Accepted Manuscript Prenatal maternal immune activation increases anxiety- and depressive-like behaviors in offspring with experimental autoimmune encephalomyelitis Jafar Majidi-Zolbanin, Mohammad-Hossein Doosti, Morteza Kosari-Nasab, Ali-Akbar Salari PII: DOI: Reference:
S0306-4522(15)00238-9 http://dx.doi.org/10.1016/j.neuroscience.2015.03.016 NSC 16123
To appear in:
Neuroscience
Accepted Date:
7 March 2015
Please cite this article as: J. Majidi-Zolbanin, M-H. Doosti, M. Kosari-Nasab, A-A. Salari, Prenatal maternal immune activation increases anxiety- and depressive-like behaviors in offspring with experimental autoimmune encephalomyelitis, Neuroscience (2015), doi: http://dx.doi.org/10.1016/j.neuroscience.2015.03.016
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.
Prenatal maternal immune activation increases anxiety- and depressive-like behaviors in offspring with experimental autoimmune encephalomyelitis
Jafar Majidi-Zolbanin a, Mohammad-Hossein Doosti a, Morteza Kosari-Nasab b, Ali-Akbar Salari b,c,*
a Immunology
b
c
Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Laboratory of Neuropsychopharmacology and Psychoneuroimmunology, Hayyan Research Institute,
University of Tabriz, Tabriz, Iran
*Corresponding author at: Laboratory of Neuropsychopharmacology and Psychoneuroimmunology, Hayyan Research Institute, University of Tabriz, P.O. Box. 51666-16471, Tabriz, Iran; Tel.: +98-9194099673; Fax: +98-411-3368208; E-mail: [email protected]
Running title: Maternal immune activation and Multiple Sclerosis Abstract: 242 words Body: 5663 words Figures: 6
1
Abstract
Multiple sclerosis (MS) is thought to result from a combination of genetics and environmental factors. Several lines of evidence indicate that significant prevalence of anxiety and depressionrelated disorders in MS patients can influence the progression of the disease. Although we and others have already reported the consequences of prenatal maternal immune activation on anxiety and depression, less is known about the interplay between maternal inflammation, MS and gender. We here investigated the effects of maternal immune activation with Poly I:C during mid-gestation on the progression of clinical symptoms of experimental autoimmune encephalomyelitis (EAE; a mouse model of MS), and then anxiety- and depressive-like behaviors in non-EAE and EAE-induced offspring were evaluated. Stress-induced corticosterone and tumor necrosis factor-alpha (TNF-α) levels in EAE-induced offspring were also measured. Maternal immune activation increased anxiety and depression in male offspring, but not in females. This immune challenge also resulted in an earlier onset of the EAE clinical signs in male offspring and enhanced the severity of the disease in both male and female offspring. Interestingly, the severity of the disease was associated with increased anxiety/depressive-like behaviors and elevated corticosterone or TNF-α levels in both sexes. Overall, these data suggest that maternal immune activation with Poly I:C during midpregnancy increases anxiety- and depressive-like behaviors, and the clinical symptoms of EAE in a sex-dependent manner in non-EAE or EAE-induced offspring. Finally, the progression of EAE in offspring seems to be linked to maternal immune activation-induced dysregulation in neuroimmune-endocrine system.
Key words: Maternal inflammation, Sex differences, Anxiety, Depression, Poly I:C, EAE.
