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Behavioural Brain Research 216 (2011) 576–584

Contents lists available at ScienceDirect

Behavioural Brain Research

journa l homepage: www.e lsev ier .com/ locate /bbr

esearch report

he effects of repeated social interaction stress on behavioural andhysiological parameters in a stress-sensitive mouse strain

élène M. Savignaca,c, Niall P. Hylanda,b, Timothy G. Dinana,d, John F. Cryana,b,c,∗

Alimentary Pharmabiotic Centre, University College Cork, Cork, IrelandDepartment of Pharmacology and Therapeutics, University College Cork, Cork, IrelandSchool of Pharmacy, University College Cork, Cork, IrelandDepartment of Psychiatry, University College Cork, Cork, Ireland

r t i c l e i n f o

rticle history:eceived 14 July 2010eceived in revised form 24 August 2010ccepted 29 August 2010vailable online 6 September 2010

eywords:rain-gut axisocial stressocial statusouse model

a b s t r a c t

Stress can impair the immune, endocrine and nervous systems. Such perturbations can also affectbrain-gut axis communication and lead to functional gastrointestinal disorders such as irritable bowelsyndrome (IBS). IBS is a common yet poorly understood disorder which is often co-morbid with anxietyand depression. As there are few mouse models of IBS, this study aimed to investigate if a short andintense social stress which involved bouts of physical interaction could induce behavioural and phys-iological changes similar to those observed in IBS patients in the innately anxious BALB/c mice. MaleBALB/c mice were exposed for 2 h to an aggressive male intruder for acute (one-day) or chronic (six-day)stress. Behaviour was analyzed and weight monitored. Two hours post stress, trunk blood and tissueswere collected. Plasma was analyzed for inflammatory cytokines and corticosterone and morphologicaldamage to the colon was also assessed. Mice displayed either dominant or submissive status following

orticosteronero-inflammatory cytokinesrain-gut axis disorders

repeated intruder exposure. Behavioural status correlated with an increase in corticosterone and pro-inflammatory cytokines in both acute and chronic submissive groups. Mice from both status groups hadbody weight loss coupled with mild damage to the colon. Together these data show that short-term socialinteraction stress exposure was able to induce behavioural and physiological impairments similar to thatobserved in patients with dysregulated brain-gut axis function. Moreover, these data demonstrate that

mod

social stress-based mousedisorders.

. Introduction

The brain-gut axis refers to bi-directional communicationetween the central nervous system (CNS) and the gastrointestinalGI) tract [1]. Interactions occur at multiple levels via the immuneystem, the autonomic nervous system and the hypothalamic-ituitary-adrenal (HPA) axis [2–4]. Stress can cause brain-gut axisisruption, activating the HPA-axis and thereby increasing gluco-orticoids and impairing immune function. Accumulating evidenceuggests that such stress-induced changes may result in functionalI disorders such as irritable bowel syndrome (IBS) [5,6]. Moreover,iven that stress is a major predisposing factor for psychiatric dis-

rders such as anxiety and depression it is perhaps not surprisinghat there is a substantial co-morbidity between psychiatric and GIisorders [7–9].

∗ Corresponding author at: Cavanagh Pharmacy Building, University College Cork,ollege Rd., Cork, Ireland. Tel.: +353 21 490 1676; fax: +353 21 490 1656.

E-mail address: j.cryan@ucc.ie (J.F. Cryan).

166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2010.08.049

els may be appropriate for interrogating the mechanisms underlying such

© 2010 Elsevier B.V. All rights reserved.

IBS is a highly debilitating functional GI disease of uncertain aeti-ology that affects 10–20% worldwide [10]. It is mainly characterisedby altered bowel habits and visceral sensitivity without obvious tis-sue damage and is strongly associated with increased anxiety andbehavioural impairment such as social avoidance [11,12]. Althoughclear biomarkers for the disease remain elusive [13], recent stud-ies have highlighted some key biological features in IBS patientsincluding HPA-axis dysfunction and elevated pro-inflammatorycytokine levels compared with healthy controls [14,15]. Animalmodels are essential for the development of novel therapeuticstrategies and understanding the pathophysiology of the diseasestate. Most potential animal models of IBS have been developed inthe rat and many employ stress-based paradigms such as mater-nal separation [16–18]. Whilst rat models are very useful in basicresearch especially in terms of their size and having a well charac-terised behavioural repertoire, the upsurge in the use of genetically

modified mice has intensified the need for developing murine mod-els of disease [19]. Animal models require both an inducing factorwhich has similar construct to the idiopathic disease and a reliablebiological or behavioural readout that can be easily assayed [20].Putative mouse models of IBS use inflammation (trinitrobenzene

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ulfonic acid, TNBS model) or infection (Trichuris muris) [21–24] asnducing agents. Thus, stress-induced IBS mouse models need to beeveloped to mimic the natural history of the disease in humansnd to develop novel therapeutic agents.

