Begin by wearing a mask, hair cover, and clean lab coat or scrubs. Next, place the anesthetized mouse on a clean surface separated from the surgery area and fit it with a nose cone delivering 1.5%isoflurane with 21%oxygen. Then inject buprenorphine subcutaneously into the dorsal cervical thoracic area.
Also inject heparin intraperitoneally to prevent thrombus formation during the period of occlusion. Apply ophthalmic ointment to the eyes to prevent corneal damage and remove hair from the ventral abdomen using clippers. Move the mouse to the heated water blanket in the surgery area.
Again, fit it with a nose cone delivering isoflurane. Next, position the mouse in dorsal recumbency and secure the limbs to the table with surgical tape. Monitor the body temperature of the animal using a rodent-specific rectal thermometer.
After thoroughly disinfecting the abdomen, perform a toe pinch test to ensure the animal is fully anesthetized. Finally, wear sterile gloves and aseptically drape the surgical site. First, make a three to five-centimeter ventral midline abdominal incision in the skin using a number 15 scalpel blade.
Separate 0.5 centimeters of the skin edge from the underlying muscle fascia to allow for staple placement later. Then continue the incision through the abdominal wall along the linear alba using spring-loaded micro scissors and place a retractor. Place sterile gauze pads moistened with warm sterile saline around the operating area.
Remove the small intestine from the abdominal cavity. Flip it cranially and to the animal's left, and place it on the moistened pads. Place another moistened gauze pad over the tissues to prevent desiccation.
Locate the superior mesenteric artery, or SMA, ventral to the inferior vena cava caudal to the celiac artery and cranial to the renal artery. Dissect it away from the surrounding connective tissue. Place an atraumatic microvascular clip along the base of the SMA where it branches off the abdominal aorta.
Verify ischemia of the small intestine by noting the color change from pink to pale white. Return the viscera to its original position inside the abdominal cavity for the duration of the ischemic period. Remove the retractor and cover the incision with moist gauze.
After a 45-minute period of ischemia, remove the occluding clip. Verify the restoration of blood flow by observing a mesenteric pulsation and flushed color. Apply warm sterile saline intraperitoneally to maintain appropriate hydration.
Then close the abdominal muscles with a 6-0 polyglactin 910 suture. Administer bupivacaine topically along the muscle incision line for pain relief. Close the skin with surgical staples or wound clips.
Return the mouse to a warm chamber on a circulating water blanket. Let the mouse recover for 90 minutes and monitor the mouse for signs of pain or distress. After performing humane euthanasia, collect the desired tissues such as the liver lobes, including the left lateral, left median, and right median lobes.
Then collect both kidneys. Cut the left kidney longitudinally and the right kidney as a cross-section. Finally, collect the entire length of the small and large intestine.
To divide the small intestinal segments into sections of equal length, fold the small intestines into a Z-shape where the top line is the duodenum, the middle line is the jejunum, and the bottom line is the ileum. The remaining part of the intestine is the colon. Flush the lumen of the intestinal segments with saline using a 10-milliliter syringe affixed with a 20-gauge angiocatheter.
Next, cut down the length of the intestines using micro dissecting scissors and lay each intestinal segment flat with the luminal side facing up. Using a three-milliliter syringe affixed with a 27-gauge needle, generously apply 10%buffered formalin dropwise to coat the entire length of the mucosa. Then roll each segment circumferentially around a toothpick, ensuring that the proximal portion forms the inner part of the roll and the lumen faces the inside.
After rolling, the intestine should look like a Swiss roll. Place the individual Swiss roll's spiral face up inside the separate labeled tissue cassettes. Finally, place the tissues in labeled vials filled with 10%buffered formalin to fix them at room temperature.
After intestinal IRI, H&E staining of the jejunum and ileum from mice in the sham group showed long and thin villi without distortion. Whereas sections from the mice in the IRI group displayed areas of necrosis and hemorrhage with blunting and distortion of the remaining villi. Further, microscopic damage to all three small intestinal segments for animals undergoing IRI was significantly increased compared to those that underwent sham laparotomy.
