Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, part of the National Institutes of Health, under Award Number R01-AR063084. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This animal protocol was approved by and is in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee (IACUC) at The Ohio State University. All policies developed by the local IACUC are in compliance with the Animal Experimentation Guide developed by Office of Laboratory Animal Welfare at the National Institutes of Health.
1. Anesthesia and Endotracheal Intubation
<ol>
	<li>Autoclave all instruments and surgical supplies before use. Wear sterile, single use surgical gloves throughout the procedure. Maintain a sterile field throughout the procedure.&#160;Use of a sterile drape is suggested but not shown in the video to allow for better visualization of anatomical landmarks on the mouse.</li>
	<li>Place each mouse individually in an induction chamber and provide anesthesia using 5% isoflurane and oxygen with a flow rate of 0.4 L/min until loss of righting reflex and then maintain the animal with 2% isoflurane in 100% oxygen with a flow of 0.4 L/min by means of a nosecone tube connected to the anesthesia apparatus until the tracheal tube is in place. The isoflurane anesthesia machine used should be appropriately vented and equipped with charcoal filters to minimize exposure of the surgeon to isoflurane fumes during the procedure.&#160;The nosecone is noted but not shown in the video to allow for visualization of the manipulations to intubate the mouse.</li>
	<li>Shave the animal&#8217;s chest with an animal hair clipper in a different location than the surgery platform to avoid contamination of the surgery location.</li>
	<li>Place the mouse in a supine position on surgery platform for subsequent intubation. A simple small polystyrene foam platform can serve as an operating platform. Cover the platform with a pre-sterilized drape to provide a sterile surface. Place a heating pad between the platform and drape to maintain the body temperature of the mice in surgical procedures.</li>
	<li>Attach a length of 2&#8211;0 silk suture of at least 10 cm to the platform with tape and then loop the suture around the front upper incisors. Position the cone in close proximity (2-3 cm) to the edge of the platform over the nose of the mouse. Pull the mouse taut and secure it to the platform by the tail with a piece of tape.</li>
	<li>Secure the legs to the sides of the body with strands of tape. It is important that the front limbs are not over-stretched as this can compromise respiration.</li>
	<li>Prepare the shaved surgical sites with Betadine and alcohol before the neck and chest incisions are made.</li>
	<li>Place the platform with the mouse head pointing in the direction of the operator. Cut a 0.5 cm median cervical skin incision. Separate the lobes of the thyroid gland at their isthmus to expose the sternohyoideus muscle where the trachea can be seen under the muscle.</li>
	<li>Remove the inner needle of an 18 gauge trocar so it can be used as an intubation tube. The needle point can serve as a holder and 1 cm of the outer tube can serve as the tracheal tube.</li>
	<li>Hold the tongue of the mouse with curved forceps in one hand and move it slightly upwards. View the trachea through the cervical skin incision. Use the other hand to gently insert the intubation tube until the tube is seen inside the trachea.</li>
	<li>As soon as the tube is in the tracheal, move the curved forceps in other hand towards the tube and quickly remove the inner needle. If the tube cannot be inserted into the tracheal, the tube should be pulled out to avoid producing respiratory problems. It is important to point the tip of the tube up when it is close to the throat in order to avoid inserting the tube into the esophagus instead of the trachea.</li>
</ol>
2. Ventilation and Fixation
<ol>
	<li>Provide artificial ventilation with an animal respirator venting 2% isoflurane in oxygen with a flow rate of 0.4 L/min. Use a modified Y-shape connector to connect the intubation tube with the ventilator. The correct positioning of the tracheal tube can be confirmed by judging the symmetrical chest expansion.</li>
	<li>Set the tidal volume at 260 &#956;l/stroke and ventilation rate is 130 strokes per minute, which can be adjusted to the body weight of a particular mouse if necessary.</li>
	<li>Remove the tape on the tail and turn the mouse gently to place it in a right lateral decubitus position for the subsequent surgery. Use tape to secure the tail and legs to the platform again.</li>
	<li>Insert the rectal probe to monitor the body temperature and adjust the warming pad to maintain the temperature around 37 &#186;C.</li>
	<li>Secure the probe to the platform using tape. Inject bupivacaine subcutaneously at the incision site to numb the area before the incision is made.</li>
</ol>
3. Thoracotomy
<ol>
