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Guanosine Exerts Neuroprotective Effect in an Experimental Model of Acute Ammonia Intoxication

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The nucleoside guanosine (GUO) increases glutamate uptake by astrocytes and acts as antioxidant, thereby providing neuroprotection against glutamatergic excitotoxicity, as we have recently demonstrated in an animal model of chronic hepatic encephalopathy. Here, we investigated the neuroprotective effect of GUO in an acute ammonia intoxication model. Adult male Wistar rats received an intraperitoneal (i.p.) injection of vehicle or GUO 60 mg/kg, followed 20 min later by an i.p. injection of vehicle or 550 mg/kg of ammonium acetate. Afterwards, animals were observed for 45 min, being evaluated as normal, coma (i.e., absence of corneal reflex), or death status. In a second cohort of rats, video-electroencephalogram (EEG) recordings were performed. In a third cohort of rats, the following were measured: (i) plasma levels of glucose, transaminases, and urea; (ii) cerebrospinal fluid (CSF) levels of ammonia, glutamine, glutamate, and alanine; (iii) glutamate uptake in brain slices; and (iv) brain redox status and glutamine synthetase activity in cerebral cortex. GUO drastically reduced the lethality rate and the duration of coma. Animals treated with GUO had improved EEG traces, decreased CSF levels of glutamate and alanine, lowered oxidative stress in the cerebral cortex, and increased glutamate uptake by astrocytes in brain slices compared with animals that received vehicle prior to ammonium acetate administration. This study provides new evidence on mechanisms of guanine-derived purines in their potential modulation of glutamatergic system, contributing to GUO neuroprotective effects in a rodent model of by acute ammonia intoxication.
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Guanosine Exerts Neuroprotective Effect in an Experimental
Model of Acute Ammonia Intoxication
G. F. Cittolin-Santos
1
&A. M. de Assis
1
&P. A. Guazzelli
1
&L. G. Paniz
1
&J. S. da Silva
1
&
M. E. Calcagnotto
1,2
&G. Hansel
1
&K. C. Zenki
1,3
&E. Kalinine
1,3
&M. M. Duarte
4
&
D. O. Souza
1,2
Received: 6 March 2016 /Accepted: 28 March 2016
#Springer Science+Business Media New York 2016
Abstract The nucleoside guanosine (GUO) increases gluta-
mate uptake by astrocytes and acts as antioxidant, thereby
providing neuroprotection against glutamatergic
excitotoxicity, as we have recently demonstrated in an animal
model of chronic hepatic encephalopathy. Here, we investi-
gated the neuroprotective effect of GUO in an acute ammonia
intoxication model. Adult male Wistar rats received an intra-
peritoneal (i.p.) injection of vehicle or GUO 60 mg/kg, follow-
ed 20 min later by an i.p. injection of vehicle or 550 mg/kg of
ammonium acetate. Afterwards, animals were observed for
45 min, being evaluated as normal, coma (i.e., absence of
corneal reflex), or death status. In a second cohort of rats,
video-electroencephalogram (EEG) recordings were per-
formed. In a third cohort of rats, the following were measured:
(i) plasma levels of glucose, transaminases, and urea; (ii) ce-
rebrospinal fluid (CSF) levels of ammonia, glutamine, gluta-
mate, and alanine; (iii) glutamate uptake in brain slices; and
(iv) brain redox status and glutamine synthetase activity in
cerebral cortex. GUO drastically reduced the lethality rate
and the duration of coma. Animals treated with GUO had
improved EEG traces, decreased CSF levels of glutamate
and alanine, lowered oxidative stress in the cerebral cortex,
and increased glutamate uptake by astrocytes in brain slices
compared with animals that received vehicle prior to
ammonium acetate administration. This study provides new
evidence on mechanisms of guanine-derived purines in their
potential modulation of glutamatergic system, contributing to
GUO neuroprotective effects in a rodent model of by acute
ammonia intoxication.
Keywords Acute ammonia intoxication .Glutamate
excitotoxicity .Hyperammonemia .Guanosine
Introduction
Encephalopathy is a global disturbance of cerebral function
characterized by an altered mental state also known as deliri-
um. These alterations in mental state range from mild disori-
entation to severe coma [1]. The underlying disease etiologies
are diverse and include metabolic and systemic causes (e.g.,
hepatopathies and nephropathies), brain ischemia, hyperten-
sion, intoxication, traumatic brain injury (TBI), epilepsy, and
infections [2]. Encephalopathy is associated with high mor-
bidity and mortality unless it is promptly treated, and in some
cases, the only option for stabilizing the patient is liver trans-
plantation [3].
Hepatic encephalopathy (HE) is considered one of the main
subtypes of the encephalopathy syndrome, with a characteris-
tic alteration in cerebral function that occurs due to liver fail-
ure (both acute and chronic) or portacaval shunt [46]. It is
G. F. Cittolin-Santos and A. M. de Assis contributed equally to this work.
*D. O. Souza
diogo@ufrgs.br
1
Postgraduate Program in Biological Sciences: Biochemistry, ICBS,
Federal University of Rio Grande do Sul, Porto
Alegre, RS 90035-003, Brazil
2
Department of Biochemistry, Federal University of Rio Grande do
Sul, Porto Alegre, RS 90035-003, Brazil
3
Department of Physiology, Federal University of Sergipe, São
Cristovão, SE 49100-000, Brazil
4
Health Sciences Center, Lutheran University of Brazil (ULBRA),
Campus Santa Maria, Santa Maria, RS 97020-001, Brazil
Mol Neurobiol
DOI 10.1007/s12035-016-9892-4
known that approximately 40 % of cirrhotic patients will pres-
ent with overt HE during their clinical course and that these
patients have a high chance of HE recurrence within months
[7,8]. Therefore, HE is one of the main reasons for hospital-
ization in patients with liver dysfunction, and its management
represents a high economic burden [9].
It is likely that ammonia plays a central role in episodic
(i.e., acute) HE. The hyperammonemia caused by liver failure
leads to increased levels of ammonia in the brain, with conse-
quent brain edema that induces behavioral changes, coma, and
death [1012]. High levels of ammonia in the brain lead to
activation of NMDA receptors, thereby triggering the
glutamate-nitric oxide-cGMP pathway [13]. This effect acti-
vates the glutamatergic system excitotoxicity mechanism
through decreased glutamate transporter activity and levels,
as well as increased extracellular glutamate levels, resulting
in hyperactivation of NMDA receptors and consequent brain
oxidative stress damage [1416].
Previous studies have shown that neuroprotective strategies
against glutamatergic excitotoxicity contribute to decreasing
the deleterious effect of hyperammonemia on the central ner-
vous system (CNS) [1719]. One such strategy can be
achieved through the use of MK801, which reduces gluta-
matergic activity [18,19]. Accordingly, the nucleoside guano-
sine exhibits several neuroprotective effects in experimental
models of brain injuriesinvolvingglutamatergic excitotoxicity,
such as HE, seizures, brain ischemia, and pain [17,2022].
We hypothesize that the intraperitoneal (i.p.) injection
of ammonia at a high concentration induces neurological
alterations followed by brain disorders and that the
nucleoside guanosine could exert a neuroprotective effect
in this situation. To test our hypothesis, we evaluated the
effects of acute ammonium exposure and the neuroprotec-
tive effects of guanosine administration on (i) the mortal-
ity and coma rates, (ii) electrophysiological recordings,
(iii) glutamatergic brain parameters, (iv) brain redox
status, and (v) biochemical parameters in cerebrospinal
fluid (CSF) and serum.
