ORAL PRESENTATION
(Oenarto et al. 2016, Sci Rep 6:18493), which is known to SESSION 3: PATHOGENESIS – 3 (BRAIN
liberate iron from heme. The aim of the present study was
to examine the effects of ammonia on the intracellular
content of free ferrous iron [Fe2+]i and its relevance for
ammonia-induced astrocyte senescence.
Results: As shown by superresolution microscopy, treat-
ment of astrocytes with NH4Cl (5 mmol/l) for up to 72 h
strongly changed the fluorescence emission of the ferrous
iron-chelating reporter dyes RhoNox1 and Rhodamine B-
[(2,20 -bipyridine-4-yl)-aminocarbonyl]benzyl ester (RDA) in
vital astrocytes indicative for an elevation of [Fe2+]i. By using
organelle specific fluorescent dyes and live cell imaging, RDA
fluorescence was detected in mitochondria and RhoNox1
fluorescence was found in lysosomal compartments. NH4Cl
(5 mmol/l, 72 h)-induced RhoNox1 and RDA fluorescence
changes were abolished by the iron chelators 2,20 -bipyridine
(BIP) or pyridoxal isonicotinoyl hydrazone (PIH), respec-
tively. NH4Cl (5 mmol/l, 72 h) exposure also strongly
changed mRNA expression levels of genes involved in iron
metabolism in astrocytes. Whereas transcription of the iron
exporter ferroportin (FPN) was enhanced, mRNA levels of
the FPN accessory factors ceruloplasmin and hephaestin
were decreased by NH4Cl (5 mmol/l) in astrocytes.
NH4Cl-induced mRNA expression changes of ferroportin,
ORAL PRESENTATION
ceruloplasmin and hephaestin were mimicked by treating
astrocytes with ferrous iron (25 mmol/l, 18 h) and were
completely prevented by the glutamine synthetase inhibitors
methionine-sulfoximine (3 mmol/l) or phosphinothricin
(100 mmol/l) as well as by the ferrous iron chelators BIP
(150 mmol/l) or PIH (100 mmol/l). Intracellular chelation of
ferrous iron using BIP or PIH fully prevented NH4Cl
(5 mmol/l, 72 h)-induced astrocyte proliferation-inhibition.
Expression levels of several genes involved in iron metabo-
lism such as natural resistance-associated macrophage pro-
tein (Nramp) 1, ceruloplasmin or hepcidin were also
changed in post mortem brain samples of patients with liver
cirrhosis and HE but not in those without HE.
Conclusion: The present study suggests that ammonia
increases the concentration of free ferrous iron in mito-
chondria and autophagosomes/lysosomes thereby pro-
moting astrocyte senescence. Altered expression of genes
involved in iron metabolism in cerebral cortex in patients
with liver cirrhosis and HE suggests that iron metabolism
is also disturbed in brain in HE.
CONFLICTS OF INTEREST
The authors have none to declare.
Corresponding author: Vinoth Boopathy.
E-mail: boris.goerg@uniuesseldorf.de
http://dx.doi.org/10.1016/j.jceh.2017.01.006
S4
EDEMA)
5
ROLE OF ENDOTHELIAL NMDA
RECEPTORS IN THE PATHOGENESIS OF
HEPATIC ENCEPHALOPATHY
Marta Skowronska 1,2,3,4, Arumugam R. Jayakumar 1,2,3,4,
Jan Albrecht 1,2,3,4, Michael D. Norenberg 1,2,3,4
1
Department of Biochemistry and Molecular Biology, Laboratory of
Neuropathology, Department of Neurotoxicology, University of Miami,
Miami, United States, 2Veterans Affairs Medical Center, Miami, United
States, 3Mossakowski Medical Research Center, Warsaw, Poland,
4
Department of Pathology, University of Miami, Miami, United States
Background: Excessive accumulation of ammonia in the
brain is the main etiological factor in hepatic encephalop-
athy (HE), a neuropsychiatric syndrome resulting from
acute or chronic liver failure (ALF/CLF). Rapid progression
of ALF is associated with brain edema, which often leads to
death as a consequence of increased intracranial pressure
and brain herniation. The brain edema is mainly cytotoxic,
resulting from astrocytic swelling and ammonia was
shown to be a key pathogenic factor. Recent findings,
however, suggest that ammonia also impairs brain endo-
thelial cells which may lead to a subtle increase in blood
brain barrier (BBB) permeability, possibly contributing to
the formation of brain edema. Even though, the precise
mechanisms involved in ammonia-induced endothelial
cell alterations are not completely understood, we recently
observed that treatment of endothelial cells with ammonia
resulted in their increased permeability by a process medi-
ated by oxidative/nitrosative stress (ONS). One potential
mechanism by which ammonia induces ONS in astrocytes
is over-stimulation of NMDA receptors (NMDA-R) that
are known to be also present on brain endothelial cells.
Objectives: In the present study, we hypothesize that
ammonia activates endothelial NMDA receptors, resulting
in ONS and leading to a selective BBB permeability, which
ultimately contributes to cytotoxic brain edema.
Methods: To examine whether ammonia activates
endothelial NMDA-R, we measured intracellular Ca2+ lev-
els in primary rat microvascular endothelial cells
(RBMECs), in the presence or absence of NMDA-R block-
ers (MK-801, memantine). Employing fluorescent probes,
we assessed whether the ammonia induced NMDA-R acti-
vation stimulates ONS. Further, different signaling path-
ways involved in NMDA-R mediated effects were
determined by in vitro permeability assay. To examine
the role of activation of NMDA-R in the HE-related
BBB permeability, we induced ALF in rats by administra-
tion of a hepatotoxin, thioacetamide (TAA). Memantine
was administered intraperitoneally alone or together with
TAA. Different molecular weight markers were injected
© 2017, INASL
 JOURNAL OF CLINICAL AND EXPERIMENTAL HEPATOLOGY
intravenously, and the extravasation of tracers was
analyzed.
