We thank Daniel Duerr for his advice regarding perfusion-fixation technique.

1. Induction of Anesthesia
<ol>
	<li>For induction with isoflurane, place the mouse into an induction box which is connected to the vapor set to 3.0 Vol% and oxygen flow of 0.5 L/min.</li>
	<li>After unconsciousness is achieved with tactile stimulus failing to induce a response and the forelimb or hindlimb pedal withdrawal reflex being absent, place the mouse on a temperature-controlled operating table in a supine position. Maintain anesthesia over a nasal cone which is connected to the vapor via the induction box. Recline the head by fixing the upper incisors with a 5-0 nylon suture to facilitate intubation.</li>
	<li>Insert the rectal temperature probe to maintain body core temperature at 37 &deg;C. Fix the extremities with tape. Cross the lower left leg over the right leg to open up the left chest and expose the heart better.</li>
	<li>Apply depilatory cream to the neck and left chest. Wipe off the cream after 1 minute. Apply povidone-iodine for local skin disinfection. Inject buprenorphine 0.05 mg/kg body weight for pain relief subcutaneously.</li>
	<li>Make a midline neck skin incision with a small scissor. Blunt dissect the glands and muscles that cover the trachea. Turn on the ventilator. Ventilator settings should be adjusted to physiological parameters. We use a Minivent, Hugo Sachs Elektronik, Harvard Apparatus with a respiratory rate of 105/min and a tidal volume of 200 &mu;l. Pull the tongue with a forceps and gently insert a 22 G metal tube. Confirm intubation by direct visualisation of the tube inside the trachea and chest movement.</li>
	<li>Bypass the induction box by switching to waste gas tubing to prevent contamination of laboratory space. Adjust vapor to 2.0 Vol%.</li>
</ol>
2. Thoracotomy
<ol>
	<li>Make a skin incision in the left midclavicular line. Blunt dissect the subcutaneous tissue towards the axilla. Identify the border of the major pectoralis muscle and blunt dissect it from the minor pectoralis muscle underneath. Pull the minor pectoralis muscle to the right. You will gain a direct view of the rib cage.</li>
	<li>Identify and bluntly pentetrate the 4th intercostal space with a forceps. Let the tips of the forceps span the intercostal space to allow yourself to insert the retractors which are adjusted with rubber bands attached to the operation table. You should have a clear view of the heart including the left auricle. This access is usually achieved without any blood loss and thus without the need of electric coagulation.</li>
</ol>
3. Preparation of the Heart
<ol>
	<li>Gently pull off the pericardium without injuring the heart.</li>
	<li>Identify the left anterior descending artery (LAD) by lifting off the left atrial auricle from the anterior wall of the left ventricle. LAD will be seen only over a short straight course with blurred borders and bright red color as compared to the veins.</li>
</ol>
4. Coronary Artery Instrumentation
<ol>
	<li>Prepare an 8-0 prolene suture with a tapered needle tip by forming it into a U-shape. Pass the needle through the myocardium deep enough underneath the LAD.</li>
	<li>Cut off the needle end of the suture to have 1 cm of suture at each side.</li>
	<li>Cut off a 1 mm PE-10 tubing section as an occluder preventing any sharp corners. Allow the occluder to soak in alcoholic desinfection and tap out before use. Note: The tubing should be soaking in 100% ethanol for 24 hours to sterilize it appropriately4.</li>
	<li>Thread both suture ends through the occluder.</li>
	<li>Use a size 3 Kalt suture needle to guide both suture ends out of the upper intercostal space.</li>
</ol>
5. Chest Closure
<ol>
	<li>Tie the upper and lower rib of the opened intercostal space together with a 6-0 prolene suture. Before you close the chest you should proceed to 5.2.</li>
	<li>Hyperinflate the lungs for a few respiratory cycles to open up atelectasis by clamping the expiratory tube. This maneuver may also depend on the type of ventilator you use. Set the tidal volume to 300 &mu;l until the chest is closed.</li>
	<li>Set the ventilator tidal volume back to 200 &mu;l.</li>
	<li>Tie both ends of the 8-0 LAD ligation suture to have them form a loop.</li>
	<li>Attach an ECG.</li>
	<li>Hook up the weights to the 8-0 loop and let the weights carefully hang. You should see a significant ST-elevation within a few heartbeats. Release the weights. Note: These experiments use a total of 5.5 g of weight, but weight could vary based on strain and body weight of mice. </li>
	<li>Place the loop in a subcutaneous pocket and close the skin with 6-0 single knot sutures.</li>
	<li>Let the mouse recover after extubation underneath a warming lamp.</li>
</ol>
6. Myocardial Ischemia and Reperfusion
<ol>
	<li>After a recovery period of at least 5 days induce anesthesia with a mixture of ketamine, xylazine and atropine (4 ml/kg BW, ketamine 10 mg/ml, xylazine 2 mg/ml, atropine 0.06 mg/ml,1.</li>
	<li>Intubate and ventilate with room air for ischemia and reperfusion experiments.</li>
	<li>Open the sutured skin of the chest. Prepare the 8-0 suture loop. Attach an ECG.</li>
	<li>Hook up the weights and let them hang. Follow your ischemia protocol. Watch ECG for potential dissolution of ST-elevation 2.</li>
	<li>Release the weights at the end of ischemia. Close the skin, extubate the mouse and let it recover.</li>
</ol>
7. Infarct Size Assessment with Reperfusion Time up to 3 Days
<ol>
	<li>Anesthetize and intubate the mouse at the end of desired reperfusion time.</li>
	<li>Cut the chest skin in the midline to the xyphoid. Open the abdomen and cut the diaphragm below the rib cage. Cut the chest open on both sides of the midclavicular line.</li>
	<li>Fix the flapped anterior chest wall with a suture to gain unobstructed access to the heart.</li>
	<li>Carefully prepare the 8-0 suture loop. Cut the loop and tie a knot to occlude the LAD.</li>
	<li>Inject 10% phthalo blue into the left atrium. To prevent cardiac volume overload, inject dye slowly and aspirate from time to time.</li>
	<li>Inject potassium chloride into the left atrium. This will arrest the heart in diastole for equal infarct size assessment.</li>
	<li>Cut out the heart, leaving as much of extracardial tissue as possible to ease cutting the heart.</li>
	<li>Wash the heart in phosphate buffer solution.</li>
	<li>Freeze the heart in isopentane and liquid nitrogen. Alternatively, hearts can be placed into a freezer until lightly frozen.</li>
	<li>Cut the heart in 1 mm slices. We have a slicing device made of razor blades to cut the heart in equal slices (Figure 4). Make sure the heart is correctly aligned to cut perpendicular to the long axis of the heart.</li>
