Accepted Manuscript

Effect of folic acid on oxidative stress and behavioral changes in the animal model of
schizophrenia induced by ketamine.

Alexandra I. Zugno, Lara Canever, Alexandra S. Heylmann, Patrícia G. Wessler,

Amanda Steckert, Gustavo A. Mastella, Mariana B. de Oliveira, Louyse S. Damázio,
Felipe D. Pacheco, Octacílio P. Calixto, Flávio P. Pereira, Tamires P. Macan, Thayara
H. Pedro, Patrícia F. Schuck, João Quevedo, Josiane Budni

PII: S0022-3956(16)30120-0
DOI: 10.1016/j.jpsychires.2016.06.013
Reference: PIAT 2887

To appear in: Journal of Psychiatric Research

Received Date: 29 September 2015

Revised Date: 2 June 2016
Accepted Date: 10 June 2016

Please cite this article as: Zugno AI, Canever L, Heylmann AS, Wessler PG, Steckert A, Mastella GA,
de Oliveira MB, Damázio LS, Pacheco FD, Calixto OP, Pereira FP, Macan TP, Pedro TH, Schuck
PF, Quevedo J, Budni J, Effect of folic acid on oxidative stress and behavioral changes in the animal
model of schizophrenia induced by ketamine., Journal of Psychiatric Research (2016), doi: 10.1016/
j.jpsychires.2016.06.013.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
 1
ACCEPTED MANUSCRIPT

Effect of folic acid on oxidative stress and behavioral changes in the animal model of

schizophrenia induced by ketamine.

*
Alexandra I. Zugnoa, Lara Canevera, Alexandra S. Heylmanna, Patrícia G.Wesslera, Amanda

Steckerta, Gustavo A.Mastellaa, Mariana B. de Oliveiraa, Louyse S. Damázioa, Felipe D.

PT
Pachecoa, Octacílio P. Calixtoa, Flávio P. Pereiraa, Tamires P. Macanc, Thayara H. Pedroc,

Patrícia F. Schuckc, João Quevedoa,b, Josiane Budnia.

RI
a

SC
Laboratório de Neurociências, Programa de Pós-Graduação em Ciências da Saúde, Unidade

Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC,

U
Brazil;
AN
b
Center for Experimental Models in Psychiatry, Department of Psychiatry and Behavioral

Sciences, Medical School, The University of Texas Health Science Center at Houston,
M

Houston, TX, USA;

c
D

Laborátorio de Erros Inatos do Metabolismo, Programa de Pós-Graduação em Ciências da

Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense,

TE

Criciúma, SC, Brazil;

EP

*Corresponding author:
C
AC

Prof. Alexandra Ioppi Zugno, MDH, PhD

Laboratório de Neurociências, Programa de Pós-Graduação em Ciências da Saúde

Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense

88806-000 Criciúma, SC, Brazil

Fax: +55 48 3431-2618

E-mail: alz@unesc.net
 2
ACCEPTED MANUSCRIPT

ABSTRACT:

Recent studies have shown benefits for the supplementation of folic acid in schizophrenic
patients. The aim of this study was to evaluate the effects of folic acid addition on adult rats,
over a period of 7 or 14 days. It also sets out to verify any potential protective action using an
animal model of schizophrenia induced by ketamine, in behavioral and biochemical
parameters. This study used two protocols (acute and chronic) for the administration of

PT
ketamine at a dose of 25 mg/kg (i.p.). The folic acid was given by oral route in doses of 5, 10
and 50 mg/kg,once daily, for 7 and / or 14 days in order to compare the protective effects of
folic acid. Thirty minutes after the last administration of ketamine, the locomotor and social

RI
interaction activities were evaluated, and immediately the brain structure were removed for
biochemical analysis. In this study, ketamine was administered in a single dose or in doses
over the course of 7 days increasing the animal’s locomotion. This study showed that the

SC
administration of folic acid over 7 days was unable to prevent hyper locomotion. In contrast,
folic acid (10 and 50 mg/kg) administrated over a period of 14 days, was able to partially
prevent the hyper locomotion. Our data indicates that both acute and chronic administrations
of ketamine increased the time to first contact between the animals, while the increased

U
latency for social contact was completely prevented by folic acid (5, 10 and 50mg/kg).
Chronic and acute administrations of ketamine also increased lipid peroxidation and protein
AN
carbonylation in brain. Folic acid (10 and 50 mg/kg) supplements showed protective effects
on the oxidative damage found in the different brain structures evaluated. All together, the
results indicate that nutritional supplementation with folic acid provides promising results in
M

an animal model of schizophrenia induced by ketamine.

Keywords: Schizophrenia; folic acid; ketamine; behavioral; oxidative stress.

D
TE
C EP
AC
 3
ACCEPTED MANUSCRIPT

1. Introduction

Folic acids one of the B complex vitamins which participates in the metabolism of
one-carbon (C1) compounds (Coppen and Boulander-Gouaille, 2005; Mattson and Shea,
2003). This vitamin plays a neuro protective role against impairments in the central nervous
system (CNS),and promotes neuronal growth and repair (Budni et al., 2011; Iskandar et al.,

PT
2004). Folic acid is also involved in the metabolism and functioning of compounds essential
to the CNS, which includes the purines, pyrimidines, DNA, RNA, amino acids, phosphate

RI
compounds, vitamin B-12, methionine, S-adenosylmethionine, dopamine, adrenaline,
noradrenaline and serotonin (Coppenand and Bolander-Gouaille, 2005; Kronenberg et al.,

SC
2009; Lucock, 2011; Matson and Shea, 2003). Thus, alterations in the metabolism of folic
acid can directly affect the CNS (Coppenand and Bolander-Gouaille, 2005; Krebs et al., 2009;

U
Miller, 2008; Stahl, 2007).
AN
The presence of neuropsychiatric symptoms in patients with B deficiency vitamins
complex and in children conceived following a short inter-pregnancy interval indicates a role
for folic acid in schizophrenia. The influence of the interval between pregnancies could be
M

explained by the reduction of the maternal reserves of folic acid when the maternal folic acid
stores are still being replenished (Dogan et al., 2009).
D

Schizophrenia is a severe, chronic and debilitating mental disorder that affects 1% of

TE

the World’s population, and is characterized by positive (e.g. hallucinations), negative (e.g.
blunted affects and social isolation) and cognitive symptoms (e.g. executive and memory
dysfunction) (Larson et al., 2010). This disorder is considered to be the result of a genetic
EP

combination and environmental factors. Although its pathophysiology has not been fully
determined, biological studies support that the involvement of several possible components,
C

including altered DNA methylation, abnormal transmission of neurotransmitters, oxidative

AC

stress, folic acid deficiencies and high maternal homocysteine levels. Each of these factors has
been separately explored, and they have all been found to involve C1 metabolism, which is a
putative target-integrating gene–environment interactions by influencing epigenetic regulation
(Krebs et al., 2009). Studies have indicated an important relationship between folic acid
metabolism and schizophrenia. In this sense, these findings support the hypothesis that the
deficiency of this vitamin during fetal development may be an important risking factor for
schizophrenia (Gunawardana et al., 2011).
Current studies utilize pharmacological tools in evaluating the effects of new
protective compounds against schizophrenia. Recent reviews have shown that ketamine is a
 4
ACCEPTED MANUSCRIPT

useful tool for studying the positive, negative and cognitive symptoms observed in acute
schizophrenia (Frohlich and Van Horn, 2014). This animal model of schizophrenia involves
the acute or repeated administration of ketamine (Becker and Grecksch, 2004; Bubenikova-
Valesova et al., 2008; Canever et al., 2010; De Oliveira et al., 2011). Ketamine is a
dissociative anesthetic which acts as an NMDA receptor non competitive antagonist, and is
largely used within research to create animal models of schizophrenia; since it induces

PT
schizophrenia-like symptoms (Dingledine et al., 1999). These animal models imitate the
behavioral changes seen in schizophrenia such as hyperactivity, social interaction and

RI
memory deficits. Moreover, ketamine induces similar biochemical alterations that are found
in schizophrenia, such NMDA receptor hypo function and oxidative stress (Chatterjee et al.,

SC
2011). Based on these previous results from our laboratory support the hypothesis that early
insults interfere with the glutamatergic system, reflecting a greater sensitivity to the effects of

U
ketamine in adulthood within an animal model of schizophrenia (Zugno etal., 2013). All these
AN
findings reinforce the validity of the ketamine model since it is capable to mimic the
phenotype of schizophrenia in both, the animal behavior as well as in the biochemical
alterations seen in brain.
M