Results: Results disclosed that treatment of RBMECs
with 5 mM ammonia increased intracellular Ca2+ accumu-
lation, while pretreatment with MK-801 or memantine
attenuated such Ca2+ accumulation. As expected, ammo-
nia increased total ROS production by 75%. Pretreatment
with MK-801 or memantine decreased ROS accumulation
in ammonia-treated cells, strongly suggesting that the
ONS induced by ammonia in RBMEC is, in part, mediated
by activation of NMDA-R. BBB permeability to 10 kDa
FITC-dextran was increased 2-fold in rats with ALF, when
compared to controls. Memantine prevented the ammo-
nia-induced changes in permeability, resulting in a return
of 10 kDa dextran extravasation to basal levels.
Conclusion: In summary, these results provide novel
and important information on the role of endothelial
NMDA receptors in BBB dysfunction associated with
HE that will bring us closer to a better understanding
of the mechanisms involved in the development of the
brain edema in acute liver failure.
CONFLICTS OF INTEREST
The authors have none to declare.
Corresponding author: Mayank Gupta.
E-mail: m.skowronska@med.miami.edu
http://dx.doi.org/10.1016/j.jceh.2017.01.007
6
NEURONAL DYSFUNCTION IN CHRONIC
HEPATIC ENCEPHALOPATHY: ROLE OF
ASTROCYTIC MATRICELLULAR PROTEINS
Michael D. Norenberg 1,2, A.R. Jayakumar 1,2
1
Miami Veterans Affairs Medical Center, Miami, FL, USA, 2Departments
of Pathology, Biochemistry & Molecular Biology, Neurology and
Neurosurgery, University of Miami School of Medicine, Miami, FL
33125, USA
Chronic hepatic encephalopathy (CHE), due to chronic
liver failure, is characterized by confusion, disorientation,
behavioral changes, impaired cognition, and motor dis-
turbances. It is associated with defective neuronal integrity,
leading to neurobehavioral and cognitive impairments.
The mechanisms responsible for the neurological abnor-
malities in CHE, however, remain largely unknown. Since a
reduction in astrocytic secretion of matricellular proteins
(MCPs), including thrombospondin-1 (TSP-1), Hevin, Gly-
picans 4 and 6, and the CCN family of proteins (CYR61/
CTGF/NOV), have been implicated in the neuronal
Journal of Clinical and Experimental Hepatology | February 2017 | Vol. 7 | No. S1 | S1–S21
dysfunction associated with various neurological condi-
tions (e.g., ischemia, Alzheimer's disease, Down's syn-
drome), we examined whether astrocytic MCP synthesis
and release may likewise be affected in CHE, and whether
this event contributes to the defective neuronal integrity
and associated neurobehavioral and cognitive impair-
ments that occur in CHE. Exposure of cultured astrocytes
to ammonia (NH4Cl, 1 mM) for 10–15 days resulted in a
decrease in intra- and extracellular levels of TSP-1, Hevin,
Glypicans 4 and 6, nerve growth factor, as well as in basic
fibroblast growth factor. These changes were associated
with a decrease in levels of specificity protein-1, activator
protein-1, Forkhead box O, and transforming growth fac-
tor-beta, factors known to enhance the synthesis and
release of MCPs. Exposure of cultured neurons to condi-
tioned media (CM) from ammonia-treated astrocytes
showed a decrease in synaptophysin, PSD95 and synapto-
tagmin levels. CM from TSP-1 overexpressing astrocytes
(by the addition of TSP-1 cDNA) that were treated with
ammonia, when added to cultured neurons, reversed the
decline in synaptic proteins. We also found a significant
decline in TSP-1, Glypicans 4 and 6, CCN1 and CCN4
levels in cortical astrocytes, as well as a reduction in levels
of synaptic proteins in an in vivo rat model of CHE (treat-
ORAL PRESENTATION
ment with the liver toxin, thioacetamide). Additionally,
treatment of rats with Metformin, an agent known to
increase levels of MCPs in other conditions, attenuated
the neurobehavioral abnormalities observed in CHE. Our
findings suggest that increasing brain levels of MCPs may
represent a useful therapeutic approach for patients with
chronic hepatic encephalopathy.
CONFLICTS OF INTEREST
The authors have none to declare.
Corresponding author: Santosh K. Yadav.
E-mail: mnorenbe@med.miami.edu
http://dx.doi.org/10.1016/j.jceh.2017.01.008
SESSION 4: WORKSHOP 1 (CLINICAL): MINI-
MAL/COVERT HEPATIC ENCEPHALOPATHY
7
THE BRAIN-MUSCLE AXIS IN MINIMAL
HEPATIC ENCEPHALOPATHY (MHE): A
PLACEBO-CONTROLLED, LONGITUDINAL
DOUBLE-BLIND TRIAL WITH L-ORNITHINE
L-ASPARTATE (LOLA) – PRELIMINARY
RESULTS
Yasmin Pasha, Robert Leech, Ines Ribeiro Violante,
Nicola Cook, Mary M.E. Crossey,
Simon D. Taylor Robinson
S5