3. Cardiogenic shock caused by acute reduction in left ventricular (LV) function is a major cause for in-hospital mortality in these patients3. This early reduction in LV function is caused by myocardial damage following ischemia and reperfusion. This so-called ischemia/reperfusion (I/R) injury is mediated by changes in cellular metabolome such as exaggerated generation of reactive oxygen species4,5.
To explore possible protective mechanisms leading to a decrease in myocardial damage in a preclinical setting, reliable mouse models are essential including methods for evaluation of post-I/R LV function6. In this setting, transthoracic echocardiography7 and magnetic resonance imaging (MRI)6 are widely used for functional phenotyping8,9. However, these methods are not suitable for the assessment of severe LV dysfunction and cardiogenic shock in an ongoing acute myocardial infarction and cannot directly show data on LV pressure. The Langendorff apparatus using isolated heart in an ex vivo assay provides information about the underlying pathomechanisms of early-phase I/R injury10. This method is limited due to its inability to reproduce in vivo adaptive mechanisms such as regulation of the autonomous nervous system or hormonal regulation and acid-base homeostasis. There is currently no method available for a complete functional phenotyping of cardiogenic shock and left ventricular dysfunction during an ongoing myocardial I/R injury.
A synchronized approach with combination of pressure-volume (PV) catheterization and transient surgical left main coronary artery (LCA) occlusion could be beneficial but technically challenging. Stable extracardiac hemodynamics during I/R injury are essential for valid results since unstable anesthesia or blood loss could heavily influence the results. A novel approach for hemodynamic phenotyping of I/R injury via LV PV catheterization and transient LCA occlusion could bring new insights on cardiogenic shock and LV dysfunction in acute myocardial infarction and serve as a method for future analysis on cardioprotection.

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			<td height="26" style="height:27px;width:237px;">LAS EZ</td>
			<td style="width:164px;">Leica</td>
			<td style="width:119px;">LAS EZ</td>
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			<td height="26" style="height:27px;width:237px;">MiniVent</td>
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			<td style="width:119px;">845</td>
			<td>ventilation</td>
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		<tr height="26">
			<td height="26" style="height:27px;width:237px;">MPVS Ultra</td>
			<td style="width:164px;">Millar instruments</td>
			<td style="width:119px;">PL3508B48/M</td>
			<td>PV catheter data acquisition device</td>
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		<tr height="26">
			<td height="26" style="height:27px;width:237px;">Octenisept</td>
			<td style="width:164px;">Sch&#252;lke</td>
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			<td height="26" style="height:27px;width:237px;">Plastipak 1ml</td>
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			<td style="width:119px;">REF 303172</td>
			<td>1ml syringe</td>
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		<tr height="26">
			<td height="26" style="height:27px;width:237px;">PowerLab 8/35</td>
			<td style="width:164px;">AD Instruments</td>
			<td style="width:119px;">PL3508</td>
			<td>analog/digital converter</td>
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			<td height="26" style="height:27px;width:237px;">Prolene 6-0</td>
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			<td style="width:119px;">XNEH7814.P31</td>
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			<td height="26" style="height:27px;width:237px;">Retraction Kit</td>
			<td style="width:164px;">FST</td>
			<td>18200-20</td>
			<td>retraction of surgical situs</td>
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			<td height="26" style="height:27px;width:237px;">Seraflex 5-0</td>
			<td style="width:164px;">Naila</td>
			<td style="width:119px;">IC108000</td>
			<td>silk suture</td>
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			<td height="26" style="height:27px;width:237px;">Small and micro-scissors</td>
			<td style="width:164px;">FST Essen</td>
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			<td style="width:119px;">6324464.00.00</td>
			<td>Xylazine hydrochloride</td>
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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.

After LV catheterization, reversible LCA ligation was performed for 45 min followed by 10 min of reperfusion. PV data was recorded at all times (Figure 1).
Correct placement of the PV catheter was confirmed by obtaining the characteristic LV PV graph (Figure 2A). LV catheter placement showed the typical ventricular pressure range with a minimum of 0 - 20 mmHg whereas fa...

Watch this Scientific Journal Video about Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice at JoVE.com

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

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 ...

