Name: murine model of intestinal ischemia-reperfusion injury
Uploaded: 2016-05-11T13:00:00
Description: Watch this Scientific Journal Video about Murine Model of Intestinal Ischemia-reperfusion Injury at JoVE.com

This work was supported by Russian Science Foundation, grant no. 14-50-00060 and LLC RUSCHEMBIO. This work was also supported by the Crohn`s and Colitis Foundation of America grant 294083 (to A.V.T.), and by NIH grant RO1 DK47700 (to C.J.).

Animal studies were performed in accordance with the National Institute of Health guidelines and were approved by the Institutional Animal Care and use Committee of the Trudeau Institute. 8-12 week old C57BL/6 mice were used for the study.
1. Preparation for Surgery
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	<li>Prepare and sterilize surgical instruments.</li>
	<li>Prepare isoflurane-based anesthesia system with nose cone, and heated pad. Make sure heated pad is not overheated (&#60;39 &#186;C).</li>
	<li>Make sure that the iso...

The development of mouse models of intestinal IR injury have greatly improved the understanding of the mechanisms of tissue injury and aided in the development of potential therapeutic strategies to minimize tissue damage 7,9,11,34. The critical steps of this protocol are proper positioning of the microvascular clips, correct timing of the ischemia and proper histologic evaluation of IR injury.
The duration of ischemia is critical for subsequent epithelial damage. The typical time r...

The intestine is very sensitive to interruption of blood flow which causes ischemia and epithelial damage. Reperfusion after ischemia provides re-oxygenation of the tissue, and can further promote pathology. Therefore, intestinal ischemia and reperfusion injury is associated with a wide range of pathologies, including necrotizing enterocolitis, allograft rejection in small bowel transplantation, complications of abdominal aortic aneurysm surgery, cardiopulmonary bypass, and inflammatory bowel disease 1,2. Intestinal IR injury, especially acute mesenteric ischemia, is a life threatening condition resulting in morbidity and mortality 3.
Although poorly understood, intestinal ischemia-reperfusion (IR) injury is thought to be associated with changes in the gut microbiota as well as the production of reactive oxygen species and inflammatory cytokines and chemokines 1,4-6. This leads to activation of both innate and adaptive immune mechanisms which promote inflammation and tissue injury 1,7,8.
Animal models are critical for understanding the mechanisms of IR injury, as they allow easy gain- and loss-of-function genetic experiments. Several animal models of IR have been developed which include complete vascular occlusion, low flow ischemia, and segmented vascular occlusion (summarized in a recent comprehensive review 9). Intestinal ischemia caused by complete vascular occlusion of superior mesenteric artery (SMA) is an easy and commonly used model of IR in large animals and rodents 9-11. However, different areas of the gut have different susceptibility to injury. In addition, the diverse range of anesthetics, analgesics, artery occlusion techniques, as well as inconsistency in the duration of ischemic injury and recovery result in variable degrees of injury confounding our understanding of the biology of IR across multiple studies. Table 1 demonstrates these inconsistencies in murine IR studies. The biggest drawback from using shorter ischemic times (30-45 min) is targeting the window of recovery upon which discernible differences between cases and controls can be observed. Mild injury to the epithelium may be resolved an hour after reperfusion, therefore specialized pathological metrics may be required to find differences in epithelial restitution. In contrast, excessive damage, as seen by 100 min of ischemic injury may result in the complete denudement of the epithelium, where restitution is no longer possible, increasing the rate of mortality, and recovery time. Therefore, here we describe the detailed procedure of intestinal IR in mice which results in reproducible injury without mortality to encourage the standardization of this technique across our field. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

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			<td style="width:379px;">Alternative: Braintree Scientific heated pad</td>
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			<td height="21" style="height:21px;">Table top research anesthesia Machine</td>
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			<td height="21" style="height:21px;">Scavenger canister and replacement cartridge</td>
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			<td height="21" style="height:21px;">Buprenorphine</td>
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			<td>Controlled substance,contact IACUC</td>
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			<td height="21" style="height:21px;">Ketaset (Ketamine HCl)</td>
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			<td>10% Povidone-Iodine</td>
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			<td height="21" style="height:21px;">Sterile saline solution</td>
			<td>Aspen</td>
			<td>46066-807-60</td>
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			<td height="21" style="height:21px;">Sterile cotton gauze pad</td>
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			<td>BIO-IRB153</td>
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			<td height="21" style="height:21px;">Micro vascular clips, 70g</td>
			<td>Roboz Surgical&#160;</td>
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			<td>Alternative: WPI 14121, for SMA occlusion</td>
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			<td height="21" style="height:21px;">Micro vascular clips, 40g</td>
			<td>Roboz Surgical&#160;</td>
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			<td height="21" style="height:21px;">Clip applying forceps</td>
			<td>World Precision Instruments</td>
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			<td height="21" style="height:21px;">Gill&#39;s 3 hematoxylin</td>
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			<td>14-390-17</td>
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			<td height="21" style="height:21px;">Surgical staples, Reflex 9 mm</td>
			<td>Cell Point Scientific</td>
			<td>201-1000</td>
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			<td height="21" style="height:21px;">Autoclip applier</td>
			<td>Beckton Dickinson</td>
			<td>427630</td>
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			<td height="21" style="height:21px;">Byopsy foam pad</td>
			<td>Simport</td>
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			<td height="21" style="height:21px;">Tissue cassette</td>
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			<td>15182701A</td>
			<td>Histosette II combination lid and base</td>
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			<td height="21" style="height:21px;">10% buffered formalin</td>
			<td>Fisher Scientific</td>
			<td>245-684</td>
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			<td height="21" style="height:21px;">Surgical iris scissors</td>
			<td>World Precision Instruments</td>
			<td>501263-G SC</td>
			<td>Alternative: Roboz RS6816</td>
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			<td height="21" style="height:21px;">Operating scissors</td>
			<td>World Precision Instruments</td>
			<td>501219-G</td>
			<td>Alternative: Roboz RS6814</td>
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			<td height="21" style="height:21px;">Dressing forceps</td>
			<td>Roboz Surgical&#160;</td>
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			<td height="20" style="height:20px;">Heparin, endotoxin free, 300 USP units/vial, 50mg</td>
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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.

