This work was supported by Merit Review awards (BX002332 and BX000640) from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development, National Institutes of Health (R15HL129140), and funds from Institutional Research and Improvement account. The project is supported in part by the National Institutes of Health grant C06RR0306551.

All surgical procedures performed on animals should be carried out in accordance with Guide for the Care and Use of Laboratory Animals13 or other appropriate ethical guidelines. Protocols should be approved by the animal welfare committee at the appropriate institution before proceeding.
1. Preparing for the ECG
NOTE: Before beginning, don personal protective equipment including gloves, eyewear and a clean laboratory coat or disposable gown.
<ol>
	<li>Clean the ECG pad using a non-alcohol and non-bleach based decontamination solution. Gently use a delicate task wipe to blot off excess solution to ensure that the electrode pad does not become damaged.</li>
	<li>If the ECG pad has a heating feature, use it. Anesthetized mice tend to lose body heat rapidly. Heat the pad to 40-42 &#176;C to maintain normothermic body temperature of 37 &#176;C throughout the surgery14. Monitor body temperature using a rectal thermometer. Adjust pad temperature as necessary to maintain the body temperature ~37 &#176;C.</li>
	<li>As most thoracotomies are performed with the mouse lying on its back (supine), ensure that the toggle is flipped to the &#34;supine&#34; setting. Many ECG pads have a function to toggle between prone and supine positions. Failure to select the right orientation can result in misrepresentation of electrocardiographic events.</li>
	<li>Anesthetize mouse using 5% inhaled isoflurane and 1 L/min oxygen. Once mouse is anesthetized, transfer mouse to ECG pad equipped with an anesthesia nose-cone and reduce isoflurane to 2% and 1 L/min oxygen. Confirm proper anesthesia by ensuring mouse does not react when the mouse&#39;s foot is pinched with forceps.</li>
	<li>Apply a thin coat of eye lubrication ointment over the mouse&#39;s eyes to prevent dryness and corneal damage while anesthetized.</li>
	<li>Clean the mouse&#39;s paws with a wet wipe to remove all visible bedding that may be stuck to the paws or may interfere with the transmission of electrical impulses from the paws to the ECG pad. Dry paws with a wipe.</li>
	<li>Apply a small amount (slightly smaller than a USD dime) of highly conductive electrolyte gel to each of the four metallic electrodes on the ECG pad. 
	Note: Be sure to only apply a small amount of gel as too much gel makes it difficult to restrain the paws to the pad using tape. Additionally, paws are likely to slip out of the restraint during surgery if they were wet before applying tape.</li>
	<li>With the mouse in supine position, use clear medical tape to restrain each paw to its corresponding electrode (Figure 1). First press each paw to its piece of tape and then adhere the tape to the ECG pad. Ensure that each restrained paw is in contact with the electrolyte gel and the electrode.</li>
</ol>
2. Acquiring the ECG
<ol>
	<li>Depending on the equipment used for ECG acquisition, configure the machine so that the ECG waveform can be visualized in real time. For ECG recordings using the physiologic monitoring settings on an echocardiography machine, a live B-mode image will have the ECG waveform running along the bottom of the screen. 
	Note: See individual machine user guides to determine how best to configure that equipment.</li>
	<li>Enable real time visualization of ECG waveform by pressing the B-mode key on an echocardiography machine or the equivalent on other ECG recording devices.
	<ol>
		<li>Adjust the resolution to account for differences in amplitude. If the peak of the R-wave or the trough (valley) of the Q-wave are out of the visual frame, adjust the resolution until the entire height of the waveform can be observed. 
