Use of a Hanging-weight System for Liver Ischemia in Mice

General remarks. All operations should be performed under an upright dissecting microscope (Leica) and by using a surgical coagulator. The mice in the experimental and sham groups should be matched in age and weight to ensure comparability of the results. Temperature, blood pressure, anesthesia and fluid administration should be stable and monitored during the entire experiment.

1. Anesthesia and Surgery

1. C57BL/6 mice should be within an age range of 10 to 16 weeks and they should be gender-matched in the respective groups.
2. Anesthetize mice with sodium pentobarbital (70 mg/kg body weight i.p.) and maintain anesthesia with approx. 10 mg/kg/h sodium pentobarbital. Overdosing can significantly lower blood pressure and thus can alter the results.
3. Place mice on a temperature-controlled heated table (RT, Effenberg, Munich, Germany) with a rectal thermometer probe attached to a thermal feedback controller to maintain body temperature at 37 °C.
4. Place mice in a supine position on the surgery table, with the upper and lower extremities attached to the table with removable tape.
5. In case of survival experiments proper aseptic techniques including clipping of hair, disinfection of skin with iodophors (e.g. Betadine), sterile handling of autoclaved instruments, and using masks a sterile gloves for the surgeon are required.

2. Portal Triad Occlusion

1. Cover the incision area with mineral oil to prevent inhalation of mouse hair by cutting the skin and thus preventing the risk of mouse hair allergies. Perform midline laparotomy and incision of the linea alba up to the upper end so that you can easily expose the liver. If possible perform the incision with a coagulation electrode (Erbe, ICC50, Tuebingen, Germany) to prevent bleeding.
2. Place the heating table with the mouse in a position that the tail is directed to the surgeon. That position provides you with the best setting to localize the triad and to place the suture.
3. Displace the intestine on the right side by using a wet cotton tip swab to expose the portal triad.
4. Slightly shift the median and left lobe towards the diaphragm and the right lobe towards the intestine. That will give you a clear view of the portal triad above the bifurcation of right, median/left lobes.
5. Carefully inspect the portal triad and hold the right lobe gently down with bland forceps. That will give you a nice view on the left portal triad.
6. Once visually identified place the needle with the suture (7/0 nylon suture; Ethicon, Norderstedt, Germany) via a needle holder under the left portal triad, including the hepatic artery, hepatic vein, and bile duct from the left to the right side of the mouse.
7. The stitch has to be closely under the triad. A deeper stitch can injure the vena vaca inferior which is located under the triad. Therefore hold the right liver lobes down with the forceps to identify the vena cava before placing the stitch.
8. Take the tip of the needle on the right side with a forceps and pull it gently out of the liver area without injuring the liver.
9. Attach to each end of the suture an Eppendorf tube (approximately 3 g, filled with water) and place both suture ends over suture holders. Place the suture holder distally of the stitch so that the suture does not interfere with the right triad.
10. By applying the weights the triad will be immediately occluded, causing interruption of blood supply to the left and median lobes of the liver. Successful occlusion can be easily confirmed by visual inspection of pale blanching of the ischemic lobes. In contrast, the change of color immediately disappears when the hanging weights are released from the poles and the liver is reperfused. This partial hepatic ischemia model avoids mesenteric congestion by preserving blood flow to the right liver lobes.
11. Replace the intestine into the abdominal cavity. Keep the liver and intestine warm with a wet swab soaked with water at 37.0 °C. In addition to keep body temperature stable cover the mice with commercially available food wrap after finishing surgery.
12. Perform ischemia as required per experimental protocol. After a defined ischemia time release the weights and reperfusion of the median and left liver lobes will start immediately. This technique allows producing reversible ischemia in mice. If no further ischemia times will be performed carefully remove the suture.
13. Add approximately 400 μl sodium chloride into the abdominal cavity 15 minutes before and directly after reperfusion. Continue to substitute fluid every 30 minutes in an amount of 100 μl to compensate fluid loss during the reperfusion time.
14. For survival experiments the surgical wound is closed using continuous suture of the muscle wall and skin with a 4/0 suture.

3. Changes in Liver Perfusion

The following outcome parameters are recommended to determine liver injury following hepatic ischemia:

Determine liver enzymes (e.g. ALT, AST) and liver histology (H&E staining) following liver ischemia and reperfusion^3,9.

Figure 1. Model of a hanging-weight system for liver ischemia: A hanging weight system has been established to induce liver ischemia of the left and median lobe³. (A) Therefore the caudate and right lobe were gently separated from the left lobe. The right lobe was then slightly shifted to clearly view the portal triad above the bifurcation of right, median and left lobes. An 8.0 nylon suture was placed under the left hepatic triad. (B) At the end of each suture a small weight (3 g Eppendorf tubes filled with water) was attached and applied. Hepatic ischemia is visible by a color change of the left and median lobe from red to pale. This experimental design allows precise occlusion and reperfusion of the left hepatic triad by applying and releasing the weight load.

Figure 2. Effect of different liver ischemia times on alanine (ALT) and aspartate (AST) aminotransferase in mice. Partial portal triad ischemia was induced as indicated (0 – 60 min ischemia). After 2 hr of reperfusion, ALT (A) and AST (B) were measured. Results are expressed as means ± SE of 6 – 7 mice/group. *P < 0.05 compared with 0 min ischemia (sham control).