Maternal immune activation induced by lipopolysaccharide triggers immune response in pregnant mother and fetus, and induces behavioral impairment in adult rats

Accepted Manuscript Maternal immune activation induced by lipopolysaccharide triggers immune response in pregnant mother and fetus, and induces behavioral impairment in adult rats Lutiana Roque Simões, Gustavo Sangiogo, Michael Hikaru Tashiro, Jaqueline S. Generoso, Cristiano Julio Faller, Diogo Dominguini, Gustavo Antunes Mastella, Giselli Scaini, Vijayasree Vayalanellore Giridharan, Monique Michels, Drielly Florentino, Fabricia Petronilho, Gislaine Zilli Réus, Felipe Dal-Pizzol, Alexandra I. Zugno, Tatiana Barichello PII:

S0022-3956(17)31182-2

DOI:

10.1016/j.jpsychires.2018.02.007

Reference:

PIAT 3305

To appear in:

Journal of Psychiatric Research

Received Date: 25 October 2017 Revised Date:

5 January 2018

Accepted Date: 8 February 2018

Please cite this article as: Simões LR, Sangiogo G, Tashiro MH, Generoso JS, Faller CJ, Dominguini D, Mastella GA, Scaini G, Giridharan VV, Michels M, Florentino D, Petronilho F, Réus GZ, Dal-Pizzol F, Zugno AI, Barichello T, Maternal immune activation induced by lipopolysaccharide triggers immune response in pregnant mother and fetus, and induces behavioral impairment in adult rats, Journal of Psychiatric Research (2018), doi: 10.1016/j.jpsychires.2018.02.007. 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.

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ACCEPTED MANUSCRIPT Maternal immune activation induced by lipopolysaccharide triggers immune response in pregnant mother and fetus, and induces behavioral impairment in adult rats

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Lutiana Roque Simões1, Gustavo Sangiogo1, Michael Hikaru Tashiro1, Jaqueline S. Generoso1, Cristiano Julio Faller1, Diogo Dominguini1, Gustavo Antunes Mastella2, Giselli Scaini5, Vijayasree Vayalanellore Giridharan5, Monique Michels3, Drielly Florentino4, Fabricia Petronilho4, Gislaine Zilli Réus2, Felipe Dal-Pizzol3, Alexandra I. Zugno2, and Tatiana Barichello1,5,6 1

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Laboratory of Experimental Microbiology, Graduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina (UNESC), Criciúma, SC, Brazil. 2

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Laboratory of Neurosciences, Graduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina (UNESC), Criciúma, SC, Brazil.

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Laboratory of Experimental Pathophysiology, Graduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina (UNESC), Criciúma, SC, Brazil.

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Laboratory of Neurobiology of Inflammatory and Metabolic Processes, Graduate Program in Health Sciences, University of South Santa Catarina (UNISUL), Tubarão, SC, Brazil.

Translational Psychiatry Program, Department of Psychiatry and Behavioral Sciences, McGovern Medical School, The University of Texas Health Science Center at Houston (UTHealth), Houston, TX, USA. 6

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Neuroscience Graduate Program, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, USA.

Corresponding author: Tatiana Barichello, Ph.D. Department of Psychiatry and Behavioral Sciences, Medical School, The University of Texas Health Science Center at Houston. 1941 East Road, Suite 3140, Houston, Texas, 77054, USA. E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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Evidence suggest that prenatal immune system disturbance contributes largely to the pathophysiology of neuropsychiatric disorders. We investigated if maternal immune activation (MIA) could induce inflammatory alterations in fetal brain and pregnant rats. Adult rats subjected to MIA also were investigated to evaluate if ketamine potentiates the effects of infection. On gestational day 15, Wistar pregnant rats received lipopolysaccharide (LPS) to induce MIA. After 6, 12 and 24 h, fetus brain, placenta, and amniotic fluid were collected to evaluate early effects of LPS. MIA increased oxidative stress and expression of metalloproteinase in the amniotic fluid and fetal brain. The blood brain barrier (BBB) integrity in the hippocampus and cortex as well integrity of placental barrier (PB) in the placenta and fetus brain were dysregulated after LPS induction. We observed elevated pro- and anti-inflammatory cytokines after LPS in fetal brain. Other group of rats from postnatal day (PND) 54 after LPS received injection of ketamine at the doses of 5, 15, and 25 mg/kg. On PND 60 rats were subjected to the memories tests, spontaneous locomotor activity, and pre-pulse inhibition test (PPI). Rats that receive MIA plus ketamine had memory impairment and a deficit in the PPI. Neurotrophins were increased in the hippocampus and reduced in the prefrontal cortex in the LPS plus ketamine group. MIA induced oxidative stress and inflammatory changes that could be, at least in part, related to the dysfunction in the BBB and PB permeability of pregnant rats and offspring. Besides, this also generates behavioral deficits in the rat adulthood's that are potentiated by ketamine.

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Keywords: maternal immune activation; cytokine; blood-brain barrier; placental barrier; ketamine; schizophrenia

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ACCEPTED MANUSCRIPT 1 Introduction

Increasing evidence suggests that maternal infection during pregnancy results in acute and chronic changes in developmental context structure and function of the central nervous system (CNS) in the fetus (Estes and McAllister, 2016). Pathogens or inflammatory stimuli are recognized to play an important etiological role in

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neuropsychiatric and neurological disorders with neurodevelopmental components (Boksa, 2010; Brown, 2011; Meyer, 2014; Simanek and Meier, 2015). Significant associations between prenatal infection during pregnancy and increased disease risk in later life have been revealed for various brain disorders, including schizophrenia

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(Brown and Derkits, 2010), autism (Patterson, 2011), bipolar disorder (Canetta et al., 2014), mental retardation (Johnson et al., 2012), and cerebral palsy (Hagberg et al.,

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2012).

A bacterial infection animal model is widely accepted and achieved by administration of lipopolysaccharide (LPS), a cell walls component of Gram-negative bacteria. LPS induces the activation of the innate immune response by increasing mRNA expression and pro-inflammatory cytokines in maternal serum, amniotic fluid and the placenta (Oskvig et al., 2012). LPS has been commonly used to mimic a

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bacterial infection through activation of Toll-like receptor 4 (TLR4), and to increase the production of pro-inflammatory cytokines, which are known to interfere with the development of the CNS (Boksa, 2010). In the fetal brain, cytokines are known to regulate the function and development of neurons, and also being involved in

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neuroprotective and neurodegenerative processes (Pujol Lopez et al., 2015). Maternal immune activation (MIA) appears to act as a “disease primer” (Meyer,

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2014) to make an individual more susceptible to the effects of genetic mutations and environmental exposures in triggering disease-related symptoms later in life (Ayhan et al., 2016). Several maternal cytokines have been identified as critical mediators of MIA on disease-related phenotypes in offspring. However, little is known about how these maternal cytokines alter brain development. One possibility is that MIA leads to longlasting changes in expression of immune molecules known to regulate neural connectivity and function in offspring (Estes and McAllister, 2015). MIA by LPS alone results in N-methyl-D-aspartate (NMDA) receptor hypofunction and a loss of hippocampal long-term plasticity in adolescent rats (Reisinger et al., 2015).

