Generating Rabbit Liver Carcinoma Model: A Surgical Procedure to Implant Tumor Tissue in Left Lobe of Liver for Induction of Hepatocellular Carcinoma

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Liver Tumor Implantation via Laparotomy

1. Prepare the recipient rabbit for laparotomy. Anesthetize the recipient rabbit using 45 mg/kg ketamine and 5 mg/kg xylazine for induction followed by intubation and maintenance with 1%–3% isoflurane as needed. When the rabbit is anesthetized, shave the surgical site using hair clippers. Clean the incision area using triplicate wash with betadine scrub, 70% ethanol solution, and betadine solution, and drape the abdomen in a sterile surgical fashion.
2. Using a #15-blade, initiate the laparotomy by making a small downward vertical midline incision through the rabbit's skin starting from the xiphoid process. This should be easily palpated and tends to be roughly the size of an American penny. See Figure 1.
3. Reflect the skin and identify the linea alba. This should be a reflective white band of tissue traveling inferiorly in the midline. Use blunt dissection to traverse the linea alba and expose the peritoneum. See Figure 2.
   NOTE: The peritoneum is easily identified since it will be directly overlying the bowel, which can be seen moving with respiration.
   CAUTION: The peritoneum tends to be adherent to the underlying bowel due to surface tension. Using blunt trauma to dissect the linea alba helps to dissipate the risk of perforating the underlying bowel.
4. Once the peritoneum is exposed, carefully dissect through it to enter the peritoneal cavity. The liver can now be identified. To better navigate the abdominal space, extend the midline incision 1‒2 cm inferiorly through the skin, muscle, and peritoneum.
   NOTE: Extending the midline incision can be easily and safely done by carefully inserting a curved hemostat into the peritoneal space with the curved tip facing superficially towards the peritoneum. Then open the hemostat slightly and use a blade to cute the tissue between the two arms of the hemostat.
5. Identify the left lobe of the liver in order to select a site for tumor implantation. The left lobe is infero-lateral to the medial lobe which sits in the midline.
6. Before attempting to draw the liver out of the peritoneal space, place a dry piece of gauze at the inferior aspect of the incision.
   NOTE: The gauze will provide an adherent surface to lay the liver on to prevent it from retracting back into the abdomen.
7. Using either atraumatic forceps or a piece of wet gauze over the fingers, carefully draw the left lobe of the liver out of the abdomen through the incision and lay it down on the dry gauze placed earlier. See Figure 3.
   CAUTION: The liver capsule is sensitive and can easily rupture. It is critical to be gentle when handling this organ to prevent capsular bleed, liver bruising, and/or eventual hemoperitoneum.
   NOTE: Usually, the liver will declare itself visually upon entering the peritoneal space; however, if the rabbit's stomach is distended, the liver can be pushed cranially out of sight. If this is the case, gently lift the abdominal wall using a blunt probe. In this scenario, the liver tends to adhere to the ventral aspect of the diaphragm due to surface tension so carefully separate the liver using a blunt probe and it should detach. Then use atraumatic forceps to draw the liver out.
8. At this point, prepare the tumor tissue for implantation and place a piece of wet gauze over the liver to protect it.
9. Select a 1‒2 mm³ piece of pre-processed tumor tissue for implantation into the liver. See Figure 4.
10. Using a #11-blade, puncture the liver tissue at a 45° angle making a 0.5 cm deep pocket, taking care not to penetrate the dorsal aspect of the liver capsule. Leave the blade in place after making the puncture. See Figure 5.
11. Gently lift the blade in a ventral direction to create a small pocket in the liver bed. Take the tumor piece using forceps, place the tumor piece in this pocket and then remove the blade.
    NOTE: The blade can be removed prior to inserting the tumor piece, however, the liver will bleed and this can obscure the puncture site making it difficult to identify.
    CAUTION: Be sure to minimize contact of the tumor with any other structures to prevent unintentional tumor seeding. This can also be avoided by setting aside any tools used to assist with implantation afterwards.
12. At this point, place a piece of hemostatic agent, such as gel foam, over the tumor pocket to promote hemostasis and to prevent ejection of the tumor piece.
13. Return the liver to the abdomen, once hemostasis is confirmed.
14. Close the abdominal wall with 3-0 polydioxanone suture on a taper needle using a simple continuous stitch and close the skin with 4-0 polyglactin 910 sutures on a cutting needle using continuous subcuticular stitch.
    CAUTION: When closing the abdominal wall, take care not to damage the omentum or other bowel structures in a suture throw.