Oral administration of d-galactose induces cognitive impairments and oxidative damage in rats.

Abstract

d-Galactose (d-gal) is a reducing sugar that can be used to mimic the characteristics of aging in rodents; however, the effects of d-gal administration by oral route are not clear. Therefore, the aim of this study was to elucidate if the oral administration of d-gal induces cognitive impairments, neuronal loss, and oxidative damage, mimicking an animal model of aging. Male adult Wistar rats (4 months old) received d-gal (100mg/kg) via the oral route for a period of 1, 2, 4, 6 or 8 weeks. The results showed cognitive impairments in the open-field test in the 4th and 6th weeks after d-gal administration, as well as an impairment in spatial memory in the radial maze test after the 6th week of d-gal administration. The results indicated increase of levels of thiobarbituric acid reactive species-TBARS-and carbonyl group content in the prefrontal cortex from the 4th week, and in all weeks of d-gal administration, respectively. An increase in the levels of TBARS and carbonyl group content was observed in the hippocampus over the entire period of d-gal treatment. In the 8th week of d-gal administration, we also observed reductions in synaptophysin and TAU protein levels in the prefrontal cortex. Thus, d-gal given by oral route caused cognitive impairments which were accompanied by oxidative damage. Therefore, these results indicate that orally administered d-gal can induce the behavioral and neurochemical alterations that are observed in the natural aging process. However, oral d-gal effect in rats deserve further studies to be better described.

Full text

Behavioural
Brain
Research
302
(2016)
35–43
Contents
lists
available
at
ScienceDirect
Behavioural
Brain
Research
jou
rn
al
hom
epage:
www.elsevier.com/locate/bbr
Research
report
Oral
administration
of
d-galactose
induces
cognitive
impairments
and
oxidative
damage
in
rats
Josiane
Budnia,b,∗,
Robson
Pachecoa,b,
Sabrina
da
Silvaa,b,
Michelle
Lima
Garceza,b,
Francielle
Minaa,b,
Tatiani
Bellettini-Santosa,b,
Jesiel
de
Medeirosa,b,
Bruna
Constantino
Vossa,b,
Amanda
Valnier
Steckerta,
Samira
da
Silva
Valvassoria,c,
João
Quevedoa,d,e,f
aLaboratório
de
Neurociências,
Programa
de
Pós-Graduac¸
ão
em
Ciências
da
Saúde,
Unidade
Acadêmica
de
Ciências
da
Saúde,
Universidade
do
Extremo
Sul
Catarinense,
Criciúma,
SC,
Brazil
bLaboratório
de
Doenc¸
as
Neurodegenerativas,
Programa
de
Pós-Graduac¸
ão
em
Ciências
da
Saúde,
Unidade
Acadêmica
de
Ciências
da
Saúde,
Universidade
do
Extremo
Sul
Catarinense,
Criciúma,
SC,
Brazil
cLaboratório
de
Sinalizac¸
ão
Neural
e
Psicofarmacologia,
Programa
de
Pós-Graduac¸
ão
em
Ciências
da
Saúde,
Unidade
Acadêmica
de
Ciências
da
Saúde,
Universidade
do
Extremo
Sul
Catarinense,
Criciúma,
SC,
Brazil
dTranslational
Psychiatry
Program,
Department
of
Psychiatry
and
Behavioral
Sciences,
The
University
of
Texas
Health
Science
Center
at
Houston
(UTHealth),
McGovern
Medical
School,
Houston,
TX,
USA
eCenter
of
Excellence
on
Mood
Disorders,
Department
of
Psychiatry
and
Behavioral
Sciences,
The
University
of
Texas
Health
Science
Center
at
Houston,
McGovern
Medical
School,
Houston,
TX,
USA
fNeuroscience
Graduate
Program,
Graduate
School
of
Biomedical
Sciences,
The
University
of
Texas
Health
Science
Center
at
Houston,
Houston,
TX,
USA
h
i
g
h
l
i
g
h
t
s
•d-Galactose
by
oral
route
induces
novelty
habituation
deficit.
•d-Galactose
by
oral
route
induces
spatial
memory
impairment.
•d-Galactose
by
oral
route
induces
high
thiobarbituric
acid
reactive
species
levels.
•d-Galactose
by
oral
route
induces
increase
of
carbonyl
group
content.
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
27
August
2015
Received
in
revised
form
20
December
2015
Accepted
25
December
2015
Available
online
31
December
2015
Keywords:
Oral
d-galactose
Aging
Cognitive
impairment
Oxidative
damage
a
b
s
t
r
a
c
t
d-Galactose
(d-gal)
is
a
reducing
sugar
that
can
be
used
to
mimic
the
characteristics
of
aging
in
rodents;
however,
the
effects
of
d-gal
administration
by
oral
route
are
not
clear.
Therefore,
the
aim
of
this
study
was
to
elucidate
if
the
oral
administration
of
d-gal
induces
cognitive
impairments,
neuronal
loss,
and
oxidative
damage,
mimicking
an
animal
model
of
aging.
Male
adult
Wistar
rats
(4
months
old)
received
d-gal
(100
mg/kg)
via
the
oral
route
for
a
period
of
1,
2,
4,
6
or
8
weeks.
The
results
showed
cognitive
impairments
in
the
open-field
test
in
the
4th
and
6th
weeks
after
d-gal
administration,
as
well
as
an
impairment
in
spatial
memory
in
the
radial
maze
test
after
the
6th
week
of
d-gal
administration.
The
results
indicated
increase
of
levels
of
thiobarbituric
acid
reactive
species—TBARS—and
carbonyl
group
content
in
the
prefrontal
cortex
from
the
4th
week,
and
in
all
weeks
of
d-gal
administration,
respectively.
An
increase
in
the
levels
of
TBARS
and
carbonyl
group
content
was
observed
in
the
hippocampus
over
the
entire
period
of
d-gal
treatment.
In
the
8th
week
of
d-gal
administration,
we
also
observed
reduc-
tions
in
synaptophysin
and
TAU
protein
levels
in
the
prefrontal
cortex.
