Behavioural Brain Research 302 (2016) 3543

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Behavioural Brain Research

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

Research report

Oral administration of d-galactose induces cognitive impairments and

oxidative damage in rats
Josiane Budni a,b, , Robson Pacheco a,b , Sabrina da Silva a,b , Michelle Lima Garcez a,b ,
Francielle Mina a,b , Tatiani Bellettini-Santos a,b , Jesiel de Medeiros a,b ,
Bruna Constantino Voss a,b , Amanda Valnier Steckert a , Samira da Silva Valvassori a,c ,
Joo Quevedo a,d,e,f
a
Laboratrio de Neurocincias, Programa de Ps-Graduaco em Cincias da Sade, Unidade Acadmica de Cincias da Sade, Universidade do Extremo Sul
Catarinense, Cricima, SC, Brazil
b
Laboratrio de Doencas Neurodegenerativas, Programa de Ps-Graduaco em Cincias da Sade, Unidade Acadmica de Cincias da Sade, Universidade
do Extremo Sul Catarinense, Cricima, SC, Brazil
c
Laboratrio de Sinalizaco Neural e Psicofarmacologia, Programa de Ps-Graduaco em Cincias da Sade, Unidade Acadmica de Cincias da Sade,
Universidade do Extremo Sul Catarinense, Cricima, SC, Brazil
d
Translational Psychiatry Program, Department of Psychiatry and Behavioral Sciences, The University of Texas Health Science Center at Houston
(UTHealth), McGovern Medical School, Houston, TX, USA
e
Center 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
f
Neuroscience 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 decit.

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-eld 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 speciesTBARSand 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.
2015 Elsevier B.V. All rights reserved.

Corresponding author at: Laboratrio de Neurocincias, Programa de Ps-Graduaco em Cincias da Sade, Unidade Acadmica de Cincias da Sade, Universidade do
Extremo Sul Catarinense, 88806-000 Cricima, 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.

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J. Budni et al. / Behavioural Brain Research 302 (2016) 3543

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 administration over long periods of time can lead to an enzymatic overload,
which impairs the bodys 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 radicals (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 Alzheimers disease [8].
Therefore, it has been postulated that d-gal may induce behavioral alterations that reproduce the natural aging processes in rats
and mice [9,10]. Several studies have suggested that chronic systemic administration of d-gal could be used as a model of cognitive
disorders and aging [1114]. 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 central 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 eld in creating biochemical abnormalities in experimental animals, such
as; accumulations of reactive oxygen species, reductions of
antioxidant enzymes, mitochondrial decits and neuroinammation/apoptosis. These changes in rodents are similar to those that
occur in the aging human brain [11,13,2022].
Moreover, chronic systemic (intraperitoneal or subcutaneous)
administrations of d-gal can induce alterations like the ones
observed in Alzheimers disease (AD) [23,24]. Lin et al. [24] found
that d-gal given via intraperitoneal administration signicantly
increased the content of amyloid beta (A) in the hippocampus
of mice. A previous study showed that intraperitoneal administrations 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 neuroinammation, as well as activation of the NFB signaling pathway in
the brain of rodents [11,2730]. -Amyloid peptide, as AGEs, can
activate the receptor for advanced glycation end products (RAGE),
leading to oxidative stress and to the activation of the transcription 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 decits 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 sufcient 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 350500 g) were
used in this research (total of 150 rats). The animals were acclimatized 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 Universidade 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 Committee on Animal UseCEUA of the Universidade do Extremo Sul
Catarinense).
2.2. Drugs and treatment
d-Gal (d-galactose, SigmaAldrich, 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 eld 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-eld 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 rst 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 tted with a frontal glass wall. The oor
of the open eld 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) activities performed by each rat during a 5 min observation period were

J. Budni et al. / Behavioural Brain Research 302 (2016) 3543

counted by an expert observer. Twenty-four hours after the training session, one new exposition (test session) to the open eld was
carried out for a period of 5 min.
2.4. Radial maze
Training was conducted in an elevated plastic maze with a center 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 habituation 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 nd 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 nd 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. Malondialdehyde 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 measured 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 dinitrophenylhidrazine (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 SDSPAGE, using polyacrilamide gels (10%), followed by transfer to PVDF
Immobilon-FL transfer membranes (Millipore, USA). Protein loading and blot transfer efciency 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; SigmaAldrich, USA; cod. A5441);
anti-TAU (1:1000; Millipore Temecula, USA; cod. #05-348); or antisynaptophysin (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 HealthCare, UK) as described by the manufacturer. Densitometric analysis
was performed using ImageJ software (version Java 1.6.0 20,

