The overall goal of this surgical procedure is to induce morphologic liver regeneration via partial lobular hepatectomy. This method can answer key questions in the fields of stem cell biology and regenerative medicine about morphologic liver regeneration and liver specific stem cells. The main advantages of this technique are that it is minimally invasive and does not require chemical insults or cauterization.
Prior to touching the animals, rub the bedding from the mother's cage onto the gloves. Then transfer all of the pups and some bedding from the mother's cage into an empty cage on top of a heating pad. Next, place a pup under a dissecting microscope.
After confirming the appropriate level of sedation by lack of response to toe pinch, use a small gauze pad to disinfect the posterior abdominal wall of the animal with betadine. Use micro dissecting scissors and forceps to make a left midclavicular half centimeter incision immediately below the ribcage. Use the forceps to gently separate the skin.
Then make a second deeper incision into the peritoneal cavity and use the back blunt edge of the micro dissecting scissors and forceps to apply gentle lateral pressure to both sides of the abdomen, forcing the apex of the left lobe out of the peritoneal cavity. When the left apex of the left lobe is visible, amputate the appropriate experimental amount of tissue from the lobe and place the tissue into a 1.5 milliliter tube of PBS on ice. Using a rolled piece of gauze, gently return the left lobe to the peritoneal cavity.
Remove the gauze when the bleeding stops and wet the surgical site and surrounding area with gauze soaked with PBS. Close the surgical site with sutures and a running stitch. Then gently but thoroughly roll a PBS soaked gauze over the pup and wound site ensuring no blood or betadine remains.
Allow the pup to recover near gauze pads exposed to the mother's feces and urine. When all of the pups have been hepatectomized return all of the animals and bedding to the mother's cage. After weighing the lobes, fix tissue samples in 2%paraformaldehyde for one hour at room temperature.
At the end of the fixation, transfer the tissues into individual freezing molds containing optimal cutting temperature medium for freezing over dry ice. And acquire 7-10 micrometer sections of each lobe using any standard cryostat. Check the pups daily for at least 56 days to confirm that their wounds remain closed and inject analgesia 24 and 48 hours post surgery.
At the appropriate experimental end point harvest and weigh the liver from each pup on block. Then carefully separate each individual lobe at its proximal attachment and compare the mass of the amputated left lobe to the mass of the whole liver to determine the extent of regeneration in each lobe. Subtle regeneration of the left lobe can be observed 7-14 days post surgery and full regeneration is often observed after 56 days.
With no significant difference in hepatocyte areas in the regenerated liver revealed by histological analysis. Further, neonatal mouse injections with EdU indicates a significant increase in the number of proliferating cells in the injured regenerating left lobe compared to uninjured controls suggesting that cell proliferation contributes to left lobe regeneration in neonates. Hepatectomy in post natal day 14 juvenile mice results in a decreased regeneration in the left lobe with an increased compensation from the uninjured lobes, however.
Indicating that by 14 days the injury response to acute resection switches from lobe specific regeneration to global compensation. Once mastered, this technique can be completed within 15 minutes if performed properly. While attempting this procedure, it is important to be as careful as possible so not to cause extra stress to the mother or the pups.
Following this procedure, other methods such as serial hepatecomies or chronic liver injuries can be done to further assess the regenerative potential of the liver. After its development, this technique paved the way for researchers in the field of stem cell biology to explore liver regeneration in the neonatal mouse. After watching this video, you should have a good understanding of how to complete a lobe specific controlled amputation of the neonatal mouse liver.
Don't forget that working with isoflurane is extremely hazardous and precautions such as a face mask and gloves should always be taken when performing this procedure.
