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(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 EDEMA)
to examine the effects of ammonia on the intracellular 5
content of free ferrous iron [Fe2+]i and its relevance for
ammonia-induced astrocyte senescence. ROLE OF ENDOTHELIAL NMDA
Results: As shown by superresolution microscopy, treat- RECEPTORS IN THE PATHOGENESIS OF
ment of astrocytes with NH4Cl (5 mmol/l) for up to 72 h HEPATIC ENCEPHALOPATHY
strongly changed the fluorescence emission of the ferrous Marta Skowronska 1,2,3,4, Arumugam R. Jayakumar 1,2,3,4,
iron-chelating reporter dyes RhoNox1 and Rhodamine B- Jan Albrecht 1,2,3,4, Michael D. Norenberg 1,2,3,4
[(2,20 -bipyridine-4-yl)-aminocarbonyl]benzyl ester (RDA) in
vital astrocytes indicative for an elevation of [Fe2+]i. By using 1
Department of Biochemistry and Molecular Biology, Laboratory of
organelle specific fluorescent dyes and live cell imaging, RDA Neuropathology, Department of Neurotoxicology, University of Miami,
fluorescence was detected in mitochondria and RhoNox1 Miami, United States, 2Veterans Affairs Medical Center, Miami, United
States, 3Mossakowski Medical Research Center, Warsaw, Poland,
fluorescence was found in lysosomal compartments. NH4Cl 4
Department of Pathology, University of Miami, Miami, United States
(5 mmol/l, 72 h)-induced RhoNox1 and RDA fluorescence
changes were abolished by the iron chelators 2,20 -bipyridine Background: Excessive accumulation of ammonia in the
(BIP) or pyridoxal isonicotinoyl hydrazone (PIH), respec- brain is the main etiological factor in hepatic encephalop-
tively. NH4Cl (5 mmol/l, 72 h) exposure also strongly athy (HE), a neuropsychiatric syndrome resulting from
changed mRNA expression levels of genes involved in iron acute or chronic liver failure (ALF/CLF). Rapid progression
metabolism in astrocytes. Whereas transcription of the iron of ALF is associated with brain edema, which often leads to
exporter ferroportin (FPN) was enhanced, mRNA levels of death as a consequence of increased intracranial pressure
the FPN accessory factors ceruloplasmin and hephaestin and brain herniation. The brain edema is mainly cytotoxic,
were decreased by NH4Cl (5 mmol/l) in astrocytes. resulting from astrocytic swelling and ammonia was
NH4Cl-induced mRNA expression changes of ferroportin,
ORAL PRESENTATION
S4 © 2017, INASL
JOURNAL OF CLINICAL AND EXPERIMENTAL HEPATOLOGY
intravenously, and the extravasation of tracers was dysfunction associated with various neurological condi-
analyzed. tions (e.g., ischemia, Alzheimer's disease, Down's syn-
Results: Results disclosed that treatment of RBMECs drome), we examined whether astrocytic MCP synthesis
with 5 mM ammonia increased intracellular Ca2+ accumu- and release may likewise be affected in CHE, and whether
lation, while pretreatment with MK-801 or memantine this event contributes to the defective neuronal integrity
attenuated such Ca2+ accumulation. As expected, ammo- and associated neurobehavioral and cognitive impair-
nia increased total ROS production by 75%. Pretreatment ments that occur in CHE. Exposure of cultured astrocytes
with MK-801 or memantine decreased ROS accumulation to ammonia (NH4Cl, 1 mM) for 10–15 days resulted in a
in ammonia-treated cells, strongly suggesting that the decrease in intra- and extracellular levels of TSP-1, Hevin,
ONS induced by ammonia in RBMEC is, in part, mediated Glypicans 4 and 6, nerve growth factor, as well as in basic
by activation of NMDA-R. BBB permeability to 10 kDa fibroblast growth factor. These changes were associated
FITC-dextran was increased 2-fold in rats with ALF, when with a decrease in levels of specificity protein-1, activator
compared to controls. Memantine prevented the ammo- protein-1, Forkhead box O, and transforming growth fac-
nia-induced changes in permeability, resulting in a return tor-beta, factors known to enhance the synthesis and
of 10 kDa dextran extravasation to basal levels. release of MCPs. Exposure of cultured neurons to condi-
Conclusion: In summary, these results provide novel tioned media (CM) from ammonia-treated astrocytes
and important information on the role of endothelial showed a decrease in synaptophysin, PSD95 and synapto-
NMDA receptors in BBB dysfunction associated with tagmin levels. CM from TSP-1 overexpressing astrocytes
HE that will bring us closer to a better understanding (by the addition of TSP-1 cDNA) that were treated with
of the mechanisms involved in the development of the ammonia, when added to cultured neurons, reversed the
brain edema in acute liver failure. 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
CONFLICTS OF INTEREST
of synaptic proteins in an in vivo rat model of CHE (treat-
ORAL PRESENTATION
The authors have none to declare. ment with the liver toxin, thioacetamide). Additionally,
treatment of rats with Metformin, an agent known to
Corresponding author: Mayank Gupta. increase levels of MCPs in other conditions, attenuated
E-mail: m.skowronska@med.miami.edu the neurobehavioral abnormalities observed in CHE. Our
http://dx.doi.org/10.1016/j.jceh.2017.01.007 findings suggest that increasing brain levels of MCPs may
represent a useful therapeutic approach for patients with
chronic hepatic encephalopathy.
CONFLICTS OF INTEREST
6
The authors have none to declare.
NEURONAL DYSFUNCTION IN CHRONIC
HEPATIC ENCEPHALOPATHY: ROLE OF Corresponding author: Santosh K. Yadav.
ASTROCYTIC MATRICELLULAR PROTEINS E-mail: mnorenbe@med.miami.edu
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 SESSION 4: WORKSHOP 1 (CLINICAL): MINI-
33125, USA MAL/COVERT HEPATIC ENCEPHALOPATHY
7
Chronic hepatic encephalopathy (CHE), due to chronic
liver failure, is characterized by confusion, disorientation, THE BRAIN-MUSCLE AXIS IN MINIMAL
behavioral changes, impaired cognition, and motor dis- HEPATIC ENCEPHALOPATHY (MHE): A
turbances. It is associated with defective neuronal integrity, PLACEBO-CONTROLLED, LONGITUDINAL
leading to neurobehavioral and cognitive impairments. DOUBLE-BLIND TRIAL WITH L-ORNITHINE
The mechanisms responsible for the neurological abnor- L-ASPARTATE (LOLA) – PRELIMINARY
malities in CHE, however, remain largely unknown. Since a
RESULTS
reduction in astrocytic secretion of matricellular proteins
(MCPs), including thrombospondin-1 (TSP-1), Hevin, Gly- Yasmin Pasha, Robert Leech, Ines Ribeiro Violante,
picans 4 and 6, and the CCN family of proteins (CYR61/ Nicola Cook, Mary M.E. Crossey,
CTGF/NOV), have been implicated in the neuronal Simon D. Taylor Robinson
Journal of Clinical and Experimental Hepatology | February 2017 | Vol. 7 | No. S1 | S1–S21 S5