Metab Brain Dis DOI 10.1007/s11011-014-9502-y

ORIGINAL PAPER

Brain edema in acute liver failure: mechanisms and concepts Kakulavarapu V. Rama Rao & Arumugam R. Jayakumar & Michael D. Norenberg

Received: 7 January 2014 / Accepted: 5 February 2014 # Springer Science+Business Media New York 2014

Abstract Brain edema and associated increase in intracranial pressure continue to be lethal complications of acute liver failure (ALF). Abundant evidence suggests that the edema in ALF is largely cytotoxic brought about by swelling of astrocytes. Elevated blood and brain ammonia levels have been strongly implicated in the development of the brain edema. Additionally, inflammation and sepsis have been shown to contribute to the astrocyte swelling/brain edema in the setting of ALF. We posit that ammonia initiates a number of signaling events, including oxidative/nitrative stress (ONS), the mitochondrial permeability transition (mPT), activation of the transcription factor (NF-κB) and signaling kinases, all of which have been shown to contribute to the mechanism of astrocyte swelling. All of these factors also impact iontransporters, including Na+, K+, Cl− cotransporter and the sulfonylurea receptor 1, as well as the water channel protein aquaporin-4 resulting in a perturbation of cellular ion and water homeostasis, ultimately resulting in astrocyte swelling/ brain edema. All of these events are also potentiated by inflammation. This article reviews contemporary knowledge regarding mechanisms of astrocyte swelling/brain edema

The authors are most pleased to contribute this article in honor of Prof. Roger F. Butterworth, who has so greatly added our understanding of the pathogenesis of hepatic encephalopathy. K. V. Rama Rao (*) : A. R. Jayakumar : M. D. Norenberg (*) Department of Pathology, University of Miami Miller School of Medicine, PO Box 016960, Miami, FL 33101, USA e-mail: [email protected] e-mail: [email protected] M. D. Norenberg Department of Biochemistry & Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, USA A. R. Jayakumar : M. D. Norenberg Veterans Affairs Medical Center, Miami, FL 33125, USA

formation which hopefully will facilitate the identification of therapeutic targets capable of mitigating the brain edema associated with ALF. Keywords Acute liver failure . Ammonia . Aquaporin-4 . Astrocytes . Brain edema . Endothelial cells . Hepatic encephalopathy . Inflammation . Ion transporters . Microglia . Oxidative/nitrative stress . Vasogenic edema

Introduction Acute hepatic encephalopathy [acute liver failure (ALF); fulminant hepatic failure; acute HE] occurs in the setting of severe liver disease generally associated with viral hepatitis, acetaminophen overdose, mushroom poisoning and exposure to other hepatotoxins. Symptoms of ALF rapidly progress from initial confusion and agitation, to the development of delirium, seizures and coma which is associated with 60 % mortality (Lee 1994; Jalan et al. 2004; Larson et al. 2005). The only effective treatment currently available for ALF is an emergency liver transplantation (Bismuth et al. 1988; Hoofnagle et al. 1995).

Brain edema in ALF The cardinal feature of ALF is the development of brain edema and associated increase in intracranial pressure leading to brain herniation (Blei 1991), which along with multiorgan failure, chronic internal bleeding, as well as sustained infection contribute to the death of these patients (Ede and Williams 1986; Bernal et al. 2010; Lee et al. 2011). Brain edema in ALF was first described by Ware et al. (1971), and it was subsequently established as a characteristic feature of ALF (Ede and Williams 1986). The wealth of evidence

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suggests that the edema in ALF is cytotoxic, i.e., an intracellular accumulation of water (for reviews, see Traber et al. 1987; Blei 1991; Detry et al. 2006; Larsen and Wendon 2008). The principal neural cell that undergoes swelling in ALF is the astrocyte. Such swelling has been documented in experimental animals with ALF (Norenberg 1977; Traber et al. 1987; Matkowskyj et al. 1999), and in humans with ALF (Traber et al. 1987; Kato et al. 1992). Diffusion weighted MR sequences (MRI) conducted in patients with ALF strongly indicate a reduction in the size of the extracellular space (as calculated by decreased ADC values), which is highly indicative of an intracellular accumulation of water (Ranjan et al. 2005; Rai et al. 2008; Chavarria et al. 2011).