37

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 Windows. 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 Students t-test. Data from the radial maze tests were analyzed using
repeated-measures analyses, followed by the Bonferroni post-hoc
test when the Mauchleys test of sphericity result was signicant (assumption of sphericity violated). The data was reported
as means SEM, and p values <0.05 were considered statistically
signicant.
3. Results
Fig. 1 shows habituation to a novel environment assessed in
the open-eld 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 displayed a reduction in the number of crossings (Fig. 1A) and rearings
(Fig. 1B), when re-exposed 24 h later (test session) to the apparatus. 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 statistical difference when observing the number of crossings between
the training and test sessions, and in the 4th week when observing 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 number 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 analysis of variance. In the 4 weeks after treatment with d-gal, there
were signicant differences for the number of behavioral repetitions when evaluating the latency time to nd 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 nd the food in the 4 arms containing this reward within
the control group, when comparing the rst 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 nd food on the rst test day
compared to the second (p = 0.036); third (p = 0.006); and fourth
days of testing (p < 0.001). There were signicant differences for the
number of behavioral repetitions when comparing the latency time
to nd 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 animals treated with water showed no signicant difference between
the rst day to the second (0.076), but there was a decrease in the
latency times between the rst, 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 nd food only
in the rst day when compared to the third day (p = 0.025), but not
when comparing the rst 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

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J. Budni et al. / Behavioural Brain Research 302 (2016) 3543

Fig. 1. The effect of d-gal (100 mg/kg) administration via the oral route in male rats subjected to the open-eld habituation task. The open-eld 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 rst 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 signicant differences for the
behavioral repetitions when evaluating the latency time to nd
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 nd 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 rst 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 rst 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 rst and second day (p = 0.792), and there was a decrease in
the total number of errors to nd food when comparing the rst
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 sessions. 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 number of errors when comparing the rst 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 rst day when comparing it to the second (p = 0.049),
third (p = 0.020) and fourth days (p = 0.009).

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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 nd 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 nd food when comparing the rst day to the subsequent days in the animals that received water, except when
comparing the rst day to the 2nd day during week 6 of treatment. In the animals that received d-gal, there were no differences between the rst 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 nd food only in the 3rd and 4th days when compared to the rst day after 4 weeks of treatment, and showed no reduction of errors in any of the test days
when compared to the rst day in the 6th week of testing. Data are the mean SEM of latency time to nd food (A), and total errors to nd food (B). Data were analyzed by
repeated-measures analyses followed by the Bonferroni post-hoc test, n = 910 animals per group. *p < 0.05 compared to the respective rst 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 thiobarbituric 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 carbonyl 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 proteins. 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 differences 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 neurotoxicity 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.

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J. Budni et al. / Behavioural Brain Research 302 (2016) 3543

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 eld 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 eld test. After 6 weeks, the
animals displayed only reduced levels of novelty-induced locomotor activity, but no alterations in their novelty-induced exploratory
behavior. Performance in the open-eld task (habituation to a novel
environment) is one of the most elementary forms of nonassociative 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 memory 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 specic 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 examined. 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 memory impairment, which may lead to losses in cognitive function
[4042]. Several studies have reported that chronic administration
of d-gal (50500 mg/kg) via the intraperitoneal or subcutaneous
routes for a period of 48 weeks induces both cognitive and memory impairments [11,20,21,4345]. In our study, d-gal given via the
oral route also induced memory decits in rats, suggesting that prolonged 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 bodys natural ability to catalyze 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

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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 diseases, such as diabetes [6], amyotrophic lateral sclerosis [7], and
Alzheimers 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
signicant 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 (carbonyl 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 reactive 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 [4851], 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 activitydependent plasticity, which culminates in learning and memory
impairment [52]. Moreover, oxidative stress is also involved in
the pathophysiology of age-related diseases such as Alzheimers
disease, Huntingtons disease and Parkinsons disease. In these age-

42

J. Budni et al. / Behavioural Brain Research 302 (2016) 3543

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 [21,5559].
Specically, chronic systemic d-gal administration induces neurodegeneration, 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 elucidate 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 synaptophysin 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 protein 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. Therefore, in this case, the behavioral abnormalities are not accompanied
by a decrease in synaptophysin.
In conclusion, the present study showed for the rst time some
of the changes induced by d-gal administered via the oral route. dGal given by oral route for a period of 4 or 6 weeks induced novelty
habituation and spatial memory impairments, respectively. Oxidative 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 addition, we will be using antioxidants to try to reverse the changes
observed.
Conict of interest
The authors declare that there is no conict of interests regarding the publication of this paper.
Acknowledgments
This study was supported in part by grants from the Conselho
Nacional de Desenvolvimento Cientco e Tecnolgico (CNPqBrazilJQ), from the Instituto Crebro e Mente (JQ) and UNESC
(JB, JQ and SSV). JQ is a recipient of the CNPq (Brazil) Productivity
Fellowship.

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