	<li>Make an oblique incision that is approximately 1 cm long at a site 2 mm away from the left sternal border in the direction of where the left front leg meets the body (approximately 1-2 mm below where the leg and body join). The superficial thoracic vein is near this site and the incision should be made so that the lateral end of the incision goes up to, but does not cut into, the vein.</li>
	<li>Cut though the thoracic muscle to expose the ribs underneath. During this step avoid accidental injury of the vessel. If bleeding does occur, use cotton applicators to stop any bleeding before proceeding to the next step7.</li>
	<li>Visualize the ribs and inflating lung through the thin and semitransparent chest wall. Open the chest cavity using surgical scissors to make a 6-8 mm incision in the third intercostal space. This incision should be a minimum of 2 mm from the sternal border where the internal thoracic artery is located. Damage to the artery will produce heavy bleeding that is difficult to control.</li>
	<li>Insert the pre-sterilized homemade chest retractors into the incision and gently pull back to open the incision so that it is about 8-10 mm wide while being careful to avoid the lung.&#160;The retractors should be attached to the surgical platform with pins.&#160;</li>
	<li>At this point the heart should be visible, however, the lung will still cover a portion of the heart. Pick up the pericardium gently with curved forceps, pull it apart, and slide the tissue behind the retractors. During this manipulation the lung will lift up and away from the heart.</li>
</ol>
4. Positioning LAD
<ol>
	<li>Locate the LAD on the surface of the heart through a dissection microscope. The LAD runs down the middle of the heart wall from near the apex of the heart down through the left ventricle. The LAD appears bright red and will be pulsing strongly. The vein here is sometimes mistaken for the LAD, however proper lighting can help distinguish the two vessels. If the lighting is too bright it can be difficult to appreciate the color differences between the vessels.</li>
	<li>Use a sterile cotton ball fragment with a diameter of approximately 1-2 mm to prepare the LAD for ligation. Place the cotton between the left atrium and left ventricular, which will lift the left atrium and help expose the LAD and clarify its position. If the LAD cannot be located, the fragment can be slid further in so the left atrium is lifted even higher to reveal the aorta where the LAD originates.</li>
</ol>
5. LAD Ligation
<ol>
	<li>The ideal positioning for the ligature is approximately 2 mm lower than the tip of the left auricle. The pulmonary trunk can be used as a marker to help identify the left auricle. Alternatively, the ligation position can be visualized as a point 1-2 mm away from the branching of the left circumflex. Use curved forceps to gently apply pressure at a site immediately below the intended ligation point. This will make it easier to see the artery and will also help hold the heart in place and simplify tying the ligature. Do not apply pressure with the forceps for more than 5 seconds at a time and avoid compression of the heart that might alter pumping.</li>
	<li>Use a tapered needle to pass a 6&#8211;0 silk suture underneath the LAD while observing with a dissecting microscope. Insert the needle under the artery with precision as the needle will enter the left ventricle chamber if placed too deeply or damage the LAD if the needle is too shallow. If the LAD is injured remove the needle and suture the LAD to control bleeding, however if bleeding cannot be controlled it is preferable to euthanize the&#160;animal.</li>
	<li>Make a loose double knot with the suture, leaving a 2-3 mm diameter loop through which a 2-3 mm long piece of PE-10 tubing is placed8.</li>
	<li>Tighten the loop around the artery and tubing then secure the loop by tying one additional slipknot, taking care not to damage the ventricle wall. For permanent ligation, directly tie the LAD with a knot9. Confirm the occlusion of LAD by checking for appearance of a paler color in the anterior wall of the LV that should appear within a few seconds after ligation.</li>
	<li>Remove the retractor and close the wound temporarily by pinching the skin together with a bulldog clamp. The length of time that ischemia is maintained depends on the experiment design, but is frequently 20, 30, 45 or 60 min. The mouse remains on the ventilator for the duration of the LAD artery occlusion.</li>
</ol>
6. Reperfusion
<ol>
	<li>After the ischemia period remove the bulldog clip and insert the chest retractors to expose the ligature. Untie the knot and remove the PE-10 tubing. Confirm reperfusion by observing a return of the pink-red color of the anterior wall of the LV after 15-20 sec.</li>
	<li>Leave the suture in place if 2% triphenyl tetrazolium chloride (TTC) and blue staining will be performed after reperfusion. If staining is not necessary, the suture can be removed.</li>
	<li>The reperfusion time will depend on the experiment design, usually spanning from 1 hr to 24 hr.</li>
</ol>
