Department of Biology

Department of Biology

Prenatal Exposure to LPS Leadsto Long-Lasting PhysiologicalConsequences in MaleOffspring

Zaharia, & Meaney, 1998; Bachmanov, Reed, Beau-champ, & Tordoff, 2002). LPS and some cytokines canactivate the hypothalamic–pituitary–adrenal (HPA) axiswhich leads to increase in plasma concentrations ofadrenocorticotropic hormone (ACTH), glucocorticoidsand also corticotrophin releasing factor (CRF) (Banet al., 1993; Barker et al., 1993; Becskei et al., 2008;Bell & Hallenbeck, 2002), causing changes in the brainneurochemistry (Betancur, Lledo, Borrell, & Guaza,1994; Chen, Zhou, Beltran, Malellari, & Chang, 2005).This immune and neuroendocrine activation couldinfluence the effective state including anxiety-relatedbehavior, mood, and cognition that have clinical impor-tance (Degroot, Kashluba, & Treit, 2001; Delrue, Dele-planque, Rougepont, Vitiello, & Neveu, 1994). Animaland human studies showed that there is an increaseof above-mentioned cytokines both in systemic andmRNA levels, especially in the brain of rodents follow-ing peripheral exposure to LPS (Dunn, 1988, 1989;Dunn & Berridge, 1990).

Developmental Psychobiology

Masoud Asiaei1

Jalal Solati1Ali-Akbar Salari2

1

Faculty of Science, Karaj BranchIslamic Azad University

P.O. Box 31485-313, Karaj, IranE-mail: solati@kiau.ac.ir

2

Islamic Azad UniversityNorth Tehran Branch, Tehran, Iran

ABSTRACT: Growing evidence suggests that early life events are criticaldeterminants for disorders later in life. According to a comprehensive number ofepidemiological/animal studies, exposure to lipopolysaccharide, causes alterationin pro-inflammatory cytokine levels, hypothalamic–pituitary–adrenal functioningand the hormonal system which may contribute to behavioral and neurologicalinjuries. In this study we investigated the effects of lipopolysaccharide adminis-tration on physiological parameters in pregnant dams and their male offspringaged 9 weeks. In gestational Day 10, pregnant mice were injected intraprito-neally with Salmonella enterica lipopolysaccharide to model prenatal exposureto infection. The following results were obtained for offspring from damsstressed during pregnancy: (a) reduced anxiety-related behavior in the elevatedplus maze; (b) reduced food and water intake; (c) reduced body weight frombirth up to postnatal Day 40. The observed data provide experimental evidenceshowing that prenatal stress can have complex and long-lasting physiological/behavioral consequences in offspring. ß 2011 Wiley Periodicals, Inc. DevPsychobiol 53: 828–838, 2011.

Keywords: lipopolysaccharide; prenatal stress; pro-inflammatory cytokine; corti-costerone; body weight; food intake; water intake; C57BL/6 mice

INTRODUCTION

Lipopolysaccharide (LPS), an endotoxin produced fromthe cell walls of gram-negative bacteria, acts as a non-specific immunostimulant to induce a severe inflamma-tory response by initiating multiple intracellularsignaling events, including the activation of nuclearfactor kb (NF-kb), which ultimately leads to the syn-thesis and release of cytokines, such as pro-inflamma-tory cytokine tumor necrosis factor alpha (TNF-a),interleukin 1b (IL-1b) and interleukin 6 (IL-6) frommacrophages (Anisman, Hayley, Turrin, & Merali,2002; Anisman, Kokkinidis, & Merali, 2002; Anisman,

Received 8 December 2010; Accepted 25 April 2011Correspondence to: J. SolatiPublished online 31 May 2011 in Wiley Online Library

(wileyonlinelibrary.com). DOI 10.1002/dev.20568

ß 2011 Wiley Periodicals, Inc.

