The overall goal of this surgical intervention is to observe the effects of localized obstructive cholestasis. This method can help answer key questions in the hepatology field about how to identify mechanisms of liver regeneration. The main advantage of this technique is that meaningful comparisons can be made between the ligated experimental lobe and the unligated control lobe within the same animal.
Demonstrating the procedure today will be Dr.Toshimasa Nakao, a post-doc from the transplant surgery laboratory. After confirming a lack of response to toe pinch in an anesthetized eight to 12-week-old C57 black 6 mouse, shave the abdominal region with hair clippers and place the animal under a surgical microscope. Secure the limbs to the table and use sterile swabs to sequentially disinfect the surgical area with 0.3 to 0.5 milliliters of povidone-iodine and 70%ethanol.
Next, make a midline incision from the xiphoid to the pubis and use a micro retractor to open the abdominal cavity. Use mosquito forceps to retract the xiphoid toward the head of the mouse and use soft clay to hold the forceps and xiphoid in this position. Hydrate the abdominal cavity with one to two milliliters of sterilized 37 degrees Celsius PBS and use wet cotton swabs to gently move the small intestine toward the head of the mouse.
Cut the ligaments between the small intestine and the retroperitoneum to avoid damaging the tissue and wrap the small and large intestines with a wet non-woven gauze sponge. Place the bowel tissue on the caudal side of the abdominal cavity and use a small wet non-woven gauze sponge to gently retract the entire liver toward the xiphoid to expose the hepatic hilum and the hepatoduodenal ligament. Locate the hepatic hilum, the common bile duct on the ventral side of the portal vein, and the left and right hepatic bile ducts which drain into the main biliary confluence at the hepatic hilum.
Using a 10-0 nylon suture and the blunt side of a suture needle, ligate the left hepatic bile duct lobe. When the suture has been placed, return the small and large intestines to their anatomical location and confirm a lack of intestinal torsion or intraabdominal bleeding. Then remove the mosquito forceps, separately close the muscle and skin layers with continuous 4-0 Vicryl sutures, and place the mouse under a heat lamp with monitoring until full recovery.
Comparison of the serum liver biochemistries between partial bile duct ligated mice and complete bile duct ligated mice transgenic for insoluble liver protein aggregation reveals minimal liver injury or cholestasis in the partial bile duct ligated mice and severe injury and enzyme buildup in the complete bile duct ligated transgenic animals. As mice subjected to partial bile duct ligation developed lobe-specific obstructive cholestasis over time, the ligated lobe appears slightly paler in color 14 days after surgery compared to the unligated lobes which maintained their normal dark reddish brown color. The ligated lobe also demonstrates an increase in bile duct proliferation and fibrosis compared to the unligated lobes in response to ligation-induced localized cholestasis.
Once mastered, this technique can be completed in 10 to 12 minutes if it is performed properly. Following this procedure, other methods like lineage tracing can be used to answer additional questions about stem cell homing to sites of liver injury and regeneration. After watching this video, you should have a good understanding of how to make meaningful comparisons using partial bile duct ligation to identify local versus systemic effects.