2
1. Introduction
Multiple sclerosis (MS) is known as one of the most common neuroinflammatory diseases in humans that is more prevalent in women (Tomassini and Pozzilli, 2009). In recent years, there has been an increasing interest in investigating the prevalence of affective disorders in MS patients. Although, previous studies have indicated a significant prevalence of anxiety and depression among MS patients, as compared with the general population (Beiske et al., 2008, Brown et al., 2009, Dahl et al., 2009, Arnason, 2010, Feinstein, 2011, Giordano et al., 2011, Wood et al., 2012). However, it has been reported that anxiety is more common than depression in patients with MS (Noy et al., 1995, Feinstein et al., 1999, Wood et al., 2012), and also depression in MS is evident even early on, and absent disability (Arnason, 2010). A number of studies have also suggested that there is a significant association between affective disorders such as depression and anxiety and suicidal intent or completed suicide in patients with MS in comparison with the general population (Stenager and Stenager, 1992, Korostil and Feinstein, 2007, Wood et al., 2012). Although, hypothalamic-pituitary-adrenal (HPA) axis dysregulation is associated with anxiety and depression in non-MS population (Young et al., 2004, Stetler and Miller, 2011) and MS patients (Wallin et al., 2006, Wood et al., 2012), clinical studies have provided evidence that HPA axis hyperactivity in patients with MS may contribute to the progression of disease (Bergh et al., 1999, Heesen et al., 2002, Schumann et al., 2002, Gold et al., 2005) and subtle HPA axis alterations during the earlier phases of MS may be a marker for depressive symptomatology (Gold et al., 2010). It is also important to note that there is a considerable relationship between proinflammatory cytokines and anxiety/depression (Raison et al., 2006, Peruga et al., 2011, Haji et al., 2012, Kaster et al., 2012). Therefore, the importance of evaluating such neuropsychiatric diseases as two of the most
3
important determining factors of the general health and the quality of life in MS patients is unquestionable. Over the past few decades, “fetal programming” has been one of the major interesting research subjects for neuroscientists to investigate the fetal origins of many neurological disorders including schizophrenia, autism, cerebral palsy, epilepsy, Alzheimer, Parkinson, anxiety, depression, and multiple sclerosis (Bale et al., 2010, Del Giudice, 2012, Harvey and Boksa, 2012, Solati et al., 2012, Faa et al., 2014, Zager et al., 2014). In addition, there is now compelling evidence that maternal adverse experiences including stress and infection during pregnancy affect normal development of the brain and behavior in offspring in adulthood. For instance, we and others have demonstrated that prenatal maternal infection/immune activation with Lipopolysaccharide (LPS, a component of gram negative bacteria) or Polyinosinic: Polycytidylic acid (Poly I:C, a synthetic double stranded RNA) enhances the risk for a number of neuropsychiatric-like behaviors such as anxiety, depression, schizophrenia and autism in adult mice offspring (for review see (Harvey and Boksa, 2012, Babri et al., 2014a)). Interestingly, in light of this evidence, some studies indicated that prenatal maternal immune activation results in earlier onset of the clinical symptoms and more severe neurological deficits in experimental autoimmune encephalomyelitis (EAE), an animal model which mimics many aspects of MS (Solati et al., 2012, Mandal et al., 2013, Zager et al., 2014). Considering that, on the one hand, one of the most significant current discussions in this research area is that whether or not interaction between two environmental risk factors during prenatal and postnatal periods is associated with greater vulnerability to neuropsychiatric disorders in offspring in later life. And on the other hand, although there have been strong connections between depression/anxiety and multiple sclerosis in clinical studies, to date there are no studies that directly investigated possible links between the effects of prenatal maternal immune activation and
4
the prevalence of anxiety- and depressive-like behaviors in EAE-induced mice offspring. Therefore, the aim of this study was to examine and validate the presence of these behavioral disorders following prenatal maternal immune activation in mice offspring with EAE, which may provide insights into the fetal potential origins involved in the disease in order to increase our understanding of the effects of environmental risk factors on anxiety and depression in people with multiple sclerosis. 2. Materials and methods 2.1. Subjects, General housing conditions and Ethics Male and female C57BL/6 mice (70–80 days) were obtained from the animal house of Pasteur Institute (Tehran, Iran). Animals were housed in standard polycarbonate cages in a temperaturecontrolled room (23 ± 1°C) with a fixed 12 h light–dark cycle (08:00–20:00) and free access to food and water. These conditions were kept as a standard housing condition in all stages of experiments. All procedures of the study were performed in accordance with the ethical guidelines set by Research and Ethics Committee of Tabriz University of Medical Sciences (GN-90.2-2.5) which completely coincides with the ‘‘National Institutes of Health NIH Guide for the Care and Use of Laboratory Animals (NIH; Publication No. 85-23, revised 1985). 2.2. Breeding The breeding procedure and the verification of gestational day (GD) 0 have previously been described elsewhere (Enayati et al., 2012). Briefly, the breeding began after a 2-week period of acclimatization to the new animal holding room. In order to facilitate of mating, male and female mice were kept together one-by-one in a cage. Successful mating was confirmed next morning (8:00 A.M.) with the presence of vaginal plug, and that day was referred as GD 0. Once a pregnant
