This work was supported by the Administration of Traditional Chinese Medicine [SLJ0204], the Jiangsu Provincial Hospital of Chinese Medicine (Y21017), the National Natural Science Foundation of China [81973763, 81973824,82004239].

The study was approved by the Animal Care and Use Committee of the Nanjing University of Chinese Medicine (permission no. 202004A002). The study strictly followed the National Institutes of Health (NIH) guidelines on the Use of Laboratory Animals (NIH publication No. 85-23, revised 2011). Thirty male Sprague-Dawley rats (weight, 300 &#177; 50 g; age, 12 &#177; 14 weeks) were used in this work.
1. Animal preparation
<ol>
	<li>Deprive the rats of food and water for 12 h before surgery. Preoperative fasting aims to prevent pulmonary aspiration14.</li>
	<li>Sterilize all instruments before surgery using a high-pressure steam sterilizer.</li>
	<li>Anesthetize the rats by administering pentobarbital sodium (1.5%, 75 mg/kg) via intraperitoneal injection (see Table of Materials).</li>
	<li>Assess the effectiveness of anesthesia by performing the pinch-toe test. 
	NOTE: The rat is considered sufficiently anesthetized if no reflexes are observed when its hind paw is held by the tweezers.</li>
	<li>Straighten the middle section of two paper clips to form an &#34;S&#34; shape. Pull down the wide section of each &#34;S&#34; to form a small retractor.</li>
	<li>Cut a 2 mm diameter polyvinyl chloride (PVC) tube into 7 mm-length pieces. Insert a 10 cm long 4-0 suture into the PVC tube, and tie its ends.</li>
	<li>Ligate the left anterior descending (LAD) coronary artery and the PVC tube together using a 6-0 suture. Cut a groove in the middle of the PVC tube using ophthalmic scissors, and use the groove to thread the 6-0 suture through the tube to prevent it from falling off. 
	NOTE: The PVC tube and &#34;S&#34; shape retractors are shown in Supplementary Figure 1.</li>
</ol>
2. Surgery procedure
<ol>
	<li>Perform surgery to generate the improved MIRI rat model following the steps below. 
	NOTE: The animal model group generated by the improved MIRI method is referred to as the experimental model group throughout the article.
	<ol>
		<li>After anesthesia (step 1.2), fix the limbs of the rat with tape by placing the rat on the surgical board in the supine position. Shave the neck and left anterior chest area with depilatory cream, and clean the skin with 75% alcohol and iodophor scrub.</li>
		<li>Cut the skin of the neck lengthwise along the median cervical line using ophthalmic scissors.</li>
		<li>Separate the neck muscles using ophthalmic tweezers, and place a retractor (step 1.4) on each side to retract them further. 
		NOTE: It is necessary to expose the trachea adequately, as it is critical for preventing bleeding from the thyroid gland during this step.</li>
		<li>After exposing the trachea, identify the space between the fourth and fifth tracheal rings. This space is the puncture point.</li>
		<li>Mark this point using the blunt edge of a needle tip. Make a 3 mm incision parallel to the cricoid cartilage at this point.</li>
		<li>Insert a suction trocar (see Table of Materials) into the trachea via the incision (step 2.1.5), and mechanically ventilate the rat to maintain normal respiration at a rate of 80&#8239;breaths/min and a tidal volume of 8 mL/kg.</li>
		<li>Next, make a 4-5 cm incision from the xiphoid to the middle of the second left intercostal space while holding the scalpel at a 45&#176; angle. Gently and slowly, separate the pectoralis major and serratus anterior muscles using ophthalmic tweezers to access the intercostal space.</li>
		<li>Make a 1.5 cm incision transversely between the left third and fourth ribs using ophthalmic scissors.</li>
		<li>If required, cut out the fourth rib to expose the heart covered by the left lung. This gives better visibility.</li>
		<li>To prevent injuries, place cotton balls soaked in the physiological saline solution above the lungs in the thoracic cavity. Dissect the pericardium using ophthalmic tweezers, lift the left atrial appendage by tweezers, and identify the coronary ostium present at the root of the aortic artery.</li>
		<li>In the section between the left lung and auricle, ligate the LAD and the pre-prepared short tube (step 1.6) together using a 6-0 surgical suture, and tie it using a slipknot. Place the slipknot in the groove of the PVC tube, and tighten the ligated tube and LAD using a second slipknot for 45 min15&#160;(Figure 1A,B).</li>
		<li>Record the color change in the anterior part of the left ventricle and ST-segment elevation on electrocardiogram (ECG) during the ischemia period. 