On the other hand, a growing body of scientific liter-ature of animal and epidemiological studies suggeststhat besides genetic factors, environmental factors, likematernal stress can have long-lasting effects onphysical development, neurochemistry, behavior andimmunocompetence of the offspring, hence thisphenomenon has been denoted as ‘‘fetal programming’’(Barker et al., 1993; File, 1996, 2001). According toseveral scientific literatures, these maternal stresses arevery important elements in the provocation or exacer-bation of a wide range of physiological/behavioral andpsychological/mental disturbances (Fride & Weinstock,1988; Golan, Lev, Hallak, Sorokin, & Huleihel, 2005;Harbuz & Lightman, 1992; Hava, Vered, Yael, Morde-chai, & Mahoud, 2006). Maternal stress can lead toelevated levels of maternal stress hormones, notably, itis well established that HPA hormones play a criticalrole in the stress response (Johnson, Kamilaris, Chrou-sos, & Gold, 1992). In rodents and non-human primatespecies, it was found that prenatal stress, includingexposure to endotoxin, can alter HPA axis and brainneurotransmitter systems in the offspring (Kapoor,Dunn, Kostaki, Andrews, & Matthews, 2006; Karrow,2006), and these effects are mediated by cytokineinduction within the maternal circulation and placenta(Kirsten, Taricano, Flo´rio, Palermo-Neto, & Bernardi,2010; Klein & Rager, 1995; Kofman, 2002). Thesephysiological and neurochemical/hormonal changes caninfluence some other behaviors like eating and drink-ing. Food and water intakes correlated positively, andthis may be due to their mutual dependence on bodysize, but an additional mechanism directly linking foodand water intakes may also be involved (Bachmanovet al., 2002; Kraly, 1984).It should be noted that the nature and the persistence

of prenatal stress effects probably depend on the timeof application of maternal stress relative to the fetalstage of development (Merlot, Couret, & Otten, 2008).There is emerging evidence suggesting that inflamma-tory events associated with immunological events inearly/middle fetal life (e.g., GD 8-10 in rats and mice)are likely to have more stronger neurodevelopmentalimpacts than late-pregnancy inflammations. Thesematernal immune activations during early/middle preg-nancy impede with cell proliferation, differentiation,migration, target selection, and synapse maturation,finally leading to several brain and behavioral abnor-malities in adulthood (Kirsten et al., 2010). The presentstudy proceeded to investigate some of the neuroendo-crine and behavioral consequences of prenatal exposureto LPS on both pregnant C57BL/6 mice in gestationalday (GD) 10 and their male offspring aged 9 weeks.Therefore, we assessed the effect of prenatally adminis-tered LPS on the concentration of corticosterone and

pro-inflammatory cytokines, anxiety-related behavior inthe elevated plus maze (EPM), food and water intakeof both pregnant dams and their male offspring atadulthood and also the body weight of offspring frombirth to postnatal day (PND) 40.

METHODS

Animals

Male and female C57BL/6 mice (Pasteur Institute of Iran)aged 6–8 weeks upon arrival and were left to acclimatize tothe laboratory for 10 days prior to testing. Mice were main-tained in groups of 5 in standard polypropylene cages, toavoid behavioral changes that may result from single housingwhich can usually be an increase in aggressive and fear-likebehavior. The animals were allowed free access to food andwater at all times and were maintained on a 12 hr light/darkschedule (lights on 07:00 hr) in a controlled temperature

(23 Æ 18C). For mating purposes, three females were housedovernight with two males starting at 19:00 hr. Each femalemouse was visually inspected for the presence of a vaginalplug the next morning at 07:00 hr. The presence of plug was

designed as day 0 of gestation. The pregnant mice (N ¼ 80)were divided randomly into four groups. Forty of these mice

(N ¼ 10 in each group) were scarified for measuring cyto-kines and corticosterone and others were left to labor

(N ¼ 10 in each group). Following delivery, litters remainedintact to avoid confounding changes in maternal behavior.The litters remained with their mothers until weaning (Day21) and were separated according to sex on Day 36. The maleoffspring maintained in groups of 3–4 in the above-mentionedconditions. The male offspring were distributed into control

and experimental groups (N ¼ 10/group; two pups wereselected from each mother for the next experiments). Thestudy was approved by the Ethics Committee of Karaj IslamicAzad University and experimental protocol is in compliancewith the National Institutes of Health Guide for Care andUse of Laboratory Animals (Publication No. 85-23, revised1985).