5
female was identified, it was removed from the breeding cage and housed individually in a standard cage. 2.3. Prenatal maternal immune activation Mice were given intraperitoneal injections of 20 mg/kg/100µl Poly I:C (Sigma Co, USA; dissolved in sterile PBS) on GD 12, because previous studies found this dose to be most effective with intraperitoneal delivery for inducing a robust immune response in C57BL/6 pregnant mice and behavioral abnormalities in their offspring in adulthood (Shi et al., 2003, Smith et al., 2007, Hsiao and Patterson, 2011, Garay et al., 2013, Khan et al., 2014). Pregnant mothers were returned to their housing immediately following PBS or Poly I:C treatment. In order to perform the behavioral tests, in each sex, mice from both prenatal treatment conditions were divided into 3 clusters (each cluster only used for two behavioral tests or the EAE clinical signs , with a 2-days interval between each test; N=10 or 15/group male or female, see Fig.1). All pregnant animals were allowed to have normal delivery and the first day of birth was considered as PND 0 (MajidiZolbanin et al., 2014). One day after the birth, all litters were culled to 4 pups per mother (2 males and 2 females). On PND 23, litters were weaned by removal of the mother and then were housed with the same sex litter-mates (2 animals per cage). Only one offspring from each litter was randomly assigned for each of the experiments to avoid litter-effects. 2.4. Induction of experimental autoimmune encephalitis For EAE-induction in C57BL/6 offspring on PND 80, animals were anaesthetized by an i.p. injection of ketamine hydrochloride (50 mg/kg; Alfasan, Woerden-Holland) plus Xylazine (5 mg/kg; Alfasan, Woerden-Holland). 300 µg of Myelin oligodendrocyte glycoprotein peptide (MOG35–55; Amino acid sequence-Purity ≥95% (HPLC): Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-
6
Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys; KJ Ross-Petersen ApS, Copenhagen, Denmark) was emulsified with an equal volume of complete Freund's adjuvant (CFA; Sigma, F5881, USA) containing 500 µg of heat-killed Mycobacterium tuberculosis (100 µl total volume), then this emulsion was injected subcutaneously in both hind flanks of each mouse. Mice also received intraperitoneal injections of pertussis toxin (300 ng in 100 µl PBS; List Biological Lab, Campbell, CA, USA) at the time of immunization and again 48 h later. Each injection was performed with a 1ml insulin syringe needle. The EAE clinical symptoms in the offspring were evaluated daily on a scale from 0 to 7 according to the following criteria: 0 = no symptoms; 1 = distal limp tail; 2 =complete limp tail; 3=one hind limb paralyzed; 4 = both hind limbs paralyzed; 5=Hind limbs and one forelimb paralyzed; 6 =Hind limbs and both forelimbs paralyzed; 7 =moribund/death. 2.5. Behavioral testing We assessed anxiety- and depression-related behaviors at 7 dpi and 9 dpi by means of the elevated plus maze (EPM), light-dark box (LDB), tail suspension test (TST) and forced swim test (FST) based on the onset of clinical signs in EAE-induced offspring (Fig. 2) and previous studies have also measured anxiety-like behavior in EAE-induced mice during 7-9 days post immunization (dpi) before the onset of motor deficits (Haji et al., 2012). Behavioral assessments began at PND 87. All behavioral parameters were recorded by observers blind to the treatment and the observers have recorded all parameters for each of the behavioral tests by using a stopwatch. In order to avoid any confounding effect of motor deficits on the offspring’s behavior in the tests, the locomotor activity was assessed in an open field for 3 min before each behavioral testing session as previously described (de Paiva et al., 2010). In addition, all behavioral tests were conducted in a quiet room during the light period (between 14:00 and 18:00 h) under illumination of 75 lux and the mice were kept in the room for at least 1 h before the assessment. At the end of each test
7
session, the arena was carefully cleaned with 70 % ethanol and after test the cage was transported back to the colony room. In all experiments, each male or female offspring was tested only once in the one test. 2.5.1. Elevated plus-maze The EPM test, which is one of the well-known methods for testing of anxiety, was performed as previously described (Amani et al., 2013). The EPM was a plus-shaped apparatus, constructed from grey wood, elevated to a height of 50 cm above the floor. This apparatus was consisted of a central platform (5×5cm), two open arms (30×5cm), and two equal closed (30×5×15cm) arms opposite to each other with an open roof. The offspring were placed individually at the center of the EPM, facing one of the open arms and allowed 5 min of free exploration. A relatively dark box was used to hold the mice in before placing on the maze in order to increase their exploratory behavior. The observers measured: (a) time spent in the open arms, (b) time spent in the closed arms, (c) number of entries into the open arms, and (d) number of entries into the closed arms during the 5-min test period. An entry was defined as all four paws on the arm. For the purpose of analysis, percent of open-arm time [OAT %: time in open arm/ time in open + closed arm × 100], and percent of open arm entries [OAE %: number of open arm entries/number of open arm + closed arm entries × 100] were defined and employed as indexes of anxiety-like behaviors in rodents. 2.5.2. Light-dark box The light–dark box apparatus consisted of a white-black wooden rectangular box (length 46 cm, width 27 cm, and height 30 cm), which divided into two compartments (light and large: 27cm×27cm, dark and small: 18cm×27cm) by a partition. These areas connected by a small central
