Synopsis
Chronic
liver disease leads to Hepatic Encephalopathy - spectrum of neuropsychiatrical
disorders. We investigated potential neurometabolic differences between two key
brain regions (hippocampus and cerebellum). Cerebellum shows similar increase
of glutamine but lower tNAA, Tau, Cr, Asc and different osmotic response
indicating that these regions are influenced unequaly.Purpose
of the study:
Chronic hepatic encephalopathy (CHE) is a severe
liver-disease-induced neuropsychiatric disorder, seriously affecting patients' quality
of life
1. The pathophysiology of CHE is still incompletely
understood but ammonia is believed to be a main toxin playing crucial role in
the development and progress of the disease, evoking glutamine (Gln) increase
in the brain and subsequent diminution of other osmolytes as an osmoregulatory
answer
2-4. Previous studies have shown differences in neurometabolic
profile between brain regions in a hyperammonemia
5 model, and that
net glutamine synthesis rates differ between the cortex and hippocampus
6.
Therefore, the aim of this study was to investigate
potential metabolic differences between the hippocampus and cerebellum, the
rationale being that they are two key brain regions to investigate in CHE since
they are implicated in its symptoms (cognitive and fine motor deficits
7).
Methods:
Wistar male adult rats (n=3) underwent bile duct
ligation (BDL), recognized animal model to study chronic liver disease (CLD)
induced HE
8,9. Longitudinal in
vivo
1H-MRS and blood sampling (bilirubin, alanine aminotransferase
(ALAT), glucose) were performed before BDL-surgery (scan0) and after every two
weeks (scan2, 4, 6, 8). Open field tests (to evaluate motor activity and the
degree of HE
10) took place at scan 4, 6 and 8. All MR experiments
were performed on a 9.4T system (Varian/Magnex Scientific) using home-built
14mm diameter quadrature
1H-surface coil as a transceiver and ultra-short-echo
time SPECIAL spectroscopy sequence
11 (TE=2.8ms, TR=4s, 160
averages). Two volumes-of-interest, hippocampus (2×2.8×2mm
3) and cerebellum
(2.5×2.5×2mm
3) were studied. First and second order shims were
adjusted using FASTMAP (linewidth of 9-11Hz in hippocampus, 14-19Hz in
cerebellum). Concentrations of metabolites were calculated by LCModel using
water as reference.
Results
and discussion:
Differences in metabolic profile between the hippocampus
and cerebellum are presented in Fig.1. In healthy animals (before BDL-surgery)
cerebellum showed significantly higher Cr, PCr, Gln, NAA, tNAA and tCr. 8 weeks
after BDL this pattern changed, the only remained significant changes were for
Cr, PE and Tau (Fig.1A-B).
All BDL rats showed increase in plasma bilirubin,
correlated with the increase in ALAT (r=0.82, p=0.0003, Fig.2A) proving the
presence of CLD. Bilirubin further correlated with brain Gln both in the hippocampus
and cerebellum (r=0.7, p=0.01, Fig.2B).
In both regions Gln reached ~200% increase at scan8
(Fig.3A-C). However the osmoregulatory response was different. Both brain
regions showed similar decrease in mIns and tCho (Fig.3D-F, Fig.4A-B) with
non-significant differences between regions. In contrast, the cerebellum showed
significantly stronger decrease in Tau (Fig.3G-I) and Cr (Fig.3J-L) leading to a
constant sum of osmolytes (Gln+mIns+Tau+tCho+Cr) in cerebellum compared to a
tendency toward an increase in the hippocampus (Fig.3M-O). Tau is considered as
neuronal osmolyte
12, shown to increase/protect cell viability in
cerebellum
13. Cr is a metabolite involved in energy metabolism but its
involvement in osmoregulation and neuroprotection was recently reported
14,15.
Additionally, the cerebellum showed a trend toward a decrease in tNAA (-5%, ns,
Fig5A) in agreement with cerebellar damage and neuronal cell loss
16
in patients with end-stage liver disease. This decrease in Cr and tNAA in the cerebellum
had a tendency to correlate with performance in behavioural tests (Fig.5B,C).
Among neurotransmitters Glu (-11%, ns) and GABA (-20%,
ns) showed a similar decrease in both brain regions with a stronger and negative
correlation with Gln in cerebellum (Fig.4E,F).
Moreover, there was a different antioxidant response
to BDL between the cerebellum and hippocampus with a stronger decrease in Asc
in the cerebellum (Fig.4G) while GSH dropped more markedly in the hippocampus
(Fig.4H).
Conclusions:
We analysed for the first time in vivo and longitudinally the metabolite changes in cerebellum and
compared them to those in hippocampus during CHE. Based on our measurement we
can conclude that hippocampus and cerebellum show similar increase in glutamine
but differences in some other metabolites (tNAA, Tau, Cr, Asc, GSH) demonstrating
that these regions are influenced differently by CLD-induced HE. These changes
in cerebellum could stand behind the observed deterioration in locomotor
activity.
Acknowledgements
Supported by CIBM of the UNIL,
UNIGE, HUG, CHUV, EPFL, the Leenaards and Jeantet Foundations. EU:
FP7-PEOPLE-2012-ITN project 316679 TRANSACT; The authors thank Prof Carmen
Sandi (Laboratory of behavioral
genetics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Vaud,
Switzerland,) for her support in behavioural part.References
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2009; 8Biecker et al, J Pharmacol Exp Ther 2005;
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Reson Med 2006; 12Lei et al,
J Cereb Blood Flow Metab 2009; 13Boldyrev et al, Neurosci Lett.
1999; 14Rae, Neurochem Res 2014; 15Braissant
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