Prenatal Administration of LPS

Lipopolysaccharide (Salmonella enterica serotype entritidis,L6011, Sigma Aldrich, Saint Louis, MO) was dissolved in ster-ile pyrogen-free saline before use. The dams were randomlyassigned to a saline control group and LPS groups. The damsin the LPS groups were administered a single intraperitoneal(i.p.) injection of 50, 100, or 150 mg/kg LPS on day 10 ofpregnancy. The dams in the control group were administered asingle i.p. injection of saline on day 10 of pregnancy. It isimportant to note that although maternal exposure to LPScauses some disturbances, it can vary depending on doses ofLPS, potency of LPS, age, sex, and interactions with environ-mental and genetic risk factors (Bell & Hallenbeck, 2002;Urakubo, Jarskog, Lieberman, & Gilmore, 2001). The dosageof LPS we chose could induce systemic inflammation, resulting

Developmental Psychobiology Prenatal LPS Exposure and Mice Behaviors 829

in a low percentage of fetal anomalies, but not abortion andpossible intra-uterine fetal death (IUFD) (Bell & Hallenbeck,2002). The rationale for choosing gestational day 10 was thatthis period corresponds to the first-to-second trimesters ofhuman pregnancy, with respect to developmental biology andpercentage of gestation from mice to human (Kaufman, 1992).Other scientific literatures state that this time phase is theperiod of early fetal brain development (Wei, Li, & Zhou,2007), cerebral organogenesis in mice, especially neural-plateformation (Kirsten et al., 2010) and also embryonic stem cellformation (gestational period 0.38–0.53 in rodents) which isone of the main periods of vulnerability of the immune systemto environmental insults (Merlot et al., 2008). In addition, otherinvestigators suggest that maternal infection from early to midpregnancy is more likely to be related to long-lasting develop-mental brain and behavioral abnormalities in the offspring(Mednick, Machon, Huttunen, & Bonett, 1988; Rodier &Hyman, 1998).

Each dam was administered 50 ml saline or LPS solution.

Following injection with LPS or saline, the dams continued tobe housed in the above-mentioned conditions. As we know,LPS can disrupt the blood–brain barrier (BBB) if adminis-tered topically to the cerebral microcirculation or intracister-nally, and it is possible that high doses of LPS cross theplacenta into the fetal circulation (Urakubo et al., 2001).Low doses of LPS were selected in order to prevent possibledirect exposure of fetus to LPS. With this dose and route ofapplication we observed no abortion in our endotoxin groups.All injections were given between 13:00 and 14:00 hr.

Measurement of Serum Corticosteroneand Cytokine Concentration

Maternal serum from trunk blood of pregnant dams was pre-

pared 1.5 hr after injection of LPS/saline (N ¼ 10 in eachgroup) by centrifugation at 5,000g for 12 min, aliquoted and

then stored at À808C until the cytokine/corticosterone assayswere performed. Concentrations of corticosterone (KT-510,Kamiya Biomedical Company, Seattle, WA), IL-1b (IB49700,Immuno-Biological Laboratories, Minneapolis, MN), IL-6(BioSource International, CA), and TNF-a (CT 302, Ucytech,Utrecht, The Netherlands) were determined by using commer-cial ELISA kits according to the manufacturer’s protocol.Trunk blood was collected for preparation of blood serum ofmale offspring in their 9th week (PND 62) and measurementof serum corticosterone and cytokine concentration wascarried out again in the same way. All samples and standardswere assayed in duplicate. The pregnant mice and their maleoffspring were selected randomly.

Anxiety Test

One of the most popular tests of anxiety-like behavior inmice and rats is the EPM, in which the reduced number ofentries or time spent in the open arms of the EPM suggeststhe operation of anxiety-like processes. This wooden, plus-shaped apparatus was elevated to the height of 50 cm above

the floor and consists of two open arms (30 cm  5 cm),two enclosed arms (30 cm  5 cm  15 cm), and central

platform (5 cm  5 cm) each with an open roof. The mazewas placed in the center of a quiet and dimly lit room. Themice behavior was directly observed using a mirror, sus-pended at an angle above the maze. Behavioral data werecollected by a ‘‘blind’’ observer who quietly sat 1 m behindone of the closed arms of the maze, using a chronometer. Theanxiety test of the offspring was carried out at postnatal Day61. It was repeated three times, three separate cohorts of maleoffspring were used for tests and each mouse was only usedon EPM once. We repeated this test three times due to ourdifferent results on EPM from other investigations and theresults represent an average of the three test sessions. For test-ing purpose, male offspring were placed individually in thecenter portion of the plus-maze, facing one of the open arms.The observer measured: (1) the time spent in the open arms,(2) the time spent in the closed arms, (3) the number ofentries into the open arms, and (4) the number of entries intothe closed arms during the 5-min test period. An entry wasdefined as all four paws in the arm. The elevated plus-mazewas thoroughly cleaned with distilled water following thetesting of each animal to avoid possible biasing effects due toodor clues left by previous mice. For the purpose of analysis,open-arm activity was quantified as the amount of time thatthe mice spent in the open arms (OAT) relative to the total

