Journal of Chemical Neuroanatomy 106 (2020) 101797

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Journal of Chemical Neuroanatomy

journal homepage: www.elsevier.com/locate/jchemneu

SIRT1 activation by resveratrol reverses atrophy of apical dendrites of T

hippocampal CA1 pyramidal neurons and neurobehavioral impairments in
moderate grade hepatic encephalopathy rats
Archita Khannaa, Anamikaa, Suwarna Chakrabortyb, Sunil Jamuna Tripathib, Arup Acharjeea,
Shankaranarayana Rao BSb, Surendra K. Triguna,*
a
Biochemistry Section, Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi, 221005, India
b
Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, Bengaluru, 560029, India

A R T I C LE I N FO A B S T R A C T

Keywords: A preliminary observation about resveratrol (RSV) dependent normalization of inflammatory and apoptotic
Hepatic encephalopathy factors in the cortex of hyperammonemic rat model of moderate grade hepatic encephalopathy (MoHE) led us to
SIRT1 evaluate whether RSV is ultimately able to confer neuroprotection against MoHE pathogenesis and that it does so
CA1 by activating its bonafide molecular target SIRT1. The present study compared the profile of relevant neuro-
Dendritic atrophy
behavioral pattern vs neuromorphometry of hippocampal CA1 neurons and SIRT1 activity in the hippocampus of
Resveratrol
the chronic liver failure (CLF) model of moderate grade HE (MoHE) rats induced by administration of 100 mg/kg
body weight of thioacetamide i.p. for 10 days and in the CLF/MoHE rats treated with 10 mg/kg body weight RSV
i.p. for 7 days. As compared to the control group rats, the MoHE rats showed significantly deranged pattern of
memory and motor functions on MWM and rota rod tests, respectively. These behavioural deficits were asso-
ciated with a significant reduction in apical dendrite length and number of branching points in the CA1 pyr-
amidal neurons. Interestingly, all these parameters were found to be recovered back to their normal levels in the
MoHE rats treated with RSV. Concordantly, MoHE associated declined SIRT1 activity in the hippocampus could
be normalized back due to RSV treatment to those MoHE rats. Our findings suggest that RSV is able to normalize
MoHE associated memory impairments and motor deficits vis a vis reversal of CA1 dendritic atrophy via SIRT1
activation.

1. Introduction
Resveratrol (RSV) is a much-acclaimed plant polyphenol that, due
to its proven cardioprotective, anti-inflammatory and antioxidant
properties (Salehi et al., 2018), has drawn special attention for its ef-
ficacy as a natural neuroprotectant. Indeed, some studies demonstrated
its neuroprotective activities in the models of neurodegenerative dis-
eases and in case of some neurological complications like ischemic in-
sult and dementia (Wong and Tang, 2016; Cao et al., 2017). In many
such cases, antioxidant properties of RSV, including its ROS scavenging
activities and up regulating endogenous antioxidant enzymes were
mainly considered accountable for bringing neurological improvements
(Salehi et al., 2018; Truong et al., 2018).
During the recent past, Sirtuin1 (SIRT1), an evolutionary conserved
protein deacetylase, has got much attention as one of the important
epigenetic regulators of protein functions at cellular level. Since SIRT1
protein contains a conserved allosteric site for RSV (Anamika et al.,
2019; Revollo and Li, 2013), it is argued that RSV could impart its cell
protective actions via activating SIRT1 which, in turn, may implicate
various downstream factors like; Nrf2 and NFκB pathways in normal-
izing redox milieu of a challenged cell (Truong et al., 2018). Though
information is limited, SIRT1 activation by RSV has been reported to
confer neuroprotection in case of ischemic insult and Alzheimer’s dis-
ease (AD) (Wong and Tang, 2016). Also, RSV has been described to
improve the cognitive functions of animals with neurodegenerative
brain disorders (Cao et al., 2017). Additionally, SIRT1 activation has
been found associated with controlling stress responses by deacety-
lating FOXO group of transcription factors involved in expressing an-
tioxidant enzymes; Catalase, manganese superoxide dismutase
(MnSOD) and thioredoxin (Brunet et al., 2004; Truong et al., 2018).
Thus, evaluating RSV-SIRT1 activation mediated neuroprotection in a
relevant in vivo brain disorder model is of high scientific merit.
Hepatic encephalopathy (HE) is a metabolic brain disorder devel-
oped in the patients with liver dysfunction and characterized by a