Considering that the etiology of schizophrenia is not clear, current researches indicate
that the accumulation of reactive oxygen species (ROS) is associated with the
D

pathophysiology of the disorder (Ciobica et al., 2011;Ruiz-Litago et al., 2012; Yao and
TE

Keshavan, 2011). Oxidative stress occurs because of the increased levels of ROS, reactive
nitrogen species (RNS) or by an imbalance in the activity of the endogenous antioxidant
system (Berg et al., 2004; Kwon et al., 2003). As a consequence of the increased levels of
EP

ROS and the failure of the endogenous antioxidant system, damage to DNA, proteins and
membrane lipids can occur (Konat 2003; Kwon et al., 2003).
C

The link between schizophrenia and oxidative stress was recently demonstrated when
AC

it was shown that the activity of glutathione peroxides (GPx) decreased in both treated and
untreated patients (Miljevic et al., 2010). Also, increased levels of markers for lipid per
oxidation were observed in a similar population (Dadheech et al., 2008; Padurariu et al.,
2010). Additionally, a study performed by our group suggested that the animal model of
schizophrenia induced by ketamine showed changes in the activity of superoxide dismutase
(SOD), catalase (CAT) and GPx, resulting in protein and lipid damage (De Oliveira et al.,
2009).
Folic acid is involved in the metabolism of homocysteine that can be remethylated to
methionine by enzymes that require folic acid, and it can also be catabolized by cystathionine-
 5
ACCEPTED MANUSCRIPT

B-synthase, an enzyme dependent on the vitamin B6, that forms cysteine, a precursor of
glutathione (Micle et al., 2012). It is known that hyperhomocysteinemia is directly related to
oxidative stress. The autoxidation of homocysteine and other disulfides, releases oxygen (O2)
and hydrogen peroxide (H2O2), both of them impair neuronal function and predispose the
neuronal tissue to neurodegenerative and psychiatric disorders (White et al., 2001).
Furthermore, the deficiency of folic acid induces the high levels of homocysteine and the

PT
formation of ROS that lead to decreases in both antioxidant potential and the activity of GPx,
increasing oxidative tissue damage (McCully, 2009).

RI
Although, studies have emphasized the association between folic acid deficiency and
schizophrenia, few preclinical studies have been conducted to study the supplementation of

SC
this vitamin, or to evaluate its effect in an animal model of schizophrenia (Godfrey et al.,
1990, Levine et al., 2006). In this sense, the aim of the study was to evaluate the effect of diet

U
supplementation with folic acid over a period of seven or fourteen days in adult rats, and also
AN
to verify potential protective actions in an animal model of schizophrenia induced by acute or
chronic administrations of ketamine on the animals behavioral and biochemical parameters.
M

2. Methods
D

2.1 Experimental Procedures

TE

The animals included in this study were handled according to the NIH Guide for the
Care and Usage of Laboratory Animals, and also in accordance with the rules of the Brazilian
Society for Neuroscience and Behavior (SBNeC). The experiments were performed at the
EP

Universidade do Extremo Sul Catarinense (UNESC) Brazil, in the Laboratory of

Neurosciences, and in partnership with the Laboratory of Pathophysiology. All experimental
C

procedures were performed in accordance with international recommendations for the care
AC

and use of laboratory animals, and additionally in compliance with the recommendations for
the use of animals as set out by the Brazilian Society of Neuroscience and Behavior (SBNeC).

2.2 Animals
Sixty day old male Wistar rats weighing around 250–300g were obtained from our
breeding colony. They were kept in cages (41 x 34 cm and 16 cm high) in groups of five, with
free access to food and water, and were maintained on a 12-h light–dark cycle (lights on at
7:00 am), at a temperature of 23 ± 1°C. These conditions were maintained constantly
throughout the experiments. Our study used 80 animals for each experimental protocol (7 and
 6
ACCEPTED MANUSCRIPT

14 days) (n=10) animals per group, consisting of 160 animals in the study. All experimental
procedures were performed in accordance with, and with the approval of, the local Ethics
Committee for the Use of Animals (Protocol 49/2012). All efforts were made to minimize
animal suffering and to reduce the number of animals used in this study, utilizing alternatives
to in vivo techniques when possible.
Male animals were chosen in this study because females show a major variability in

PT
their responses, within, both the behavioral and biochemical tests due to their specific
hormonal profile.

RI
2.3 Protocols used for folic acid supplementation and the administration of ketamine

SC
This study utilized two protocols (acute and chronic) for the administration of
ketamine at a dose of 25 mg/kg to evaluate the immediate and long-term actions of this

U
anesthetic in inducing an animal model of schizophrenia. Folic acid administered in doses of
AN
5, 10 and 50 mg/kg was also used for 7 or 14 days in order to compare the protective effects
of folic acid over different periods of supplementation.
Protocol 1:
M

Folic acid (FA) in doses of 5, 10 or 50mg/kg was given by the oral route once daily for
7 days. Water, which was used as a vehicle, was administered by gavage. On the eighth day of
D

the experiment, the animals were injected with ketamine (CU ChemieUetikon, Germany) at a
TE

dose of 25 mg/kg intraperitoneally (i.p.), to mimic an animal model of schizophrenia. The

experimental design is shown in Figure 1.
EP

Protocol 2:
Folic acid (FA) in doses of 5, 10 or50mg/kg was given by the oral route once for 14
C

days. Water, which was used as a vehicle, was administered by gavage. From the eighth day
AC

of the experiment, saline or ketamine were administered intra peritoneally (i.p.) at a dose of
25 mg/kg, for 7 days (CU ChemieUetikon, Germany). On the last day of the experiment,
ketamine was injected thirty minutes before taking behavioral tests in order to mimic an
animal model of schizophrenia. The experimental design is shown in Figure 2.

2.4 Animal model of schizophrenia and experimental groups

Folic acid (Sigma Chemical Co., St. Louis, U.S.A.) was dissolved in distilled water
and administered orally (p.o.) by gavage in doses of 5, 10 or 50 mg/kg for periods of 7 and 14
days. Ketamine was prepared in saline at a volume of 1mL/100g (Becker and Grecksch, 2004;
 7
ACCEPTED MANUSCRIPT

Imre et al., 2006; Tomiya et al., 2006). Ketamine (Ket), administered at a dose of 25 mg/kg
was used to reproduce some of the psychotic symptoms found in schizophrenia, such as the
blunted affect and hyper locomotion (Hunt et al., 2006). The control group was administered
with saline (sal). The animals were divided into eight groups, according to each experimental
protocol (n=10) as follows: 1) Water+salgroup; 2) FA 5mg/kg+salgroup; 3) FA 10mg/kg+sal
group; 4) FA 50mg/kg+sal; 5) Water + ket group; 6) FA 5mg/kg+ketgroup; 7) FA

PT
10mg/kg+ket; 8) FA 50mg/kg+ket.
After a period of thirty minutes from the last injection of ketamine or saline, the

RI
animals were subjected to the locomotor test activity, then, each animal was socially isolated
for 6 hours to achieve the social interaction. The animals were killed by decapitation right

SC
after the social interaction test. The rats brain structures (prefrontal cortex, hippocampus and
striatum) were then carefully dissected for biochemical analysis.

U
AN
2.5 Behavioral evaluation

2.5.1 Locomotor activity

M

Thirty minutes after the last injection of ketamine or saline, the animal’s locomotors
activity were measured by using an activity monitor (40x60cm). The activity monitor is
D

surrounded by 50 cm high acrylic walls containing 6 parallel bars, each bar containing 16
TE

infrared sensors that can detect a rat’s exact position and movement, making it possible to
undertake a detailed analysis of an animal’s behavior.
The information detected by the sensors during the 15 minutes tests was transmitted to
EP

a computer by the Open Source software Interbase 6.01 (Activity Monitor – Insight
Laboratory Equipment, Ribeirão Preto, SP). The distance covered by an animal was
C

considered as the sum of the changes in position monitored by the activity arena; the software
AC

calculates the distance between two locations, plus the previously traveled distances (De
Oliveira et al., 2011).