Thus,
d-gal
given
by
oral
route
caused
cognitive
impairments
which
were
accompanied
by
oxidative
damage.
Therefore,
these
results
indicate
that
orally
administered
d-gal
can
induce
the
behavioral
and
neurochemical
alterations
that
are
observed
in
the
natural
aging
process.
However,
oral
d-gal
effect
in
rats
deserve
further
studies
to
be
better
described.
©
2015
Elsevier
B.V.
All
rights
reserved.
∗Corresponding
author
at:
Laboratório
de
Neurociências,
Programa
de
Pós-Graduac¸
ão
em
Ciências
da
Saúde,
Unidade
Acadêmica
de
Ciências
da
Saúde,
Universidade
do
Extremo
Sul
Catarinense,
88806-000
Criciúma,
SC,
Brazil.
E-mail
address:
josiane.budni@unesc.net
(J.
Budni).
http://dx.doi.org/10.1016/j.bbr.2015.12.041
0166-4328/©
2015
Elsevier
B.V.
All
rights
reserved.

36
J.
Budni
et
al.
/
Behavioural
Brain
Research
302
(2016)
35–43
1.
Introduction
d-Galactose
(d-gal)
is
a
reducing
sugar
or
monosaccharide
which
is
abundantly
present
in
milk
products,
fruits
and
vegetables
[1],
and
is
usually
converted
into
glucose
by
galactose-1-phosphate
uridyltransferase
and
galactokinase
[2].
However,
d-gal
adminis-
tration
over
long
periods
of
time
can
lead
to
an
enzymatic
overload,
which
impairs
the
body’s
natural
ability
to
catalyze
galactose
into
glucose,
so
causing
an
increase
of
galactitol
and
an
activation
of
aldose
reductase.
This
in
turn
causes
a
depletion
in
NADPH,
which
leads
to
an
accumulation
of
hydrogen
peroxide
and
other
free
rad-
icals
(Lai,
2009),
causing
oxidative
damage
to
the
cells
[3,4].
In
addition,
at
high
levels,
d-gal
may
react
with
the
amino
groups
of
proteins
and
peptides
to
form
advanced
glycation
end
products
(AGE)
in
vivo
[5].
AGE
are
increased
during
aging
and
have
been
associated
with
the
pathogenesis
of
many
diseases,
such
as
diabetes
[6],
amyotrophic
lateral
sclerosis
[7],
and
Alzheimer’s
disease
[8].
Therefore,
it
has
been
postulated
that
d-gal
may
induce
behav-
ioral
alterations
that
reproduce
the
natural
aging
processes
in
rats
and
mice
[9,10].
Several
studies
have
suggested
that
chronic
sys-
temic
administration
of
d-gal
could
be
used
as
a
model
of
cognitive
disorders
and
aging
[11–14].
Aging
is
a
natural
process
of
changes
that
culminates
in
a
progressive
decline
in
both
physiological
and
behavioral
ability.
The
progression
of
aging
tends
to
compromise
the
entire
organism,
showing
particular
severity
within
the
cen-
tral
nervous
system
[15,16].
It
is
characterized
by
a
gradual
loss
of
cognitive
performance,
memory,
and
spatial
ability
[17].
These
symptoms
are
accompanied
by
structural
and
functional
changes
within
the
brain,
such
as
a
decline
in
mitochondrial
function
[18]
characterized
by
a
decrease
in
ATP
synthesis
and
oxidative
damage
[19].
These
changes
play
a
crucial
role
in
the
neurodegenerative
disorders
associated
with
the
pathogenesis
of
age-related
diseases.
According
to
data
from
studies,
d-gal
leads
the
field
in
cre-
ating
biochemical
abnormalities
in
experimental
animals,
such
as;
accumulations
of
reactive
oxygen
species,
reductions
of
antioxidant
enzymes,
mitochondrial
deficits
and
neuroinflamma-
tion/apoptosis.
These
changes
in
rodents
are
similar
to
those
that
occur
in
the
aging
human
brain
[11,13,20–22].
Moreover,
chronic
systemic
(intraperitoneal
or
subcutaneous)
administrations
of
d-gal
can
induce
alterations
like
the
ones
observed
in
Alzheimer’s
disease
(AD)
[23,24].
Lin
et
al.
[24]
found
that
d-gal
given
via
intraperitoneal
administration
significantly
increased
the
content
of
amyloid
beta
(A␤)
in
the
hippocampus
of
mice.
A
previous
study
showed
that
intraperitoneal
adminis-
trations
of
d-gal
also
increased
the
expression
of
the
brains
A␤
precursor
protein
[25].
It
has
been
well
described
in
literature
that
the
aggregation
and
deposition
of
A␤
in
the
brain
is
a
key
step
in
the
pathogenesis
of
AD,
and
that
this
process
elicits
a
cascade
of
cellular
events
that
ultimately
leads
to
neuronal
loss
and
dementia
[26].
In
addition,
intraperitoneal
or
subcutaneous
injections
of
d-gal
lead
to
spatial
learning
impairments,
oxidative
stress
and
neuroin-
flammation,
as
well
as
activation
of
the
NF␬B
signaling
pathway
in
the
brain
of
rodents
[11,27–30].
␤-Amyloid
peptide,
as
AGE´
ıs,
can
activate
the
receptor
for
advanced
glycation
end
products
(RAGE),
leading
to
oxidative
stress
and
to
the
activation
of
the
transcrip-
tion
factor
NF␬-B
signaling
pathways,
causing
the
transcription
of
inducible
nitric
oxide
synthase
and
a
variety
of
cytokines
[8].
On
the
other
hand,
there
is
compelling
evidence
showing
that
the
oral
administration
of
d-gal
induces
protective
effects
in
an
animal
model
of
AD
induced
by
streptozotocin.
A
recent
study
compared
both
systemic
and
oral
chronic
administrations
of
d-gal,
and
the
results
demonstrated
that
the
oral
administration
route,
unlike
the
systemic
method,
can
reverse
cognitive
deficits
in
a
streptozotocin-induced
model
of
AD,
thus
the
protective
effects
of
this
sugar
may
well
be
concentration
or
administration
route
dependent
[31].