		NOTE: The anterior part of the left ventricle turns pale during the ischemia period.</li>
		<li>Clamp the chest muscles and skin using an artery clip, and cover the wound with moist saline gauze.</li>
		<li>Loosen the slipknot, and remove the pre-prepared short tube after 45 min15&#160;(Figure 1C).</li>
		<li>Keep the rats anesthetized during reperfusion for 2 h.</li>
	</ol>
	</li>
	<li>Perform surgery to generate the rat model following the previously published procedure16. 
	NOTE: This animal model group is referred to as the existing model group throughout the article.
	<ol>
		<li>Before ligation of the LAD coronary artery, perform the same steps as the experimental model group.</li>
		<li>During the ischemic period, ligate each rat&#39;s proximal LAD coronary artery with a slipknot only using a 6-0 surgical suture at the same position as the experimental model group and tie a slipknot for 45 min.</li>
		<li>After the ligation, loosen the slipknot with tweezers, suture the incisions of the rat with a suture needle and tweezers, and keep the animal in deep anesthesia of 1.5% pentobarbital sodium throughout the period of reperfusion17,18,19&#160;for 2 h before harvesting the rat&#39;s hearts.</li>
	</ol>
	</li>
</ol>
3. Assessment of triphenyltetrazolium chloride staining
<ol>
	<li>At the end of reperfusion, the rats&#160;are euthenized while still deeply anesthetized.&#160;Sacrifice the rats and harvest their hearts16,20&#160;immediately. Wash the hearts in PBS solution, and store them at &#8722;20 &#176;C for ~20 min to harden the tissues.</li>
	<li>Subsequently, cut the hearts into 2 mm slices with a microtome blade, incubate them with 2% triphenyltetrazolium chloride (TTC) (see Table of Materials) at 37 &#176;C for ~30&#8239;min, and fix them in 10% neutral formalin.</li>
	<li>Photograph the heart slices, and calculate the infarct areas using an image processing program of ImageJ software (see Table of Materials). 
	&#8203;NOTE: Due to the staining, the infarct sites appear pale white, whereas normal tissues appear dark red.</li>
</ol>
4. Histological staining
<ol>
	<li>Harvest the hearts under deep anesthesia of 1.5% pentobarbital sodium at the end of the reperfusion period.</li>
	<li>Fix the hearts in 10% formalin at 4 &#176;C for 48 h.</li>
	<li>Subsequently, section the hearts with a microtome into at least 6 slices (5 &#181;m thick) and ensure at least three slices for hematoxylin and eosin (H&#38;E) and Masson staining20,21.</li>
	<li>Observe the slides under a light microscope, and photograph them.</li>
</ol>
5. ECG assessment
<ol>
	<li>Randomly divide animals into experimental or existing MIRI model groups or sham groups to assess the ECG changes.</li>
	<li>Anesthetize all rats during the surgical ligations and assess standard limb lead II tracing20,21&#160;to identify ECG changes and confirm myocardial ischemia.</li>
	<li>Store all images in a digital library.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Perform statistical analyses using scientific graphing and statistics software (see Table of Materials).</li>
	<li>Express all data as mean &#177; standard error of the mean. After normality and lognormality tests of each group, perform a one-way analysis of variance and t-tests22&#160;to determine significant differences among the groups. Consider p-value &#60;0.05 as statistically significant.</li>
</ol>

<ol>
	<li>Mozaffarian, D., 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=Heart+disease+and+stroke+statistics-2016+update:+a+report+from+the+American+heart+association.">Heart disease and stroke statistics-2016 update: a report from the American heart association.</a> Circulation. 133 (4), 38 (2016).</li><li>Allen, D. G., Orchard, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+contractile+function+during+ischemia+and+hypoxia.">Myocardial contractile function during ischemia and hypoxia.</a> Circulation Research. 60 (2), 153-168 (1987).</li><li>Ashraf, M. I., 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=A+p38MAPK/MK2+signaling+pathway+leading+to+redox+stress,+cell+death+and+ischemia/reperfusion+injury.">A p38MAPK/MK2 signaling pathway leading to redox stress, cell death and ischemia/reperfusion injury.</a> Cell Communication and Signaling. 12, 6 (2014).</li><li>Hernandez-Resendiz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+redox+dysregulation+in+the+inflammatory+response+to+acute+myocardial+ischaemia-reperfusion+injury+-+adding+fuel+to+the+fire.">The role of redox dysregulation in the inflammatory response to acute myocardial ischaemia-reperfusion injury - adding fuel to the fire.</a> Current Medicinal Chemistry. 25 (11), 1275-1293 (2018).