		Note: This can be done under the physiological settings tab on an echocardiography machine. Click the increase or decrease arrows until the entire waveform is visible.</li>
	</ol>
	</li>
	<li>Any time that an image is to be obtained, clear the ECG pad of tools. Touching the mouse during ECG recording with forceps or fingers will disturb the waveform. Ensure that the mouse is still and untouched on the ECG pad before recording any ECGs.</li>
	<li>Use the machine&#39;s &#34;record&#34; or &#34;store&#34; feature before making any surgical incisions on the mouse. This image will be used as a baseline for comparison later on.</li>
</ol>
3. Surgical Procedure and Recording ECG
<ol>
	<li>Inject anesthetized mouse with analgesic (buprenorphine, 1.5 &#181;g, intraperitoneal) before beginning. The details of the ischemia/reperfusion surgical procedure can also be found elsewhere5.</li>
	<li>Remove hair around the surgical site chemically or mechanically and disinfect the area with betadine solution. Use a scalpel to make a vertical incision parallel to the esophagus and trachea. Gently move the lymph nodes to each side of the incision until the thin tissue covering the trachea is exposed. Using forceps, gently separate the tissue until the white cartilage rings of the trachea are visible. 
	Note: We generally use Nair hair removal lotion. The lotion is applied on the surgical site for ~1 min. Nair is then thoroughly washed off using saline or water. This method is preferred in our laboratory because high resolution echocardiography (which can detect hair follicles) is performed before and after surgery. However, caution should be taken to avoid sensitive areas such as genitals, and then thoroughly washing the Nair off to avoid potential skin burns.</li>
	<li>Quickly remove the mouse&#39;s nose from the nose-cone and insert ventilation tubing into the mouth of the mouse and towards the throat. When the tip of the ventilation tubing is visible through the exposed neck area, align the tube with the start of the trachea. Gently wiggle the tube side to side while applying upward pressure until the tubing slides into the trachea which can be confirmed visually through the translucent trachea.</li>
	<li>Ensure that the mouse remains anesthetized during the intubation procedure. Pause from intubation and return the mouse to the nose-cone if it begins to stir.</li>
	<li>Using a loop of string, hook the mouse&#39;s two front teeth through the loop and tape the string ends to the ECG pad to steady the head of the mouse and to ensure the ventilation tubing does not move during surgery. Quickly attach ventilation tubing to rodent ventilator and adjust ventilation settings according to the weight of the mouse. Tape ventilation tubing in place.</li>
	<li>Cover the mouse&#39;s exposed trachea with a gauze soaked in warm saline to keep tissue from drying.</li>
	<li>Make a vertical incision using a scalpel along the left side of the sternum.</li>
	<li>Using forceps, gently separate the fascia layer from the muscle layer. Carefully cut the underlying muscle layers without cutting visible blood vessels.</li>
	<li>Using forceps, grab the third rib and pull upwards gently. Maintain grip on the rib with one hand and use surgical scissors to carefully cut the intercostal tissue between the third and fourth rib. Ensure that the lungs are not damaged. 
	Note: Lungs will retract deep into the chest cavity almost immediately after the chest cavity is punctured by the surgical incision due to the loss of the pressure gradient. Wait until the lungs have retracted before continuing.</li>
	<li>Use forceps to grab and gently separate the thin layer of pericardium which surrounds the heart.</li>
	<li>Insert retractors or manually use forceps as rib retractors to move the ribs into a position where the heart is visible between the ribs. 