Etiological factors responsible for astrocyte swelling/brain edema in ALF Ammonia While the means by which astrocytes undergo swelling in ALF is not completely clear, a major factor in its pathogenesis is ammonia, whose levels are known to be increased in blood and CSF in experimental animals, as well as in patients with ALF (Blei et al. 1994; Clemmesen et al. 1999; Kramer et al. 2000; Ong et al. 2003). Moreover, astrocytes are the cells in brain that detoxify ammonia by converting glutamate into glutamine through a reaction mediated by glutamine synthetase, an enzyme in brain that is principally present in astrocytes (Martinez-Hernandez et al. 1977). A close correlation between increased levels of blood or brain ammonia and worsening of HE have been documented, while procedures that reduce blood or brain ammonia levels has been reported to alleviate HE (Clemmesen et al. 1999). Ammonia, at patho-physiologically relevant levels (5 mM NH4Cl), causes significant cell swelling when added to cultured astrocytes for more than 12 h (Norenberg et al. 1991; Olson et al. 1992; Faff-Michalak et al. 1994; Zwingmann et al. 2000; Konopacka et al. 2009). Lower concentrations of ammonia (0.5 and 1 mM) given for longer exposure periods (3 and 5 days) result in a similar degree of cell swelling in cultured astrocytes (Rao et al. 2013). At 3–5 mM for 24–48 h ammonia also causes astrocyte swelling in rat cerebral cortical slices and organotypic slice cultures from mouse forebrain (Ganz et al. 1989; Back et al. 2011). In summary, overwhelming evidence indicates a critical role of ammonia in the induction of astrocyte swelling/brain edema in the setting of ALF. A recent study (Rangroo Thrane et al. 2013), however, reported that exposure of cultured astrocytes to ammonia (10–30 mM for 30–60 min) did not result in astrocyte swelling, thereby questioning the role of ammonia in the production of cell swelling. It must be noted that the concentrations of ammonia employed in this study were exceptionally high and not pathophysiologically relevant. Also, the presence of

astrocyte swelling was examined only at 30–60 min following exposure to ammonia, time points too short to be significant as astrocyte swelling is not observed earlier than 12 h after ammonia treatment (Norenberg et al. 1991). While there are many interesting aspects of this study, its relevance to the pathogenesis of ALF is unclear. Infection/inflammation While ammonia continues to hold a primary role in the astrocyte swelling associated with ALF, systemic infection and inflammation have also been shown to contribute to the astrocyte swelling/brain edema in ALF (Wilkinson et al. 1974; Wyke et al. 1982; Odeh et al. 2004). Infections are a frequent complication of ALF as these patients display lowered resistance to infections (Mookerjee et al. 2007) and gram-negative bacteria were frequently identified in patients with worsening of ALF, which was associated with high blood levels of the bacterial endotoxin lipopolysaccahride (LPS) (Garcovich et al. 2012). Additional evidence suggests that proinflammatory cytokines, likely derived from liver necrosis and/or sepsis (Wilkinson et al. 1974; Wyke et al. 1982; Odeh et al. 2004), also plays an important role in the brain edema formation in ALF. Consistent with the role of inflammation in the astrocyte swelling/brain edema in ALF, we previously found that treatment of cultured astrocytes with inflammatory cytokines (TNF-α, IL-1β, IL-6 and IFN-γ) caused astrocyte swelling (Rama Rao et al. 2010a). Notably, the swelling caused by these cytokines was markedly potentiated when astrocytes were pre-treated with ammonia for 24 h, and then exposed to cytokines for an additional 24 h strongly suggesting a synergistic interaction between ammonia and inflammatory cytokines in the astrocyte swelling/brain edema associated ALF (Rama Rao et al. 2010a). A compelling role of inflammatory cytokines in the induction of brain edema in ALF was also established when transgenic mice deficient in receptors for TNF-α and IL-1β, IFN-γ were found to be resistant to ALF-associated brain edema (Jiang et al. 2009a, b). While the means by which cytokines (CKs) induce astrocyte swelling is not completely clear, it was shown that CKs activate NF-κB in cultured astrocytes (discussed below), while BAY 11-7082 and SN50, inhibitors of NF-κB, significantly mitigated the astrocyte swelling caused by CKs (Rama Rao et al. 2010a), suggesting that NF-κB-dependent mechanisms contribute to the astrocyte swelling produced by CKs.