</li><li>Heidrich, F., 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=The+role+of+phospho-adenosine+monophosphate-activated+protein+kinase+and+vascular+endothelial+growth+factor+in+a+model+of+chronic+heart+failure.">The role of phospho-adenosine monophosphate-activated protein kinase and vascular endothelial growth factor in a model of chronic heart failure.</a> Artificial Organs. 34 (11), 969-979 (2010).</li><li>Shen, Y., Liu, X., Shi, J., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+Nrf2+in+myocardial+ischemia+and+reperfusion+injury.">Involvement of Nrf2 in myocardial ischemia and reperfusion injury.</a> International Journal of Biological Macromolecules. 125, 496-502 (2019).</li><li>Hinkel, 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=AntimiR-21+prevents+myocardial+dysfunction+in+a+pig+model+of+ischemia/reperfusion+injury.">AntimiR-21 prevents myocardial dysfunction in a pig model of ischemia/reperfusion injury.</a> Journal of the American College of Cardiology. 75 (15), 1788-1800 (2020).</li><li>Torrado, 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=Sacubitril/Valsartan+averts+adverse+post-infarction+ventricular+remodeling+and+preserves+systolic+function+in+rabbits.">Sacubitril/Valsartan averts adverse post-infarction ventricular remodeling and preserves systolic function in rabbits.</a> Journal of the American College of Cardiology. 72 (19), 2342-2356 (2018).</li><li>Guan, 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=MCU+Up-regulation+contributes+to+myocardial+ischemia-reperfusion+Injury+through+calpain/OPA-1-mediated+mitochondrial+fusion/mitophagy+Inhibition.">MCU Up-regulation contributes to myocardial ischemia-reperfusion Injury through calpain/OPA-1-mediated mitochondrial fusion/mitophagy Inhibition.</a> Journal of Cellular and Molecular Medicine. 23 (11), 7830-7843 (2019).</li><li>Fan, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dectin-1+contributes+to+myocardial+ischemia/reperfusion+injury+by+regulating+macrophage+polarization+and+neutrophil+infiltration.">Dectin-1 contributes to myocardial ischemia/reperfusion injury by regulating macrophage polarization and neutrophil infiltration.</a> Circulation. 139 (5), 663-678 (2019).</li><li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+myocardial+ischemic+preconditioning+on+ischemia-reperfusion+stimulation-induced+activation+in+rat+thoracic+spinal+cord+with+functional+MRI.">Effect of myocardial ischemic preconditioning on ischemia-reperfusion stimulation-induced activation in rat thoracic spinal cord with functional MRI.</a> International Journal of Cardiology. 285, 59-64 (2019).</li><li>Li, D., 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=Cardioprotection+of+CAPE-oNO2+against+myocardial+ischemia/reperfusion+induced+ROS+generation+via+regulating+the+SIRT1/eNOS/NF-&#954;B+pathway+in+vivo+and+in+vitro.">Cardioprotection of CAPE-oNO2 against myocardial ischemia/reperfusion induced ROS generation via regulating the SIRT1/eNOS/NF-&#954;B pathway in vivo and in vitro.</a> Redox Biology. 15, 62-73 (2018).</li><li>Cui, Y., Wang, Y., Liu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+Barbaloin+in+a+rat+model+of+myocardial+ischemia+reperfusion+injury+through+the+regulation+of+the+CNPY2PERK+pathway.">Protective effect of Barbaloin in a rat model of myocardial ischemia reperfusion injury through the regulation of the CNPY2PERK pathway.</a> International Journal of Molecular Medicine. 43 (5), 2015-2023 (2019).</li><li>Lin, M. W., 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=Prolonged+preoperative+fasting+induces+postoperative+insulin+resistance+by+ER-stress+mediated+Glut4+down-regulation+in+skeletal+muscles.">Prolonged preoperative fasting induces postoperative insulin resistance by ER-stress mediated Glut4 down-regulation in skeletal muscles.</a> Int J Med Sci. 11 (5), 1189-1197 (2021).</li><li>Wu, 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=Sevoflurane+alleviates+myocardial+ischemia+reperfusion+injury+by+inhibiting+P2X7-NLRP3+mediated+pyroptosis.">Sevoflurane alleviates myocardial ischemia reperfusion injury by inhibiting P2X7-NLRP3 mediated pyroptosis.</a> Frontiers in Molecular Biosciences. 26 (8), 768594 (2021).</li><li>Wu, Y., Yin, X., Wijaya, C., Huang, M. H., McConnell, B. K. <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+in+rats.">Acute myocardial infarction in rats.</a> Journal of Visualized Experiments. (48), e2464 (2011).</li><li>Zhang, C. 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=Mitochondria-targeted+cyclosporin:+A+delivery+system+to+treat+myocardial+ischemia+reperfusion+injury+of+rats.">Mitochondria-targeted cyclosporin: A delivery system to treat myocardial ischemia reperfusion injury of rats.