	Note: It is common practice to move the mouse&#39;s lower left paw so that it is overlapping the lower right paw during the placement of the ligature. This helps to position the heart so that the left atrial appendage, or auricle, is easily visible during placement of the ligature. Be aware that valid ECG waveforms will not be obtained while the lower left paw is off of the electrode. For this reason it is advisable to return the paw to its electrode after the suturing ligature is passed through the myocardial tissue but before a knot is tightened.</li>
	<li>Locate the LAD visually, beneath the left auricle. Swiftly insert a 7-0 silk tapered suturing needle into the myocardium deeply enough to pass under the LAD but not so deep as to penetrate the LV cavity. Pull the suturing ligature through until there is about 4 cm of suturing silk left on the free (non-needle) end of the suturing ligature.</li>
	<li>Begin to tie a simple suture knot. Once the free end of the suturing silk has been pulled through the loops to form the knot, pause.</li>
	<li>Holding both the free and needle ends of the suturing silk with forceps, insert a ~1 cm section of PE-10 tubing underneath the forming knot and atop the epicardial surface.</li>
	<li>If the mouse&#39;s left paw is crossed, return the paw to its proper electrode. Tighten knot so that the PE-10 tubing is sutured to the heart. Release all physical contact with the mouse to allow ECG to be recorded.</li>
	<li>Allow ECG waveform to cycle through for ~10 sec. Check ECG waveform visually and record waveform as &#34;Time of Occlusion&#34;. If the T-wave does not increase in amplitude within 1 min, reassess placement of ligature.
	<ol>
		<li>If the T-wave amplitude does not increase, either discard the animal from the study or attempt to correct the ligature placement.</li>
	</ol>
	</li>
	<li>Visually check the color of the myocardium to confirm ischemic paling of the LV.</li>
	<li>If ECG changes and myocardial color changes indicate ischemia, double knot the suture around the PE-10 tubing.</li>
	<li>Cover the open chest cavity with warm saline gauze.</li>
	<li>Record ECG every 5-10 min for the duration of the ischemic period.</li>
</ol>
4. Confirmation of Reperfusion Using ECG
<ol>
	<li>Remove saline gauze covering the chest cavity and visualize the heart.</li>
	<li>Use a blade to cut the suturing silk atop the PE-10 tubing. Once the ligature is cut, remove the section of PE-10 tubing and gently remove the suturing ligature from the myocardium.</li>
	<li>Release all physical contact with the mouse and allow the ECG waveform ~10 sec to cycle. Record waveform as &#34;Time of Reperfusion.&#34; Continue to record ECG waveforms every 5-10 min until the desired experimental time point is reached.</li>
	<li>Adjust resolution for changes in amplitude as needed. If the T-wave does not change upon removal of the PE-10 tubing and ligature, reperfusion is not confirmed.
	<ol>
		<li>If the T-wave does not change upon removal of tubing, either discard the animal from the study or attempt to correct the ligature placement.</li>
	</ol>
	</li>
	<li>Visually inspect myocardium to additionally confirm reperfusion by return to red color.</li>
	<li>Close chest cavity by suturing the intercostal space with a 5-0 silk suture while applying gentle pressure to the mouse&#39;s chest to expel excess air that has entered during surgery. Then suture the muscle layers and finally, skin. Note: Applying pressure to the chest cavity may not be sufficient to evacuate the chest cavity of air in all mice.&#160; Therefore, the syringe and needle method of evacuation should be employed to ensure that all air has been expelled.</li>
	<li>Record the last ECG before turning inhaled anesthesia off and removing the mouse&#39;s paws from the electrodes. Increase oxygen to 2 L/min and maintain ventilation until the mouse regains consciousness.</li>
	<li>Allow mouse to recover in a constant temperature controlled environment, e.g. heating pad or warm incubator, to avoid infarct variability. Treat mouse with buprenorphine 24 hr after the surgery and then as needed as indicated by the mouse grimace scale. 
	Note: Procedure for reperfusion is also discussed in detail by Xu et al.5</li>
</ol>