Materials and Methods
Animals
Adult male Wistar rats (90 days old), from the Central Animal
House of the Department of BiochemistryUFRGS, were
maintained under a standard light/dark cycle (light between
7:00 a.m. and 7:00 p.m.) at room temperature (22± 2 °C). The
rats were housed in plastic cages (five rats per cage), with tap
water and commercial food available ad libitum. These con-
ditions were kept constant throughout the experiments.
Drugs
Guanosine, ammonium acetate, and all other chemicals were
from Sigma-Aldrich (St. Louis, MO, USA). L-[3,4-
3
H]-
Glutamic acid (50 Ci/mmol) was from Perkin Elmer Life
Sciences (Boston, MA, USA).
Ammonium acetate (150750 mg/ml) was dissolved in
distilled water and administered via i.p. injection.
Concentrations of ammonium acetate solutions were adjusted
to reach the desired dose by injecting 3 mL/kgof body weight,
as previously described [10]. Guanosine was dissolved in
0.1 mM NaOH and used as a pretreatment 20 min before the
ammonium acetate injection. A vehicle solution of 0.1 mM
NaOH was used as a control. Both solutions were adjusted to
pH 7.4. The guanosine solutions and the vehicle solution were
administered by i.p. injection at a dose of 2 mL/kg of body
weight.
Experimental Design
The experiments were divided into three protocols (Fig. 1).
Protocol 1Neurological Parameters
For evaluation of neurological parameters, the animals were
divided into four groups. Each group received a combination
of pretreatment (i.p. injection of either guanosine or vehicle)
and insult (i.p. injection of either ammonium acetate or dis-
tilled water). The four groups of animals were as follows:
control (vehicle + distilled water), ammonium (vehicle + am-
monium acetate), guanosine (guanosine + distilled water),
and guanosine + ammonium (guanosine + ammonium ace-
tate). The animals were monitored for 20 min after the pre-
treatment injection and 45 min after the insult injection.
Neurological evaluation was performed every 3 min during
the 45 min after ammonium acetate injection. The time point
at which the animals entered the comatose state (i.e., loss of
corneal reflex), as well as the duration of coma and the time of
death, was recorded.
Dose Curve of Ammonium Acetate and Guanosine
Administration
The established dose of i.p. ammonium acetate was 550 mg/
kg according to a dose response curve (Fig. 2a). The animals
were pretreated using different doses of guanosine 20 min
before the insult to evaluate the effect of guanosine on the
mortality rate (Fig. 2b). The dose of i.p. guanosine 60 mg/kg
was used in the further experiments.
Mol Neurobiol
Protocol 2Electroencephalographic Analysis
For EEG analysis, the animals were divided into two groups.
The animals received either vehicle or GUO as a pretreatment,
and 20 min later both groups received an ammonium acetate
injection (Fig. 1). Both groups were monitored by recording
the video EEG at 15 min before pretreatment administration
(i.e., baseline EEG), 20 min after guanosine (60 mg/kg) or
vehicle injection, and 60 min after the ammonium acetate
injection. The animals used for the EEG analysis were not
included in the clinical evaluation or the neurochemical
analysis.
Epidural electrodes were implanted 1 week before the
video-EEG recording. To place the electrodes on the cortical
surface, the animals were anesthetized with i.p. injections of
ketamine (80 mg/kg0.8 mL/kg) and xylazine (10 mg/kg
0.5 mL/kg) and placed on a stereotaxic instrument. The three
stainless steel screw electrodes (1.0-mm diameter) were
placed according to the coordinates from Paxinos and
Wats o n [ 23]. Two of them were placed LL +/2.0 mm, AP
1.0 mm from bregma; the third (the reference) electrode was
placed on the midline of the occipital bone and kept in contact
with cerebrospinal fluid. The ground (small screw) was placed
over the frontal bone and was used for fixation of the dental
acrylic helmet to the skull [17].
One week after implanting the electrodes, each animal was
individually transferred to an observation cage to perform the
video-EEG recordings. The EEG was recorded with a stan-
dard data acquisition system (Multichannel Plexon
Acquisition Processor System). EEG signals were filtered at
0.01100 Hz, followed by digitization at 1 kHz for posterior
analysis. All analyses were performed using built-in and
custom-written routines in MATLAB (Mathworks, Inc.).
The power density spectra were obtained to calculate the
EEG left index at baseline, after injection of guanosine or
vehicle, after ammonium acetate injection and at the end of
the video-EEG recording. The EEG left index was calculated
as the logarithm of the ratio between the power of the low
frequency (17.4 Hz) and the high frequency (13.5
26.5 Hz). Normal rats have an EEG left index of approximate-
ly 0.60, and rats in coma have an index of approximately
0.800.90 [24].
Protocol 3Biochemical Analyses
For biochemical analyses, we used the four groups as de-
scribed above for protocol 1 (control, ammonium, guanosine,
and guanosine + ammonium). At the 40-min time mark, the
animals were euthanized to collect cerebrospinal fluid (CSF)
and blood samples, as well as cerebral cortex tissues.
CSF Analysis
The animals were anesthetized with inhaled isoflurane and
placed on a stereotaxic apparatus to collect CSF samples
(4080 μL per rat) by direct puncture of the cisterna magna
with an insulin syringe (27 gauge × 1/2-in. length) [25]. The
CSF was centrifuged at 1000×gfor 10 min, and the superna-
tant was stored at 80 °C for further evaluation of (i) ammo-
nia, measured using a commercial kit (Sigma-Aldrich, St.
Protoc ol 1 - Neurologica l param eters
Protocol 2 - Electroencephalography analysis
Protoc ol 3 - Biochemic al para meter s
Guanosine
or Vehicle
020 65
Guanosine
or Vehicle
015 95
Ammo nium
Acetate
35
Time (minutes)
Time (minutes)
0 20 40
Sample
Harvesting
EEG
Monitoring
Guanosine
or Vehicle
Ammonium Acetate
or Distille d Water
Time (minutes)
End of
analysis
End of
analysis
Ammonium Acetate
or Distille d Water
Fig. 1 Protocol 1 Pretreatment (with guanosine or vehicle) set the time at
0. Ammonium acetate or distilled water was injected 20 min later for
neurological evaluation during a 45-min period. Protocol 2 The EEG
monitoring set the time at 0. Pretreatment (with guanosine or vehicle)
was injected 15 min later of the beginning of recording. Ammonium
acetate was injected 20 min later of pretreatment. EEG was recorded
15 min before pretreatment and 80 min after the first injection
(pretreatment). Protocol 3 Pretreatment (with guanosine or vehicle) set
the time at 0. Ammonium acetate or distilled water was injected 20 min
later. The sample harvesting happened after 40 min after the first injection
(pretreatment)
Mol Neurobiol
Louis, MO, USA) according to the manufacturersprotocol
and (ii) glutamine, glutamate, and alanine levels, measured by
HPLC [26].
Plasma Biochemical Analysis
Blood samples were drawn into EDTA tubes, followed by
centrifugation at 5000×gfor 10 min. Plasma was stored at
80 °C for further evaluation of alanine aminotransferase
(ALT) and aspartate aminotransferase (AST) activities, as well
as the levels of glucose and urea. These evaluations were
performed using commercial kits (Labtest, MG, Brazil).