⁎
Corresponding author.

https://doi.org/10.1016/j.jchemneu.2020.101797
Received 1 January 2020; Received in revised form 20 March 2020; Accepted 15 April 2020
Available online 22 April 2020
0891-0618/ © 2020 Elsevier B.V. All rights reserved.
A. Khanna, et al.
spectrum of neuropsychiatric complications like; attention deficits,
working memory impairment, motor dysfunction, stupor and coma
(Felipo, 2009; Ferenci, 2017). Since, chronic liver dysfunctions, de-
veloped due to viral hepatitis, drug intoxication and alcoholism, is more
common in general population, the prevalence of chronic type mod-
erate-minimum grade HE becomes an inevitable situation in those cir-
rhotic patients (Poordad, 2007). Indeed, approximately 30–50 % of
cirrhotic patients are reported to develop overt HE while around 80 %
of them suffer from minimal HE (Raphael et al., 2016). Moreover, it is
now suggested that the patients suffering from chronic type prolonged
HE become susceptible to develop Parkinsonism at the later stage of
their life (Granado et al., 2013). Thus, understanding cerebral patho-
genesis of HE in a relevant animal model is of high neurological con-
cern.
Out of the many animal models tested, thioacetamide (TAA) in-
duced liver failure rat model is considered the most suitable one as, it
causes hepatocytes to undergo concentration dependent damage and
thus, it has been reported to develop neurobehavioral characterized
different grades of HE (Singh and Trigun, 2014, 2010). The TAA in-
duced moderate grade of HE (MoHE) in rats is argued to represent a
true liver failure model of overt HE; histopathologically characterized
with moderate level centrilobular hepatocytes necrosis, ∼2 fold in-
crease in liver function test (LFT) parameters and ∼3 fold enhanced
serum ammonia level (Singh and Trigun, 2010). Such a persistent hy-
perammonemia (HA) facilitates rise of brain ammonia level leading into
brain edema, neuroinflammation, and a number of other neurochemical
aberrations (Felipo, 2009; Ferenci, 2017). These alterations, in general,
induce glutamate excitotoxicity via over activating NMDA receptors
(Felipo, 2009; Mondal and Trigun, 2015). As reviewed previously
(Felipo, 2009), different brain regions then undergo series of neuro-
chemical changes leading to deranged mitochondrial bioenergetics and
thereby making brain cells deprived of adequate energy production
which is accompanied by increased ROS damage (Aldridge et al., 2015;
Liere et al., 2017; Khanna and Trigun, 2016). Since, RSV has been found
to modulate ROS induced pathophysiology of many metabolic disorders
including neuropathology of AD, Parkinson's disease (PD) and dementia
(Wong and Tang, 2016; Cao et al., 2017); it is speculated to impart
neuroprotection during HE as well.
Recovery of neuropsychiatric complications is considered as the
endpoint assay to determine the neuroprotective efficacy of a phar-
macological agent. Similarly, recording morphological alterations in
the susceptible neurons could provide a neuroarchitectural basis of
neuroprotection by that agent. Several studies suggest that cognitive
deficits and/or motor dysfunctions in neurodegenerative conditions
such as AD, PD, etc. are strongly associated with a decrease in SIRT1
level (Elibol and Kilic, 2018) attributing towards impaired long-term
potentiation (LTP) and loss of dendritic arborization in the cortical
neurons (Grabowska et al., 2017). Moreover, administration of RSV was
found to promote neuronal plasticity by increasing the spine density
and dendritic length in the CA1 and CA3 regions of the aged rats
(Codocedo et al., 2012; Michán et al., 2010). Additionally, the total
dendritic length has been found to be significantly less in SIRT1-KO
mice in comparison to the wild type (Monserrat Hernández‐Hernández
et al., 2016). Codocedo et al. (2012) further demonstrated that SIRT1
over expression and SIRT1 activation by RSV could enhance dendritic
branching in hippocampal neurons with concordant improvement in
learning and memory functions in normal and Aβ toxic animals. In our
previous reports, it has been demonstrated that MWM and rota-rod tests