</a> Journal of Nanobiotechnology. 17 (1), 18 (2019).</li><li>Liu, X. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNA+MALAT1+modulates+myocardial+ischemia-reperfusion+injury+through+the+PI3K/Akt/eNOS+pathway+by+sponging+miRNA-133a-3p+to+target+IGF1R+expression.">Long non-coding RNA MALAT1 modulates myocardial ischemia-reperfusion injury through the PI3K/Akt/eNOS pathway by sponging miRNA-133a-3p to target IGF1R expression.</a> European Journal of Pharmacology. 916, 174719 (2022).</li><li>Li, 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=Ginsenoside+Rg3-loaded,+reactive+oxygen+species-responsive+polymeric+nanoparticles+for+alleviating+myocardial+ischemia-reperfusion+injury.">Ginsenoside Rg3-loaded, reactive oxygen species-responsive polymeric nanoparticles for alleviating myocardial ischemia-reperfusion injury.</a> Journal of Controlled Release. 317, 259-272 (2020).</li><li>Mickelson, J. 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=Streptokinase+improves+reperfusion+blood+flow+after+coronary+artery+occlusion.">Streptokinase improves reperfusion blood flow after coronary artery occlusion.</a> International Journal of Cardiology. 23 (3), 373-384 (1989).</li><li>Verscheure, Y., Pouget, G., De Courtois, F., Le Grand, B., John, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+by+R+56865,+a+novel+cytoprotective+drug,+of+regional+myocardial+ischemia-+and+reperfusion-induced+electrocardiographic+disturbances+in+anesthetized+rabbits.">Attenuation by R 56865, a novel cytoprotective drug, of regional myocardial ischemia- and reperfusion-induced electrocardiographic disturbances in anesthetized rabbits.</a> Journal of Cardiovascular Pharmacology. 25 (1), 126-133 (1995).</li><li>Fan, M. 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=Animal+model+of+coronary+microembolization+under+transthoracic+echocardiographic+guidance+in+rats.">Animal model of coronary microembolization under transthoracic echocardiographic guidance in rats.</a> Biochemical and Biophysical Research Communications. 568 (3), 174-179 (2021).</li><li>Lim, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+injection+of+allogeneic+umbilical+cord-derived+multipotent+mesenchymal+stromal+cells+reduces+the+infarct+area+and+ameliorates+cardiac+function+in+a+porcine+model+of+acute+myocardial+infarction.">Intravenous injection of allogeneic umbilical cord-derived multipotent mesenchymal stromal cells reduces the infarct area and ameliorates cardiac function in a porcine model of acute myocardial infarction.</a> Stem Cell Research &amp; Therapy. 9 (1), 129 (2018).</li><li>Trautner, H., 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=Heart+innervation+after+ligation+of+the+left+anterior+descending+coronary+artery+(LAD).">Heart innervation after ligation of the left anterior descending coronary artery (LAD).</a> Histochemistry. 92 (2), 103-108 (1989).</li><li>Han, C., Wang, X. A., Fiscus, R. R., Gu, J., McDonald, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+cardiac+neuropeptide+Y+after+experimental+myocardial+infarction+in+rat.">Changes in cardiac neuropeptide Y after experimental myocardial infarction in rat.</a> Neuroscience Letters. 104 (1-2), 141-146 (1989).</li><li>Huang, W., 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=Deletion+of+neuropeptide+Y+attenuates+cardiac+dysfunction+and+apoptosis+during+acute+myocardial+infarction.">Deletion of neuropeptide Y attenuates cardiac dysfunction and apoptosis during acute myocardial infarction.</a> Frontiers in Pharmacology. 10, 1268 (2019).</li><li>Kalla, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cardiac+sympathetic+co-transmitter+neuropeptide+Y+is+pro-arrhythmic+following+ST-elevation+myocardial+infarction+despite+beta-blockade.">The cardiac sympathetic co-transmitter neuropeptide Y is pro-arrhythmic following ST-elevation myocardial infarction despite beta-blockade.</a> European Heart Journal. 41 (23), 2168-2179 (2020).</li><li>Cuculi, F., 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=Relationship+of+plasma+neuropeptide+Y+with+angiographic,+electrocardiographic+and+coronary+physiology+indices+of+reperfusion+during+ST+elevation+myocardial+infarction.">Relationship of plasma neuropeptide Y with angiographic, electrocardiographic and coronary physiology indices of reperfusion during ST elevation myocardial infarction.</a> Heart (British Cardiac Society). 99 (16), 1198-1203 (2013).</li><li>Vuorio, T., Tirronen, A., Yl&#228;-Herttuala, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Lymphatics+-+a+new+avenue+for+therapeutics.">Cardiac Lymphatics - a new avenue for therapeutics.