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is

a

dissociative

anesthetic

drug

that

affects

glutamate

neurotransmission by blocking the NMDA receptor (Laruelle et al., 2000). Many experimental studies have been used ketamine to induce schizophrenic-like behavior (Reus et al., 2018; Reus et al., 2017). In fact, rats subjected to 25 mg/kg of ketamine, have increased spontaneous locomotor activity, a positive schizophrenia symptom

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(Canever et al., 2010). In addition, rats exposed to immune activation in the early life, presented a negative influence to ketamine induced-behavior, and elevated oxidative stress and inflammatory parameters in the brain (Réus et al., 2017). In human, ketamine use has been linked to impairment in cognition, specifically by inducing a larger deficit

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in recall memory in people with schizophrenia compared to controls (Lahti et al., 1995). Acute doses of ketamine in healthy volunteers induce schizophrenic-like positive and negative symptoms, and may also lead to impairments in cognitive function that

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resemble schizophrenia (Deakin et al., 2008; Stone et al., 2012).

Based on the hypothesis that prenatal exposure to immune challenges may be a vulnerability factor for neurodevelopmental brain disorders rather than a diseasespecific risk factor (Harvey and Boksa, 2012; Miller et al., 2013), we investigated if MIA could induce oxidative stress and inflammatory alterations in fetal brain and

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pregnant rats. Moreover, in adult rats who were subjected to MIA it was investigated if ketamine in different doses could potentiate the behavioral effects of infection or if MIA could affect the response of ketamine.

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2 Materials and Methods

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2.1 Maternal immune activation

All procedures were approved by the Animal Care and Experimentation

Committee of UNESC/114/2013-2 and 066/2016-1 (Brazil) and by the Institutional Animal Welfare Committee of the Center for Laboratory Animal Medicine and Care. Protocol number AWC 15/0187, UTHealth, Texas, USA. All possible efforts were made to reduce animal suffering and the number of animals used. The total number of pregnant rats used for both experiment 1 and 2 was 156. To total number of fetus used for experiment 1 was 108. The total number of adult male Wistar rats used in the experiment 2 was 192. On gestational day 15, pregnant Wistar rats received intraperitoneal (i.p.) injection of either Escherichia coli O55:B5 LPS (LPS; 0.25 mg/kg;

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Sigma, St. Louis, MO), diluted (1 mg/ml) in phosphate-buffered saline (PBS), or equivalent volume of PBS (Oskvig et al., 2012). 2.2 Experimental design In experiment 1 (figure 1A), pregnant Wistar rats on the 15th day of gestation

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were divided into two groups: control and LPS groups and received injection of PBS (1 mg/ml) or LPS (0.25 mg/kg) i.p., respectively. At 6, 12 and 24 h after LPS or PBS (n = 6 per group: LPS and PBS and each time: 6, 12 and 24) administration the pregnant rats were anesthetized with ketamine (6.6 mg/kg) and xylazine (0.3 mg/kg) (Barichello et

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al., 2014; Grandgirard et al., 2007) then amniotic liquid (n = 6 per group) and fetus brain (n = 6 per group) were collected to evaluate the oxidative damage, enzymatic antioxidant defenses, matrix metalloproteinase (MMP) 2 and 9 activities (animal total

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for analysis = 36). Pregnant rats were anesthetized and decapitated, a midline incision was made into the peritoneal cavity to expose the embryos. Embryos were removed, and amniotic fluid was collected from each amniotic sac using a 1 mL syringe with a 23 g needle and collected in 0.2 mL and placed either in empty 1.5 mL microcentrifuge tubes. Fetal brains were removed and placed either in empty 1.5 mL microcentrifuge

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tubes. For BBB and PB analysis were used a total of 36 animals. The hippocampus and cerebral cortex were used for analysis of BBB integrity. Additionally, cytokine levels were evaluated only in fetal brains at 6, 12 and 24 h after LPS or PBS administration (n = 6 per group) (animal total for analysis n = 36).

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In experiment II (figure 1), 48 pregnant Wistar rats on the 15th day of gestation were divided into two groups: control and LPS groups and received injection of PBS or LPS i.p., respectively. After injection pregnant rats were returned to their primary cages

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where they remained until the birth and weaning of offspring, that held on the 21st postnatal day. The mothers were killed by decapitation, and only the offspring were used after completing 54 days of life, where then adult male rats were subjected to experimental animal model of schizophrenia induced by ketamine. We used male rats based in our previous study investigating the effects of both LPS and ketamine (Reus et al., 2017). They were divided into eight groups: control/saline, control/ketamine 5 mg/kg, control/ketamine 15 mg/kg, control/ketamine 25 mg/kg,

LPS/saline,

LPS/ketamine 5 mg/kg, LPS/ketamine 15 mg/kg and LPS/ketamine 25 mg/kg (n = 12 per group). After seven days of ketamine treatment (54th - 60th), we performed the behavioral tests as following: open field, object recognition, locomotor activity (animal

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total for these tests, n = 96), and pre-pulse inhibition (PPI) (animal total for this test, n = 96). Then the animals were euthanized, the hippocampus and prefrontal cortex removed for the evaluation of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) levels.

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2.3 Animal model of schizophrenia induced by ketamine

Ketamine (5, 15 and 25 mg/kg doses, once daily, i.p., CU Chemie Uetikon, Germany) was administered at a volume of 1 mL/100 kg in male adult offspring for

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seven days prior to behavioral tests. On the last day of treatment, ketamine was administered 30 min before behavioral tests (Zugno et al., 2013).

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2.4 Cytokines

The cytokines levels were assayed using multiplex fluorescent immunoassay kits (Bio-Plex Pro™ Rat Cytokine 24-Plex Assay). The xMAP platform used here was based on the Rules-Based Medicine (RBM) fluorescent beads and antibody pairs. These

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are sensitive, specific and widely used reagents, sourced by numerous manufacturers, and data collected using xMAP multiplex beads are widely reported in the literature in studies in which multiple proteins are assayed simultaneously. Total cellular extracts from the brain was prepared by lysing the cells in lysate dilution buffer according to the

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manufacturer’s instructions, followed by centrifugation at 4ºC for 10 min at 10,000 x g. Tissue lysate were prepared according to the instructions provide by Bio-Plex Cell Lysis kit (#171304011) with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis,

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MO, USA). The assays were conducted in 96-well polystyrene, round-bottom microplates. Initially, 50 µL aliquot of the working bead mixture was transferred into the wells, and the plate was washed 2 times by adding 100 µL of assay buffer into each well. After, 50 µL of the standard, control or total extracts were added to each well, as indicated. The plate was incubated on a plate shaker (850 rpm) in the dark at RT for 60 min. The plate was then placed in the magnetic separator and incubated for separation for 60 s. The supernatant was carefully removed from each well by manual inversion. Beads were washed 3 times by adding 100 µL of assay buffer into each well to ensure the absence of any undesirable or non-specifically bound antibodies. After this protocol, 25 µL of a detection antibody were added to each well. Incubation was again conducted

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in darkness and at RT on a plate shaker (850 rpm) for 30 min, and washing was performed as previously described. Finally, 50 µL of streptavidin-PE was added to each well. The plate was incubated on a plate shaker (850 rpm) in the dark at RT for 10 min. The supernatant was carefully removed after magnetic separation of the beads by manual inversion, and washing was performed as previously described. Assay buffer

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(125 µL) was added into each well, and the plate was placed onto a plate shaker for approximately 30 s in order to achieve gentle agitation of the beads. Samples were run in duplicate using a Bioplex system (Bio-Plex 200 Systems, BioRad, Hercules, CA) and data analysis was conducted in Bio-Plex Manager 4.0 using a 5-parameter logistic

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regression model.