2.5.2 Social Interaction

Immediately after the locomotor activity, each animal was socially isolated for 6 hours
in order to prior the experiment, without access to food and water, for the social interaction
moment. The isolation of 6 hours was followed as described in the protocol. The test
consisted in placing two animals from different cages, but from the same group, in the open
field arena (50 × 25 × 50 cm) for 15 minutes (Schneider and Przewlocki, 2005). During this
 8
ACCEPTED MANUSCRIPT

period, three criteria were evaluated: the latency to the first contact between animals, the
number of contacts between animals and the total interaction time that the animals remained
together (Gama et al., 2012; Niesink and Van Ree, 1989; Schneider and Przewlocki, 2005).

2.6 Biochemical evaluation

PT
2.6.1 Oxidative damage to lipids and proteins
The effects of ketamine and/or folic acid upon the formation of thiobarbituric acid

RI
reactive species (TBARS) were measured in the animal’s cerebral structures to verify
oxidative damage. As described by Draper and Hadley (1990), samples of striatum, pre-

SC
frontal cortex and hippocampus were mixed in 1ml of10% trichloroacetic acid and 1ml of
0.67% thiobarbituric acid, and then heated in a bath of boiling water for 30min. The

U
equivalents of malondialdehyde (MDA) were determined spectrophoto metrically using
AN
absorbance at 532nm.
The determination of carbonyl groups content based on the reaction with
dinitrophenylhidrazine (DNPH) was made to assess the damage to proteins. This protocol was
M

previously described by Levine and colleagues (1994). Briefly, proteins were precipitated by
the addition of 20% trichloroacetic acid, and were redissolved in DNPH. Absorbance was
D

monitored spectrophotometrically at 370nm.

TE

2.6.2 Activity of antioxidant enzymes

The activity of SOD was defined as the capacity of pyrogallol to autoxidize, a process
EP

highly dependent on O2−2; a substrate for SOD (Bannister and Calabrese, 1987). Briefly, the
presence of SOD influences the inhibition of pyrogallolautoxidation, thus, enzyme activity
C

can be indirectly tested. The activity was performed in a double beam spectrophotometer
AC

(420nm). Also, to define the calibration, a standard curve was developed using purified SOD.
Values of SOD activity were expressed as units per mg of protein, and 1 unit of SOD is
defined when there is a 50% autoxidation of pyrogallol. For the analysis of CAT, the protocol
of Laemmli (1970) was applied using a spectrophotometer at 240nm. The method is based on
the disappearance of H2O2 in areaction medium containing 20 mM H2O2, 0.1% Triton X-100,
10 mM potassiumphosphate buffer, pH 7.0, and 0.1-0.3mg protein / ml. One CAT unit was
defined as 1mol of hydrogen peroxide consumed per minute.
GPx enzyme activity was measured using tert-butyl hydroperoxide, the substrate
according to the protocol described previously (Wendel, 1981). Enzymeactivity was measured
 9
ACCEPTED MANUSCRIPT

by monitoring the rate of disappearance of NADPH at 340 nm in a 50 mM potassium

phosphate buffer, pH 7.0, containing 1.0 mM EDTA, 2.0 mM glutathione (GSH), 0.2 U / mL
GSH reductase, 1.0 mM azide, 0.2 mM tertbutylhydroperoxide, 0.2 mM NADPH, and
supernatant containing 0.2-0.3 mg protein/mL. The unit of GPx activity was expressed as
nmol of NADPH oxidized per minute per mg of protein, using an extinction coefficient of Ɛ =
6.220M-1cm-1 for NADPH.

PT
All measurements relating to antioxidant enzyme activities (SOD, CAT and GPx)
were normalized using the values of total protein (bovine serum albumin) from the samples,

RI
as described (Lowry et al., 1951).

SC
2.7 Statistical analysis

U
All data are presented as mean and standard error of mean. Differences among
AN
experimental groups were determined by Two-way analysis of variance (ANOVA) tests,
followed by Newman-Keulspost hoc test. In all comparisons, statistical significance was set at
p <0.05.
M

3. Results
D
TE

3.1 Behavioral tests

The results in Figure 3A and 3B show the effect of folic acid administration over a
period of 7 and / or 14 days on the locomotor activity of rats in an animal model of
EP

schizophrenia induce dby ketamine. Acute and chronic ketamine administration at a dose of
25 mg/kg increased the locomotor activity in these animals. Treatments with folic acid over a
C

period of 7 days at doses of 5, 10 and 50 mg/kg were notable to prevent the hyper locomotor
AC

effect. In contrast, folic acid (10 and 50 mg/kg) administered over 14 days was able to
partially prevent ketamine-induced hyperlocomotion, while AF (5 mg/kg) was not able to
reverse the increase in locomotor activity. Two-way ANOVA indicated a significant
difference between ketamine treatment [F(1,113)=34.74, p<0.01], folic acid treatment
[F(3,113) =12.98, p <0.01] andfolic acidversusketamineinteraction[F (3,113) =3.20, p <0.05].
Ketamine also induces social deficits, as observed by social interaction in the open
field test. The results in Figure 4A and 5A show that both acute and chronic ketamine
administration (25mg/kg) increased the time of the first contact between the animals, inducing
social deficit. However, folic acid (5, 10 and 50mg/kg )administrated over a period of 7 days
 10
ACCEPTED MANUSCRIPT

completely prevented the increased in latency induced by ketamine. Two-way ANOVA

indicated a significant difference between ketamine treatment [F (1,35) =15.06, p <0.01], folic
acid treatment [F(3,35)=6.31, p<0.01] and folic acid versus ketamine interaction [F(3,35)
=5.87, p<0.01].Treatment with folic acid (5, 10 and 50 mg/kg) administered over 14 days
showed a protective effect by increasing the latency induced by ketamine. Two-way ANOVA
revealed significant differences for latency between ketamine treatment [F(1,54)=9.29,

PT
p<0.01], folic acid treatment [F(3,54)=4.97, p<0.01] and folic acid versus ketamine
interaction[F (3,54) =3.87, p <0.05].

RI
No change was observed in the number of social contacts made by the animals
exposed to acute ketamine and/or folic acid administration over 7 days (Figure 4B). Chronic

SC
ketamine treatments decreased the number of social contacts and treatment with folic acid for
14 days at the doses of 10 and 50 mg/kg partially increased the number of contacts (Figure

U
5B). Two-way ANOVA revealed differences only for folic acid treatment [F(3,47)=6.31,
AN
p<0.01] and folic acid versus ketamine interaction [F (3,47) =6.51, p <0.01].
Acute administration of ketamine decreased the total of social contact time, and the
administration of folic acid (50mg/kg) over a period of 7 days significantly increased the
M

interaction time, preventing the effects of ketamine in these animals (Figure 4C). Two-way
ANOVA showed a significant difference only for the folic acid versus ketamine interaction
D

[F(3,30) =4.04, p <0.05].No change was observed in the total time of social contacts (Figure
TE

5C) for chronic ketamine administration and/or folic acid supplementation over 14 days.

3.2 Biochemical measurements

EP

In Figures 6 and 7, TBARS and protein carbonylation levels are shown respectively.
TBARS levels were measured because malondialdehyde (MDA) is a late product of lipid
C

peroxidation. Figures 6A and 6B illustrate that all of the experimental groups that received
AC

only acute and / or chronic ketamine administration showed significant increasement in lipid
peroxidation in the prefrontal cortex, hippocampus and striatum. Figure 6A shows that
supplements of folic acid (5, 10 and 50mg/kg) over a period of 7 days were unable to prevent
the lipid damage induced by ketamine in prefrontal cortex. However, folic acid given at doses
of 5, 10 and 50mg/kg was able to completely prevent the increase in lipid peroxidation caused
by ketamine in the hippocampus and striatum. Two-way ANOVA indicates significant
differences in the prefrontal cortex (ketamine treatment [F(1,32)=16.16, p<0.01], folic acid
treatment [F(3,32)=1.99 p<0.05] and interaction [F(3,32)=2.96, p<0.05); hippocampus
ketamine treatment [F(1,32)=5.40, p <0.05], folic acid treatment [F(3,32)=3.02, p <0.05] and
 11
ACCEPTED MANUSCRIPT

interaction [F(3,32)=4.01, p<0.05) and striatum (ketamine treatment [F(1,32)=12.45, p<0.01],

folic acid treatment [F(3,32)=1.41, p<0.05] and interaction [F(3,32)=3.58, p <0.05).
According to Figure 6B, chronic administration of ketamine showed significant increases in
lipid peroxidationin the prefrontal cortex, hippocampus and striatum. However, it was found
that the administration of folic acid over a period of 14 days did not prevent the lipid damage
induced by ketamine in the prefrontal cortex. Two-way ANOVA showed significant