Therefore,
there
is
some
controversy
surrounding
the
use
of
d-gal
via
the
oral
route.
Considering
that
many
studies
related
to
aging
focus
on
the
animal
model
of
d-gal
administered
by
the
intraperitoneal
and
subcutaneous
routes,
the
administration
of
this
carbohydrate
by
the
oral
route
has
not
received
sufficient
attention.
Therefore,
in
this
study
we
are
investigating
if
the
oral
administration
of
d-gal
induces
cognitive
and
biochemical
abnormalities,
since
the
oral
route
can
be
used
as
an
alternative
way
of
administrating
d-gal
over
longer
periods
of
time.
2.
Material
and
methods
2.1.
Animals
4
month
old
adult
male
Wistar
rats,
(weighing
350–500
g)
were
used
in
this
research
(total
of
150
rats).
The
animals
were
accli-
matized
to
the
laboratory
conditions
at
room
temperature
prior
to
any
experimentation.
The
animals
were
kept
under
standard
lab
conditions
of
a
12
h
light/dark
cycle,
with
food
and
water
available
ad
libitum,
and
were
housed
in
plastic
cages
with
soft
bedding.
All
manipulations
were
performed
between
8:00
a.m.
and
5:00
p.m.
The
project
was
approved
by
the
ethical
committee
of
the
Univer-
sidade
do
Extremo
Sul
Catarinense
and
all
experimental
procedures
were
performed
according
to
the
NIH
Guide
for
the
Care
and
Use
of
Laboratory
Animals,
as
well
as
under
the
Brazilian
Society
for
Neuroscience
and
Behavior
recommendations
for
animal
care.
This
study
was
approved
by
the
local
ethics
committee
(Ethics
Com-
mittee
on
Animal
Use—CEUA
of
the
Universidade
do
Extremo
Sul
Catarinense).
2.2.
Drugs
and
treatment
d-Gal
(d-galactose,
Sigma–Aldrich,
St.
Louis,
MO,
USA)
solution
was
used.
It
was
dissolved
in
water
for
administration
at
the
dose
of
100
mg/kg
[9,14,32,33]
of
body
weight,
and
given
by
oral
gavage,
once
a
day,
over
a
period
of
1,
2,
4,
6
or
8
weeks.
Animals
were
randomized
into
two
groups:
control
animals
(receiving
water
by
oral
gavage)
or
d-gal
animals
(receiving
d-gal
by
oral
gavage).
The
behavioral
tests
and
biochemical
analysis
were
undertaken
on
the
1st,
2nd,
4th,
6th
and
8th
weeks
after
the
last
administration
of
d-gal.
Twenty-four
hours
after
the
last
administration
of
d-gal
in
each
period
of
treatment,
the
animals
were
weighed
and
subjected
to
the
behavioral
tests.
After
the
completion
of
the
open
field
task,
or
72
h
after
the
last
administration
of
d-gal,
the
rodents
were
killed
by
decapitation
without
the
use
of
anesthesia
(the
procedure
was
approved
by
the
Ethics
Committee)
and
their
brain
tissues
were
collected
for
use
in
the
molecular
studies.
2.3.
Open-field
test
Long-term
retention
of
habituation
in
a
novel
environment
can
be
considered
a
non-associative,
non-aversive
type
of
learning,
which
can
be
measured
by
a
decrease
in
the
amount
of
exploratory
activity
undertaken
by
the
test
subject.
In
rodents,
it
is
assessed
by
the
number
of
rearings
performed
in
a
test
session
carried
out
24
h
after
the
first
exploration
session
[34].
This
apparatus
consists
of
a
45
cm
×
60
cm
brown
plywood
arena
which
is
surrounded
by
50
cm
high
wooden
walls
and
fitted
with
a
frontal
glass
wall.
The
floor
of
the
open
field
was
divided
into
nine
rectangles
(15
cm
×
20
cm
each)
by
black
lines.
The
animals
were
gently
placed
on
the
left
rear
quadrant
and
then
left
to
explore
the
arena.
To
investigate
the
effects
of
any
drug
treatment
on
spontaneous
locomotor
activity,
the
numbers
of
horizontal
(crossings)
and
vertical
(rearings)
activ-
ities
performed
by
each
rat
during
a
5
min
observation
period
were

J.
Budni
et
al.
/
Behavioural
Brain
Research
302
(2016)
35–43
37
counted
by
an
expert
observer.
Twenty-four
hours
after
the
train-
ing
session,
one
new
exposition
(test
session)
to
the
open
field
was
carried
out
for
a
period
of
5
min.
2.4.
Radial
maze
Training
was
conducted
in
an
elevated
plastic
maze
with
a
cen-
ter
platform
(40
cm
in
diameter)
that
was
connected
to
eight
60
cm
by
9
cm
arms
extending
radially.
Twenty-four
hours
after
the
last
administration
of
d-gal
in
each
period
of
treatment,
the
animals
were
subjected
to
the
maze,
but
only
for
the
purpose
of
habitua-
tion
to
the
apparatus.
Subsequently,
food-rewarded
training
trials
began
on
day
2
[35].
During
the
habituation
sessions,
the
animals
were
allowed
to
explore
the
eight
maze
arms
for
10
min,
and
then
returned
to
their
home
cages.
After
this,
10
fruit
loops
per
cage
were
given
over
a
period
of
2
h.
On
the
second
day,
each
rat
was
returned
to
the
maze
with
all
eight
arms
open,
and
fruit
loops
were
placed
in
only
four
of
the
arms.
The
animals
were
placed
in
the
central
portion
of
the
maze
and
allowed
to
find
the
rewards,
the
test
period
either
lasting
a
total
of
10
min,
or
ending
when
the
animal
had
found
the
food
in
all
4
arms.
The
training
periods
were
performed
over
four
consecutive
days.
The
total
time
to
find
the
food
in
the
4
arms
was
recorded.
Entries
into
arms
that
did
not
contain
fruit
loops,
or
into
arms
in
which
the
animal
had
previously
consumed
the
food
were
recorded
as
total
errors.
2.5.