This method can help answer key questions in the field of cardiovascular research such as identifying early protective mechanisms in acute myocardia infraction. The main advantage of this technique is that an advanced hemodynamic characterization can be performed real time in mice undergoing myocardial infarction followed up by reperfusion. Endotracheal intubation and ischemia reperfusion surgery will be performed by Pia Stock, a biologist from our laboratory.After anesthetizing the animal, remove the neck and chest hair using electric clippers. Next, clean the skin with three alternating scrubs of Betadine and 70%alcohol. Now, using small surgical scissors, make a 10-millimeter longitudinal median incision five millimeters beneath the bottom lip and towards the sternum.Next, bluntly dissect the left and right part of the submandibular gland using forceps. Then, separate the muscle and fat tissue in the right paratracheal region to access the right commune corroded artery. Using curved forceps, carefully bluntly dissect the vessel to mobilize and separate five to 10 millimeters of it.Always avoid moving the vagus nerve in the corroded body, as this can lead to severe hypotension and bradycardia. After the vessel mobilization, pass the two prepared 5/0 silk sutures under the vessel. Then ligate the distal vessel using a tight knot, and place a loose knot on the proximal suture where the catheter will go.Next, place a hemostat vascular clamp on the proximal end of the exposed vessel, proximal to the suture. This will be used to reversibly block blood flow. Now, using microscissors, open the vessel with the wedge-shaped incision one millimeter proximal to the cranial knot.A small drop of blood should be seen. Then, stretch the incision and insert the catheter 10 millimeters into the vessel. Now, start recording the data collected by the catheter probe.Next, extract the vascular clamp, then add one or two drops of saline into the incision to help slide the catheter another 10 millimeters down the vessel. Then, tighten the knot to prevent blood reflux alongside the catheter without locking the catheter in place. Now, gently continue advancing the catheter until the pressure analysis shows an arterial blood pressure profile when the catheter has reached the aorta.At the aortic valve, light resistance and pull synchronized movement should also be felt. When trying to advance the catheter through the aortic valve, it can help to pull it back five millimeters and advance again. Proper left ventricular catheterization will be indicated by a change in diastolic pressure.Any change in volume is also an indicator of advancing the sensor to the left ventricle. Once certain that the catheter is in the left ventricle, fasten the proximal knot more tightly to prevent it from moving. After the catheterization, prepare the 45 minute ischemia.First, incise the skin from the caudal sternum to the left axilla. Then proceed with the blunt preparation of the two muscle layers to expose the ribs. Next, open the thorax with an incision between the third and fourth left rib.Make use of surgical hooks to access the pericardium, and then resect the pericardium covering the heart. Before continuing with the left main coronary artery ligation, wait 30 seconds without touching the animal to record pressure and volume data for a valid analysis. Now, identify the left main coronary artery.It emerges under the left oracle and descends at the left side of the heart towards the apex. Then, loop a 6/0 polypropylene suture around the artery two millimeters under the left oracle. Next, place a small silicon tube under the loop and secure it with a knot.If the distal myocardium starts to turn gray, then the left main coronary artery has been occluded. To proceed, cut the suture at one millimeter length, and then release the surgical hooks and manually close the muscle layers above the incision. Then wait 45 minutes while continuously recording pressure and volume data.After 45 minutes, reopen the incision and remove the silicon tube to induce reperfusion. Then record data for another 20 minutes. When the heart turns back to being red, the reperfusion is successful.After the reperfusion period, loosen the knot and extract the catheter while quickly fastening the knot again to prevent loss of blood if blood is needed for further studies. After completing the calibration steps, which are detailed in the text protocol, perform a software-guided data analysis. For the baseline data, highlight at least 10 cycles taken before the passing of the catheter into the aortic valve.As needed, exclude any cycles with deviations due to ventilation or manipulation. Next, analyze 10 cycle blocks on five minute intervals through the ischemia and reperfusion. The various parameters calculated by this analysis are sufficient for characterization of the left ventricular function.At the end of the experiment, after the catheter has been retracted from the left ventricle, take a final analysis of the pressure data. Following the protocol, after the left ventricular catheterization, reversible left main coronary artery ligation was performed for 45 minutes followed by 20 minutes of reperfusion. Correct placement of the volume and pressure catheter was confirmed by obtaining the characteristic left ventricular pressure-volume graph.Successful occlusion of the left main coronary artery was visually confirmed by blanching of the distal left ventricular myocardium. After the left main coronary artery occlusion, pressure and volume data was analyzed on five minute intervals. Pressure data showed no changes in maximum left ventricular systolic pressure, indicating preserved peripheral perfusion and stable anesthesia.Analysis of left ventricular volume revealed that during the early phase of the ischemia, there was a significant decrease in ejection fraction and in absolute stroke volume. The maximum change in pressure, a parameter of left ventricular contractility, showed a 30%reduction in mice undergoing myocardial ischemia. The minimum change in pressure significantly decreased as well, indicating impaired left ventricular compliance.After watching this video, you should have a good understanding on how to characterize left ventricular systolic and diastolic function in mice undergoing acute myocardial infarction using pressure-volumes catheter technique.

real-time-pressure-volume-analysis-acute-myocardial-infarction

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8.9K 조회수.  University Hospital Essen. This method can help answer key questions in the field of cardiovascular research such as identifying early protective mechanisms in acute myocardia infraction. The main advantage of this technique is that an advanced hemodynamic characterization can be performed real time in mice undergoing myocardial infarction followed up by reperfusion. Endotracheal intubation and ischemia reperfusion surgery will be performed by Pia Stock, a biologist from our laboratory.After anesthetizing the an...