Glutamate Uptake
After decapitation, the cortex was immediately dissected on
ice (4 °C). Frontoparietal cortical slices (0.2-mm thickness)
were rapidly obtained using a McIlwain Tissue Chopper and
immersed at 4 °C in HBSS buffer ph 7.2. Frontoparietal cor-
tical slices were preincubated with HBSS at 37 °C for 15 min.
The incubation was started by the addition of 0.33 μCi/mL of
L-[
3
H] glutamate, and stopped after 7 min with two ice-cold
washes using 1 mL of HBSS. After washing, 0.5 N NaOH was
immediately added to the slices and they were stored over-
night. The Na
+
-independent uptake of glutamate was mea-
sured. Na
+
-dependent uptake of glutamate was measured as
the difference between the total uptake and the Na
+
-indepen-
dent uptake [27].
Glutamine Synthetase Activity
The glutamine synthetase (GS) enzymatic activity assay was
performed as described previously by Quincozes-Santos et al.
[28]. Synthetic γ-glutamylhydroxamate was used as the stan-
dard. GS activity was expressed as micromole per hour per
milligram of protein of formed product.
Redox Parameters
The oxidative stress parameters were evaluated in the cerebral
cortex. The cortices were homogenized in PBS (20 mM, pH
7.4) for analysis of the following redox parameters.
Dichlorodihydrofluorescein Oxidation
The levels of reactive oxygen species were measured as de-
scribed before [17]. A calibration curve was calculated with
standard dichlorodihydrofluorescein DCFH, and the levels of
reactive species were expressed as micromole of DCF formed
per milligram of protein.
Thiobarbituric Acid Reactive Species
To assess the extent of lipoperoxidation, the levels of thiobar-
bituric acid reactive species (TBARS) were measured accord-
ing to Paniz et al. [17]. The results are expressed as nanomole
of TBARS per milligram of protein.
0
20
40
60
80
100
250 400 550 700
Ammonium Acetate (mg/kg)
Coma
Lethality
Coma and lethality rate dose
response curve (%)
03060120
0
25
50
75
100
**
Lethality
(%)
Ammonium acetate 550mg/kg
Guanosine (mg/kg)
A
B
C
060120
0
10
20
30
40
50
Guanosine (mg/kg)
**
Ammonium acetate 550mg/kg
Coma duration
(minutes)
Fig. 2 Ammonium acetate effect on rates of coma and death (a),
guanosine effect on lethality rate (b), and on coma duration of animals
entering in comatose state and did not die (c). The Spearman correlation
coefficient for coma is r=1 and for mortality is r=0.9747 (a). Asterisk
indicates a difference from 0 to 30 mg/kg guanosine groups, p<0.05,
one-way ANOVA (b).Theresultsareexpressedasthemedianand5
95 % range (minutes), Asterisk indicates a difference from the control
group, p< 0.05, KruskalWallis test with Dunns multiple comparison
test (c)
Mol Neurobiol
Antioxidant Enzyme Activities
Superoxide dismutase (EC 1.15.1.1) activity was assessed as
described previously [29]. The results were expressed as units
of SOD per milligram of protein. The glutathione peroxidase
(EC 1.11.1.9) [30] (GSH-Px) activity was measured according
to Wendel. One unit of GSH-Px activity was defined as
1μmol of NADPH consumed per minute, and the specific
activity was expressed as units per milligram of protein.
Western Blotting of Glutamine Synthetase
Cerebral cortex was homogenized in Laemmli sample buffer
(20 μg) and separated by SDS-PAGE on 10 % (w/v)acrylam-
ide and 0.275 % (w/v) bisacrylamide gels. The proteins were
then electrotransferred onto nitrocellulose membranes. The
membranes were incubated for 1 h at 25 °C in Tris/saline
buffer Tween-20 (TSB-T 20 mM Tris-HCl, pH 7.5, 137 mM
NaCl and 0.05 % (v/v) Tween 20) that contained 1 % (w/v)
non-fat milk powder. The membranes were subsequently in-
cubated for 12 h with the appropriate primary antibody (glu-
tamine synthetase, 1:10,000). After washing in TBS-T, the
blots were incubated with HRP-linked anti-immunoglobulin
G (IgG) antibodies for 1.5 h at 25 °C [31]. Chemiluminescent
bands were detected, and a densitometric analysis was per-
formed with ImageJ software (NIH, Bethesda, MD, USA).
Statistical Analysis
Data are expressed as the means ± S.E.M. To compare the
groups, we used the following statistical methods, as appro-
priate: one-way ANOVA followed by Tukey test, chi-square
test, Spearman correlation coefficient, NewmanKeuls post-
test, KruskalWallis test, Dunns multiple comparison test,
and Studentsttest, when mentioned, using GraphPad Prism
vs. 5 (La Jolla, CA, USA). The level of significance was
considered to be p<0.05.
Results
Neurological Evaluation
There were no neurological alterations in the control or gua-
nosine groups. However, guanosine had a strong effect on
mortality in the groups that received ammonium acetate. The
lethality rate of the ammonium acetate group was 36 %, but 60
and 120 mg/kg guanosine (in the guanosine +ammonium ac-
etate group) decreased this rate to 15 % (n= 52) (Fig. 2b,
p< 0.05). There was no difference in the percentage of animals
entering in comatose state between the guanosine+ ammoni-
um acetate group (n= 41) and the ammonium acetate group
(n= 42), 75 and 69 %, respectively. However, guanosine
B
Ammonium Acetate
(minute 30)
Guanosine
or Vehicle
(minute 10)
Vehicle + Ammonium Acetate
Guanosine + Ammonium Acetate
1: 05-10 min of baseline
2: 10-20 min post Guanosine or Vehicle
3: 10-20 min post Ammonium Acetate
4: 20-30 min post Ammonium Acetate
A
Fig. 3 EEG analysis: aEEG left
index values for ammonium
acetate group and guanosine +
ammonium acetate group at four
different times. bRepresentative
EEG traces for each analyzed
period of both groups. Asterisk
Indicates a difference from the
guanosine+ ammonium acetate
other group, p< 0.01, Students
ttest
Mol Neurobiol
significantly reduced the coma duration of animals that sur-
vived in the time evaluated (ammonium acetate group, 29.1
± 1.5 min. vs. guanosine + ammonium acetate group, 22.8
± 1.9 min, Fig. 2c,p<0.005, ttest). Concerning the mortality
rates of this experimental model, the rats were only evaluated
neurologically for up to 45 min following ammonium acetate
administration, as after this period, no animal died. However,
at this time mark, most of the ammonium group maintained
the same neurological status, which was coma. In contrast, the
guanosine + ammonium group had already begun to show
neurological improvement at 3335 min by starting to walk
again. Thus, it can be inferred that the reduction of coma
duration (Fig. 2c,p< 0.05) that we observed would be even
more pronounced if the rats had been assessed for longer than
45 min.
EEG
Guanosine per se had no effect on the EEG left index (gua-
nosine or vehicle, Fig. 3), which became abnormally
higher in the first 10 min after ammonium administration
(Fig. 3a) in both groups (guanosine + ammonium group,
0.90 + 0.05, n= 9; ammonium group, 0.95 + 0.03, n=9).
However, during the 2030-min interval after ammonium
acetate administration, the EEG left index in the ammoni-
um group remained high up to the end of the recording
time (0.93 + 0.05, n=8; p< 0.005), but guanosine reversed
this ammonium effect because the EEG left index returned
to normal values i n the guanosine + ammonium group
(0.69 + 0.05, n= 9). This finding could be related to the fact
that the animals in the guanosine + ammonium group
remained in severe coma for a shorter duration than those
in the ammonium group. One animal in the ammonium
group died before the 20-min time mark after ammonium
acetate administration. Figure 3b shows the representative
EEG traces of both groups.
