This model was developed with the support of the Swiss National Science Foundation (Grants 310030_162629 to LL) and departmental funds from the Services of Thoracic Surgery and Intensive Care Medicine of the Lausanne University Hospital. JL is recipient of a grant from the Emma Muschamp Foundation. We acknowledge the crucial support of veterinarians and animal facility staff of the Faculty of Biology and Medicine of the Lausanne University. We thank Dr. Giuseppina Milano from the Service of Cardiac Surgery of Lausanne University Hospital and Dr. Alexandre Sarre from Cardiovascular Assessment Facilty of Lausanne University for their technical hints.

Animal experiments described in this protocol were reviewed and approved by Animal Ethics Committee of Canton of Vaud.
NOTE: For these experiments, we used male C57Bl/6J mice weighing between 25 g and 30 g and an age of 8-12 weeks. Mice were fed chow pellets and water ad libitum and bred under conventional conditions. Surgical equipment was previously sterilized. The experimenter should wear sterile surgical gloves and a surgical mask to limit contamination and post-operative infections.
1. Anesthesia and tracheal cannulation.
<ol>
	<li>Weigh the mouse to determine the dosage of anesthetic drugs, post-operative analgesic medication and tidal volume of the ventilator. Pre-warm the heating pad at 37 &#176;C. The surgical setup is depicted in Figure 1.</li>
	<li>Inject mouse intraperitoneally with a mix of ketamine and xylazine at a dose of 80 mg/kg and 10 mg/kg respectively.</li>
	<li>Quickly shave the mouse fur on the throat and the left side of the rib cage using an electric razor.</li>
	<li>Check depth of anesthesia by pinching tail and/or hind feet and settle the animal in a supine position on the heating pad. Place a small gauze compress under the head of the animal to avoid overheating of the eyes. Apply ocular gel to avoid eye dryness.</li>
	<li>Secure the four limbs with adhesive tape on the surface of the heating pad. Pass a loop of 5-0 silk suture under the upper incisors and stick the extremity of the loop with adhesive tape onto the heating pad. This will keep the mouth of the animal open and facilitate cannulation.</li>
	<li>Apply hair removal cream on the pre-shaved areas and gently massage with a cotton swab for 1 min. Wipe the excess of fur and cream with a gauze. Use drops of 0.9% saline solution and gauze to clean the incision areas. Apply pieces of sterile gauze to the shaved throat and thorax and soak them in iodopovidone. 
	NOTE: We recommend application of a local anesthetic drug (lidocaine or bupivacain) to incisions sites.</li>
	<li>Set the ventilator at a tidal volume of 7 mL/kg and ventilation rate of 140 strokes/min. 
	NOTE: From now on work under a microsurgery stereomicroscope.</li>
	<li>Hold the skin on the center of the throat and perform an incision of 0.5 cm following a caudal/cephalic line using small scissors. Separate the lobes of salivary gland, then gently separate fascia of sternohyoid muscle with curved dissecting forceps until larynx and trachea are visible. Secure edges of the opening with retractors attached to elastic bands. 
	NOTE: Do this step without incision of the muscles.&#160;A trained operator will be able to intubate the animal via oral cavity without visualization of the trachea making this step optional.</li>
	<li>Hold gently the tongue sideways. With forceps, insert the blunted inner needle of a 16 G cannula into the trachea. Visualize correct insertion into the trachea through the throat incision.</li>
	<li>Connect the cannula to the ventilator and ensure correct ventilation by placing the exhaust tubing into water. The presence of bubbles indicates correct intubation. 
	NOTE: In order to keep tissues wet during operation place sterile gauze soaked with 0.9% saline solution and iodopovidone on the throat incision. Control moisture during the procedure.</li>
</ol>
2. Ligation of LAD coronary artery
<ol>
	<li>Release left anterior paw from duct tape and carefully move the mouse to right side decubitus position. Secure the left anterior limb once animal is in the correct position.</li>
