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 to monitor the body temperature. Clean and sterilize all surgical instruments.</li>
	<li>Prepare 2 pieces of 10 cm 5-0 silk thread for vessel ligation, 5 cm of 6-0 polypropylene thread, and 2 mm silicon tube for ligation of the LCA.</li>
	<li>Prepare the PV catheter calibration cuvette by pre-heating it to 37 &#176;C and prepare a 100 &#181;L Hamilton syringe filled with 15% sodium chloride (NaCl) in H2O for saline calibration.</li>
	<li>Place the PV catheter (3 cm, 1.4 F) in 37 &#176;C pre-heated 0.9% NaCl in H2O (saline) at least 30 min before measurement. Connect the catheter to the data acquisition device and connect the device to an analog/digital converter. Connect both devices to a computer.</li>
	<li>Set up the software. Perform software-guided pressure calibration and conductance calibration as demanded by the software-guided workflow8.</li>
</ol>
2. Anesthesia and Analgesia
<ol>
	<li>Anesthetize the mouse using ketamine 100 mg/kg body weight and xylazine hydrochloride 10 mg/kg body weight by intraperitoneal injection. At the beginning of I/R surgery, administer 0.05 mg/kg body weight buprenorphine intraperitoneally to maintain analgesia.</li>
	<li>After 10 min, perform an endotracheal intubation using a 20 G intravenous (iv) catheter and ventilate the mouse with 40% oxygen (O2) and 2% (v) isoflurane. Set appropriate ventilation parameters (e.g., 220 &#181;L stroke volume, 150/min for a 25 - 30 g C57BL/6JRj mouse).</li>
	<li>Continuously monitor body temperature via rectal probe. Fasten mouse on the heated plate with the head pointing towards the investigator.&#160;Mouse normal body temperature is 36.5 - 38 &#176;C. Maintain body temperature within 1 degree by adjustment of heated plate temperature.</li>
</ol>
3. Left Ventricular Catheterization
<ol>
	<li>Disinfect chest and neck&#160;with three alternating scrubs of betadine and 70% alcohol.&#160; Wait for skin disinfectant to dry.&#160;Remove the chest hair by using a small animal shaving system.&#160;</li>
	<li>Perform a 10 mm longitudinal median incision 5 mm beneath the bottom lip towards the sternum using small surgical scissors.</li>
	<li>Dissect the left and right part of the submandibular gland via blunt preparation using a forceps. Separate muscle and fat tissue in the right paratracheal region to expose the right commune carotid artery. Mobilize and separate the vessel for a total length of 5 - 10 mm from connective tissue via careful blunt preparation alongside the vessel with a bent forceps. 
	NOTE: Avoid mechanical manipulation of the vagus nerve or the carotid body at all time as this can cause severe hypotension and bradycardia.</li>
	<li>Pass the two prepared silk threads under the vessel. Ligate the distal vessel with a tight knot and place a loose knot on the proximal exposed region that still allows passage of the catheter.</li>
	<li>Fix the threads of the cranial (tight) knot next to the head of the mice to apply a light tension on the vessel as this will facilitate the introduction of the catheter. Place a hemostat vascular clamp on the proximal vessel proximal of the loose knot to reversibly block blood flow.</li>
	<li>Perform a wedge-shaped incision 1 mm proximal to the cranial knot to open the vessel with micro scissors. 
	NOTE: A small drop of blood will indicate proper execution of this step.</li>
	<li>Insert the catheter carefully for 10 mm. Start recording of catheter data. 
	NOTE: Stretching the incision with a forceps can make this process easier.</li>
	<li>Extract the vascular clamp. Add 1 - 2 drops of saline to the incision to facilitate catheter movement. Continue introducing the catheter for approximately another 10 mm. After passing the proximal knot with the sensor tip, fasten the knot carefully just enough to prevent blood reflux alongside the thinner parts of the catheter without impairing catheter movement. 
	NOTE: The size of the sensor at the tip of the catheter prevents reflux of blood when extracting the vascular clamp.</li>
	<li>Gently continue inserting the catheter until pressure analysis shows arterial blood pressure profile indicating that the catheter is placed in the aorta (Figure 3A). 
	NOTE: The catheter reaching the aortic valve will be indicated by light resistance and pulse-synchronized motion of the catheter.</li>
	<li>When experiencing resistance trying to advance through the aortic valve, pull the catheter back 5 mm and advance again until LV catheterization will be indicated in a change in PV analysis as diastolic pressure will reach 0 - 20 mmHg (Figure 3B). Note changes in volume monitoring to further confirm left ventricular placement of the sensor tip (Figure 3C). Fasten the proximal knot more tightly to prevent catheter movement.</li>