2.5.1. Malondialdehyde equivalents

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2.5 Oxidative stress parameters

To determine oxidative damage in lipid, we measured the formation of thiobarbituric acid reactive species (TBARS) during an acid-heating reaction, as

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previously described (Draper and Hadley, 1990). The samples were mixed with 1 mL of trichloroacetic acid 10% and 1 mL of thiobarbituric acid 0.67%, and then heated in a boiling water bath for 30 min. Malondialdehyde (MDA) equivalents were determined in tissue and in sub-mitochondrial particles of the rat brain spectrophotometrically by the

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absorbance at 532 nm.

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2.5.2. Carbonyls protein formation

The oxidative damage to proteins was assessed by the determination of carbonyl

groups content based on the reaction with dinitrophenylhidrazine (DNPH), as previously described (Levine et al., 1990). Proteins were precipitated by the addition of 20% trichloroacetic acid and were re-dissolved in DNPH. The absorbance was monitored spectrophotometrically at 370 nm.

2.5.3. Superoxide dismutase activity

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This method for the assay of superoxide dismutase (SOD) activity is based on the capacity of pyrogallol to autoxidize, a process highly dependent on O2

−2

; a

substrate for SOD (Bannister and Calabrese, 1987). The inhibition of autoxidation of this compound thus occurs when SOD is present, and the enzymatic activity can be then indirectly assayed spectrophotometrically at 420 nm, using a double-beam

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spectrophotometer with temperature control. A calibration curve was performed using purified SOD as the standard, in order to calculate the specific activity of SOD present in the samples. A 50% inhibition of pyrogallol autoxidation is defined as 1 unit of SOD,

2.5.4. Catalase activity

catalase

(CAT)

activity

was

assayed

using

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The

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and the specific activity is represented as units per mg of protein.

a

double-beam

spectrophotometer with temperature control. This method is based on the disappearance of H2O2 at 240 nm in a reaction medium containing 20 mM H2O2, 0.1% Triton X-100, 10 mM potassium phosphate buffer, pH 7.0, and 0.1-0.3 mg protein/ml (Aebi, 1984). One CAT unit is defined as 1 mol of hydrogen peroxide consumed per minute, and the

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specific activity is reported as units per mg protein.

2.6 Western Blotting for expression MMP-2 and MMP-9

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For evaluation of the MMP-2 and MMP-9 expression the animals were separated in control and LPS group. The animals were killed at 6, 12 and 24 h after the injection of PBS or LPS. In brief, structures homogenized in 4 mL ice cold PBS and the pellets

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were re-suspended in 15% dextran T-500 and then added onto 20% dextran T-500, followed by centrifugation at 25,000 g for 10 min at 4°C. Gelatinase content was determined using a standardized Western Blot analyses. In brief, proteins (50 µg/lane) were separated using 10% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. Blots were washed with PBS-T (150mmNaCl, 10mm Na2HPO4, 1.5mmNaH2PO4, and 0.1% Tween-20, pH 7.5) and incubated overnight at 4°C with a MMP-2 antibody (Abcam - 86607) and MMP-9 antibody from (Abcam 137867), the integrated optical density of bands was quantitated using the Image J v.1.34 software and expressed as fold- values of the average optical density. For MMP-

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2 and MMP-9, the optical densities were expressed as the ratio of active/total enzyme (total equals the sum of the optical densities of the bands) (Planas et al., 2000).

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2.7 Blood-brain barrier and placental barrier permeability to Evan’s blue

To evaluate the BBB and PB integrities, control and/or LPS injected animals were killed at 6, 12, and 24 h (Smith and Hall, 1996). The animals were injected

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intravenously with 1 mL of 1 % Evan’s blue 1 h before being killed. The anesthesia consisted of an i.p. administration of ketamine (6.6 mg/kg), xylazine (0.3 mg/kg), and acepromazine (0.16 mg/kg) (Barichello et al., 2012). The hippocampus, cerebral cortex,

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placenta, and fetus brain were weighed and placed in a 50% trichloroacetic solution. The samples were homogenized and then centrifuged for 20 minutes at 10,000 rpm, the extracted dye was diluted with ethanol (1:3), and its fluorescence was determined (excitation at 620 nm and emission at 680 nm) with a luminescence spectrophotometer (Hitachi 650-40, Tokyo, Japan). Calculations were based on the external standard (62.5-

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500 ng/mL) in the same solvent. The Evan’s blue tissue content was quantified from a linear standard line derived from known amounts of the dye, and it was expressed per gram of tissue (Smith and Hall, 1996).

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2.8 Behavioral tests

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2.8.1 Spontaneous locomotor activity and habituation to open field test

The open field apparatus was used in spontaneous locomotor activity, positive

symptom of schizophrenia, and memory habituation to evaluate cognitive impairment induced by MIA and/or ketamine. The apparatus is a 40 × 60 cm open field surrounded by 50-cm high walls made of brown plywood with a frontal glass wall. The floor of the open field is divided by black lines into 9 rectangles. Animals were gently placed on the left rear quadrant and allowed to explore the arena for 5 min; the number of crossings (the number of times that animals crossed the black lines, assessing the locomotor activity) and rearings (the exploration behavior observed in rats when placed in a new environment) were measured. In the first exposition was measured the spontaneous

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locomotor activity (number of crossings and rearing by 5 min). Twenty-four h after the same animals were exposed to the apparatus to the memory habitation test by 5 min. The behavioral tests was performed by the same person (manual analyses) who was blind to the group treatment (Vianna et al., 2000).