Social stress is one of the main and potent sources of stress inumans inducing a strong immune and endocrine reaction [25,26].ocial stress-based mouse models have been successfully used tonvestigate the effects of stress per se on behaviour and on phys-ological functions and have been used as models of depression25,27,28]. The social disruption paradigm, consisting of the intro-uction of an aggressive intruder in a group of siblings to defeathe resident mice, induces the release of very high corticosteroneevels in the plasma and is associated with an exaggerated immuneesponse within only 6–7 days of defeat [29,30]. Therefore, animalodels of social stress, and in particular the social disruption pro-

edure, have the potential to induce symptoms and physiologicalatterns observed in IBS patients.

Accordingly, this study aimed to investigate the effects of aodified version of the social disruption paradigm on physiol-

gy and behaviour in the innately anxious inbred BALB/c mousetrain [31]. The impact of chronic, and for comparison acute, stressxposure was assessed on behavioural, neuroendocrine, immune,nd gastrointestinal parameters. Moreover, given that social statusdominant or submissive) has been shown to differentially affecttress-induced changes [32–34], where appropriate we also sub-nalysed the parameters measured by status.

. Materials and methods

.1. Animals

39 anxiety-prone male BALB/cOlaHsd (n = 13 per group, test mice) and 28ale C57BL/6JOlaHsd (aggressors) [31], aged 12–13-week-old, were purchased

rom Harlan Laboratories, UK and used in this study. Mice arrived in the ani-al facility at 8 weeks old and were kept under standard controlled conditions

temperature 20–21 ◦C, 55–60% humidity) on a 12 h light/dark cycle (lights on.00 am). Mice were provided with standard laboratory diet and water ad libi-um.

Animals were allowed 1 week habituation before being singly-housed in plex-glas cages (33 cm × 15 cm × 13 cm, L × l × h cm) for 4 weeks in order to reducenter-individual variability and increase the likelihood of social interaction-inducedtress without creating overt phenotypical changes. Indeed, it has been reported thatndividual housing at adulthood for a period of time less than 40 days may increasehe excitability and arousal response to a stressful event without inducing majormmuno-endocrine alterations [35]. All experiments were conducted in accordance

ith the European Directive 86/609/EEC, the Recommendation 2007/526/65/EC andpproved by the Animal Experimentation Ethics Committee of University Collegeork.

.2. Selection of aggressive intruders

Prior to the social stress procedure, C57BL/6 mice were screened in a preliminarytudy for aggressive behaviour towards a separate cohort of BALB/c mice to ensurehe defeat of the resident experimental mice, as previously described [28]. The heav-est C57BL/6 mice were selected for the aggression test. Latency of first attack andominance status of mice were visually determined by observing key behavioursuch as tail rattling, chasing or biting for dominance and escape and upright pos-ures or immobility for submission [26,27,28]. Mice being aggressive, dominant andresenting the lowest latency to attack were then selected to be aggressive intrudersor the stress procedure.

.3. Social stress procedure

After 4 weeks of single-housing, mice were randomly assigned to the controlr stress exposure groups, with n = 13 per group. In the present study, a modifiedersion of the social disruption test was used which involves bouts of physical inter-ction [32,36]; a singly-housed resident mouse (BALB/c) received a single aggressiventruder (C57BL/6), with physical contact allowed either for a single-trial (referred tos acute) or for a consecutive period of 6 days (referred to as chronic), for 2 h, between

.30 and 12.00 am. At the end of the exposure period, intruders were returned toheir home cages.

Precautions were taken to ensure the defeat of the resident as previouslyeported [28,36], as a trained aggressive intruder should defeat the resident mouse.owever, to avoid. To avoid habituation to the intruder, aggressors were rotatedaily. An intruder displaying no aggressive behaviour within the first 5 min towards

n Research 216 (2011) 576–584 577

resident mouse was replaced by another one, as previously described [36]. However,aggressive intruders were changed only twice in case of non-aggression towards theresident-test mouse to keep consistency in testing conditions. Therefore, a residentmouse displaying continuous dominant behaviour was left with the last rotatedsubmissive intruder. Control animals remained in their home cage, to avoid anynon-specific effects of animal handling [26].