</a> Trends in Endocrinology and Metabolism: TEM. 28 (4), 285-296 (2017).</li><li>Henri, 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=Selective+stimulation+of+cardiac+lymphangiogenesis+reduces+myocardial+edema+and+fibrosis+leading+to+improved+cardiac+function+following+myocardial+infarction.">Selective stimulation of cardiac lymphangiogenesis reduces myocardial edema and fibrosis leading to improved cardiac function following myocardial infarction.</a> Circulation. 133 (15), 1484-1497 (2016).</li><li>Oliver, G., Kipnis, J., Randolph, G. J., Harvey, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lymphatic+vasculature+in+the+21st+century:+novel+functional+roles+in+homeostasis+and+disease.">The lymphatic vasculature in the 21st century: novel functional roles in homeostasis and disease.</a> Cell. 182 (2), 270-296 (2020).</li><li>Klotz, 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=Cardiac+lymphatics+are+heterogeneous+in+origin+and+respond+to+injury.">Cardiac lymphatics are heterogeneous in origin and respond to injury.</a> Nature. 522 (7554), 62-67 (2015).</li><li>Percie du Sert, N., 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=Reporting+animal+research:+Explanation+and+elaboration+for+the+ARRIVE+guidelines+2.0.">Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0.</a> PLoS Biology. 18 (7), 3000411 (2020).</li><li>Miller, D. L., Li, P., Dou, C., Armstrong, W. F., Gordon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evans+blue+staining+of+cardiomyocytes+induced+by+myocardial+contrast+echocardiography+in+rats:+evidence+for+necrosis+instead+of+apoptosis.">Evans blue staining of cardiomyocytes induced by myocardial contrast echocardiography in rats: evidence for necrosis instead of apoptosis.</a> Ultrasound in Medicine &amp; Biology. 33 (12), 1988-1996 (2007).</li><li>Deng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-Lipoic+acid+reduces+infarct+size+and+preserves+cardiac+function+in+rat+myocardial+ischemia/reperfusion+injury+through+activation+of+PI3K/Akt/Nrf2+pathway.">&#945;-Lipoic acid reduces infarct size and preserves cardiac function in rat myocardial ischemia/reperfusion injury through activation of PI3K/Akt/Nrf2 pathway.</a> PLoS ONE. 8 (3), 58371 (2013).</li></ol>

The authors have nothing to disclose.

The main difference between the already existing and improved methods was the use of PVC tubes in the ligation process. In the existing surgery method, the myocardial tissue was ligated using the 6-0 silk suture only, which induced damage to the myocardium during ligation resulting in intraoperative death. Moreover, the pulsation of the heart would loosen the slipknot. In contrast, in the improved method with the PVC tube, the slipknot placed in the groove of the tube could be tightened, and the area of the myocardium affected by ligation increased. These benefits were observed during the experimental procedure and confirmed by the TTC staining and percent survival findings.
The critical step of the improved surgery method was placing the soft tube on the proximal LAD coronary artery, accompanied by nerves, lymphatic vessels, and myocardial tissue during ligation in the ischemic period. This pre-prepared soft tube can act as a cushion that protects the peripheral tissues (nerves, myocardia, and lymphatic vessels) and decreases mortality during coronary artery ligation. The surgery performed by the already existing method was similar to the surgery for myocardial infarction. The percent survival findings indicated that rats in the existing model group mainly died during the ischemic period (two rats died at 2 min post-ligation, and two rats died at 45 min post-ligation). Otherwise, the underlying causes of death are still unclear, and there are a series of hypotheses, including additional damage to the nervous structures23, lymphatic vessels, and myocardia.
Regarding nervous damage, previous studies have indicated that during the ischemic period in the animal model, besides the direct local effects of ischemia on the nervous structures, there is also probably a significant decrease in neuropeptide Y (NPY) levels that contribute to disturbances in axoplasmic transport in the sympathetic innervation24. This finding agrees with results reported by Han et al.25, who revealed that a gradual disappearance of NPY occurred within the infarcted myocardium after ligation of the LAD coronary artery in rats. However, the role of NPY in this context remains unclear. Its deletion attenuates cardiac dysfunction and apoptosis during acute myocardial infarction26, and is associated with arrhythmia27, high blood pressure, and coronary microvascular function28.