7. Chest Closure and Postoperative Care
<ol>
	<li>Close the chest cavity by sewing shut the incision in the 3rd intercostal space with 4&#8211;0 silk suture. It is important that the lungs are clear of the suture and do not become trapped as the 3rd and 4th ribs are sutured together. While tying the suture knots it is helpful to apply slight pressure to the chest with the needle holder to minimize any room air that might be trapped in the chest cavity.</li>
	<li>Close all layers of muscle with continuous sutures using 4&#8211;0 silk. Use nylon sutures to close the skin with a continuous suture. Alternatively, the skin can be closed with interrupted suture.</li>
	<li>When suturing is complete cease the flow of isoflurane while oxygen continues to flow. Once the mouse moves its whiskers or tail it, should start making attempts to breathe spontaneously. Remove the mouse from the ventilator with the intubation tube still kept in the trachea.</li>
	<li>Observe the animal carefully until the mouse resumes a normal breathing pattern and then extubate the mouse. The tube should be removed slowly to avoid aspiration of oral cavity secretions.</li>
	<li>Confirm the mouse is not in any respiratory distress by observing it for another 3-5 min before returning it to a cage. If signs of dehydration are observed after surgery, provide up to 0.5 ml of sterile saline by intraperitoneal injection.</li>
	<li>For post-operative analgesia, administer an opioid analgesic (buprenorphine, 0.1 mg/kg) subcutaneously (SC) before&#160;the animal is ambulatory and then provide an additional dose every 4-6 hr for the next 24 hr. Check the animal signs of distress at 12 hr after surgery. Simulation of myocardial infarct using survival surgery requires assessment of pain and distress following recovery from the surgery. The current accepted best practice is to provide analgesia for the first 24 hr following an invasive procedure with additional doses given as warranted due to weight loss or signs of pain. For permanent ligation, body weight should be tracked daily to help to gauge the animal&#8217;s recovery.</li>
	<li>Ibuprofen (Motrin), a nonsteroidal anti-inflammatory drug (NSAID) with anti-inflammatory, analgesia and antipyretic activity, or other NSAIDs, may be provided in the animal&#39;s drinking water as a 0.2 mg/ml solution for two days before the surgery and up to a 7 days after surgery in along with the&#160;buprenorphine&#160; to manage any additional pain/distress.</li>
</ol>
8. Measurement of Myocardial Infarct Size
<ol>
	<li>Anesthetize and intubate the mouse at the end of desired reperfusion time. Cut the chest skin in the midline to the xyphoid. Open the abdomen and the diaphragm below the rib cage and from both sides of the midclavicular line.</li>
	<li>Expose the heart and then re-ligate the LAD in the same location. Cannulate the aorta so 10% Phthalo Blue can be slowly injected directly into the aorta to stain the heart for delineation of the ischemic zone from the nonischemic zone10.</li>
	<li>Rapidly excise the heart and wash it in 30 mM KCl (potassium chloride solution) to cease the beating of the heart and allow for more consistent sectioning. Freeze the heart for at least 4 hr at -20 &#176;C and cut the heart into slices of 1 mm using a heart matrix sectioning device11.</li>
	<li>Incubate heart slices with 2% TTC at 37 &#176;C for 40 min. The infarct area is demarcated as a white area while viable tissue stains red.</li>
	<li>Fix the stained slices with 10% formaldehyde overnight, which will help to increase the contrast between the infarct area and the normal tissue. Photograph the slices and calculate the area at risk (AAR), the nonischemic zone and the infarct area using ImageJ software.</li>
</ol>
9. Measurement of Cardiac Enzyme Levels
Measure cardiac troponin I (cTnI) levels in the serum of mice by obtaining blood from the portal vein and then isolating serum by centrifugation. Serum cTnI levels are then determined with a quantitative rapid cTnI assay12.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+artery+ligation+and+intramyocardial+injection+in+a+murine+model+of+infarction.">Coronary artery ligation and intramyocardial injection in a murine model of infarction.</a> J Vis Exp. , (2011).</li><li>Salto-Tellez, 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=Myocardial+infarction+in+the+C57BL/6J+mouse:+a+quantifiable+and+highly+reproducible+experimental+model.">Myocardial infarction in the C57BL/6J mouse: a quantifiable and highly reproducible experimental model.</a> Cardiovasc Pathol. 13, 91-97 (2004).</li><li>Kumar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+mouse+coronary+anatomy+and+myocardial+infarction+consequent+to+ligation.">Distinct mouse coronary anatomy and myocardial infarction consequent to ligation.</a> Coron Artery Dis. 16, 41-44 (2005).</li><li>Degabriele, N. 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Dr. Noah Weisleder is a Founder and Chief Scientific Officer at TRIM-edicine, Inc.

