The major role for an ALF model is to provide a more controlled experimental environment than currently exists [18]. An ideal model should satisfy the following criteria: 1) induced hepatic failure should be reversible; 2) the features of liver damage should be reproducible; 3) selective damage should lead to fatal liver failure over a period similar to that encountered in the human condition; but 4) with sufficient time to allow the option for successful treatment; 5) the model should be large enough to accommodate anthropocentric therapies; and 6) the toxins used should not be hazardous to laboratory personnel. A pig model would be preferable as it has the potential to fulfil all of these criteria, and also importantly shares very similar liver metabolic function with human liver [19].
The porcine model described here develops most of the signs typifying acute liver injury/failure, namely: tachycardia, hypotension, increased cardiac output, decreased systemic vascular resistance, coagulopathy, renal failure and altered hepatic biochemistry. Consequently, it is the only animal model of acetaminophen-induced liver injury large enough to allow monitoring at a level equivalent to intensive care. Whilst the outcome of acetaminophen-injured pigs is relatively reproducible there still remain some inherent variations highlighting the difficulty of such studies. This reinforces the need to perform large studies when using such animals to ensure that individual variations can be accounted for.
Our model showed the typical increase in cardiac output and reciprocal reductions in SVR found in the clinical setting. Of note this is accelerated within the porcine model, starting within 16 hours of the onset of liver damage. This is likely a reflection of pre-treatment with phenobarbitone. The increased PVR in the context of normal PaO2 values was in keeping with the increased requirement for positive end-expiration pressure (PEEP) during ventilation. This may have indicated the onset of an ARDS-like picture, although lung histology would be important to support this conclusion. That PAOP did not increase suggests that the ventilatory changes were not associated with pulmonary oedema secondary to left ventricular failure. A non-significant reduction in PVR Index after liver injury has been described [20]. The reason for this difference is unclear. Acute lung injury is common in patients with acetaminophen-induced fulminant hepatic failure and is associated with systemic circulatory failure, cerebral oedema and high mortality.
The marked reduction in Factor V and VII levels in the current model were consistent with severe liver injury. The Clichy criteria [21] deemed a reduction of Factor V levels to <20-30% as the trigger for liver transplantation; this was the case in most animals in this experiment. The absence of changes in Factor VIII levels indicate DIC was not the cause of altered levels of other coagulation factors.
An increase in AST levels indicated the development of liver necrosis, although these were not as high as are seen in patients. Similarly, a rise in bilirubin did not occur. This may reflect a species-specificity of the assay used, or insufficient time for bilirubin to rise. It is possible that there are subtle variations in this model compared to humans in that some systemic features prevail over a liver focused pattern of injury. Liver tissue analysis revealed severe coagulative necrosis in some animals with moderate and mild injury in others. This supports the probability that significant liver injury accounts for the clinical syndrome reported here. Although there is some variability in the liver injury seen in this model, there is still significant liver injury in the majority of animals. Indeed, this variation closely mimics what is seen clinically.
There are several explanations for the failure to observe changes in ICP. The time course may have been too brief. However, a raised ICP is not always encountered in the clinical setting, or only in association with other factors such as sepsis. It may also reflect differences in the pathophysiology of drug-induced ALF as opposed to ischaemic models of ALF. Measurements of arterial ammonia, cerebral blood flow or the use of cerebral microdialysis may help to clarify intracranial effects in future studies. Biochemical analysis suggested that animals may have been developing encephalopathy, although in the absence of ICP changes and with general anaesthesia it is difficult to be certain. Fischer's ratio [16], the ratio of the aromatic amino acid (AAA; tyrosine and phenylalanine) to branch chain amino acid (BCAA; leucine, isoleucine and valine) concentrations has a value of 3 to 4 in clinical liver failure but when this ratio is reversed and is in absolute figures <1.4 then most patients develop hepatic encephalopathy.
There was evidence of marked renal impairment, although fatal hyperkalaemia was not observed. Renal dysfunction is an important feature of acetaminophen induced liver failure [22], and the renal histology in treated animals was consistent with a toxic injury rather than the distinctive pattern of frank outer medullary tubular necrosis encountered with ischaemia, i.e., critical hypotension.
Methaemoglobinaemia
This study demonstrates the importance of continuous monitoring of acetaminophen levels, the one animal from which it was withheld died from methaemoglobinaemia. The amount of acetaminophen required to maintain levels between 200-300 mg/l for 12 hours was calculated beforehand, but it was necessary in every case to reduce the infusion rate (and the total dose administered) as acetaminophen levels exceeded this range. Levels greater than 250 mg/l increased serum methaemoglobin levels, although this usually resolved by stopping the acetaminophen infusion which allowed plasma levels to fall. Acetaminophen and its intermediates oxidise haemoglobin to methaemoglobin, which is unable to carry oxygen. Although it occurs in human beings, it is less marked than in cats, dogs, and pigs. In some species, methemoglobin is reduced to hemoglobin by the methemoglobin reductase using reduced glutathione (GHS) as a substrate. GSH itself is recycled by the glutathione reductase, using reduced nicotinamide-adenine dinucleotide phosphate (NADPH) from the pentose phosphate pathway. Due to the glucose impermeability of porcine red blood cells (compared with rats and rabbits) there is a diminution in generation of NADPH and hence decreased reduction of methaemoglobin [23]. Levels of methaemoglobinaemia in pigs [24] administered acetaminophen are only one-half those reported in cats [25] and dogs [26]. There are several measures to control methaemoglobin levels in animal models. Kelly et al [8] suspected high plasma acetaminophen levels were responsible for lethal methaemoglobinaemia and so tried to prevent them. Not having access to bedside testing and thus being unable to prevent high acetaminophen levels they administered MB at 10 mg/kg if the blood appeared brown on visual inspection. The usefulness of this strategy was not confirmed in the current study.
Methylene blue is the recognized clinical treatment for methaemoglobinaemia and acts by reducing methaemoglobin back to haemoglobin. Its action depends on the availability of adequate NADPH concentrations within the erythrocyte; a deficiency of NADPH availability leads to further methaemoglobinaemia. On the single occasion it was used in the current study the animal died of cardiovascular collapse 5 minutes later.
Presence of anaemia
Miller et al [12] found that the haematocrit fell rapidly (25% decrease from the initial packed cell volume) in sixty percent of animals 1 to 2 hours before death. The cause of anaemia in their study was unclear, and may reflect extravascular haemolysis within the spleen. The haemoglobin nor haematocrit levels did not fall in either the control nor the treatment groups in the current study.
In summary, we have described the first intensively monitored porcine model of acetaminophen-induced severe liver injury which displays most of the features seen in the human condition. This large animal model can play an important role in the evaluation of the effectiveness of liver support systems, as well as providing us with the ability to develop a much better understanding of the pathophysiology of this devastating clinical syndrome.