Plasma Biochemical Parameters
The injection of ammonium acetate decreased the plasma
levels of glucose (~30 %, Fig. 4a,p< 0.05) and increased
the plasma levels of ALT and AST (~70 %, Fig. 4b; ~70 %,
Fig. 4c,p< 0.05) without affecting plasma urea levels
(Fig. 4d). Pretreatment with guanosine had no effect on the
analyzed parameters. These results indicate that systemic and
hepatic parameters were not affected by guanosine.
0
20
40
60
*
Serum Glucose
(mg/dL)
0
10
20
30
40
50 *
Serum AST
(U/L)
0
10
20
30
40
50
*
Serum ALT
(U/L)
0
5
10
15
20
25
Serum Ureia
(mg/dL)
Control
Guanosine
Ammonium Acetate
Guanosine + Ammonium Acetate
AB
CD
20 min after injection
of ammoniun acetate
20 min after injection
of ammoniun acetate
20 min after injection
of ammoniun acetate
20 min after injection
of ammoniun acetate
Fig. 4 Plasma biochemical levels of aglucose, bALT, cAST, and d
urea. The results are expressed in milligram per decaliter (aand d)and
U/L (band c) and as the means ± S.E.M. (n= 8 per group). Asterisk
Indicates a difference among ammonium acetate groups (guanosine or
vehicle) and distilled water groups (guanosine or vehicle), p< 0.05,
one-way ANOVA
Mol Neurobiol
CSF Biochemical Parameters
Ammonia levels in the CSF (μmol/L) (Fig. 5a)increasedin
the ammonium group (6.87 ± 1.3) compared to the control
(3.06 ± 0.91) and guanosine (3.53 ± 1.20) groups (p<0.001).
This increase was not affected by guanosine administration
(guanosine + ammonium group 6.17 ± 1.47) (p< 0.001).
Interestingly, the increase in CSF levels of glutamate (4.75
±1.62, n= 11) and alanine (26.3 ± 5.4, n=6) observed in the
ammonium group was reversed by pretreatment with guano-
sine. The CSF amino acid levels in the guanosine + ammoni-
um group were 3.37 ± 0.80 and 19.6 ± 2.70, respectively
(n= 10) (Fig. 5c and d,p<0.001). CSF glutamine levels were
similar for all groups (Fig. 5b).
Brain Glutamatergic Parameters
Glutamate Uptake
The glutamate uptake activity in cortical slices decreased in
the ammonium group, compared with the control group
(~30 %, Fig. 6a,p< 0.05). Guanosine administration partially
reversed this increase.
Glutamine Synthetase Activity and Immunocontent
The GS activity decreased in the ammonium group (Fig. 6b,
p< 0.001) when compared with the control group. Guanosine
administration abolished this decrease. The GS
immunocontent was similar for all groups (Fig. 6c).
Brain Redox Parameters
Concerning the activity of antioxidant enzymes, in the ammo-
nium group compared with the control group, there was a
decrease in SOD activity and an increase in GSH-Px activity.
Pretreatment with guanosine prevented both of these effects
(Fig. 7a and b,p< 0.05). TBARS (Fig. 7c)andDCFH
(Fig. 7d) levels were similar in all groups. Guanosine per se
had no effect on any parameter investigated, which is in ac-
cordance with previous results from our group [20].
Discussion
We used an effective experimental model of acute ammonia
intoxication [13] that exhibited neurological and EEG pattern
0
2
4
6
8
10
Ammonia in CSF
(µg/ml)
#
0
200
400
600
Glutamine in CSF
(µM)
0
2
4
6
Glutamate in CSF
(µM)
*
0
10
20
30
40
Alanine in CSF
(µM)
*
20 min after injection
of ammoniun acetate
20 min after injection
of ammoniun acetate
AB
CD
20 min after injection
of ammoniun acetate
20 min after injection
of ammoniun acetate
Cont rol
Guanosine
Ammonium Acetate
Guanosine + Ammonium Acetate
Fig. 5 CSF levels of aammonia, bglutamine, cglutamate, and d
alanine. The results are expressed in microgram per milliliter (a)and
micromolar (b,c,d)asthemeans±S.E.M.(n= 8 per group).
Octothorpe indicates difference among ammonium acetate groups
(guanosine or vehicle) and distilled water groups (guanosine or vehicle)
(a); Asterisk indicates difference from all other groups (c,d), p<0.05,
one-way ANOVA
Mol Neurobiol
alterations similar to those in human acute HE [32]. This ex-
perimental model of ammonium acetate injection repro-
duces the increase in CSF ammonia levels, which is likely
the main alteration described in HE pathophysiology [33].
The increase in total brain extracellular glutamate due to
high ammonia levels is well described in HE [34], includ-
ing the rise in brain extracellular glutamate after ammoni-
um acetate administration [13]. Moreover, it has been
shown that preventing the increase in brain extracellular
glutamate using MK-801 [13]ormemantine[24]could
diminish the clinical and electrophysiological alterations
following ammonium acetate administration.
Furthermore, Rose and colleagues [15] have suggested that
the pathophysiology of hyperammonemic encephalopathy
in acute hepatic dysfunction could be related to a decrease
in glutamate uptake in the CNS. Because MK-801 and
memantine alter glutamate metabolism routes by decreas-
ing the brain extracellular glutamate pool, it is reasonable
that the use of guanosine, previously shown to exert neu-
roprotective effects against glutamatergic excitotoxicity in
experimental models of brain injury [17,22,2528,31,
35], could potentially alter the course of ammonium ace-
tate intoxication in vivo.
To our knowledge, this is the first report that indicates the
neuroprotective effects of guanosine in acute
hyperammonemic encephalopathy. The systemic administra-
tion of guanosine reversed/diminished the following effects of
ammonium administration: mortality rate and coma duration
(protocol 1), alterations in the EEG left index (protocol 2);
increase in alanine CSF levels and alterations in various glu-
tamatergic parameters (increased CSF glutamate levels, de-
creased ex vivo brain glutamate uptake (partially reversed),
decreased brain GS activity) (protocol 3); and alterations in
some brain redox parameters (increased SOD activity and
decreased GSH-Px activity) (protocol 3). These findings rein-
force previous data from our group and others showing that
guanosine exerts neuroprotective effects in various in vivo
animal models of glutamate excitotoxicity, such as seizures,
brain ischemia, glutamatergic nociception, and chronic HE
[17,22,26,3537]. It is also well known that guanosine has
the ability to increase glutamate uptake by astrocytes, al-
though the specific protein carriers have not yet been
identified.
Some systemic metabolic parameters, such as plasma
levels of glucose, ALT, and AST, were affected by ammo-
nium acetate administration, but guanosine did not reverse
0
50
100
150
Glutamate Uptake
(% of control)
#
0
2
4
6
8
GS Activity
(µmol / h
-1.mg protein)
*
0.0
0.5
1.0
1.5
GS Immunocontent
(-tubulin density/GS density)
V A G+A
GS
B-tubulin
43 kDa
50 kDa
G
20 min after injection
of ammoniun acetate
20 min after injection
of ammoniun acetate
20 min after injection
of ammoniun acetate
A
BC
Control
Guanosine
Ammonium Acetate
Guanosine + Ammonium Acetate
Fig. 6 Cortical (a) glutamate uptake, (b) glutamine synthetase activity,
and (c) glutamine synthetase immunocontent. The resultsare expressed as
percent of control (control group value= 1393.5 ± 149.7 cpm/min/mg of
protein) (a), micromole per hour per milligram of protein (b), and ratio of
the control/β-tubulin (c), as the means ± S.E.M. (n= 6 per group).