Furthermore, adverse obstruction of cardiac lymph flow occurred during the ischemic period, leading to severe cardiac edema, left dysfunction, and hemorrhages29, which might be another cause of death in rats. During this pathological process, the ligature of the LAD coronary artery might be attributed to the obstruction of coronary arteries or cardiac lymphatic transport within the infarct area, which can cause additional complications, such as adverse remodeling of the epicardial collector lymphatics, reduced lymphatic flow, and persistent edema30.
Therefore, circulation in lymphatic vessels plays a functional role in cardiac homeostasis31&#160;and wound healing32, and the percent survival findings in this study suggest that the improved MIRI surgical procedure might avoid lymphatic damage and promote lymphatic reperfusion by placing the soft tube on the LAD coronary artery during ligature. In comparison, the existing surgery method is more likely to tear the heart muscle and cause a massive hemorrhage during ligation of the LAD coronary artery, without the cushioning effect of the soft tube. Additionally, the pre-prepared soft tube diameter was much larger than the 6-0 silk suture, and the tube may have contracted and induced a larger infarct size when the slipknot was tied to the tube during the ischemic period.
This study had a few limitations. The infarct size of the heart was analyzed in the preliminary experiment. The substitution formula (N = 7.75) was calculated using a previously reported equation33. Considering the possible death of rats during the operation, N was raised by 25%; hence, n = 10 (ten rats for each group) was decided. Otherwise, the already existing method to generate the MIRI model had a high mortality rate. Therefore, few cases (low sample size) in the experimental model group influenced the statistical findings. Several assessments, including echocardiography30, Evans blue staining34, and the myocardial enzyme measurement35, were essential for cardiac function evaluation and analysis. Owing to the low sample size of this work, these assessments were not performed and will be described in a future study of pharmacodynamic research in MIRI. However, considering that the existing surgical procedure to generate the MIRI model is associated with extensive myocardial damage, it is worthwhile to report this present method to improve the modeling of MIRI in rats and bring light to this preclinical model that correctly simulates ischemic heart disease.
In conclusion, the improved surgery method to generate the MIRI model had a higher survival rate, an elevated ST-T segment, and a larger infarct size than the existing MIRI model generation method, suggesting that the improved model better simulates MIRI pathology.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63510">Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury</a>. The Authors section was&#160;updated.
The Authors section was updated from:
Hua-Qin Tong*1 
Man-Lu Fan*1 
Tong Sun1 
Hao-Wen Zhang2 
Jie Han3 
Meng-Xi Wang1 
Jian-Dong Chen3 
Wei-Xin Sun4 
Xiao-Hu Chen3 
Mian-Hua Wu5 
1First College of Clinical Medicine, Biological Technology Center for Innovation in Chinese Medicine, Nanjing University of Chinese Medicine 
2School of Health Preservation and Rehabilitation, Key Laboratory of Acupuncture and Medicine Research of Ministry of Education 
3Department of Cardiology, Jiangsu Provincial Hospital of Chinese Medicine 
4Department of Cardiology, Yancheng TCM Hospital Affiliated to Nanjing University of Chinese Medicine 
5Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine (TCM) Prevention and Treatment of Tumor, Nanjing University of Chinese Medicine 
* These authors contributed equally
to:
Hua-Qin Tong*1,2,3 
Man-Lu Fan*3 
Tong Sun3 
Hao-Wen Zhang4 
Jie Han5 
Meng-Xi Wang3 
Jian-Dong Chen5 
Wei-Xin Sun6 
Xiao-Hu Chen1,2 
Mian-Hua Wu7 
1Department of Cardiology, Affiliated Hospital of Nanjing University of Chinese Medicine 
2Department of Cardiology, Jiangsu Province Hospital of Chinese Medicine 
3First College of Clinical Medicine, Biological Technology Center for Innovation in Chinese Medicine, Nanjing University of Chinese Medicine 
4School of Health Preservation and Rehabilitation, Key Laboratory of Acupuncture and Medicine Research of Ministry of Education 
5Department of Cardiology, Jiangsu Provincial Hospital of Chinese Medicine 
6Department of Cardiology, Yancheng TCM Hospital Affiliated to Nanjing University of Chinese Medicine 
7Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine (TCM) Prevention and Treatment of Tumor, Nanjing University of Chinese Medicine 
* These authors contributed equally

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63510">Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury</a>. The Authors section was&#160;updated.

