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>
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			<td>80000, 80120</td>
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			<td>V711802</td>
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			<td>NDC 66794-013-10</td>
			<td>Controlled substance, contact IACUC</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>
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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>
			<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>1238</td>
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			<td height="21" style="height:21px;">IR rodent thermometer</td>
			<td>BIOSEB</td>
			<td>BIO-IRB153</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>
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			<td>RS6472</td>
			<td>Alternative:WPI 14120, for collateral vessels occlusion</td>
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			<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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			<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>
			<td>M476-1</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>
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			<td height="21" style="height:21px;">Surgical iris scissors</td>
			<td>World Precision Instruments</td>
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			<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>
			<td>RS-5228, RS-8122</td>
			<td>Alternative: World Precision Instruments 1519-G</td>
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			<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, ...

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