2.4. Behavioural analysis

Social status was scored daily by visual assessment as previously described[26,28,29]. Agonistic and non-social behaviours were also recorded for further anal-ysis. Briefly, mice were deemed dominant if they displayed aggressive behaviourtoward their opponent such as tail rattling, chasing and fight attacks. Mice weresubmissive if they displayed defending and avoidant behaviour such as escaping,defensive response (responding to attack from the aggressor by a defensive attack),upright posture and defensive immobility. Social investigation was also assessedby sniffing behaviour or stretched attend postures (SAP), reflecting risk assessment.Digging and rearing behaviour (referred to as “Mix rear-dig”) were scored, and arean assessment of non-social escape-oriented exploration. At the end of the 2 h pro-cedure, intruders were removed and returned to their home cage, while residentsstayed in their home cage until the next stress session. Fur score was also assesseddaily as previously described [29]. Briefly, clean, shiny and well groomed fur wasscored 1, dull and irregular fur with small amount of minor wounds was scored 2,fur more marked with minor wounds and less hair was denoted 3 and very ruffledfur with several minor wounds and lack of hair was 4; an intermediate score of 1.5was given for fur that had lost its shine but without any obvious signs of wounds ormarks. In addition, although many social stress experiments indicate that woundsare present, few give any clear indication of the extent to which it occurs and itis often ignored. As wounds have the potential to affect immunological status itwas important to also count the number of minor wounds present in addition tomonitoring fur coat state [30,37]. Animals displaying any severe wounding wereexcluded.

2.5. Sample collection

Mice were weighed before the beginning of the stress procedure on day 1 andat the end following the stress procedure. Two hours after the last chronic stressexposure or following a single acute episode, mice were sacrificed by cervical dis-location. This timepoint was selected to best capture the stress-induced alterationsin both inflammatory and neuroendocrine markers. Trunk blood was collected inEDTA (ethylene-diamine tetra-acetic acid) tubes and spun for 15 min at 5000 rpm.Plasma was pipetted out and stored under −80 ◦C for further corticosterone andcytokine analysis. Distal/proximal colons were harvested, the length measured andwere then stored at −80 ◦C for further histological analysis. Splenic weight was alsoroutinely recorded.

2.6. Corticosterone assay

Plasma corticosterone was determined using an Enzyme Immunoassay Kit(Assay Designs, Inc., MI, U.S.A.) according to the manufacturer’s instructions. 20 �Lplasma per sample per well was used for the assay. Samples were analysed induplicate in a single assay; threshold detection = 32 pg/mL; coefficient of variationlimit = 20%; concentration expressed in pg/ml.

2.7. Cytokine assay

Pro- and anti-inflammatory cytokines (IL-1�, IL-6, mKC, IL-10, IFN-�, TNF-�,IL-12p70) levels in plasma were measured using an ultra-sensitive electro-chemiluminescence multiplex system Sector 2400 imager from Meso ScaleDiscovery (Gaithersburg, MD) according to the manufacturer’s instructions. 10 �Lplasma per sample per well was used for the assay. Samples were analysed in dupli-cate in a single assay; threshold detection = 2.5 pg/mL (values for some animals couldnot be detected in the assay notably in control group, decreasing n per group). Coef-ficient variation limit = 20%. A number of sample duplicates failed to be within thislimit and were discarded from analysis.

2.8. Colon study

Colon length was routinely measured and segments were subsequently col-lected and fixed with paraformaldehyde (4%) followed by 20% sucrose solutionbefore freezing and storage at −80 ◦C. For histological assessment, tissue was thenembedded for cryostat sectioning at −20 ◦C. Cross-sections (10 �m) were cut andstained with haematoxylin–eosin treatment to examine cellular structure. Micro-scopic damage was assessed by an experimenter blind to conditions and pictures

obtained at 20× magnification (CellF̂ software). Different aspects of histologicaldamage were scored as previously described [38,39] using a scale adapted forthe present study for mild tissue damage and ranged from 1; no damage to 3;severe damage. As preliminary analysis suggested tissue damage in our studywas mild, the following adaptation to the above scale was applied: control slideswere referenced as 0 for no damage and 3 represented maximum impairment

578 H.M. Savignac et al. / Behavioural Brain Research 216 (2011) 576–584

Fig. 1. Evolution of the effects of repeated social stress on behaviour in BALB/c mice from day 1 to day 6. (A) Ethogram: mice displayed aggressive and active behaviouron day 1 (dominant) whereas defeated and passive on day 6 (submissive). “Resp. def”, active defensive response to attacks; “def immob.”, defensive immobility; “SAP”,stretched attend postures; “Mix rear–dig”, mix of digging and rearing behaviour; n = 13 (*p < 0.05; ***p < 0.001; day 1 vs. day 6; sniffing day 1 vs. day 6, p = 0.086). (B) Statusevolution: mice were changing behaviour over the duration of the experiment, being dominant at the start and becoming submissive at the end of stress test. (C) Fur score(grouped): fur score increase, day 1 to 6, n = 13 all groups. (D) Minor wounds (grouped): number of minor wounds increase in stress groups compared to control mice. Then .001;a ntrol* tatus)g ve ans ed as

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umber remained stable throughout the stress procedure; n = 13 (**p < 0.01; ***p < 0significant increase in fur score was observed in submissive mice compared to co

**p < 0.001 control vs. submissive group. (F) Number of minor wounds (separated sroups compared to control mice, but the effect was more pronounced in submissiubmissive mice (**p < 0.01; ***p < 0.001 control vs. stress groups). Data are express

hich equated to mild inflammation relative to overt colitic inflammation (dataot shown).