could be employed to characterize the degree of HE in the rat model
(Singh and Trigun, 2014, 2010). Also, we have seen that resveratrol is
able to normalize neuroinflammatory factors in the brain of the HA
model of HE.
Therefore, the present work was aimed to investigate whether
hippocampal CA1 pyramidal neurons undergo morphometric altera-
tions during MoHE pathogenesis, and if so, whether these changes
could be reversed due to RSV-SIRT1 activation. The study compares the
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Journal of Chemical Neuroanatomy 106 (2020) 101797
level of neurobehavioral deficits vis a vis profiling of neuronal arbor-
ization in CA1 pyramidal neurons & SIRT1 activity in the hippocampus
of normal, thioacetamide (TAA) induced moderate grade HE (MoHE)
and in MoHE rats treated with RSV.
2. Materials and methods
2.1. Animals and chemicals
Adult male Charles foster rats weighing 130–160 g were kept in
separate cages, fed with the recommended diet, and maintained at
standard conditions of 12 h light and dark period at room temperature
(25 ± 2∘C) in an animal house. All procedures on rats were performed
as approved by the institutional animal ethical committee for the care
and use of laboratory animals.
All chemicals used were of analytical grade obtained from E-Merck
and Sisco research Laboratory, Mumbai (India) except SIRT1 activity
assay kit (Fluorometric), which was purchased from Sigma Aldrich,
USA.
2.2. Induction of CLF led moderate grade HE
CLF in rats was induced by administration of TAA (in 0.9 % NaCl)
via intraperitoneal (i.p.) route at a dose of 100 mg/kg body weight once
daily for 10 days (Singh and Trigun, 2010). The control rats were ad-
ministered with 0.9 % NaCl i.p., once daily for 10 days and for RSV
treatment group (MoHE + RSV), CLF rats were administered with 10
mg/kg body weight RSV (dissolved in 1% DMSO) i.p. once daily,
starting from 8th day till 14th day post TAA treatment. To prevent from
a possible weight loss, osmolite imbalance in the TAA treated rats, all
experimental and control group rats, as employed previously (Singh
and Trigun, 2010), were supplemented with 5% dextrose solution
containing 0.45 % NaCl and 20 meq/ L KCl in drinking water. After
14th day, 6 rats from each group were subjected for neurobehavioral
study. 3–4 rats from each group were sacrificed after 24 h of the last
dose given; hippocampus was dissected out and processed for bio-
chemical and molecular study.
2.3. Neurobehavioral study
2.3.1. Morris water maze (MWM)
Following the protocol of Vorhees and Williams (2006) and as de-
scribed previously from our lab (Singh and Trigun, 2014), the MWM
task was performed to test the spatial reference memory of control and
experimental group rats. The water maze consisted of a black circular
pool (160 cm in diameter, 80 cm in height) filled with water to a depth
of 30 cm (23–25 °C). A flexi glass platform of 10 cm diameter was lo-
cated at fixed (target) quadrant submerged below 2 cm water level.
Visual cues of cardboards of different shapes (round, square, triangle
and star) and colours were placed on the cylindrical tank wall corre-
sponding to the 4 quadrant corners. MWM test was conducted between
11∶00 am to 1∶00 pm to avoid diurnal variations. Before the recording,
a free-swimming trial of 120 s was performed for habituation training
to learn finding out the invisible platform. Test was conducted for 5
days with two consecutive trials on each day with a gap of 5 min. A
video camera was mounted over the centre of the maze and activity of
rat was recorded for 120 s. The escape latency time was analyzed using
ANY-maze software (Microsoft version 4.84, USA).
2.3.2. Rota rod test
Following the reported procedure (Singh and Trigun, 2014), each
rat underwent five sessions of rota rod performance test repeated at
approximately same time on 5 consecutive days. The speed of rota rod
was fixed with the increment of 5,101,520 and 25 rpm starting from
day1−5 respectively. Each session had three trials for each rat with 10
min interval. The time, up to which the rats stayed on the rotating rod,
A. Khanna, et al.