Octothorpe indicates difference from the control and guanosine groups;
Asterisk indicates difference from all other groups, p< 0.05, one-way
ANOVA
Mol Neurobiol
these alterations. Although it could be postulated that the
decrease in serum glucose levels in groups treated with
ammonium acetate could contribute to the observed mor-
tality rate, this possibility is unlikely because guanosine
administration did not affect the decrease in serum glucose
levels but did decrease the mortality rate by approximately
50 %.
CSF analysis is extensively used in translational research to
identify some markers of brain disorders [3840]. Here, it was
observed that ammonium administration increased the CSF
levels of ammonia as well as of the amino acids glutamate
and alanine. In accord with our results, Therrien and
Butterworth [41] observed the same increase in CSF amino
acids levels (glutamate and alanine) in an experimental model
of portal systemic encephalopathy. Intriguingly, the increase
in ammonia CSF levels here observed in the ammonium
group was not reversed by guanosine administration, while
the increases in CSF levels of the amino acids glutamate and
alanine was reversed.
Ammonia is considered the major toxin responsible for
brain disorder in patients with liver disease. The role of
brain glutamine and amino acids (e.g., glutamate and ala-
nine) and consequently the role of brain transaminases and
of tricarboxylic acid cycle intermediates on ammonia
metabolization (potentially affecting the levels of amino
acids as glutamate and alanine) are controversial [4244].
Here, guanosine decreased the CSF glutamate and alanine
levels without affecting the ammonia levels, pointing that
guanosine administration affected the amino acid levels
(favoring a neuroprotective effect against glutamatergic
excitotoxicity) probably without affecting brain transami-
nases activity. Therefore, the source (if central or systemic)
of CSF glutamate and alanine and the variation in the
levels of other amino acids in CSF and BBB alterations
to amino acid permeability are, among other issues, under
investigation by our group.
Several studies have shown that EEG is the most adequate
method for detecting and monitoring oscillatory changes of
neural networks in patients with HE [24,45,46]. In this study,
the EEG analysis showed intermittent slow activity and an
increased EEG left index in animals receiving the ammonium
acetate injection, indicating a decline in the conscious level
[24,45]. However, the animals pretreated with guanosine had
a shorter period of EEG abnormalities and presented a subse-
quent normalization of the EEG profile at 20 to 30 min after
ammonium administration. These EEG findings corroborate
with the observation that the animals pretreated with guano-
sine spent less time in the comatose state.
0
10
20
30
40
SOD
(U/mg of protein)
*
0
10
20
30
40
GSH-Px
(U/mg of protein)
*
0
50
100
150
200
TBARS
(% of control)
0
50
100
150
DCFH oxidation
(% of control)
Control
Guanosine
Ammonium Acetate
Guanosine + Ammonium Acetate
20 min after injection
of ammoniun acetate
20 min after injection
of ammoniun acetate
20 min after injection
of ammoniun acetate
20 min after injection
of ammoniun acetate
A
C
B
D
Fig. 7 Oxidative stress parameters in cortical homogenate of aSOD
activity, bGSH-Px activity, cTBARS levels, and (d) DCFH levels. The
results are expressed as units per milligram of protein (aand b) and
percent of control (TBARS: control group value = 0.160 ± 0.01 nmol/
mg of protein; DCFH: control group value = 3385.1 ± 94.3 nmol/mg of
protein) (cand d) as the me ans ± S.E.M. (n= 8 per group). Asterisk
indicates difference from all other groups, p<0.05, one-way ANOVA
Mol Neurobiol
The involvement of disturbances in the redox and gluta-
matergic brain homeostasis has been demonstrated in acute
HE [6]. Our group and other groups have previously indicat-
ed, using both in vitro and in vivo protocols, that guanosine
has neuroprotective effects against glutamatergic
excitotoxicity and oxidative stress [17,21,22,28,47].
These roles have been attributed to the action of guanosine:
(i) stimulating glutamate uptake by astrocytes in experimental
in vivo and in vitro models of brain injury [48] and (ii)
exerting antioxidant effects [28,31]. Our study shows that
ammonium acetate administration decreased the ex vivo glu-
tamate uptake and caused the dysfunction of antioxidant en-
zymes in the CNS, both of which were reversed by guanosine
administration.
In an additional group of animals, we followed the
same experimental protocol as in protocol 3, with the
difference of harvesting the samples at 10 min after the
ammonium acetate injection to evaluate the earlier effect
of guanosine on biochemical parameters. We observed the
same effect of a reduction in glutamate uptake, but we
found no difference in the redox status (data not shown).
It seems that the decrease in glutamate uptake occurs be-
fore the oxidative stress alterations in the pathophysiology
of acute hyperammonemia. In accordance with our re-
sults, Butterworth and colleagues [49,50]proposedthat
the increase of ammonia levels in the brain of patients
with acute HE leads to an inhibition of glutamate removal
by astrocytes, with consequent increases in extracellular
brain glutamate levels and posterior oxidative/nitrosative
stress [49]. We can speculate that the antioxidant effect of
guanosine is a consequence of its ability to enhance glu-
tamate uptake and thereby prevent oxidative/nitrosative
stress.
In conclusion, this study shows the well-known involve-
ment of the glutamatergic system and oxidative stress in
the pathophysiology of acute ammonia intoxication, which
is probably the main factor underlying acute HE. Most
importantly, our data suggest a remarkable neuroprotective
effect of guanosine in acute encephalopathy as shown by
neurological, electrophysiological, and neurochemical pa-
rameters, with a very significant decrease in mortality rate.
Considering that HE still has high morbidity and mortality
rates unless promptly treated, here, we presented valuable
evidence to further understand the pathophysiology of this
condition and the possible use of guanosine as a treatment
for acute encephalopathy.
Acknowledgments This work was supported by Brazilian agencies
and grants: Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado
do Rio Grande do Sul (FAPERGS), and Brazilian Institute of
NeuroscienceIBN Net FINEP, INCTExcitotoxicity and
Neuroprotection (573577/2008-5).
Compliance with Ethical Standards All experiments were approved
by the Ethics Commission (CEUA/UFRGS) under project number 29468
and followed the National Institutes of Health BGuide for the Care and
Use of Laboratory Animals^(NIH publication No. 80-23, revised 1996).
Conflict of Interest The authors declare that they have no competing
financial interests.
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Mol Neurobiol
... An excessive increase in glutamate levels in the synaptic cleft leads to overstimulation of glutamatergic receptors, including NMDA receptors, which is harmful to neurons, an event called glutamatergic excitotoxicity, which is involved in many brain diseases (Trotti et al., 1998;Wang and Reddy, 2017;Zadori et al., 2018). Glutamate release by presynaptic neurons and/or reduced glutamate uptake by GLT1/GLAST astrocytic glutamatergic transporters have been reported as the main factors responsible for increasing glutamate levels in the synaptic cleft and, consequently, glutamatergic excitotoxicity (Trotti et al., 1998;Montana et al., 2014;Cittolin-Santos et al., 2017). ...