Mechanisms of astrocyte swelling A number of factors implicated in the pathogenesis of ALF have also been shown to contribute to the astrocyte swelling/ brain edema in ALF. These include oxidative/nitrative stress

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(ONS), signaling kinases, the mitochondrial permeability transition, and activation of NF-κB. All of these factors impact on one or more ion transporters, as well as the water channel protein aquaporin-4, ultimately contributing to the astrocyte swelling/brain edema in ALF. The following sections describe the means by which these factors contribute to astrocyte swelling.

Oxidative/nitrosative stress (ONS) O’Connor and Costell (1990) were the first investigators to implicate oxidative stress in the pathogenesis of HE. They found that hyperammonemic mice displayed a marked increase in lipid peroxidation, which was subsequently identified in non-synaptic mitochondria in an experimental model ALF (Reddy et al. 2004). Cultured astrocytes treated with ammonia have also been shown to cause lipid peroxidation (Murphy et al. 1992). An increase in lipofuscin pigment (evidence of lipid peroxidation) in astrocytes is a characteristic histopathological feature of HE (Norenberg 1981). The involvement of ONS in the pathogenesis of hepatic encephalopathy (HE) has previously been reviewed (Norenberg et al. 2004; Häussinger and Görg 2010; Skowronska and Albrecht 2013). Ammonia was shown to generate free radicals in a rat model of hyperammonemia (Kosenko et al. 1996, 1997), and in cultured astrocytes (Murthy et al. 2001; Görg et al. 2008). Additionally, decreased activity of antioxidant enzymes (glutathione peroxidase, superoxide dismutase and catalase), and increased superoxide production in brain were found in a rat model of hyperammonemia (Kosenko et al. 1997). Similarly, increased levels of heme-oxygenase-1 (a marker of oxidative stress) were found in brains of portacaval-shunted rats (a model of chronic HE) (Warskulat et al. 2002), as well as in rats with ALF induced by the hepatotoxin thioacetamide (TAA) (Rama Rao et al. 2010c). ONS has also been identified in postmortem brains from humans with HE (Görg et al. 2010). Oxidative stress is known to result in cell swelling in brain slices (Chan et al. 1982; Brahma et al. 2000), and in cultured astrocytes (Chan et al. 1982; Staub et al. 1994; Jayakumar et al. 2006). Moreover, antioxidants, including superoxide dismutase, catalase, vitamin E and N-tert-butyl-α-phenylnitrone (PBN), were all shown to block the ammonia-induced astrocyte swelling (Jayakumar et al. 2006). Antioxidants, particularly Nacetylcysteine, were effective in mitigating the brain edema in experimental models, as well as in patients with ALF (for review, see Vaquero and Butterworth 2007). In aggregate, these studies provide strong support for a key role of ONS in the astrocyte swelling/brain edema associated with ALF .

