Name: confirmation of myocardial ischemia and reperfusion injury in mice using surface pad electrocardiography
Uploaded: 2016-11-24T14:00:00
Description: Watch this Scientific Journal Video about Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography at JoVE.com

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.
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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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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 ...

The overall goal of this procedure is to use electrocardiography to confirm a successful myocardial ischemia and reperfusion in mice. This method can help increase the consistency and reproducibility of the ischemia reperfusion surgery. The main advantage of this technique is that it allows the success of the ischemia and reperfusion to be assessed during the procedure, as opposed to necessitating time-consuming post-surgical analysis.After preparing the ECG pad, place a smaller than dime-sized amount of highly conductive electrolyte gel onto each of the four metallic electrodes on the ECG pad. Then press each paw of an anesthetized supine mouse to a piece of tape, and attach the tape to an ECG electrode, taking care that each paw is in contact with the gel and the electrode. Next, configure the machine so that the ECG waveform can be visualized in real time, adjusting the resolution to account for the differences in amplitude as necessary.If the peak of the R wave or the trough of the Q wave is out of the visual frame, adjust the resolution until the entire height of the waveform can be observed. Then clear the ECG pad of tools, taking care that the mouse is still and untouched and obtain a baseline waveform of the animal's normal, healthy ECG. To perform the ischemia reperfusion, begin by removing the hair around the surgical site and disinfecting the area with betadine and alcohol solutions.Next, use a scalpel to make a vertical incision parallel to the esophagus and trachea, gently moving 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. Then quickly insert the ventilation tubing into the mouth towards the throat.When the tip of the 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, as confirmed visually through the translucent tissue. When the tube is in place, use a loop of string to hook the mouse's two front teeth and tape the string ends to the ECG pad to steady the head of the animal and to ensure the ventilation tubing does not move during surgery.Then quickly attach the ventilation tubing to the rodent ventilator and adjust the ventilation settings according to the weight of the animal. Cover the exposed trachea with a gauze soaked in warm saline to keep the tissue from drying. Then use a scalpel to make a vertical incision along the left side of the sternum.Using forceps, gently separate the fascia layer from the muscle layer, followed by careful cutting of the underlying muscles layers without damaging any visible blood vessels. Use the forceps to gently pull the third rib upwards. Then, maintaining the grip on the rib with one hand, use surgical scissors to carefully cut the intercostal tissue between the third and fourth rib.When the lungs have retracted, use the forceps to gently separate the thin layer of pericardium surrounding the heart. Then move the ribs into a position where the heart is visible between the ribs. To position the heart so that the left atrial auricle is easily visible, move the mouse's lower left paw so that it overlaps the lower right paw.Locate the left anterior descending coronary artery, or LAD, beneath the left auricle, and swiftly insert a 7-0 silk tapered suturing needle into the myocardium deeply enough to pass under the LAD, but not so deeply as to penetrate the left ventricle cavity. Hold the suturing ligature until there is about four centimeters of suturing silk left on the free end, and 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, grasp both the free and needle ends of the suturing silk with forceps and insert an approximately one-centimeter section of PE10 tubing under the forming knot atop the epicardial surface.When the tubing is in place, return the lower left paw to its original electrode and tighten the knot so that the PE10 tubing is sutured to the heart. Release all physical contact with the mouse to allow the ECG waveform to stabilize for 10 seconds, visually checking the waveform and recording it as the time of occlusion. Then, if the ECG and myocardial color changes indicate ischemia, double-knot the suture around the PE10 tubing and cover the open chest cavity with warm saline gauze.At the end of the ischemic period, remove the gauze covering the chest cavity and visualize the heart. Cut the suturing silk atop the PE10 tubing and remove the tubing, followed by the gentle removal of the ligature from the myocardium. Release all physical contact with the mouse and allow the ECG waveform to cycle for 10 seconds, recording the waveform as the time of reperfusion.At the experimental endpoint, adjust the resolution for any changes in the amplitude, as necessary. If the T wave changes and the myocardium returns to red, indicating a successful reperfusion, suture the intercostal space with a 5-0 silk suture, while applying gentle pressure to the mouse's chest to expel any excess air that entered the cavity during surgery to close the chest cavity. Then suture the muscle layers and skin, allowing the mouse to fully recover in a constant temperature-controlled environment with monitoring.This figure follow the progression of one mouse from a baseline rating all the way through reperfusion, with the first waveform displaying a normal sinus rhythm at the recorded baseline. The second waveform displays the ECG one minute after the ligature was tied and the artery became occluded, with an elevated, hyperacute T wave peak observed, compared to the baseline, preligature waveform. The third waveform shows a complete ST segment elevation at the five-minute time point, with the S portion of the complex not reaching as deeply negatively as in the baseline waveform, before progressing into the J and T waves.At 20 minutes of ischemia, the QT interval has widened, and the ST segment is still elevated, with both sections remaining as such up to the 45-minute mark. After five minutes of reperfusion, evidence of deep, significant Q waves begin to appear, and the T wave returns to the isoelectric line. After 30 minutes, however, the negative Q waves still remain, and are likely indicative of both permanent damage and that the dying heart tissue is deflecting the electrical currents around the damaged area.Further, continuous ischemia and the progression to injury and infarction leads to an enhanced negative T wave projection that is usually permanent. While attempting this procedure, it's important to remember to obtain a baseline ECG before making any marks on the animal. ECG analysis can be applied to detect any electrical changes or abnormalities.The heart can be further processed for protein analysis to investigate proteomic changes or tissue staining to investigate proteomic or morphometric changes. After watching this video, you should have a good understanding of how to use electrocardiography to confirm successful myocardial ischemia and reperfusion in a mouse.

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

Research

JoVE Journal

Medicine

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, ...

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, ...

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, ...

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...