</ol>
4. Ischemia/Reperfusion Surgery
<ol>
	<li>Perform skin incision from caudal sternum towards the left axilla for a total length of 15 mm. Proceed with blunt preparation of the two muscle layers until the ribs can be visualized.</li>
	<li>Open the thorax via incision between the third and fourth left rib. Use surgical hooks to gain access to the pericardium. Resect the pericardium above the heart. Before continuing with LCA ligation, wait 30 s without touching the animal to record PV data for valid analysis.</li>
	<li>Localize the LCA emerging under the left auricle and descending at the left side of the heart towards the apex. Use a 6-0 polypropylene suture to encircle the artery with a loop 2 mm underneath the left auricle. Place a small silicon tube under the loop and place a tight knot above. 
	NOTE: Distal myocardium turning grey serves as positive control for LCA occlusion6. I/R surgery should be performed within 5 min independently from the operating investigator.</li>
	<li>Cut the suture at 1 mm length. Release the surgical hooks and manually close the muscle layers above the incision. Wait 45 min while continuously recording PV data.</li>
	<li>After 45 min, re-open the incision and remove the silicon tube to induce reperfusion. Record data for another 20 min. 
	NOTE: A change to a red color as seen before ischemia indicates successful reperfusion.</li>
</ol>
5. Calibration
NOTE: The calibration of the PV catheter system consists of 4 mandatory steps, two of which have to be performed after the measurement. Calibration should be repeated after every experiment to ensure valid results.
<ol>
	<li>Perform pressure calibration and conductance calibration before the experiment as described in Step 1.5.</li>
	<li>Perform saline calibration when the catheter is still placed in the left ventricle after the experiment itself is finished. Localize the right jugular vein lateral of the carotid artery in the prepared area. Inject 10 &#181;L 25% NaCl in H2O via a Hamilton syringe while recording data.</li>
	<li>Calculate the calibration using the acquisition software by highlighting the ascending phase in volume curve (Figure 5C). Repeat this process for a total of 3 times. Avoid loss of blood after syringe extraction by using a vascular clamp to rapidly compress the puncture.</li>
	<li>Perform volume calibration to calibrate volume data acquisition by analysis of standardized volumes. Obtain approximately 500 &#181;L mouse blood from cardiac puncture with a slightly heparinized 1 mL syringe (e.g., 5 &#181;L per 200 IE heparin). Pull back the PV catheter 10 - 15 mm to avoid damaging the catheter.</li>
	<li>Fill the obtained blood into the 37 &#176;C pre-heated calibration cuvette (Figure 5A). Avoid bubbles as it may interfere with the results. Add the catheter tip into each well and record data. Obtain a standard curve by software-guided analysis (Figure 5B). Repeat the process for a total of 3 times.</li>
	<li>Euthanize mice by exsanguination or cervical dislocation while sufficient isoflurane anesthesia is maintained at all time.</li>
</ol>
6. Data Analysis
<ol>
	<li>After completing the calibration steps, perform software-guided data analysis. Therefore, highlight the appropriate section (at least ten cycles) within the Analyze section of the PV Workflow and perform baseline analysis. Exclude cycles with deviations due to ventilation or manipulation if necessary (Figure 3D).</li>
	<li>Perform PV baseline analysis before passage of the aortic valve (arterial pressure only), immediately before and after LCA occlusion. Proceed by conducting PV baseline analysis and in intervals of 5 min. while ischemia and after reperfusion. At the end of the experiment, perform analysis of pressure data after retraction of the catheter from the left ventricle (arterial pressure).</li>
	<li>Analyze at least 10 consecutive cycles to avoid sampling error. When experiencing strong interference of obtained values with the ventilation, transient interruption of the ventilation for a maximum of 5 s can be considered.</li>
	<li>Use the following parameters that are calculated in Baseline Analysis (Figure 3D) to characterize LV function:
	<ol>
		<li>Stroke volume (&#181;L)</li>
		<li>Ejection fraction: Stroke volume / end-diastolic volume (%)</li>
		<li>Cardiac output: Stroke volume * heart rate (&#181;L/min)</li>
		<li>Cardiac index: Cardiac output / body surface area (&#181;L/(min*cm&#178;)</li>
		<li>Stroke work: Inner area of PV curve (mmHg*&#181;L)</li>
		<li>Maximum pressure (Pmax); Mean pressure (Pmean)</li>
		<li>max dP/dt (mmHg/s) as a parameter of LV systolic function</li>
		<li>min dP/dt (mmHg/s) as a parameter for LV compliance</li>
		<li>Time constant of isovolumetric relaxation: Tau (ms)</li>
	</ol>
	</li>
</ol>