Nuclear factor-kappaB (NF-κB) The transcription factor NF-κB was shown to be activated in cultured astrocytes exposed to ammonia (Schliess et al. 2004; Sinke et al. 2008), as well as in experimental ALF (Jayakumar et al. 2011; Shah et al. 2013). Inflammatory cytokines (TNF-α, IL-1β, IL-6 and IFN-γ) were found to markedly potentiate the ammonia-mediated activation of NF-κB (Rama Rao et al. 2010a). Such activation is known to induce oxidative stress, additional inflammation [production of cytokines, activation of phospholipase A2 (PLA2), cyclooxygenase 2 (COX2), as well as the inducible form of nitric oxide synthase (iNOS)], all factors well-known to induce astrocyte swelling (for review see Zemtsova et al. 2011). It was previously reported that inhibitors of NF-κB (BAY 11-7082, SN 50) blocked the ammonia-induced astrocyte swelling in cultures (Sinke et al. 2008). Likewise, transgenic mice with a functional inactivation of astrocytic NF-κB were resistant to the development of brain edema in experimental ALF induced by TAA (Jayakumar et al. 2011). Mitogen-activated protein kinases (MAPKs) Ammonia was been shown to activate MAPKs, including ERK1/2, JNK1/2/3 and p38-MAPK (Schliess et al. 2002; Jayakumar et al. 2006; Bodega et al. 2007) and inhibition of these kinases differentially blocked the ammonia-induced astrocyte swelling (Jayakumar et al. 2006), suggesting an important role of these kinases in the mechanism of astrocyte swelling. The mitochondrial permeability transition (mPT) The mPT is a Ca+2-dependent process characterized by opening of the permeability transition pore present in the inner mitochondrial membrane. Opening of the pore results in increased permeability to protons, ions and other solutes ≤1,500 Da leading to a collapse of the mitochondrial inner membrane potential, ultimately resulting in decreased oxidative phosphorylation and bioenergetic failure (Zoratti and Szabo 1995). The induction of the mPT can also lead to a secondary oxidative stress (Zorov et al. 2000; Votyakova and Reynolds 2005). A specific inhibitor of the mPT is cyclosporine A (CsA) (Broekemeier et al. 1989). We previously reported that treatment of cultured astrocytes with ammonia (5 mM NH4Cl) resulted in the induction of the mPT (Bai et al. 2001). The mPT was also induced in the TAA rat model of ALF (Rama Rao et al. 2010c). Inflammatory cytokines, including TNF-α, IL-1β, IL-6 and IFN-γ, also induced the mPT in cultured astrocytes (Alvarez et al. 2011). The induction of the mPT in cultured astrocytes displayed a marked synergism when cells were exposed to

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ammonia followed by treatment with cytokines (Alvarez et al. 2011). The mPT was shown to contribute to the astrocyte swelling by ammonia as CsA completely blocked such swelling (Rama Rao et al. 2003b). CsA also partially (35 %) mitigated the brain edema in experimental ALF induced in rats by the administration of TAA (Norenberg et al. 2007). A recent study, however, reported CNS toxicity after treatment of portacaval-shunted rats with CsA. The investigators also showed that CsA did not mitigate the brain edema in these animals (Larsen et al. 2013). We similarly identified increased mortality associated with the use of CsA in rats with ALF after TAA treatment (unpublished observation). In addition to CsA, other agents capable of blocking the mPT include trifluoparazine, magnesium, pyruvate and Lhistidine (Norenberg and Rao 2007). These agents were recently examined for their effect on ammonia-induced astrocyte swelling. We found that all of these agents inhibited the ammonia-induced cell swelling in cultured astrocytes to a differential degree (Reddy et al. 2009). Additionally, Lhistidine also blocked the brain edema in rats with ALF induced by TAA (Rama Rao et al. 2010b). Together, these studies implicate the mPT in the mechanism of astrocyte swelling/brain edema in ALF. The means by which the mPT contributes to brain edema is not known. However, the mPT represents an additional source of free radicals (Zorov et al. 2000; Votyakova and Reynolds 2005), which is known to cause brain edema (Ringel et al. 2006). It is also possible that bioenergetic failure following the induction of the mPT, may compromise the activity of iontransporters necessary for cell volume regulation. Ion transporters and aquaporin-4 While ONS, the mPT, signaling kinases, and NF-κB have been shown to contribute to the astrocyte swelling/brain edema in ALF, these factors ultimately must impact on ion transporters as well as aquaporins required for proper volume regulation (for reviews, see Hoffmann 1992; PasantesMorales 1996; Hoffmann et al. 2009; Pasantes-Morales and Cruz-Rangel 2010). Various studies have examined the importance of ion transporters and water channels including the Na–K–2Cl cotransporter (NKCC), and the SUR1-regulated nonselective cation channel (NCCa-ATP) and aquaporin-4 (AQP4), in the mechanism of astrocyte swelling/brain edema in ALF. NKCC is involved in the regulation of cell volume and ion gradients (Kaplan et al. 1996). NKCC is an electroneutral transporter involved in the influx of Na+, K+, and Cl− into cells with a stoichiometry of 1Na+, 1K+, and 2Cl− (Isenring and Forbush 2001). The transport of these ions is associated with obligatory water entry leading to the regulation of cell volume, while over-activation