Thiobarbituric
acid
reactive
species
levels
The
hippocampus
and
prefrontal
cortex
were
mixed
with
1
mL
of
trichloroacetic
acid
10%
and
1
mL
of
thiobarbituric
acid
0.67%,
and
then
heated
in
a
bath
of
boiling
water
for
30
min.
Malondialde-
hyde
equivalents
(a
marker
of
lipid
peroxidation)
were
determined
spectrophotometrically
at
532
nm.
Formation
of
thiobarbituric
acid
reactive
species
(TBARS)
during
an
acid-heating
reaction
was
mea-
sured
as
previously
described
[36].
2.6.
Carbonyls
protein
content
The
oxidative
damage
to
proteins
was
assessed
by
the
determination
of
carbonyl
groups
content
based
on
a
dinitro-
phenylhidrazine
(DNPH)
reaction
[37].
The
hippocampus
and
prefrontal
cortex
were
precipitated
by
the
addition
of
20%
trichloroacetic
acid,
and
resuspended
in
DNPH.
The
absorbance
was
monitored
spectrophotometrically
at
370
nm.
2.7.
Immunoblot
analysis
The
hippocampus
and
prefrontal
cortex
were
removed
for
immunoblot
analysis
72
h
after
the
last
administration
of
d-gal.
Protein
samples
of
hippocampal
tissue
were
separated
by
SDS-
PAGE,
using
polyacrilamide
gels
(10%),
followed
by
transfer
to
PVDF
Immobilon-FL
transfer
membranes
(Millipore,
USA).
Protein
load-
ing
and
blot
transfer
efficiency
were
monitored
by
staining
with
Ponceau
S
(0.5%
ponceau:
1%
acetic
acid).
Membranes
were
blocked
for
1
h
with
TBS-T
(tris-buffered
saline
and
0.1%
Tween-20;
pH
7.4)
and
milk
(0.5%).
Membrane
blots
were
incubated
with
primary
antibody
anti-␤-actin
(1:1000;
Sigma–Aldrich,
USA;
cod.
A5441);
anti-TAU
(1:1000;
Millipore
Temecula,
USA;
cod.
#05-348);
or
anti-
synaptophysin
(1:750;
Millipore,
USA;
cod.
#MAB368)
diluted
in
TBS-T
and
stored
overnight
at
4◦C.
After
washing,
the
membranes
were
incubated
for
1
h
with
goat
anti-mouse
(1:5000;
Santa
Cruz
Biotechnology,
USA)
horseradish
peroxidase
(HRP)—conjugated
secondary
antibodies.
Immunocomplexes
were
visualized
using
the
enhancing
chemiluminescence
detection
system
(GE
Health-
Care,
UK)
as
described
by
the
manufacturer.
Densitometric
analysis
was
performed
using
ImageJ
software
(version
Java
1.6.0
20,
USA).
The
total
protein
concentrations
were
determined
using
the
method
described
by
Lowry
et
al.
[38].
2.8.
Statistical
analysis
Statistical
analyses
were
performed
using
SPSS
20.0
for
Win-
dows.
Data
from
the
habituation
test
and
immunoblot
analysis
were
reported
as
means
±
SEM.
Oxidative
damage
was
reported
as
means
±
SD.
These
data
were
analyzed
using
the
paired
Stu-
dent’s
t-test.
Data
from
the
radial
maze
tests
were
analyzed
using
repeated-measures
analyses,
followed
by
the
Bonferroni
post-hoc
test
when
the
Mauchley’s
test
of
sphericity
result
was
signifi-
cant
(assumption
of
sphericity
violated).
The
data
was
reported
as
means
±
SEM,
and
p
values
<0.05
were
considered
statistically
significant.
3.
Results
Fig.
1
shows
habituation
to
a
novel
environment
assessed
in
the
open-field
task.
The
control
rats
in
all
treatment
protocols
in
the
1st
(crossings:
p
<
0.001;
rearings:
p
<
0.001),
2nd
(crossings:
p
<
0.001;
rearings:
p
=
0.0012),
4th
(crossings:
p
=
0.002;
rearings:
p
<
0.001),
6th
(crossings:
p
=
0.013;
rearings:
p
=
0.017)
and
8th
(crossings:
p
=
0.0055;
rearings:
p
=
0.0042)
weeks
of
treatment
dis-
played
a
reduction
in
the
number
of
crossings
(Fig.
1A)
and
rearings
(Fig.
1B),
when
re-exposed
24
h
later
(test
session)
to
the
appara-
tus.
The
animals
that
received
d-gal
administration
produced
the
same
pattern
of
response
as
displayed
by
the
control
rats
in
the
1st,
2nd,
and
8th
weeks.
However,
in
the
4th
(crossings:
p
=
0.86)
and
6th
(crossings:
p
=
0.26)
weeks,
they
did
not
present
a
statis-
tical
difference
when
observing
the
number
of
crossings
between
the
training
and
test
sessions,
and
in
the
4th
week
when
observ-
ing
the
number
of
rearings
(p
=
0.3),
suggesting
an
impairment
in
the
habituation
memory.
In
addition
to
this,
the
animals
treated
with
d-gal
for
a
period
of
8
weeks
displayed
an
increased
num-
ber
of
rearings
in
the
training
sessions
compared
with
the
saline
group.
Thus,
this
indicates
that
treatment
with
d-gal
altered
the
level
of
spontaneous
exploration
in
rats.
The
analysis
of
the
radial
maze
data
(Fig.
2)
was
undertaken
by
repeated
measures
analy-
sis
of
variance.
In
the
4
weeks
after
treatment
with
d-gal,
there
were
significant
differences
for
the
number
of
behavioral
repe-
titions
when
evaluating
the
latency
time
to
find
food
(Fig.
2A)
[F(3.54)
=
20.99,
p
<
0.001],
but
there
was
no
statistical
difference
in
d-gal
administration
[F(1.18)
=
0.452,
p
=
0.665].
