In ALF, it is generally considered that failure of cell volume regulation in astroglia underpins the phenomenon of cytotoxic brain edema that so frequently leads to increased intracranial pressure, brain herniation and patient death. Various mechanisms have been proposed to explain this failure of neuroglial cell volume regulation in ALF that include as described in two subheadings below.

High affinity transport of amino acids is a major function of astroglia. Such transport mechanisms exist to maintain the supply of key intermediates required for cellular energy metabolism , synthetic processes and for the termination of action of neurotransmitters such as glutamate and γ-aminobutyric acid (GABA) that form the basis of the so-called “glial-neuronal metabolic interactions “. Astroglial glutamate transporters are essential components of the glutamate-glutamine cycle and are responsible for the removal of excess glutamate from the synaptic cleft. Expression of the sodium-dependent, high affinity glutamate transporters EAAT-1 and EAAT-2 (in human; in rodents GLAST and GLT-1, respectively) have been reported in animal models of both acute and chronic liver failure (Knecht et al. 1997; Suarez et al. 2000) resulting in increases in extracellular brain glutamate concentrations (Vaquero and Butterworth 2006) . It has been suggested that limitations in the availability of glutamate in astroglia could limit ammonia-removal capacity since glutamate is the obligate precursor for GS, and, if so, could limit the synthesis of glutamine.

Decreases in expression of the high affinity glycine transporter (GLYT-1) have been reported in cerebral cortical extracts from rats with ALF resulting from hepatic devascularisation (Zwingmann et al. 2002) . Given the predominantly astroglial localization of GLYT-1 in cerebral cortex, it was proposed that these changes might relate to increases in availability of extracellular glycine with the potential to activate the glycine neuromodulatory site on the N-methyl-D aspartate (NMDA) subclass of glutamate receptor. NMDA receptor activation has been proposed to explain the hyperexcitability and nitrosative stress that occur in ALF (Vaquero and Butterworth 2006) .

Following the cloning and characterization of high affinity glutamine transporters (small neutral amino acid transporters or SNATs) and given the consistent finding of increased brain glutamine in HE, a study was undertaken to assess SNATs in an animal model of ALF (Desjardins et al. 2012) . Coma/edema stages of encephalopathy were accompanied by a selective decrease in expression of SNAT-5. Given the astroglial localization of SNAT-5 (Cubelos et al. 2005) it was proposed that down-regulation of transporter expression in liver failure could result in effective “trapping” of glutamine in the cell, an action that is consistent with cell swelling due to glutamine accumulation in the astrocyte as had previously been widely proposed. Moreover, since astroglial glutamine functions as immediate precursor of releasable (transmitter) glutamate, a limit upon its availability following decreased release from the astroglial cell has the potential to result in impairment of glutamatergic transmission an action that could result in excessive inhibition that is also characteristic of HE.

Translocator protein (TSPO), previously known as the “peripheral-type benzodiazepine receptor” is a mitochondrial protein responsible for the transport of cholesterol into the mitochondrion. The protein is expressed predominantly by neuroglia with both astroglia and microglia exhibiting high levels of expression in mammalian systems. Increased expression of TSPO has been reported in a wide range of hyperammonemic disorders including urea cycle enzymopathies, ALF, animals with end-to-side portacaval anastomoses and in patients with end-stage chronic liver failure (see (Ahboucha and Butterworth 2008) for review). In one study in humans using PET and the TSPO ligand 11C-PK11195, increased signals were observed in anterior cingulate cortex where the magnitude of the increased signals correlated with the degree of cognitive impairment (Cagnin et al. 2006) . The origin of these increased signals was considered to relate to microglial activation . Interest in TSPO in HE relates to the well-established relationship of the protein to the GABA system. Activation of neuroglial TSPO results in increased transport of cholesterol into the mitochondrion followed by increased synthesis of so-called “neurosteroids”, one of which, allopregnanolone is a very high affinity agonist for the neuronal GABA-A receptor. Increases in concentration of allopregnanolone were reported in autopsied brain tissue from patients who died in hepatic coma (Ahboucha and Butterworth 2005) . Based upon these findings, it was proposed that the increase in “GABAergic tone” resulting from activation of TSPO sites and the subsequent increase in synthesis of allopregnanolone could also contribute to the excessive neuroinhibition in HE.