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2.8.2 Novel object recognition

To evaluate the recognition to a novel object, a type of memory test, it was used the novel object recognition test. This test was also used to evaluate cognitive

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impairment induced by MIA and/or ketamine. The apparatus and procedures for the novel object recognition test have been described elsewhere (de Lima et al., 2005). Briefly, the test took place in a 40 x 50 cm open field surrounded by 50-cm high walls

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made of plywood with a frontal glass wall. The floor of the open field was divided by black lines into 12 equal rectangles. All animals were submitted to a habituation session where they were allowed to freely explore the open field for 5 min; no objects were placed in the box during the habituation trial. The number of times the black lines were crossed and the numbers of rearings performed in this session were evaluated as

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indicators of locomotor and exploratory activity, respectively. At different times following habituation, training was conducted by placing individual rats in the field for 5 min. Two identical objects (objects A1 and A2, both cubes) were positioned in two adjacent corners, 10 cm from the walls. In the long-term recognition memory test that

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was given 24 h after training, the rats explored the open field for 5 min in the presence of one familiar (A) and one novel (B, a pyramid with a square-shaped base) object. All of the objects had similar texture (smooth), color, and size (weight 150-200 g) but with

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distinctive shapes. A recognition index was calculated for each animal and reported as the ratio TB/ (TA + TB) (TA = time spent exploring the familiar object A; TB = time spent exploring the novel object B). Exploration was defined as sniffing (exploring the object 3-5 cm away from it) or touching the object with the nose or forepaws.

2.8.3 Pre-pulse inhibition

The quantification of inhibition promoted by a pre-pulse startle response induced by pulse was performed, based on a previous study performed (Levin et al., 2011). This behavioral test was used to investigate negative symptoms of schizophrenia. Boxes with

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seal sound were used to measure the startle (Insight - EP 175). At first, the animals remained for a period of 5 min of habituation in these boxes. After that, 10 pulses were applied to the habituation of the animals (this number was not considered for the calculations). During the session, 3 types of stimuli were presented 10 times and randomly distributed in intervals of 20 s: (1) pulses of 120 dB for 40 ms (able to

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produce a startle response), (2) pre-pulses of 65, 70 or 75 dB for 20 ms presented 80ms before the pulse, (3) absence of stimulus. The mean startle amplitude after the sessions pulse (P) as well as the mean amplitude of startle response sessions after the pre-pulse

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(PP) was calculated for each animal.

2.9 BDNF and NGF

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The BDNF and NGF levels were determined in frontal cortex and hippocampus. Brain structures were homogenized in phosphate buffer solution with a protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA), centrifuged at 3,000 g for 5 min and the supernatant was used for the determinations. The levels of these neurotrophins in brain tissues were determined using BDNF (R & DSystems, Inc., USA, DY248) and

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NGF (R & DSystems, Inc., DY556) commercial ELISA kits following the manufacturer's recommendations. The protein levels were determined using the Lowry method (1951), with bovine serum albumin used as a standard (Lowry et al., 1951).

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2.10 Statistical analysis

The data were analyzed for normality using the Shapiro-Wilk test and for

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homogeneity using the Levene’s test. The data were normal and homogeneity of variance was confirmed, parametric tests were used. For oxidative stress, MMP-2 and 9 expressions, BBB and PB integrities and cytokines were reported as the mean ± SEM, and the groups were compared using independent Student’s t-test. Data from the habituation to open field, locomotor activity, and pre-pulse inhibition were reported as two-way Analysis of variance (ANOVA) followed by Tukey post-hoc test and expressed as mean ± SEM. For the comparisons between the training and test sessions in the test of habituation open field habituation, was used the t-test for paired samples. Comparisons among groups for the object recognition test were performed using a Mann-Whitney U-test. The intra-group comparisons were performed using Wilcoxon’s

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tests. In all comparisons, p<0.05 indicated statistical significance. All analyses were performed using the Statistical Package for the Social Science (SPSS) software version

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20.0.

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ACCEPTED MANUSCRIPT 3. Results

3.1 Effects of MIA on cytokine levels in the fetus brains

In the table 1 are shown the cytokines levels in the brain of fetus. There was an

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increase of IL-1β (p = 0.032; p = 0.006), IL-2 (p = 0.012; p < 0.001), IL-5 (p = 0.015; p < 0.001), IFN-γ (p = 0.015; p = 0.025) and M-CSF (p = 0.019; p < 0.001) levels at 6 and 24 h in the LPS groups when compared with control groups. The IL-4 level was decreased at 12 h (p = 0.002) and increased at 24 h (p = 0.003) in the LPS group. The

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IL-6 level was increased at 12 (p = 0.011) and 24 h (p < 0.001) after LPS injection. There was an increase of IL-18 and MIP-3a levels at 6 (p = 0.006; p = 0.018, respectively) and 24 h (p < 0.001; p < 0.001, respectively) and a decrease at 12 h in the

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LPS group (p = 0.002; p = 0.018, respectively). In the TNF-α (p = 0.001), IL-7 (p = 0.001), IL-10 (p < 0.001), MIP-1a (p = 0.001), EPO (p = 0.030) levels there was an increase only at 24 h in the LPS groups when compared with control groups.

3.2 Effects of MIA on oxidative stress parameters in the amniotic fluid and fetus

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brains

The figure 2 shows oxidative stress parameters in amniotic fluid of pregnant Wistar rats submitted to LPS injection. The MDA levels were increased at 12 and 24 h

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after LPS injection when compared with control group (p = 0.039; p = 0.001, respectively; figure 2A). Protein carbonylation levels increased only at 12 h after LPS injection (p = 0.023; figure 2B). The SOD activity was increased at 6, 12, and 24 h in

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the LPS group when compared with control group (p < 0.001; p = 0.003; p = 0.007, respectively; figure 2C) and the CAT activity was increased only 12 h in the LPS group (p = 0.023; figure 2D).

In the fetal brain the MDA (figure 3A) and carbonylation protein (figure 3B)

levels were increased at 24 h in the LPS group (p = 0.004; p = 0.026, respectively). The SOD (figure 3C) and CAT (figure 3D) activity were increased at 12 h in the LPS group (p < 0.001; p = 0.040, respectively).

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3.3 Effects of MIA on MMP 2 and 9 expressions in the amniotic fluid and fetus brains

In the figure 4, the expression of MMP 2 was increased at 12 and 24 h after LPS

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injection in the amniotic fluid of pregnant Wistar rats (p = 0.037; p = 0.010, respectively; figure 4A). However, the MMP 9 expression was increased only at 6 h in the LPS group when compared with control group (p = 0.002; figure 4B).

In the fetus brain, the MMP 2 expression was increased at 12 h (p = 0.008;

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figure 5A) and the MMP 9 expression at 6 and 24 h (p = 0.012; p = 0.001, respectively; figure 5B) in the LPS groups.

integrity placenta and fetal brain

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3.4 Effects of MIA on BBB integrity in the hippocampus and cerebral cortex and PB

We investigated the BBB integrity in the hippocampus and cerebral cortex and PB integrity in the placenta and fetal brain. There were BBB breakdown in the

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hippocampus at 6 (p = 0.047), 12 (p = 0.018) and 24 h (p = 0.048) (figure 6A) and cerebral cortex at 6 (p = 0.040), 12 (p < 0.001) and 24 h (p = 0.0047) (figure 6B) of pregnant Wistar rats after LPS injection. In the PB there was breakdown in the placenta at 6 h in the LPS group when compared with control group (p = 0.010; figure 6C).