.9. Statistical analysis

Data were analyzed using Graph Pad Prism 4 software. For the ethogram,aired t-test was used to compare every parameter’s difference between day 1 anday 6. For analysis of fur score, injuries number, corticosterone levels, inflamma-ory cytokines levels, body weight, spleen weight, and histological score, one-wayNOVA was used to compare groups, followed by Neuman–Keuls post hoc test for

ultiple comparisons. When variances were not equal as assessed by a Bartlett

est, non-parametric statistics were conducted using a Kruskal–Wallis test followedy Dunn’s multiple comparison test. t-Test or Mann–Whitney test were used for-group comparison where appropriate. For data correlations, Pearson correlationest was used. Statistical significance was set at p < 0.05. Data are expressed as

ean ± SEM.

stress vs. control group). (E) Fur score (separated status) following 6 days of stress:animals; n = 13 control and acute group, n = 6 dominant and n = 7 submissive mice,following 6 days of stress: An increase in minor wounds was observed in all stress

imals from chronic group; n = 13 control and acute group, n = 6 dominant and n = 7mean ± SEM.

3. Results

3.1. Effect of repeated social stress on behaviour

Mouse behaviour was analysed during the stress procedure foraggressive or defensive behaviour and data are presented in anethogram (Fig. 1A) representing the range of key behaviours forthe first (day 1) and the last day of stress (day 6). The ethogramshowed an evolution in behaviour across the duration of the pro-

cedure, with mice displaying statistically significant differences inbehaviour between day 1 and day 6. Mice were more active, anddisplayed dominant, fighting and defending-aggressive behaviouron day 1 compared with day 6. Indeed, across social exposure,mice displayed more and more avoidant and submissive behaviour,

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ith higher immobility. Mice also showed a trend toward higherocial investigation on day 1 compared with day 6, correspond-ng to a trend toward higher non-social investigation (Mix ofearing–digging) on day 6. Indeed, there was a significant decreasen fight attacks (p < 0.05), active responses to attacks by intrud-rs (p < 0.05), stretched attend postures (p < 0.05), and a significantncrease in defensive immobility (p < 0.001).

During the course of the chronic stress exposure we observedwo different types of social status, dominant and submissive. Miceisplaying active and aggressive behaviours were categorised asominant and mice displaying passive and defending behaviours asubmissive. Over time (1–6 days), the proportion of mice display-ng dominant behaviour declined (Fig. 1B). Therefore, physiologicalata were analysed collectively for all animals or were separatedy social status i.e. dominant or submissive.

The physical state of the animals was also assessed by fur scorend by the number of minor wounds, for grouped status duringtress procedure (Fig. 1C and D) and separated into status by domi-ance/submission for the last day of social stress (Fig. 1E and F). (Nonimal was excluded from the analysis due to wound severity as allounds were superficial skin wounds caused by biting and scratch-

ng. When data were analysed altogether, repeated (chronic) stressesulted in a significantly higher fur score than non-stressed con-rols (H(df = 7) = 39.64, p < 0.0001; from day 3, p < 0.01 to day 6,< 0.001; vs. control group; Fig. 1C). Control animals did not displayny injuries, therefore the number of minor wounds was signif-cantly higher in both acute and chronic stress groups compared

ith controls (F(7,96) = 5.098, p < 0.0001; acute, days 1–6, p < 0.01;s. control group, Fig. 1D). However, the number of minor woundsas not statistically significantly higher on day 6 compared withay 1 (p > 0.05), whereas fur score was for these two time-pointsp < 0.05), showing that although minor wounds appeared at thetart of the stress procedure, these remained superficial and did notranslate into major injury. Analysis of separated status betweenominant and submissive animals (Fig. 1E), data for day 6 revealedur score was significantly increased on day 6 (p < 0.001) in sub-

issive mice compared with controls. Moreover, this score wasignificantly different from day 1 to day 6 for submissive micep < 0.05), reflecting a better adaptation to stress in dominant thanubmissive animals that were chronically stressed.