... Indeed, glutamatergic excitotoxicity is relevant to HE (Monfort et al., 2002;Vaquero and Butterworth, 2006;Montana et al., 2014). Our group found a significant ex vivo reduction in glutamate uptake by cerebral cortex slices caused by in vivo acute hyperammonemia (Cittolin-Santos et al., 2017), which was normalized by the in vivo administration of guanosine. Furthermore, a reduction in the expression of the main astrocytic glutamate transporters (GLT-1 and GLAST) has been reported in different models of hyperammonemia and HE (Knecht et al., 1997;Chan et al., 2000;Suarez et al., 2000). ...
... Recently, our group has studied two animal models of HE: one caused by intraperitoneal ammonium acetate injection (Cittolin-Santos et al., 2017) and the other by chronic HE caused by bile duct ligation (BDL) (Paniz et al., 2014). Although easily reproducible and appropriate for studying acute brain dysfunction, the intraperitoneal ammonium acetate injection model does not contain all pathophysiological factors involved in ALF. ...
Article
Full-text available
Hepatic encephalopathy (HE) represents a brain dysfunction caused by both acute and chronic liver failure, and its severity deeply affects the prognosis of patients with impaired liver function. In its pathophysiology, ammonia levels and glutamatergic system hyperactivity seem to play a pivotal role in the disruption of brain homeostasis. Here, we investigate important outcomes involved in behavioral performance, electroencephalographic patterns, and neurochemical parameters to better characterize the well-accepted animal model of acute liver failure induced by subtotal hepatectomy (92% removal of tissue) that produces acute liver failure (ALF). This study was divided into three cohorts: (1) rats clinically monitored after hepatectomy every 6 hours for 96 hours or until death; (2) rats tested in an open field task (OFT) before and after surgery and had blood, cerebrospinal fluid, and brain tissue collected after the last OFT; and (3) rats that had continuous EEGs recorded before and after surgery for 3 days. The hepatectomized rats presented significant motor behavioral changes accompanied by important abnormalities in classical blood laboratory parameters of ALF, and EEG features suggestive of HE and deep disturbances in the brain glutamatergic system. Using an animal model of ALF induced via subtotal hepatectomy, this work provides a comprehensive and reliable experimental model that increases the opportunity for studying the effects of new treatment strategies to be explored in an unprecedented way. It also presents insights into the pathophysiology of HE in a reproducible model of ALF, which correlates important neurochemical and EEG aspects of the syndrome.
... This happens both by an increase in extracellular glutamate levels and by direct ammonium activation of NMDA receptors. The NMDA receptor overstimulation is a crucial factor in the development of oxidative stress (Sathyasaikumar et al., 2007;Cittolin-Santos et al., 2017) due to the increase of calcium influx into the cell, which in turn increases ROS production (Hermenegildo et al., 2000;Montes-Cortes et al., 2019). The hepatectomy group presented elevated levels of ROS and decreased activity of SOD and GSH-Px (Figures 6B,C). ...
... The hepatectomy group presented elevated levels of ROS and decreased activity of SOD and GSH-Px (Figures 6B,C). Similar results regarding SOD activity were previously described in acute ammonia intoxication in rats by our group and others (Kosenko et al., 1998;Görg et al., 2013;Cittolin-Santos et al., 2017). This means that ammonia may cause an imbalance in brain redox status through antioxidant enzymes inhibition as well as glutamatergic overstimulation. ...
... Differences between groups were analyzed by t-test and are indicated as * p < 0.05; * * p < 0.01 and * * * p < 0.001. excitotoxicity there is a normalization of brain redox status and a decrease in lethality under acute ammonia intoxication (Cauli et al., 2014;Paniz et al., 2014;Cittolin-Santos et al., 2017). ...
Article
Full-text available
Acute liver failure (ALF) implies a severe and rapid liver dysfunction that leads to impaired liver metabolism and hepatic encephalopathy (HE). Recent studies have suggested that several brain alterations such as astrocytic dysfunction and energy metabolism impairment may synergistically interact, playing a role in the development of HE. The purpose of the present study is to investigate early alterations in redox status, energy metabolism and astrocytic reactivity of rats submitted to ALF. Adult male Wistar rats were submitted either to subtotal hepatectomy (92% of liver mass) or sham operation to induce ALF. Twenty-four hours after the surgery, animals with ALF presented higher plasmatic levels of ammonia, lactate, ALT and AST and lower levels of glucose than the animals in the sham group. Animals with ALF presented several astrocytic morphological alterations indicating astrocytic reactivity. The ALF group also presented higher mitochondrial oxygen consumption, higher enzymatic activity and higher ATP levels in the brain (frontoparietal cortex). Moreover, ALF induced an increase in glutamate oxidation concomitant with a decrease in glucose and lactate oxidation. The increase in brain energy metabolism caused by astrocytic reactivity resulted in augmented levels of reactive oxygen species (ROS) and Poly [ADP-ribose] polymerase 1 (PARP1) and a decreased activity of the enzymes superoxide dismutase and glutathione peroxidase (GSH-Px). These findings suggest that in the early stages of ALF the brain presents a hypermetabolic state, oxidative stress and astrocytic reactivity, which could be in part sustained by an increase in mitochondrial oxidation of glutamate.
... An excessive increase in glutamate levels in the synaptic cleft leads to overstimulation of glutamatergic receptors, including NMDA receptors, which is harmful to neurons, an event called glutamatergic excitotoxicity, which is involved in many brain diseases (Trotti et al., 1998;Wang and Reddy, 2017;Zadori et al., 2018). Glutamate release by presynaptic neurons and/or reduced glutamate uptake by GLT1/GLAST astrocytic glutamatergic transporters have been reported as the main factors responsible for increasing glutamate levels in the synaptic cleft and, consequently, glutamatergic excitotoxicity (Trotti et al., 1998;Montana et al., 2014;Cittolin-Santos et al., 2017). ...
... Indeed, glutamatergic excitotoxicity is relevant to HE (Monfort et al., 2002;Vaquero and Butterworth, 2006;Montana et al., 2014). Our group found a significant ex vivo reduction in glutamate uptake by cerebral cortex slices caused by in vivo acute hyperammonemia (Cittolin-Santos et al., 2017), which was normalized by the in vivo administration of guanosine. Furthermore, a reduction in the expression of the main astrocytic glutamate transporters (GLT-1 and GLAST) has been reported in different models of hyperammonemia and HE (Knecht et al., 1997;Chan et al., 2000;Suarez et al., 2000). ...
... Recently, our group has studied two animal models of HE: one caused by intraperitoneal ammonium acetate injection (Cittolin-Santos et al., 2017) and the other by chronic HE caused by bile duct ligation (BDL) (Paniz et al., 2014). Although easily reproducible and appropriate for studying acute brain dysfunction, the intraperitoneal ammonium acetate injection model does not contain all pathophysiological factors involved in ALF. ...
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Hepatic encephalopathy (HE) represents a brain dysfunction caused by both acute and chronic liver failures, and its severity deeply affects the prognosis of patients with impaired liver function. In its pathophysiology, ammonia levels and glutamatergic system hyperactivity seem to play a pivotal role in the disruption of brain homeostasis. Here, we investigate important outcomes involved in behavioral performance, electroencephalographic patterns, and neurochemical parameters to better characterize the well-accepted animal model of acute liver failure (ALF) induced by subtotal hepatectomy (92% removal of tissue) that produces ALF. This study was divided into three cohorts: (1) rats clinically monitored after hepatectomy every 6 h for 96 h or until death; (2) rats tested in an open-field task (OFT) before and after surgery and had blood, cerebrospinal fluid, and brain tissue collected after the last OFT; and (3) rats that had continuous EEGs recorded before and after surgery for 3 days. The hepatectomized rats presented significant motor behavioral changes accompanied by important abnormalities in classical blood laboratory parameters of ALF, and EEG features suggestive of HE and deep disturbances in the brain glutamatergic system. Using an animal model of ALF induced via subtotal hepatectomy, this work provides a comprehensive and reliable experimental model that increases the opportunity for studying the effects of new treatment strategies to be explored in an unprecedented way. It also presents insights into the pathophysiology of HE in a reproducible model of ALF, which correlates important neurochemical and EEG aspects of the syndrome.
