Development of an invasively monitored porcine model of acetaminophen-induced acute liver failure

Abstract

Background: The development of effective therapies for acute liver failure (ALF) is limited by our knowledge of the pathophysiology of this condition, and the lack of suitable large animal models of acetaminophen toxicity. Our aim was to develop a reproducible invasively-monitored porcine model of acetaminophen-induced ALF.

Method: 35kg pigs were maintained under general anaesthesia and invasively monitored. Control pigs received a saline infusion, whereas ALF pigs received acetaminophen intravenously for 12 hours to maintain blood concentrations between 200-300 mg/l. Animals surviving 28 hours were euthanased.

Results: Cytochrome p450 levels in phenobarbital pre-treated animals were significantly higher than non pre-treated animals (300 vs 100 pmol/mg protein). Control pigs (n = 4) survived 28-hour anaesthesia without incident. Of nine pigs that received acetaminophen, four survived 20 hours and two survived 28 hours. Injured animals developed hypotension (mean arterial pressure; 40.8 +/- 5.9 vs 59 +/- 2.0 mmHg), increased cardiac output (7.26 +/- 1.86 vs 3.30 +/- 0.40 l/min) and decreased systemic vascular resistance (8.48 +/- 2.75 vs 16.2 +/- 1.76 mPa/s/m3). Dyspnoea developed as liver injury progressed and the increased pulmonary vascular resistance (636 +/- 95 vs 301 +/- 26.9 mPa/s/m3) observed may reflect the development of respiratory distress syndrome.Liver damage was confirmed by deterioration in pH (7.23 +/- 0.05 vs 7.45 +/- 0.02) and prothrombin time (36 +/- 2 vs 8.9 +/- 0.3 seconds) compared with controls. Factor V and VII levels were reduced to 9.3 and 15.5% of starting values in injured animals. A marked increase in serum AST (471.5 +/- 210 vs 42 +/- 8.14) coincided with a marked reduction in serum albumin (11.5 +/- 1.71 vs 25 +/- 1 g/dL) in injured animals. Animals displayed evidence of renal impairment; mean creatinine levels 280.2 +/- 36.5 vs 131.6 +/- 9.33 mumol/l. Liver histology revealed evidence of severe centrilobular necrosis with coagulative necrosis. Marked renal tubular necrosis was also seen. Methaemoglobin levels did not rise >5%. Intracranial hypertension was not seen (ICP monitoring), but there was biochemical evidence of encephalopathy by the reduction of Fischer's ratio from 5.6 +/- 1.1 to 0.45 +/- 0.06.

Conclusion: We have developed a reproducible large animal model of acetaminophen-induced liver failure, which allows in-depth investigation of the pathophysiological basis of this condition. Furthermore, this represents an important large animal model for testing artificial liver support systems.

Figure 1

Acetaminophen concentrations (A) and Methaemoglobin levels (B) in pigs following acetaminophen administration. Panel A. Acetaminophen levels for individual pigs are noted, with the corresponding methaemoglobin levels illustrated in panel B. Acetaminophen levels of between 200 300 mg/l were aimed for although even with frequent measurements and reductions in the infusion rate there were rises often to 400 mg/l. Prolonged elevation of levels above 300 mg/l did lead to a rise in methaemoglobin. For pig 7 facilities to measure acetaminophen levels were unavailable and this led to a marked rise in methaemoglobin levels.

Figure 2

Kaplan Meier survival curve for control and acetaminophen injured pigs. (A) The control group (Group 0) as demonstrated by the uninterrupted line all survived, and indeed appeared healthy when euthanased. The injury group (Group 1) as demonstrated by the interrupted line died as denoted in the Kaplan-Meier curve. Two animals* were euthanased at 25 hours as they appeared particularly unwell, and another further two were euthanased at the end of the experiment with no obvious ill effects from acetaminophen administration. (B) This table outlines the likeliest cause of death along with a correlation with the amount of liver and renal injury. In most cases the cause of death was multi-organ failure (MOF) with evidence of hypotension, marked oedema, oliguria and ventilatory difficulties. In two animals organisms were identified in routine blood cultures. In pig 2 a cavitating lung abscess was identified although this was in addition to liver and renal injury. However in pig 9 Pseudomonas paucimobilis was identified in blood cultures with little evidence of significant renal or liver damage. +++ covered severe hepatic coagulative necrosis, ++ covered moderate hepatic coagulative necrosis and + covered mild hepatic coagulative necrosis. Similarly the scoring scale for renal injury covered the range from mild (+) to moderate (++) to severe (+++) tubular necrosis. Pig 7 developed significant methaemoglobinaemia (>10%). Administration of Methylene blue led to circulatory collapse and death within 5 minutes. Pig 8 had moderately elevated levels of methaemoglobinaemia (>4%), and shortly before death dropped its oxygen saturations to 40%. No additional contributory cause to death was identified.

Figure 3

Profile of Pulse, Mean Arterial pressure, Cardiac Output and Systemic Vascular Resistance in control and acetaminophen injured pigs. Chart A demonstrates a progressive increase in pulse and concomitant reduction in MAP following liver injury. Furthermore, chart B demonstrates a marked increase in cardiac output (CO) and decreased systemic vascular resistance (SVR) following liver injury. These haemodynamic changes are in keeping with the multi-organ failure seen in acute liver injury.

Figure 4

Profile of Pulmonary Artery Occlusion Pressure and Pulmonary Vascular Resistance in control and acetaminophen injured pigs. Fluid resuscitation was guided by Pulmonary Artery Occlusion Pressure (PAOP), and therefore this was similar between the two groups. Pulmonary Vascular Resistance (PVR) became noticeably higher in the acetaminophen injured pigs. This was also seen in an increased ventilatory requirement in the latter stages of the injury experiments.

Figure 5

Profile of serum AST, serum Albumin, arterial pH and plasma Prothrombin time in control and acetaminophen injured pigs. Panel A depicts the increase in serum AST and concomitant reduction in serum albumin in acetaminophen injured pigs. This pattern of liver injury was also seen in panel B, where arterial pH drops and Prothrombin time increases.

Figure 6

Profile of the plasma coagulation factors V and VII in control and acetaminophen injured pigs. The levels of plasma coagulation factors V and VII can be seen to markedly reduce in acetaminophen injured pigs in keeping with the previously observed prolongation of prothrombin time.

Figure 7

Profile of serum Creatinine and Potassium in control and acetaminophen injured pigs. This panel depicts the development of renal injury in acetaminophen injured pigs as assessed by serum Creatinine and Potassium.

Figure 8

Fischer's ratio in acetaminophen injured pigs. Fischer's ratio, a ratio of the concentrations of aromatic amino acids (tyrosine and phenylalanine) to branch chain amino acids (leucine, isoleucine and valine) is demonstrated in the panel. There was no change in the control animal group, but after liver injury there was a marked reduction.

Figure 9

Histology. This figure depicts representative photographs of liver and renal sections. Panel a, represents liver tissue taken from a control pig. Panel b represents liver tissue taken from pig 4 and demonstrates moderate injury. There is diffuse microvesicular change, with moderately severe centrilobular necrosis. Panels c and d (higher power) represents liver tissue taken from pig 7 demonstrating more severe coagulative centrilobular necrosis. Panel e represents renal tissue taken from a control pig. Panel f represents liver tissue taken from pig 7 demonstrating severe vacuolar injury to the cortical tubules in keeping with the development of acute tubular necrosis.