of NKCC will lead to cell swelling and tissue edema. Specific inhibitors of NKCC include bumetanide or furosemide (Isenring and Forbush 2001). Ammonia was shown to activate NKCC in cultured astrocytes and bumetanide significantly mitigated the ammoniainduced astrocyte swelling (Jayakumar et al. 2008). NKCC was also found to be activated in rats treated with TAA, and the administration of bumetanide to TAA-treated rats significantly lessened the severity of the brain edema, implicating NKCC in the brain edema associated with ALF. Another ion transporter involved in the mechanism of astrocyte swelling/brain edema is the ATP-dependent, nonselective cation channel (NCCa-ATP channel) which regulates the transport of most inorganic cations in brain (for review, see Walcott et al. 2012). The sulfonylurea receptor type 1 protein (SUR1), is coupled to and regulates the pore-forming region of the NCCa-ATP channel in astrocytes (Simard et al. 2007). An increase in SUR1 gene expression provides a valid measure of NCCa-ATP channel activity (Simard et al. 2007). Gliblenclamide is a specific inhibitor of this channel (Simard et al. 2007). Ammonia was found to upregulate both mRNA and protein expression of SUR 1 (Jayakumar et al. 2014). A similar upregulation was found in brains of rats with ALF induced by TAA. Gliblenclamide significantly inhibited the ammoniainduced astrocyte swelling in culture, as well as the brain edema in rats with ALF (Jayakumar et al. 2014), implicating the NCCa-ATP channel in the mechanism of astrocyte swelling/brain edema in ALF. Activation of ion-channels and the subsequent transport of various ions into cells require an obligatory entry of water into cells, a process mediated by aquaporin water channels (King and Agre 1996). AQP4, the principal water channel in astrocytes (Nielsen et al. 1997), has been implicated in the development of brain edema in various neurological conditions, including ischemic stroke, traumatic brain injury, brain neoplasms and hyponatremia (for review, see Papadopoulos and Verkman 2013). Knock-out mice lacking AQP4 are resistant to the development of cytotoxic brain edema following hyponatremia and ischemic stroke (Manley et al. 2000). AQP4 was shown to be upregulated in the plasma membrane of cultured astrocytes exposed to ammonia (Rama Rao et al. 2003a; Bodega et al. 2012). Likewise, increased brain plasma membrane levels of AQP4 were found in rats with ALF induced by TAA (Rama Rao et al. 2010b) or galactosamine (Eefsen et al. 2010). A similar upregulation of AQP4 was observed in human postmortem brain tissue from patients with of ALF (Thumburu et al. 2013). We recently examined the role of AQP4 in ALF by using transgenic mice deficient in AQP4 (AQP4-null mice) (Rama Rao et al. 2013). These mice displayed a marked resistance to the development of brain edema following the induction of ALF with TAA or acetaminophen. These mice also showed a

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marked reduction in neurological symptoms as compared to wild-type mice (Rama Rao et al. 2013). Ammonia-induced factors, including ONS, the mPT, signaling kinases and NF-κB, are known to impact on NKCC, SUR 1 and AQP4 in cultured astrocytes as well as in experimental ALF, ultimately leading to astrocyte swelling/brain edema. A schematic diagram is provided in Fig. 1 illustrating interactions among these factors.

Cell-cell interactions in the mechanism of astrocyte swelling/brain edema in ALF Although ammonia is capable of directly impacting astrocytes and causing cell swelling, other neural cells (especially endothelial cells and microglia) may also contribute to the astrocyte swelling in ALF. Endothelial cells (ECs, the resident neural cells first exposed to toxins from blood, e.g., ammonia) and microglia are both known to evoke inflammatory responses, and to contribute to the neuroinflammation associated with ALF (Shawcross and Jalan 2005; Butterworth 2013). An increase in inflammatory mediators, including CKs and lipopolysaccahride (LPS) may activate ECs in the presence of necrosis of liver or sepsis. ECs are known to stimulate the production of inflammatory mediators following injury, including inducible-nitric oxide synthase (iNOS), phospholipase A2 (PLA2) and cyclooxygenase 2 (COX 2) (del Zoppo 2009). As noted above, the products of these factors (nitric oxide, arachidonic acid and prostaglandin E) are all well known to cause astrocyte swelling (Norenberg et al. 2009).

