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Behavioural Brain Research
journal homepage:www.elsevier.com/locate/bbr
Research report
Two-hit model of schizophrenia induced by neonatal immune activation and
peripubertal stress in rats: Study of sex di
ff
erences and brain oxidative
alterations
Aline Santos Monte
a, Bruna Stefânia Ferreira Mello
a, Vládia Célia Moreira Borella
a,
Tatiane da Silva Araujo
a, Francisco Eliclécio Rodrigues da Silva
a, Francisca Cléa F de Sousa
a,
Antônio Carlos Pinheiro de Oliveira
b, Clarissa Severino Gama
c,d, Mary V. Seeman
e,
Silvânia Maria Mendes Vasconcelos
f, David Freitas De Lucena
a,f, Danielle Macêdo
a,g,⁎ aDrug Research and Development Center, Department of Physiology and Pharmacology, Universidade Federal do Ceará, Fortaleza, CE, Brazil bDepartment of Pharmacology and Department of Physiology–ICB, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil cLaboratório de Psiquiatria Molecular, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil dPrograma de Pós-Graduação em Psiquiatria e Ciências do Comportamento, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil eDepartament of Psychiatry, University of Toronto, Toronto, Ontario, Canada
fNational Institute for Developmental Psychiatry (INCT
–INPD, CNPq), Brazil gNational Institute for Translational Medicine (INCT-TM, CNPq), Brazil
A R T I C L E I N F O
Keywords:
Schizophrenia Sex differences Two-hit animal model Neonatal poly(I:C) Oxidative imbalance
A B S T R A C T
Schizophrenia is considered to be a developmental disorder with distinctive sex differences. Aiming to simulate the vulnerability of the third trimester of human pregnancy to the developmental course of schizophrenia, an animal model was developed, using neonatal poly(I:C) as afirst-hit, and peripubertal stress as a second-hit, i.e. a two-hit model. Since, to date, there have been no references to sex differences in the two-hit model, our study sought to determine sex influences on the development of behavior and brain oxidative change in adult rats submitted to neonatal exposure to poly(I:C) on postnatal days 5–7 as well as peripubertal unpredictable stress (PUS). Our results showed that adult two-hit rats present sex-specific behavioral alterations, with females showing more pronounced deficits in prepulse inhibition of the startle reflex and hyperlocomotion, while males showing more deficits in social interaction. Male and female animals exhibited similar working memory deficits. The levels of the endogenous antioxidant, reduced glutathione, were decreased in the prefrontal cortex (PFC) of both male and female animals exposed to both poly(I:C) and poly(I:C) + PUS. Only females presented decrements in GSH levels in the striatum. Nitrite levels were increased in the PFC of male and in the striatum of female poly(I:C) + PUS rats. Increased lipid peroxidation was observed in the PFC of females and in the striatum of males and females exposed to poly(I:C) and poly(I:C) + PUS. Thus, the present study presents evidence for sex differences in behavior and oxidative brain change induced by a two-hit model of schizophrenia.
1. Introduction
Schizophrenia is a severe mental disorder affecting more than 21 million people worldwide[1]. Epidemiological studies have provided substantial evidence for an association between prenatal infections and increased risk for the development of some neuropsychiatric disorders,
such as schizophrenia and autism[2]. Because these disorders begin in fetal life, they are classified as neurodevelopmental disorders, although some components of schizophrenia may be neurodegenerative [3]. Developmental disorders often show profound sex differences in their expression. For example, autism is 4–5.1 times more prevalent in males than in females and shows other important differences between the
http://dx.doi.org/10.1016/j.bbr.2017.04.057
Received 26 October 2016; Received in revised form 9 April 2017; Accepted 10 April 2017
⁎Corresponding author at: Department of Physiology and Pharmacology, Universidade Federal do Ceará, Rua Cel. Nunes de Melo 1000, 60430-270 Fortaleza, CE, Brazil. E-mail addresses:alinesmonte@yahoo.com.br(A.S. Monte),bubs_mello@hotmail.com(B.S.F. Mello),vladiacmborella@hotmail.com(V.C.M. Borella),
tatianedrc@gmail.com(T. da Silva Araujo),elicleciors@gmail.com(F.E.R. da Silva),cleasousa@yahoo.com.br(F.C.F.d. Sousa),acpoliveira@gmail.com(A.C.P. de Oliveira),
clarissasgama@gmail.com(C.S. Gama),Mary.Seeman@utoronto.ca(M.V. Seeman),daviddelucena@me.com(D.F.D. Lucena),
daniellesilmacedo@gmail.com,danielle.macedo@ufc.br(D. Macêdo).