PT
differences between ketamine treatment [F(1,28)=18.72, p<0.01] and folic acid versus
ketamine interaction [F(3,28)=4.06, p <0.05]. In the hippocampus and striatum, folic acid (5,

RI
10 and 50 mg/kg) showed a neuroprotective effect against lipid peroxidation induced by
ketamine. Two-way ANOVA indicates significant differences in the hippocampus between

SC
ketamine treatment [F(1,32)=5.40, p <0.05], folic acid treatment [F(3,32)=3.02, p<0.05] and
interaction [F(3,32)=4.00, p<0.05) and in the striatum, ANOVA revealed differences only

U
between ketamine treatment [F(1,32)=12.44, p<0.01] and folic acid versus ketamine
AN
interaction [F(3,32)=3.58, p <0.05).
Figure 7A shows a significant increase in protein damage induced by acute ketamine
administration in the prefrontal cortex, hippocampus and striatum. Folic acid supplementation
M

at a dose of 5mg/kg over7 days partially prevented protein carbonylation in the prefrontal
cortex. In contrast, doses of 10 and50mg/kg of folic acid completely prevented the protein
D

damage induced by the acute administration of ketamine. In the hippocampus and striatum,
TE

supplementation of folic acid (5, 10, 50mg/kg) resulted in a significant decrease in protein
carbonylation, preventing an increase in the damage caused by ketamine. Two-way ANOVA
indicates significant differences in the prefrontal cortex (ketamine treatment [F(1,32)=50.33,
EP

p<0.01], folic acid treatment [F(3,32)=17.31, p<0.01] and interaction [F(3,32)=22.62,

p<0.01); hippocampus (ketamine treatment [F(1,32)=44.66, p<0.01], folic acid treatment
C

[F(3,32)=12.13, p<0.01] and interaction [F(3,32)=14.18, p<0.01) and striatum (ketamine

AC

treatment [F(1,32)=14.80,p <0.01], folic acid treatment [F(3,32)=4.43, p<0.05] and

interaction [F(3,32)=5,86, p<0.01). Figure 7B shows that chronic administration of ketamine
also increased protein damage in the prefrontal cortex and hippocampus. Supplementation
with folic acid (5, 10 and 50 mg/kg) over a period of 14 days showed a protective effect in
these brain structures. In contrast, no effect for ketamine and / or folic acid was observed in
the striatum. Two-way ANOVA indicates significant differences in the prefrontal cortex
(ketamine treatment [F(1,32)=50.33,p <0.01], folic acid treatment [F(3,32)=17.30,p <0.01]
and interaction [F(3,32)=22.62,p <0.01) and hippocampus (ketamine treatment [F(1,32)
 12
ACCEPTED MANUSCRIPT

=44.66,p <0.01], folic acid treatment [F(3,32)=12.12,p <0.01] and interaction [F(3,32)=14.18,
p <0.01).
We also evaluated the activity of antioxidant enzymes GPx, SOD and CAT in the
prefrontal cortex, hippocampus and striatum, the results of which are shown in Figure 8 and 9.
According to figures 8A, 8B and 8C respectively, no significant difference was observed in
the activity of antioxidant enzymes GPx, SOD and CAT in the prefrontal cortex,

PT
hippocampus or striatum for the acute administration of ketamine and / or folic acid over a 7
days period. Chronic administrations of ketamine did not demonstrate any effect on the

RI
activity of the antioxidant enzymes GPx, SOD and CAT in the brain structures studied as
shown in figure 9A, 9B and 9C, respectively. According to Figure 9B, only in the

SC
hippocampus, folic acid (5mg/kg) increased of SOD activity in relation to the ketamine group.
Two-way ANOVA showed significant differences between ketamine treatment [F(1,27)

U
=4.38,p <0.05] and folic acid versus ketamine interaction [F(3,27)=4.01,p <0.05]. Figures 9C
AN
showed that folic acid supplements (5 mg/kg) for 14 days increased the activity of the
antioxidant enzyme CAT in the prefrontal cortex, as well as folic acid (50 mg/kg) that also
increased CAT activity in the hippocampus. Two-way ANOVA indicates significant
M

differences in the prefrontal cortex (ketamine treatment[F (1,29) =11.87,p <0.01], folic acid
treatment [F(3,29)=2.50, p <0.05] and interaction [F(3,29)=4.0, p<0.05) and hippocampus
D

(ketamine treatment [F(1,31)=1.85, p <0.05] ,folic acid treatment [F(3,31)=5.84, p <0.01] and
TE

interaction [F(3,31)=3.27, p <0.05).

4. Discussion
EP

In this study, ketamine administered in a single dose or over a period of 7 days

increased the test animal’s levels of locomotion, corroborating previous studies proposed by
C

Gama et al (2012) and Zugno and colleagues(2013).Therefore, the administration of folic acid
AC

(5, 10 and 50 mg/kg) during a period of 7 days was unable to prevent hyperlocomotion in this
experiment. It is possible that folic acid was not able to prevent the hyperlocomotion seen in
this study due to the short period of time that the treatments were administered for. In
contrast, folic acid (10 and 50 mg/kg) administrated during a period of 14 days was able to
partially prevent ketamine-induced hyperlocomotion, while folic acid (5 mg/kg) was not able
to reverse the increase in locomotor activity.
It has been suggested that, there is a relationship between folic acid and negative
symptoms in schizophrenia (Hill et al., 2011). It is well known that ketamine impairs the
ability of animals to interact socially with other members of their own species (Torgalsbøen
 13
ACCEPTED MANUSCRIPT

and Rund, 1998). Thus, evidence also indicates that negative and cognitive symptoms are the
main cause of functional impairment in schizophrenic individuals (Shamsi et al., 2011;
Tandon et al., 2009). Our data indicates that acute and chronic administration of ketamine
increased the time to the first contact between the animals. These results corroborated
previous studies from our laboratory (Zugno etal., 2013). A study performed by Gama et al.
(2012) found that the animals had fewer contacts with each other, as well as a lower total time

PT
of interaction, showing a interactions decrease between these animals.
Taken together, our study shows that the increase in latency for social contact was

RI
completely prevented by the administration of folic acid (5, 10 and 50mg/kg) over a period of
7 days, as well as for folic acid supplements over 14 days (5, 10 and 50 mg/kg). This result is

SC
similar, at least in part, to findings of Roffman et al. (2013a) which showed that the
supplementation of acid folic plus vitamin B12 for 16 weeks may ameliorate the negative

U
symptoms of schizophrenia. Evidence suggests that folic acid supplementation may hold
AN
promise for the treatment of negative symptoms in schizophrenia. Folic acid plays a key role
in one-carbon metabolism and is essential for gene transcription, homocysteine metabolism
and the synthesis of several neurotransmitters (Hill et al., 2011). Folic acid deficiency has
M

been associated with several neuropsychiatric disorders including depression and

schizophrenia (Koren et al., 2002), and more recently, with the severity of negative symptoms
D

(Goff et al., 2004).

TE

This preclinical study showed some limitations (e.g. social interaction is the only
behavioral test to assess negative symptoms in the animal model of schizophrenia; the number
of animals in this test may vary, due to the necessity of many animals to obtain the
EP

results). In social interaction, animals are evaluated in pairs: two animals from different
cages and the same group are collocated in the open field to assess the social interaction
C

parameters. Thus, each pair the animals corresponds to a result, which can limit the number of
AC

animals in the study. This can interfere in the total sample and on the results at the end of the
study. Previous studies proposed by Gama et al (2012) and Zugno and colleagues (2013)
evaluated the social interaction and use different number of animals (n=12) and (n=5-7),
respectively, according to availability for the study.
Based on this research, in relation to social interaction, this study demonstrated that
folic acid administered at dose of 50 mg/kgover a period of 7 days was able to increase the
total of social contact time, completely preventing the damage caused by ketamine
administration. This finding corroborates a study performed by Budni et al. (2013) which
suggests a possible antidepressant effect for folic acid at the same dose. The antidepressant
 14
ACCEPTED MANUSCRIPT

effect of folic acid can be related to its protective effect in schizophrenia, since one of the
biggest causes of disability in schizophrenic patients are the negative symptoms,which are
very similar to the symptoms of depression. These symptoms have been found to remain
throughout the patient's life with currently available treatments (Ponizovsky et al., 2013).
Moreover, it has been shown that deficiencies in the level of folic acid can result in
low levels of monoamines (serotonin, norepinephrine, and dopamine), which could contribute