amount of time spent in any arm (open/total  100), and thenumber of entries into the open arms (OAE) was quantifiedrelative to the total number of entries into any arm (open/

total  100). The total number of open arms entered, as wellas the total number of closed arms entered were used asindexes of general locomotor activity (LMA) (Solati, Zarrindast,& Salari, 2010; Zarrindast, Solati, Oryan, & Parivar, 2008).All testing was conducted between 13:00 and 16:00 hr.

Body Weight

The body weights of male offspring (Æ0.1 g) were regularlymonitored at 13:00 hr every 10 days during the experimentfrom birth (PND 0) to PND 40.

Food and Water Intake

In order to find out whether i.p. exposure to LPS during preg-nancy affects food/water intake in dams or in their male off-spring under our experimental conditions, a feeding/drinkingstudy was conducted. Mice had free access to food and waterbefore the experiments began. Each cage contained 3 or 4mice which were given with the same amount of food andwater. Their food intake was measured the following day bysubtracting the uneaten food manually. The amount of wateringested in our experiment was measured with 0.2 ml gradu-ated glass burettes adapted with a metal drinking spout.Immediately after injection of LPS/saline in pregnant dams,each mouse was returned to its cage and we measured thecumulative water intake manually the following day. Thesemeasurements were done in 3 days after injection of the LPS/

saline in respect to pregnant dams (N ¼ 10 in each group)and in 5 days in PND 56-60 in male offspring (N ¼ 10 ineach group) and it was calculated as food (in grams Æ0.1 g)/water (in ml Æ0.2 ml) intake per mice per day. Measurements

830 Asiaei, Solati and Salari Developmental Psychobiology

p < 0.01, and ÃÃÃp < 0.001, when compared to the saline

challenge, TNF-a (F3,36 ¼ 161.825, p < 0.001), IL-1b(F3,36 ¼ 39.468, p < 0.000) and IL-6 (F3,36 ¼ 9.968,p < 0.001) in maternal serum were increased 1.5 hrafter LPS administration (except for IL-6 in 50 mg/kgLPS; Fig. 1). Table 1 shows that there were no signifi-cant differences between prenatally LPS and saline-treated offspring in serum concentrations of TNF-a(F3,36 ¼ 1.971, p < 0.145), IL-1b (F3,36 ¼ 1.403,p < 0.266) and IL-6 (F3,36 ¼ 1.496, p < 0.241) intheir 9th week (PND 62).

Effects of LPS on Serum Concentrationsof Corticosterone in Pregnant Mice andTheir Male Offspring

The effects of LPS treatment on serum corticosteronelevel of pregnant dams and their male offspring areshown in Figure 1 and Table 1. One way ANOVAconfirmed that in pregnant dams, high doses of LPS(100 or 150 mg/kg) increased serum corticosteronelevels relative to saline-treated animals (F3,36 ¼ 8.937,p < 0.002) (Fig. 1). But prenatal LPS exposure doesnot have any significant effect on serum concentrationsof corticosterone in male offspring in their 9th week(PND 62) (F3,36 ¼ 3.442, p < 0.052) (Tab. 1).

Effects of LPS on Anxiety-Related Behaviors inMale Offspring

Figure 2 shows data on the effects of prenatal exposureto LPS on anxiety-related behavior of male offspring inthe EPM. One-way ANOVA revealed that prenatalexposure to LPS has increased the percentage of open

FIGURE 1 Effect of intraperitoneal injection of saline(50 ml/mouse) or LPS (50, 100, or 150 mg/kg) on serum levelof TNF-a, IL-1b, IL-6 (a) and corticosterone (b) in pregnant

dams. Each bar is mean Æ SEM. N ¼ 10. Ãp < 0.05,ÃÃ

treated group.

arm time (F3,36 ¼ 49.396, p < 0.000) and open armentries (F3,36 ¼ 8.843, p < 0.000). No differenceswere found in the total number of arm entries in theplus-maze (open-arms þ closed-arms) (F3,36 ¼ 1.604,p < 0.215). Considering all of these effects, the pres-ently observed behavioral data suggest low levels ofanxiety in prenatally LPS-treated offspring which wasnot reported like this before.