Fig. 1. Morris water maze test suggests RSV dependent reversal of impaired spatial learning and reference memory in MoHE rats. (a) represents the video recorded
track plots, analyzed by ANYMAZE software, for control, MoHE and MoHE group treated with resveratrol. (b) shows Escape latency score during the learning period.
(c) Rota rod performance test suggests impairment in motor coordination function of control, MoHE and MoHE rats treated with resveratrol. Statistical analysis
applied was One-way ANOVA followed by Tukey’s post-hoc test. Values are represented as mean ± SD; n = 6; p < 0.05(*control vs MoHE group; # MoHE vs MoHE
+ RSV group).
was recorded automatically. Each trial continued for 180 s. The mean
riding time on rotating rod was recorded.
2.4. Golgi Cox histology and quantification of dendritic arborization in CA1
pyramidal neurons
The Golgi staining was performed following the method of Zaqout
and Kaindl (2016) with some modifications. Decapitated brain was
immediately kept in the impregnation solution prepared by mixing 5%
(w/v) solution of K2Cr2O7, K2CrO4, HgCl2 and ddH2O in the ratio of
5:5:4:10. After 24 h, solution is replaced with a fresh impregnation
solution and left at RT in dark for 7–10 days. Tissue was transferred to
tissue protectant solution made up of 0.1 M phosphate buffer (pH7.2)
containing 30 % sucrose, 0.1 % PVP40 and 30 % v/v ethylene glycol for
7 days. Later, 150 μm floating sections of the brain were cut using vi-
brotome. Slices were then developed and mounted using DPX.
All slides were coded and analyzed in a blinded manner. After
scanning the sections at lower magnification, neurons were randomly
selected from the CA1 subfield of the hippocampus. The neurons which
fulfilled the following criteria were used for reconstruction; 1) fully
impregnated neurons; 2) consistent staining in all dendrites; 3) den-
drites without truncated branches, and 4) presence of neuron in relative
isolation from other neighbouring neurons (Rao et al., 2001; Titus et al.,
2007). Dendritic trees of selected neuron were reconstructed at 400×
magnification using a BX51 microscope (Olympus, Tokyo, Japan)
equipped with a MAC 5000 XYZ motorized stage, camera, Ludl’s joy-
stick with focus control (Ludl Electronic Products, Hawthorn, NY, USA)
and controlled through a Dell workstation installed with Neurolucida
software (MBF Bioscience, Williston, Vermont, USA). After 3D tracing,
the Sholl’s analysis (Sholl, 1953, 1956) was performed on Neuroex-
plorer software (MBF Bioscience, Williston, Vermont, USA). The con-
centric spheres radiating from the centre of soma, with a diameter of 50
μm were positioned on the tracing. The number of branching points and
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Journal of Chemical Neuroanatomy 106 (2020) 101797
length of dendrites in each successive segment were estimated by the
software. The apical and basal dendritic branching were analyzed up to
a length of 350 and 150 μm distance from the center of the soma, re-
spectively (Rao et al., 2001; Titus et al., 2007). Six neurons satisfying
the criteria were selected from each animal and the data were averaged.
A total of 30 neurons were studied from each of the three experimental
groups (n = 5 per group).
2.5. SIRT1 activity assay
SIRT1 assay was performed using the hippocampal extracts (15 %)
prepared in NETN buffer composed of 20 mM TrisCl (pH 8.0), 100 mM
NaCl, 1 mM EDTA, 0.5 % NP-40, 5 mM NaF and protease inhibitor
cocktail. The extract was then incubated at 4 °C for 20 min under
constant shaking and then centrifuged at 10,000 x g for 10 min at 4 °C.
The supernatant collected was used for the assay. The fluorometric
assay was performed as per instructions given in the SIRT1 activity
assay kit.
2.6. Statistical analysis
Data from the Golgi-Cox study was plotted as the segmental distance
from the soma and analyzed using repeated measure Two-way ANOVA
followed by Sidak's multiple comparisons test. Data of total length and
number of branching points and neurobehavioral study were analyzed
using One-way ANOVA followed by Tukey’s post-hoc test. Data were
expressed as mean ± SD and values of p < 0.05 were considered sta-