Further
analysis
with
the
Bonferroni
post-hoc
test
showed
a
decrease
in
the
latency
time
to
find
the
food
in
the
4
arms
containing
this
reward
within
the
control
group,
when
comparing
the
first
day
to
the
second
(p
=
0.007);
third
(p
<
0.001)
and
fourth
days
(p
<
0.001);
likewise,
the
animals
treated
for
a
period
of
4
weeks
with
d-gal
showed
decreases
in
their
latency
times
to
find
food
on
the
first
test
day
compared
to
the
second
(p
=
0.036);
third
(p
=
0.006);
and
fourth
days
of
testing
(p
<
0.001).
There
were
significant
differences
for
the
number
of
behavioral
repetitions
when
comparing
the
latency
time
to
find
food
[F(3.42)
=
6.20,
p
=
0.002],
but
there
was
no
statistical
difference
in
d-gal
administration
[F(1.14)
=
0.388,
p
=
0.735]
in
the
6th
week.
Further
analysis
with
the
post-hoc
test
observed
that
ani-
mals
treated
with
water
showed
no
significant
difference
between
the
first
day
to
the
second
(0.076),
but
there
was
a
decrease
in
the
latency
times
between
the
first,
third
(p
=
0.017)
and
the
fourth
days
(0.007).
However,
the
rats
that
received
d-gal
over
a
period
of
6
weeks,
showed
a
decrease
in
their
latency
times
to
find
food
only
in
the
first
day
when
compared
to
the
third
day
(p
=
0.025),
but
not
when
comparing
the
first
day
to
the
second
(p
=
0.077)
or
the
fourth
days
(p
=
0.150).
When
evaluating
the
animals’
spatial
memory
8
weeks
after
the
treatment
with
water,
it
was
observed
that
there

38
J.
Budni
et
al.
/
Behavioural
Brain
Research
302
(2016)
35–43
Fig.
1.
The
effect
of
d-gal
(100
mg/kg)
administration
via
the
oral
route
in
male
rats
subjected
to
the
open-field
habituation
task.
The
open-field
test
was
carried
24
h
after
the
last
training
session,
and
lasted
for
a
period
of
5
min.
The
tests
were
performed
on
the
1st,
2nd,
4th,
6th
and
8th
weeks
of
treatment.
Data
are
the
mean
±
SD
number
of
crossings
(A)
and
rearings
(B).
The
control
rats
in
all
treatment
protocols
in
the
1st,
2nd,
4th
and
6th
weeks
of
treatment
were
observed
to
have
a
reduced
number
of
crossings
(A)
and
rearings
(B),
when
re-exposed
24
h
later
(test)
to
the
apparatus.
The
animals
that
received
d-gal
administration
produced
the
same
pattern
of
response
in
the
1st,
2nd
and
8th
weeks.
However,
in
the
4th
and
6th
weeks
there
were
no
statistical
differences
when
observing
the
number
of
crossings,
and
in
the
4th
week
when
observing
the
number
of
rearings
(suggesting
impairments
in
the
habituation
memory).
Data
were
analyzed
by
paired-samples
t-test
to
compare
the
test
session
with
training
session,
and
compare
the
control
test
with
d-gal
test,
n
=
9
animals
per
group.
*p
<
0.05
compared
to
the
respective
test
session
of
the
group.
was
a
decrease
in
the
latency
time
in
the
first
day
when
compared
to
the
second,
third
and
fourth
days
of
the
test
(p
=
0.008,
p
<
0.001,
p
<
0.001)
respectively,
and
also
in
the
animals
that
were
treated
with
d-gal
for
a
period
of
8
weeks
(p
=
0.003,
p
=
0.001,
p
<
0.001)
respectively,
and
that
there
were
significant
differences
for
the
behavioral
repetitions
when
evaluating
the
latency
time
to
find
food
[F(3.54)
=
56.18,
p
=
p
<
0.001],
but
that
there
was
no
statistical
difference
in
d-gal
administration
[F(1.18)
=
0.603,
p
=
0.717].
These
dates
suggest
impairments
in
the
spatial
memory
of
the
animals
that
received
d-gal,
however,
only
in
the
6
weeks
of
the
treatment.
When
evaluating
the
total
errors
to
find
food
(Fig.
2B),
there
were
differences
for
the
number
of
behavioral
repetitions
after
4
weeks
of
treatment
[F
(3.54)
=
18.94,
p
<
0.001],
but
there
was
no
statistical
difference
in
d-gal
administration
[F(1.18)
=
0.576,
p
=
0.247],
and
analysis
with
the
Bonferroni
post-hoc
test
showed
that
there
was
a
decrease
when
comparing
the
first
day
to
the
second
(p
=
0.044),
third
(p
=
0.001)
and
fourth
days
(p
<
0.001)
of
the
test
in
the
animals
that
received
water;
the
animals
that
received
d-gal
also
showed
a
decrease
in
the
total
number
of
errors
when
comparing
the
first
day
to
the
third
(p
=
0.022)
and
fourth
(p
<
0.001),
but
not
when
compared
to
the
second
day
(p
=
0.130).
In
the
period
6
weeks
after
treatment,
there
were
differences
in
the
number
of
behavioral
repetitions
[F(3.42)
=
5.037,
p
=
0.049],
but
there
was
no
statistical
difference
in
d-gal
administration
[F(1.14)
=
0.732,
p
=
0.546],
and
the
post-hoc
test
showed
that
in
the
animals
that
received
water,
there
were
no
differences
between
the
first
and
second
day
(p
=
0.792),
and
there
was
a
decrease
in
the
total
number
of
errors
to
find
food
when
comparing
the
first
day
to
the
third
day
(p
=
0.006)
and
fourth
day
(p
=
0.010);
however,
in
the
animals
treated
with
d-gal,
there
were
no
decreases
in
the
total
number
of
errors
between
the
days,
demonstrating
that
these
animals
did
not
learn
the
location
of
food
during
the
training
ses-
sions.
In
the
eighth
week,
there
were
differences
in
the
numbers
of
behavioral
repetitions
[F(3.54)
=
19.61,
p
=
0.001],
but
there
were
no
statistical
differences
in
d-gal
administration
[F(1.18)
=
0.171,
p
=
0.835].