	<li>Incubate the slices in 1.5% TTC at 37 &deg;C for 20 min. We use a 96 well plate where each slice is put into one well. This will save TTC and spare you the use of a Whatman filter to prevent artifacts.</li>
	<li>Fix the slices with 4% formaldehyde overnight. This will shrink the slices but enhances contrast of the dye.</li>
	<li>Put the slice on a microscope slide. Cover with another slide. Use 1 mm metal spacers at each end of the slide and hold the slides together with paper clips.</li>
	<li>Take a digital image from both sides of each slice. Always use the same settings and do not zoom in for smaller slices.</li>
	<li>Use a software for planimetry. We use ImageJ by NIH. Always use the same criteria for infarcted areas, e.g. only white areas are infarcted. White pinkish areas are not infarcted. We have blinded investigators for interventions and planimetry as well.</li>
</ol>
8. Alternative Heart Preparation for Histology
<ol>
	<li>Follow steps 7.1 to 7.2. and proceed to 8.2. Reliable infarct assessment with TTC staining can be done within 72 hours of reperfusion because of scar shrinkage.</li>
	<li>Prepare off any extracardial tissue and blunt dissect the thymus covering the aortic root.</li>
	<li>Grab the ascending aorta with the forceps and cut out the heart with as less extracardiac tissue as possible.</li>
	<li>Wash and squeeze the heart gently in cardioplegic solution.</li>
	<li>Place the heart into a p35 dish filled with cardioplegic solution.</li>
	<li>Prepare the ascending aorta.</li>
	<li>Cannulate the ascending aorta with a cannula that is prefilled with formaldehyde. We use zinc-formalin fixative and a 24 G iv line 3.</li>
	<li>Cut a hole between the left auricle and left atrium.</li>
	<li>Insert a 26 G catheter into the left atrium with a 16 cm long tubing attached to it. You can also use a PE50 tubing.</li>
	<li>Perfuse the heart for ten minutes with the formalin fixative.</li>
	<li>Place the heart into a tube filled with fixative for a maximum of 24 hours at 7 &deg;C.</li>
	<li>Continue with preparation for histology/immunohistochemistry.</li>
</ol>
9. Representative Results
Chronic coronary artery ligation is a complex technique with multiple pitfalls. However, once it is mastered it can be performed with very low mortality rates and highly reliable results. Optimal positioning of the mice and access to the heart are crucial for successful identification and instrumentation of the LAD. The position of the ligation will obviously influence infarct size, thus requiring to have a standardized ligation site. Also, if septal branches are affected this could lead to a bundle branch block instead of ST elevation. Bleeding from epimyocardial veins or from ventricle, if ligation is too deep, can occur and mice should be excluded if bleeding is excessive. Pericard should be removed as completely as possible. Leaving the pericard will aggravate pushing the needle into the myocard for ligation. Also, it will cause pericarditis, eventually induce adhesions and will make histological examination difficult. The PE-occluder should be as short as possible without any sharp corners to minimize trauma to myocard. Hyperinflation of the lungs is absolutely crucial to prevent tension pneumothorax after chest closure. There is no need for a chest drainage. Testing of a correct position of the ligation in the open chest by pulling the suture ends should be omitted because pulling tension is difficult to control. If instrumentation of the LAD fails, further attempts should be avoided because this will add trauma and edema to the myocardium.
In order to achieve reliable results, protocol parameters should be standardized. Therefore, mice are intubated and ventilated with room air and body temperature is tightly controlled with a feedback system. The use of hanging weights has been already emphasized. Other pulling devices have the disadvantage of tension loss and non-standardized pulling tension. Ischemic pre- and postconditioning protocols with multiple cycles of reperfusion and occlusion are more easily performed with hanging weights because they just need to be lifted and let hung (Figure 1).
Infarct areas (white) should be distinguishable from areas at risk (red) and non area at risks (blue) (Figure 2A-B). Infarct sizes are dependent on duration of ischemia. Reperfusion time should be at least 2 hours to allow successful TTC staining (Figure 1 and 2). Most importantly, cytokine RNA expression is low in sham operated animals which had all surgical procedures except ischemia and reperfusion as compared to animals which underwent myocardial infarction (Figure 3A-C).
<img alt="Figure 1" src="/files/ftp_upload/3896/3896fig1.jpg" /> 
Figure 1. Infarct size in percentage of area at risk (IS/AAR%). Mice underwent 30 minutes ischemia followed by 120 minutes of reperfusion (I/R, n=10). IPC: Ischemic postconditioning, mice underwent 30 minutes of ischemia followed by 3 cycles of reperfusion/occlusion 20 sec each (n=6, *indicates p&lt;0.05).
<img alt="Figure 2A" src="/files/ftp_upload/3896/3896fig2A.jpg" /> 
Figure 2A. Representative TTC-stained heart slice. White: infarct area, Red: area at risk, Blue: non-occluded area.
<img alt="Figure 2B" src="/files/ftp_upload/3896/3896fig2B.jpg" /> 
Figure 2B. Representative slice of an infarcted (white) area. Note that due to the conical shape of the left ventricle close to the apex, the epimyocard will appear as plane area and should not be considered for planimetric measurement (pink/blue outer area). Red=TTC stained viable myocard.
<img alt="Figure 3A" src="/files/ftp_upload/3896/3896fig3A.jpg" /> 
Figure 3A. No significant difference in myocardial TNF-&alpha; mRNA expression after 30 minutes ischemia and 120 minutes reperfusion. n=4-6 per group.
<img alt="Figure 3B" src="/files/ftp_upload/3896/3896fig3B.jpg" /> 
Figure 3B. Myocardial IL-1&beta; mRNA expression after 30 minutes ischemia and 120 minutes reperfusion (I/R). There is no significant difference between control (no surgery) and sham-operated (no ischemia/reperfusion) group. n=4-6 per group, *indicates p&lt;0.05.
<img alt="Figure 3C" src="/files/ftp_upload/3896/3896fig3C.jpg" /> 
Figure 3C. Myocardial IL-6 mRNA expression after 30 minutes ischemia and 120 minutes reperfusion (I/R). There is no significant difference between control (no surgery) and sham-operated (no ischemia/reperfusion) group. n=4-6 per group, *indicates p&lt;0.05.
<img alt="Figure 4" src="/files/ftp_upload/3896/3896fig4.jpg" /> 
Figure 4A-C. Heart cutting device. A: closed with razor blades in cutting position. B: open, side view. C: open, front view. Heart will be aligned in the groove of the white plastic bed with its long axis perpendicular to the razor blades (arrow).