<ol>
	<li>Kochanek, K. D., Murphy, S. L., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deaths:+Final+Data+for+2011.">Deaths: Final Data for 2011.</a> Natl Vital Stat Rep. 63 (3), 1-120 (2015).</li><li>Klabunde, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cardiovascular Physiology Concepts 2edn. , 243 (2012).</li><li>Bhardwaj, R., Kandoria, A., Sharma, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarction+in+young+adults-risk+factors+and+pattern+of+coronary+artery+involvement.">Myocardial infarction in young adults-risk factors and pattern of coronary artery involvement.</a> Niger Med J. 55 (1), 44-47 (2014).</li><li>Xu, Z., Alloush, J., Beck, E., Weisleder, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+myocardial+ischemia-reperfusion+injury+through+ligation+of+the+left+anterior+descending+artery.">A murine model of myocardial ischemia-reperfusion injury through ligation of the left anterior descending artery.</a> J Vis Exp. (86), (2014).</li><li>Fern&#225;ndez, B., 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+coronary+arteries+of+the+C57BL/6+mouse+strains:+implications+for+comparison+with+mutant+models.">The coronary arteries of the C57BL/6 mouse strains: implications for comparison with mutant models.</a> J Anat. 212 (1), 12-18 (2008).</li><li>Thaler, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Only EKG Book You'll Ever Need. , (2003).</li><li>Poirier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise,+heart+rate+variability,+and+longevity:+the+cocoon+mystery?.">Exercise, heart rate variability, and longevity: the cocoon mystery?.</a> Circulation. 129 (21), 2085-2087 (2014).</li><li>Boudoulas, K. D., Borer, J. S., Boudoulas, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Rate,+Life+Expectancy+and+the+Cardiovascular+System:+Therapeutic+Considerations.">Heart Rate, Life Expectancy and the Cardiovascular System: Therapeutic Considerations.</a> Cardiology. 132 (4), 199-212 (2015).</li><li>Wehrens, X. H., Kirchhoff, S., Doevendans, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+electrocardiography:+an+interval+of+thirty+years.">Mouse electrocardiography: an interval of thirty years.</a> Cardiovasc Res. 45 (1), 231-237 (2000).</li><li>Boukens, B. J., Rivaud, M. R., Rentschler, S., Coronel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Misinterpretation+of+the+mouse+ECG:+'musing+the+waves+of+Mus+musculus.">Misinterpretation of the mouse ECG: 'musing the waves of Mus musculus.</a> J Physiol. 592 (21), 4613-4626 (2014).</li><li>O'Gara, P. T., 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=ACCF/AHA+guideline+for+the+management+of+ST-elevation+myocardial+infarction:+executive+summary:+a+report+of+the+American+College+of+Cardiology+Foundation/American+Heart+Association+Task+Force+on+Practice+Guidelines:+developed+in+collaboration+with+the+American+College+of+Emergency+Physicians+and+Society+for+Cardiovascular+Angiography+and+Interventions.">ACCF/AHA guideline for the management of ST-elevation myocardial infarction: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines: developed in collaboration with the American College of Emergency Physicians and Society for Cardiovascular Angiography and Interventions.</a> Catheter Cardiovasc Interv. 82 (1), E1-E27 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , (2011).</li><li>Gao, S., Ho, D., Vatner, D. 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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=In+vivo+cardioprotection+by+S-nitroso-2-mercaptopropionyl+glycine.">In vivo cardioprotection by S-nitroso-2-mercaptopropionyl glycine.</a> J Mol Cell Cardiol. 46 (6), 960-968 (2009).</li><li>Preda, M. B., Burlacu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrocardiography+as+a+tool+for+validating+myocardial+ischemia-reperfusion+procedures+in+mice.">Electrocardiography as a tool for validating myocardial ischemia-reperfusion procedures in mice.</a> Comp Med. 60 (6), 443-447 (2010).</li><li>Speerschneider, T., Thomsen, M. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+mimics+ischemic+preconditioning+via+activation+of+K(ATP)+channels:+reduction+of+myocardial+infarct+size+with+an+acute+memory+phase.">Isoflurane mimics ischemic preconditioning via activation of K(ATP) channels: reduction of myocardial infarct size with an acute memory phase.</a> Anesthesiology. 87 (2), 361-370 (1997).</li><li>Gargiulo, 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=Mice+anesthesia,+analgesia,+and+care,+Part+I:+anesthetic+considerations+in+preclinical+research.">Mice anesthesia, analgesia, and care, Part I: anesthetic considerations in preclinical research.</a> ILAR J. 53 (1), E55-E69 (2012).</li><li>Curtis, M. 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The authors have nothing to disclose.

