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 height="21" style="height:21px;width:343px;">Heated Pad</td>
			<td style="width:191px;">Sunbeam</td>
			<td style="width:249px;">E12107-819</td>
			<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>
			<td>Vasco</td>
			<td>UCAP 0001-0000171</td>
			<td>Alternative: Parkland Scientific, V3000PS</td>
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			<td height="21" style="height:21px;">Nose Cone</td>
			<td>Parkland Scientific</td>
			<td>ARES500</td>
			<td></td>
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			<td height="21" style="height:21px;">Scavenger canister and replacement cartridge</td>
			<td>Parkland Scientific</td>
			<td>80000, 80120</td>
			<td></td>
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			<td height="21" style="height:21px;">Induction Chamber</td>
			<td>Surgivet</td>
			<td>V711802</td>
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			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>NDC 66794-013-10</td>
			<td>Controlled substance, contact IACUC</td>
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			<td height="21" style="height:21px;">Animal clipper&#160;</td>
			<td>Oster&#160;</td>
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			<td>Webster</td>
			<td>8804604</td>
			<td></td>
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			<td height="21" style="height:21px;">Buprenorphine</td>
			<td>McKesson</td>
			<td>562766</td>
			<td>Controlled substance,contact IACUC</td>
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			<td height="21" style="height:21px;">Ketaset (Ketamine HCl)</td>
			<td>Pfizer</td>
			<td>NADA 45-290</td>
			<td>Controlled substance, contact IACUC</td>
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			<td height="21" style="height:21px;">Cotton tips</td>
			<td>Puritan medical products</td>
			<td>806-WC</td>
			<td>Autoclave before use</td>
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			<td height="21" style="height:21px;">Betadine</td>
			<td>Purdue Products</td>
			<td>67618-150-17</td>
			<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>
			<td>Adjust to room temperature before use</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sterile cotton gauze pad</td>
			<td>Fisher Healthcare</td>
			<td>22-415-468</td>
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			<td height="21" style="height:21px;">Non-adherent pad prepack</td>
			<td>Telfa</td>
			<td>1238</td>
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			<td height="21" style="height:21px;">IR rodent thermometer</td>
			<td>BIOSEB</td>
			<td>BIO-IRB153</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Micro vascular clips, 70g</td>
			<td>Roboz Surgical&#160;</td>
			<td>RS5424, RS5435</td>
			<td>Alternative: WPI 14121, for SMA occlusion</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Micro vascular clips, 40g</td>
			<td>Roboz Surgical&#160;</td>
			<td>RS6472</td>
			<td>Alternative:WPI 14120, for collateral vessels occlusion</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Clip applying forceps</td>
			<td>World Precision Instruments</td>
			<td>14189</td>
			<td>Alternative: Roboz #RS-5410 or&#160; #RS-5440</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Gill&#39;s 3 hematoxylin</td>
			<td>Thermo Scientific</td>
			<td>14-390-17</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Surgical staples, Reflex 9 mm</td>
			<td>Cell Point Scientific</td>
			<td>201-1000</td>
			<td></td>
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			<td height="21" style="height:21px;">Autoclip applier</td>
			<td>Beckton Dickinson</td>
			<td>427630</td>
			<td></td>
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			<td height="21" style="height:21px;">Byopsy foam pad</td>
			<td>Simport</td>
			<td>M476-1</td>
			<td></td>
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			<td height="21" style="height:21px;">Tissue cassette</td>
			<td>Fisher Healthcare</td>
			<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>
			<td></td>
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		<tr height="21">
			<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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		<tr height="21">
			<td height="21" style="height:21px;">Operating scissors</td>
			<td>World Precision Instruments</td>
			<td>501219-G</td>
			<td>Alternative: Roboz RS6814</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dressing forceps</td>
			<td>Roboz Surgical&#160;</td>
			<td>RS-5228, RS-8122</td>
			<td>Alternative: World Precision Instruments 1519-G</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Heparin, endotoxin free, 300 USP units/vial, 50mg</td>
			<td>Sigma</td>
			<td>2106</td>
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			<td height="20" style="height:20px;width:343px;">Reflex wound clip removing forceps</td>
			<td>Roboz Surgical&#160;</td>
			<td>RS-9263</td>
			<td>Alternative: World Precision Instruments: 500347</td>
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			<td height="20" style="height:20px;">Mice C57BL/6J mice&#160;</td>
			<td>Jackson Laboratory</td>
			<td>Stock No 0664</td>
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			<td height="20" style="height:20px;">Telfa non-adherent dressings, 3x4, sterile</td>
			<td>Coviden</td>
			<td>1050</td>
			<td></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.

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