Analysis of the number of minor wounds by separated status

Fig. 1F), data for day 6 revealed all stress groups displayed higherumber of injuries than control animals as those do not have any

njury (acute, p < 0.001; dominant mice, p < 0.01 and submissiveice p < 0.001). However, this number remained stable for both

tatuses on the last days of the stress procedure, as showed for

ig. 2. Effect of repeated social stress on corticosterone levels in plasma. (A) Corticosteroignificant corticosterone increase vs. controls, whereas chronic group displayed a non-sigtatus (overall groups difference, p < 0.01), chronic submissive (n = 7) displayed a nearly sign = 6) did not display any increase (*p < 0.05 acute vs. control and +p < 0.05 acute vs. ch

ean ± SEM.

n Research 216 (2011) 576–584 579

grouped status (Fig. 1D). To make sure minor wounds did not havean impact on endocrine and immune parameters measured, a cor-relation between them was further conducted (see Section 3.4).

3.2. Effect of repeated social stress on plasma corticosterone levels

Corticosterone levels were measured as an index of HPA-axis activation. Both acutely and chronically stressed groups hada 3–4-fold increase in plasma corticosterone (H(df = 2) = 9.111,p < 0.05; Fig. 2A). When separated by status (Fig. 2B), there wasno statistical difference between chronically stressed animals andthe control group. However, submissive mice displayed a non-significant increase in corticosterone levels relative to controlanimals (H(df = 3) = 13.34, p = 0.068).

3.3. Effect of repeated social stress on plasma pro- andanti-inflammatory cytokines levels

Table 1 shows results for pro-inflammatory IL-1�, IL-6,chemokine mKC (CXCL1), TNF-�, IL-12p70, IFN-� and anti-inflammatory IL-10 levels in control, acutely and chronicallystressed animals; both combined, and separated by social status.Kruskal–Wallis analysis for general group comparison indicatedthat all cytokines levels were increased in both acute and chronicstressed groups compared with controls with the exception ofIFN-�, but no difference between stressed groups was detected(H(df = 2) = 15.76, p < 0.001 for IL-1�, H(df = 2) = 17.32, p < 0.001 forIL-6, H(df = 2) = 14.36, p < 0.001 for mKC; H(df = 2) = 10.01, p < 0.01for IL-12p70, H(df = 2) = 9.61, p < 0.01 for IL-10 and H(df = 2) = 14.02,p < 0.05 for TNF-�). Further post hoc analysis revealed the effectsof acute stress resulted in a significant increase, compared to con-trol group, for IL-1� (p < 0.01), IL-6 (p < 0.01), mKC (p < 0.01), TNF-�(p < 0.05), IL-12p70 (p < 0.05) and IL-10 (p < 0.05). On the other hand,animals undergoing chronic stress had a significant increase com-pared with control groups for IL-1� (p < 0.001), IL-6 (p < 0.001), mKC(p < 0.01), TNF-� (p < 0.001), IL-12p70 (p < 0.01) and IL-10 (p < 0.05).IFN-� levels did not reach the level of statistical difference betweengroups (p = 0.06).

When separated by dominant or submissive status (Table 1), thepattern of IL-1�, IL-6 and mKC increases in parallel to that observedwith corticosterone (Fig. 2B and Table 1). Chronic submissive mice

displayed a significant increase compared with control group inIL-1� (p < 0.01), IL-6 (p < 0.01) and mKC (p < 0.001). On the con-trary, chronically stressed dominant mice displayed a much lowerincrease compared with controls (IL-1�, p < 0.05; IL-6, p < 0.01;mKC, p < 0.01). Moreover, chronically stressed submissive animals

ne levels with grouped status in chronically stressed mice: acute group displayednificant 3-fold increase vs. controls; (n = 13, p < 0.05). (B) When analysing separatednificant 4-fold increase compared with controls (n = 12), whereas chronic dominantronic dominant; p = 0.068 chronic submissive vs. controls). Data are expressed as

580 H.M. Savignac et al. / Behavioural Brain Research 216 (2011) 576–584

Table 1Effect of repeated social stress on pro- and anti-inflammatory cytokines levels in plasma-grouped and separated status.