8
open door (7.5cm×7.5 cm) located in the center of the partition at floor level. The large compartment was open at the top, illuminated by a 100 W bulb located 90cm above the apparatus and the small compartment had a removable black lid at the top. To start the test, each offspring was placed at the center of the light compartment, facing away from the door and the animal was allowed to explore freely both compartments for 5 min and their behavior was recorded during this time. The following parameters were recorded: light compartment time (LCT), light compartment entries (LCE) and latency to enter the light compartment (LL) after the first entry into the dark division. A decrease in the amount of time spent and numbers of entries into the light compartment were evaluated as indicative of anxiety-like behaviors (Majidi-Zolbanin et al., 2013). 2.5.3. Tail suspension test The TST was performed as previously described (Doosti et al., 2013). At the beginning of the experiment, each mouse was individually suspended by the tail using a clamp, 2 cm from the end, in a grey wooden enclosure (40 cm high, 30 cm wide and 20 cm deep) such that the head of mouse was about 25 cm above the floor. The total duration of immobility was recorded (in seconds) during the 5 min test period. Any animals that did climb their tails were removed from the experimental group, and were not used in the analysis. Immobility was defined as the lack of motion of the whole body, whereas mobility was defined as any movement of the body. 2.5.4. Forced swim test The FST remains one of the most widely used tools for measuring behavioral despair in rodents. To describe this behavioral model in mice, the following procedure was adopted, mice were individually placed into the transparent glass cylinders (Height: 25cm, Diameter: 10cm), filled with water to a height of 15 cm and maintained at 25±1ᵒC. The water was replaced by fresh water
9
between each test. The total duration of immobility was recorded during the last 4 min of the 6 min testing period. At the end of swimming session, the animals were removed from the cylinder, dried with towels, and placed gently near an electric heater for 15–30 min. Each mouse was judged to be immobile when it ceased struggling and remained floating motionless in the water and making only those movements necessary to keep its head above water. An increase in the duration of immobility time is known as an indicative of depressive-like behavior in mice (Babri et al., 2014b). 2.6. Corticosterone and tumor necrosis factor-alpha Stress-induced corticosterone levels or TNF-α concentration in EAE-induced offspring were measured on PND 89, 25 min after the LDB (TNF-α) or FST (corticosterone), respectively. The blood was collected using cardiac puncture method as previously described (Enayati et al., 2012) and circulating levels of TNF-α (BioLegend Co., USA) and corticosterone (Bio-Medical Assay Company, China) in the offspring serum were measured by using cytokine specific quantitative sandwich ELISA kit according to the manufacturer’s instructions. All samples and standards were assayed in duplicate. 2.7. Statistics All data were analyzed based on the analysis of variance (ANOVA) using the statistical package of SPSS (IBM, Version 21). The clinical scores were analyzed by three-way repeated measures ANOVA with treatment and sex as between subject factors and dpi as a within-subject (repeated measure) factor (Fig. 2). The anxiety and depression data in Fig. 3 to 5 were analyzed by three-way ANOVA with sex, EAE induction and treatment as main factors. The corticosterone and TNF-α data in Fig. 6 were analyzed using the two-way ANOVA with sex and treatment as main factors. All data
10
are presented as the mean ± standard error of the mean (S.E.M). A P-value less than 0.05 was considered statistically significant. 3. Results 3.1. Effects of prenatal maternal immune activation on EAE clinical symptoms in offspring Mean clinical scores are shown from day 8 to day 26 post-immunization for female and male offspring in Fig. 2. Three-way ANOVA analysis revealed the main effects of DPI [F
10.475, 586.594
=
379.879, P
S0306-4522(15)00238-9 http://dx.doi.org/10.1016/j.neuroscience.2015.03.016 NSC 16123
To appear in:
Neuroscience
Accepted Date:
7 March 2015
Please cite this article as: J. Majidi-Zolbanin, M-H. Doosti, M. Kosari-Nasab, A-A. Salari, Prenatal maternal immune activation increases anxiety- and depressive-like behaviors in offspring with experimental autoimmune encephalomyelitis, Neuroscience (2015), doi: http://dx.doi.org/10.1016/j.neuroscience.2015.03.016
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.