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While in the fetal brain there was breakdown at 6 and 24 h after infection LPS (p = 0.033; p = 0.038, respectively; figure 6D).

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3.5 Effects of MIA and ketamine on locomotor spontaneous activity

The effects of LPS and ketamine administration are illustrated in figure 7 on

locomotor activity. The administration of LPS during the prenatal period did not have any effects on locomotor activity (p = 0.152). However, the administration of the dose of ketamine 25 mg/kg increased the distance travelled by the rats that received a saline and LPS injection during the prenatal period (p < 0.001). The two-way ANOVA did not show differences for ketamine versus LPS interaction (p = 0.125).

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3.6 Effects of MIA and ketamine on memory habituation

The two-way ANOVA not showed interaction between LPS and ketamine in the

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session training crossing (p = 0.069; figure 8) and rearing (p = 0.985). However, in the test session there was interaction between LPS and ketamine in crossing (p < 0.0001) and rearing (p < 0.0001). In the test session, there was a significant reduction in both crossings and rearing in the control/saline and control/ketamine 5 mg/kg groups when

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compared with the training session, demonstrating habituation memory in these groups. However, the control groups that received dose of ketamine 15 and 25 mg/kg presented no difference in the crossing and rearing between training and test sessions. In the rats

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of LPS groups that received treatment with saline and ketamine in all doses there were no differences between training and test sessions, demonstrating habituation memory impairment in these groups.

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3.7 Effects of MIA and ketamine on novel object recognition test

In the test session of the novel object recognition test, there was a significant difference in exploring the novel object in the control/saline (p = 0.005) and control/ketamine 5 mg/kg compared with training session (p = 0.037), demonstrating

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recognition memory in these groups. Nevertheless, the control groups that received dose of ketamine 15 and 25 mg/kg presented no difference in the crossing and rearing between test and training sessions. In the rats of LPS groups that received treatment

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with saline and ketamine in all doses there were no differences between training and test sessions, demonstrating impairment in recognition memory in these groups (figure 9).

3.8 Effects of MIA and ketamine on pre-pulse test

In the pre-pulse test, we observed that the administration of LPS and ketamine revealed an effect on pre-pulse inhibition. There was an interaction between LPS and ketamine in 65 (p = 0.047) and 70 dB (p = 0.043). However, there was no interaction in 75 dB (p = 0.874). The LPS/ ketamine 25 mg/kg group showed a decreased the PPI test in the intensity of 65 dB. The control and LPS group that received ketamine 25 mg/kg

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and LPS group that received 15 mg/kg decreased the PPI in the intensity of 70 dB. In the control groups treated with ketamine 15 and 25 mg/kg there was a decreased the PPI compared with control/saline group in the intensity of 75 dB. In the LPS groups that received ketamine 15 and 25 mg/kg there was a decreased the PPI compared with

3.9 Effects of MIA and ketamine on BDNF and NGF levels

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control/saline and LPS/saline groups (figure 10).

In the hippocampus, there was effects for LPS (p = 0.002) and ketamine (p =

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0.002), as well as interaction (p = 0.002) in the BDNF levels. The doses of ketamine 15 and 25 mg/kg increased the BDNF levels in the LPS group when compared with control/saline, LPS/saline and ketamine dose control. In prefrontal cortex, there was not

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affected by LPS (p = 0.123) and ketamine (p = 0.077), but there was interaction for LPS versus ketamine (p < 0.001). In the LPS/ketamine 15 mg/kg and LPS/ketamine 25 mg/kg groups there was a decreased BDNF level as compared with LPS/saline and control/ketamine 15 and 25 mg/kg groups (figure 11A).

In the hippocampus, the two-way ANOVA showed effects for LPS (p = 0.001),

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but not for ketamine (p = 0.056). There was an interaction between LPS versus ketamine (p = 0.044). In the LPS/ketamine 25 mg/kg the NGF level was increased compared with control/saline and control/ketamine 25 mg/kg. In prefrontal cortex, there was no effects for LPS (p = 0.630) and ketamine (p = 0.573), and two-way ANOVA did

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not show interaction for ketamine versus LPS (p = 0.393) (figure 11B).

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This study investigated the immunological, molecular, and behavioral effects of MIA in the offspring of Wistar rats challenged with LPS on gestational day 15. The administration of LPS, a cell wall component of Gram-negative bacteria, is a well

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characterized and widely accepted bacterial infection model (Boksa, 2010; Gayle et al., 2004). LPS induces the activation of the innate immune response with production of cytokines that cross the BBB of the fetus with consequent effects on the CNS development, and has effects on neuronal survival, differentiation, apoptosis, and

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neurotransmitter expression (Boksa, 2010). In response to LPS, the synthesis and release of pro-inflammatory cytokines, mainly, IL-1β, IL-6 and TNF-α, are produced locally and act on fibroblasts and endothelial cells inducing their own synthesis, as well

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as IL-6 and other cytokines (Luheshi, 1998). In response to these inflammatory stimulus, cells can produce quantities of nitric oxide and superoxide anion, leading to lipid peroxidation, DNA single strand breaks, MMP activation, BBB injury, PB breakdown, and brain impairment (Klein et al., 2006; Sellner et al., 2010; Singh et al., 2012).

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Studies have reported that MMP2 and MMP9 are involved with brain injury (Shi et al., 2017), acute stroke (Kreisel et al., 2016), and ischemia (Zhang et al., 2017). Further, increased levels or expression of MMP2 and MMP9 are associated with extracellular matrix damage. MMP production could be induced by cytokines (Leppert

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et al., 2001). In addition, MMP2 and 9 could lead to dysfunctions in BBB and increase an inflammatory response from LPS, then inducing a vicious cycle (Roomi et al., 2017), as reported in the present study where was found an increase in MMP2 and 9 in the

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amniotic liquid and brain fetus time dependent. BBB and PB are the barriers that protect brain and embryo respectively, from the

migration of proteins and toxic substances from blood circulation (Mishra et al., 2009). At the same time these barriers allow the migration of essential nutrients that are useful for the development during organogenesis. BBB protective mechanism persists constitutively throughout the life where as the PB is activated after embryo implantation. These barriers also have the property to act like an insulator to protect the brain and placenta from infectious diseases. However, few virus and bacteria are known to breach these barriers (Greenwood, 1991). Previous studies demonstrate that after LPS maternal administration there is no changes in the mRNA expression of TNF-α, IL-1β

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and IL-6 in fetal brain, suggesting that cytokines are not produced by immature fetal organism. The introduction of LPS maternal substantially increases the levels of proinflammatory cytokines in maternal serum, amniotic fluid, and fetal brain, showing that the increase in fetal brain cytokines is derived exclusively from the passage of maternal serum for fetal brain (Gayle et al., 2004; Ning et al., 2008). In this study we

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observed both BBB and PB breakdown after LPS administration. Other study using LPS in mice demonstrated that the BBB was more vulnerable of LPS-induced disruption in some brain areas, and these effects were mediated by cyclooxygenase (Banks et al., 2015). Furthermore, pregnant rats injected by LPS had placental inflammation mediated

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by IL-1β 3 h after LPS induction (Girard et al., 2012).