Cytokine Control (n = 10–13) Acute (n = 10–13) Chronic stress

Grouped (n = 12–13) Dominant (n = 5–6) Submissive (n = 6–7)

IL-1� 16.2 ± 4.8 120.7 ± 59.2** 90.9 ± 20.8*** 62.4 ± 11.4* 115.4 ± 36.0**

IL-6 37.7 ± 14.3 787.6 ± 398.9** 731.5 ± 403.1*** 210.1 ± 54.1** 1252.9 ± 776.6**

mKC 103.7 ± 28.0 3377.8 ± 2224.9** 2546.7 ± 969** 455.8 ± 131.1** 4338.9 ± 1521.7$,***

IL-10 19.7 ± 7.8 95.8 ± 22.9* 98.7 ± 20.6* 90.9 ± 25.1* 105.4 ± 33.2*

TNF-� 3.4 ± 0.9 7.7 ± 2.1* 10.1 ± 1.5*** 7.7 ± 1.6* 12.2 ± 2.1***

IFN-� 2.9 ± 0.6 3.7 ± 0.9 5.7 ± 1.2 4.1 ± 0.9 7 ± 2.0IL-12p70 73.9 ± 34.0 243.3 ± 63.8* 363.6 ± 80.5** 411.8 ± 137.6 329.1 ± 104.2*

Cytokines levels were increased in both stress groups compared with controls for pro-inflammatory IL-1�, IL-6, mKC, TNF-�, IL-12p70 and anti-inflammatory IL-10, witha much stronger increase in chronic submissive mice for the 3 pro-inflammatory cytokines IL-1�, IL-6 and mKC; resembling corticosterone pattern. Data are expressed asmean ± SEM.

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*** p < 0.001 vs. controls (Mann–Whitney post hoc analysis for specific 2-group com$ p < 0.05 chronic submissive vs. chronic dominant.

isplayed a statistically significant increase in mKC levels comparedith chronic dominant group (p < 0.05). Although not significant,

he same pattern was observed for IL-1� and IL-6.Chronically stressed animals also presented a significant

ncrease in TNF-� (p < 0.001 for submissive and p < 0.05 for dom-nant) compared with control animals. Both chronically stressedroups displayed an increase in anti-inflammatory IL-10 andro-inflammatory IL-12p70 levels compared with controls (IL-0, dominant, p < 0.05, submissive, p < 0.05; IL-12p70, dominant,= 0.076, submissive, p < 0.05). Finally, chronic submissive miceisplayed a non-significant 2-fold increase compared with controlnimals in IFN-� levels (p = 0.057) (Table 1).

.4. Relationship between injury severity and physiologicalarameters

To rule out the influence of unwanted injuries on bothmmune activation and corticosterone response and to ensurebserved immune and neuroendocrine changes were be due tohe stress procedure alone, the relationship between the num-er of injuries and these parameters levels were investigated in

ig. 3. Effect of repeated social stress on colon physiology. (A) Colon length: there was nChronic D, n = 6) and chronic submissive (Chronic S, n = 7) groups. (B) Colon histology: hhe chronic group with mild infiltration (p < 0.01) (*p < 0.05 chronic dominant vs. control axpressed as mean ± SEM. (C) Microscopic pictures of colonic tissue stained with haemato

n).n).on).

chronically stressed animals (n = 12–13, data not shown). Therewas no correlation between the number of injuries (counted asnumber of minor wounds) and corticosterone levels in plasma(Pearson coefficient correlation, p = 0.98; R2 = 0.00004), or plasmalevels of pro-inflammatory cytokine IL-6 (p = 0.51, R2 = 0.0408),IL-1� (p = 0.64, R2 = 0.0208), mKC (p = 0.79, R2 = 0.0066), TNF-� (p = 0.9, R2 = 0.0014), IFN-� (p = 0.31, R2 = 0.0946), IL-12p70(p = 0.61, R2 = 0.0273), IL-10 (p = 0.23, R2 = 0.1286). Similarly whenstatus were analysed separately, also no correlation between injuryseverity and any of the parameters above cited were identified (datanot shown).

3.5. Effect of repeated social stress on colon tissue

No difference in colon length (Fig. 3A) between control ani-mals and any of the stressed groups, either grouped (F(2,36) = 1.72,

p = 0.19, data not shown), or separated by status was observed(F(3,35) = 1.19, p = 0.49, Fig. 3A). Microscopic analysis (Fig. 3C)revealed mild damage in tissues from chronically stressed miceas opposed to acute and control groups, but with no differ-ence between dominant and submissive mice. Histological scores

o significant difference between control (n = 13), acute (n = 13), chronic dominantistological score revealed repeated stress exposure induced mild tissue damage innd +p < 0.05 chronic dominant vs. acute). “D” = dominant; “S” = submissive. Data arexylin–eosin treatment at 20× magnification for control, acute and chronic groups.

H.M. Savignac et al. / Behavioural Brai

Fig. 4. Effect of repeated social stress on body weight change. All stress groups dis-played a significant decrease in body weight gain compared to control group, chronicd(m“

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ominant group presenting the bigger difference compared with other stress groupsp < 0.001). Control and acute groups n = 13, chronic dominant n = 6, chronic sub-

issive n = 7 (**p < 0.01, ***p < 0.001, p = 0.069 chronic dominant vs. acute group).D” = dominant; “S” = submissive. (Data are expressed as mean ± SEM).

