Findings of the present study indicate that TNF-α levels are increased early during AOM-induced ALF and that etanercept administration significantly delays the progression of HE by reducing hepatocellular damage, decreasing both systemic and central inflammation as well as hepatic and cerebral oxidative stress in these animals. These results add further support to the notion that TNF-α plays a major role in the pathogenesis of ALF.
TNF-α is a key cytokine that exerts pleiotropic effects ranging from proliferative responses, inflammatory effects and modulation of immune responses, to destructive cellular outcomes such as apoptotic and necrotic cell death. TNF-α is produced as a transmembrane (tmTNF-α) or a membrane-cleaved circulating cytokine (sTNF-α) by various cell types such as macrophages, lymphocytes, cerebral microglia and astrocytes, and hepatic Kupffer cells. It has been suggested that TNF-α plays a major role in the pathogenesis of HE associated with ALF where the circulating levels of this cytokine are significantly increased both in patients and animal models 
. Moreover, a direct correlation has been established between TNF-α levels, severity of ALF and prognosis 
. In the present study, plasma TNF-α levels reached statistical significance 9 h after AOM administration and clearly preceded the onset of coma and massive hepatocyte cell death which occurred only at coma stages of HE, as shown by large increases in ammonia and transaminase levels. These observations suggest that TNF-α plays a role early in the pathogenesis of AOM-induced ALF and HE. These findings are in accordance with other experimental models of ALF where elevated serum levels of TNF-α have been observed shortly after the initiating insult. For example, plasma TNF-α levels were increased as early as 4 h following injection of a single dose of acetaminophen in mice and were correlated with the onset of hepatic damage 
TNF-α performs its biological functions as a homotrimer that binds to p55 and p75 TNF receptors (TNFR 1 and 2, respectively) and induces a variety of intracellular pathways regulating the transcription of a large number of genes implicated in host defence 
. Importantly, TNF-α triggers within hours the secretion of multiple pro-inflammatory mediators, among them IL-6 
. Results of the present study demonstrated that plasma IL-6 levels were significantly increased 9 h after AOM-induced hepatotoxicity and correlated with plasma TNF-α levels, which underscores the rapid pro-inflammatory effect of TNF-α and confirms the presence of systemic inflammation in the AOM model of ALF.
Systemic administration of etanercept has been shown to be effective in neutralizing TNF-α in various animal models of central nervous system injury, thus leading to improved outcomes 
. Etanercept is a fusion protein of two TNFR2 extracellular domains linked to the Fc fragment of human immunoglobulin 1 (IgG1). It binds to a single sTNF-α trimer or tmTNF-α in a 1
1 ratio thereby acting as a competitive inhibitor of TNF-α 
. Etanercept has a 50-fold greater affinity and is 1000-fold more efficient than the endogenous monomeric sTNF-α receptor as assessed in vitro
by inhibition of TNF-α binding or bioactivity 
. The half-life of etanercept in vivo
is three days, which is five times that of monomeric sTNF-α receptor 
. These characteristics, as well as the high volume of distribution of etanercept, result in a greater ability to neutralize the biologic effects of TNF-α, suggesting that it may be a suitable anti-TNF-α therapy in our model of ALF. Results of the present study show that systemic administration of etanercept 30 min before or 3 h after AOM leads to delayed progression of HE and onset of coma as well as improved hepatic function. The protective effect of etanercept on AOM-induced liver damage was evidenced by reduced hepatocellular necrosis, microvesicular steatosis and hemorrhagic congestion, and by a preventive effect on the increases in plasma levels of ammonia and transaminases. Similar protective effects of etanercept were also observed in acetaminphen-induced liver injury, suggesting that the beneficial effects of etanercept are not specific to AOM-induced liver damage and that this drug may be useful in liver damage from various aetiologies.