Prenatal maternal immune activation increases anxiety- and depressive-like behaviors in offspring with experimental autoimmune encephalomyelitis
Jafar Majidi-Zolbanin a, Mohammad-Hossein Doosti a, Morteza Kosari-Nasab b, Ali-Akbar Salari b,c,*
a Immunology
b
c
Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Laboratory of Neuropsychopharmacology and Psychoneuroimmunology, Hayyan Research Institute,
University of Tabriz, Tabriz, Iran
*Corresponding author at: Laboratory of Neuropsychopharmacology and Psychoneuroimmunology, Hayyan Research Institute, University of Tabriz, P.O. Box. 51666-16471, Tabriz, Iran; Tel.: +98-9194099673; Fax: +98-411-3368208; E-mail: [email protected]
Running title: Maternal immune activation and Multiple Sclerosis Abstract: 242 words Body: 5663 words Figures: 6
1
Abstract
Multiple sclerosis (MS) is thought to result from a combination of genetics and environmental factors. Several lines of evidence indicate that significant prevalence of anxiety and depressionrelated disorders in MS patients can influence the progression of the disease. Although we and others have already reported the consequences of prenatal maternal immune activation on anxiety and depression, less is known about the interplay between maternal inflammation, MS and gender. We here investigated the effects of maternal immune activation with Poly I:C during mid-gestation on the progression of clinical symptoms of experimental autoimmune encephalomyelitis (EAE; a mouse model of MS), and then anxiety- and depressive-like behaviors in non-EAE and EAE-induced offspring were evaluated. Stress-induced corticosterone and tumor necrosis factor-alpha (TNF-α) levels in EAE-induced offspring were also measured. Maternal immune activation increased anxiety and depression in male offspring, but not in females. This immune challenge also resulted in an earlier onset of the EAE clinical signs in male offspring and enhanced the severity of the disease in both male and female offspring. Interestingly, the severity of the disease was associated with increased anxiety/depressive-like behaviors and elevated corticosterone or TNF-α levels in both sexes. Overall, these data suggest that maternal immune activation with Poly I:C during midpregnancy increases anxiety- and depressive-like behaviors, and the clinical symptoms of EAE in a sex-dependent manner in non-EAE or EAE-induced offspring. Finally, the progression of EAE in offspring seems to be linked to maternal immune activation-induced dysregulation in neuroimmune-endocrine system.
Key words: Maternal inflammation, Sex differences, Anxiety, Depression, Poly I:C, EAE.