Using ECG changes as a supplemental method for confirming myocardial ischemia and reperfusion ensures the accurate placement of the occluding ligature. Accuracy of ligature placement is critical to reducing data variability among animals. The LAD in a mouse heart is a difficult artery to visualize. Therefore, supplementing visual pallor with electrocardiographic changes will help ensure the correct placement of the ligature and resulting tissue damage.
Since the ECG pad offers a non-invasive v...

Heart disease remains the leading cause of death worldwide1,2. Not only is the left ventricle (LV) the most muscular chamber, responsible for pumping blood from the heart to the entire body3, it is a common cardiac injury site post-myocardial infarction4. Left ventricular tissue death often results in systolic heart failure. Animal models of heart disease are imperative for the advancement of biomedical cardiovascular research. The C57Bl/6 strain of mice have been a popular choice for animal models due to their quick breeding time, low cost and ease in genetic alterations. Most murine surgical models for the study of heart disease involve occlusion of the LAD branch of the left coronary artery. The LAD is sometimes called the left obtuse marginal5,6. The LAD supplies blood to the left ventricular anterior and antero-lateral walls. LAD occlusion studies are aimed at inducing anterior infarctions, sometimes extending into the inferior and lateral wall regions7.
Two models that are used frequently for myocardial infarction studies include chronic occlusion myocardial infarction and myocardial ischemia/reperfusion injury. The chronic occlusion is created by surgically suturing around and permanently blocking blood flow through the LAD. The ischemia/reperfusion injury is created much in the same way only with a transient, usually 30-60 min, ischemic period. To achieve transient ischemia, the occluding suture ties around the LAD and a small PE-10 tube which is placed parallel to the LAD on the epicardial surface of the heart, followed by a reperfusion period where the tubing and occluding suture is removed and blood is allowed to once again flow through the artery and into the myocardium. The ischemia/reperfusion surgery has been deemed to be clinically relevant due to the nature of reperfusion injury paralleling the treatment of human infarctions which includes prompt coronary angioplasty and stenting of the artery, or coronary artery bypass. Typically, during these surgeries, ischemia of the LV in a mouse heart is confirmed by visible pallor of the myocardial wall. However, by simply performing the surgeries on an electrocardiogram (ECG) pad under constant monitoring conditions, visible changes can be observed in the ECG waveform, thereby confirming ischemia and reperfusion of the mouse myocardium.
Although the murine heart is similar to the human heart in many respects, including its four-chambered structure, the hearts also have differences. One obvious difference is the average resting heart rate of adult mice is 600 - 700 beats per min (bpm) whereas that of adult humans is ~60-100 bpm8,9. Additionally, in mice the repolarization waves, J and T, often merge with the depolarization QRS-complex making a clear ST-segment difficult to discern10. To complicate the process of electrocardiographically confirming myocardial ischemia, it is the elevation of the T-wave and the ST-segment which are used as markers for the diagnosis of ischemia and myocardial infarction injury in humans, clinically referred to as ST elevation myocardial infarction or STEMI. One of the key differences between human and murine waveforms is that S-wave is immediately followed be a J-wave that transfers directly into a negative T-wave. During acute myocardial ischemia in mice the amplitude of S-wave decreases and is directly followed by an abnormal J-wave and an inverted T-wave11. The T-wave does not seem to represent a significant portion of the repolarization in mice11. Despite nomenclature and mouse vs. human differences, ECG confirmation of murine myocardial ischemia and reperfusion is still feasible and relatively simple. For the sake of simplifying waveform interpretation, the segment between the S-J-T is referred to as ST-segment herein.
STEMI guidelines published in 2013 recommend a patient door-to-balloon time of less than 90 min12.This means that the time frame from the identification of the patient&#39;s coronary artery occlusion until the artery is reopened should be less than 90 min. The beating heart is constantly working and therefore, has a high oxidative metabolism and a high level of oxygen consumption3. To provide for this, a network of capillaries is available to each myocyte3. It only takes a heart a few beats to exhaust its oxygen and nutrient supply. In a 90 min window, an ischemic heart region in a human will have been blocked from receiving between 5,400 and 9,000 heart beats worth of oxygen-rich blood. In that same 90 min window, a mouse would have 54,000 to 63,000 heart beats. Experimental time points for murine ischemia/reperfusion injury are typically between 30 and 60 min.
The importance of developing a supplemental method of confirming myocardial ischemia and reperfusion in a murine model has profound implications on the consistency and reproducibility of data in myocardial ischemia/reperfusion studies. The current practice of visually observing the heart for a change in tissue color is not adequate as a stand-alone diagnostic. Additionally, reperfusion after removal of the tubing and suture is not guaranteed. Although the artery is no longer tied off, the artery may have sustained damage during the procedure and may become impossible to reperfuse. It would be beneficial to have a record of electrocardiographic changes to confirm reperfusion rather than relying on observations of myocardial pallor and rubor (red color). Hearts that do not show the markers of ischemia/reperfusion injury can then quickly be flagged and a decision on how to proceed can be made by the investigators.
Lastly, establishing a record of ECG changes from baseline throughout the ischemic and reperfusion periods allows investigators to continue to monitor the heart after the initial surgery. Investigators currently lose sight of the heart as soon as the surgery is completed. ECG is a simple way to gain insight into changes occurring in the myocardium hours to days after the surgery. ECG recorded at time points after surgery could reveal late-developing Q-waves indicating continued or worsening tissue death. However, to effectively gage new or worsening electrocardiographic markers, a baseline ECG must be available for comparison.
This protocol will demonstrate how to prepare, obtain, and interpret the ECG to confirm ischemia and reperfusion of the mouse heart using 8 - 12 week old male C57Bl/6 mice.