Fig. 1 Schematic diagram illustrating interactions among ammonia-induced factors impacting on Na + ,K + 2Cl - cotransporter (NKCC), sufonylurea receptor 1 (SUR 1) and aquaporin-4 (AQP4), ultimately leading to astrocyte swelling. ONS oxidative/nitrative stress; MAPKs mitogen activated protein kinases; mPT mitochondrial permeability transition

We recently treated ECs (3–24 h) with ammonia, LPS or a mixture of CKs (TNF-α, IL-1β and Il-6, IFN-γ). Following replacement of the treatment media, conditioned media (CM) from ECs were added to cultured astrocytes and such treatment resulted in a significant degree of cell swelling (35– 50 %) (Jayakumar et al. 2012). Notably, the addition of CM from ECs exposed to a combination of ammonia, LPS and a mixture of CKs to astrocytes showed a marked potentiation in ammonia-induced astrocyte swelling (Jayakumar et al. 2012). These findings indicate that CKs and LPS impact ECs to ultimately exacerbate the ammonia-induced astrocyte swelling. Ammonia is also capable of activating microglia as documented in experimental models of ALF, as well as in a rat model of hyperammonemia (Jiang et al. 2009a, b; Bruck et al. 2011; Rodrigo et al. 2010; Görg et al. 2013; Zemtsova et al. 2011). We recently found that CM from primary cultures of microglia previously exposed to ammonia (12 and 24 h) and then replaced with fresh media (and kept for additional 24 h) caused a significant cell swelling when added to astrocytes (Rao et al. 2013), implicating microglia in the mechanism of astrocyte swelling/brain edema in ALF. One common factor responsible for the release of inflammatory mediators from ECs and microglia following exposure to ammonia is the toll-like receptor 4 (TLR4). TLR-4 is a plasma membrane receptor which in brain is predominantly present in ECs and microglia (Buchanan et al. 2010). Activation of TLR4 produces various inflammatory mediators, including free radicals, nitric oxide, arachidonic acid, as well as prostaglandins, factors that are well-known to cause astrocyte swelling. Ammonia was shown to activate TLR-4 in ECs and exposure of astrocytes to CM from TLR4-silenced ECs that were treated with ammonia resulted in a significant reduction in astrocyte swelling (Jayakumar et al. 2013b). Similar to ECs, activation of TLR-4 was also found in microglia following treatment with ammonia. When CM from ammonia-treated microglia in which the TLR-4 gene was silenced, was added to astrocytes, such treatment resulted in a significant reduction in cell swelling (unpublished observations) supporting the role of TLR-4 in the mechanism of astrocyte swelling. Upregulation of TLR4 was found in brains of rats with experimental ALF induced by TAA (Jayakumar et al. 2013b). Additionally, transgenic mice with a gene-deletion of TLR-4 (TLR-4-KO mice) showed significant resistance to the development of brain edema following ALF (Jayakumar et al. 2013b). Consistent with these finding, Shah et al. (2013) reported that STM-28, an antagonist of TLR-4, when given to mice with ALF induced by acetaminophen resulted in a significant reduction in brain edema. Likewise, TLR4-KO mice also showed resistance to development of brain edema in acetaminophen-induced ALF (Shah et al. 2013). However,

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in addition to reducing brain edema, this study also found a marked improvement of liver function in TLR-4-KO mice following induction of ALF with acetaminophen. These investigators concluded that an improvement in liver function was largely responsible for the observed reduction in brain edema in TLR-KO mice. This observation is in contrast with our recent finding (Jayakumar et al. 2013b), wherein we observed no improvement in liver function in TLR-KO mice following the administration of TAA; yet, we observed a reduction in brain edema in these mice. The reason for such differential effect between two models of ALF relative to the improvement of liver function and brain edema may be due to the nature of hepatotoxins (i.e., acetaminophen vs. TAA). Nevertheless, both in vitro and in vivo findings (Jayakumar et al. 2013b) strongly support the view that activation of TLR4 in brain in ALF contributes to the brain edema. A schematic diagram illustrating the mechanisms by which endothelial cells and microglia impact astrocytes to cause cell swelling/ brain edema in ALF is given in Fig. 2.