2
1. Introduction
Multiple sclerosis (MS) is known as one of the most common neuroinflammatory diseases in humans that is more prevalent in women (Tomassini and Pozzilli, 2009). In recent years, there has been an increasing interest in investigating the prevalence of affective disorders in MS patients. Although, previous studies have indicated a significant prevalence of anxiety and depression among MS patients, as compared with the general population (Beiske et al., 2008, Brown et al., 2009, Dahl et al., 2009, Arnason, 2010, Feinstein, 2011, Giordano et al., 2011, Wood et al., 2012). However, it has been reported that anxiety is more common than depression in patients with MS (Noy et al., 1995, Feinstein et al., 1999, Wood et al., 2012), and also depression in MS is evident even early on, and absent disability (Arnason, 2010). A number of studies have also suggested that there is a significant association between affective disorders such as depression and anxiety and suicidal intent or completed suicide in patients with MS in comparison with the general population (Stenager and Stenager, 1992, Korostil and Feinstein, 2007, Wood et al., 2012). Although, hypothalamic-pituitary-adrenal (HPA) axis dysregulation is associated with anxiety and depression in non-MS population (Young et al., 2004, Stetler and Miller, 2011) and MS patients (Wallin et al., 2006, Wood et al., 2012), clinical studies have provided evidence that HPA axis hyperactivity in patients with MS may contribute to the progression of disease (Bergh et al., 1999, Heesen et al., 2002, Schumann et al., 2002, Gold et al., 2005) and subtle HPA axis alterations during the earlier phases of MS may be a marker for depressive symptomatology (Gold et al., 2010). It is also important to note that there is a considerable relationship between proinflammatory cytokines and anxiety/depression (Raison et al., 2006, Peruga et al., 2011, Haji et al., 2012, Kaster et al., 2012). Therefore, the importance of evaluating such neuropsychiatric diseases as two of the most
3
important determining factors of the general health and the quality of life in MS patients is unquestionable. Over the past few decades, “fetal programming” has been one of the major interesting research subjects for neuroscientists to investigate the fetal origins of many neurological disorders including schizophrenia, autism, cerebral palsy, epilepsy, Alzheimer, Parkinson, anxiety, depression, and multiple sclerosis (Bale et al., 2010, Del Giudice, 2012, Harvey and Boksa, 2012, Solati et al., 2012, Faa et al., 2014, Zager et al., 2014). In addition, there is now compelling evidence that maternal adverse experiences including stress and infection during pregnancy affect normal development of the brain and behavior in offspring in adulthood. For instance, we and others have demonstrated that prenatal maternal infection/immune activation with Lipopolysaccharide (LPS, a component of gram negative bacteria) or Polyinosinic: Polycytidylic acid (Poly I:C, a synthetic double stranded RNA) enhances the risk for a number of neuropsychiatric-like behaviors such as anxiety, depression, schizophrenia and autism in adult mice offspring (for review see (Harvey and Boksa, 2012, Babri et al., 2014a)). Interestingly, in light of this evidence, some studies indicated that prenatal maternal immune activation results in earlier onset of the clinical symptoms and more severe neurological deficits in experimental autoimmune encephalomyelitis (EAE), an animal model which mimics many aspects of MS (Solati et al., 2012, Mandal et al., 2013, Zager et al., 2014). Considering that, on the one hand, one of the most significant current discussions in this research area is that whether or not interaction between two environmental risk factors during prenatal and postnatal periods is associated with greater vulnerability to neuropsychiatric disorders in offspring in later life. And on the other hand, although there have been strong connections between depression/anxiety and multiple sclerosis in clinical studies, to date there are no studies that directly investigated possible links between the effects of prenatal maternal immune activation and
4
the prevalence of anxiety- and depressive-like behaviors in EAE-induced mice offspring. Therefore, the aim of this study was to examine and validate the presence of these behavioral disorders following prenatal maternal immune activation in mice offspring with EAE, which may provide insights into the fetal potential origins involved in the disease in order to increase our understanding of the effects of environmental risk factors on anxiety and depression in people with multiple sclerosis. 2. Materials and methods 2.1. Subjects, General housing conditions and Ethics Male and female C57BL/6 mice (70–80 days) were obtained from the animal house of Pasteur Institute (Tehran, Iran). Animals were housed in standard polycarbonate cages in a temperaturecontrolled room (23 ± 1°C) with a fixed 12 h light–dark cycle (08:00–20:00) and free access to food and water. These conditions were kept as a standard housing condition in all stages of experiments. All procedures of the study were performed in accordance with the ethical guidelines set by Research and Ethics Committee of Tabriz University of Medical Sciences (GN-90.2-2.5) which completely coincides with the ‘‘National Institutes of Health NIH Guide for the Care and Use of Laboratory Animals (NIH; Publication No. 85-23, revised 1985). 2.2. Breeding The breeding procedure and the verification of gestational day (GD) 0 have previously been described elsewhere (Enayati et al., 2012). Briefly, the breeding began after a 2-week period of acclimatization to the new animal holding room. In order to facilitate of mating, male and female mice were kept together one-by-one in a cage. Successful mating was confirmed next morning (8:00 A.M.) with the presence of vaginal plug, and that day was referred as GD 0. Once a pregnant
