Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

The overall goal of this procedure is to induce severe injury in the rodent adult pancreas to establish an environment that allows beta cell neogenesis and proliferation. This is accomplished by first performing a midline laparotomy to allow exposure of the pancreas. The main pancreatic duct in the neck region of the pancreas is then ligated using two surgical knots.

In the final step, the partial duct ligation or PDL pancreas is harvested. Ultimately, immunohistochemical analysis and Q-R-T-P-C-R are used to study the factors involved in the tissue remodeling, endocrine proliferation, and neogenesis induced by the partial duct ligation. The main advantage of this technique over other pancreatic injury models, such as pancreatectomy and chemical beta cell ablation is that partial duct ligation is a simple procedure that does not involve the removal of pancreatic tissue or the destruction of beta cells.

Partial duct ligation due to pancreas of rats, has been introduced to our institute more than 20 years ago. We have translated this technique to mice to facilitate genetic cell lineage tracing, as well as loss and gain of function experiments. To evaluate the mechanism behind the generation of beta cells under these experimental conditions, Before beginning the procedure, disinfect the thorax, an abdomen of an eight week old anesthetized mouse with an antiseptic chlorhexidine solution.

Next, shave a 2.5 by 1.5 centimeter section of the abdomen and disinfect the surgical site with alternating scrubs of the antiseptic and ethanol. After the last scrub, confirm the appropriate depth of anesthesia by toe. Pinch then drape the animal.

Then use a sterile blade to make an upper midline incision in the skin, extending from the xiphoid process to the umbilicus using sterile scissors. Separate the underlying linear elbow and the peritoneum to expose the upper abdominal quadrant. Then retract the stomach superiorly to expose the spleen.

Then expose the splenic lobe or tail region of the pancreas. Next, gently retract the duodenum and part of the upper jejunum to the right upper abdominal quadrant to expose the head, neck, and body of the pancreas. Locate the pancreatic main duct in the neck region of the pancreas.

The body and tail region of the pancreas are now exposed to ligate the pancreatic duct. Carefully place a 6.0 suture needle under the neck region and ligate at the left side of the portal vein, separating the gastroduodenal and splenic lobes. Place a second ligation in close proximity to the first to ensure that the lobes are adequately separated.

Then return the organs to the abdominal cavity and use four zero poly glycol filamentous threads to close the muscle layer in a continuous suture pattern. Finally, close the skin with a discontinuous suture and monitor the animal until it is fully recovered. The PDL pancreas will be reduced in size and appear almost translucent.

With the eyelets appearing as small white dots, the head of the pancreas will appear in opaque pink color, allowing identification of the distinct exocrine LOI using sterile scissors. Cut along the spleen to separate the ligated tail of the pancreas from the spleen and the connective tissue connecting the tail of the pancreas to the internal organs. Then cut the PD L tail immediately in front of the ation.

Isolate the sham tail tissue in the same way. Note however, that both the tail and head region of the sham pancreas are opaque pink. To isolate both parts separately, cut the sham ligated pancreas in the neck region.

When PDL is performed correctly, the mice appear healthy and do not exhibit a significant difference in body weight or glycemia. Compared to the sham operated animals, the exocrine asar tissue is lost gradually after PD L surgery resulting in a reduction in the size and weight of the ligated PD L tail three days post PDL Thein R tissue morphology becomes disrupted with apparent asar cells apoptosis. After one week, the pancreatic tissue becomes engulfed by infiltrating CD 45 positive immune cells.

After two weeks, many ASIN r lobules are replaced by fibrotic and adipose tissue, causing the translucent appearance of the PD L tail pancreas and allowing the eyelets to become visible to the naked eye. Coincidence with the initiation of the asar apoptosis, an increase in the cell activity of the ductile epithelium is also observed. Moreover, two weeks after PDL, the total beta cell volume in the PDL tail doubles compared to that observed in the non ligated sham tail, which is accompanied by an increase in beta cell proliferation, an activation of the embryonic endocrine progenitor marker neurogen in three.

While attempting this procedure, it's important to use the proper aseptic technique for every step of the surgery and to perform the ligation of the pancreatic duct with as minimal damage as possible to the underlying arteries and the surrounding tissues. Following this procedure, other methods such as lineage tracing in partial duct ligated transgenic mice can be performed in order to answer additional questions on the fate and origin of cells. After PD L.