PT
to the development of these disorders (Morris et al., 2008).Folic acid may also be directly
involved in the regulation of neurotransmitter metabolism. The direct mechanism by which

RI
this occurs remains unknown, however, it has been postulated that tetrahydrobiopterin (BH4)
metabolism, a cofactor for the synthesis of monoamine neurotransmitters which has some

SC
structural similarities to folic acid, could be involved (Bottiglieri, 2005). It has been suggested
that supplementation with B vitamins, in particular folic acid, could be a beneficial strategy

U
for schizophrenic patients since they mainly attenuate the negative symptoms of the disorder
AN
(Moustafa et al., 2014).
The results of the present study also indicate an oxidative imbalance induced by acute
and chronic administrations of ketamine, alterations of markers for oxidative damage to lipids
M

and protein, and antioxidant defense in the brain structures analyzed. However, folic acid was
able to prevent lipid and protein damage in some of the structures analyzed. Oxidative stress
D

has been implicated in the pathophysiology of many neuropsychiatric disorders such as

TE

schizophrenia, bipolar disorder and major depression (Pandya et al., 2013). In this context, it
has already been reported that free radicals are elevated in patients diagnosed with
schizophrenia (Frendi et al., 2006; Yao et al., 1998). Our study shows that an acute or chronic
EP

administration of ketamine induces changes in some of the oxidative stress parameters, such
as increases in TBARS levels within the pré-frontal cortex, hippocampus and striatum. Da
C

Silva et al. (2010) also reported an increase in lipid peroxidation after a single dose of
AC

ketamine (20 mg/kg). Our findings are also consistent with the results of Gazal et al. (2014),
which showed lipid damage induced by chronic administration of ketamine (25 mg/kg) in an
animal model of mania.Finally, these results corroborate studies by Dietrich-Muszalska and
Kontek (2010) which showed that schizophrenic patients have increased levels of TBARS in
their platelets. Genetic and environmental factors have been found to cause increased cellular
levels of reactive oxygen species (ROS) beyond the impair capacity of antioxidant defense in
patients with psychiatric disorders. These factors trigger oxidative cellular damage to lipids
and proteins, leading to abnormal neural growth and differentiation.
 15
ACCEPTED MANUSCRIPT

Chen and colleagues (2011) reported that folic acid deficiency induces an increase in
the levels of TBARS in the hippocampus. Kale et al. (2010) observed low levels of folic acid
and vitaminB12in plasma, and low levels of folic acid in the red blood cells collected from
patients before their first psychotic episodes. In the same study, these reductions were
associated with significant increases in homocysteine and cortisol levels. It is assumed that
there is a common pathophysiological mechanism involving disorders of folic acid, vitamin

PT
B12 and fatty acid membranes (Kale et al., 2010). This present study, only in the prefrontal
cortex, similarly, folic acid supplements (5, 10 and 50mg/kg) administered over a period of 7

RI
days or 14 days did not prevent the lipid peroxidation induced by acute or chronic
administration of ketamine, respectively. Some studies have indicated that oxidative stress is a

SC
common pathogenic mechanism underlying psychiatric disorders, sincewithin the brain there
is a high level of oxygen consumption and a lipid-rich environment.This environment is

U
considered highly susceptible to oxidative stress or redox imbalances. Therefore, the fact that
AN
oxidative stress is implicated in several mental disorders is not surprising (Salim, 2014). Folic
acid (5, 10 and 50mg/kg) administered over a period of 7 daysor 14 days was able to
completely prevent the increase in lipid peroxidation within the hippocampus and striatum
M

caused by the acute or chronic administration of ketamine, respectively. Brocardo et al.

(2010) showed a protective effect for folic acid (50 mg/kg) administered over a period of 7
D

days in the lipid damage induced by ouabain, corroborating the findings of the present study.
TE

Protein carbonylation may be involved in the pathogenesis of at least some of the

schizophrenic patients that have low levels of vitamin B (Arai et al., 2012). Protein oxidation
by ROS can lead to losses of sulfhydryl groups and modifications of amino acids that render
EP

proteins nonfunctional (Stadtman and Levine, 2003). Young et al. (2007) reported that there
was an increase in protein carbonyl groups as well as damage to DNA in schizophrenic
C

patients, suggesting that an oxidative alteration had occurred. Thus, this study observed that in
AC

the prefrontal cortex, hippocampus and striatum, the acute ketamine administration increased
the level of protein damage. In theprefrontal cortex, protein damage was partially prevented
by folic acid supplements given at the dose of 5mg/kg, and totally prevented by folic acid
given at the dose 10 and 50 mg/kg over a period of 7 days. Also, we observed that the effects
of acute dose ketamine in the hippocampus and striatum were completely prevented by the
prior administration of folic acid (5, 10 and 50 mg/kg). Chronic ketamine administration
increased the amount of protein damage in the prefrontal cortex and hippocampus. Folic acid
(5, 10 and 50 mg/kg) supplements given over14 days showed a protective effect in these brain
structures. In contrast, no effect was observed for ketamine and/or folic acid in the striatum.
 16
ACCEPTED MANUSCRIPT

Our findings suggest a neuroprotective effect for folic acid at different doses on lipid
peroxidation and protein carbonylation in both protocols used in the study.The prevention of
lipid peroxidation and protein damage by folic acid maybe due to the antioxidant effect of this
vitamin per se,and also in its ability to act as a scavenger of ROS (Joshi et al., 2001). It is
known that NMDA receptors are sensitive to the oxidative stress level of the cell, and
decreasing this level activates the NMDA receptor, so becoming less subjected to the

PT
inhibitory action of ketamine (Yangetal.,2010). In addition, folic acid can modify the
expression of GABA receptors through epigenetic modulation, which has an inhibitory action,

RI
thus preventing psychotic symptoms following the administration of ketamine (Vasquez etal.,
2013).

SC
Finally, our study did not find any significant statistical difference in the activity of
antioxidant enzymes GPx, SOD and CAT in the prefrontal cortex, hippocampus and striatum

U
of animals treated with acute ketamine and folic acid supplements for a period of 7 days. The
AN
chronic administration of ketamine also was not able to alter the activity of the antioxidant
enzymes GPx, SOD and CAT in these structures. In the hippocampus, folic acid (5mg/kg)
increased of SOD activity in relation to the ketamine group, as folic acid supplements (5
M

mg/kg) and folic acid (50 mg/kg) for 14 days increased the activity of the antioxidant enzyme
CAT in the prefrontal cortex and in the hippocampus, respectively. Currently, there is some
D

understanding for benefits of folic acid supplementation in schizophrenic patients. Moreover,

TE

the efficacy of supplementing a nutrient antioxidant such as folic acid in improving the
enzymatic defense system demonstrates its potential application as a prophylactic treatment
(Wu et al., 2013). New research in both humans and animals is needed to establish precise
EP

protocols, better costs, and thus identify patients who will actually benefit from this
supplementation (Roffman et al., 2013b).
C

In this study, acute and chronic administrations of ketamine were able to reproduce the
AC

positive and negative symptoms of schizophrenia. Folic acid (10 and 50 mg/kg), administered
over a period of 14 days, demonstrated a neuroprotective effect for positive symptoms. For
negative symptoms, both treatment regimens utilizing folic acid (7 and 14 days) were
effective in preventing these signals. Chronic and acute administrations of ketamine also
increased lipid peroxidation and protein carbonylation in the prefrontal cortex, hippocampus
and striatum. Folic acid (10 and 50 mg/kg) supplements administered for periods of 7 and 14
days showed the best protective effects on the oxidative damage found in the different brain
structures evaluated. On the other hand, no resultswere found for the activity of the
antioxidant enzymes GPx, SOD or CAT in the prefrontal cortex, hippocampus or striatum of
 17
ACCEPTED MANUSCRIPT

the animals treated with acuteand / or chronic administration of ketamine andfolic acid over
7and/or 14 days.
All together, these results indicate that folic acid (10 and 50 mg/kg) given as a
nutritionalsupplement provides promising results in an animal model of schizophrenia
induced by ketamine, and could be an adjuvant strategy for the treatment of this
disorder.Moreover, fundamental new preclinical and clinical studies involving folic acid are

PT
needed to confirm the best dose and the most appropriate time of administration for this
vitamin as adjuvant therapy in schizophrenia.

RI
Role of the funding source

SC
There are no conflict of interest. The funding agencies had no role in design and conduct of
the study, the collection, management, analysis and interpretation of the data, or the

U
preparation, review or approval of the manuscript. The authors were not paid to write this
AN
article by a pharmaceutical company or other agency.