5
female was identified, it was removed from the breeding cage and housed individually in a standard cage. 2.3. Prenatal maternal immune activation Mice were given intraperitoneal injections of 20 mg/kg/100µl Poly I:C (Sigma Co, USA; dissolved in sterile PBS) on GD 12, because previous studies found this dose to be most effective with intraperitoneal delivery for inducing a robust immune response in C57BL/6 pregnant mice and behavioral abnormalities in their offspring in adulthood (Shi et al., 2003, Smith et al., 2007, Hsiao and Patterson, 2011, Garay et al., 2013, Khan et al., 2014). Pregnant mothers were returned to their housing immediately following PBS or Poly I:C treatment. In order to perform the behavioral tests, in each sex, mice from both prenatal treatment conditions were divided into 3 clusters (each cluster only used for two behavioral tests or the EAE clinical signs , with a 2-days interval between each test; N=10 or 15/group male or female, see Fig.1). All pregnant animals were allowed to have normal delivery and the first day of birth was considered as PND 0 (MajidiZolbanin et al., 2014). One day after the birth, all litters were culled to 4 pups per mother (2 males and 2 females). On PND 23, litters were weaned by removal of the mother and then were housed with the same sex litter-mates (2 animals per cage). Only one offspring from each litter was randomly assigned for each of the experiments to avoid litter-effects. 2.4. Induction of experimental autoimmune encephalitis For EAE-induction in C57BL/6 offspring on PND 80, animals were anaesthetized by an i.p. injection of ketamine hydrochloride (50 mg/kg; Alfasan, Woerden-Holland) plus Xylazine (5 mg/kg; Alfasan, Woerden-Holland). 300 µg of Myelin oligodendrocyte glycoprotein peptide (MOG35–55; Amino acid sequence-Purity ≥95% (HPLC): Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-
6
Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys; KJ Ross-Petersen ApS, Copenhagen, Denmark) was emulsified with an equal volume of complete Freund's adjuvant (CFA; Sigma, F5881, USA) containing 500 µg of heat-killed Mycobacterium tuberculosis (100 µl total volume), then this emulsion was injected subcutaneously in both hind flanks of each mouse. Mice also received intraperitoneal injections of pertussis toxin (300 ng in 100 µl PBS; List Biological Lab, Campbell, CA, USA) at the time of immunization and again 48 h later. Each injection was performed with a 1ml insulin syringe needle. The EAE clinical symptoms in the offspring were evaluated daily on a scale from 0 to 7 according to the following criteria: 0 = no symptoms; 1 = distal limp tail; 2 =complete limp tail; 3=one hind limb paralyzed; 4 = both hind limbs paralyzed; 5=Hind limbs and one forelimb paralyzed; 6 =Hind limbs and both forelimbs paralyzed; 7 =moribund/death. 2.5. Behavioral testing We assessed anxiety- and depression-related behaviors at 7 dpi and 9 dpi by means of the elevated plus maze (EPM), light-dark box (LDB), tail suspension test (TST) and forced swim test (FST) based on the onset of clinical signs in EAE-induced offspring (Fig. 2) and previous studies have also measured anxiety-like behavior in EAE-induced mice during 7-9 days post immunization (dpi) before the onset of motor deficits (Haji et al., 2012). Behavioral assessments began at PND 87. All behavioral parameters were recorded by observers blind to the treatment and the observers have recorded all parameters for each of the behavioral tests by using a stopwatch. In order to avoid any confounding effect of motor deficits on the offspring’s behavior in the tests, the locomotor activity was assessed in an open field for 3 min before each behavioral testing session as previously described (de Paiva et al., 2010). In addition, all behavioral tests were conducted in a quiet room during the light period (between 14:00 and 18:00 h) under illumination of 75 lux and the mice were kept in the room for at least 1 h before the assessment. At the end of each test
7