Erratum: Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Ischemic heart disease is the leading cause of mortality worldwide. Cardiovascular mortality has a crucial role in public health and epidemiology globally1. Myocardial ischemia and reperfusion injury play essential functions in ischemic heart disease, which refers to a complex pathophysiological process that includes depletion of adenosine triphosphate2, excessive generation of reactive oxygen species3, inflammatory reactions4, and mitochondrial dysfunction owing to calcium overload5, which triggers acute myocardial infarction via metabolic dysfunction and structural damage6.
However, the detailed mechanisms underlying myocardial ischemia and reperfusion injury (MIRI) remain unknown. The present work aims to develop a unique animal model that adequately simulates the clinical presentation and treatment of MIRI. Otherwise, in the process of MIRI model research, large animals7 (such as pigs) require interventional surgery, which is expensive. Small animals (such as rabbits8, mice9,10,11,12, and rats13) require delicate surgery under microscopy10, remote-controlled saccules8,11, or squeezing the heart out of the cavity9, which requires a high level of technology and may cause several postoperative complications that disturb the accuracy of findings. An ideal MIRI model with a higher survival rate and lower cost will play a crucial role in pathological research.
This study aimed to combat these issues by establishing a more accessible and feasible model of MIRI in rats to facilitate the research on the pathology of MIRI, which could lead to the discovery of clinical therapies for MIRI.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Neutral Formalin</td>
			<td>Chunyu, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-Triphenyl-2H-Tetrazolium Chloride</td>
			<td>Solarbio, China</td>
			<td>T8107</td>
			<td></td>
		</tr>
		<tr>
			<td>75% Alchol</td>
			<td>SCR, China</td>
			<td>10009261</td>
			<td></td>
		</tr>
		<tr>
			<td>Artery Clip</td>
			<td>Zhonglin Dongsheng, China</td>
			<td>6.5cm</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Olympus Corporation, Japan</td>
			<td>EPL5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton ball</td>
			<td>Huachen, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Dpilatory cream</td>
			<td>Veet, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye speculum</td>
			<td>Shanghai Jingzhong, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Zhonggan, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad</td>
			<td>GraphPad Software, USA</td>
			<td>8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>H&#38;E Kit</td>
			<td>Solarbio, China</td>
			<td>G1120</td>
			<td></td>
		</tr>
		<tr>
			<td>High-pressure steam sterilizer</td>
			<td>TOMY, Japan</td>
			<td>SX-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH, USA</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson Kit</td>
			<td>Solarbio, China</td>
			<td>G1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Tape</td>
			<td>Mr.Song, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus Corporation, Japan</td>
			<td>CKX31</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopy</td>
			<td>TEKSQRAY, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica, Germany</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome Blade</td>
			<td>Leica, Germany</td>
			<td>819</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Shanghai Jingzhong, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic scissors</td>
			<td>Shanghai Jingzhong, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic tweezers</td>
			<td>Shanghai Jingzhong, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper clip</td>
			<td>Chenguang, China</td>
			<td>ABS91613</td>
			<td></td>
		</tr>
		<tr>
			<td>Physiological saline solution</td>
			<td>Kelun, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Powerlab ECG</td>
			<td>ADINSTRUMENTS ,China</td>
			<td>4/35</td>
			<td></td>
		</tr>
		<tr>
			<td>PVC tube</td>
			<td>Guanzhijia, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal ventilator</td>
			<td>TECHMAN, China</td>
			<td>HX-101E</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pentobarbital</td>
			<td>SIGEMA, USA</td>
			<td>1030001</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction trocar</td>
			<td>TECHMAN, China</td>
			<td>HX-101E</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture line</td>
			<td>Lingqiao, China</td>
			<td>4-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture needle with thread</td>
			<td>Shanghai Pudong Jinhua Medical Products Co LTD, China</td>
			<td>6-0</td>
			<td></td>
		</tr>
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</table>

improved rodent model of myocardial ischemia and reperfusion injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

TTC staining 
Heart sections from rats that underwent either the existing or improved MIRI procedure or sham surgery were stained with TTC, and the images were stored digitally and analyzed using ImageJ. Rats that underwent either the already existing or improved MIRI procedures had myocardial infarctions, while rats from the sham group did not (Figure 2B). Compared to rats in the sham group, rats in the existing (p &#60; 0.0001) and experimental (p &#60; 0.0001) MIRI model groups had a significant difference in myocardial infarct size, and the experimental model group had a larger myocardial infarct size than the existing model group (p = 0.0176) (Figure 3B).
