Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Acute liver failure or ALF is generally defined as rapid development of liver injury in a healthy liver and is accompanied by severe impairment of its major functions. Orthotopic liver transplant is the only available cure in most of the cases. But due to shortage of liver donors, the rate of mortality in ALF patients is very high.

To study the potential of alternative therapeutic approaches and to better understand the pathophysiology of ALF, animal models of the disease are employed. Many of the ALF models are based on the hepatotoxic effects of acetaminophen, carbon tetrachloride, Concanavalin A, lipopolysaccharide, D-Galactosamine and thioacetamide. However, these have several shortcomings such as poor reproducibility and reversibility, toxicity to other vital organs, poor reflection of human disease pathology and species variation.

Hence, there is a need for a better model system that is reproducible and allows sufficient time interval to study effects of a therapeutic intervention. In the current study, an ALF model in rats has been created by combining effects of partial hepatectomy and acetaminophen hepatotoxicity. Median and left lateral lobe of the liver are removed to excise 70%liver mass, and further, acetaminophen is given to cause ALF.

The development of ALF in the model has been characterized by biochemical and histological analysis and reversibility of liver damage has been confirmed by transplantation of syngeneic healthy rat hepatocytes. Wistar Rats having body weight within the range of 180 to 250 grams were used in the current study. The following steps describe the surgical procedure for induction of ALF in a rat.

The animal is anesthetized by injecting Ketamine-Xylazine mixture intraperitoneally. Complete anesthetization is confirmed by pinching the toe of the animal, and further procedures are carried out only when there is no pedal reflex. To prevent corneal desiccation, a carboxy methyl-cellulose based eye drop is applied to both the eyes.

The animal is restrained to the surgical board using a white tape. Hair is removed from the upper right abdominal surgical area using an electric clipper. Disinfect the surgical site by three alternating scrubs of 70%ethanol and povidone iodine using sterilized cotton pads in circular motion.

The skin to be cut is marked just beneath the sternum perpendicular to the xiphoid and parallel to ribcage. A sterile drape sheet having an opening of around three centimeter by one centimeter is placed over the marked skin. A transverse incision of around two to three centimeters is made along the marked line with the help of a scalpel.

The attachment of skin with underlying muscle layer in the vicinity of the incised area is gently removed by sterile moistened cotton tips. Next, a transverse incision is made through peritoneal layer just beneath the xiphoid process. The left lobe of the liver is exposed by giving a gentle pressure to the thorax with the help of two saline moistened cotton tips.

A sterile nylon thread loop is slipped around the exposed liver lobe. The loop is carefully taken to the base of the lobe with the help of micro dissecting forceps or cotton buds. With the help of microsurgery needle holder and micro forceps, the two ends of the loop are tied, placing the knot as close to the base of the lobe as possible.

This is to constrict the blood vessels and reduce bleeding after the liver lobe is removed. Two additional knots are tied on the other side. The tied lobe is cut just above the knot using microsurgery scissors which leaves an ischemic stump.

Similarly, the median lobe of the liver is lifted using cotton tips. Recognize the unique shape of median lobe of the liver. Place a nylon thread loop over the lobe, firmly tie its ends.

Two additional knots are tied on the other side and resect the lobe. After removing the lobes, the peritoneum is sutured using absorbable catgut chromic 4/0 absorbable suture with continuous stitches followed by skin suturing with simple interrupted suture. After skin closure, povidone iodine solution is applied around the surgical site.

The drape sheet is removed and the animal is taken off from the surgery board. An antibiotic dose of 12 milligram cefotaxime in one ml of 5%glucose solution is given intraperitoneally to the animal to protect it from the risk of postoperative infection. For pain relief after surgery, the analgesic meloxicam, typically 1 mg per kg body weight is given subcutaneously to the animal, followed by a dose each day for two days.

The animal is moved to a warm recovering cage. The animal becomes conscious within 30 to 40 minutes and begins to move. The operated animal is kept in isolation until surgical wounds are completely healed, which may take three to four days.

For inducing ALF, 750 milligram per kg body weight of acetaminophen and the second dose of meloxicam is injected intraperitoneally 24 hours after partial hepatectomy procedure. This dose regimen is repeated after 24 hours. After induction of ALF, survival in animals was found to be severely decreased.

To characterize the development of ALF, operated animals were euthanized after second dose of acetaminophen, and various biochemical and histological studies were done. The results are described ahead in the video. Survival percentage in the ALF induced group was found to be severely decreased and up to 80%mortality was observed within 24 hours of second dose of acetaminophen.

Liver damage was apparent at morphological level in ALF induced group and only 70%partial hepatectomy group. Whereas the control group and only acetaminophen treated group had healthy appearance. Enzyme levels of alanine amino transferase, aspartate amino transferase and alkaline phosphatase were found to be highly elevated in ALF induced group in comparison to other groups.

Analysis of gene expression profile by qPCR showed elevated expression of genes involved in apoptosis, inflammation and progression of liver injury. Hematoxylin and eosin staining of liver tissue sections revealed mostly normal hepatocytes in the control group. In the ALF group, mostly moderate macro vesicular fatty degeneration was observed.

Prothrombin time and International Normalized Ratio were found to be elevated in ALF induced group, indicating hindered blood coagulation process. Survival percentage was found to be restored in ALF induced group after transplantation of syngeneic healthy rat hepatocytes. Serum levels of enzymes alanine amino transferase, aspartate amino transferase and alkaline phosphatase were found to be restored in ALF induced animals after cell transplantation.

This method of inducing ALF in rats is reproducible, shows good reversibility and provides a suitable time window to test new therapies. Hence, this model can serve as an efficient disease model for evaluation of potential therapeutic approaches to treat ALF.