... brain damage when administrated up to 3 h after stroke [27] involving (i) modulation of astrocyte functions, by modulating the glutamatergic system (decreasing the glutamatergic excitotoxicity) [28][29][30]; (ii) modulating the adenosinergic system [31,32]; (iii) repercussions on inflammatory cascade and on oxidative stress [26,32]; and (iv) modulation of electrophysiological parameters [33][34][35][36]. The involvement of kinase pathways on these Guo effects has been also demonstrated [37,38]. ...
... In this study, the effect of In-Guo administration on brain oscillations during ischemia was associated with a decrease in synchrony, suggesting an improvement in global brain functionality apparently caused by a modulation of ipsilateral and contralateral peri-infarct area and in those unrelated to the ischemic lesion (as E1). Since it has been shown that abnormal cortical oscillations affect behavioral parameters, the maintenance of brain rhythms similar to the normal/Sham pattern in both hemispheres seems to contribute to the better and long-lasting behavioral outcome of ischemic animals treated with Guo [33][34][35]. ...
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Ischemic stroke is a major cause of morbidity and mortality worldwide and only few affected patients are able to receive treatment, especially in developing countries. Detailed pathophysiology of brain ischemia has been extensively studied in order to discover new treatments with a broad therapeutic window and that are accessible to patients worldwide. The nucleoside guanosine (Guo) has been shown to have neuroprotective effects in animal models of brain diseases, including ischemic stroke. In a rat model of focal permanent ischemia, systemic administration of Guo was effective only when administered immediately after stroke induction. In contrast, intranasal administration of Guo (In-Guo) was effective even when the first administration was 3 h after stroke induction. In order to validate the neuroprotective effect in this larger time window and to investigate In-Guo neuroprotection under global brain dysfunction induced by ischemia, we used the model of thermocoagulation of pial vessels in Wistar rats. In our study, we have found that In-Guo administered 3 h after stroke was capable of preventing ischemia-induced dysfunction, such as bilateral suppression and synchronicity of brain oscillations and ipsilateral cell death signaling, and increased permeability of the blood-brain barrier. In addition, In-Guo had a long-lasting effect on preventing ischemia-induced motor impairment. Our data reinforce In-Guo administration as a potential new treatment for brain ischemia with a more suitable therapeutic window.
... Intracellularly, GUO is a relevant regulatory and structural molecule [9]. Additionally, it has been shown that GUO acts as a neuroprotective agent against experimental models of brain diseases involving glutamatergic excitotoxicity [10][11][12][13], like ischemic stroke [14], oxidative stress [15], hepatic encephalopathy [16,17], and anxiety [18]. ...
... ** p < 0.05, *** p < 0.001 and **** p < 0.0001 when compared the percentage of astrocytes ramified and no-ramified in the same group. Animals/group: 3. CL contralateral; IPSI ipsilateral brain ischemia [2,14,61,62], Parkinson's disease [63,64], Alzheimer's disease [65], hepatic encephalopathy [16,17,66] and sepsis [13]. In an in vitro model of ischemia (oxygen glucose deprivation), it was also observed the neuroprotective effects of guanosine. ...
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Guanosine (GUO) has neuroprotective effects in experimental models of brain diseases involving glutamatergic excitotoxicity in male animals; however, its effects in female animals are poorly understood. Thus, we investigated the influence of gender and GUO treatment in adult male and female Wistar rats submitted to focal permanent cerebral ischemia in the motor cortex brain. Female rats were subdivided into non-estrogenic and estrogenic phase groups by estrous cycle verification. Immediately after surgeries, the ischemic animals were treated with GUO or a saline solution. Open field and elevated plus maze tasks were conducted with ischemic and naïve animals. Cylinder task, immunohistochemistry and infarct volume analyses were conducted only with ischemic animals. Female GUO groups achieved a full recovery of the forelimb symmetry at 28–35 days after the insult, while male GUO groups only partially recovered at 42 days, in the final evaluation. The ischemic insult affected long-term memory habituation to novelty only in female groups. Anxiety-like behavior, astrocyte morphology and infarct volume were not affected. Regardless the estrous cycle, the ischemic injury affected differently female and male animals. Thus, this study points that GUO is a potential neuroprotective compound in experimental stroke and that more studies, considering the estrous cycle, with both genders are recommended in future investigation concerning brain diseases.
... Considering a neuroprotective therapy, guanosine (GUO), an endogenous guanine-based purine, has shown protective effects with in vivo and in vitro models of brain diseases, including ischemic stroke, AD, hepatic encephalopathy, and seizures [16][17][18][19][20]. In these studies, GUO modulated several neurochemical processes and behavioral parameters, reducing inflammation, oxidative stress, and glutamate excitotoxicity [21]. ...
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... Cittolin-Santos GF et al. 2017 [8] Neuroprotection against glutamatergic excitotoxicity GUO dramatically reduced the lethality rate and the duration of the coma. Oxidative stress in the cerebral cortex and increased glutamate uptake by astrocytes in slices of the brain compared to animals that received a vehicle before the administration of ammonium acetate. ...
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Guanosine (GUO) is an endogenous guanine nucleoside to which several neuroprotective and neurotrophic effects have been attributed in experimental in vitro and/or in vivo models of central nervous system (CNS) diseases, including ischemic stroke, Alzheimer's, Parkin-son's disease, spinal cord injury, nociception and depression. The objective of this work is to systematically review the neuroprotective effects of guanosine in experimental models in the CNS. The research was conducted through PubMed, Science direct, Scopus, Cochrane Library and SciELO (Scientific Electronic Library Online) and databases, where a number of relevant articles were found. Central nervous system (CNS) astrocytes release guanosine extracellularly, that has trophic effects. In the CNS, extracellular guanosine (GUO) stimulates mitosis, the synthesis of trophic factors and cell differentiation, including neuritogenesis, is neuroprotective and reduces apoptosis due to various stimuli. In this work we demonstrate that guanosine has a neuroprotective effect, through the survey of data that we carry out.
... Hippocampus is the most vulnerable brain region associated with cognitive functions for learning and memory during cerebral ischemia injury [2][3][4][5][6]. Until now, no effective treatment is available to improve brain repair and neurological recovery [7]. ...