Further,
the
Bonferroni
post-hoc
test
showed
that
for
oral
water
administration,
there
was
a
decrease
in
the
total
num-
ber
of
errors
when
comparing
the
first
day
to
the
second
(p
=
0.011),
third
(p
=
0.002)
and
fourth
days
(0.002),
and
there
was
a
decrease
in
the
number
of
total
errors
in
the
animals
treated
with
d-gal
at
8
weeks
for
the
first
day
when
comparing
it
to
the
second
(p
=
0.049),
third
(p
=
0.020)
and
fourth
days
(p
=
0.009).

J.
Budni
et
al.
/
Behavioural
Brain
Research
302
(2016)
35–43
39
Fig.
2.
The
effects
of
d-gal
(100
mg/kg,
v.o)
administration
via
the
oral
route
in
male
rats
subjected
to
the
radial
maze
one
day
after
habituation.
(B)
The
same
apparatus
was
used
for
the
training
sessions
and
testing.
When
the
tests
were
initiated,
each
rat
had
10
min
to
find
the
food,
the
tests
being
performed
in
the
end
of
4th,
6th
and
8th
weeks
of
treatment.
There
was
a
decrease
in
the
latency
time
to
find
food
when
comparing
the
first
day
to
the
subsequent
days
in
the
animals
that
received
water,
except
when
comparing
the
first
day
to
the
2nd
day
during
week
6
of
treatment.
In
the
animals
that
received
d-gal,
there
were
no
differences
between
the
first
and
the
2nd
days,
and
the
4th
day,
demonstrating
that
these
animals
did
not
learn
the
location
of
food
when
analyzing
the
total
errors,
however,
the
animals
treated
with
d-gal
showed
decreases
in
the
errors
to
find
food
only
in
the
3rd
and
4th
days
when
compared
to
the
first
day
after
4
weeks
of
treatment,
and
showed
no
reduction
of
errors
in
any
of
the
test
days
when
compared
to
the
first
day
in
the
6th
week
of
testing.
Data
are
the
mean
±
SEM
of
latency
time
to
find
food
(A),
and
total
errors
to
find
food
(B).
Data
were
analyzed
by
repeated-measures
analyses
followed
by
the
Bonferroni
post-hoc
test,
n
=
9–10
animals
per
group.
*p
<
0.05
compared
to
the
respective
first
test
day
session
of
the
group.
The
oxidative
damage
is
represented
in
Fig.
3.
The
thiobarbituric
acid
reactive
species
levels
(Fig.
3A)
were
found
to
have
increased
with
d-gal
treatment
in
the
prefrontal
cortex
when
compared
to
the
control
group
after
four
weeks
(p
=
0.042),
six
weeks
(p
=
0.020)
and
eight
weeks
(p
=
0.019).
d-Gal
also
induced
an
increase
of
thio-
barbituric
acid
reactive
species
levels
in
the
hippocampus
after
one
week
(p
=
0.029),
two
weeks
(p
=
0.022),
four
weeks
(p
=
0.002),
and
six
weeks
(p
=
0.008).
The
carbonyl
protein
content
is
shown
in
Fig.
3B.
The
results
showed
that
there
was
an
increase
of
car-
bonyl
groups
in
the
prefrontal
cortex
of
rats
treated
with
d-gal
when
compared
to
the
control
group
in
every
week
of
treatment
(week
one:
p
=
0.021),
(week
two:
p
=
0.002),
(week
four:
p
=
0.020),
(week
six:
p
=
0.001)
and
(week
eight:
p
=
0.003),
and
that
the
same
increase
also
occurred
in
the
hippocampus
of
the
animals
within
the
d-gal
group
(week
one:
p
=
0.011),
(week
two:
p
=
0.004),
(week
four:
p
=
0.006),
(week
six:
p
=
0.048)
and
(week
eight:
p
=
0.031).
These
results
indicated
that
d-gal
treatment
can
lead
to
oxidative
damage
in
lipids
and
proteins.
Fig.
4
shows
the
content
of
synaptophysin
and
TAU
total
pro-
teins.
A
decrease
in
synaptophysin
protein
content
in
the
prefrontal
cortex
was
only
observed
8
weeks
after
treatment
in
animals
that
had
been
treated
with
d-gal
(Fig.
4A)
when
compared
to
the
control
group,
(p
=
0.024)
while
in
the
hippocampus,
there
were
no
differ-
ences
among
the
groups.
TAU
content
(Fig.
4B)
was
decreased
in
rats
treated
with
d-gal
when
compared
to
the
control
group
only
in
the
prefrontal
cortex
at
8
weeks
of
treatment
(p
=
0.003),
while
in
the
hippocampus,
there
were
no
differences
among
the
groups.
4.
Discussion
The
present
study
has
been
conducted
to
investigate
if
d-gal
(100
mg/kg)
administered
via
the
oral
route,
can
induce
neurotox-
icity
in
rats
after
1,
2,
4,
6
or
8
weeks
of
treatment.
We
choose
the
dose
of
d-gal
(100
mg/kg)
based
upon
what
is
currently
being
used
in
intraperitoneal
and/or
subcutaneous
routes
to
induce
an
animal
model
of
aging
[9,14,32,33].
We
performed
a
time-curve
analysis
of
d-gal
(100
mg/kg)
administered
via
the
oral
route
to
evaluate
the
quickest
time
feasible
to
cause
damage
in
Wistar
rats.
The
oral
route
can
minimize
the
levels
of
stress
and
damage
caused
to
the
animals
while
using
intraperitoneal
and/or
subcutaneous
routes
in
the
administration
of
this
carbohydrate
over
long
periods
of
time.

40
J.
Budni
et
al.
/
Behavioural
Brain
Research
302
(2016)
35–43
Fig.
3.
The
effects
of
d-gal
(100
mg/kg,
v.o)
administration
via
the
oral
route
on
male
rats
showing
the
levels
of
oxidative
damage
in
the
prefrontal
cortex
and
hippocampus.
Data
are
the
mean
±
SD
of
the
hippocampus
and
prefrontal
cortex
taken
from
animals
treated
with
either
water
or
d-gal
at
the
end
of
1,
2,
4,
6
and
8
weeks.