We optimized the experimental protocol of IR surgery to obtain reproducible IR induced injury of the ileum in mice. Representative results are demonstrated in this section.
Figure 1 shows examples of microvascular clips position to induce ischemia of the ileum. Black arrows show position of the main clips occluding first order branches of superior mesenteric artery. Green arrows show the position of additional c...

Watch this Scientific Journal Video about Murine Model of Intestinal Ischemia-reperfusion Injury at JoVE.com

Murine Model of Intestinal Ischemia-reperfusion Injury

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, ...

The overall goal of this procedure is to perform reproducible Intestinal Ischemia Reperfusion Induced Injury in the mousedistalilium region by temporarily occluding the peripheral and terminal collateral branches of the superior mesenteric artery. This method can help to understand a basic methods in the field of gastroenterology and immunology such as understanding the mechanism of patella injury and regeneration. The advantage of this technique is that it can be done by any trained individual in a lab.Another advantage is that the procedure itself doesn't cause mortality to mice. Prepare the surgical site as specified in the text protocol. Next, use surgical scissors to make a three to five mid line centimeter laparotomy.Then cover the surgical area with a sterile non adherent pad moistened with saline. Isolate the cecum and ilium, and use saline moistened cotton swabs to expose the superior mesenteric artery. Then to facilitate the clip application use stressing forceps to gently raise the intestine and find iris scissors to cut the mesentary on both sides of the superior mesenteric artery at the desired clipped position.Holding the intestine in the raised position, gently squeeze a clip applier with the other hand to open the jaws of a high quality 70 G-Force microvascular vessel clip. Then position the clip on the vessel, and gently release the jaws, occluding the vessel. Place one to two more microvascular clips in the same way, until a five to seven centimeter region of the ischemic ilium adjacent to the cecum is created by the occlusion of the first order branches of the superior mesenteric artery.Now place two 40 G-Force microvascular clips across the intestine to block the collateral blood flow, and to demark the ischemic intestine region. When all of the clips are in place, apply a sterile delicate wipe moistened with 37 degree Celsius saline to the surgical area, and maintain the ischemia for 60 minutes with close monitoring. If the ischemia procedure has been performed correctly, the region will change to wine red in color in approximately 30 minutes, and the blood vessels distal to the clip position will become enlarged, indicating a successful occlusion.At the end of the ischemia, mark the edges of the ischemic area with ten micro liters of Gill's Hematoxylin, to facilitate the harvesting of the ischemic and adjacent healthy tissues. At the end of the ischemia, add a few drops of saline on the clip area, and gently remove the microvascular clips with a clip applier. Then, gently push the intestine back into the abdominal cavity using saline moistened cotton tips.Remove the non adherent pad, and close the abdominal wall and skin using nine millimeter stainless steal wound clips. Maintain the animals in a heated, clean cage for the desired reperfusion period, checking the mice every 30 minutes to confirm their stability. At the end of the reperfusion, euthanize the mouse, open the abdominal cavity and harvest the ischemic and adjacent healthy normal tissues.Next, use a 30 milliliter syringe equipped with a gavage needle filled with saline to wash out the intestinal contents, and then cut the intestines longitudinally. If a sample of intestine is required for gene express analysis, reserve a one point five to two millimeter longitudinal fragment. For histological analysis use a pair of forceps to roll the intestine into a Swiss roll, and place the pieces of intensive between biopsy foam pads into the cassette.Finally, place the cassettes in ten percent buffered formalin to fix the ischemic and healthy intestinal tissues. Here, a representative tissue cassette containing Swiss rolls prepared from control, and ischemic regions of the ilium after one hour of ischemia and one hour of reperfusion is shown. Note the color difference between the control and ischemic tissues.A piece of spleen was also included to facilitate the positioning of the control and ischemia reperfusion intestine during the processing and staining. In these images, representative hematoxylin and eosin staining of control, and ischemic regions of the ilium after one hour of ischemia, or one hour of ischemia followed by two hours of reperfusion, are shown. Note the severe damage of the epithelium observed after one hour of ischemia, characterized by the hemorrhagic VILI, epithelium denudement with partial to complete ablation of crips, and immune cell infiltration.After a two hour reperfusion the VILI damage and inflammation persist, but there is no tissue hemorrhage. Analysis of the inflammatory sinica expression at one and two hours after ischemia reperfusion in ischemic and control intestines demonstrates an up regulation in the TNF alpha, IL one beta, IL six sideacine, as well as an increase in Cxcl two chemokine mRNA expression under both ischemia reperfusion conditions compared to control healthy tissue. Once mastered this technique, it can be completed in three and a half hours to be performed properly.After watching this video you should have a good understanding of how to perform reproducible Intestinal Ischemia Reperfusion Injury.

murine-model-of-intestinal-ischemia-reperfusion-injury

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 ...

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 ...

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, ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

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

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

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

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

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, ...

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 ...

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 ...