In general, cytokines are expected to regulate the expression of other classes of immune molecules on neurons, including major histocompatibility complex I (MHCI)

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molecules. In the immune system, MHCI levels are controlled by cytokines, an important early step in the immune response. In the healthy brain, MHCI is found on neurons, where it negatively regulates synapse formation and the synaptic plasticity required for activity-dependent synaptic pruning (Estes and McAllister, 2015; Glynn et al., 2011). Alterations in synaptogenesis and pruning are associated with a range of

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neurodevelopmental disorders and are thought to play a central role in the etiology of autism and schizophrenia (Sekar et al., 2016; Tang et al., 2014). Similarly, if molecules act through similar pathways in the brain, then it is possible that immune signaling in neurons may converge upon mammalian target of rapamycin (mTOR) signaling. In the

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immune system, mTOR acts as a regulatory hub integrating inputs from numerous upstream intracellular signaling pathways, including cytokines, trophic factors, and

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synaptic scaffolding proteins, which are altered in the brains of MIA offspring, as well as in individuals with schizophrenia (Caccamo et al., 2014; Sahin and Sur, 2015). Usually the fetus is highly protected by placenta which has a barrier that does

not permit the migration of microorganism. However, in some cases, this barrier is breached (Singh et al., 2012). In this study, integrity of the BBB was evaluated by means of Evan's blue dye extravasation method, in the maternal hippocampus and cerebral cortex, as well as it was evaluated the PB in the placenta and fetus brain at 6, 12 and 24 h after the injection of LPS, suggesting that the breakage occurred in the four compartments of the BBB analyzed with greater magnitude extravasation in the placenta followed by the progressive decrease in maternal and fetal brain. Our data are consistent with other studies, which showed that LPS increased maternal levels of

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cytokines in these structures through the BBB breakdown and quantitative differences between the compartments of cytokines (Singh et al., 2012). This dye measureable difference and cytokines may indicate that the placenta serve as a filter between the maternal serum and fetal brain protecting the fetus (Oskvig et al., 2012). Changes in the cytokines levels in CNS cause long-term behavioral changes in adult rats following

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maternal infection during pregnancy. In fact, prenatal exposure to LPS can impair memory and learning (Boksa, 2010). The results of the present study demonstrated that ketamine (25 mg/kg) increased locomotor activity in the offspring of rats that were not exposed to LPS. Similar to these results, adult rats exposed to the pharmacological

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model induced by ketamine at the dose of 25 mg/kg for seven days had an increase in the distance traveled in the locomotor activity test (Canever et al., 2010; Ghedim et al., 2012). Injection of LPS in the prenatal period did not alter locomotor activity in adult

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life; however, administration of ketamine at a dose of 25 mg/kg for 7 days induced hyperlocomotion. Our results are in accordance with Zhu et al. (2014), where they showed that intra-hippocampal injection of LPS on the 7th postnatal day did not show difference in locomotor activity of adulthood rats (Zhu et al., 2014). Moreover, previous research from our group revealed that rats exposed to neonatal LPS injection and

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ketamine in adult life did not change spontaneous locomotor activity (Reus et al., 2017), these differences could be associated to differences in the LPS time administration. In our study through the open field test there was a significant reduction in crossings and rearing’s numbers in the control group and control/ketamine 5 mg/kg

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when compared to the training session, demonstrating a loss of habituation in these groups. However, in the LPS groups receiving saline or ketamine as a treatment there was no difference in motor and exploratory activity between training sessions and test,

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showing the occurrence of impaired habituation in these groups due to damage caused by LPS-induced MIA (Chlodzinska et al., 2011). Regarding recognition memory tests, control and control/ketamine 5 mg/kg groups showed difference between training sessions and test. In all LPS groups there was no difference between sessions, showing that animals spend the same time exploring new and familiar objects (Wang et al., 2009). The startle reflex PPI test is commonly used as a measure of sensorimotor gating and it is evaluated by measuring the ability of a weak pre-stimulus to reduce the startle response to a high acoustic stimulus (Braff et al., 2001). Ketamine leads to loss of PPI probably due to changes in structures of the limbic system, such as the amygdala and

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hippocampus (Neill et al., 2010). Similar to our findings, a study by Dickerson and Bilkey (2013) showed that animals exposed to MIA did not have difference in the PPI compared to the control group. Though there was a significant reduction in long-range synchronization in the group of MIA compared to the control group (Dickerson and Bilkey, 2013), suggesting that dB intensity could influences the results.

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It is known that neurotrophic factors are important signaling molecules during stages of neurodevelopment, including proliferation, differentiation and cerebral migration, as well as maintaining neuronal health and synaptic maintenance until adulthood (Rybakowski, 2008). Changes in neurotrophin levels, such as BDNF and

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NGF, could produce lasting effects on neurotrophic processes that influence neuronal maturation and plasticity in late life (Vicario-Abejon et al., 2002). Stressful factors during gestation, such as maternal infection, may cause changes in neurotrophin levels

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and predispose to the emergence of psychiatric disorders in adult offspring (Malaeb and Dammann, 2009). The results show that MIA during pregnancy influences the development of the offspring immune system, an effect that persists until adulthood. Specifically, the offspring of rats exposed to LPS and ketamine-induced model at the doses of 15 and 25 mg/kg had increased levels of BDNF and NGF in the hippocampus.

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In general, these findings suggest, at least in part, that MIA was able to alter neurotrophic factors in the late life of offspring after exposure, possibly interfering in neurogenic processes, since the expression of BDNF in the hippocampus is correlated with neurogenesis (Xia et al., 2014). Lazar et al. (2008) demonstrated that injections of

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NGF, into the brain of neonatal rats, resulted in a reduction in the social interaction, a negative symptom of schizophrenia (Lazar et al., 2008). In addition, the up-regulation of NGF is described in inflamed tissues (Minnone et al., 2017). Also, NGF receptors,

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including, tropomyosin receptor kinase A (TrkA) and p75 neurotrophin receptor (p75NTR) are regulated by immune cells (Minnone et al., 2017). Thus, we suggest that increased levels of NGF as reported in the present study could be due inflammation induced by MIA, and then could be, at least in part, responsible for schizophrenic-like behavior. It is well established in the literature that pre-natal exposure to infection is characterized as a risk factor for neurodevelopmental disorders. However, the actual mechanisms by which maternal infection acts in the developing brain to induce such disorders need to be elucidated (Dammann et al., 2002). Studies using experimental models of MIA, especially LPS and polyriboinosinic polyribocytidylic acid (Poly I:C)

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suggest that the cytokines produced by the mother act as likely mediators of abnormal brain development, leading to long-term behavioral changes as well as neurochemical changes in adult offspring (Monji et al., 2009). In addition, evidence indicates the presence of interactions between proinflammatory cytokines and neurotrophins in the CNS, since MIA alters both BDNF and NGF in the developing brain (Gilmore et al.,

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2003, 2005). In summary, such findings were also confirmed in the present study, where MIA altered brain levels of neurotrophins in adult offspring in the hippocampus. In the present study we did not evaluate neurotrophin and cytokines receptors or signaling cascade involved with its activation. This can be considered a limitation of this study

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since more specifically results could be explained evaluating these pathways. In addition, in the adult rats were not evaluated oxidative stress and inflammatory parameters. Futures studies are suggested to include these analyses to better investigate

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the effects of MIA associated with ketamine.