Effects of Prenatal Exposure to LPS onBody Weight in Male Offspring

During postnatal development, all the pups gainedbody weight continuously, but in different ways amongour experimental groups. The birth weight (PND 0) ofprenatally LPS-treated offspring (LPS 50: 1.31 Æ 0.33 g;LPS 100: 1.20 Æ 0.33 g; LPS 150: 1.20 Æ 0.32 g) waslower than prenatally saline-treated offspring (1.62 Æ0.31 g) (F3,36 ¼ 36.282, p < 0.000). Furthermore, thetotal body weight of prenatally LPS-treated offspringwas lower than the prenatally saline-treated offspringduring 40 days of age [PND 10 (F3,36 ¼ 26.399,

Developmental Psychobiology

were performed by the same experimenter and we used separ-ate cohorts of pregnant dams for food/water intake, but thesame groups of male offspring were used for food/waterintake and anxiety test on EPM.

Statistical Analysis

Data were analyzed using SPSS. Since data displayed normaldistribution and homogeneity of variance, one-way ANOVAwas used for comparison between the effects of different

doses of LPS with vehicle. Differences with p < 0.05between experimental groups at each point were consideredstatistically significant.

RESULTS

Effects of LPS on Serum Concentrations ofTNF-a, IL-1b, and IL-6 in Pregnant Miceand Their Male Offspring

The effects of LPS treatment on TNF-a, IL-1b, and IL-6in maternal serum were analyzed. In response to LPS

Prenatal LPS Exposure and Mice Behaviors 831

activity (LMA). N ¼ 10. p < 0.05, p < 0.01, andp < 0.001 when compared to the saline treated group. p < 0.001 when compared to the saline treated group.

p < 0.000), PND 20 (F3,36 ¼ 25.849, p < 0.000), PND30 (F3,36 ¼ 8.174, p < 0.001), PND 40 (F3,36 ¼ 5.366,p < 0.006)] (Fig. 3). It is important to note that weobserved no miscarriage or fetal loss among our groupsduring experiment.

Effects of LPS on Food and Water Intake inPregnant Mice and Their Male Offspring

As can be clearly seen in Figure 4, i.p. administrationof LPS reduced food intake in the first day after injec-tion in pregnant dams (F3,36 ¼ 13.465, p < 0.000).During the 2nd (F3,36 ¼ 2.375, p < 0.095) and 3rd(F3,36 ¼ 1.685, p < 0.197) day after challenge, therewere no significant differences between LPS andsaline-treated dams in food intake. In other words,feeding then returned to normal over the next 48–72 hras the LPS effect dissipated. It is also important to notethat there were significant decreases in food intake

between prenatally LPS or saline-treated male offspringat postnatal Day 56–60 [PND 56 (F3,36 ¼ 37.029,p < 0.000), PND 57 (F3,36 ¼ 20.638, p < 0.000), PND 58(F3,36 ¼ 30.582, p < 0.000), PND 59 (F3,36 ¼ 45.155,p < 0.000), PND 60 (F3,36 ¼ 36.505, p < 0.000)](Fig. 4).The intraperitoneal injection of small amounts of

endotoxin taken from Salmonella enterica significantlydecreased and almost completely interrupted the waterintake of pregnant dams. One day after LPS exposurethere was a significant decrease in water intake amongall dams (F3,36 ¼ 47.734, p < 0.000). In other words,in the experiment presented in Figure 5, 50 mg/kg ofendotoxin was sufficient to alter water intake. Therewere no significant differences in the water intake ofdams which received the smallest dose of endotoxin,compared to the control group, but the inhibition per-sisted longer as the dose was increased (F3,36 ¼20.714, p < 0.000). With 100 or 150 mg/kg of endo-toxin, the daily water intake in pregnant dams did not

Table 1. Effect of Prenatal Exposure to LPS on Serum Level of Pro-Inflammatory Cytokines and Corticosterone in Male

Offspring in PND 62 (Mean Æ SEM, N ¼ 10 in Each Group)

Control (Saline) LPS 50 mg/kg LPS 100 mg/kg LPS 150 mg/kg

TNF-a (pg/ml)IL-1b (pg/ml)IL-6 (pg/ml)