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		<tr height="42">
			<td height="42" style="height:42px;width:129px;">Vevo 1100</td>
			<td style="width:191px;">Fujifilm 
			Visual Sonics</td>
			<td style="width:124px;"></td>
			<td style="width:275px;">Echocardiography Machine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:129px;">Mouse Handling Plate</td>
			<td style="width:191px;">Fujifilm 
			Visual Sonics</td>
			<td style="width:124px;"></td>
			<td>Heated ECG plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:129px;">Signa-Gel&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:124px;"></td>
			<td>Highly Conductive Multi-</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:129px;">Electrode Gel</td>
			<td style="width:191px;">Parker</td>
			<td style="width:124px;">15-25</td>
			<td>Purpose Electrolyte</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:129px;">Transpore Medical Tape</td>
			<td style="width:191px;">3M</td>
			<td style="width:124px;">1527-0</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:129px;">PI-Spray II</td>
			<td style="width:191px;">Pharmaceutical Innovations</td>
			<td style="width:124px;">NDC 36-2013-25</td>
			<td>Cleaning agent for ECG plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:129px;">C57Bl6 Mice</td>
			<td style="width:191px;">The Jackson Laboratory</td>
			<td style="width:124px;">000664</td>
			<td>Male, 8-12 wk</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:129px;">IsoThesia-Isoflurane</td>
			<td style="width:191px;">Henry Schein</td>
			<td style="width:124px;">NDC 1169-0500-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:129px;">Excel</td>
			<td style="width:191px;">Microsoft</td>
			<td style="width:124px;"></td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:129px;">Systane Nighttime Lubricant Eye Ointment</td>
			<td style="width:191px;">Alcon</td>
			<td style="width:124px;">65050935</td>
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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.

A normal murine ECG is displayed in Figure 2 with alphabetic markers for electrical events P, Q, R, S, J and T. P is the initial atrial depolarization. QRS is the wave of depolarization over the ventricles. J is early repolarization and T represents heterogeneous repolarization also known as recovery11. It should be noted that many labs do not use the J-wave nomenclature and instead refer to the SJT-segment as the ST-segment10,15-17. Here, results an...

Watch this Scientific Journal Video about Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography at JoVE.com

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

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 ...

confirmation-myocardial-ischemia-reperfusion-injury-mice-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

13.2K Views.  East Tennessee State University. 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.

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

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 ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

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, ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

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 ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

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, ...