2.1. Animals

• The model should be performed on 50 male Wistar rats weighing an average of 200 g and aged between 8 and 10 weeks.
• One week before the experiment, the animals must be housed for acclimatization at a temperature of 22 ± 2 °C, humidity 55 ± 10% and light/dark cycles for 12 h.
• They must have free access to water and food pellets.

2.2. Anesthetic protocol

• Animals should be premedicated by intramuscular injection of tiletamine/zolazepam (Zoletil) at a dose of 20 mg/kg.
• They must then be relocated to the original house, maintain a quiet environment with soft light until the animals reach a state of deep sedation.
• The achievement of a deep anesthesia level must be maintained by gaseous anesthesia: the rat, under sedation, must be placed on a heated carpet and a mask must be applied to the animal's muzzle for the administration of a mixture of oxygen, air and 2% isoflurane.
• During anesthesia, the heart rate and body temperature must be monitored by special equipment (in our case: Indus Instruments-Visualsonic), the maintenance of anesthesia must be followed by the veterinarian through the evaluation of clinical parameters such as increased breathing and heart rate.
• At the end of the procedure, recovery should be obtained by interrupting the administration of isoflurane.
• The animal must then be placed in a special heated room with soft light where it will be followed by the veterinarian until it is fully awakened.
• The anesthetic procedures for animals to be subjected to bile duct ligation must be integrated with analgesic therapy by administering Carprofen (Non-Steroidal Anti-Inflammatory Drug) 5 mg/kg, performed immediately after the end of the operation and every 24 hours for two days, and antibiotic therapy with the administration of Enrofloxacin at a dose of 5 mg/kg diluted in 0.5 ml of physiological solution every 24 hours for 5 days.

2.3. Euthanasia

2.4. Ligature of the Common Bile Duct

• Bile duct ligation surgeries are performed under deep anesthesia.
• Shave the abdominal wall of the animals and disinfect the skin with chlorhexidine solution (0.5%).
• Make a midline abdominal incision (approximately 5 cm long) to expose the xiphoid process.
• Retract the severed abdominal wall bilaterally (laparotomy) using double-curved forceps to hold the peritoneal cavity open and expose the liver.
• Using a saline-moistened cotton tip, gently pull down on the middle lobe of the liver and cut the falciform ligament using curved microsurgical scissors.
• Pull the duodenum to the left with a saline-moistened cotton tip to visualize the hepatic hilum.
• Using the microdissection forceps, place a 5-0 silk thread around the common bile duct (Figure 4).
• Make a second ligature at a distance of about 1 cm (Figure 5).
• Use the microsurgical curved scissors to cut the common bile duct between the two ligatures (Figure 6).
• Carefully reposition the middle and left lateral lobes to their anatomical position using moistened cotton swabs.
• Gently return the intestinal loops to the abdominal cavity.
• Close the muscle layer of the abdominal wall using a running suture with a 3-0 polyglycatin suture and needle holder.
• Close the skin incision using a running stitch with a 4-0 silk suture.
• After closing the abdominal cavity, clean the skin around the suture with chlorhexidine.
• Administer 1 ml of saline (0.9%) subcutaneously.
• Place the animal in a heated recovery chamber for 15 minutes.

2.5. Blood Sampling

• Anesthetize the animals and place them in sternal recumbency.
• Place a pre-heparinized 24G peripheral venous catheter into the lateral tail vein. This procedure is facilitated by a tourniquet at the base of the tail.
• Collect a 600 μl blood sample in a tube with a separating gel.
• Then centrifuge the sample at 2700 rpm for 15 minutes at 4°C.
• Aliquot the collected plasma in 2 ml Eppendorf and use it for the determination of GOT, GPT and total bilirubin.

2.6. Microdialysis

• Anesthetize the animals and place them on the operating table in dorsal recumbency.
• Proceed with trichotomy of the abdominal region with a special clipper, then disinfect the skin with surgical betadine.
• Make an incision along the linea alba proceeding in the craniocaudal direction for a length of approximately 2 cm, involving the skin, subcutaneous tissue, muscle and peritoneum.
• Once in the abdominal cavity, use a special abdominal retractor for proper visualization of the cavity bodies.
• Then proceed to remove the intestine from the liver parenchyma.
• Into the left and right lobes of the liver insert two CMA20 Elite microdialysis probes (CMA / Microdialysis AB, Stockholm, Sweden) and perfuse them at a rate of 1.0 mL/min with T1 perfusion fluid (147 mM Na, K 4 mM, 2, 3 mm Ca, 156 mM Cl, pH 6, osmolality 290 mOsm/kg) (CMA/Microdialysis AB, Stockholm, Sweden) via a microdialysis pump (CMA/Microdialysis AB). (Figure 7)
• Subsequently secure the probes to the muscular component of the abdominal wall using a 3/0 Vicryl suture (Figure 8).
• Collect two samples at 30 minute intervals.
• Use the Iscus CMA analyzer microdialysis for glucose, glycerol, lactate and pyruvate analysis.
• Finally, restore the planes by surgical suturing from the muscle and skin using a 3/0 vicryl suture.

Critical steps in the Protocol

Modifications and Troubleshooting of the Method

The Significance of the Method with Respect to Existing/Alternative Methods

Future Applications or Directions of the Method