26.00 Æ 1.41484.25 Æ 3.705285.5 Æ 3.662

88.50 Æ 3.379

118.75 Æ 2.750297.00 Æ 5.552

114.25 Æ 4.211123.75 Æ 4.871317.00 Æ 6.819

130.50 Æ 4.113130.50 Æ 3.617320.50 Æ 5.781

return to normal until the end of the 3rd day after injec-tion (F3,36 ¼ 4.975, p < 0.008), (see Fig. 5). The water

FIGURE 2 Effects of prenatal exposure to LPS on anxiety-related behavior in offspring in the elevated plus-maze. Male

offspring prenatally exposed to saline (50 ml/mouse) or LPS(50, 100, or 150 mg/kg) and anxiety test of these offspring

carried out at postnatal Day 61. Each bar is mean Æ SEM.Open arm time (OAT), open arm entries (OAE) or locomotor

à ÃÃ

ÃÃÃ

FIGURE 3 Effect of prenatal exposure to saline (50 ml/mouse) or LPS (50, 100, or 150 mg/kg) on body weight ofmale offspring from birth day to PND 40. The endotoxinoffspring had a lower body weight from birth day up to

PND 40. Each bar is mean Æ SEM. N ¼ 10. ÃÃp < 0.01 andÃÃÃ

832 Asiaei, Solati and Salari Developmental Psychobiology

intake in prenatally LSP-treated male offspring waslower compared to prenatally-saline treated offspringin PND 56–60 [PND 56 (F3,36 ¼ 78.004, p < 0.000),PND 57 (F3,36 ¼ 109.789, p < 0.000), PND 58(F3,36 ¼ 137.651, p < 0.000), PND 59 (F3,36 ¼ 161.790,

p < 0.000), PND 60 (F3,36 ¼ 6.552, p < 0.002)](Fig. 5). Different studies showed that the inhibition ofwater intake was essentially the same, despite the strainof Gram-negative species from which the endotoxinwas prepared.

FIGURE 4 Effect of intraperitoneal injection or prenatal exposure to saline (50 ml/mouse)or LPS (50, 100, or 150 mg/kg) on food intake in pregnant dams and their male offspring.Endotoxin dams ate less than saline animals just in first day after LPS injection, while endo-

toxin offspring ate less than saline offspring during experiment. Each bar is mean Æ SEM.N ¼ 10. ÃÃÃp < 0.001 when compared to the saline treated group.

FIGURE 5 Effect of intraperitoneal injection or prenatal exposure to saline (50 ml/mouse)or LPS (50, 100, or 150 mg/kg) on water intake in pregnant dams and their male offspring.

Each bar is mean Æ SEM. N ¼ 10. Ãp < 0.05, ÃÃp < 0.01, and ÃÃÃp < 0.001, when com-pared to the saline treated group.

Developmental Psychobiology Prenatal LPS Exposure and Mice Behaviors 833

DISCUSSION

There is a consensus that severe environmental orpsychological stress, including exposure to LPS, resultsin the release of glucocorticoids, pro-inflammatorycytokines, ACTH and CRF in maternal blood stream.These changes could operate directly or indirectly byregulating the activation of HPA axis and changes inplacental metabolism on fetal development. Amongthese candidates, maternal glucocorticoids and pro-inflammatory cytokines are the main candidates for themediation of prenatal stress to the fetus (Harbuz &Lightman, 1992; Johnson et al., 1992; Weinstock, Pol-tyrev, Schorer-Apelbaum, Men, & McCarty, 1998).Glucocorticoids are known for their central roles inimmunocompetence in anxiety circumstances (Dunn,1989; Dunn & Berridge, 1990; Merlot et al., 2008;Weinstock et al., 1998) and they can apply many organ-izational effects on prenatal tissue development, includ-ing HPA development, leading to changes in the HPAaxis function that persist throughout life (Merlot et al.,2008; Muglia, Jacobson, Dikkes, & Majzoub, 1995).In this study, we measured the level of corticosterone,major murine glucocorticoid hormone, and pro-inflam-matory cytokines in both pregnant dams and their maleoffspring.As described in the results, we observed an

