Chronic pancreatitis is a complex disease involving pancreatic inflammation, fibrosis, abdominal pain, and other symptoms. The bile duct TNBS infusion model replicates the neuropathic pain observed in chronic pancreatitis patients. This bile duct TNBS infusion protocol has been optimized to induce the generation of a chronical pancreatitis model in mice.
As bile duct drug fusion in mice is technically challenging, its visualization will be helpful to researchers new to the method. Before and two and three weeks after the surgery, use Von Frey monofilaments to apply different levels of force in ascending order to the upper abdominal area of an eight to 10 week old, C57 black six mouse, 10 times for one to two seconds per application. Consider raising retraction or licking of the abdomen as a positive response.
Next, load an insulin syringe equipped with a 31 gauge needle with 50 microliters of a freshly prepared 0.75 percent TNBS solution. Load a second syringe with 50 microliters of 10 percent ethanol in saline as a vehicle control and place both syringes on ice protected from light. After confirming a lack of response to pedal reflex, remove the hair from the abdominal region of the anesthetized mouse by shaving or applying hair removal cream and place the mouse on a heated surgical pad under a dissecting microscope.
Apply veterinary ointment to each eye, and disinfect the surgical site with three sequential two percent iodine and three 70 percent ethanol wipes use micro scissors to make a 0.5 to one centimeter incision in one continuous motion and use cotton swabs to gently expose the duodenum and to locate the common bile duct. To prevent the flow of the treatment into the liver and gallbladder, use the microscope to place a straight micro hemoclip over the proximal common duct, and insert the needle of the TNBS loaded syringe through the papilla of vater into the pancreatic duct. Use a curved micro hemoclip to secure the needle, and infuse the solution into the pancreatic duct over the course of one minute.
When all of the solution has been delivered, carefully remove the micro clamps and confirm that the duodenum has returned to its original position. Before closure of the abdominal cavity, add 500 microliters of 36 to 37 degrees Celsius sterile saline to help the duodenum return to its original position and to assist with the recovery of peristalsis. Use a continuous suture with a five-O stitch to close the incision in the muscle layer, and close the skin using an interrupted suture with a four-O stitch.
Then place the mouse in a recovery cage on a heating pad with monitoring until full recumbency. Monitor mouse health and behavior daily during the first week post-surgery for signs of distress, such as vocalizing, a hunched back posture or reduce locomotion and measure the body weight every other day. As observed, the injection of 50 microliters of ink as demonstrated results in the infiltration of the entire pancreas, allowing the induction of a more homogenous disease phenotype.
In this representative analysis, in the first three days after surgery, the mice in the control group lost around six percent of their original body weight before gradually recovering and gaining over 100%of their original body weight by day 21. In contrast, mice that received TNBS lost about 15 percent of their original body weight during the first five days after surgery and regained just under 100 percent of their original body weight by day 21. Compared to control mice, the TNBS-injected animals showed an increased abdominal mechanical hypersensitivity during the second and third weeks after surgery.
The size and weight per body weight of the pancreases, were significantly reduced in TNBS mice compared to pancreases harvested from control animals, suggesting a marked pancreatic atrophy in TNBS-treated animals consistent with the findings in humans with severe and longterm chronic pancreatitis. Histological analysis of pancreatic tissue sections from control ethanol-treated mice showed a normal morphology while tissue sections from TNBS-treated mice exhibited vacuous realization with a massive loss of acinar cells that were replaced by fat cell infiltration and fibrosis. Preventing the injected TNBS from entering the gal bladder and the intestine is important as a TNBS has tissue damaging effects.
During the infusion, keep the hand as stable as possible to avoid puncture-acting that bile duct. In addition to inducing chronical pancreatitis development, this bile duct infusion mice art can be used for the development of the other pancreatic disease models. This TNBS chronic pancreatitis mouse model can be used to study both the pathogenesis and treatment of chronic pancreatitis as well as other pancreatic diseases.