	<li>Identify the line between left pectoralis minor and major muscles and make an oblique skin incision on 1 cm with scissors following the line. With dissecting blunt micro scissors, separate fascia of pectoralis muscles without incision. Maintain pectoralis muscles separated with retractors attached to elastic bands.</li>
	<li>Set the ventilator with a positive end-expiratory pressure (PEEP) of 3 cm H2O.</li>
	<li>Open the chest cavity by using blunt forceps at the 3rd intercostal space between 3rd and 4th ribs. Avoid touching internal thoracic artery as there is danger of bleeding. Do not touch heart or lung. Apply two retractors into the ribcage, one on each rib (Figure 2A).</li>
	<li>With a curved fine forceps, carefully remove the pericardium and pull it apart without harming the heart and lungs.</li>
	<li>Locate left anterior descending (LAD) coronary artery. LAD artery appears as a superficial bright red line running from the edge of the left auricle toward the apex.</li>
	<li>Use a needle holder to pass a 7-0 silk suture under the LAD 2 to 3 mm below left atria. Pull the silk slowly to avoid a tearing of heart tissue. Tie the ligature with three knots. The lower left part of the left ventricle will instantly turn pale upon ligation (Figure 2B-E). 
	NOTE: It is important to not go too deep into the ventricular cavity or to stay too superficial. For sham-operated animals, pull the suture silk under the LAD and remove it slowly avoiding tissue tearing.</li>
	<li>Release the rib retractors, hold the 3rd rib with forceps and make two passes with a 6-0 silk suture under the 3rd and 4th ribs. 
	CAUTION: Do not the perforate heart or lung. Do not tighten knots yet.</li>
	<li>Put three drops of 37 &#176;C 0.9% saline solution onto the opening and shut the expiration exhaust tube for 2 or 3 respiratory cycles to properly inflate lungs. Tighten the suture and secure with two throws.</li>
	<li>Release retractors holding muscles and help them retrieve their correct place.</li>
	<li>Close thoracic skin with two stitches of 5-0 suture silk and secure with two throws. Close throat skin with one stitch of 5-0 suture silk and secure with two throws.</li>
</ol>
3. Post-operative procedures and follow-up.
<ol>
	<li>Remove adhesive tape bands from limbs. Put a compress on the heating pad on the right side of the animal. 
	NOTE: The overall procedure from anesthesia to this point should not take longer than 40-45 min. Optionally inject IP 0.2 mL of atipamezole at a concentration of 0.1 mg/mL to speed up the waking up process.</li>
	<li>Intraperitoneally inject 0.3 mL of 5% glucose solution pre-warmed at 37 &#176;C.</li>
	<li>Carefully turn the animal on ventral decubitus onto the compress pad.</li>
	<li>Stop ventilator; if the mouse spontaneously breathes, cautiously remove cannula.</li>
	<li>Inject subcutaneous (SC) 0.1 mg/kg buprenorphine and put mice in a pre-warmed cage heated at 30 &#176;C and ventilated with a 100% O2 for a minimum of 1 h. Monitor mice for any life-threatening condition such as excessive dyspnea or hemorrhage.</li>
	<li>During the two first days following surgery, monitor mouse twice daily. Inject SC 0.1 mg/kg buprenorphine twice daily. Intraperitoneally inject 0.3 mL 0 of 5% glucose solution twice daily. Provide mice with soft diet and water ad libitum. Warm up the animal if necessary. 
	NOTE: In addition to opioids, animals should be provided with nonsteroidal anti-inflammatory drugs mixed in diet or diluted in drinking water.</li>
	<li>From day three, inject SC 0.1 mg/kg buprenorphine twice daily if the animal exhibits any unusual signs concerning general appearance, respiration or behavior. Intraperitoneally inject 0.3 mL of 5% glucose solution twice daily if the animal is still losing weight. Warm the animal if necessary. 
	NOTE: Strictly apply predefined interruption criteria when necessary to avoid excessive suffering. Usually mice lose weight up to day 3 and 4 and then gain weight. After seven days, mice usually retrieve pre-operation weight.</li>
</ol>