Abbreviations:GSH, reduced glutathione; NO, nitric oxide; TBARS, thiobarbituric-acid reacting substances; PFC, prefrontal cortex; PN, postnatal day; poly(I:C), polyinosinic:polycy-tidylic acid; PPI, prepulse inhibition of the startle reflex; PUS, peripubertal unpredictable stress
Available online 17 May 2017
0166-4328/ © 2017 Elsevier B.V. All rights reserved.
– –
to postnatal day (PN) 7–10 in rodents. Therefore, PN5-7 in rodents is equivalent to the end of the third-trimester of pregnancy in humans[7]. It has been theorized that, for schizophrenia to occur, two“hits”are required, one in fetal life and one in adolescence[9]. The vulnerability of the adolescence period resides in the neuromaturational processes taking place in this age, like increased hypothalamic–pituitary–adrenal axis activity, as well as a normative decline in cortical gray matter volume, that are also posited to play an important role in the clinical expression of schizophrenia [10]. Accordingly, exposure to traumatic life events during adolescence appears to increase the risk of psychosis [9]. In our experimental condition our second hit was based on the exposure of animals from PN40-48, a period corresponding to human age 12–18 [7], to different stressful situations, namely peripubertal unpredictable stress (PUS), as previously proposed[6].
What sparked our interest in sex differences in animal models of schizophrenia was the recent observation in our laboratory that rats submitted to a neonatal first-hit using ketamine presented adult behavioral changes analogous to schizophrenia behaviors that were influenced by the sex[11]. These behavioral alterations were accom-panied by oxidative changes in the levels of glutathione (GSH), lipid peroxidation and nitrite in the hippocampus[11].
As far as we know, there are no studies addressing sex influences in the two-hit [combination of neonatal poly(I:C) and PUS] model of schizophrenia. Our intention to search for oxidative alterations under-lying these sex differences was based on recent findings pointing towards sex influences in oxidative stress biomarkers[12], including increased lipid peroxidation[13]and decreased fatty acid levels[14]in males compared to females with schizophrenia. In addition distinct brain regions implicated in schizophrenia, such as the prefrontal cortex (PFC), are known to be modulated by sex in laboratory animals[15].
Thus, here we hypothesized that male and female rats submitted to either one neonatal hit with poly(I:C) or PUS or to a two-hit model of schizophrenia [poly(I:C) combined with PUS] would present distinct behavioral phenotypes in adulthood. We planned to evaluate this by using tests capable of predicting “positive symptoms” − (prepulse
inhibition of the startle reflex − PPI and open field tests) [16], “negative symptoms” − (social interaction) [17] and “cognitive symptoms”(Y-maze test)[18]. We also hypothesized that sex-distinct oxidative alterations in brain areas related to schizophrenia pathophy-siology underlay these differences and would be associated with hit exposure. We hoped our results would contribute to the understanding of the neurobiological alterations underpinning hit exposure and sex differences in schizophrenia.
2. Materials and methods
2.1. Animals
A total of 70 adult Wistar rats (male:female = 1:2) were used to breed, generating a total of 64 male and 64 female Wistar rat pups. Pregnant females were monitored for the parturition day, which was counted as postnatal day 0 (PN0). All animals were obtained from the
2.3. Experimental design
The animals were submitted to no-hit [three intraperitoneal (i.p.) administration of sterile saline (Sal) during PN5-7]; one-hit [three administration of poly(I:C) 2 mg/kg, i.p. during PN5-7 or PUS, for more details see supplementary material] or two-hit [neonatal poly(I:C) combined with PUS]. Poly(I:C) or Sal were administered at 0.1 ml/ 10 g body weight. The pups were maintained with their respective dams in individual cages until weaning on PN21. Between PN40 and 48 the PUS protocol was implemented (supplementary methods). These distinct stressors were used to simulate stressful life events. The behavioral determinations were conducted on PN60 (adulthood) by raters blinded to animal sex and hit exposure protocol. Sixteen animals per group were assigned for the behavioral experiments.