session, the arena was carefully cleaned with 70 % ethanol and after test the cage was transported back to the colony room. In all experiments, each male or female offspring was tested only once in the one test. 2.5.1. Elevated plus-maze The EPM test, which is one of the well-known methods for testing of anxiety, was performed as previously described (Amani et al., 2013). The EPM was a plus-shaped apparatus, constructed from grey wood, elevated to a height of 50 cm above the floor. This apparatus was consisted of a central platform (5×5cm), two open arms (30×5cm), and two equal closed (30×5×15cm) arms opposite to each other with an open roof. The offspring were placed individually at the center of the EPM, facing one of the open arms and allowed 5 min of free exploration. A relatively dark box was used to hold the mice in before placing on the maze in order to increase their exploratory behavior. The observers measured: (a) time spent in the open arms, (b) time spent in the closed arms, (c) number of entries into the open arms, and (d) number of entries into the closed arms during the 5-min test period. An entry was defined as all four paws on the arm. For the purpose of analysis, percent of open-arm time [OAT %: time in open arm/ time in open + closed arm × 100], and percent of open arm entries [OAE %: number of open arm entries/number of open arm + closed arm entries × 100] were defined and employed as indexes of anxiety-like behaviors in rodents. 2.5.2. Light-dark box The light–dark box apparatus consisted of a white-black wooden rectangular box (length 46 cm, width 27 cm, and height 30 cm), which divided into two compartments (light and large: 27cm×27cm, dark and small: 18cm×27cm) by a partition. These areas connected by a small central
8
open door (7.5cm×7.5 cm) located in the center of the partition at floor level. The large compartment was open at the top, illuminated by a 100 W bulb located 90cm above the apparatus and the small compartment had a removable black lid at the top. To start the test, each offspring was placed at the center of the light compartment, facing away from the door and the animal was allowed to explore freely both compartments for 5 min and their behavior was recorded during this time. The following parameters were recorded: light compartment time (LCT), light compartment entries (LCE) and latency to enter the light compartment (LL) after the first entry into the dark division. A decrease in the amount of time spent and numbers of entries into the light compartment were evaluated as indicative of anxiety-like behaviors (Majidi-Zolbanin et al., 2013). 2.5.3. Tail suspension test The TST was performed as previously described (Doosti et al., 2013). At the beginning of the experiment, each mouse was individually suspended by the tail using a clamp, 2 cm from the end, in a grey wooden enclosure (40 cm high, 30 cm wide and 20 cm deep) such that the head of mouse was about 25 cm above the floor. The total duration of immobility was recorded (in seconds) during the 5 min test period. Any animals that did climb their tails were removed from the experimental group, and were not used in the analysis. Immobility was defined as the lack of motion of the whole body, whereas mobility was defined as any movement of the body. 2.5.4. Forced swim test The FST remains one of the most widely used tools for measuring behavioral despair in rodents. To describe this behavioral model in mice, the following procedure was adopted, mice were individually placed into the transparent glass cylinders (Height: 25cm, Diameter: 10cm), filled with water to a height of 15 cm and maintained at 25±1ᵒC. The water was replaced by fresh water
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between each test. The total duration of immobility was recorded during the last 4 min of the 6 min testing period. At the end of swimming session, the animals were removed from the cylinder, dried with towels, and placed gently near an electric heater for 15–30 min. Each mouse was judged to be immobile when it ceased struggling and remained floating motionless in the water and making only those movements necessary to keep its head above water. An increase in the duration of immobility time is known as an indicative of depressive-like behavior in mice (Babri et al., 2014b). 2.6. Corticosterone and tumor necrosis factor-alpha Stress-induced corticosterone levels or TNF-α concentration in EAE-induced offspring were measured on PND 89, 25 min after the LDB (TNF-α) or FST (corticosterone), respectively. The blood was collected using cardiac puncture method as previously described (Enayati et al., 2012) and circulating levels of TNF-α (BioLegend Co., USA) and corticosterone (Bio-Medical Assay Company, China) in the offspring serum were measured by using cytokine specific quantitative sandwich ELISA kit according to the manufacturer’s instructions. All samples and standards were assayed in duplicate. 2.7. Statistics All data were analyzed based on the analysis of variance (ANOVA) using the statistical package of SPSS (IBM, Version 21). The clinical scores were analyzed by three-way repeated measures ANOVA with treatment and sex as between subject factors and dpi as a within-subject (repeated measure) factor (Fig. 2). The anxiety and depression data in Fig. 3 to 5 were analyzed by three-way ANOVA with sex, EAE induction and treatment as main factors. The corticosterone and TNF-α data in Fig. 6 were analyzed using the two-way ANOVA with sex and treatment as main factors. All data
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are presented as the mean ± standard error of the mean (S.E.M). A P-value less than 0.05 was considered statistically significant. 3. Results 3.1. Effects of prenatal maternal immune activation on EAE clinical symptoms in offspring Mean clinical scores are shown from day 8 to day 26 post-immunization for female and male offspring in Fig. 2. Three-way ANOVA analysis revealed the main effects of DPI [F
10.475, 586.594
=
379.879, P
