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>

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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><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&amp;amp;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></tbody></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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5.8K Views.  Affiliated Hospital of Nanjing University of Chinese Medicine. Dit myocardiale ischemie reperfusiemodel wordt verbeterd door een zelfgemaakt retractor, polyvinylchloridechloride buis en een unieke knoopmethode. Deze techniek gaat gepaard met een hogere overlevingskans en een grotere impactgrootte. Deze methode zal de overlevingskans van myocardiale ischemie reperfusie letsel bij ratten verbeteren, wat licht zal brengen in de klinische therapie van myocardiale ischemie reperfusie letsel in de toekomst.Nadat u de rat hebt verdoofd, maakt u het midde...

Video: Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

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 ...

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 ...

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 ...

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 ...

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

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

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

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

A New Model of Heart Failure Post-Myocardial Infarction in the Rat

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial infarction (MI). However, the disadvantages of this model include high mortality rate after ligation and larger variations both in the infarct size and the degree of impaired cardiac function. In addition, a ventilator or exteriorization of the heart is indispensable for the previous models, which complicates the procedure during the ligation. In this study, we developed a reliable and reproducible model without the ventilator or exteriorization of the heart by ligating the LAD coronary artery. Four weeks after the procedure, we found that the serum concentrations of CK-MB, NT-proBNP, and Renin, which were used to assist diagnoses of MI and HF, were significantly higher in the MI group compared to the sham group. In contrast, the value of left ventricle ejection fraction (LVEF) in the MI group was obviously less than in the sham group. Furthermore, the infarct size and cardiac fibrosis area were individually confirmed and quantitatively analyzed by TTC staining and Masson's trichrome staining. Smaller variations were found in either infarct size or fibrosis area in the MI group, which helped to develop a reliable and reproducible model of HF post-MI. This new model of HF post-MI in the rat is vital for studying the potential mechanisms of MI and HF. This new method can be used to develop the new drug for treatment of MI and HF in rats by using pharmacological strategies.

We successfully developed a reliable and reproducible model of heart failure post-myocardial infarction in the rat without ventilation or exteriorization of the heart. This simplifies the procedure and benefits the further studies about the potential mechanisms behind the heart failure.

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Biology

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

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

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

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

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

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