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 astro-
cytes. 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 mito-
chondrial permeability transition (mPT), activation of the
transcription factor (NF-B) and signaling kinases, all of
which have been shown to contribute to the mechanism of
astrocyte swelling. All of these factors also impact ion-
transporters, 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
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); ful-
minant 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 infec-
tion 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
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: vkakulavarapu@med.miami.edu
e-mail: mnorenbe@med.miami.edu
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
Metab Brain Dis
DOI 10.1007/s11011-014-9502-y
suggests that the edema in ALF is cytotoxic, i.e., an intracel-
lular 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 strong-
ly indicate a reduction in the size of the extracellular space (as
calculated by decreased ADC values), which is highly indic-
ative 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 ALFis
not completely clear, a major factor in its pathogenesis is ammo-
nia, 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 docu-
mented, 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
NH
4
Cl), 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 35 mM for 2448 h ammonia also causes
astrocyte swelling in rat cerebral cortical slices and organotypic
slice cultures frommouse 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
(1030 mM for 3060 min) did not result in astrocyte swell-
ing, 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 3060 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 astro-
cyte swelling associated with ALF, systemic infection and
inflammation have also been shown to contribute to the astro-
cyte 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 resis-
tance 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 proinflamma-
tory cytokines, likely derived fromliver 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 treat-
ment of cultured astrocytes with inflammatory cytokines
(TNF-, IL-1, IL-6 and IFN-) 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 inflam-
matory cytokines in the induction of brain edema in ALF was
also established when transgenic mice deficient in receptors
for TNF- and IL-1, IFN- 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-Bin
cultured astrocytes (discussed below), while BAY 11-7082
and SN50, inhibitors of NF-B, significantly mitigated the
astrocyte swelling caused by CKs (Rama Rao et al. 2010a),
suggesting that NF-B-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
Metab Brain Dis
(ONS), signaling kinases, the mitochondrial permeability
transition, and activation of NF-B. 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)
OConnor and Costell (1990) were the first investigators to
implicate oxidative stress in the pathogenesis of HE. They
found that hyperammonemic mice displayed a marked in-
crease in lipid peroxidation, which was subsequently identi-
fied 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 in-
volvement of ONS in the pathogenesis of hepatic encepha-
lopathy (HE) has previously been reviewed (Norenberg et al.
2004; Hussinger and Grg 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; Grg et al.
2008). Additionally, decreased activity of antioxidant en-
zymes (glutathione peroxidase, superoxide dismutase and cat-
alase), 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 (Grg 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--phenylnitrone (PBN), were all shown to
bl ock t he ammoni a-i nduced ast rocyt e swel l i ng
(Jayakumar et al. 2006). Antioxidants, particularly N-
acetylcysteine, 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-B)
The transcription factor NF-B 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-, IL-1, IL-6 and IFN-) were found to markedly
potentiate the ammonia-mediated activation of NF-B
(Rama Rao et al. 2010a). Such activation is known to induce
oxidative stress, additional inflammation [production of cyto-
kines, activation of phospholipase A2 (PLA2), cyclooxygen-
ase 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 previ-
ously reported that inhibitors of NF-B (BAY 11-7082, SN
50) blocked the ammonia-induced astrocyte swelling in cul-
tures (Sinke et al. 2008). Likewise, transgenic mice with a
functional inactivation of astrocytic NF-B 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 as-
trocyte swelling (Jayakumar et al. 2006), suggesting an im-
portant 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 open-
ing of the permeability transition pore present in the inner
mitochondrial membrane. Opening of the pore results in in-
creased permeability to protons, ions and other solutes
1,500 Da leading to a collapse of the mitochondrial inner
membrane potential, ultimately resulting in decreased oxida-
tive 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 cyclospor-
ine A (CsA) (Broekemeier et al. 1989).
We previously reported that treatment of cultured astro-
cytes with ammonia (5 mM NH
4
Cl) resulted in the induction
of the mPT (Bai et al. 2001). The mPTwas also induced in the
TAA rat model of ALF (Rama Rao et al. 2010c).
Inflammatory cytokines, including TNF-, IL-1, IL-6 and
IFN-, 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
Metab Brain Dis
ammonia followed by treatment with cytokines (Alvarez et al.
2011).
The mPTwas 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 CsAin rats with ALF after
TAA treatment (unpublished observation).
In addition to CsA, other agents capable of blocking the
mPT include trifluoparazine, magnesium, pyruvate and L-
histidine (Norenberg and Rao 2007). These agents were re-
cently examined for their effect on ammonia-induced astro-
cyte 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, L-
histidine 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 mPTcontributes to brain edema is
not known. However, the mPTrepresents 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 ion-
transporters necessary for cell volume regulation.
Ion transporters and aquaporin-4
While ONS, the mPT, signaling kinases, and NF-B have
been shown to contribute to the astrocyte swelling/brain ede-
ma in ALF, these factors ultimately must impact on ion
transporters as well as aquaporins required for proper volume
regulation (for reviews, see Hoffmann 1992; Pasantes-
Morales 1996; Hoffmann et al. 2009; Pasantes-Morales and
Cruz-Rangel 2010). Various studies have examined the im-
portance of ion transporters and water channels including the
NaK2Cl 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
i on gradi ent s (Kapl an et al . 1996). NKCC i s 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 astro-
cytes and bumetanide significantly mitigated the ammonia-
induced 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 signifi-
cantly 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, non-
selective 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 mea-
sure 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 pro-
tein 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 ammonia-
induced 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 astro-
cytes (Nielsen et al. 1997), has been implicated in the devel-
opment of brain edema in various neurological conditions,
including ischemic stroke, traumatic brain injury, brain neo-
plasms 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 mem-
brane 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 galactos-
amine (Eefsen et al. 2010). A similar upregulation of AQP4
was observed in human postmortem brain tissue frompatients
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
Metab Brain Dis
marked reduction in neurological symptoms as compared to
wild-type mice (Rama Rao et al. 2013).
Ammonia-induced factors, including ONS, the mPT, sig-
naling kinases and NF-B, are known to impact on NKCC,
SUR 1 and AQP4 in cultured astrocytes as well as in exper-
imental 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 endo-
thelial 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, in-
cluding inducible-nitric oxide synthase (iNOS), phospholi-
pase 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).
We recently treated ECs (324 h) with ammonia, LPS or a
mixture of CKs (TNF-, IL-1 and Il-6, IFN-). Following
replacement of the treatment media, conditioned media (CM)
from ECs were added to cultured astrocytes and such treat-
ment 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 docu-
mented 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; Grg 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 inflam-
matory mediators fromECs 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 media-
tors, 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 expo-
sure 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 devel-
opment 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,
Fig. 1 Schematic diagram illustrating interactions among ammonia-in-
duced 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
Metab Brain Dis
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 in-
vestigators 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 viewthat 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-B, MAPKs and the mPT) as
well as ion transporters (NKCC, SUR 1) and AQP4 have been
implicated in the mechanism of astrocytes swelling in exper-
imental ALF. All of these represent potential therapeutic tar-
gets 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
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 compen-
satory 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 confound-
ing 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 astrocyte-
endothelial 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
Fig. 2 Schematic diagram
depicting endothelial cell-
astrocyte 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
Metab Brain Dis
fluorescein isothiocyanate, indicating a compromised integri-
ty 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 etiologi-
cal 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 signal-
ing kinases (MAPKs) and NF-B. Such activation perturbs
astrocytic ion and water homeostasis by altering the expres-
sion of ion-transporters and water channel protein aquaporin-
4, 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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