Contributors
M

Drs Zugno, Budni, Quevedoand Schuck had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the data analysis.
D

Study concept and design: Zugno and Budni

TE

Acquisition of data:Wessler, Steckert, Mastella, De Oliveira, Damázio, Calixto,Pereira, Pedro

Analysis and interpretation of data: Canever, Heylmann,Wessler and Pacheco
Drafting of the manuscript: Canever and Budni
EP

Critical revision of the manuscript for importante intelectual content: Zugno and Budni
Statistical analysis: Canever, Heylmann, Pacheco and Macan
C

Obtained funding: Zugno, Budni and Quevedo

AC

Administrative, technicalor material support: Zugno, Budni, Quevedo and Schuck

Study supervision: Zugno and Budni
All authors have approved the final article.

Acknowledgments: Laboratory of Neurosciences (Brazil) is one of the centers of the

National Institute for Translational Medicine (INCT-TM) and one of the members of the
Center of Excellence in Applied Neurosciences of Santa Catarina (NENASC). This research
was supported by grants from CNPq, InstitutoCérebro e Mente and UNESC. JQ and
AIZ are CNPq fellows.
 18
ACCEPTED MANUSCRIPT

References

Arai, M., Miyashita, M., Ichikawa, T., Itokawa, M., 2012. Carbonyl stress-related
schizophrenia perspective on future therapy and hypotheses regarding pathophysiology of
schizophrenia. SeishinShinkeigakuZasshi 114(3):199-208.
Becker, A., Grecksch, G., 2004. Ketamine-induced changes in rat behaviour: a possible
animal model of schizophrenia. Test of predictive validity Prog.Neuropsychopharmacol. Biol.

PT
Psychiatry 28(8): 1267-1277.
Berg, D.,Youdim, M.B., Riederer, P., 2004. Redox imbalance. Cell. Tissue Res.
318(1):201-13.

RI
Brocardo, O.S., Budni, J., Pavesi, E., Franco, J.L., Uliano-Silva, M., Trevisan, R.,
Terenzi, MG., Dafre, AL., Rodrigues, AL., 2010. Folic acid administration prevents ouabain-
induced hyperlocomotion and alterations in oxidative stress markers in the rat brain. Bipolar

SC
Disorder 12(4): 414-424.
Bottiglieri, T., 2005. Homocysteine and folate metabolism in depression. Prog
NeuroPsychopharmacol Biol. Psychiatry29:1103–12.

U
Bubeníková-Valesová, V., Horácek, J., Vrajová, M., Höschl, C,. 2008. Models of
schizophrenia in humans and animals based on inhibition of NMDA receptors.
AN
NeurosciBiobehav. Rev. 32(5): 1014-23.
Budni, J.,Zomkowski, A.D.,Engel, D., Santos, D.B., dos Santos, A.A.,Moretti,
M.,Valvassori, S.S.,Ornell, F., Quevedo, J., Farina, M., Rodrigues, A.L., 2013. Folic acid
prevents depressive-like behavior and hippocampal antioxidant imbalance induced by
M

restraint stress in mice. Exp. Neurol. 240:112-21.

Budni J, Romero A, Molz S, Martín-de-Saavedra M.D, Egea J, Del Barrio L, Tasca C.I,
Rodrigues A.L, López M.G.2011. Neurotoxicity induced by dexamethasone in the human
D

neuroblastoma SH-SY5Y cell line can be prevented by folic acid. Neuroscience 190:346-53.
Canever, L., Oliveira, L., D'altoé, De Luca R., Correa, P.T., Fraga, D.B., Matos, M.P.,
TE

Scaini, G., Quevedo, J., Streck, E.L., Zugno, A.I., 2010. A rodent model of schizophrenia
reveals increase in creatine kinase activity with associated behavior changes. Oxid. Med.
Cell.Longev. 3(6): 421-427.
Chatterjee, M., Ganguly, S., Srivastava, M., Palit, G., 2011. Effect of 'chronic' versus
EP

'acute' ketamine administration and its 'withdrawal' effect on behavioural alterations in mice:
implications for experimental psychosis. Behav. Brain Res. 216(1): 247-254.
Ciobica, A., Padurariu, M., Dobrin, I., Stefanescu, C., Dobrin, R., 2011. Oxidative stress
C

in schizophrenia - focusing on the main markers. Psychiatr.Danub. 23(3): 237-245.

Coppen, A., Bolander-Gouaille, C., 2005. Treatment of depression: time to consider folic
AC

acid and vitamin B12. J.Psychopharmacol. 19(1): 59-65.

Chen, J.G., Wang, F., 2011.Reversal of aging-associated hippocampal synaptic plasticity
deficits by reductants via regulation of thiol redox and NMDA receptor function. Aging Cell.
9(5): 709-721.
Dadheech, G., Mishra, S., Gautam, S., Sharma, P., 2008. Evaluation of antioxidant deficit
in schizophrenia. Indian J. Psychiatry 50(1): 16-20.
Da Silva, F.C., do Carmo de Oliveira Cito, M., da Silva, M.I., Moura, B.A., de Aquino,
M.R., Feitosa, M.L., de Castro Chaves, R., Macedo, D.S., de Vasconcelos, S.M., de
França,M.M., de Sousa, FC., 2010. Behavioral alterations and pro-oxidant effect of a single
ketamine administration to mice. Brain Res. Bull. 30;83(1-2):9-15.
De Oliveira, L., Fraga, D.B., De Luca, R.D., Canever, L., Ghedim, F.V., Matos, M.P.,
Streck, E.L., Quevedo, J., Zugno, A.I., 2011. Behavioral changes and mitochondrial
 19
ACCEPTED MANUSCRIPT

dysfunction in a rat model of schizophrenia induced by ketamine. Metab. Brain Dis. 26(1):
69-77.
De Oliveira, L., Spiazzi, C.M., Bortolin, T., Canever, L., Petronilho, F., Mina, F.G., Dal-
Pizzol, F., Quevedo, J, Zugno, A.I., 2009. Different sub-anesthetic doses of ketamine increase
oxidative stress in the brain of rats. Prog.Neuropsychopharmacol. Biol. Psychiatry 33(6):
1003-1008.
Dietrich-Muszalska, A., Kontek, B., 2010. Lipid peroxidation in patients with
schizophrenia. Psychiatry Clin.Neurosci. 64(5): 469-475.

PT
Dingledine, R., Borges, K., Bowie, D., Traynelis, S.F., 1999. The glutamate receptor ion
Channels Pharmacol. Rev. 51(1): 7-61.
Dogan, M., Ozdemir, O., Sal, E.A., Dogan, S.Z., Ozdemir, P., Cesur, Y., Caksen,

RI
H.,2009. Psychotic disorder and extrapyramidal symptoms associated with vitamin B12 and
folate deficiency. J Trop. Pediatr. 55(3):205-7.
Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as index of lipid

SC
peroxidation. Methods Enzymol. 186:421-31.
Fendri, A., Mechri, G., Khiari, A., Othman, A., Kerkeni, L. Gaha. 2006. Oxidative stress
involvement in schizophrenia pathophysiology: a review, Encephale 32 244–252.
Frohlich, J., Van Horn, J.D., 2014. Reviewing the ketamine model for schizophrenia.

U
J.Psychopharmacol. 28(4): 287-302.
Gama, C.S., Canever, L., Panizzutti, B., Gubert, C., Stertz L, Massuda, R., Pedrini, M.,
AN
de Lucena, D.F., Luca, R.D., Fraga, D.B., Heylmann, A.S., Deroza, P.F., Zugno, A.I., 2012.
Effects of omega-3 dietary supplement in prevention of positive, negative and cognitive
symptoms: A study in adolescent rats with ketamine-induced model of schizophrenia.
M

Schizophr. Res. 141(2-3): 162-167.

Gazal, M., Valente, M.R., Acosta, B.A., Kaufmann, F.N., Braganhol. E., Lencina, C.L.,
Stefanello, F.M., Ghisleni, G., Kaster, M.P., 2014. Neuroprotective and antioxidant effects of
D

curcumin in a ketamine- induced model of mania in rats. Eur. J.Pharmacol. 5;724:132-9

Godfrey, P.S., Toone, B.K., Carney, M.W., Flynn, T.G., Bottiglieri, T., Laundy, M.,
Chanarin, I., Reynolds, E.H., 1990. Enhancement of recovery from psychiatric illness by
TE

methylfolate. Lancet 336(8712): 392-395.