<ol>
	<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 simultaneously providing measures of LV systolic performance (contractility dP/dt, stroke work) and LV compliance (-dP/dt, Tau) as a parameter for diastolic function.
As acute heart failure in patients with acute myocardial infarction is a critical predictor for early in-hospital morbidity and mortality2, monitoring of acute hemodynamic impairment and cardiogenic shock in acute myocardial infarction could serve as a valuable tool for identifying possible protective mechanisms in an experimental setting.
Several factors turned out to be critical for successful data acquisition. Stable anesthesia was crucial for valid PV data since isoflurane showed a strong cardiodepressive effect with drops in pressure, LV EF, and stroke volume. Atraumatic preparation of the carotid artery was important to avoid hypovolemia due to blood loss. Furthermore, compression or injury of the vagus nerve and the carotid body could result in severe impairment of hemodynamics.
Saline calibration and cuvette calibration appeared to be another critical step to maintain valid data. For saline calibration, injection of 15% NaCl solution led to increased conductance indicated by a temporary increase in volume level (Figure 5C). Maintaining the same speed when injecting was crucial for stable data. When conducting cuvette calibration, it was important to avoid bubbles within the cuvettes to ensure valid results.
The obtained PV data furthermore indicate the importance of a simultaneous acquisition of pressure and volume data for a valid hemodynamic characterization since pressure data alone did not show significant changes throughout the experiment (Figure 4A). The combined PV analysis offered both baseline parameters for LV systolic function (e.g., ejection fraction) as well as parameters for LV contractility (dP/dt) and LV relaxation (-dP/dt, Tau).
Interestingly, acute occlusion of the LCA in patients usually causes a severe deficit of LV function with immediate necessity for mechanical hemodynamic support and is associated with a high mortality rate11,12. LCA occlusion in mice showed less hemodynamic impairment and LCA occlusion-associated death during the procedure was not observed. As a sign of persisting hemodynamic stability during ischemia, systolic LV blood pressure was stable at all time (Figure 4A). However, this effect could be caused by more distal ligations in mice compared to LCA occlusions in humans.
Taken together, real-time hemodynamic monitoring of acute myocardial infarction in mice could serve as a new method for studying cardioprotective mechanisms in severe LV dysfunction aiming to improve early-phase treatment of patients undergoing acute myocardial infarction.