The study comprised eight groups: Sal male and female (Sal PUS−),
Sal combined with PUS male and female (Sal PUS+), poly(I:C) male and female (poly(I:C) PUS−), poly(I:C) combined with PUS male and female
(poly(I:C) PUS+). Subgroups of eight animals were submitted to PPI and openfield or to sociability and social novelty preference and Y-maze task. In order to minimize potential confounds associated with litter effects, offsprings derived from a given dam were subjected to saline treatment (and further divided into male or female groups of Sal PUS−or Sal PUS+, after weaning) or to Poly I:C treatment (and further divided into male or female groups of poly(I:C) PUS− or poly(I:C)
PUS+, after weaning). Thus, each experimental group consisted of pups from at least six different litters. No mortality was observed in offspring treated with Poly(I:C). Since previous results of our research group found no differences in behavioral parameters between animals neona-tally treated with Sal or left undisturbed during the 60 days period of the experiment, in order to reduce the number of animals we decided to use as controls Sal-treated rats. Immediately after the behavioral assays (PN60), the brain areas, PFC and striatum, were removed and stored at
−80 °C for neurochemical assays. To avoid litter effect, only one offspring from each dam was randomly allocated in each experimental group.Fig. 1presents an overview of the experimental design.
2.4. Behavioral assessments
2.4.1. Prepulse inhibition of the startle response (PPI)
This test was used to determine deficits in sensorimotor gating, an endophenotype of schizophrenia[19]. The rats were tested for their acoustic startle reactivity (ASR) and PPI levels in a startle chamber (Insight, São Paulo, Brazil) as described previously[20]. The test was initiated with a 5 min acclimatization to the startle chamber in the presence of 65 dB background noise. Next, the animals received nine 120 dB pulses alone (startle amplitude) and eighteen pulses preceded by 100 ms by a prepulse (PP) of 70, 75 or 80 dB intensity. The amount of PPI was calculated as a percentage score for each acoustic pre-pulse trial type: %PPI = 100−startle amplitude with PP × 100 startle
2.4.2. Locomotor activity
The apparatus used to evaluate the exploratory activity of the animals was a 50 × 50 cm openfield divided into four squares of equal areas by black lines. The evaluation was performed during 5 min in a room under red lights and controlled noise[21]. The software Panlab SMART video tracking system version 3.0 analyzed the total distance travelled by the animal.
2.4.3. Y-maze task
This test was used to assess spatial working memory by spontaneous alternation performance, which allows the evaluation of cognitive searching behavior [22]. A Y-maze apparatus made of black acrylic consisted of three arms with 425 mm (length), 145 mm (width), and 225 mm (height) mounted symmetrically (120° between arms) to an equilateral triangular center compartment. Each rat was placed at the end of one arm and allowed to freely move through three arms of the maze during 8 min. The series of arm entries was recorded visually. The number of maximum alternations was the total number of arms entered minus 2, and the percent alternation was calculated as (actual alterna-tions/maximum alternations) X 100. An alternation was considered correct if the animal visited a new arm and did not return to a previously visited arm (example of correct alternation: 1, 2, 3 arms; example of incorrect alternation: 1, 2, 1 arms).
2.4.4. Sociability and social novelty preference
The testing apparatus consisted of a 60 × 40 cm Plexiglas box divided into three chambers. Animals were able to move between chambers through a small opening (6 × 6 cm) in the dividers. Iron cages in each of the two side chambers contained, in one side, an unfamiliar, same-sex probe rat from the same experimental group, whereas in the other side, the cage was empty. Test animals were placed in the center chamber and allowed 5 min of exploration time in the box. Iron cages in each of the two side chambers contained, in one side, an unfamiliar, same-sex probe rat from the same experimental group, whereas in the other side, the cage was empty. The time spent in each of the three chambers was clocked, and social preference was defined as follows: (time spent in the social chamber)−(time spent in the opposite chamber)[23].
2.5. Neurochemical determinations
2.5.1. Determination of reduced glutathione (GSH) levels
GSH was evaluated to estimate endogenous defenses against oxidative stress. The method was based on Ellman’s reagent (DTNB) reaction, as described elsewhere[24]. GSH levels were determined by the absorbance at 412 nm and were expressed as ng GSH/g wet tissue.