Therapeutic implications A number of factors (ONS, NF-κB, MAPKs and the mPT) as well as ion transporters (NKCC, SUR 1) and AQP4 have been implicated in the mechanism of astrocytes swelling in experimental ALF. All of these represent potential therapeutic targets in ALF. Recent reports as noted above, have indeed shown beneficial effects of agents that inhibit these factors including bumetanide, gliblenclamide, L-histidine, as well as the antioxidant N-acetylcysteine (NAC) and inhibitors of Fig. 2 Schematic diagram depicting endothelial cellastrocyte and microglial interactions in the mechanism of astrocyte swelling. Ammonia, cytokines, CKs and lipopolysaccahride, LPS, all activate toll-like receptor 4 (TLR4) in both endothelial cells and microglia, and such activation results in the release of inflammatory mediators (reactive oxygen and nitrogen species, RONS; cytokines, CKs; arachidonic acid; prostaglandins; nitric oxide) that ultimately contribute to astrocyte swelling

MAPKs. These agents appear promising candidates for the treatment of patients with ALF.

Vasogenic edema in ALF While the vast literature strongly supports the cytotoxic nature of brain edema associated with ALF (Norenberg 1977; Kato et al. 1989; Traber et al. 1989; Szumanska and Albrecht 1997), some reports also implicate the coexistence of vasogenic edema in ALF (Cauli et al. 2011, 2013; Nguyen et al. 2006). Such vasogenic edema might indeed occur when secondary complications of ALF, including sepsis, hypotension and brain hypoxia are present in ALF. These complications need to be excluded when invoking a vasogenic mechanism for the brain edema in experimental animals as well as in patients with ALF. Additionally, reports showing alterations in tight junctional proteins (e.g., claudin, occludin) in ALF do not by themselves necessarily indicate a breakdown of blood brain barrier (BBB) and subsequent vasogenic edema, as compensatory mechanisms may have been triggered in response to changes in tight-junctional proteins. Caution should also be exercised when using hepatotoxins in experimental models of ALF since an important confounding factor in drug-induced experimental ALF is the possibility of a direct effect of the hepatotoxin on brain endothelial cells. This possibility has recently been examined by using cell culture model of the BBB (co-cultures of primary astrocyteendothelial cells) (Jayakumar et al. 2013a). This study found that exposure of these cultures with a concentration of azoxymethane equivalent to that given to mice caused a marked increase (220 %) in endothelial cell permeability to

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fluorescein isothiocyanate, indicating a compromised integrity of the BBB (Jayakumar et al. 2013a). Accordingly, agents known to directly impact on the cerebral vasculature (in addition to causing liver injury) should not be employed in studies of ALF. It is therefore important to exclude such vascular adverse agents in models of ALF.

Concluding remarks Brain edema in the setting of ALF remains a challenging clinical problem. Ammonia represents the dominant etiological factor, although complicating events, particularly infection/inflammation may aggravate the ammonia-induced brain edema. A set of reactions triggered by ammonia leading to astrocyte swelling/brain edema in ALF include oxidative/ nitrative stress, induction of the mPT (an additional source of free radicals), as well as the activation of intracellular signaling kinases (MAPKs) and NF-κB. Such activation perturbs astrocytic ion and water homeostasis by altering the expression of ion-transporters and water channel protein aquaporin4, eventually resulting in astrocyte swelling/brain edema. We anticipate that agents capable of mitigating these signaling factors will reduce ALF-related brain edema. Acknowledgments This work was supported by NIH Grant DK06331 and by a Department of Veterans Affairs Merit Review Award.

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