Fig. 3B) demonstrated that both chronically stressed animals dis-layed a mild inflammatory cell infiltration, loss of goblet cellsnd increased muscle thickness compared with control and acutelytressed animals (F(3,30) = 4.51, p < 0.01; chronic dominant vs. con-rol, p < 0.05 and vs. acute, p < 0.05; chronic submissive vs. controls,< 0.05).

.6. Effect of repeated social stress on spleen weight

As 2-h repeated social stress has been shown to induceplenomegaly [32,37], spleen weight was assessed. There was notatistical significance between the three groups, when animalsere grouped collectively (F(2,36) = 0.54, p = 0.59, data not shown)

r separated by status (F(3,35) = 1.03, p = 0.39, data not shown).

.7. Effect of repeated social stress on body weight change beforend after stress exposure

Body weight changes were investigated as chronic social stresss often associated with body weight loss [33,34,41]. Both stressxposures significantly induced body weight loss compared to non-tressed animals (grouped data F(2,36) = 13.97, p < 0.0001, data nothown; separated by status F(3,35) = 1.03, p < 0.001, Fig. 4). Post hocnalysis revealed that all stressed groups, irrespective of status,ignificantly lost body weight compared to controls (controls vs.cute, p < 0.01; controls vs. chronic dominant, p < 0.001; controlss. chronic submissive, p < 0.01).

.8. Relationship between neuroendocrine and immunearameters

To investigate whether the observed changes in cytokinemmune profile were related to the repeated social stress, cor-elations were made between corticosterone and cytokine levelseasured in plasma. In the first instance, correlations were

one with all groups to include basal and stress-induced lev-

able 2ummary of the effects of repeated social stress on behaviour and physiology.

Acute

Behaviour Chase fight/active defenceInjuries IncreaseFur score NSPlasma corticosterone IncreasePlasma cytokines IncreaseColon length NSSpleen weight NSBody weight change Decrease

n Research 216 (2011) 576–584 581

els and then after with chronic group only to assess stresslevels only. When all groups were analysed together, therewas a positive correlation between corticosterone levels andthat of the pro-inflammatory cytokines: IL-1� (n = 36, Pear-son correlation coefficient, p < 0.0001, R2 = 0.5465), IL-6 (n = 33,coef. correlation, p < 0.05, R2 = 0.1447), mKC (n = 35, coef. correla-tion, p < 0.0001, R2 = 0.7240) and TNF-� (n = 37, coef. correlation,p < 0.0001, R2 = 0.3504). There was also a correlation betweencorticosterone levels and the anti-inflammatory cytokine IL-10(n = 38, coef. correlation, p < 0.01, R2 = 0.2393). On the other hand,there was no correlation between corticosterone and IFN-� (n = 37,coef. correlation, p = 0.07, R2 = 0.0908) or IL-12p70 (n = 38, coef.correlation, p = 0.77, R2 = 0.0022) (data not shown). Moreover,when analysing data from chronically stressed animals only,there was a positive correlation between corticosterone and pro-inflammatory cytokines levels that are found to be increased inIBS and depressed patients [13,42,43]: IL-6 (n = 12, coef. correla-tion, p < 0.05, R2 = 0.369), mKC (n = 13, coef. correlation, p < 0.0001,R2 = 0.862) and a nearly significant correlation for TNF-� (n = 13,coef. correlation, p = 0.051, R2 = 0.304). However, there was no cor-relation for IL-1� (n = 13, coef. correlation, p = 0.106, R2 = 0.220),IFN-� (n = 13, coef. correlation, p = 0.393, R2 = 0.067), IL-10 (n = 13,coef. correlation, p = 0.501, R2 = 0.042) and IL-12p70 (n = 11, coef.correlation, p = 0.296, R2 = 0.121) (data not shown).

4. Discussion

In these studies, our aim was to investigate the effects ofrepeated social exposure produced by the introduction of anaggressive intruder in the home cage of an anxiety-prone mousestrain. The protocol used here aimed at reproducing a very shortand intense social and physically interactive stress in the innatelyanxiety-prone BALB/c mice, using a single mouse in its homecage (Table 2). The behavioural analysis shows BALB/c mice havebeen physically and psychologically challenged by the introduc-tion of an aggressive mouse, as manifested by responding in adominant fashion displaying active, aggressive behaviour or beingsubmissive and displaying passive, defeated behaviours. Both acuteand chronic submissive groups had elevations in corticosteroneand all stress groups had changes in pro- and anti-inflammatorycytokines, which were more marked in submissive animals. More-over, repeated stress exposure also induced mild microscopiccolonic damage without altering colon length or spleen weight.