2.5.2. Nitrite determination
In order to assess alterations in nitric oxide (NO) production, nitrite levels were determined in the rat brain homogenates. The production of NO was determined based on the Griess reaction [25,26]. The absorbance was measured at 550 nm via a microplate reader. The standard curve was prepared with several concentrations of NaNO2 (ranging from 0.75 to 100μM) and was expressed asμmol/g of protein.
2.5.3. Measurements of lipid peroxidation
Lipid peroxides formation was analyzed by measuring the thiobar-bituric-acid reacting substances (TBARS) in the homogenates[27]as an index of reactive oxygen species (ROS) production. Lipid peroxides were read at the absorbance at 535 nm. Results are expressed asμmol malondialdehyde (MDA)/mg tissue[28].
2.6. Statistical analysis
All results are expressed as means ± standard error of the mean (SEM). Data analyses were performed using IBM SPSS Statistics version 21 for Mac. The results were analyzed by three-way ANOVA followed by Bonferronipost hoctest considering as between-subject the factors “sex” (male and female),“neonatal treatment” (saline and poly(I:C)) and“PUS exposure”(PUS+and PUS−). Significance level was set at
P≤0.05.
3. Results
3.1. Sex differentially affects the development of behavioral alterations in animals submitted to one-hit or two-hit models of schizophrenia
exposed to poly(I:C) combined with PUS presented significant increase in the total distance travelled when compared to sex-matched non-stressed-, stressed-controls or poly(I:C) groups. Of note, the hyperloco-motion in female animals was 1.5-fold higher when compared to male counterparts.
Regarding social preference (Fig. 3A), significant two-way interac-tions between “sex” and “PUS” [F(1,50) = 8.326, P = 0.006] and “neonatal treatment”x “PUS” [F(1,50) = 3.965, P = 0.05] were ob-served. Significant deficits in social preference were observed in male animals exposed to PUS when compared to nonstressed-control (P < 0.0001). The combination of poly(I:C) + PUS potentiated the deficit in social interaction observed in male rats being this decrease significant when compared to stressed-, nonstressed-controls or poly (I:C) male rats (P < 0.0001). Females exposed to poly(I:C) + PUS presented deficits in social preference when compared to stressed-control (P < 0.05). The comparison between male and female rats showed that male rats exposed to poly(I:C) + PUS presented significant deficits in social interaction when compared to female counterparts (P < 0.05).
In the evaluation of working memory (Fig. 3B) a significant three-way interaction between “sex”, “PUS” and “neonatal treatment” [F (1,50) = 10.307, P = 0.002] was observed. In this task, PUS exposure caused no alteration in male and female rats when compared to nonstressed-controls. Poly(I:C) when administered to male animals decreased the% correct alternations when compared to male
non-stressed- (P < 0.001), non-stressed-controls (P < 0.0001) or to poly(I:C) female rats (P < 0.001). On the other hand, the combination of Poly (I:C) + PUS decreased the% correct alternations in both male and female animals when compared to nonstressed- or stressed-controls in the case of males (P < 0.01) and to nonstressed-, stressed-controls or poly(I:C) in the case of females (P < 0.0001).
3.2. Sex influences in brain oxidative alterations in rats submitted to submitted to one-hit or two-hit models of schizophrenia
We next tested the hypothesis that oxidative imbalance in distinct brain areas related to the neurobiology of schizophrenia could mediate the sex differences in the behavioral alterations following hit exposure. The analysis of GSH levels in the PFC revealed a significant two-way interaction between “PUS” and “neonatal treatment” [F(1,40) = 39.647, P < 0.0001]. In this brain area the antioxidant GSH was approximately three-fold decreased in both male and female rats exposed to PUS, poly(I:C) and poly(I:C) + PUS compared to sex-matched nonstressed-control rats (P < 0.01) (Fig. 4A). In the striatum we observed a significant three-way interaction between“sex”,“PUS” and “neonatal treatment” [F(1,40) = 12.742, P = 0.001]. Female animals exposed to PUS, poly(I:C) and poly(I:C) + PUS presented an approximately five-fold reduction in GSH levels compared to female nonstressed-control (P < 0.01) (Fig. 4B). It is noteworthy that the striatal levels of GSH in female nonstressed-control rats was almost three-fold higher than in their male counterparts.