tistically significant.
A. Khanna, et al.
3. Results
3.1. Effect of resveratrol on neurobehavioral scores
MWM performance is a widely accepted behavioral test to measure
learning- memory and cognition functions in the rodent models.
According to Fig. 1a, as compared to control group rats, CLF rats not
only showed an erratic pattern taking longer time to find out the hidden
platform but also exhibited a significant increase in escape latency score
on last 3 days of the test period (p < 0.05). Thus, suggesting impaired
spatial reference memory in the CLF rats. As these data matched with
previously reported MoHE pattern (Singh and Trigun (2010)), these
CLF rats were also referred to as MoHE rats. Moreover, administration
of resveratrol to those MoHE rats brought escape latency score at
control level which also coincided with adopting a straight path taking
shorter time to locate hidden platform (Fig. 1a & b). Together, MWM
test scores suggested markedly improved learning-memory ability of
MoHE rats due to RSV treatment.
Motor function deficit, analyzed by rota-rod performance test in
rodents, is considered another common tool to characterize HE. As
depicted in Fig. 1c, with increasing rod speed, there is a significant
decline (p < 0.05) in the time stayed by the MoHE rats on the rotating
rod as compared to the control group rats. However, this timing could
be recovered back to the normal level due to the treatment of RSV to
the MoHE rats.
3.2. Effect of resveratrol on the morphology of CA1 pyramidal neurons
We analyzed the dendritic branching pattern in hippocampal CA1
pyramidal neurons following MoHE and RSV treatment. Fig. 2a shows
the representative photomicrographs of Golgi-Cox stained CA1 pyr-
amidal neurons and their respective tracing from control, MoHE and
MoHE group treated with resveratrol. It is evident that there is a con-
siderable decrease in the total length of CA1 pyramidal neurons of
MoHE rats in comparison to control; while a notable recovery in the
length of CA1 pyramidal neurons on resveratrol treatment is also dis-
cernible (Fig. 2b & c).
Results from the morphometric analysis have been presented in
(Fig. 2a-b & supplementary Fig S1). In comparison to control, the length
of apical dendrites showed a significant decrease (p < 0.05) at the distal
350 μm segment in the MoHE group. We observed a trend of decrease in
the number of branching points in the MoHE group, albeit statistically
non-significant. Moreover in Fig. 2b & c, MoHE rats had a significant
reduction in total length (p < 0.05) and the number of branching points
(p < 0.05) in CA1 pyramidal neurons. Notably, treatment with RSV
reversed the MoHE-associated decrease in length of apical dendrites at
the distal segment (p < 0.05). RSV treatment also restored the total
length (p < 0.05) and number of branching points (p < 0.05) as well in
CA1 neurons of MoHE rats. In addition to apical dendrites, we also
analyzed the arborization in basal dendrites of CA1 pyramidal neurons.
As observed in supplementary Fig S2, the length and number of
branching point’s of basal dendrites of those CA1 neurons showed no
significant changes across the groups.
3.3. Effect of resveratrol on SIRT1 activity
Resveratrol has been reported to be an activator of SIRT1 both in
vivo and in vitro (Borra et al., 2004). As illustrated in Fig. 3, SIRT1
activity is significantly declined (p < 0.05) in the hippocampus of
MoHE rats. However, administration of resveratrol led to the significant
recovery in SIRT1 activity in the hippocampus of those MoHE rats
(p < 0.01).
4. Discussion
In this study, we intended to evaluate the neurobehavioral and
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Journal of Chemical Neuroanatomy 106 (2020) 101797
neuromorphological basis of neuroprotection by resveratrol (RSV) in
the MoHE rat model. As far as characterizing HE is concerned, cognitive
and motor dysfunction are the two main features of the cirrhotic pa-
tients (Mechtcheriakov et al., 2006; Bajaj, 2008) which can be eval-
uated with reproducibility in the rodent models by performing MWM
and Rota-rod tests (Singh and Trigun, 2014). Using these tests, it has