Thiobarbituric
acid
reactive
species
levels
(TBARS)
are
shown
in
(A)
and
carbonyl
protein
content
in
(B).
(A)
The
TBARS
levels
were
found
to
have
increased
in
the
prefrontal
cortex
with
d-gal
treatment
when
compared
to
the
control
group
after
four,
six
and
eight
weeks
of
treatment,
and
d-gal
also
induced
increases
in
TBARS
levels
in
the
hippocampus
after
one,
two,
four
and
six
weeks.
(B)
The
levels
of
carbonyl
groups
in
the
prefrontal
cortex
and
hippocampus
were
increased
in
rats
treated
with
d-gal
compared
to
the
control
group
in
every
week
of
treatment,
indicating
that
d-gal
treatment
can
lead
to
oxidative
damage
in
lipids
and
proteins.
Data
were
analyzed
using
the
paired-samples
t-test.
n
=
7
animals
per
group.
*p
<
0.05
compared
to
the
respective
test
session
of
the
group.
First,
we
evaluated
the
effects
of
d-gal
administration
given
via
the
oral
route
for
1,
2,
4,
6
or
8
weeks
on
the
habituation
task
in
the
open
field
test.
The
results
showed
that
d-gal
given
via
the
oral
route
can
induce
impairments
in
the
habituation
memory
after
4
weeks
of
treatment,
as
evaluated
in
the
open
field
test.
After
6
weeks,
the
animals
displayed
only
reduced
levels
of
novelty-induced
locomo-
tor
activity,
but
no
alterations
in
their
novelty-induced
exploratory
behavior.
Performance
in
the
open-field
task
(habituation
to
a
novel
environment)
is
one
of
the
most
elementary
forms
of
nonassocia-
tive
learning
[34].
In
this
study,
the
results
indicated
that
d-gal
induced
impairment
of
habituation
to
novelty
after
4
weeks
and
partial
impairment
to
this
task
after
6
weeks.
In
addition,
the
radial
maze
test
was
performed
to
evaluate
the
effect
of
d-gal
given
via
the
oral
route
on
the
animals’
spatial
mem-
ory
after
4,
6
or
8
weeks
of
administration.
d-Gal
induced
spatial
memory
impairment
when
administered
for
6
weeks.
The
radial
maze
task
is
an
important
tool
in
evaluating
spatial
working
and
reference
memory.
In
this
test,
the
animal
has
to
remember
the
location
of
food
localized
in
four
specific
arms
(out
of
a
total
of
eight
arms)
of
the
radial
maze,
avoiding
previously
visited
arms
that
contain
no
food
[39].
The
results
were
not
identical
in
the
two
tasks
that
were
exam-
ined.
The
present
study
showed
that
the
main
effects
of
d-gal
administration
via
oral
route
occurred
only
after
4
or
6
weeks:
and
these
were
impairments
in
the
animals’
habituation
to
novelty
spaces
and
in
their
spatial
memory,
respectively.
These
different
responses
can
be
related
to
the
different
mechanisms
and
brain
regions
responsible
for
the
formation
of
nonassociative
learning
(habituation
to
novelty)
and
spatial
memory
[34].
An
important
hallmark
of
aging
and
age-related
neurological
disorders
is
mem-
ory
impairment,
which
may
lead
to
losses
in
cognitive
function
[40–42].
Several
studies
have
reported
that
chronic
administration
of
d-gal
(50–500
mg/kg)
via
the
intraperitoneal
or
subcutaneous
routes
for
a
period
of
4–8
weeks
induces
both
cognitive
and
mem-
ory
impairments
[11,20,21,43–45].
In
our
study,
d-gal
given
via
the
oral
route
also
induced
memory
deficits
in
rats,
suggesting
that
pro-
longed
administration
by
the
oral
route
can
cause
impairments
in
behavior.
d-Gal
administration
over
long
periods
of
time
can
lead
to
an
enzymatic
overload,
which
impairs
the
body’s
natural
ability
to
cat-
alyze
galactose
into
glucose,
so
causing
an
increase
of
galactitol
and
an
activation
of
aldose
reductase.
This
causes
oxidative
damage
to
the
cells
[3,4]
and
form
AGE
[5].
This
product
is
closely
related
with

J.
Budni
et
al.
/
Behavioural
Brain
Research
302
(2016)
35–43
41
Fig.
4.
The
effect
of
d-gal
(100
mg/kg,
v.o)
administration
via
the
oral
route
on
male
rats
subjected
to
immunoblot
analysis.
The
hippocampus
and
prefrontal
cortex
were
removed
from
animals
for
immunoblot
analysis
48
h
following
oral
water
or
oral
d-gal
administration
at
the
end
of
1,
2,
4,
6
and
8
weeks
of
treatment.
Data
are
the
mean
±
SEM
of
the
optical
density
(D.O)
of
the
synaptophysin
(A)
and
TAU
total
protein
bands
(B)
divided
by
␤-actin
protein.
(A)
8
weeks
after
treatment
with
d-gal,
there
was
a
decrease
of
synaptophysin
protein
in
the
prefrontal
cortex
and
(B)
TAU
content.
Data
were
analyzed
using
the
paired-samples
t-test.
n
=
4
animals
per
group.
*p
<
0.05
compared
to
the
respective
test
session
of
the
group.
aging
and
have
been
associated
with
the
pathogenesis
of
many
dis-
eases,
such
as
diabetes
[6],
amyotrophic
lateral
sclerosis
[7],
and
Alzheimer’s
disease
[8].
In
fact,
previous
studies
have
shown
that
the
chronic
administration
of
d-gal
injected
via
the
subcutaneous
or
intraperitoneal
routes
at
the
dose
of
100
mg/kg
induced
cognitive
impairments
after
8
weeks
[14,46],
or
12
weeks
of
treatment
[24].
In
the
present
study,
no
cognitive
damage
was
observed
after
1,
2
or
8
weeks
of
d-gal
(100
mg/kg)
administration.
However,
a
study
performed
by
Cardoso
et
al.