A significant interaction between“PUS”and“neonatal treatment” was observed in the analysis of nitrite levels in the PFC [F(1,40) = 11.686, P = 0.002]. (Fig. 4C). Increased levels of nitrite were detected in both male (P < 0.01) and female (P < 0.05) rats exposed to the combination of poly(I:C) + PUS being this increase significant in relation to sex-matched nonstressed-, stressed-controls or poly(I:C) Fig. 2.Effect of poly(I:C) and poly(I:C) + PUS on mean% PPI (A), acoustic startle
response (B) and total distance travelled (C). The rats were injected at PND5-7 with saline or poly(I:C). At PND40-48 the animals were exposed to PUS (PUS+) or not (PUS−). Bars
are means ± SEM (n = 6-8 animals/group). Data were analyzed using three-way ANOVA followed by Bonferroni post hoc test. *P < 0.05, ***P < 0.001, ****P < 0.0001. Abbreviations: Sal = Saline; PND = Postnatal day; PUS = peripubertal unpredictable stress; M = Male; F = Female.
Fig. 3.Effect of poly(I:C) and poly(I:C) + PUS on% social preference (A) and% correct alternations in the Y-maze task (B). The rats were injected at PND5-7 with saline or poly (I:C). At PND40-48 the animals were exposed to PUS (PUS+) or not (PUS−). Bars are
groups. In the striatum (Fig. 4D) no significant interaction was detected. In this brain area females exposed to poly(I:C) + PUS presented significantly higher levels of nitrite compared to non-stressed-, stressed-controls or poly(I:C) females (P < 0.01).
We did not observe significant interaction between factors in the analysis of lipid peroxidation in the PFC (Fig. 5A) and striatum (Fig. 5B). This means that both male and female rats exposed to poly (I:C) and poly(I:C) + PUS presented increased levels of TBARS in the PFC when compared to sex-matched nonstressed-controls, while the groups poly(I:C) + PUS were also significant when compared to stressed controls. This increase was approximately six-fold in females poly(I:C) + PUS compared to female nonstressed-control. Similarly, in the striatum lipid peroxidation was increased in both male and female rats submitted to poly(I:C) + PUS compared to sex-matched non-stressed-controls.
4. Discussion
Our results revealed that the neonatal challenge with poly(I:C) combined or not with PUS triggers long-term sex-specific behavioral and brain-region-specific oxidative alterations in male and female rats. In this regard we observed that male and female rats when exposed to one-hit with poly(I:C) presented deficits in PPI (females with higher deficits than males) and working memory impairment (only males) accompanied by decreased levels of GSH in the PFC (both sexes), striatum (only females) and increased levels of lipid peroxidation in the PFC (both sexes). After two-hit exposure the following changes were observed: PPI deficits (only females), hyperlocomotion (females with higher hyperlocomotion than males), social impairment (males with
higher impairment than females) working memory deficits (both sexes) accompanied by changes in the PFC observed in both sex: decreased GSH, increased nitrite and increased lipid peroxidation. Only females presented alterations in the striatum, i.e., decreased GSH and increased nitrite. These results might reflect a tendency for the development of worse“positive” schizophrenia-like symptoms by females exposed to one-hit [poly(I:C)] and two-hit, while males exposed to two-hit present worse“negative”schizophrenia-like symptoms.“Cognitive” schizophre-nia-like symptoms were observed in male animals exposed to one-hit with poly(I:C) and with both males and females exposed to two-hit. The exposure to PUS alone caused deficits in PPI (both sexes) and social impairment in male animals accompanied by decreased levels of GSH in the PFC (both sexes) and decreased GSH in the striatum (only females). The original two-hit model of schizophrenia was based on maternal exposure to physiologically relevant dose of poly(I:C) at gestational day (GD)9, considered as thefirst-hit, followed by PUS as the second-hit [6]. However, the rodent brain still undergoes significant development and maturation in early postnatal life [7]. Thus, here we decided to introduce the first-hit during neonatal life in order to simulate an immune challenge that, translated to humans, represents the end of the third trimester of pregnancy [7] since viral infection at the end of pregnancy in humans is associated with an increased risk for psychosis among adult offspring[30].