increased level of pro-inflammatory cytokines (exceptfor IL-6 in 50 mg/kg LPS) and corticosterone in preg-nant dams which received 100 or 150 mg/kg LPS, butnot in their male offspring. These elevations ofmaternal cytokines and glucocorticoids can result ingrowth retardation and behavioral alterations in the off-spring (Anisman, Hayley, et al., 2002; Anisman, Kokki-nidis, et al., 2002). The possible mechanism is thatprenatal stress such as exposure to high level of gluco-corticoids has long-term consequences on child, includ-ing on neurodevelopment (Talge, Neal, & Glover,2007; Van den Bergh, Mulder, Mennes, & Glover,2005). Glucocorticoids inhibit hippocampal cell pro-liferation (Gould, Cameron, Daniels, Woolley, & McE-wen, 1992; Reul et al., 1994) and exert their action viadirect influence on ontogeny of fetal cells by reachingthe fetal organs. In this respect human studies showthat increased cortisol exposure affects the expressionof over a thousand genes in fetal brain cells and thisexposure affect the function of the placenta, includingthe expression and activity of 11beta-hydroxysteroiddehydrogenase 2 (11b-HSD2), the main barrier to theplacental passage of glucocorticoids (Glover, Bergman,Sarkar, & O’Connor, 2009; Mairesse et al., 2007;Salaria et al., 2006; Welberg, Thrivikraman, & Plotsky,2005). In humans lower placental levels of 11b-HSD2have been found to be associated with intrauterine

growth restriction (Dy, Guan, Sampath-Kumar,Richardson, & Yang, 2008; Glover et al., 2009). Otherhuman studies show that synthetic glucocorticoids thatcross the placenta can affect infant neurodevelopment(Sizonenko et al., 2006). On the other hand, animalstudies also demonstrate long-term effects of prenataladministration of synthetic glucocorticoids, such asdexamethasone, on offspring brain development andbehavior. For example, some animal works show thatprenatal exposure to raised glucocorticoids can damagethe brain and caused a reduction in hippocampal vol-ume in rodents and monkeys (Bergman, Sarkar, Glover,& O’Connor, 2010; Mitra & Sapolsky, 2008; Unoet al., 1994). Thus, maternal hormones seem to be goodcandidates for communication between the dam anddeveloping fetus (Joffe, 1969; Merlot et al., 2008).Although direct evidence for these causal links is lack-ing, and the mechanisms by which infection-inducedelevation of cytokine and glucocorticoid levels duringfetal life can interfere with neurodevelopmental eventsremain poorly understood.It has been suggested that infections and inflamma-

tory processes may be causative factors in emotionaldisorders, including anxiety (Belzung & Griebel, 2001;File, 2001; Hogg, 1996). To explore this hypothesis, wemeasured the anxiety-related behavior of male off-spring on EPM. Prenatal LPS exposure without loco-motor impairment in the elevated plus maze, increasedthe percentage of open arm times and open arm entriesin adult male offspring, indicating the induction ofanxiolytic response by prenatal immune challenge,which do not agree with others’ obtained in similarcontexts (Dunn & Berridge, 1990; Fride & Weinstock,1988, 1989). Though, at first these results appear con-tradictory, the discrepancy may be related to the g-ami-nobutyric acid (GABA) system. Prenatal stress hasbeen found to affect the GABA system in differentbrain regions, however, different stress paradigmshave different effects on the GABA system (Bowers,Cullinan, & Herman, 1998; Gruen, Wenberg, Elahi, &Friedhoff, 1995; Montpied et al., 1993). GABA, whichis the main inhibitory neurotransmitter of brain, has awell known and very important role in the modulationof anxiety. In addition, it is well known that GABA-ergic agents and drugs have strong anxiolytic effects inhumans and animals. One feasible mechanism is thatprenatal exposure to endotoxin changes the sensitivityof GABAA receptor and finally modifies the behavioralresponses to stress in adulthood (Jaiswal & Bhatta-charya, 1993; Kellogg, Taylor, Rodriguez-Zafra, &Pleger, 1993). In fact, LPS may have negated theeffects of stress on neonate mice, for example, throughsuppression of a stress-induced surge of corticosteroneand altering the composition and function of the