In conclusion, infections during pregnancy activate the innate immune system of the mother and alter the fetal environment, and could be a risk factor to the development of neuropsychiatric disorders, including schizophrenia and autism. In the present study, it was concluded that MIA increased the permeability of BBB and PB, the circulation of

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inflammatory mediators in fetal brains, such as cytokines and reactive oxygen species. This immune response in the prenatal period may be responsible for the behavioral changes in the adult life of offspring induced by MIA.

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Acknowledgments

The Translational Psychiatry Program is funded by the Department of Psychiatry

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and Behavioral Sciences, McGovern Medical School, The University of Texas Health Science Center at Houston (UTHealth). Research from Laboratório de Microbiologia Experimental and Laboratório de Neurociências (Brazil) is supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC), and Universidade do Extremo Sul Catarinense (UNESC).

Statement of Author Contributions All authors participated in the design and interpretation of the studies, analysis of the data and review of the manuscript; Sangiogo, Tashiro, Generoso, and Faller

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conducted the experiments; Scaini, Giridharan, Michels, Florentino, Petronilho, and Dal-Pizzol were responsible the biochemical analyzes; Dominguini, Mastella and Zugno, were responsible for the behavioral tests; and Simões, Réus and Barichello wrote the manuscript. Conflict of interest

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The authors declare that they have no conflict of interest.

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Singh, P., Agnihotri, S.K., Tewari, M.C., Kumar, S., Sachdev, M., Tripathi, R.K., 2012. HIV-1 Nef breaches placental barrier in rat model. PLoS One 7(12), 11. Stone, J.M., Dietrich, C., Edden, R., Mehta, M.A., De Simoni, S., Reed, L.J., Krystal, J.H., Nutt, D., Barker, G.J., 2012. Ketamine effects on brain GABA and glutamate

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levels with 1H-MRS: relationship to ketamine-induced psychopathology. Mol Psychiatry. 2012 Jul;17(7):664-5. doi: 10.1038/mp.2011.171. Epub 2012 Jan 3. Tang, G., Gudsnuk, K., Kuo, S.H., Cotrina, M.L., Rosoklija, G., Sosunov, A., Sonders, M.S., Kanter, E., Castagna, C., Yamamoto, A., Yue, Z., Arancio, O., Peterson, B.S.,

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Xia, Y., Qi, F., Zou, J., Yang, J., Yao, Z., 2014. Influenza vaccination during early pregnancy contributes to neurogenesis and behavioral function in offspring. Brain Behav Immun 42, 212-221. Zhang, H.T., Zhang, P., Gao, Y., Li, C.L., Wang, H.J., Chen, L.C., Feng, Y., Li, R.Y., Li, Y.L., Jiang, C.L., 2017. Early VEGF inhibition attenuates blood-brain barrier

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inhibition and microglial activation in rats: Implication for a new schizophrenia animal model. Brain Behav Immun 38, 166-174.

Zugno, A.I., de Miranda, I.M., Budni, J., Volpato, A.M., Luca, R.D., Deroza, P.F., de

Cipriano,

A.L.,

Quevedo,

J.,

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Oliveira, M.B., Heylmann, A.S., da Rosa Silveira, F., Wessler, P., Antunes Mastella, G., 2013.

Effect

of

maternal

deprivation

on

acetylcholinesterase activity and behavioral changes on the ketamine-induced animal

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model of schizophrenia. Neuroscience 248, 252-260.

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Figure 1. Schematic representation of timeline and experimental design. Experiment 1. Pregnant rats received an injection of PBS (1 mg/ml) or LPS (0.25 mg/kg) on the 15th day of gestation. Six, 12 and 24 hours after injection the animals were anesthetized and

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samples from fetus and pregnant rats were removed for the analyzes of cytokines, oxidative stress parameters, MMP 2 -9, and integrity of BBB and PB. Experiment 2. Pregnant rats received a PBS (1 mg/ml) or LPS (0.25 mg/kg) injection on the 15th day of gestation. After birth on the 21st day of life the offspring were weaned and the male rats

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were separated to perform the behavioral tests. On the 54th day of life, the animals were submitted to different doses of ketamine (5, 15 and 25 mg/kg) by 7 days, and the animals were subjected to the behavioral tests (habituation to the open field, locomotor

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activity, novel object recognition, and inhibition of pre-pulse).

Figure 2. Oxidative stress parameters in the amniotic fluid of pregnant Wistar rats on gestational day 15 after injection with PBS (1 mg/ml) or LPS (0.25 mg/kg). TBARS (A), protein carbonyl (B), SOD (C) and CAT (D). Data were reported as the mean ±

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S.E.M. (n = 6) and the groups were compared using independent Student’s t-test. *p < 0.05 indicates statistically significant differences as compared to the control group.

Figure 3. Oxidative stress parameters in the fetus brain of Wistar rats after injection

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with PBS (1 mg/ml) or LPS (0.25 mg/kg) on gestational day 15. TBARS (A), protein carbonyl (B), SOD (C) and CAT (D). Data were reported as the mean ± S.E.M. (n = 6) and the groups were compared using independent Student’s t-test. *p < 0.05 indicates

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statistically significant differences as compared to the control group.

Figure 4. Evaluation of MMP expression 2 (A) and 9 (B) in the amniotic fluid of pregnant Wistar rats after injection with PBS (1 mg/ml) or LPS (0.25 mg/kg) on gestational day 15. Data were reported as the mean ± S.E.M. (n = 6) and the groups were compared using independent Student’s t-test. Representative images of each protein MMP2 (A), MMP9 (B), and β-actin, respectively are shown in the upper panels. *p < 0.05 indicates statistically significant differences as compared to the control group.

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Figure 5. Evaluation of MMP expression 2 (A) and 9 (B) in the fetus brain of pregnant Wistar rats after injection with PBS (1 mg/ml) or LPS (0.25 mg/kg) on gestational day 15. Data were reported as the mean ± S.E.M. (n = 6) and the groups were compared using independent Student’s t-test. Representative images of each protein MMP2 (A), MMP9 (B), and β-actin, respectively are shown in the upper panels. *p < 0.05 indicates

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statistically significant differences as compared to the control group.

Figure 6. Blood-brain barrier integrity in the hippocampus (A) and cerebral cortex (B); also placental barrier integrity in the placenta (C) and fetus brain (D) of pregnant Wistar

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rats after injection with PBS (1 mg/ml) or LPS (0.25 mg/kg) on gestational day 15. Data were reported as the mean ± S.E.M. (n = 6) and the groups were compared using

compared to the control group.