Mouse myocardial ischemia-reperfusion models are an effective method for cardiovascular research to simulate clinical acute or chronic heart disease13,14. Significant effort has been applied to develop and refine surgical approaches that produce ischemic events and reperfusion damage in the hearts of several different animal types. While there are particular advantages to the use of different animals systems, the mouse has characteristics that have led to extensive interest in producing myocardial I/R in the m...

Animal models of myocardial infarction (MI) are important in research of the complex pathophysiology of ischemic heart disease1. Ischemia-reperfusion (I/R) injury is a major contributor the myocardial damage generated during MI. The initial ischemia injury produced by occlusion of the coronary circulation can be minimized in MI patients by the use of angioplasty to restore perfusion in a timely fashion. While this intervention has greatly reduced the number of deaths due to acute MI, restoration of blood flow into the ischemic area results in I/R injury that leads to death of cardiomyocytes. This loss of myocardial mass contributes to decreased cardiac output and progression towards heart failure. Thus, study of the mechanisms that result in cardiomyocyte death from I/R injury is an important line of inquiry in cardiovascular research. Surgical coronary ligation is a useful experimental technique to induce models of MI in various animal types, including the rat, dog and pig. Publications in different laboratories have introduced various methods on the establishment of the mice heart model of I/R injury2,3. In order to gain insight into these mechanisms we must have access to reliable animal models that can reproduce several aspects of MI pathology. Development of such models is also essential for testing therapeutic approaches for treatment of MI and associated I/R injury.
Most of the currently available surgical techniques to simulate MI in experimental animals involve surgical dissection into the chest cavity to expose the left anterior descending artery (LAD) that is then occluded by a ligature for defined period in time to produce the ischemic event. Then that ligature can be removed to allow for reperfusion of the ischemic area and generation of I/R injury. One major limitation of these approaches in that the position of the literature on the LAD is not always accurately reproduced, which can lead to variation in the severity of the MI induced by this approach. Most available techniques only generally described the approximate location of the LAD in the anterior wall of the heart. As the branching and direction of the LAD can vary in individual animals the location is not always fixed and can be easily confused4,5, leading to potential complications during surgery6. The consequences of improper placement of the ligature can run from variability in the size of the infarct induced in the left ventricle to completely compromising the specificity of the model. Here we present a modified method for myocardial I/R and permanent ligation in mice that allows for improved accuracy of placement of the ligature on the LAD. By applying specific approaches for the initial incision and internal dissection, as well as the use of manipulations to lift the atria to allow better appreciation of the LAD and the site where it emerges from the aorta. Establishing the position on the LAD and its origin provides the opportunity to ligate the LAD in a reproducible fashion. This model of myocardial I/R and permanent ligation not only decreases the variation in infarct size following surgery, it can also decrease the incidence of excessive bleeding during the operation.