been described (Singh and Trigun, 2014, 2010) that increasing grades
of TAA induced CLF in rats coincides with a similar trend of the severity
of memory deficits and motor dysfunction in rat model. Thus, MWM
and rota-rod performance scores were used as an endpoint assay to
evaluate the efficacy of RSV as a neuroprotectant. Results of MWM tests
(Fig. 1 (a) & b) and of rota rod performance test (Fig. 1 c) clearly
suggest a significantly impaired spatial reference memory coinciding
with a marked decline in rota-rod performance tests shown by the CLF
rats. Since, the data of escape latency scores and time spent by these
CLF rats on rotating rod matched with the pattern shown by moderate
grade HE rats (Singh and Trigun, 2010), these rats were also categor-
ized as MoHE rats. Notably, all these MoHE, associated deranged neu-
robehavioral scores are observed to be recovered back following RSV
treatment. Though this is the first demonstration of RSV dependent
recovery in MoHE associated memory deficit and motor dysfunction,
these are in concordance with earlier studies that demonstrate similar
neurobehavioral recovery following RSV treatment in case of other
brain disorders. For instance, RSV treatment reversed behavioural
deficits in animal models of diabetic neuropathy (Cao et al., 2017),
ischemic insult and dementia (Wong and Tang, 2016; Cao et al., 2017).
In general, altered neurobehavioral outputs are resulted from
changes in the fine-tuning of specific neuronal connections. So far
memory functions are concerned; CA1 pyramidal cells and their den-
dritic structures play crucial roles in the trisynaptic circuit that is im-
portant for memory acquisition and LTP (Michán et al., 2010). In this
respect, quantification of dendritic arborization and morphology of
spines provide data for the neuro-structural basis of synaptic plasticity.
Therefore, we performed a morphometric analysis of the apical and
basal dendrites of CA1 pyramidal neurons using Golgi-Cox staining.
Tracings of Golgi stained CA1 neurons from the MoHE brain showed an
overall decrease in the apical length and number of branching points as
compared to the control group rats; while resveratrol treatment sig-
nificantly reversed the MoHE-induced decrease in apical dendritic ar-
borization (Fig. 2a–c). The restorative effect of RSV on the morphology
of CA1 pyramidal neurons was comparable to that of the control. In the
present study, we did not observe any changes in the branching pattern
of CA1 basal dendrites. Taken together, the findings of Figs. 2 and 1
suggest a cause and effect relationship between RSV dependent re-
versible morphometric changes in CA1 pyramidal neurons with that of
the RSV dependent recovery in MWM & rota-rod scores. To our
knowledge, this is the first report to provide a neuromorphometric basis
for reversal of neurobehavioral deficits in case of MoHE pathogenesis.
Of note, RSV has been shown to significantly increase the dendritic
length and spine density in pyramidal neurons of the prefrontal cortex
and hippocampus of aged rats (Monserrat Hernández‐Hernández et al.,
2016).
There could be more than one biochemical mechanism by which
RSV could prevent neuronal derangements. Previously, ROS scavenging
activities and modulating endogenous antioxidant enzymes due to RSV
treatment (Truong et al., 2018) have drawn much attention as main
mechanism of RSV led neuroprotection. In case of HE, however, report
on RSV led neuroprotection is scanty. Nonetheless, some findings sug-
gest that exposure of astroglial and neuronal cells to increased ammonia
concentration in culture could enhanced oxidative stress and proin-
flammatory factors with concordant decline of brain ammonia meta-
bolizing enzyme, which, however, could be reversed due to resveratrol
treatment (Bobermin et al., 2012, 2015). In the present MoHE model,
however, antioxidant role of RSV seems to be a less likely option, be-
cause, ROS level and profile of most of the antioxidant enzymes have
been reported to remain unaltered in the cortex (region of memory
A. Khanna, et al. Journal of Chemical Neuroanatomy 106 (2020) 101797