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.

<table><tbody><tr><td>Betaisodona Loesung</td><td>Mundipharma</td><td>4162-1606/89x30mm</td><td>Povidon-Iod</td></tr><tr><td>Calibration cuvette</td><td>Millar instruments</td><td>910-1049</td><td>Calibration cuvette</td></tr><tr><td>Contura professional hair trimmer</td><td>Wella</td><td>HS-60</td><td>Small animal shaving system</td></tr><tr><td>Eclipse Needle 27G</td><td>BD</td><td>REF 305770</td><td>27G needle</td></tr><tr><td>Forceps</td><td>FST</td><td>11203-25, 11069-08, 11616-15, 11506-12, 11051-10</td><td>Surgical forceps</td></tr><tr><td>Forceps</td><td>Aesculap Braun</td><td>BN731R, BD 311R</td><td>Surgical forceps</td></tr><tr><td>Foris FS2434</td><td>Eizo</td><td>0FTD2033</td><td>Monitor</td></tr><tr><td>Hamilton Syringe 100 &amp;micro;l needle</td><td>Hamilton</td><td>80621</td><td>100&amp;micro;l syringe with needle</td></tr><tr><td>Heated Small animal OP table</td><td>Harvard Apparatus</td><td>15001</td><td>Heated OP table</td></tr><tr><td>Heparin-Natrium 25000</td><td>Ratiopharm</td><td>N68542.04</td><td>Heparin</td></tr><tr><td>Ketamin 10% 100 mg/ml</td><td>bela-pharm</td><td>FS1670041</td><td>Ketamin</td></tr><tr><td>Labchart Pro 8 + Pro modules</td><td>AD Instruments</td><td>MLS260/8</td><td>PV data analysis software</td></tr><tr><td>LAS EZ</td><td>Leica</td><td>LAS EZ</td><td>Microscope camera software</td></tr><tr><td>Leica IC80 HD</td><td>Leica</td><td>IC80 HD</td><td>Microscope camera</td></tr><tr><td>Leica M80</td><td>Leica</td><td>M80</td><td>Microscope</td></tr><tr><td>Micro-tip catheter transducer</td><td>Millar instruments</td><td>SPR-839</td><td>PV catheter</td></tr><tr><td>MiniVent</td><td>Harvard Apparatus</td><td>845</td><td>ventilation</td></tr><tr><td>MPVS Ultra</td><td>Millar instruments</td><td>PL3508B48/M</td><td>PV catheter data acquisition device</td></tr><tr><td>Octenisept</td><td>Sch&amp;uuml;lke</td><td>20000832-A</td><td>disinfectant</td></tr><tr><td>Plastipak 1ml</td><td>PD</td><td>REF 303172</td><td>1ml syringe</td></tr><tr><td>PowerLab 8/35</td><td>AD Instruments</td><td>PL3508</td><td>analog/digital converter</td></tr><tr><td>Prolene 6-0</td><td>Ethicon</td><td>XNEH7814.P31</td><td>Polypropylene suture</td></tr><tr><td>Retraction Kit</td><td>FST</td><td>18200-20</td><td>retraction of surgical situs</td></tr><tr><td>Seraflex 5-0</td><td>Naila</td><td>IC108000</td><td>silk suture</td></tr><tr><td>Small and micro-scissors</td><td>FST Essen</td><td>14059-11, 15007-08, 14064-11</td><td>Surgical scissors</td></tr><tr><td>Small silicon tube</td><td>Reichelt Chemietechnik</td><td></td><td>tube for LCA occlusion</td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td>Sodium Chloride</td></tr><tr><td>testo 108</td><td>testo</td><td>5631080</td><td>rectal thermometer</td></tr><tr><td>Thinkcentre desktop computer</td><td>Lenovo</td><td>PC0EJS2V</td><td>Computer</td></tr><tr><td>Vasofix Safety 20G</td><td>Braun</td><td>4269110S-01</td><td>intubation catheter</td></tr><tr><td>Windows 10</td><td>Microsoft</td><td>KW9-00240</td><td>Operating system</td></tr><tr><td>Xylazin 2%</td><td>Ceva</td><td>6324464.00.00</td><td>Xylazine hydrochloride</td></tr></tbody></table>

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 false placement of the PV-catheter in the aorta would have shown a typical arterial pressure curve with a minimum pressure of 30 - 60 mmHg (diastolic blood pressure) and a small excursion at the end of systole indicating the aortic valve closing (Figure 3B and 3C). Successful occlusion of the LCA was visually confirmed by blanching of the distal LV myocardium (Figure 2B).
After LCA occlusion, PV data was acquired in 5 min intervals. Pressure data analysis demonstrated no changes in maximum LV systolic pressure indicating preserved peripheral perfusion and stable anesthesia (Figure 4A). Analysis of LV volume revealed a significant decrease in both EF (52% vs. 40%, p&#160;= 0.008) and absolute stroke volume (Figure 4B and 4C). These changes happened within the early phase of ischemia and LV functional data remained unchanged in the later phase of ischemia. Maximum dP/dt as a parameter of LV contractility showed a 30% reduction in mice undergoing myocardial ischemia. Stroke work was 30% reduced (Figure 4D and 4E). As a parameter for diastolic function, minimum dP/dt was significantly decreased indicating impaired LV compliance (Figure 4F). Reperfusion by extraction of the silicon tube was visually validated. Reperfusion did not show significant changes in PV data analysis within a period of 20 min (Figure 4A-4D). Sham-operated animals did not show a significant reduction in LV systolic or diastolic parameters (Figure 4I-4J).
At the end of data acquisition, cuvette calibration and saline calibration were performed (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57621/57621fig1v3.jpg" /> 
Figure 1: Scheme of the method. Sequence of left ventricular (LV) catheterization, left main coronary artery (LCA) occlusion and reperfusion.&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57621/57621fig2.jpg" /> 
Figure 2: Surgical procedures. (A) Left ventricular catheterization placed via the right common carotid artery. (B) Left main coronary artery occlusion with polypropylene suture and silicon tube. <a href="/files/ftp_upload/57621/57621fig2large.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/57621/57621fig3.jpg" /> 
Figure 3: Representative pressure-volume data. (A) Representative arterial pressure indicated by a minimal pressure of &#62;30 mmHg and a typical excursion at the end of systole indicating closing of the aortic valve. (B) Representative left ventricular pressure data showing diastolic values &#60;20 mmHg. (C) Representative left ventricular pressure-volume diagram. (D) Screenshot of software-based PV baseline analysis. <a href="/files/ftp_upload/57621/57621fig3large.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/57621/57621fig4.jpg" /> 
Figure 4: Pressure/volume data in mice undergoing ischemia/reperfusion. (A) Systolic left ventricular blood pressure (Pmax). (B) Left ventricular (LV) ejection fraction (EF) (%). (C) LV stroke volume (&#181;L). (D) Maximum dP/dT (max dP/dt) (mmHg/s). (E) LV Stroke work (SW). (F) Minimum dP/dt (min dP/dt) (mmHg/s). (G) Time constant of isovolumetric relaxation Tau (ms). (H) Pressure/volume diagram before and 45 min after induction of myocardial ischemia. (I-J) Stroke volume (SV) (&#181;L) and maximum dP/dt (max dP/dt) (mmHg/s) in sham-operated animals compared to animals after 15/30 min ischemia. Data (A-G) are presented as mean &#177; SEM. * p &#60;0.05 via Student&#39;s t-test or ratio-paired t-test, n&#160;= 4 mice/group (A+D-G)) or n&#160;= 3 mice/group (B, C). +45: 45 min ischemia; rep.: reperfusion. <a href="/files/ftp_upload/57621/57621fig4large.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/57621/57621fig5.jpg" /> 
Figure 5: Post-hoc calibration. (A) Schematic of calibration cuvette. Volumes in &#181;L. (B) Representative linear regression analysis of obtained volume data to perform cuvette calibration. (C) Representative volume data after injection of 10 &#181;L of 25% sodium chloride in H2O into the right jugular vein to perform saline calibration. <a href="/files/ftp_upload/57621/57621fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

9.1K Views.  University Hospital Essen. Acuut myocardinfarct in muizen induceert acute maar onvolledig gekarakteriseerd veranderingen in functie van left ventriculaire (LV). LV catheterisatie in muizen ondergaan coronaire occlusie fungeert als een nieuwe methode voor een real-time evaluatie van LV functie.

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

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 ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Bioengineering

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 ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

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 ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI. 
The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson's trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected. 
In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson's trichrome.

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. ...

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

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