Innate and adaptive immune responses occur during acute liver injury and are accompanied by the secretion of large amounts of pro-inflammatory cytokines 
. In the present study, we focused on plasma IL-6 and CD40L, two pro-inflammatory mediators implicated in the inflammatory cascade mediated by TNF-α, in order to evaluate the biological effects of TNF-α inhibition. Etanercept administration prevented the increases of IL-6 and CD40L in mice with AOM-induced ALF confirming that the protective effects of etanercept are the consequence of the peripheral inhibition of the biological action of TNF-α. Importantly, previous studies have demonstrated the implication of these cytokines in the pathogenesis of ALF. Indeed, plasma IL-6 levels are significantly increased in patients with fulminant hepatitis and are indicative of a poor prognosis 
. On the other hand, the dyad of the TNF family members CD40/CD40L is implicated in the inflammatory cascade that leads to ALF and induces the synthesis of pro-inflammatory cytokines such as TNF-α and IL-6 
. In liver, increased expression of CD40L and its receptor CD40 constitute an early mechanism for liver cell damage in both human and murine fulminant hepatic failure 
and a recent study has demonstrated that overexpression of hepatic CD40L in mice induces ALF 
. Moreover, serum levels of CD40L are higher in patients with fulminant hepatitis than in patients with acute hepatitis or controls and are associated with a poor prognosis 
Interestingly, administration of etanercept 6 h after AOM treatment had no effect on the progression of HE, suggesting that the inflammatory cascade driven by TNF-α might already be too extensive at this stage to be reversed by etanercept. Indeed, although only a slight and non-significant increase in plasma TNF-α is observed 6 h after AOM administration, it is possible that the activation of Kupffer cells and the secondary recruitment of immune cells such as monocytes and neutrophils may already have led to an irreversible inflammatory response locally, accompanied by hepatocellular damage 
Oxidative stress is a component of the innate inflammatory response that develops as a consequence of injury. Our unit has previously demonstrated that oxidative stress plays a key role in the physiopathology of ALF in mice resulting from AOM hepatotoxicity, and that the protective effects of mild hypothermia or N-acetylcysteine in this model is mediated by an attenuation of plasma levels of pro-inflammatory cytokines, and a reduction of both the depletion of liver glutathione stores and of the GSH/GSSG ratio 
. Results of the present study also demonstrate that systemic sequestration of TNF-α by etanercept attenuates the oxidative stress response associated with inflammatory pathways by preventing the depletion of liver glutathione stores and attenuating the decrease in hepatic GSH/GSSG ratio. Maintenance of hepatic glutathione stores thus appears to be an important mechanism by which peripheral inhibition of TNF-α prevents further hepatocellular damage.
Acute liver failure is frequently associated with serious neurological complications including HE and cerebral herniation. For decades, a great deal of attention has been focused on ammonia as the main agent responsible for the central nervous system complications of ALF. However, ammonia-lowering strategies are of limited value in preventing the cerebral complications of ALF 
, and recent studies strongly suggest that inflammation acting alone or in concert with ammonia plays an important role in the pathogenesis of HE 
. Results of the present study demonstrated that systemic administration of etanercept also attenuated the neuroinflammatory response associated with AOM-induced ALF, as shown by lower cerebral IL-6 levels, attenuated microglial activation and a preserved GSH/GSSG ratio. It is important to note that OX-42 is a marker of both activated microglia and macrophages, therefore it cannot be excluded that OX-42 positive cells may in part result from monocyte infiltration as shown in another model of liver damage 
. Moreover, analysis of cerebral cortex extracts from etanercept-treated ALF mice failed to show the presence of significant amount of etanercept in the brain, suggesting that the 150 kDa recombinant complex does not cross the blood-brain barrier (BBB) following AOM administration and that BBB integrity was maintained. These results suggest that the beneficial effects of etanercept on HE in AOM-treated ALF mice are primarily mediated peripherally via
an improvement of liver function. However, it cannot be excluded that etanercept is also acting indirectly by blocking the transduction of pro-inflammatory signalling to the brain. Immune-to-brain signalling can indeed occur via
mechanisms such as i) entry of cytokines through brain regions lacking a functional BBB; ii) active cytokine transport at the BBB; iii) activation of brain endothelial cells resulting in release of second messengers into the brain; iv) signalling through afferent nerve fibres in the periphery; v) recruitment of activated immune cells into the brain parenchyma 
. Moreover, there is evidence that systemic pro-inflammatory cytokines such as IL-6 and TNF-α increase the permeability of cerebrovascular endothelial cells to ammonia 
suggesting that beneficial effects of etanercept on neuroinflammation during ALF may also result from the prevention of an increase of BBB permeability to ammonia.
In conclusion, the present study demonstrates that TNF-α plays a key role in the pathogenesis of ALF and HE following AOM-induced hepatotoxicity in the mouse. Systemically administrated etanercept delayed the onset and progression of HE by attenuating systemic inflammation and its deleterious consequences i.e hepatocellular damage, oxidative stress and neuroinflammation. These results suggest that etanercept has the potential to provide a promising therapeutic approach for the management of ALF in patients awaiting liver transplantation.