Fig. 2. (a) Photomicrographs and neurolucida tracings of CA1 pyramidal neurons in the hippocampus. Representative photographs of Golgi-Cox stained CA1 neurons
and their respective tracings from control, MoHE and MoHE treated with resveratrol. Scale bar =100 μm. (b) Quantification of branching pattern in the apical
dendrites of CA1 pyramidal neurons. (a) total length as a function of radius from the soma, (b)total number of branching points as a function of radius from the soma.
Statistical analysis applied was One-way ANOVA followed by Tukey’s post-hoc test. Values are represented as mean ± SD; n = 30 neurons per group; p < 0.05
(*control vs MoHE group; # MoHE vs MoHE + RSV group).

HA model of MoHE (Khanna and Trigun, 2016), further suggests a

Fig. 3. SIRT1activity assay. The activity of SIRT1 in the hippocampus of con-
trol, MoHE and MoHE rats treated with SIRT1 activator resveratrol. Statistical
analysis applied was student’s t-test. Values are represented as mean ± SD,
where n = 5, p < 0.05 (*control vs MoHE group; # MoHE vs MoHE + RSV
group).
functions) of moderate grade HA rats (Singh et al., 2008) and in the
cortical slices exposed to moderate HA (Mehrotra and Trigun, 2012).
Moreover, it is now becoming clearer that SIRT1 activation happens
to be the main integrator of RSV dependent neurochemical homeostasis
at neuronal cells level. It has been suggested that resveratrol prevents
neuroinflammation by declining ROS level and also by inactivating
NFκB, however, via activating SIRT1 signalling pathways
(Malaguarnera et al., 2018; Magaji et al., 2017). A similar pattern of
RSV dependent declined expression of TNFα and NfκB, in case of the
possible role of SIRT1 activation in RSV mediated normalization of
neuro-inflammation in MoHE rats. This is because; SIRT1 possesses an
allosteric activation site for resveratrol (Revollo and Li, 2013) which
upon activation regulates expression of many antioxidant and in-
flammation-related proteins mainly by deacetylating certain transcrip-
tion factors (Anamika et al., 2019; Revollo and Li, 2013).
Though limited but there are some reports that demonstrate the role
of RSV vis a vis SIRT1 activation in maintaining neuroarchitecture. For
example, RSV-SIRT1 activation has been found accountable for proper
neuronal differentiation (Mehrotra and Trigun, 2012). Also, SIRT1-KO
mice have been shown to exhibit significantly reduced hippocampal
dendritic arborizations (Michán et al., 2010). Further, resveratrol
treatment to BALB/c mice improves memory and increases spine den-
sity in the hippocampus (Torres-Pérez et al., 2015). There are some
direct evidences as well, wherein, RSV mediated SIRT1 activation has
been demonstrated to improve dendritic arborizations in hippocampal
neurons challenged with Aβ neurotoxicity (Gomes et al., 2018). In the
present context as well we observed significantly declined dendritic
length with less arborization of the CA1 pyramidal neurons of MoHE
rats, which could be considered accountable for declined LTP at CA1
synapses leading to memory deficit in those rats (Figs. 1,2). Moreover,
these neuromorphological changes were consistent with significantly
declined activity of SIRT1 in the hippocampus of those MoHE rats
(Fig. 3). Thus, suggesting a role of SIRT1 in maintaining normal sy-
naptic plasticity required for memory consolidation. Indeed, a con-
cordant reversal of both, CA1 neuronal arborizations (Fig. 2 and

5
A. Khanna, et al.
supplementary Figs S1 & S2) and neurobehavioral scores (Fig. 1 a–c) vis
a vis SIRT1 activation (Fig. 3), following RSV treatment, provide a
cause and effect relationship between SIRT1 activity and prevention of
neuronal derangements in the hippocampus of MoHE rats. Thus, SIRT1
activation could be argued as a target to improve cognitive deficits and
motor dysfunction in liver cirrhotic MoHE conditions. Indeed, SIRT1
has been proposed as a potential therapeutic target for the cognitive
impairment induced by type 2 diabetes mellitus (Cao et al., 2017) and
that RSV, in particular, is advocated as a treatment regimen for de-
pression, anxiety, and MHE (Malaguarnera et al., 2018; Magaji et al.,
2017).
5. Conclusion
The findings highlight about neuroarchitectural basis of resveratrol-
SIRT1 activation-dependent reversal of neurobehavioral deficits of
MoHE pathogenesis and thus, advocate for SIRT1 activation as a po-
tential target to prevent neuronal derangements of MoHE.
6. Contribution
Study plan: SK Trigun, Archita Khanna; Experiments:
Neurobehavioral and SIRT1 activity by Archita Khanna, Golgi-cox
staining; Anamika, Arup Acharjee, Archita Khanna; MS writing: Archita
Khanna, SK Trigun, Anamika; Morphometric analysis of Golgi-cox
stained sections and fine tuning of MS: Suwarna Chakraborty, Sunil
Jamuna Tripathi and Shankaranarayana Rao BS.
Ethical declaration
The study plan was approved by the Institutional ethical Committee
for the care and use of laboratory animals.
Authors contributions included in the manuscript
Study plan: SK Trigun, Archita Khanna; Experiments:
Neurobehavioral and SIRT1 activity by Archita Khanna, Golgi-cox
staining; Anamika, Arup Acharjee, Archita Khanna; MS writing: Archita
Khanna, SK Trigun, Anamika; Morphometric analysis of Golgi-cox
stained sections and fine tuning of MS: Suwarna Chakraborty, Sunil
Jamuna Tripathi and Shankaranarayana Rao BS.
Declaration of Competing Interest
The authors declare no conflict of interest about this paper.
Acknowledgements
This work was financially supported by ICMR (Indian Council of
Medical Research) project (54/38/CFPGER/2011/NCD-II) to SKT and
Lady Tata Memorial Trust- JRF/SRF’ award to Archita Khanna. Authors
are thankful to the DST FIST, UGC-CAS (Department of Zoology) &
UGC-UPE, DST Purse and DBT-BHU ISLS programmes in Institute of
Science and Morphometric analysis facility with Shankaranarayana Rao
BS at Department of Neurophysiology, NIMHANS, Bengaluru.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jchemneu.2020.
101797.
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