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No conflicts of interest declared.

We have modified a murine closed-chest model by accessing the heart through a left lateral intercostal thoracotomy and leading out the LAD sutures to the chest in the left midclavicular line. Leaving the bony rib cage intact will minimize trauma, need for pain medication, surgical site infection and thus, facilitate recovery. By preserving the left internal mammal artery there is no need for electrocautery. We leave the suture loop in the subcutaneous tissue for later easy access and use a hanging weight system fo...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Vapor</td>
 <td>Dr&auml;gerwerk AG</td>
 <td>Isoflo</td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td>Leica</td>
 <td>M80</td>
 </tr>
 <tr>
 <td>Light source</td>
 <td>Schott</td>
 <td>KL 1500 LCD</td>
 </tr>
 <tr>
 <td>Homeothermic Blanket Control Unit</td>
 <td>Harvard Apparatus</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MiniVent Type 845</td>
 <td>Hugo Sachs Elektronik</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>8-0 Prolene</td>
 <td>Ethicon</td>
 <td>BV130-5 6.5mm 3/8c</td>
 </tr>
 <tr>
 <td>6-0 Prolene</td>
 <td>Ethicon</td>
 <td>BV-1 9.3 mm 3/8c</td>
 </tr>
 <tr>
 <td>Kalt suture needle size 3</td>
 <td>FST</td>
 <td>12050-03</td>
 </tr>
 <tr>
 <td>Triphenyltetrazolium</td>
 <td>Sigma Aldrich</td>
 <td>93145</td>
 </tr>
 <tr>
 <td>Phthalo blue</td>
 <td>Heucotech LTD</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PowerLab</td>
 <td>ADInstruments</td>
 <td>&nbsp;</td>
 </tr>
</tbody> 
</table>

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.

Watch this Scientific Journal Video about A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion at JoVE.com

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

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 ...

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28.6K Views.  University of Bonn, Germany. 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.

Video: A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

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 ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

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 ...

A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes &#8211; but is not limited to &#8211; hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.

This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic ...

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, ...

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

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

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

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

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

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 ...

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, ...