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independent Student’s t-test. *p < 0.05 indicates statistically significant differences as

Figure 7. Spontaneous locomotor activity test in Wistar rats submitted to MIA in the prenatal period and exposed to the administration of ketamine in different doses 5, 15 and 25 mg/kg in adulthood. Data were expressed as mean ± S.E.M. and reported as two-

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way ANOVA followed by Tukey’s post-hoc tests (n = 12). *p < 0.05 indicate statistically significant when compared to the control group.

Figure 8. Habituation to open-field test in Wistar rats submitted to MIA in the prenatal

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period and exposed to the administration of ketamine in different doses 5, 15 and 25 mg/kg in adulthood. Data were expressed as mean ± S.E.M. and reported as two-way

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ANOVA followed by Tukey’s post-hoc tests. The intra-group comparisons were performed using t-test for paired samples (n = 12). *p < 0.05 indicate statistically significant when compared the training and test sessions.

Figure 9. Novel object recognition test in Wistar rats submitted to MIA in the prenatal period and exposed to the administration of ketamine in different doses 5, 15 and 25 mg/kg in adulthood. Comparisons among groups for the object recognition test were performed using a Mann-Whitney U-test. The intra-group comparisons were performed using Wilcoxon’s tests (n = 12). *p < 0.05 indicate statistically significant when compared the training and test sessions.

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Figure 10. Pre-pulse inhibition test in Wistar rats submitted to MIA in the prenatal period and exposed to the administration of ketamine in different doses 5, 15 and 25 mg/kg in adulthood. Data were expressed as mean ± S.E.M. and reported as two-way ANOVA followed by Tukey’s post-hoc tests (n = 12) *p < 0.05 indicate statistically significant when compared to control/sal group,

#

p < 0.05 indicate statistically

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significant when compared to the LPS/sal group.

Figure 11. Effects of MIA in BDNF and NGF levels in the hippocampus and prefrontal cortex of offspring with 60 days of life after administration of ketamine 5, 15 and 25

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mg/kg. BDNF (A), and NGF (B) were expressed as mean ± S.E.M. and reported as twoway ANOVA followed by Tukey’s post-hoc tests (n = 6) *p < 0.05 indicate statistically significant when compared to the control/saline group, #p <0.05 indicate statistically

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significant when compared to the LPS/saline group and &p < 0.05 indicate statistically

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significant when compared to ketamine dose control.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

Figure 5.

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Optical density MMP9 expression/ -actin

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Evans blue (ng/mg tissue)

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* 30000

Control/ket 25 mg/kg LPS/saline

*

LPS/ket 5 mg/kg 20000

LPS/ket 15 mg/kg

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Covered distance (cm)

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LPS/ket 25 mg/kg

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Figure 8.

80

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Number of crossing and rearing

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5 mg/Kg

15 mg/Kg 25 mg/Kg

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Figure 9.

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5 mg/Kg LPS

Crossing - Training Crossing - Test Rearing - Training Rearing - Test

15 mg/Kg 25 mg/Kg

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Figure 10. 80 Control/saline

60

Control/ket 15 mg/kg

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* *

* *

Control/ket 25 mg/kg LPS/saline

*

20

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Mean percent PPI

Control/ket 5 mg/kg

*/# */#

*

LPS/ket 15 mg/kg

0

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Figure 11. A

* /#/&

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LPS/ket 25 mg/kg

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Prefrontal cortex

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B

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Control/saline Control/ket 5 mg/kg

* /&

Control/ket 15 mg/kg

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Control/ket 25 mg/kg LPS/saline

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LPS/ket 5 mg/kg 100

LPS/ket 15 mg/kg LPS/ket 25 mg/kg

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Table 1. Cytokine levels in the fetus brain of Wistar rats after injection with LPS (0.025 mg/kg) or PBS (1 mg/ml) on gestational day 15.

IL-6

IL-7

IL-10

IL-18

EPO

INF-γ

M-CSF

0.035 ± (0.003)

0.030 ± (0.004)

0.035 ± (0.002)

LPS

0.045 ± (0.003)a

0.037 ± (0.002)

0.061 ± (0.007)a

Control

0.039 ± (0.005)

0.034 ± (0.002)

0.038 ± (0.002)

a

0.035 ± (0.003)

0.110 ± (0.013)a

LPS

0.058 ± (0.004)

Control

0.016 ± (0.004)

0.010 ± (0.0004)

0.009 ± (0.0003)

LPS

0.013 ± (0.001)

0.008 ± (0.0002)

a

0.022 ± (0.003)a

Control

0.076 ± (0.006)

0.059 ± (0.005)

0.051 ± (0.003)

LPS

0.098 ± (0.006)

a

0.052 ± (0.003)

0.201 ± (0.016)a

Control

0.065 ± (0.006)

0.057 ± (0.003)

LPS

0.079 ± (0.005)

0.073 ± (0.004)

a

Control

0.016 ± (0.002)

0.016 ± (0.002)

LPS

0.021 ± (0.002)

Control

0.162 ± (0.002)

LPS

0.171 ± (0.007)

Control

0.188 ± (0.009)

0.016 ± (0.001)

0.016 ± (0.002)

0.048 ± (0.006)a

0.114 ± (0.008)

0.116 ± (0.004)

0.119 ± (0.006)

0.251 ± (0.018)a

0.130 ± (0.012)

0.076 ± (0.007)

a

0.628 ± (0.081)a

0.267 ± (0.034)

0.237 ± (0.012)

Control

0.492 ± (0.035)

LPS

0.518 ± (0.031)

0.319 ± (0.011)

0.424 ± (0.035)a

Control

0.018 ± (0.003)

0.026 ± (0.004)

0.012 ± (0.002)

0.020 ± (0.004)

0.041 ± (0.009)

a

0.079 ± (0.006)

a

LPS

0.033 ± (0.005)

Control

0.050 ± (0.002)

0.061 ± (0.004)

0.062 ± (0.004)

a

0.070 ± (0.004)

0.448 ± (0.035)a

0.041 ± (0.004)

0.046 ± (0.004)

0.052 ± (0.006)

0.052 ± (0.004)

0.044 ± (0.005)

0.250 ± 0.040)a

0.025 ± (0.003)

0.025 ± (0.003)

0.019 ± (0.002)

LPS

0.034 ± (0.002)

a

0.018 ± (0,001)

a

0.113 ± (0.013)

a

Control

0.038 ± (0.003)

0.037 ± (0.005)

0.043 ± (0.004)

LPS

0.047 ± (0.003)

0.042 ± (0.005)

0.060 ± (0.002)a

Control

Control

0.060 ± (0.003)

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TNF-α

0.184 ± (0.015)a

0.248 ± (0.017)

LPS MIP-3a

0.068 ± (0.004)

LPS

LPS MIP-1a

a

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6 hours

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Cytokines

es statistically significant differences vs. control group.

a

p < 0 . 0 5 i n d i c a t

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The authors declare that they have no conflict of interest.