834 Asiaei, Solati and Salari Developmental Psychobiology

GABAA receptor complex by means of a transcriptionalmechanism (Stone et al., 2001). Stone et al. (2001)demonstrated that elevation in the level of glucocorti-coids can affect the GABAergic system and change theexpression of GABAA receptor subunit mRNA levels inthe brain regions that are involved in modulationanxiety, like hippocampus. This elevation also affectsmRNAs for glutamic acid decarboxylase (GAD) iso-forms, the enzyme that converts glutamate to GABA.Prenatal increase of corticosterone level increasesGAD67 mRNA in hippocampus. Increased inhibitoryinput by local GABA neurons may discuss the effectsof prenatal stresses on reduced anxiety-related behav-iors in adulthood. Therefore, there is a possibility thatprenatal activation of HPA axis by LPS may increasethe activity of GABAergic system in the brain regionslike hippocampus and in this way decrease the anxietylevel of offspring (Stone et al., 2001). In sum, it is clearthat our results cannot directly prove this phenomenonand surely need to be replicated before firm conclusionscan be drawn.In other parts of our study we examined the effects

of LPS exposure on ingestive behaviors of pregnantdams and their male offspring. In this part of the studywe observed the reduction of food and water intakein both pregnant dams and their male offspring (PND56–60). The central mechanisms underlying anorexiaduring exposure to bacterial components are not fullyunderstood. Several studies confirmed that all systems,signals, and switches (physiologic, metabolic, neuro-logic, anatomic, endocrine, behavioral, etc.) can influ-ence food and water intake (Bachmanov et al., 2002;Kraly, 1984) and Moyer, Herrenkohl, and Jacobowitz(1978) suggested that prenatal stress may cause perma-nent changes in noradrenaline hypothalamic contentwith a possible anorexic effect (Moyer et al., 1978).Our results, which concur with other reports in theliterature (Maccari et al., 2003; Maccari & Morley-Fletcher, 2007; Seckl, 2001; Tamashiro, Terrillion,Hyun, Koenig, & Moran, 2009), demonstrated thatexposure to increased levels of corticosterone and pro-inflammatory cytokines during prenatal stage, induceadverse effects on ingestive behaviors in later life.These results thus highlight the long-term influencesof prenatal exposure to LPS (which leads to maternalmalnutrition, anxiety and hormonal imbalance duringpregnancy) on ingestive behaviors in mice. Althoughit needs replication in larger cohorts before firm con-clusions can be drawn.Previous reports on the effects of prenatal stresses

on body weight are contradictory. Some authorsreported that prenatal stress leads to reduced bodyweight of pups (Armario, Restrepo, Castellanos, &Balasch, 1985; Klein & Rager, 1995; Patin et al., 2002;

Pollard, 1984; Salgado, Martinez, & Tarres, 1977;Valle´e, Mayo, Maccari, Le Moal, & Simon, 1996),whereas other studies failed to find an effect (Fride,Dan, Feldon, Halevy, & Weinstock, 1986; Glo¨ckner &Karge, 1991; Hughes & Beveridge, 1990). The discrep-ancies between studies could be due to differences inthe prenatal stress protocols employed. Our studyindicates that prenatal exposure to Salmonella endo-toxin decreases fetal body weight at term, indicating afetal growth restriction. In addition, the male offspringwith prenatal exposure to LPS showed decrease inbody weights when they grew up, in which the durationof the effects was directly related to the dose injected.The possible mechanism(s) is that decrease in bodyweight of the offspring might have been induced bylong-term effects of maternal nutritional deficiency,hyperactivity of the HPA axis (Levitt et al., 2000; Phil-lips et al., 1998; Reynolds & Phillips, 2000), excessiveglucocorticoid level in fetal environment (Krieg et al.,1992) and/or alteration in organ maturation (Novy &Walsh, 1983; Reinisch, Simon, Karow, & Gandelman,1978). Although the mechanisms underlying thesephenomena are still unclear, further understandingcould be gained by investigating the consequences ofprenatal manipulations.

CONCLUSION

In summary, this study demonstrated that exposure toendotoxin during fetal life can lead to long-lastingphysiological consequences most of them detrimentalfor the animal during adulthood. This conclusion wasbased on the following data observed in dams exposedto LPS in GD10 during pregnancy and their male off-spring versus the control group: (1) Increased pro-inflammatory cytokine and corticosterone levels inmaternal serum, (2) Decrease in anxiety-related behav-ior in male offspring, (3) Decrease in food and waterintake in pregnant dams, and interestingly in their maleoffspring, (4) Restriction in the body weight of maleoffspring up to PND 40.

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