Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

The overall goal of this procedure is to induce experimental hepatic fibrosis in mice. This is accomplished by first performing a midline laparotomy to open the animal's abdomen. Next, the liver is lifted to expose the bile duct.

Then the bile duct is obstructed using two surgical knots. Ultimately, liver damage, hepatic fibrosis and cirrhosis are measured by monitoring the deposition of collagen through histological analysis. The main advantage of this experimental model compared to other models, such as the chronic application of carbon tetrachloride, is that this model is highly reproducible and has an overall low mortality rate.

This method helps to answer key questions in the field of liver fibrosis progression. For instance, the contribution of different immune cell infiltration or the regulation of signaling pathways in the liver. The bile duct ligation model shares many characteristics with human pathogenesis.

So we believe that this model is very valuable in translating knowledge from mouse to human disease. Though this method provides an insight into general aspect of liver fibrogenesis, it can be also applied to other animal models such as hamsters or rats. The critical part of the surgical procedure for the beginner is to separate the B duct from the franking port.Wind.

Demonstration of this missiles will be done by K and civilians, our line two technicians of our lab bodies. All experiments were approved by the official state animal care and use committee. All experiments were conducted in accordance with the German federal law regarding the protection of animals and guide for the care and use of laboratory animals to prepare for surgery.

Assemble the following sterile instruments and solutions as outlined in the list of specific materials and equipment in the text protocol. After anesthetizing a mouse, according to the text protocol, ensure that the animal shows no response to pain With an electric shaver, shave the abdominal fur and apply an eye and nose ointment to protect the eyes from drying out. Place the mouse on a 37 degrees Celsius heated hot plate.

Insert the mouse snout in the fluvac mask of a fluvac anesthesia system, and use a silk tape to fix the legs of the animal. Maintain anesthesia by inhalation of 1.5 to 3%ISO fluorine in 100%oxygen at a flow rate of one liter per minute. Finally, disinfect the surgical area.

Cover the operational area overall with fluid impermeable self-adhesive drapes. To begin surgery, use surgical scissors to perform a midline laparotomy approximately two centimeters in length by simultaneously cutting the cuts plus the fascia. Use the scissors as a spreader to dissect the connective tissue on top of the peritoneum.

Cut the peritoneum along the linear alba to open the peritoneal cavity. Then enlarge the cavity by inserting a holding suture in the sternum, raising the filament of the suture and fixing it on top of the fluvac mask. Add the perioperative analgesia containing of 0.1 milligrams per kilogram of buprenorphine in the peritoneal cavity.

Next, insert a Cree retractor into the peritoneal cavity to spread the operation area. Then use a saline moisture, cotton swab to lift the liver so that the ventral side of it sticks to the diaphragm and the hilum is clearly visible. Now to expose the bile duct, move the gut in the coddle direction, then use micro serration forceps to carefully separate the bile duct from the flanking portal vein and hepatic artery.

Place a five to zero suture around the bile duct and secure it with two surgical knots. Continuously increasing the attractive force to ensure effective obstruction without severing the bile duct. To prevent the knot from potentially leaking and the animal developing severe peritonitis, add a second cranial ligament in the same manner without dissecting the bile duct in between, cut the ends of the sutures, lower the sternum, and remove the retractor.

Use 0.9%saline to rinse the peritoneal cavity and replace the abdominal organs to their physiological positions. Use six zero merc silk running sutures to close both the peritoneum and the cutest plus fascia. Use antiseptic solution to disinfect the operation area.

After surgery, allow the mouse to recover in a cage warmed by an infrared lamp. When the mouse is fully awake and active, move it to a normal cage and provide ad liptum access to food and water bile duct ligation was carried out to investigate hepatic fibrogenesis in the initiation phase during progression and long term. As demonstrated in this table, pers sinusoidal fibrosis has already developed on day 10 after the surgery, while periportal fibrosis that permanently increased up to the end of the experiment was fully developed after 20 days, both a LT and a ST serum markers for hepatic injury rapidly increased and peaked during day seven and day 20.

After BDL thereafter, a LT and A ST decreased steadily until day 30 and remained stable until 60 days after surgery. In line with the scholastic injury, serum concentrations of total bilirubin were steadily elevated and reached a plateau after seven days. Typically, the livers of sham operated animals still look smooth at the end of the experiment, while the animals that received BDL develop edema and fibrotic nodules on the surface of the livers and hydrops of the gallbladder filled with bile.

Morphological changes are also seen through histological analysis. The development of fibrosis is highly reproducible and demonstrates a time dependent increase in intrahepatic collagen expression and deposition as a consequence of ongoing fibrogenesis, additional indicators of fibrogenesis are increased expression of alpha smooth muscle actin vimentin, and the inflammation associated marker lipin two. Once master, this procedure can be done in 10 to 50 minutes if performed properly While attempting the procedure.

It's important to remember to carry out oil procedures under clean, but non-sterile conditions Following this procedure. Other methods like the administration of carbon tetrachloride can be performed in order to identify whether the mechanisms are specific to the bile duct ligation model or generally relevant to liver fibrogenesis After its development. This methodology paved the way for many researchers working in the field of hepatology to analyze and explore the different signaling pathways evolved in hepatic fibrogenesis.

After watching this video, you should have a good understanding of how to perform product ligation in mice with high reproducibility. Don't forget that working with animals requires responsible handling and that VA of inhalation narcotics can be extremely hazardous during the performance of the procedure.