We have recently demonstrated that neonatal challenge with poly (I:C) from PN5-7 in rats caused PPI and working memory deficits in adulthood followed by microglial activation and oxidative imbalance (i.e. decreased levels of GSH and increased lipid peroxidation) in brain areas such as PFC and ST[8]. Accordingly, a previous report showed that the neonatal challenge with poly(I:C) from PN2-6 caused, in adult Fig. 4.Effect of poly(I:C) and poly(I:C) + stress on GSH (A) and nitrite (B) levels in the prefrontal cortex (A) and striatum (B). The rats were injected at PND5-7 with saline or poly(I:C). At PND40-48 the animals were exposed to PUS (PUS+) or not (PUS−). Bars are means ± SEM (n = 6-8 animals/group). Data were analyzed using three-way ANOVA followed by
animals, anxiety-like behavior, sensorimotor gating deficits, impair-ment of object recognition memory and social behavior accompanied by impaired depolarization-evoked glutamate release in the hippocam-pus[31].
Regarding sex differences, we had observed that male and female adult rats submitted to a neonatal first-hit with ketamine presented behavioral and oxidative alterations that resemble sex differences in schizophrenia. In the case of females, the alterations varied across the estrous cycle[11]. Thesefindings added evidence for the protective role of estrogen in schizophrenia [32]. In addition, the sex influences observed in our ketamine developmental model of schizophrenia had already revealed that animal models might be useful tools for the study of sex differences in schizophrenia[11].
To date, no study on the two-hit model of schizophrenia evaluated the consequences of neonatal poly(I:C), as the first-hit; neither has anyone investigated sex influences on the development of schizophre-nia-like alterations in this model. Here, after submitting male and female animals to neonatal poly(I:C), PUS or to the two-hit, we observed that the most behavioral and oxidative alterations occurred in animals exposed to poly(I:C) + PUS. Of note, early and late risk factors are not simply additive, rather, the theory is that the first-hit increases the individual’s vulnerability to the effects of the second-hit [33].
Our behavioral results of poly(I:C) + PUS seem to be in accordance with clinical observations of substantial sex differences in the experi-ence of schizophrenia, with women tending to present with more affective and positive psychotic symptoms, and men tending to present with more negative symptoms [34]. In fact, both male and female schizophrenia patients present PPI deficits [29], but in our experi-mental condition we observed that females poly (I:C) + PUS presented higher deficits in PPI when compared to male counterparts. We also observed that females poly(I:C) + PUS presented a pattern of hyperlo-comotion more intense than males, which may be suggestive of worse positive-like symptoms in females exposed to the two-hit. The reasons
interact with specific genetic factors to produce oxidative stress and enhance the risk of long-term neurodevelopmental brain dysfunction [38]. Importantly, oxidative stress is a well-known mechanism long implicated in the pathogenesis of schizophrenia [39]. Furthermore, oxidative pathways (e.g. GSH and lipid peroxidation) are known to be influenced by sex[40,41]. To test this hypothesis, we determined the levels of GSH, nitrite and lipid peroxidation in brain areas related to positive and negative/cognitive symptoms, respectively, in humans, straitum striatum[42]and PFC[43,44].
In our study, GSH, the most abundant antioxidant thiol present in mammalian cells, was decreased in rats exposed to PUS, neonatal poly (I:C) and poly(I:C) + PUS. Previous experiments have shown that immobilization stress in rodents induces generation of reactive oxygen species and decreases endogenous antioxidant defenses, which can be attenuated by extracellular administration of antioxidant GSH [45]. Decreased brain levels of GSH is also a core brain alteration observed in animal models of schizophrenia[8,46]and in schizophreniapostmortem
patients[39].
GSH biosynthesis is under the direct control of N-methyl-D-aspartate receptors (NMDAR)[47]. A previous study showed that the prenatal activation of toll-like receptors-3 by poly(I:C) resulted in decreased levels of the NMDA receptor-associated protein GluN1 in the brains of PN21 offspring [48]. Indeed, NMDAR hypofunction is another core brain alteration observed in schizophrenia[49]. Interestingly, in our results, the decrease in GSH levels observed in the PFC of male and female animals and in the striatum of female animals exposed to PUS, neonatal poly(I:C) and poly(I:C) + PUS was not directly related to behavioral change since we did not detect behavioral alterations in animals exposed only to peripubertal stress. Thus, it seems that this parameter is sensitive to environmental insults that do not correlate with the behavioral phenotype we observed.