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			<td height="19" style="height:19px;width:347px;">PhysioSuite with RightTemp Homeothermic Warming</td>
			<td style="width:192px;">Kent Scientific Corp</td>
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			<td height="21" style="height:21px;width:347px;">Mouse Heart Slicer Matrix</td>
			<td style="width:192px;">Zivic Miller</td>
			<td style="width:139px;">HSMS001-1</td>
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			<td height="21" style="height:21px;width:347px;">Micro Tray - Base, Lid, &#38; Mat (6.0 x 10 x 0.75)</td>
			<td style="width:192px;">Fine Science Tools</td>
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			<td height="21" style="height:21px;width:347px;">2,3,5-Triphenyltetrazolium chloride</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:139px;">T8877</td>
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			<td height="21" style="height:21px;">Buprenorphine (Buprenex Injectable)</td>
			<td>Reckitt Benkiser Healthcare</td>
			<td>NDC 12496-0757-1</td>
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			<td height="21" style="height:21px;">bupivacaine</td>
			<td style="width:192px;">Hospira</td>
			<td>NDC 0409-1163-01</td>
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			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Abbott</td>
			<td>NDC 5260-04-05</td>
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			<td height="21" style="height:21px;">Betadine Soultion&#160;</td>
			<td>Purdue Pharma</td>
			<td>25655-41-8</td>
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			<td height="21" style="height:21px;">Mouse Cardiac Troponin T(cTnT) ELISA</td>
			<td>Kamiya Biomedical Company</td>
			<td>KT-58997</td>
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			<td height="21" style="height:21px;width:347px;">Dumont #5 Forceps</td>
			<td style="width:192px;">Fine Science Tools</td>
			<td style="width:139px;">11251-30</td>
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			<td height="21" style="height:21px;width:347px;">Dumont #3 Forceps</td>
			<td style="width:192px;">Fine Science Tools</td>
			<td style="width:139px;">11231-30</td>
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			<td height="21" style="height:21px;width:347px;">Castroviejo Micro Needle Holders</td>
			<td style="width:192px;">Fine Science Tools</td>
			<td style="width:139px;">12060-01</td>
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			<td height="21" style="height:21px;width:347px;">Slim Elongated Needle Holder</td>
			<td style="width:192px;">Fine Science Tools</td>
			<td style="width:139px;">12005-15</td>
			<td></td>
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			<td height="21" style="height:21px;width:347px;">Round Handled Needle Holders</td>
			<td style="width:192px;">Fine Science Tools</td>
			<td style="width:139px;">12075-12</td>
			<td></td>
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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.

Following 24 hours of reperfusion, analysis of infarct size and the area-at-risk (AAR), by phthalo blue dye and triphenyl tetrazolium chloride (TTC), ligation of the LAD can be confirmed by observing blanching of myocardial tissue distal to the suture as well as dysfunction of the anterior wall. Reperfusion can be verified by the return of red color to the myocardial tissue and the demonstration of some recovery of anterior wall motion.
Infarct areas (white) should be distinguishable from area...

Watch this Scientific Journal Video about A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery at JoVE.com

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

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 ...

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Research

JoVE Journal

Medicine

39.1K Views.  The Ohio State University. 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.

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

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 ...

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

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

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

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

Murine Model of Intestinal Ischemia-reperfusion Injury

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

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

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

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

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

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

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

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

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

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

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

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

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 ...

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 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 ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

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

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

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

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