Goff, D.C., Bottiglieri, T., Arning, E., Shih, V., Freudenreich, O., Evins, A.E.,
Henderson, D.C., Baer, L., Coyle, J.,2004. Folate, homocysteine, and negative symptoms in
EP

schizophrenia. Am. J.Psychiatry 161: 1705-1708.

Gunawardana, L., Smith, G.D., Zammit, S., Whitley, E., Gunnell, D., Lewis, S.,
Rasmussen, F., 2011. Pre-conception inter-pregnancy interval and risk of schizophrenia. Br. J.
Psychiatry 199(4): 338-339.
C

Hill, M., Shannahan, K., Jasinski, S., Macklin, E.A., Raeke, L., Roffman, J.L., Goff,
AC

D.C., 2011.Folatesupplementation in schizophrenia: a possiblerole for MTHFRgenotype.

Schizophr. Res. 127(1-3): 41-45.
Hunt, M.J., Raynaud, B., Garcia, R., 2006. Ketamine dose-dependently induces high-
frequency oscillations in the nucleus accumbens in freely moving rats. Biol. Psychiatry
60(11):1206-14.
Imre, G., Fokkema, D.S., Den Boer, J.A., Ter Horst, G.J., 2006. Dose-response
characteristics of ketamine effect on locomotion, cognitive function and central neuronal
activity. Brain Res. Bull. 69(3): 338-345.
Iskandar, B.J., Nelson, A.,Resnick, D., Skene, J.H.,Gao, P.,Johnson, C.,Cook,
T.D.,Hariharan, N., 2004. Folic acid supplementation enhances repair of the adult central
nervous system. Ann. Neurol. 56(2): 221-227.
 20
ACCEPTED MANUSCRIPT

Joshi, R., Adhikari, S., Patro, B.S., Chattopadhyay, S., Mukherjee, T., 2001. Free radical
scavenging behavior of folic acid: evidence for possible antioxidant activity. Free Radic. Med.
30(12): 1390-9.
Kale, A., Naphade, N.,Sapkale, S., Kamaraju, M.,Pillai, A.,Joshi, S.,Mahadik, S., 2010.
Reduced folic acid, vitamin B12 and docosahexaenoicacidand increased homocysteine and
cortisol in never-medicated schizophrenia patients: implications for altered one-carbon
metabolism. Psychiatry Res. 175(1-2): 47-53.
Konat, G.W., 2003. H2O2-induced higher order chromatin degradation: a novel

PT
mechanism of oxidative genotoxicity. J.Biosci. 28(1): 57-60.
Koren, G., Cohn, T., Chitayat, D., Kapur, B., Remington, G., Reid, D.M., Zipursky, R.B.,
2002.Use of atypical antipsychotics during pregnancy and the risk of neural tube defects in

RI
infants. Am. J. Psychiatry 159 (1), 136–137.
Krebs, M.O., Bellon, A., Mainguy, G., Jay, T.M., Frieling, H., 2009.One carbon
metabolism and schizophrenia current challenges and future directions. Trends Mol Med

SC
15(12): 562-570.
Kwon, Y.W., Masutani, H., Nakamura, H., Ishii, Y., Yodoi, J., 2003. Redox regulation of
cell growth and cell death. Biol. Chem. 384(7): 991-996.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of

U
bacteriophage T4. Nature 227(5259): 680-685.
Larson, M.K., Walker, E.F., Compton, M.T., 2010. Early signs, diagnosis and
AN
therapeutics of the prodromal phase of schizophrenia and related psychotic disorders.Expert
Rev.Neurother.10(8): 1347–1359.
Levine, J., Stahl, Z., Sela, B.A., Ruderman, V., Shumaico, O., Babushkin, I., Osher, Y.,
M

Berdusky, Y., Belmaker, R.H., 2006. Homocysteine-reducing strategies improve symptoms in

chronic schizophrenic patients with hyperhomoysteinemia. Biol. Psychiatry 60(3): 265-269.
Levine, R.L., Williams, J.A., Stadtman, E.R., Shacter, E., 1994. Carbonyl assays for
D

determination of oxidatively modified proteins. Methods Enzymol. 233: 346-357.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951.Protein measurement with
the Folin phenol reagent. J. Biol. Chem. 193(1): 265-275.
TE

Mattson, M.P.,Shea, T.B., 2003.Folateand homocysteine metabolism in neural plasticity

and neurodegenerative disorders.Trends Neurosci. 26(3): 137-146.
McCully, K.S., 2009.Chemical pathology of homocysteine. IV. Excitotoxicity, oxidative
EP

stress, endotelial dysfunction, andinflammation. Ann ClinLabSci. 39(3): 219-32.

Micle, O., Muresan, M., Antal, L., Bodog, F., Bodog, A., 2012.
The influence of homocysteine and oxidative stress on pregnancy outcome. J. Med
Life. 5(1):68-73.
C

Miljevic, C., Nikolic, M., Nikolic-Kokic, A., Jones, D.R., Niketic, V., Lecic-Tosevski,
AC

D., Spasic, M.B., 2010. Lipid status, anti-oxidant enzyme defence and haemoglobin content in
the blood of long-term clozapine-treated schizophrenic patients. Prog.Neuropsychopharmacol.
Biol. Psychiatry 34(2): 303-307.
Miller, A.L., 2008. The methylation, neurotransmitter, and antioxidant connections
between folate and depression. Altern. Med. Rev. 13(3): 216-226.
Morris, D.W., Trivedi, M.H., Rush, A.J., 2008. Folate and unipolar depression. J. Altern.
Complement. Med. 14, 277–285.
Moustafa, A.A., Hewedi, D.H., Eissa, A.M., Frydecka, D., Misiak, B.,2014.
Homocysteine levels in schizophrenia and affective disorders-focus on cognition.Front.
Behav. Neurosci. 6;8:343.
Niesink, R.J.M., Van Ree, J.M., 1989. Involvement of opioid and dopaminergic systems
in isolation-induced pinning and social grooming of young rats. Neuropharmacology 28(4):
411-418.
 21
ACCEPTED MANUSCRIPT

Stadtman, E.R., Levine, R.L., 2003. Free radical-mediated oxidation of free amino acids
and amino acid residues in proteins. Amino Acids. 25(3-4) 207-18.
Padurariu, M., Ciobica, A., Dobrin, I., Stefanescu, C., 2010. Evaluation of antioxidant
enzymes activities and lipid peroxidation in schizophrenic patients treated with typical and
atypical antipsychotics. Neurosci.Lett. 479(3): 317-320.
Pandya, C.D., Howell, K.R., Pillai, A., 2013. Antioxidants as potential therapeutics for
neuropsychiatric disorders. Prog.Neuropsychopharmacol. Biol. Psychiatry 46: 214–223.
Ponizovsky, A.M., Finkelstein, I., Poliakova, I., Mostovoy, D., Goldberger, N., Rosca, P.,

PT
2013. Interpersonal distances, coping strategies and psychopathology in patients with
depression and schizophrenia.World J. Psychiatry 3(3): 74-84.
Roffman, J.L., Lamberti, J.S., Achtyes, E., Macklin, E.A., Galendez, G.C., Raeke, L.H.,

RI
Silverstein, N.J., Smoller, J.W., Hill, M., Goff, D.C., 2013ª. Randomized multicenter
investigation of folate plus vitamin B12 supplementation inschizophrenia. JAMA Psychiatry
70(5):481-489.

SC
Roffman, J.L., Brohawn, D.G., Nitenson, A.Z., Macklin, E.A., Smoller, J.W., Goff, D.C.,
2013b. Genetic variation throughout the folate metabolic pathway influences negative
symptom severity in schizophrenia. Schizophr. Bull. 39(2): 330-338.
Ruiz-Litago, F., Seco, J., Echevarría, E., Martínez-Cengotitabengoa, M., Gil, J., Irazusta,

U
J., González-Pinto, A.M., 2012. Adaptive response in the antioxidant defence system in the
course and outcome in first-episode schizophrenia patients: a 12-months follow-up study.
AN
Psychiatry Res. 200(2-3): 218-222.
Salim, S., 2014. Oxidative stress and psychological disorders.Curr.Neuropharmacol.
12(2): 140-147.
M

Schneider, T., Przewlocki, R., 2005. Behavioral alterations in rats prenatally exposed to
valproic acid: animal model of autism. Neuropsychopharmacol. 30(1): 80-89.
Shamsi, S., Lau, A., Lencz, T., Burdick, K.E., DeRosse, P., Brenner, R., Lindenmayer,
D

J.P., Malhotra, A.K., 2011.Cognitive and symptomatic predictors of functional disability in

schizophrenia. Schizophr. Res.126(1-3): 257–264.
Stahl, S.M.,2007. Novel therapeutics for depression: L-methylfolate as a trimonoamine
TE

modulator and antidepressant-augmenting agent. CNS Spectr. 12(10): 739-744.