We also observed increased levels of nitrite in the PFC of male and in the striatum of female rats exposed to poly(I:C) + PUS. Indeed, NO can react with molecules like superoxide anion and form reactive nitrogen oxide species (RNOS). These RNOS can modify biological macromolecules playing a pivotal role in cell death mediated by NO [50]. Since poly(I:C) + PUS animals also presented decreased levels of GSH, further studies need to be designed in order to determine if this effect is related to an accumulation of hydrogen peroxide forming the free-radical peroxynitrite, resulting in oxidative and nitrosative stress (O & NS). Indeed, O & NS seems to be a core response related to the course and treatment of schizophrenia [51]. As mentioned earlier, nitrite levels increased only in animals exposed to poly(I:C) + PUS, the group that showed the worst behavioral alterations.
Also deserving mention is the fact that both male and female poly (I:C) + PUS rats had deficits in the Y-maze task and increased levels of nitrite in the PFC, while females poly(I:C) + PUS showed more hyperlocomotion and PPI deficits and increased levels of nitrite in the striatum. The relevance of this observation is that PFC is a brain area involved with working memory performance[44], while striatum has been related to psychosis[52].
Lipid peroxidation, in turn, is the metabolic process by which Fig. 5.Determination of lipid peroxidation in the prefrontal cortex (A) and striatum (B) of
rats exposed to poly(I:C) or poly(I:C) + stress. The rats were injected at PND5-7 with saline or poly(I:C). At PND40-48 the animals were exposed to PUS (PUS+) or not (PUS−).
reactive oxygen species (ROS) result in the oxidative deterioration of lipids. This may significantly affect cell membrane structure and function[53]. We found increments in lipid peroxidation in the ST of male and female rats exposed to poly(I:C) + PUS as well as in the PFC of male and female rats exposed to poly(I:C) and poly(I:C) + PUS. A recent study showed that male and female mice exposed to neonatal poly(I:C) followed by juvenile restraint stress presented oxidative alterations mainly in the PFC[54]. The difference between this work and ours is that Deslauriers et al. did not divide male and female animals into separate groups whereas we did and we did not observe sex influences in lipid peroxidation levels in the PFC and striatum.
4.1. Limitations
Our experimental design did not address two of the main sex differences in schizophrenia, i.e. differential onset age in males and females and illness severity[5,55]. Our model used perinatal infection as thefirst hit whereas other possibilities, such as maternal stress, for example, were not evaluated[2]. Finally, based on the importance of GSH [56], NO[57]and lipid peroxidation [58]in schizophrenia we decided to measure these parameters and not others, for example, catalase and superoxide dismutase enzymes that despite being related to oxidative defenses, present controversial findings in schizophrenic patients[56].
5. Conclusion
Here we provide evidence of sex-specific patterns of behavior that are analogous to symptoms of adult schizophrenia in adult rats exposed to neonatal poly(I:C) + PUS. Specifically, we observed that male animals exposed to poly(I:C) + PUS presented greater social impair-ment when compared to females being this behavioral alteration associated with “negative” schizophrenia-like symptoms [17], while females presented greater PPI deficits and hyperlocomotion that model “positive” schizophrenia-like symptoms [16]. Both sexes presented cognitive deficits. The behavioral alterations in females poly(I:C) + PUS were accompanied by oxidative changes (decreased levels of GH and increased nitrite) in the striatum, a brain area associated with positive symptoms of schizophrenia [52] whereas, in the PFC, the changes were equivalent in male and female animals. Thus, the present study advances knowledge about the two-hit animal model of schizo-phrenia by showing evidence of adult sex differences in induced behavior and in oxidative brain changes. Furthermore, in rats exposed to one-hit with poly(I:C) we also observed greater PPI deficits in female than in males, while only males presented working memory deficits. Taken together, in our results we observed that both one-hit and two-hit models of schizophrenia present sex-specific patterns of behavior.
Funding and disclosure
CNPq, grant number 466724/2014-4, funded this study. 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 technical assistance of Maria Vilani Bastos.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, athttp://dx.doi.org/10.1016/j.bbr.2017.04.057.
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