[47]
showed
that
d-gal
(300
mg/kg)
administered
intraperitoneally
for
a
period
of
8
weeks
induced
no
significant
behavioral
alterations.
These
results
are
controversial;
therefore,
additional
investigations
will
need
to
be
performed
to
clear
this
point.
Moreover,
we
evaluated
the
possible
oxidative
damage
induced
by
d-gal
which
had
been
administered
via
the
oral
route
for
a
period
of
1,
2,
4,
6
or
8
weeks.
The
results
of
the
present
study
showed
that
d-gal
increased
the
level
of
oxidative
damage
to
proteins
(car-
bonyl
group
content)
in
the
prefrontal
cortex
and
hippocampus
for
all
of
the
treatment
schedules
analyzed
here.
In
addition,
d-gal
increased
the
levels
of
lipid
peroxidation
(thiobarbituric
acid
reac-
tive
species
levels)
in
the
hippocampus
after
1,
2,
4
and
6
weeks
of
treatment
as
well
as
in
the
prefrontal
cortex
after
4,
6
and
8
weeks
of
treatment.
d-Gal
administered
for
a
period
of
8
weeks
increased
the
levels
of
lipid
peroxidation
in
the
prefrontal
cortex,
but
not
in
the
hippocampus.
Biological
aging
is
closely
related
to
oxidative
stress
[48–51],
which
involves
shifts
in
redox
balance,
leading
to
lipid
and
protein
oxidation.
In
turn,
this
event
induces
reductions
in
the
levels
of
neuronal
excitation,
leading
to
reduction
of
activity-
dependent
plasticity,
which
culminates
in
learning
and
memory
impairment
[52].
Moreover,
oxidative
stress
is
also
involved
in
the
pathophysiology
of
age-related
diseases
such
as
Alzheimer’s
disease,
Huntington’s
disease
and
Parkinson’s
disease.
In
these
age-

42
J.
Budni
et
al.
/
Behavioural
Brain
Research
302
(2016)
35–43
related
diseases,
the
generation
of
reactive
oxygen
species
leads
to
central
nervous
system
oxidative
stress,
microvascular
dysfunction
and
neuronal
damage
[53,54].
The
animal
model
of
aging
has
also
shown
an
increase
in
the
levels
of
oxidative
stress
in
various
brain
regions
[21,55–59].
Specifically,
chronic
systemic
d-gal
administration
induces
neu-
rodegeneration,
oxidative
damage
and
mitochondrial
dysfunction
in
both
mice
and
rats
[10,11,14,46].
The
present
study
showed
increases
in
lipid
and
protein
oxidation,
indicative
of
oxidative
stress
like
aging.
In
fact,
the
most
oxidative
damage
occurred
after
4
and
6
weeks
of
treatment
with
d-gal,
which
can
help
to
explain
the
cognitive
and
memory
impairments
observed
after
4
and
6
weeks
of
d-gal
treatment.
Therefore,
additional
investigations
were
performed
to
eluci-
date
the
effect
of
d-gal
administered
via
the
oral
route.
For
this,
the
present
study
evaluated
the
protein
content
of
synaptophysin
and
total
TAU.
However,
our
results
showed
a
reduction
in
the
synap-
tophysin
and
TAU
total
contents
only
in
the
prefrontal
cortex
after
8
weeks
of
d-gal
treatment.
TAU
protein
is
abundant
in
neurons
and
plays
an
important
role
to
the
assembly
and
stabilization
of
microtubules,
and
maintains
the
cytoskeletal
structure
[60].
The
microtubule-associated
protein
TAU
promotes
axonal
outgrowth,
and
it
is
also
necessary
for
maintaining
axonal
morphology
and
axonal
transport
[61].
Similarly,
synaptophysin
is
one
of
major
pro-
tein
components
that
are
present
in
the
synaptic
vesicles
possibly
responsible
for
neuronal
transmission
[62].
Robinson
et
al.
[63]
observed
that
synaptophysin
was
reduced
in
aged
individuals
with
cognitive
impairments
and
dementia,
suggesting
that
synaptic
loss
is
a
major
contributor
to
dementia
in
the
elderly
[63].
Ullah
et
al.
also
show
that
d-gal
(120
mg/kg/day
intraperitoneally
for
60
days)
induces
reductions
of
synaptophysin
in
the
hippocampus
of
rats
[21].
Our
results
indicate
that
d-gal
administered
via
the
oral
route
does
not
alter
synaptophysin
after
4
or
6
weeks
of
treatment.
There-
fore,
in
this
case,
the
behavioral
abnormalities
are
not
accompanied
by
a
decrease
in
synaptophysin.
In
conclusion,
the
present
study
showed
for
the
first
time
some
of
the
changes
induced
by
d-gal
administered
via
the
oral
route.
d-
Gal
given
by
oral
route
for
a
period
of
4
or
6
weeks
induced
novelty
habituation
and
spatial
memory
impairments,
respectively.
Oxida-
tive
damage
was
observed
during
each
period
of
treatment.
These
results
indicated
that
d-gal
administered
via
the
oral
route
over
long
periods
of
time
can
induce
the
behavioral
and
neurochemical
alterations
observed
in
aging.
However,
further
studies
are
required
to
better
understand
the
effects
of
oral
d-gal
administration
in
rats.
During
the
next
study,
we
intend
to
add
different
ages
and
include
a
washout
period
to
detect
if
the
changes
are
reversible.
In
addi-
tion,
we
will
be
using
antioxidants
to
try
to
reverse
the
changes
observed.
Conflict
of
interest
The
authors
declare
that
there
is
no
conflict
of
interests
regard-
ing
the
publication
of
this
paper.
Acknowledgments
This
study
was
supported
in
part
by
grants
from
the
‘Conselho
Nacional
de
Desenvolvimento
Científico
e
Tecnológico’
(CNPq-
Brazil—JQ),
from
the
‘Instituto
Cérebro
e
Mente
(JQ)’
and
UNESC
(JB,
JQ
and
SSV).
JQ
is
a
recipient
of
the
CNPq
(Brazil)
Productivity
Fellowship.
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