Many reports assessing the extrahepatic biliary system reported that they obtained the samples but not how they obtained them, raising issues regarding comparability. The main advantage of this technique is that the entire extrahepatic biliary system can be dissected and contaminating cells can be removed atraumatically. Our dissection protocol can be generally applied to experimental approaches working on murine neonatal bile duct disorders, aiming for the dissection of the extrahepatic biliary system.
Some features of the protocol, for example, leaving the stomach attached to the duodenum, might be useful in research of duodenal atresia to allow correct orientation by the partially-digested milk expressed by the stomach. At the beginning, the technique may necessitate some time for training. Practicing and following the step-by-step approach will increase speed and outcome.
To begin, place the disinfected and autoclaved instruments on a sterile surface next to the operation table. After making a cut in the peritoneum, expand the cut to the location of the decapitation, following the left frontal axillary line. Remove the skin from the left to the right side with atraumatic forceps.
Under a microscope, hold the peritoneum surrounding the spleen. Gently lift it up until the peritoneum resembles a tent-like structure, and cut a one-millimeter hole in the center. Wait for the peritoneal tent to be filled with air.
Cut off the peritoneum in a window framed by the lower ribs, both lateral abdominal regions, and the lower bladder area. Ensure full access to the liver, bile duct system, stomach, small intestines, and colon. Examine the gallbladder and bile ducts by carefully pulling the xiphoid process in a cranial ventral position.
Avoid tearing of the falciform ligament that can lead to tearing of the gallbladder from the bile duct system. Then, release the pull of the xiphoid process. Gently pull down the duodenum to free the bile duct system.
Then, perform the lower En-bloc mobilization by identifying the duodenal papilla, which connects the bile duct system to the duodenum. Cut through the duodenum about two centimeters to the right lateral side of the papilla. Then, cut through the pyloric area.
Ensure that the contents of the stomach are present in the pyloric area between the cutting location and the duodenal papilla. To perform the upper En-bloc mobilization, gently pull the xiphoid process and get access to the falciform ligament. Make a one-centimeter cut through the falciform ligament between the gallbladder and close to the xiphoid process.
Cut through the connecting structures between the liver and the thorax, such as the esophagus, inferior vena cava, thoracic aorta, and all ligaments that surround the bare area of the liver. Also, cut all dorsally-remaining tissue connections. Under 20 times microscopic magnification, place the En-bloc sample on a foam pad.
Assemble the sample by reorganizing it in the correct anatomical position. Using gentle movement, flatten the oral and aboral part of the duodenum. Using atraumatic forceps, start the flattening movement at the duodenal papilla and continue to the cutting edges.
Then, smooth out the white pulpy contents of the stomach in the oral part of the duodenum. Identify the oral and the aboral part of the duodenum to rule out probable bile duct rotation. Cut away large remnants of hepatic tissue before dissecting the bile duct.
After a few scraping movements, transfer the sample to a cleaner position on the foam mat. Use the less-squeezed liver tissue in the background to optimize the view for differentiation between EBDS and unwanted cells. Process the hepatoduodenal ligament until the isolated EBDS remains.
Observe the hepatic artery and portal vein arising as white and very delicate filament, approximately three to five millimeters orally off the duodenal papilla. Carefully, remove this delicate filament using a gentle pull to the left lateral side. Ensure that the scraping movements start from the bile duct system and lead to the liver boundaries.
Using this protocol, EBDS was dissected from nine-day-old murine neonates. The length of the isolated EBDS from gallbladder to duodenal papilla was less than 10 millimeters, and the diameter of the delicate ductus choledochus varied from 0.05 to 0.2 millimeters. Hematoxylin eosin staining was performed on the longitudinal section of the EBDS with an open lumen.
The cholangiocytes were identified surrounding the lumen as a monolayer and were dyed darker. Researchers should share and standardize operating techniques such as dissection and injection protocols to improve comparability and rapid usability of their studies and results. Preceding dissection will be advantageous for histological and single-cell approaches, such as the isolation of intra and extraperi-cholangiocytes, because it results in the reduction of cell culture contamination.