Tandon, R., Nasrallah, H.A., Keshavan, M.S., 2009. Schizophrenia, ‘Just the Facts’
4.Clinical features and conceptualization. Schizophr. Res.110(1-3): 1–23.
EP

Tomiya, M., Fukushima, T., Kawai, J., Aoyama, C., Mitsuhashi, S., Santa, T., Imai, K.,
Toyo'oka, T., 2006. Alterations of plasma and cerebrospinal fluid glutamate levels in rats
treated with the N-methyl-D-aspartate receptor antagonist, ketamine. Biomed Chromatogr.
20(6-7): 628-633.
C

Torgalsbøen, A.K., Rund, B.R., 1998. "Full recovery" from schizophrenia in the long
AC

term: a ten-year follow-up of eight former schizophrenic patients. Psychiatry 61(1): 20-34.
Vasquez, K.,Kuizon, S., Junaid, M., Idrissi, A.E., 2013. The effect of folic acid on
GABA(A)-B 1 receptor subunit.Adv. Exp. Med. Biol. 775: 101-109.
Wendel, A., 1981. Glutathioneperoxidase. MethodsEnzymol77: 325-333.
Yang, Y.J.,Wu, P.F.,Long, L.H.,Yu, D.F., Wu, W.N.,Hu, Z.L.,Fu, H.,Xie, N.,Jin, Y.,Ni,
L., Wang, J.Z., Yao, J.K., Keshavan, M.S., 2010. Antioxidants, redox signaling, and
pathophysiology in schizophrenia: an integrative view. Antioxid. Redox Signal. 15(7): 2011-
35.
Yao, J.K.,Keshavan, M.S., 2011. Antioxidants, redox signaling, and pathophysiology in
schizophrenia: an integrative view. Antioxid. Redox Signal 15(7): 2011-35.
Yao, R., Peddy, L.G., Mcelhinny, D.P., 1998. Van Kammen, Reduced status of plasma
total antioxidant capacity in schizophrenia, Schizophr. Res. 32:1–8.
 22
ACCEPTED MANUSCRIPT

Young, S.B., McKinney, B.M., Ross, K.W., Wahle, S.P., Boyle. 2007. Biomarkers of
oxidative stress in schizophrenic and control subjects, Prostag. Leukotr. Essent. Fat. Acids 76;
73–85.
White, A.R., Huang, X., Jobling, M.F., Barrow, C.J., Beyreuther, K., Masters, C.L.,
Bush, A.L., Cappai, R., 2001. Homocysteine potentiates copper-andamyloid beta peptide-
mediated toxicity in primary neuronal cultures possible risk factors in the Alzheimer’s-type
neurodegenerative pathways. J Neurochem. 76(5):1509–20.
Wu, B.T., Innis, S.M., Mulder, K.A., Dyer, R.A., King, D.J., 2013. Low plasma vitamin

PT
B-12 is associated with a lower pregnancy-associated rise in plasma free choline in Canadian
pregnant women and lower post natal growth rates in their male infants. Am. J.Clin. Nutr.
98(5):1209-17.

RI
Zugno,A.I., de Miranda, I.M., Budni, J.,Volpato, A.M.,Luca, R.D.,Deroza, P.F.,de
Oliveira, M.B., Heylmann, A.S.,da Rosa Silveira, F.,Wessler, P., Cipriano, A.L., Quevedo, J.,
2013.Effect of maternal deprivation on acetylcholinesterase activity and behavioral changes

SC
on the ketamine-induced animal model of schizophrenia. Neuroscience 248C: 252-260.

U
Legend to figures AN
Figure 1. Experimental design – protocol 1: Folic acid (FA) at doses of 5, 10 and 50mg/kg,
administeredfor a period of 7 days, and acute ketamine administration at a dose of 25 mg/kg
M

intraperitoneally (i.p.).
D

Figure 2. Experimental design – protocol 2: Folic acid (FA) at doses of 5, 10 and 50mg/kg
administered for 14 days, and chronic ketamine administration at a dose of 25 mg/kg (i.p.).
TE

Figure 3.The effect of folic acid supplementation (5, 10 and 50mg/kg) for a period of 7 days
EP

and treatment with acute ketamine (25 mg/kg) (panel A), and the effect of folic acid
supplementation (5, 10 and 50mg/kg) for a period of 14 days and treatment with chronic
C

ketamine (25 mg/kg) (panel B) on the locomotor activity of rats. Data are expressed as
AC

Mean±EPM (n=10). *Differences from Water+Sal (Saline) group; # Differences from

Water+Ket (Ketamine) group.

Figure 4. The effect of folic acid supplementation (5, 10 and 50 mg / kg) for a period of 7
days and treatment with acute ketamine (25 mg/kg) on the social interaction of rats (panel A:
latency tothe first contact; panel B: number of interactions; panel C: total contact time). Data
are expressed as Mean ± EPM (n=10). *Differences from Water+Salgroup;#Differences from
Water+Ket group.
 23
ACCEPTED MANUSCRIPT

Figure 5.The effect of folic acid supplementation (5, 10 and 50 mg / kg) for a period of 14
days and treatment with chronic ketamine (25 mg/kg) on the social interaction of rats (panel
A: latency to the first contact; panel B: number of interactions; panel C: total contact time).
Data are expressed as Mean ± EPM (n=10). *Differences from Water+Sal group; #
Differences from Water+Ket group.

PT
Figure 6.The effect of folic acid supplementation (5, 10 and 50mg/kg) for a period of 7 days
and treatment with acute ketamine (25 mg/kg) (panel A) and effect of folic acid

RI
supplementation (5, 10 and 50mg/kg) for a period of 14 days and treatment with chronic
ketamine (25 mg/kg) (panel B) on the formation of thiobarbituric acid reactive species

SC
(TBARS) in the prefrontal cortex, hippocampus and striatum of rats. Data are expressed as
Mean±EPM (n=5). *Differences from Water+Sal (Sal) group; #Differencesfrom Water+Ket.

U
AN
Figure 7.The effect of folic acid supplementation (FA, 5, 10 and 50mg/kg) for a period of 7
days and treatment with acute ketamine (25 mg/kg) (panel A) and effect of folic acid
supplementation (5, 10 and 50mg/kg) for a period of 14 days and treatment with chronic
M

ketamine (25 mg/kg) (panel B) on the carbonyl protein contents in the prefrontal cortex,
hippocampus and striatum of rats. Data are expressed as Mean ± EPM (n=5). * Differences
D

from Water+Sal (Sal) group; #Differencesfrom Water+Ket.

TE

Figure8.The effect of folic acid (5, 10 and 50mg/kg) supplementation for a period of 7 days
andtreatment with acute ketamine (25 mg/kg) on theactivity of antioxidant enzymesin the
EP

prefrontal cortex, hippocampus and striatum of rats: Glutathione peroxidase (GPx: panel A);
Superoxide dismutase (SOD: panel B) and Catalase(CAT: panel C). Data are expressed as
C

Mean±EPM (n=5).
AC

Figure 9.The effect of folic acid (5, 10 and 50mg/kg) supplementation for a period of 14 days
andtreatment with chronic ketamine (25 mg/kg) on the activity of antioxidant enzymes in the
prefrontal cortex, hippocampus and striatum of rats: Glutathione peroxidase (GPx: panel A);
Superoxide dismutase (SOD: panel B) and Catalase (CAT: panel C). Data are expressed as
Mean ± EPM (n=5). *Differences from Water+Sal (Sal) group; #Differences from
Water+Ket.
 ACCEPTED MANUSCRIPT

Criciúma, September 29th, 2015.

PT
RI
Acknowledgments

SC
Laboratory of Neurosciences (Brazil) is one of the centers of the National Institute for
Translational Medicine (INCT-TM) and one of the members of the Center of Excellence
in Applied Neurosciences of Santa Catarina (NENASC). This research was supported

U
by grants from CNPq, Instituto Cérebro e Mente and UNESC. JQ and AIZ are CNPq
AN
fellows.
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC