Journal of Neuroimmunology 320 (2018) 133–142

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Journal of Neuroimmunology
journal homepage: www.elsevier.com/locate/jneuroim

Sex influences in behavior and brain inflammatory and oxidative alterations T

in mice submitted to lipopolysaccharide-induced inflammatory model of
depression
Bruna Stefânia Ferreira Melloa, Adriano José Maia Chaves Filhoa, Charllyany Sabino Custódioa,
Rafaela Carneiro Cordeiroa, Fabio Miyajimaa, Francisca Cléa Florenço de Sousaa,
⁎
Silvânia Maria Mendes Vasconcelosa, David Freitas de Lucenaa, Danielle Macedoa,b,
a
Neuropharmacology Laboratory, Drug Research and Development Center, Department of Physiology and Pharmacology, Faculty of Medicine, Universidade Federal do
Ceará, Fortaleza, CE, Brazil
b
National Institute for Translational Medicine (INCT-TM, CNPq), Ribeirão Preto, Brazil

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

Keywords: Peripheral inflammation induced by lipopolysaccharide (LPS) causes a behavioral syndrome with translational
Depression relevance for depression. This mental disorder is twice more frequent among women. Despite this, the majority
Lipopolysaccharide of experimental studies investigating the neurobiological effects of inflammatory models of depression have been
Inflammation performed in males. Here, we sought to determine sex influences in behavioral and oxidative changes in brain
Sex
regions implicated in the pathophysiology of mood disorders (hypothalamus, hippocampus and prefrontal cortex
Oxidative stress
- PFC) in adult mice 24 h post LPS challenge. Myeloperoxidase (MPO) activity and interleukin (IL)-1β levels were
Lipid peroxidation
measured as parameters of active inflammation, while reduced glutathione (GSH) and lipid peroxidation as
parameters of oxidative imbalance. We observed that male mice presented behavioral despair, while females
anxiety-like alterations. Both sexes were vulnerable to LPS-induced anhedonia. Both sexes presented increased
MPO activity in the PFC, while male only in the hippocampus. IL-1β increased in the PFC and hypothalamus of
animals of both sexes, while in the hippocampus a relative increase of this cytokine in males compared to
females was detected. GSH levels were decreased in all brain areas investigated in animals of both sexes, while
increased lipid peroxidation was observed in the hypothalamus of females and in the hippocampus of males after
LPS exposure. Therefore, the present study gives additional evidence of sex influence in LPS-induced behavioral
alterations and, for the first time, in the oxidative changes in brain areas relevant for mood regulation.

1. Introduction more recurrent episodes (Schuch et al., 2014).

Depression is a common chronic-recurrent mental disorder.
Recently, the World Health Organization reported that globally > 300
millions of people of all ages suffer from depression, highlighting de-
pression as the leading cause of disability worldwide (WHO |
Depression, 2015). Depression causes great social and economic costs.
For example, in the United States a survey revealed that 8.3% of all
years lived with disability (YLDs) were associated to depression, with
an enormous economic burden in the order of 210.5 billions of dollars
each year (Greenberg et al., 2015). Furthermore, there is a well-es-
tablished overall gender difference in the prevalence of depression,
with women outnumbering men 2:1. Additionally, the onset of de-
pression in women occurs at younger ages with women also presenting
In the last decades, compelling evidences revealed an important
contribution of immune-inflammatory alterations to the pathophy-
siology of depression (Miller et al., 2009; Rosenblat et al., 2014). In-
deed, recent meta-analyses have confirmed that depressed patients
present increased serum levels of inflammatory markers, such as in-
terleukin-1β (IL-1β), IL-6, tumor necrosis factor (TNF)-alpha and IL-10,
and markers of cell activation, such IL-2 receptors (sIL-2Rs) and
neopterin (Farooq et al., 2016; Köhler et al., 2017). Furthermore, the
systemic injection of cytokines or of the bacterial endotoxin lipopoly-
saccharide (LPS) induce depressive symptoms in health humans and
depressive-like behavior in rodents (Suarez et al., 2004; Vogelzangs
et al., 2016).
In preclinical approaches, LPS-based models of depressive-like

⁎
Corresponding author at: Drug Research and Development Center, Department of Physiology and Pharmacology, Universidade Federal do Ceará, Rua Cel. Nunes de Melo 1000,
Fortaleza 60431-270, CE, Brazil.
E-mail address: danielle.macedo@ufc.br (D. Macedo).

https://doi.org/10.1016/j.jneuroim.2018.04.009
Received 21 November 2017; Received in revised form 12 April 2018; Accepted 12 April 2018
0165-5728/ © 2018 Elsevier B.V. All rights reserved.
B.S.F. Mello et al.
behavior are accompanied by time-dependent alterations related to
sickness behaviors (behavioral inhibition, anorexia, weight loss, fa-
tigue, hyperalgesia, malaise symptoms, neurocognitive impairment and
anxiety) (Dantzer et al., 2008; Maes et al., 2012) and depressive-like
behavior. In this regard, some studies (Custódio et al., 2013; Dantzer
et al., 2008; Frenois et al., 2007) have demonstrated that most beha-
vioral alterations elicited by the acute (1.5–2 h) exposure to LPS com-
pose the spectrum characteristic of sickness behavior, in which a
marked pyrexia, anorexia and locomotor inhibition occurs. On the
other hand, 24 h later, the emergence of depressive-like behavior
characterized by despair-, anhedonia- and anxiety-like behaviors takes
place. Twenty-four h after LPS exposure motor activity, food and drink
consumption returns to normal (Dantzer et al., 2008).
The influence of sex in the behavioral responses to LPS is not fully
understood. In this regard, there are studies pointing to a greater sen-
sibility of female animals to LPS and cytokine effects in some behavioral
aspects, such as sexual activity as well as sucrose and food consumption
(Avitsur and Yirmiya, 1999; Merali et al., 2003). On the other hand,
other studies reported better coping strategies of females in despair-like
conditions, such as forced swimming test (Pitychoutis et al., 2009).
Furthermore, the findings in the literature about sex influences in LPS-
induced behavioral effects changes markedly accordingly to the dose of
LPS used, time of assessment and murine specie tested (Badalà et al.,
2008; Cai et al., 2016).
It is well estabished that depression is accompanied by increased
levels of reactive oxygen- (ROS) and nitrogen- (RNS) species, such as
superoxide, peroxynitrite and hydrogen peroxide, and oxidative da-
mage (Maes et al., 2011a). These pro-oxidative alterations are triggered
in some patients by inflammation and mitochondrial metabolic pro-
cesses. In clinical samples, some studies pointed that sex could be a
determining factor for ROS and RNS levels in health subjects (Bellanti
et al., 2013; Massafra et al., 2002), however little is known about the
influence of sex in a depressive subset of patients, that is, those patients
with depression induced by inflammatory alterations. Also, regarding
animal models, several studies demonstrated the contribution of oxi-
dative stress in the molecular signature of depressive-like alterations
(Lucca et al., 2009; Zhang et al., 2009). However, most of them were
conducted in male animals, thus not assessing the possible influence of
sex in the oxidative response triggered by depressive-like conditions
induced by inflammatory challenge. This issue regarding sex influences
in depression deserves a better comprehension, since it will contribute
to the development of personalized treatments against this mental
disorder.
Therefore, taking into account the important and poorly understood
influence of sex in the manifestation of depressive-like phenomenology
by LPS immune challenge, in this study, we investigated the behavioral
(despair-, anhedonia- and anxiety-like) alterations in male and female
mice 24 h post-LPS administration, a time related to the emergence of
depressive-like phenotype without locomotor bias (Custódio et al.,
2013). Also, we evaluated the influence of sex in brain inflammatory
alterations, namely myeloperoxidase (MPO) activity and IL-1β, and, to
our knowledge for the first time, in the oxidative stress parameters
reduced glutathione (GSH) and lipid peroxidation in relevant brain
areas for mood regulation: prefrontal cortex (PFC), hippocampus and
hypothalamus.
2. Materials and methods
2.1. Drugs
Lipopolysaccharide (LPS) from Escherichia coli, strain 055:B5 (Sigma
e Aldrich Corp., St Louis, (USA) was used. The drugs were made up
freshly for the study. All other chemicals used were of analytical grade.
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Journal of Neuroimmunology 320 (2018) 133–142
2.2. Animals
Male and female adult Swiss mice (weighing 25 to 30 g) obtained
from the Animal house of the Federal University of Ceara were used.
Animals were housed 10 per cage under standard polycarbonate cages
(42 × 20.5 × 20 cm) and at normal environmental conditions
(22 ± 1 °C, humidity of 60 ± 5%; reversed 12 h light cycle/darkness
with lights on at 18:00) with access to food (FRILAB Mouse II, FRIRibe)
and water ad libitum. All experimental procedures were performed
between 8:00 AM and 02:00 PM and were conducted in accordance
with the NIH Guide for the Care and Use of Laboratory Animals (NIH,
2011) and the Brazilian law for scientific use of animals from the
Brazilian College of Animal Experimentation (COBEA). This research
protocol was approved by the local ethics committee of the Federal
University of Ceara.
2.3. Experimental protocol
The animals were randomly divided into four experimental groups:
male LPS-challenged group, male control-group, female LPS-challenged
group, female control-group (n = 24 mice/group). LPS-challenged
groups received an intraperitoneal injection of 0.5 mg/kg LPS dissolved
in 0.2 ml sterile endotoxin-free SF 0.9% - saline (as vehicle). Control
animals received saline, 0.1 ml/10 g weight. Twenty-four hours after
LPS or saline exposure, the behavioral tests and sample collection were
performed. The behavioral tests were conducted in the order of the less
stressful to the more stressful, i.e., open field, elevated plus maze and
forced swimming test. A different set of animals was used to perform
sucrose preference test due the potential stressful conditions of food and
water deprivation involved in this test. In all behavioral determina-
tions, two raters blinded to the experimental treatment performed the
tests. Two independent experiments were performed to ensure the re-
producibility of the data. To avoid some potential confounding effect of
the behavioral testing in the neurochemical parameters, a different set
of animals was used to the removal of brain structures. The animals
were killed by decapitation, and the brain areas were quickly dissected
namely prefrontal cortex (PFC), hippocampus and hypothalamus. All
samples were immediately stored −70 °C until assay. The dose of LPS
used and the time of assessment was based on previous studies de-
monstrating depressive-like behavior and neuroinflammatory altera-
tions induced by LPS in mice (Custódio et al., 2013; Frenois et al.,
2007).
2.4. Behavioral determinations
2.4.1. Forced swimming test (FST)
The animals were placed individually in an acrylic cylinder (25 cm
of height × 10 cm of diameter) containing 8 cm of water at 24 °C. After
a habituation period of 1 min, the immobility time (in seconds) of the
animals was evaluated for 5 min. Immobility was defined as the absence
of targeted escape behavior, such as swimming, jumping, lifting, smell
or diving (Porsolt et al., 1977). Any mouse seeming to have trouble
keeping its head out of the water was removed from the cylinder and
excluded from the analysis. In this study, two experienced evaluators
blinded to the treatment group independently assessed the mice beha-
vior.
2.4.2. Sucrose preference test (SPT)
The test was performed as described previously (Mao et al., 2014).
Briefly 72 h before testing, rats were trained to adapt sucrose solution at
10% (w/v). Two sucrose solution bottles were placed in each cage, and
24 h later one of them was replaced by a bottle with water for 24 h.
After adaptation, mice were deprived of food and water for 24 h. The
test section was conducted to 9:00 AM in which the rats were housed in
B.S.F. Mello et al. Journal of Neuroimmunology 320 (2018) 133–142

individual cages and had free access to two bottles containing 100 ml of
sucrose solution (10% w/v) and 100 ml of water, respectively. After 1 h,
the volumes of sucrose solution and water consumed were recorded and
preferably sucrose consumption was calculated by the following for-
mula:
Sucrose preference = (sucrose consumption ×100%)
/(water consumption + sucrose consumption)
2.6. Statistical analyses
Data are present in scatter dot plot with lines representing
mean ± S.E.M. (standard errors of the mean) and were compared by
regular two-way ANOVA with Tukey's as post hoc test. The factors used
were “sex” (male and female) and “LPS treatment” (LPS and saline
treatment). The significance level was set at P ≤ 0.05. The statistical
program used was GraphPad Prism 6.0 Version for Windows, San
Diego, CA, USA.

2.4.3. Open field test (OFT)

The open field arena was made of acrylic (30 × 30 × 15 cm) with
transparent walls and a black floor, divided into nine squares of equal
areas. The open field test was used to evaluate the locomotor and ex-
ploratory activity of the animal during 5 min (Archer, 1973). The
parameters number of squares crossed by the animal (crossings
number), number of rearings (vertical locomotion), number of self-
cleaning grooming behavior, and total time in the center of arena were
observed over 5 min and were registered in seconds.
2.4.4. Elevated plus maze test (EPM)
The elevated plus-maze consisted of two open (30 × 5 cm) and two
darkened, closed arm (30 × 5 × 15 cm) emanating from a common
central platform (5 × 5 cm), to form a plus shape (Pellow and File,
1986). The entire apparatus was raised 45 cm above the base, and the
test was performed under dim red light (60 W × 2). The test started by
placing a mouse on the central platform, facing an open arm. An ob-
servation period of 5 min was used, during which the total arms entries,
and the amount of time spent by animals in the open and closed arms of
the maze was measured. These data were used to calculate the number
of total entries, % of open entries (i.e., open entries/total entries × 100)
and % of time in the open arms (time in open arms/total time of the
test × 100).
3. Results
3.1. Male mice are more susceptible to despair-like behavior while both
sexes present anhedonia without motor alterations 24 h post-LPS
In the evaluation of despair-like behavior using the FST, two-way
ANOVA revealed a significant interaction between the factors “sex” and
“LPS challenge” [F(1, 27) = 4.989, P = 0.0340], with a significant
main effect “sex” [F(1, 27) = 59.21, P < 0.0001] and “LPS challenge”
[F(1, 27) = 8.867, P = 0.0061]. Post hoc test revealed a significant
increase in the immobility time in LPS-challenged male mice when
compared to male controls (P < 0.0001). No significant alterations
were observed in female animals exposed to LPS. We also observed
increased immobility time in male control animals in relation to female
ones (P < 0.0001), being this sex-related increase in immobility time
also observed in LPS-challenged males when compared to females
(P < 0.0001) (Fig. 1A).

2.5. Neurochemical determinations

2.5.1. Analysis of myeloperoxidase (MPO) activity

Myeloperoxidase is a highly oxidative enzyme. The extracellular
activity of this enzyme gives an estimate of the oxidative stress in in-
flammatory conditions (Pulli et al., 2013). Peroxidase activity with
3,30,5,50-Tetramethylbenzidine (TMB) was measured as described
elsewhere (Suzuki et al., 1983). Absorbance was determined at 450 nm
in two time-points, 0 and 3 min, to estimate MPO activity (U MPO/min/
mg tissue).

2.5.2. Immunoassay for IL-1β

The brain areas were homogenized in 8 volumes of PBS buffer with
protease (EMD Biosciences) inhibitor and centrifuged (10.000 rpm,
5 min). The concentration of the cytokines in 50 ml samples was de-
termined by the immunoenzymatic assay ELISA (R&D systems,
Minneapolis, MN, USA) according to the manufacturer's protocol and
expressed in pg/g tissue.

2.5.3. Determination of reduced glutathione (GSH) levels

The levels of GSH were evaluated to estimate endogenous defenses
against oxidative stress. The method was based on Ellman's reagent
(DTNB) reaction with free thiol groups (Sedlak and Lindsay, 1968). The
reaction was read in the absorbance of 412 nm, and the product was
expressed as ng of GSH/mg wet tissue.
2.5.4. Measurement of lipid peroxidation
Lipid peroxides formation was analyzed by measuring the thio-
barbituric-acid reacting substances (TBARS) in the brain homogenates
(Mihara and Uchiyama, 1978), as an index of reactive oxygen species
(ROS) production. Lipid peroxidation was assessed by the absorbance at
532 nm and expressed as μmol of malonaldehyde (MDA)/mg of tissue.
Fig. 1. Effect of LPS (0.5 mg/kg, i.p.) in the duration of immobility (in seconds)
in the forced swimming test (A) and in the % of sucrose preference in the su-
crose consumption test (B). Male and female mice were injected with LPS
(0.5 mg/kg, i.p) and, 24 h later, the behavioral tests were performed. Each dot
represents individual values, n = 7–8/group, with horizontal and vertical lines
representing mean ± S.E.M., respectively. Data are representative of two in-
dependent experiments and were analyzed by two-way ANOVA followed by
Tukey's post hoc test. ***P < 0.001, ****P < 0.0001. Abbreviations:
LPS = Lipopolysaccharide, Saline = vehicle.

135
B.S.F. Mello et al.
In the evaluation of anhedonia with the SPT (Fig. 1B), we detected a
significant main effect of “LPS challenge” in the percent of sucrose
consumption [F(1, 25) = 29.05, P < 0.0001], without significant in-
teraction between factors. Post hoc analysis revealed a significant de-
crease in the percent of sucrose consumption in LPS-challenged male
(P < 0.001) and female (P < 0.001) mice in relation to sex-matched
controls.
The number of crossings in the OFT was used as a parameter of
horizontal locomotor activity. As shown in Fig. 2A, LPS immune chal-
lenge did not induced significant alterations in the number of crossings
in both male and female animals when compared to their sex-matched
controls, as well no significant difference was noted when comparing
each treatment group in relation to sex. In the exploratory behavior, we
did not observe significant differences in the number of rearings post-
LPS challenge in animals of both sexes (Fig. 2B).
Regarding grooming behavior and the time spent in the center of the
arena, both parameters related to anxiety-like behavior, we observed in
grooming behavior (Fig. 2C) a significant main effect of “LPS challenge”
[F(1, 27) = 5.984, P = 0.0212] without significant interaction between
factors. In this behavioral parameter, we observed that LPS-challenged
female mice presented increased grooming behavior in relation to fe-
male controls. On the other hand, in the analysis of the time spent in the
center of the arena, two-way ANOVA demonstrated no significant al-
terations (Fig. 2D).
3.2. Female mice seem to be more susceptible to anxiety-like behavior in the
EPM test 24 h post-LPS
In the EPM test, we did not detect significant differences in the total
number of entries (Fig. 3A) as well as in the percent of time in the open
arms (Fig. 3C). Nevertheless, in the percent of entries in the open arms,
a significant main effect of “LPS challenge” [F(1, 21) = 7.863,
P = 0.0106], without a significant interaction between factors was
observed. In the post hoc test, a significant decrease in the percentage
of open arms entries was noted in LPS-challenged female mice when
compared to female controls (P < 0.05) (Fig. 3B).
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Journal of Neuroimmunology 320 (2018) 133–142
Fig. 2. Effect of LPS (0.5 mg/kg, i.p.) in the number
of crossings (A), number of rearings (B), number of
groomings (C) and time in the center of arena (in
seconds) (D) in the open field test. Male and female
mice were injected with LPS (0.5 mg/kg, i.p) and,
24 h later, the behavioral tests were performed. Each
dot represents individual values, n = 7–8/group,
with horizontal and vertical lines representing
mean ± S.E.M., respectively. Data are re-
presentative of two independent experiments and
were analyzed by two-way ANOVA followed by
Tukey's post hoc test. *P < 0.05. Abbreviations:
LPS = Lipopolysaccharide, Saline = vehicle.
3.3. Male and female mice present brain alterations in MPO activity, IL-1β
levels and oxidative parameters 24 h post-LPS
Regarding MPO activity in the PFC (Fig. 4A), two-way ANOVA
demonstrated a significant main effect of each factor alone ([F(1,
26) = 23.26, P < 0.0001], for “sex”; [F(1, 26) = 36.48, P < 0.0001],
for “LPS challenge”), without significant interaction between factors.
Post hoc comparisons revealed a significant increase in MPO activity in
the PFC of both male and female subjects challenged with LPS com-
pared to their sex-matched controls (P < 0.0001 for males and fe-
males). Furthermore, male control animals presented higher MPO ac-
tivity when compared to female control ones (P < 0.05). In the
hippocampus (Fig. 4B), ANOVA analysis revealed a significant inter-
action between factors [F(1, 24) = 8.064, P = 0.0091], with a sig-
nificant main effect of each one alone: [F(1, 24) = 8.900, P = 0.0065],
for “sex”; [F(1, 24) = 8.523, P = 0.0075], for “LPS challenge”. In the
post hoc comparisons, we evidenced a significant increase in MPO ac-
tivity in the hippocampus of female mice after LPS challenge
(P < 0.01). We also observed higher MPO activity in the hippocampus
of male control mice when compared to female controls (P < 0.01). In
the hypothalamus ANOVA analysis showed no significant interaction
nor main effect of any of the factors analyzed (Fig. 4C).
The levels of IL-1β in the PFC (Fig. 4D) were significantly increased
in both male (P < 0.05) and female (P < 0.01) mice challenged with
LPS in relation to sex-matched controls (two-way ANOVA: main effect
of “LPS challenge” [F(1, 24) = 24.11, P < 0.0001]. In the hippo-
campus (Fig. 4E), two-way ANOVA revealed no interaction between
factors, but a significant main effect of “sex” [F(1, 23) = 39.30,
P < 0.0001] and “LPS challenge” [F(1, 23) = 9.269, P = 0.0058]. Post
hoc comparisons showed a significant increase in IL-1β levels in male
controls when compared to female controls (P < 0.01) and in LPS-
challenged males when compared to LPS-challenged females
(P < 0.01). In the hypothalamus (Fig. 4F), ANOVA analysis demon-
strated main effect of “sex” [F(1, 24) = 27.34, P < 0.0001] and “LPS
challenge” [F(1, 24) = 30.16, P < 0.0001], without significant inter-
action between factors. In the post hoc comparisons, it was evidenced a
significant increase in IL-1β levels in LPS-challenged mice of both sexes
B.S.F. Mello et al.
Fig. 3. Effect of LPS (0.5 mg/kg, i.p.) in the number of total entries (A), in the %
of open entries (B) and in the % of time in open arms (C) in the elevated plus
maze test. Male and female mice were injected with LPS (0.5 mg/kg, i.p.) and,
24 h later, the behavioral tests were performed. Each dot represents individual
values, n = 7–8/group, with horizontal and vertical lines representing
mean ± S.E.M., respectively. Data are representative of two independent ex-
periments and were analyzed by two-way ANOVA followed by Tukey's post hoc
test. *P < 0.05. Abbreviations: LPS = Lipopolysaccharide, Saline = vehicle.
in relation to sex-matched controls (P < 0.001 for males and P < 0.05
for females). Also, male control mice presented increased levels of IL-1β
in relation to female ones (P < 0.05).
Regarding the levels of the main endogenous antioxidant, GSH, we
observed in the PFC, a significant main effect of both factors alone: [F
(1, 26) = 10.44, P = 0.0033], for “sex” and [F(1, 26) = 26.93,
P < 0.0001], for “LPS challenge”, without significant interaction. In
the post hoc test, we identified a significant decrease in GSH contents in
the PFC of both male (P < 0.05) and female (P < 0.01) mice chal-
lenged with LPS when compared to their sex-matched controls. Female
control mice presented higher levels of GSH when compared to control
male mice (P < 0.05) (Fig. 5A). In the hippocampus, two-way ANOVA
revealed a significant main effect of “sex” [F(1, 27) = 1.626,
P = 0.2131] and “LPS challenge” [F(1, 27) = 54.69, P < 0.0001], and
a significant interaction between factors [F(1, 27) = 5.290,
P = 0.0294]. In post hoc test, we detected a significant decrease in GSH
levels in the hippocampus of animals of both sexes challenged with LPS
137
Journal of Neuroimmunology 320 (2018) 133–142
when compared to their same sex controls (P < 0.0001, for males;
P < 0.001, for females) (Fig. 5B). In the hypothalamus, there was a
significant main effect of “LPS challenge” [F(1, 25) = 88.34,
P < 0.0001]. Post hoc comparisons demonstrated a significant de-
crease in GSH levels in the hypothalamus of animals of both sexes
challenged with LPS compared to sex-matched saline controls
(P < 0.0001, for males; P < 0.0001, for females) (Fig. 5C).
Lipid peroxidation represents the damage of lipid membranes
caused by oxidative imbalance (Ramos-Loyo et al., 2013). In this con-
text, we observed no lipid peroxidation in the PFC of LPS-challenged
mice when compared to controls (Fig. 5D). In the hippocampus, two-
way ANOVA analysis demonstrated a significant interaction between
“sex” and “LPS challenge” [F(1, 23) = 6.131, P = 0.0211]. In the post
hoc test, we evidenced a significant increase in MDA levels in the
hippocampus of LPS-challenged male mice in relation to their same sex
controls (P < 0.05) (Fig. 5E). In the hypothalamus (Fig. 5F), a sig-
nificant increase in MDA levels was evidenced in LPS-challenged female
mice in relation to their same sex controls (P < 0.05) (two-way
ANOVA: main effect of “LPS challenge” [F(1, 21) = 8.211,
P = 0.0093].
4. Discussion
In this study, we demonstrated that at the time-point of 24 h post
systemic challenge with LPS, male and female mice present a distinctive
spectrum of behavioral accompanied by proinflammatory and oxidative
alterations. Of note, male animals presented behavioral despair and
anhedonia, while females presented anhedonia and anxiety-like al-
terations. Both sexes presented brain proinflammatory and pro-oxida-
tive alterations. The main strengths of the present study were de-
termining sex influences in anxiety-like behavior as well as
proinflammatory and pro-oxidative alterations in brain areas relevant
to mood regulation in mice submitted to an inflammatory (LPS) model
of depressive-like behavior.
Men and women differ in the occurrence and presentation of several
psychiatric disorders. This is specifically relevant for major depressive
disorder (MDD), which is twice as common in females than in males.
Interestingly, prior to puberty, subjects of both genders are equally
affected by depression (Marcus et al., 2005). Many biological, psycho-
social, and sociological theories attempted to explain this dramatic in-
crease in the prevalence of depression among women, but none of them
is fully satisfactory. In this context, animal models despite their lim-
itations, represent a useful tool for the investigation of sex differences in
the neurobiology of depression and antidepressant response (Dalla
et al., 2010).
In line with the limited knowledge regarding sex influences in de-
pression, only few studies were conducted aiming to determine sex
influences in the behavioral and neurochemical alterations induced by
LPS challenge. In this context, Pitychoutis et al., 2009 reported that
male and female rats presented different vulnerability to sickness be-
havior at the time-point of 2 h post-LPS administration. In their study,
LPS-challenged female rats showed a better coping strategy, evidenced
by the increased time of swimming, while males presented a more ro-
bust locomotor suppression. The other behavioral parameters eval-
uated, such as sucrose consumption, social exploration and food intake
were equally affected in animals of both sexes (Pitychoutis et al., 2009).
Additionally, a recent study focusing on the sex-specific behavioral
effects of LPS at the time-points of 6 and 24 h post LPS (Sens et al.,
2017) found that the immobility duration was equally affected in ani-
mals of both sexes. The same results were obtained when mice were
tested for anhedonia by sucrose consumption. Notably, they found that
the grooming behavior in the splash test, an index of motivation and
self-care behavior, was persistently impaired in female mice, while
anorexic behavior showed to be more pronounced in male counterparts
(Sens et al., 2017).
In our experimental conditions, male and female mice showed a
B.S.F. Mello et al.
distinct behavioral pattern in the forced swimming test 24 h post LPS
challenge. Our results pointed to an increased vulnerability of LPS
challenged male animals to the development of despair behavior in the
FST. Of note, we also observed that male control animals presented
increased immobility time in relation to female ones. Taken together,
these results obtained in male animals corroborates previous findings
showing the ability of female animals in the development of coping
strategies in the stressful FST (Pitychoutis et al., 2009). It is important
to highlight that this previous study was conducted in Sprague–Dawley
(SD) rats with LPS serotype 026:B6, i.e., different from the serotype
used in the present study, and that the enhanced coping abilities ob-
served in females were detected at the time-point of 2 h post LPS
(Pitychoutis et al., 2009). This sex influence in the immobility time in
the FST observed in our results was not observed at the time-point of
24 h post LPS serotype 026:B6 (Sens et al., 2017), revealing, thus, that
the effects of LPS are not only time, but also specie and serotype de-
pendent (Felgner et al., 2017).
In addition, we observed that anhedonia assessed by SPT was
equally established in both male and female animals. As expected, 24 h
post-LPS administration, no locomotor impairment was observed in
male and female mice. This result is in line with previous ones showing
that both sexes are vulnerable to anhedonia (Sens et al., 2017).
A well-established concept is that anxiety and inflammation walk
together. In this regard, Bassi et al., 2012. described the anxiogenic-like
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Journal of Neuroimmunology 320 (2018) 133–142
Fig. 4. Effect of LPS (0.5 mg/kg, i.p.) in MPO activity
and IL-1β levels in the prefrontal cortex (A, D), hip-
pocampus (B, E) and hypothalamus (C, F). Male and
female mice were injected with LPS (0.5 mg/kg, i.p.)
and, 24 h later, the brain areas were collected for
neurochemical analysis. Each dot represents individual
values, n = 7–8/group, with horizontal and vertical
lines representing mean ± S.E.M., respectively. Data
are representative of two independent experiments
and were analyzed by two-way ANOVA followed by
Tukey's post hoc test. *P < 0.05, **P < 0.01,
***P < 0.001, ****P < 0.0001. Abbrevia-
tions: LPS = Lipopolysaccharide, Saline = vehicle,
IL = interleukin, MPO = myeloperoxidase.
effects of LPS in male rats in several doses and in several behavioral
tests (elevated plus maze, black-white box, open field, elevated T maze,
emission of ultrasonic vocalizations) 3–4 h after administration. They
found that LPS induced a dose-dependent increase in anxiety-like be-
haviors, forming an inverted U curve which peaks in 200 μg/kg dose
(Bassi et al., 2012). Several other studies demonstrated the anxiogenic-
like effects of LPS in the sickness spectrum of alterations (few hours
after injection) as well as in depressive-like ones (24 h after) (Li et al.,
2017; Salazar et al., 2012; Savignac et al., 2016). We also observed, in a
previous study, that LPS 0.5 mg/kg caused anxiety-like behavior in
male animals at the time-point of 1.5 h post LPS, while at the time-point
of 24 h no alteration was observed (Custódio et al., 2013). Furthermore,
the intravenous administration of Salmonella abortus equi endotoxin to
20 healthy male volunteers caused a transient significant increase in the
levels of anxiety and depressive mood at the time-points of 1, 3 and 9 h
post administration. To the best of our knowledge, no previous study
evaluated sex influences in anxious-like behavior in LPS-challenged
animals (Reichenberg et al., 2001).
Our results showed that LPS-challenged female mice presented with
a more robust anxiety-like phenotype. This evidence came from two
different behavioral tasks, i.e., increased grooming behavior in the OFT,
and reduced percentage of open entries in the EPM. In general, anxiety
disorders are much more prevalent in women than in men (McLean
et al., 2011). Indeed, in animal studies females usually demonstrate less
B.S.F. Mello et al.
anxiety-like behavior in basal situations (Rodgers and Cole, 1993;
Võikar et al., 2001). However, when exposed to stressful-aversive sti-
muli, remarkable anxiety-like alterations are observed.
It is worth mentioning that adrenocorticotropic hormone (ACTH) and
corticosterone responses are greater in female subjects than in male ones
during the exposure to a variety of acute stressors (Doremus-Fitzwater
et al., 2009; Iwasaki-Sekino et al., 2009). More recently, Vieira et al.,
2017, confirmed this proposition by demonstrating that female rodents
showed increased anxiety-like behavior and plasma corticosterone levels
when exposed to chronic unpredictable stress and chronic homotypic
stress (repeated restraint stress) (Vieira et al., 2017). In this context, our
results revealing enhanced vulnerability of females to anxiety-like phe-
notype in LPS inflammatory model of depressive-like behavior are in
accordance with the previous findings of female susceptibility to anxiety-
like behavior against environmental stressful situations. It is important to
mention that there is a discussion in the literature about the possible
relation of inflammatory models of depression with one specific subtype
of depression, namely atypical depression (Remus and Dantzer, 2016). In
fact, atypical depression is the most common form of depression, four
times more prevalent in women presenting high comorbidity with an-
xiety disorder (Singh and Williams, 2006).
It is consistently recognized that LPS activates toll-like receptor 4
(TLR4). The TLR4 pathway is coupled to the activation inflammasome
signaling, in special nucleotide-binding domain leucine-rich repeat
(NLRP) 3 (Scholz and Eder, 2017). NLRP3 mediates, in large part, the
processing and maturation of IL-1β (Cullen et al., 2015; Jo et al., 2016).
139
Journal of Neuroimmunology 320 (2018) 133–142
Fig. 5. Effect of LPS (0.5 mg/kg, i.p.) in reduced
glutathione (GSH) levels and lipid peroxidation in
the prefrontal cortex (A, D), hippocampus (B, E) and
hypothalamus (C, F). Male and female mice were
injected with LPS (0.5 mg/kg, i.p.) and, 24 h later,
the brain areas were collected for neurochemical
analysis. Each dot represents individual values,
n = 7–8/group, with horizontal and vertical lines
representing mean ± S.E.M., respectively. Data are
representative of two independent experiments and
were analyzed by two-way ANOVA followed by
Tukey's post hoc test. *P < 0.05,
**P < 0.01, ****P < 0.0001. Abbreviat-
ions: LPS = lipopolysaccharide, Saline = vehicle,
MDA = malonaldehyde.
IL-1β acts as a pleotropic proinflammatory cytokine, inducing its own
synthesis and the synthesis of other cytokines that potentiate its effect,
such as IL-18 and IL-6 (Weber et al., 2010). Mitochondria ROS is both a
major trigger and product of NLRP3 inflammasome activation (Han
et al., 2015).
Oxidative stress and inflammation are closely related pathophysio-
logical processes. Inflammatory cells release reactive species at the site
of inflammation leading to exaggerated oxidative stress. On the other
hand, a number of reactive species can initiate intracellular signaling
cascades that enhances proinflammatory gene expression (Biswas,
2016). In line with this evidence, MPO is an important enzyme of innate
immune system that is activated in phagocytic cells during in-
flammatory conditions (Arnhold and Flemmig, 2010). MPO generates
strong oxidant radicals, in special, hypochlorous acid (HOCl), that can
covalently modify lipids and/or proteins causing local damage and
amplification of inflammatory response (Arnhold and Flemmig, 2010;
Pattison et al., 2012).
Relevant studies reported increased MPO expression and activity in
serum samples of depressive patients. For example, Vaccarino et al.,
2008, demonstrated in 178 monozygotic and dizygotic twins, a strong
association between serum MPO levels and depression scores in stan-
dard scales (Vaccarino et al., 2008). Additionally, it was found that
MPO mRNA and protein levels are markedly enhanced in peripheral
blood cells of patients with recurrent depression, and present a positive
correlation with the impaired performance in several cognitive tasks
(Talarowska et al., 2015).
B.S.F. Mello et al.
In the present study, we observed a brain region-dependent increase
in MPO activity in animals submitted to LPS immune challenge. Indeed,
MPO activity was increased in the PFC of male and female mice, while
in the hippocampus this increase was observed only in female animals.
Animal submitted to chronic stress presented higher MPO activity in
selected brain areas, namely hippocampus and cortex, which was ac-
companied by additional proinflammatory alterations such as micro-
glial activation and NO release (Kasımay Çakır et al., 2017; Lisowski
et al., 2013). Currently, as far as we know there is no available data
about brain MPO activity in rodents submitted to LPS-based depressive-
like model. Therefore, our results demonstrated not only the effects of
LPS as an activator of MPO activity in the brain, as well as advocate for
a possible sex related brain region-dependent response.
Still regarding inflammatory alterations triggered by LPS challenge,
in the present study we assessed the levels of IL-1β in relevant brain
areas related to mood regulation. We found 24 h post LPS increased
levels of IL-1β in the PFC and hypothalamus of male and female mice.
We also observed higher levels of IL-1β in the hippocampus of LPS-
challenged male mice when compared to female ones, although this
increase was not significant in relation to male control mice. It is im-
portant to highlight that despite being classified as a cytokine, IL-1β is
also a neuroactive molecule that interferes with several mechanisms
related to depression: i) decreases hippocampi cell proliferation, being
an antineurogenic mediator (Koo and Duman, 2008) and ii) is a potent
stimulant of the serotonin metabolism (Ramamoorthy et al., 1995).
Considering sex influences in LPS-induced inflammatory alterations,
some authors reported an increased inflammatory response in females
24 h post nasal LPS administration (Badalà et al., 2008), while others
showed increased sickness-behavior and serum cytokine levels in male
mice 10 h post intraperitoneal LPS injection (Cai et al., 2016).
Regarding oxidative alterations in depression, considerable evi-
dence reported diminished levels of GSH and/or impaired activity of
the enzymes involved in GSH synthesis and restoration in the serum and
post-mortem brain of depressed patients (Gawryluk et al., 2011; Maes
et al., 2011b). Among the endogenous antioxidant systems, GSH is the
most abundant thiol antioxidant present in mammalian cells protecting
cells against ROS generated by the mitochondrial respiratory chain
(Dringen and Hirrlinger, 2003). Accordingly, several preclinical studies
showed that rodents submitted to environmental or pharmacological
models of stress presented a marked reduction in total GSH levels and
glutathione peroxidase activity (GPX) in brain areas related to mood
regulation (Silva et al., 2016; Tao et al., 2016; Todorović and Filipović,
2017).
Lipid peroxidation is an autoxidation process promoted by radical
species against phospholipids and unsaturated fatty acids of the cellular
membranes. This process severely hampers the membrane functions,
increasing membrane rigidity and allowing the leakage of calcium ions
(Fuchs et al., 2014). Malondialdehyde (MDA) is a byproduct of poly-
unsaturated fatty acid and arachidonic acid peroxidation consistently
employed as a measure of lipid peroxidation (Gaweł et al., 2004). There
are numerous studies supporting the involvement of lipid peroxidation
in depression, and a lot of them demonstrating increased MDA levels in
biological samples of depressive patients [for a detailed Review, see
(Maes et al., 2011a)].
In our study, we found that male and female mice exposed to LPS
immune challenge equally presented impaired GSH levels in all brain
areas investigated, and a sex- and brain region-dependent increase in
lipid peroxidation. Notably, male mice showed increased levels of MDA
in the hippocampus, while females showed increased levels of MDA
only in the hypothalamus. Despite previous studies have demonstrated
important oxidative damage (lipid peroxidation, increased nitrite and
PGE2 levels) and impairment in antioxidant systems (reduced GSH le-
vels) in the brain of mice submitted to LPS challenge (Custódio et al.,
2013; Mello et al., 2013; Sayd et al., 2014), all of them investigated
oxidative changes only in male rodents. Therefore, as far as we know,
140
Journal of Neuroimmunology 320 (2018) 133–142
our study gives the first evidence of sex influences in brain oxidative
status in LPS-induced depressive-like model in mice.
It is an important to mention that the neural circuitries related to
anxiety situations or reward-pleasure stimuli involves basolateral
amygdala and bed nucleus of the stria terminalis (BNST) (Duval et al.,
2015; Russo and Nestler, 2013). BNST recruits projections from the
ventral hippocampus and hypothalamic nuclei, promoting the pitui-
tary-adrenal response (Duval et al., 2015). In reward-promoting situa-
tions, orbitofrontal cortex (OFT) projects reward information to ante-
rior cingulate cortex, that, in turn, send projections to anterior
ventromedial PFC, dorsolateral PFC and ventral hippocampus (Russo
and Nestler, 2013). These nuclei are key regulators of decision-making
based on reward value and the reinforcement for future actions
(Gorwood, 2008).
Taking this above mecioned cirtuitry into consideration, it is pos-
sible to suggest, in our experimental condition, that the presence of a
pronounced inflammatory-oxidative damage in hippocampal-hypotha-
lamic areas of female mice is in accordance with the emergence of an
increased anxiety-like phenotype, since these neurocircuits are involved
in the regulation of anxiety and stress response (Duval et al., 2015). On
the other hand, in male animals, the predominance of oxidative damage
in the PFC and hippocampal regions, which are key areas for the reg-
ulation of brain reward circuitry (Russo and Nestler, 2013), can be a
plausible factor for the development of a robust despair-like/anhedonia
behavior in these animals. Finally, the hippocampus, as a region in-
trinsically involved in the regulation of both circuits, was impaired in
animals of both sexes. On the other hand, in our experimental condition
it seems that hippocampus was more affected in males than in females.
This study presents some limitations. Firstly, we did not assess the
influence of estrous cycle changes in the behavioral and neurochemical
effects of LPS in females. This is an interesting point for future research,
since ovarian hormones, in special estrogens, are known to modulate
not only female behavior, as well as the activity of several cellular
antioxidant systems (Bellanti et al., 2013). Additionally, we decided to
measure the parameters of oxidative damage: MPO, GSH, and lipid
peroxidation, and not others, for example, catalase and superoxide
dismutase enzymes, that despite being relevant for redox status, present
controversial evidence in depressive patients (Maes et al., 2011a).
In conclusion, this study provides additional evidence of a sex-
specific pattern of behavior in adult mice submitted to LPS-induced
inflammatory model of depression. Notably, in our results, male ani-
mals presented a greater vulnerability to despair-like behavior, while
females developed anxiety-related alterations. Both sexes were equally
vulnerable to LPS-induced anhedonia. The behavioral alterations in
females were followed by increased oxidative changes in all brain areas
investigated here, but especially in the hypothalamus, a brain area as-
sociated to stress response and anxiety. Male animals presented a pre-
ponderance for the involvement of oxidative damage in the PFC and
hippocampal regions, which are key areas for the regulation of reward.
Therefore, this study advances the knowledge about sex influence in
inflammatory-based model of depression by presenting evidences of
sex-specific pattern of behavioral alterations and, for the first time, of
brain oxidative changes.
Disclosure
The authors declare no conflict of interests.
Acknowledgements
The authors thank the Brazilian Institutions CAPES, FUNCAP and
CNPq for the financial support. The authors are grateful for the tech-
nical assistance of Maria Vilani Bastos.
B.S.F. Mello et al.
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