<ol>
	<li>GBD 2016 Causes of Death Collaborators. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=regional,+and+national+age-sex+specific+mortality+for+264+causes+of+death,+1980-2016:+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study.">regional, and national age-sex specific mortality for 264 causes of death, 1980-2016: a systematic analysis for the Global Burden of Disease Study.</a> Lancet. 390 (10100), 1151-1210 (2017).</li><li>Reed, G. W., Rossi, J. E., Cannon, C. P. <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> Lancet. 389 (10065), 197-210 (2017).</li><li>Frangogiannis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+response+in+myocardial+injury,+repair,+and+remodelling.">The inflammatory response in myocardial injury, repair, and remodelling.</a> Nature Reviews Cardiology. 11 (5), 255-265 (2014).</li><li>Lugrin, J., 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=Cutting+edge:+IL-1alpha+is+a+crucial+danger+signal+triggering+acute+myocardial+inflammation+during+myocardial+infarction.">Cutting edge: IL-1alpha is a crucial danger signal triggering acute myocardial inflammation during myocardial infarction.</a> Journal of Immunology. 194 (2), 499-503 (2015).</li><li>Hashmi, S., Al-Salam, S. <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+and+myocardial+ischemia-reperfusion+injury:+a+comparison.">Acute myocardial infarction and myocardial ischemia-reperfusion injury: a comparison.</a> International Journal of Clinical and Experimental Pathology. 8 (8), 8786-8796 (2015).</li><li>van Zuylen, V. 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=Myocardial+infarction+models+in+NOD/Scid+mice+for+cell+therapy+research:+permanent+ischemia+vs+ischemia-reperfusion.">Myocardial infarction models in NOD/Scid mice for cell therapy research: permanent ischemia vs ischemia-reperfusion.</a> Springerplus. 4, 336 (2015).</li><li>Yan, X., 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=Temporal+dynamics+of+cardiac+immune+cell+accumulation+following+acute+myocardial+infarction.">Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction.</a> Journal of Molecular and Cellular Cardiology. 62, 24-35 (2013).</li><li>Xu, Z., Alloush, J., Beck, E., Weisleder, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+myocardial+ischemia-reperfusion+injury+through+ligation+of+the+left+anterior+descending+artery.">A murine model of myocardial ischemia-reperfusion injury through ligation of the left anterior descending artery.</a> Journal of Visualized Experiments. (86), (2014).</li><li>Reichert, K., 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=Murine+Left+Anterior+Descending+(LAD)+Coronary+Artery+Ligation:+An+Improved+and+Simplified+Model+for+Myocardial+Infarction.">Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction.</a> Journal of Visualized Experiments. (122), (2017).</li><li>Kolk, M. V., 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=LAD-ligation:+a+murine+model+of+myocardial+infarction.">LAD-ligation: a murine model of myocardial infarction.</a> Journal of Visualized Experiments. 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X., Weisleder, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Murine+Model+of+Myocardial+Ischemia-Reperfusion+Injury.">A Murine Model of Myocardial Ischemia-Reperfusion Injury.</a> Methods in Molecular Biology. 1717, 145-153 (2018).</li><li>Parapanov, R., 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=Toll-like+receptor+5+deficiency+exacerbates+cardiac+injury+and+inflammation+induced+by+myocardial+ischaemia-reperfusion+in+the+mouse.">Toll-like receptor 5 deficiency exacerbates cardiac injury and inflammation induced by myocardial ischaemia-reperfusion in the mouse.</a> Clinical Science. 129 (2), 187-198 (2015).</li><li>Curaj, A., Simsekyilmaz, S., Staudt, M., Liehn, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+invasive+surgical+procedure+of+inducing+myocardial+infarction+in+mice.">Minimal invasive surgical procedure of inducing myocardial infarction in mice.</a> Journal of Visualized Experiments. (99), e52197 (2015).</li><li>van den Bos, E. J., Mees, B. M., de Waard, M. C., de Crom, R., Duncker, 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=A+novel+model+of+cryoinjury-induced+myocardial+infarction+in+the+mouse:+a+comparison+with+coronary+artery+ligation.">A novel model of cryoinjury-induced myocardial infarction in the mouse: a comparison with coronary artery ligation.</a> American Journal of Physiology-Heart and Circulatory Physiology. 289 (3), H1291-H1300 (2005).</li><li>Duerr, G. 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The authors have nothing to disclose.

The first critical step of this procedure is certainly intubation. We use the blunted inner needle of a 16 G catheter as a tracheal tube. We do not recommend using this setup with mice that weight less than 22 g. With this setup, it may be difficult to intubate mice properly with smaller bodyweight without damaging the trachea. Another critical point is to limit incisions made to the muscle while exposing the trachea and ribcage. Reducing tissue damage is of major importance, especially when studying inflammatory processes subsequent to MI. That is why we prefer gentle spreading of muscle and ribs with forceps and retractors8,9. We do not use electric cauterizer to control bleeding10. This may cause iatrogenic burns and favor infections. Both trauma and infections may bias inflammatory read-outs. Application of an extrinsic PEEP of 3 cm H2O by plunging the ventilation exhaust into a water tube limits end-expiratory alveolar collapse during thoracotomy. Localization of LAD is another critical step and one should keep in mind that the anatomy of the coronary arteries may vary depending on the strain and the genotype of the mouse11. It requires some experience to visualize the LAD, however placing the suture directly 2-3 mm below the left atria as described in the procedure shall allow correct positioning of the ligation. Instant discoloration of large portions of the left ventricle under the suture confirm the accuracy. Finally, artificially applying auto-PEEP by blocking ventilation exhaust for 2-3 respiratory cycles during chest closure allows a transient hyperinflation of the lung that will help chase the air from thoracic cavity12. We purposely do not perform a thoracentesis as shown in 9,10. This way, we limit the risk of lung and heart injuries and avoid excessive tissue damage or perforation.
Myocardial ischemia-reperfusion (I/R) is a related surgical model that mimics the restoration of coronary blood flow that is done to MI patients in clinics. During the I/R model a transient occlusion of the coronary artery is done by tightening a piece of tubing onto the LAD for a duration of 20 to 45 min8,13. Then the occlusion is released to allow reperfusion of the myocardium for the desired duration. This simple modification applied to our protocol can easily turn it into an I/R model4,8,14,15. The infarction can be confirmed by a blood test for cardiac troponin T8,10 or by echocardiography15.
MI differs from I/R model because reperfusion by itself induces an injury. MI induces more tissue necrosis and apoptosis is more pronounced in reperfused myocardium5. Kinetics of inflammatory cells infiltration is also different between in MI and IR with a delayed myocardial infiltration of immune cells in MI7. The size and the position of the infarcted area will also differ between permanent ligation and I/R models15. Keeping this in mind, one must be cautious to choose a relevant model since I/R and permanent MI models are not equivalent. Another murine model of myocardial infarction is the cryoinfarction model. Application of a cryogenic probe on the LV anterior wall induces the freezing of ventricular tissue and blood flow arrest in the LAD artery. This technique however differs from MI and I/R techniques regarding timing and amplitude of remodeling and inflammatory responses16,17.
Variability is a limitation as for any surgical procedure. This variability relies on biological differences. A good example is the variation in coronary arterial arrangement in mice11. It also relies on experimenter skills. It is worthwhile mentioning that adequate training of the experimenters is mandatory in order to reach stable outcomes with this model. A well-trained experimenter can easily produce infarct sizes that are reproducible (Figure 3A-B). The mortality of the model depends on the position of the LAD, duration of the experiments (days, weeks), mouse strain and genotypes. The types of anesthetic and analgesic drugs may also affect the outcome of the experiments with putative cardioprotective or cardiodepressant effects. In our hands, this model has a global mortality rate of 25-30%. This mortality rate comprises spontaneous deaths and sacrifices before the end of the experiment, regardless strains and experiment duration. Most of the deaths or sacrifice are between the second and fourth days post-surgery. Applying a strict pain management and follow up of the animals can reduce mortality.
Here we present representative results of infarct size analyzed using TTC staining and expression of protein and genes involved in inflammatory or fibrotic processes in LV by western blot and real-time PCR respectively (Figure 3C-G). It is also possible to measure many of these parameters by enzyme-linked immunosorbent assay (ELISA) or enzymatic assays. Of course, in accordance with hypothesis that needs to be tested, this method can be followed by any functional analysis by ultrasound, MRI or intraventricular catheter measurement of pressure and volume. It is also possible to extract heart and further investigate cardiac cell biology on isolated cells. Overall, the MI model with permanent ligation of the LAD coronary artery is particularly useful to evaluate inflammatory and fibrotic processes, wound healing and changes in cardiac function subsequent to myocardial infarction.

Acute myocardial infarction (MI) is the most severe expression of ischemic heart diseases (IHD). IHD are the leading cause of morbidities and death worldwide, especially in western countries1. Consequently, it has an enormous economic impact on healthcare systems2. MI is characterized by the occlusion of a coronary artery by atherosclerotic plaque and the subsequent arrest of blood flow in large parts of the myocardium. Lack of oxygen supply in the myocardium leads to ischemic death of cardiomyocytes. This pathological condition triggers responses in the ventricular tissue that ultimately leads to deficiencies in ventricular functions, remodeling and heart failure3. MI is a complex pathophysiological condition that involves multiple and intricate biological processes comprising regulated cell death, response to oxidative stress, inflammation, wound healing, fibrosis and ventricular remodeling. Some of these biological responses are modeled as individual processes in vitro like necrosis-induced release of damage-associated molecular patterns and associated inflammatory responses4. These simplified models are essential to understanding MI. However only an in vivo model can provide a realistic image of the biological processes complexity engaged in response to MI.
Even though models of MI in larger animals like swine may more closely relate to human pathophysiology of MI, the power of the murine models resides in the possibilities offered by genetic engineering that is more advanced than in any other mammal species. Other non-negligible aspects are the relative low cost and the simplicity of the surgical setup.
It is worth to mention that models of ischemia-reperfusion of the myocardium can exhibit different outcomes than permanent MI models. Biological processes like the type of cell death engaged, quality/amplitude or kinetics of inflammatory and wound healing responses in the myocardial tissue might vary according to the model5,6,7. However, this protocol of permanent coronary occlusion can easily be adapted to obtain an ischemia-reperfusion model.
This method is relevant for studies related to the physiopathology of MI without reperfusion and allows monitoring of pathological processes occurring from coronary occlusion (minutes) to late stage heart failure (weeks) at the local heart tissue and systemic levels.

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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.

Mice were euthanized seven days after surgery. Animals were anesthetized with 80 mg/kg ketamine and 10 mg/kg xylazine. Under anesthesia, blood was drawn from vena cava and heart was sampled. Atria were removed, myocardium were washed in ice-cold PBS. For measurements of ischemic areas, hearts were frozen at -20 &#176;C for 40 min, then sliced and stained for 20 min at 37 &#176;C in PBS containing 2% triphenyltetrazolium chloride (TTC). Heart slices were fixed overnight in 4% buffered paraformaldehyde solution at room temperature. Ischemic areas remained unstained whereas live tissue was stained in red due to the presence of dehydrogenases. Ischemic areas were calculated as percentage of white area of the left ventricle (LV) with an imaging software (Figure 3A, B). For biochemical and molecular biology analyses, hearts were frozen in liquid nitrogen. After grinding hearts on liquid nitrogen the organ powder was used for protein and mRNA extraction. The extent of fibrosis in the myocardial tissue of infarcted hearts was assessed by western blot analysis of alpha smooth-muscle actin (&#945;SMA) and SMAD2 phosphorylation, which are respectively major read-outs of myofibroblasts and of TGF&#946; signaling activation (Figure 3C). mRNA expression of Tgfb, and downstream targets Ctgf, Postn and Il11 are all indicators of myocardial fibrosis. This was shown by real-time polymerase chain reaction (PCR) analysis (Figure 3D).
Pro-inflammatory signaling pathways and expression of pro-inflammatory genes were typically found activated within the first week following myocardial infarction. Phosphorylation of NF-&#954;B p65 transcription factor is a hallmark of inflammation and was observed in whole myocardium extracts of the MI mice (Figure 3E). mRNA expression of pro-inflammatory genes Il1b, Il6 and Cxcl10 (Figure 3F) and monocytes/macrophages markers Cd14 and Mertk were analyzed by real-time PCR (Figure 3G). Note that there was a variability in the extent of NF-&#954;B p65 and SMAD2 phosphorylation (Figure 3C,E, lanes 4-7). This variability depends largely on the size of the infarct.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59591/59591fig1.jpg" /> 
Figure 1: Description of the surgical setup. (A) Surgical setup comprises a modified heating pad, a ventilator and retractors attached to elastic bands. (B) Set of scissors, forceps and needle holder used during the surgery. (C) Close-up of the mini-retractors. Not shown: surgical stereo microscope. <a href="https://www.jove.com/files/ftp_upload/59591/59591fig1large.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/59591/59591fig2.jpg" /> 
Figure 2: Representative images of the surgery and LAD ligation. (A) Opened chest with retractors. The left ventricle was apparent. Top, left and bottom retractors held the ribcage and right retractor held the pectoralis muscle. (B) The needle was passed under the LAD. (C) Suture silk was passed under the LAD, into the left ventricle. (D)&#160;Single stitch on the LAD. (E) End of the ligation procedure, the suture was secured with three knots. (F) Representation of an anterior view of the heart. The position of LAD ligation was 2-3 mm below left atria and above diagonal branch of the LAD. <a href="https://www.jove.com/files/ftp_upload/59591/59591fig2large.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/59591/59591fig3.jpg" /> 
Figure 3: Fibrosis and inflammation in whole myocardium extracts seven days post-surgery. (A) Representative images of TTC staining of a sliced infarcted heart seven days post-surgery. Pale ischemic areas remained unstained and white whereas live tissue was stained red. The ligation was visible on the third slice from the left. (B) The size of the ischemic areas of five infarcted hearts were measured using TTC staining technique. Results were the percentage of white area of the left ventricle (LV). (C) Western blot analysis of SMAD2 phosphorylation and alpha-SMA expression in whole myocardium as indicators of fibrosis. (D)&#160;mRNA expression of Tgfb, Ctgf, Postn and Il11 in whole myocardium extracts. (E) Western blot of NF-&#954;B p65 phosphorylation in whole myocardium extracts. (F) mRNA expression of pro-inflammatory genes Il1b, Il6 and Cxcl10 in whole myocardium extracts. (G) mRNA expression of Cd14 and Mertk as indicators of the presence in the myocardium of monocytes/macrophages and phagocytic macrophages respectively. N = 3 in sham and N = 4 in MI group. For mRNA expression analysis, expression was relative to the endogenous control Rps18 and group comparisons were unpaired Student&#39;s T-tests, *p &#8804; 0.05, **p &#8804; 0.01, *** p &#8804; 0.001. In panels B, D, F and G error bars represent standard deviations.&#160;&#160;<a href="https://www.jove.com/files/ftp_upload/59591/59591fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery at JoVE.com

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

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 ...

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27.2K Views.  Lausanne University. 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.

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

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 ...

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

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 ...

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 ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery (ALCAPA)

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ischemia and infarction in children. If left untreated, it results in a 90% mortality rate in the first year of life. In patients who survive to the adulthood, the coronary steal phenomenon and retrograde left-sided coronary flow provide a substrate for chronic subendocardial ischemia, which may lead to left ventricular dysfunction, ischemic mitral regurgitation, malignant ventricular arrhythmias, and sudden cardiac death. The average age of life-threatening presentation is 33 years and of sudden cardiac death 31 years. Therefore, surgical correction is highly recommended as soon as the diagnosis is made, regardless of age. In adult-type ALCAPA originating from the right-facing sinus of the pulmonary artery, direct re-implantation of the ALCAPA into the aorta is the more physiologically sound repair technique to re-establish the dual-coronary perfusion system and is recommended. This protocol describes the technique of direct re-implantation of adult-type ALCAPA into the aorta.

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery ALCAPA

Surgical correction of ALCAPA is highly recommended, regardless of age or the degree of intercoronary collateralization. This protocol presents a technique for the direct re-implantation of adult-type ALCAPA into the aorta to re-establish the dual-coronary perfusion. Whenever feasible, direct re-implantation is preferred to other surgical correction techniques.

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...