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.
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.