Histological staining 
Analysis of specimens stained using H&#38;E and Masson stains22,23&#160;showed that compared to the sham group, the cardiomyocytes of both the experimental and the existing model groups had experienced critical damage and nucleolysis and were infiltrated by numerous neutrophils (Figure 3).
ECG test 
The ECG ST-T segments of rats in the existing and experimental MIRI model groups were elevated compared with those of rats in the sham group (Figure 4A), and the differences between the experimental model and sham groups (p &#60; 0.0001) or the existing model and sham groups (p &#60; 0.0001) were significant (Figure 4B). Furthermore, the ST-T segment was more elevated in the experimental model group than in the existing model group (p = 0.0274) (Figure 4C).
Percent survival 
The survival rate was significantly different between the two MIRI model groups (Figure 4D). Four of the ten rats died in the existing model group. The mortality rate was 40% during the reperfusion period. In contrast, none of the rats in the experimental model group died during surgery, demonstrating that the current improved model had a higher survival rate (p = 0.0291).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63510/63510fig01.jpg" /> 
Figure 1: Key steps of the myocardial ischemic and reperfusion injury (MIRI) model surgery. Green points indicate the protocol of ligature during the ischemic period, including placing the soft tube on the coronary arteries (A), hooking the suture line into the groove of the pre-prepared soft tube (B), loosening the slipknot, and removing the soft tube when the reperfusion period was started (scale bar = 1 cm) (C). LAA: Left Atrial Appendage, RAA: Right Atrial Appendage, LAD: Left Anterior Descending, RCA: Right Coronary Artery, IVC: Inferior Vena Cava, SVC: Superior Vena Cava, AO: Aorta Artery, PA: Pulmonary Artery. <a href="https://www.jove.com/files/ftp_upload/63510/63510fig01large.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/63510/63510fig02.jpg" /> 
Figure 2: The whole surgery procedure and differences in triphenyltetrazolium chloride (TTC) staining between different groups. The pre-prepared small retractor (scale bar = 15 mm), soft tube (scale bar = 10 mm), and the whole surgery (scale bar = 15 mm) are shown (A). Thirty rats were randomly divided into the experimental (n = 10), sham group (n = 10), and existing model (n = 10) groups. TTC staining indicated that both the experimental and existing models&#39; groups had significant changes compared to the sham group (B). The anterior wall of the myocardium in the experimental and the lateral wall in the existing model groups turned pale white, confirming the ischemic area&#39;s location (scale bar = 5 mm). The &#34;existing model&#34; is depicted as the &#34;old model&#34; in the figure. <a href="https://www.jove.com/files/ftp_upload/63510/63510fig02large.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/63510/63510fig03.jpg" /> 
Figure 3: Differences in H&#38;E and Masson staining between groups. Thirty male Sprague Dawley rats were randomly divided into the experimental (n = 10), sham group (n = 10), and existing model (n = 10) groups, and the comparison of cell morphological changes between groups is shown (scale bar = 2 mm). Hematoxylin and Eosin (H&#38;E), and Masson staining show that myocardial cells of the experimental model and existing model groups have critical damage, nucleolysis, and are infiltrated by numerous neutrophils compared to those of the sham group (scale bar = 100 &#181;m). The &#34;existing model&#34; is depicted as the &#34;old model&#34; in the figure. <a href="https://www.jove.com/files/ftp_upload/63510/63510fig03large.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/63510/63510fig04.jpg" /> 
Figure 4: Differences in statistical results between groups.Thirty male Sprague Dawley rats were randomly divided into the experimental (n = 10), sham group (n = 10), and existing model (n = 10) groups. Electrocardiogram findings show that compared to the already existing model group, the experimental model group has a larger myocardial infarct size (****p &#60; 0.0001, *p = 0.0176) (A), a higher ST-segment elevation (****p &#60; 0.0001, *p = 0.0274) (B), and a higher survival percentage (p = 0.0291) (C). Especially, rats of the existing model group were more likely to die at the beginning of the ischemia period and the beginning of the reperfusion period (D). The &#34;existing model&#34; is depicted as the &#34;old model&#34; in the figure. <a href="https://www.jove.com/files/ftp_upload/63510/63510fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: The details of the pre-prepared retractor and PVC tube. The pre-prepared retractor (A) and PVC tube (B) are shown. <a href="https://www.jove.com/files/ftp_upload/63510/Supplementary Figure 1.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury at JoVE.com

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group.

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence ...

improved-rodent-model-of-myocardial-ischemia-and-reperfusion-injury

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5.5K Views.  Affiliated Hospital of Nanjing University of Chinese Medicine. Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group. 

Video: Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

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 ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...