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Radix Astragali (AR) is a commonly used medicinal herb for post-stroke disability in Traditional Chinese Medicine but its active compounds for promoting neurogenic effects are largely unknown. In the present study, we tested the hypothesis that Astragaloside VI could be a promising active compound from AR for adult neurogenesis and brain repair via targeting epidermal growth factor (EGF)-mediated MAPK signaling pathway in post-stroke treatment. By using cultured neural stem cells (NSCs) and experimental stroke rat model, we investigated the effects of Astragaloside VI on inducing NSCs proliferation and self-renewal in vitro, and enhancing neurogenesis for the recovery of the neurological functions in post-ischemic brains in vivo. For animal experiments, rats were undergone 1.5 h middle cerebral artery occlusion (MCAO) plus 7 days reperfusion. Astragaloside VI (2 μg/kg) was daily administrated by intravenous injection (i.v.) for 7 days. Astragaloside VI treatment promoted neurogenesis and astrogenic formation in dentate gyrus zone, subventricular zone, and cortex of the transient ischemic rat brains in vivo. Astragaloside VI treatment enhanced NSCs self-renewal and proliferation in the cultured NSCs in vitro without affecting NSCs differentiation. Western blot analysis showed that Astragaloside VI up-regulated the expression of nestin, p-EGFR and p-MAPK, and increased neurosphere sizes, whose effects were abolished by the co-treatment of EGF receptor inhibitor gefitinib and ERK inhibitor PD98059. Behavior tests revealed that Astragaloside VI promoted the spatial learning and memory and improved the impaired motor function in transient cerebral ischemic rats. Taken together, Astragaloside VI could effectively activate EGFR/MAPK signaling cascades, promote NSCs proliferation and neurogenesis in transient cerebral ischemic brains, and improve the repair of neurological functions in post-ischemic stroke rats. Astragaloside VI could be a new therapeutic drug candidate for post-stroke treatment.
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The available medications for the treatment of major depressive disorder have limitations, particularly their limited efficacy, delayed therapeutic effects, and the side effects associated with treatment. These issues highlight the need for better therapeutic agents that provide more efficacious and faster effects for the management of this disorder. Ketamine, an N-methyl-D-aspartate receptor antagonist, is the prototype for novel glutamate-based antidepressants that has been shown to cause a rapid and sustained antidepressant effect even in severe refractory depressive patients. Considering the importance of these findings, several studies have been conducted to elucidate the molecular targets for ketamine’s effect. In addition, efforts are under way to characterize ketamine-like drugs. This review focuses particularly on evidence that endogenous glutamatergic neuromodulators may be able to modulate mood and to elicit fast antidepressant responses. Among these molecules, agmatine and creatine stand out as those with more published evidence of similarities with ketamine, but guanosine and ascorbic acid have also provided promising results. The possibility that these neuromodulators and ketamine have common neurobiological mechanisms, mainly the ability to activate mechanistic target of rapamycin and brain-derived neurotrophic factor signaling, and synthesis of synaptic proteins in the prefrontal cortex and/or hippocampus is presented and discussed.
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Accumulating evidences indicate that endogenous modulators of excitatory synapses in the mammalian brain are potential targets for treating neuropsychiatric disorders. Indeed, glutamatergic and adenosinergic neurotransmissions were recently highlighted as potential targets for developing innovative anxiolytic drugs. Accordingly, it has been shown that guanine-based purines are able to modulate both adenosinergic and glutamatergic systems in mammalian central nervous system. Here, we aimed to investigate the potential anxiolytic-like effects of guanosine and its effects on the adenosinergic and glutamatergic systems. Acute/systemic guanosine administration (7.5 mg/kg) induced robust anxiolytic-like effects in three classical anxiety-related paradigms (elevated plus maze, light/dark box, and round open field tasks). These guanosine effects were correlated with an enhancement of adenosine and a decrement of glutamate levels in the cerebrospinal fluid. Additionally, pre-administration of caffeine (10 mg/kg), an unspecific adenosine receptors’ antagonist, completely abolished the behavioral and partially prevented the neuromodulatory effects exerted by guanosine. Although the hippocampal glutamate uptake was not modulated by guanosine (both ex vivo and in vitro protocols), the synaptosomal K+-stimulated glutamate release in vitro was decreased by guanosine (100 μM) and by the specific adenosine A1 receptor agonist, 2-chloro-N 6-cyclopentyladenosine (CCPA, 100 nM). Moreover, the specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM) fully reversed the inhibitory guanosine effect in the glutamate release. The pharmacological modulation of A2a receptors has shown no effect in any of the evaluated parameters. In summary, the guanosine anxiolytic-like effects seem closely related to the modulation of adenosinergic (A1 receptors) and glutamatergic systems.
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In addition to its intracellular roles, the nucleoside guanosine (GUO) also has extracellular effects that identify it as a putative neuromodulator signaling molecule in the central nervous system. Indeed, GUO can modulate glutamatergic neurotransmission, and it can promote neuroprotective effects in animal models involving glutamate neurotoxicity, which is the case in brain ischemia. In the present study, we aimed to investigate a new in vivo GUO administration route (intranasal, IN) to determine putative improvement of GUO neuroprotective effects against an experimental model of permanent focal cerebral ischemia. Initially, we demonstrated that IN [(3)H] GUO administration reached the brain in a dose-dependent and saturable pattern in as few as 5 min, presenting a higher cerebrospinal GUO level compared with systemic administration. IN GUO treatment started immediately or even 3 h after ischemia onset prevented behavior impairment. The behavior recovery was not correlated to decreased brain infarct volume, but it was correlated to reduced mitochondrial dysfunction in the penumbra area. Therefore, we showed that the IN route is an efficient way to promptly deliver GUO to the CNS and that IN GUO treatment prevented behavioral and brain impairment caused by ischemia in a therapeutically wide time window.
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The concept of synergistic mechanisms as the pathophysiologic basis of hepatic encephalopathy started with the pioneering work of Les Zieve in Minneapolis some 60 years ago where synergistic actions of the liver-derived toxins ammonia, methanethiol, and octanoic acid were described. More recently, synergistic actions of ammonia and manganese, a toxic metal that is normally eliminated via the hepatobiliary route and shown to accumulate in brain in liver failure, on the glutamatergic neurotransmitter system were described. The current upsurge of interest in brain inflammation (neuroinflammation) in relation to the CNS complications of liver failure has added a third dimension to the synergy debate. The combined actions of ammonia, manganese and pro-inflammatory cytokines in brain in liver failure result in oxidative/nitrosative stress resulting from activation of glutamate (NMDA) receptors and consequent nitration of key brain proteins. One such protein, glutamine synthetase, the sole enzyme responsible for brain ammonia removal is nitrated and inactivated in brain in liver failure. Consequently, brain ammonia levels increase disproportionately resulting in alterations of brain excitability, impaired brain energy metabolism, encephalopathy and brain swelling. Experimental therapeutic approaches for which proof-of-principle has been established include the NMDA receptor antagonist memantine, N-acetyl cysteine (recently shown to have antioxidant properties at both hepatic and cerebral levels) and probiotics.
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Hepatic encephalopathy (HE) is a major complication of cirrhosis resulting in significant socioeconomic burden, morbidity, and mortality. HE can be further subdivided into covert HE (CHE) and overt HE (OHE). CHE is a subclinical, less severe manifestation of HE and requires psychometric testing for diagnosis. Due to the time consuming screening process and lack of standardized diagnostic criteria, CHE is frequently underdiagnosed despite its recognized role as a precursor to OHE. Screening for CHE with the availability of the Stroop test has provided a pragmatic method to promptly diagnose CHE. Management of acute OHE involves institution of lactulose, the preferred first-line therapy. In addition, prompt recognition and treatment of precipitating factors is critical as it may result in complete resolution of acute episodes of OHE. Treatment goals include improvement of daily functioning, evaluation for liver transplantation, and prevention of OHE recurrence. For secondary prophylaxis, intolerance to indefinite lactulose therapy may lead to non-adherence and has been identified as a precipitating factor for recurrent OHE. Rifaximin is an effective add-on therapy to lactulose for treatment and prevention of recurrent OHE. Recent studies have demonstrated comparable efficacy of probiotic therapy to lactulose use in both primary prophylaxis and secondary prophylaxis.
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