A range of hepatotoxic agents have been used to uncover basic mechanisms responsible for the CNS complications of liver failure . This topic was reviewed by a panel of experts nominated by The International Society for Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) who, after careful deliberation, recommended two toxic models based upon the extent of the characterization. The two models of ALF were the azoxymethane (AOM) mouse model and the thioacetamide (TAA) rat model (Butterworth et al. 2009) . The AOM mouse model of ALF exhibits many of the pathophysiological characteristics of human HE due to acute liver failure . These features include (1) a clear pattern of neurological behaviors starting with the prodromal phase due to liver failure, where neurological symptoms are not yet evident followed by a number of distinct phases of neurological decline that rapidly progress to stupor andcoma; (2) the presence of cerebral edema; and (3) high levels of ammonia in the blood and brain. Very elegant and detailed analyses of the morphological changes in microglia and real-time analysis of microglial dysmotility after AOM have been demonstrated (Rangroo Thrane et al. 2012) . Both microglia activation (as demonstrated by an ameboidal phenotype) and motility (as demonstrated by analysis of the turnover rate) were shown to be altered in the cerebral cortex at late stages of HE when severe neurological symptoms were evident, coinciding with the appearance of brain edema (Fig. 15.4). Increased OX-42/CD11b immunoreactivity was also demonstrated in the cerebral cortex of AOM-injected mice (Chastre et al. 2012) , which was attenuated by treatment with the tumor necrosis factor (TNF)-α neutralizing molecule etanercept. Furthermore, there was a concomitant attenuation of AOM-induced liver injury and decreased expression of neuroinflammatory molecules in the brain after etanercept treatment (Chastre et al. 2012) .

Ischemic liver failure, although uncommon, is encountered clinically. Experimental ALF can be induced by the performing an end-to-side portacaval anastomosis followed by hepatic artery ligation. Rats undergoing this surgery exhibit key clinical features of HE, including cerebral edema and hyperammonemia, which ultimately result in grade 4 HE (hepatic coma). An increase in the number of OX-42/CD11b positive microglia has been demonstrated in the frontal cortex, thalamus and hippocampus starting 6 h after surgery (early stage HE) and worsening at the time of coma/edema (Jiang et al. 2009a, b), which could be alleviated by either mild hypothermia (Jiang et al. 2009b) or minocycline (Jiang et al. 2009a), with a concomitant attenuation of the progression of HE and brain edema .

In a related, more subtle model of HE induced by end-to-side portacaval shunt surgery alone, without subsequent hepatic artery ligation, rats develop mild cognitive impairment over the following 3–4 weeks. Associated with this mild form of HE, currently referred to clinically as “minimal HE”, is a change in the morphology of major histocompatibility complex class II (MHCII)-positive microglia to a more ameboid, activated phenotype (Agusti et al. 2011) . Curiously, these changes were restricted to cerebellum. Chronic infusion of a p38 mitogen-activated protein kinase inhibitor reversed the morphological changes observed in microglia and prevented the cognitive impairment (Agusti et al. 2011) .

Obstruction of the common bile duct induces a reproducible model of biliary cirrhosis in rats. Bile duct-ligated (BDL) animals have liver failure , developing jaundice, portal hypertension, portal-systemic shunting, bacterial translocation and immune system dysfunction. BDL rats are hyperammonemic but show only low-grade encephalopathy (decreased locomotor activities) (Butterworth et al. 2009) . Using this model, microglia are activated predominantly in the cerebellum, with only traces of activation in the striatum and thalamus (Rodrigo et al. 2010) (Fig. 15.5a). Interestingly, no microglia activation was evident in the frontal cortex. Treatment with ibuprofen reduced the microglia activation and reversed the concomitant cognitive impairments observed (Rodrigo et al. 2010) . Similarly, microglia activation has been shown after BDL in mice, as demonstrated by morphological changes in Iba-1 positive microglia (D’Mello et al. 2009) . However, in contrast to the rat model, the activation of microglia was localized to the cerebral cortex rather than the cerebellum (Fig. 15.5b). The cause of these region-and species-selective changes remains unknown. As discussed in a separate section of this chapter, the activation of microglia in BDL mice is thought to subsequently recruit monocytes to the brain that contribute to the cognitive impairment observed (Kerfoot et al. 2006) .

It has long been accepted that ammonia plays an important role in the neurological symptoms of HE. Hyperammonemia has been demonstrated in the blood and cerebral cortex at all stages of HE in both ALF and in cirrhosis. Furthermore, injection of a bolus dose of ammonium acetate into rats in the absence of liver failure results in a transient comatose state, supporting a role for ammonia in the neurological decline of HE. In contrast, rats fed an ammonium-containing diet for up to 28 days display cognitive deficits more indicative of minimal HE (Felipo et al. 1988) . However, the role of ammonia toxicity in the activation of microglia and subsequent neuroinflammatory response is not as clearly defined. Treatment of primary microglial cultures with ammonia results in microglia swelling, migration , alterations in filipodia length, and Iba-1 expression (Zemtsova et al. 2011; Lachmann et al. 2013) . Similarly, increased Iba-1 immunoreactivity and expression was demonstrated in the cerebral cortex of the in vivo rat model of ammonia intoxication (Zemtsova et al. 2011). In contrast, injection of an acute bolus dose of ammonium acetate into mice had no effect on the morphology or turn-over rate of microglia (Rangroo Thrane et al. 2012) .

Microglial activation was evident in the cerebellum of chronic ammonium-fed rats, as demonstrated by in-depth morphometric analyses of MHCII positive cells (Rodrigo et al. 2010) . In support of a role for microglia activation in the cognitive deficits observed in this chronic hyperammonia model, treatment with ibuprofen reduced the microglia activation and reversed the concomitant cognitive impairments observed (Rodrigo et al. 2010) .