The overall goal of this pancreatic duct infusion procedure is to enable lineage tracing, gene introduction, and cell line-specific targeting in the pancreas. This method could help answer key questions about diseases affecting the pancreas, including chronic pancreatitis, diabetes, and pancreatic cancer. To begin, place an anesthetized mouse under a dissecting microscope so that its abdomen is positioned in the center of the field of view.
Immobilize the animal with a surgical tape and then apply an ophthalmic ointment to its eyes to prevent them from drying. Next, cover the abdomen of the mouse with a thick layer of hair removal cream. And after approximately three minutes, gently wipe off the cream with gauze soaked in 70%ethanol to remove all hair.
Finally, wipe the skin with 70%ethanol, followed by 10%povidone iodide solution to remove any contaminants and disinfect the skin. To begin the surgery, create with a scalpel a midline skin incision extending from the xiphoid process to the umbilicus. Then using Adson forceps, lift the peritoneum and make with scissors a small midline hole.
Expand the cut along the midline of the peritoneum so that its length is equal to the length of the skin incision. Next, use blunt retractors to pull up the liver and after exposing the common bile duct, clamp it with a curved bulldog vascular clamp. Identify the duodenum and with Arruga forceps, pull it out caudally to display the papilla visible as a white spot on the anterior surface of the second portion of the duodenum.
To identify the pancreatic duct, withdraw the duodenum caudally, then position a 30-1/5 gauge needle tangentially to the duodenum and make a puncture on its wall that directly opposites the papilla. To infuse the solution, introduce the catheter connected to an infusion pump through the created duodenotomy and slide it along the duct until it is visible within the vessel, but reaches not further than one centimeter to avoid bypassing minor ductal tributaries. Immobilize the catheter by clamping it with a second curved bulldog vascular clamp.
Then turn on the pump to initiate the infusion of 150 microliters of the solution into the duct. Next, inject to the peritoneum of the mouse approximately 1.5 milliliters of saline to avoid any loss of infused volume and then cover the exposed bowel with a moistened gauze to prevent desiccation. Once the infusion is completed, release the immobilized catheter and using the bulldog clamp, gently remove it from the pancreatic duct.
Then release the clamp from the common bile duct. Close the peritoneum and the skin by continuously suturing them in a single layer and administer the first of the two doses of analgesic, the second of which will be given the following day. Then return the mouse to a cage positioned under a heat lamp and provide the animal with food and water ad libitum.
Observe the mouse until it has regained sufficient consciousness manifested in sternal recumbency. Seven days after the surgery, excise from the euthanized animal pancreas as well as spleen, liver, and duodenum serving as controls. Immerse the entire isolated tissues in a 4%paraformaldehyde solution and fix them at four degrees Celsius overnight.
Once the tissues are fixed, cryo protect the samples by soaking them in a 30%sucrose solution at four degrees Celsius overnight. After an overnight cryo protection, snap freeze the samples in liquid nitrogen and using a microtome, cut tissue sections at six micrometers. Finally, image the sections under a fluorescent microscope.
Presented here are microscopic images of pancreas sections excised from mice seven days after sham surgery and pancreatic duct infusion of AAV8 carrying the GFP gene. Liver, spleen, and duodenum isolated from the mouse infused with AAV8 did not show any GFP signal while the pancreas revealed prominent GFP expression, confirming pancreas-specific delivery of the virus following pancreatic duct infusion. Once mastered, this technique can be done in 10 minutes when performed properly.
While performing this procedure, it's very important to perform careful dissection and make the smallest possible duodenotomy for introduction of the catheter. After its development, the technique paved the way for researchers studying the pancreas to investigate a number of pathologies that afflict the organ, including chronic pancreatitis, diabetes, and pancreatic cancer in both rodent and larger animal models. After watching this video, you should have a very good understanding of how to both effectively and selectively infuse the pancreatic duct.
