Experimental and clinical evidence show that liver ischemia reperfusion can increase the formation of metastatic foci by circulating cancer cells and the growth of micrometastatic disease. This reproducible murine model permits the relevant study of both the establishment of metastases and the growth of pre-existing tumor within the liver. Liver surgery, bleeding, and septic shock are all associated with liver ischemia reperfusion and can affect the growth of metastatic disease.
These techniques allow the study of this phenomena in an effort to better diagnose and prevent the detrimental effect of ischemia reperfusion on tumor growth in a clinical setting. After confirming a lack of response to toe pinch in an anesthetized eight to 12-week old male C57 black 6 mouse, disinfect the exposed abdominal wall skin with a povidone-iodine scrub and use toothed forceps to lift the skin. Using a surgical scalpel, make a midline incision in the skin and lift the abdominal muscle and peritoneum to facilitate the creation of a three-centimeter midline incision to expose the abdominal contents.
After placing a hemostat at both sides of the incision and under the xiphoid process, pull and tape the tail downward to extend the abdomen and use a six-inch sterile cotton tip applicator to separate and expose the spleen from the pancreatic fat tissue. Next, vortex the cancer cell suspension of interest to dissociate any cell clumps and load the cells into a 0.5-milliliter insulin syringe equipped with a 28-gauge needle. Slowly and carefully inject 100 microliters of cells into the tip of the spleen, placing a cotton tip adjacent to the side of the needle puncture to absorb any possible tumor spillage into the peritoneal cavity while adding gentle pressure to stabilize the spleen during injection.
A successful injection will be evident by a change in the color of the liver. When all of the cells have been delivered, place a piece of PBS-soaked sterile gauze over the dissected area and place the mouse onto a heating pad for 15 minutes to allow the cancer cells to circulate within the system. To induce an ischemia and reperfusion injury, use two moistened cotton tips to gently move the intestine from the cavity to expose the portal vein and place the mouse under a dissecting microscope.
Lift the median and left lateral liver lobes up against the diaphragm. Place a small moist cotton swab between the median lobe and the right lateral lobe to create sufficient space for clamping and carefully pass a vessel dilator forceps behind the portal triad and, using the vessel dilator forceps, use a 10-centimeter piece of 4-0 polypropylene suture to pass behind and lift the portal triad. Use a micro-serrifine clamp applicator with a lock to place a microvascular clamp onto the left and median liver lobes to occlude all of the structures in the portal triad.
When blanching is observed, remove the 4-0 polypropylene suture and small cotton swab and carefully return the intestine to the abdominal cavity. Cover the abdominal wall with a new piece of PBS-soaked gauze and cover the gauze with plastic wrap to minimize the evaporative loss. Then place the mouse back onto the heating pad for 60 minutes.
Throughout the ischemic interval, visualize the pale blanching of the right medial, left medial, and lateral lobes to confirm evidence of the ischemia injury. At the end of the injury period, remove the clamp. To perform a splenectomy, carefully lift the spleen with smooth forceps and use a handheld cautery device to cauterize the splenic blood vessels to avoid excessive bleeding.
Transect the vessels at the cauterized section to remove the spleen and immediately use 4-0 polypropylene sutures to close the muscle and skin incisions in a double-layer suture pattern. Then place the mouse back into its home cage with monitoring until full recovery. Surgical stress significantly increases the amount of pre-established micrometastases within the liver.
This difference is observed even two weeks after ischemia reperfusion compared to non-ischemia reperfused mice. The liver ischemia reperfusion and tumor cell injection can be affected by room temperature and cell viability, so take care to control these variables. This technique facilitates the study of the effects of surgical stress and liver injury on the growth of metastatic disease.
This will help researchers to better understand the phenomenon of surgery-induced tumor growth.