Our data are in line with previous studies which have shownthat social disruption of a group of siblings by the introductionof an aggressive intruder in C57BL/6 mice induces the defeat ofthe resident mice, produces social stress and triggers immune andphysiological changes, and this, within only seven days of defeat[25,36]. Although care was taken to maximise the opportunities

to develop a consistent and uniform phenotype in defeated mice(e.g. selection of aggressive intruder; rotation of intruders to avoidhabituation; replacement of sub-optimum intruders with moreaggressive ones) two social statuses developed. Having such diver-gent phenotypes allowed us to assess the impact of status on

Chronic dominant Chronic submissive

Chase fight–aggressive active Defence immobilityIncrease Increase2-Fold increase NS IncreaseNS 4-Fold increase NSIncrease IncreaseNS NSNS NSDecrease Decrease

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he various physiological parameters assessed. According to thethogram, on day 1, mice fought, were chasing or escaping but alsoctively defending as indicated by an equal time spent to respond tontruders bites. Active defence diminished in favour of immobilitynd passive defence by day 6. Previous findings using a similar pro-ocol showed the same in C57BL/6 mice, where attacks decreasedn favour of increased submissive behaviour [32]. Therefore, most

ice were dominant at the beginning of the interaction but eitherecame submissive or remained dominant to the end. Relativelyigh sniffing time compared to other behaviours is evidence thatice were also displaying significant levels of social investigation.

hey also displayed various non-social escape-like behaviours suchs digging and rearing, indicating they were stressed and attempt-ng to escape the stress. These two parameters did not change fromay 1 to day 6. Interestingly, dominant behaviours of outbred OF1ice have been shown to change between a 9 to a 23-days proce-

ure, with mice defending more actively after a long compared to ahort social stress period [26]. It will be of interest for future stud-es to investigate if a prolonged protocol of chronic social stress

ould induce differential and persistent behavioural changes inubmissive vs. dominant mice.

It should be noted in the present study that amongst mice withsubmissive phenotype, they evolved from engaging in an active

trategy, on day 1, to a passive and socially avoidant one, on day, which can be viewed as a coping mechanism to deal with thetressful situation. In animal studies of social stress, the inabilityor mice to escape a stressful home cage can produce a learned-elplessness-like stress [34], which is known to be a model ofepression, and is confirmed by the passive behaviour of the micen day 6 [31].

Status differences are well described in the literature, usingifferent social stress protocols or mouse strains [44–46]. Indeed,nderstanding the molecular basis of susceptibility and resilienceo social stress has been a subject of intense investigation recently33,47]. Such differences in social avoidance behaviour and cop-ng strategy are also observed in IBS patients [12,48], suggestingur behavioural data may feature some behavioural impairmentimilar to those displayed by people suffering from IBS.

Both acute and chronically stressed groups displayed a 3–4-old increase in corticosterone levels compared with control group,onfirming animals were stressed by the repeated social expo-ure. Social stress in animal models is also known to inducencreased plasma corticosterone in chronically stressed animals,s an obvious and normal response to stress [28,32,45]. Whenice were analyzed by dominance, it appeared that chronic

ominant mice displayed corticosterone levels similar to controlice, whereas although not significant, submissive mice displayed4-fold increase compared with controls. Functional HPA-axis

esponse to repeated stressor results in an adaptation and increasedegative feedback leading to reduced corticosterone response asbserved in dominant animals undergoing chronic social stressut not in submissive ones. This suggests that animals in the laterroup may have an impaired HPA-axis response. Another possi-ility to explain this blunted response in dominant mice, is thathe chronic social exposure may not have been strong enougho induce changes in these animals. However, fur score of bothhronic dominant and submissive mice was increased within thetress procedure. It has been previously described that social sta-us may not influence corticosterone response, with both dominantnd submissive mice displaying increased corticosterone levels,lthough differing in other physiological parameters [32,34]. How-

ver, another study found the contrary with corticosterone levelseing higher in subordinate mice [46]. Such discrepancies in lit-rature may be due to protocol and sample collection time-pointsifferences. In the present study, sampling occurred 2 h post stress,lthough corticosterone peaks earlier than this (approximately

n Research 216 (2011) 576–584

30–45 min post stress), studies have shown elevated levels up to12–24 h following the last social defeat [28,49]. We cannot rule outthat different effects would be seen at earlier time-points. How-ever, this timepoint allowed for analysis of many of the cytokineshypothesised to be important in stress-induced inflammatory pro-cesses which time-course studies show peak around 2 h post stress[50]. Future studies should focus on garnering a complete time-course of both the neuroendocrine and inflammatory sequelae ofchronic social defeat stress. Interestingly, patients suffering fromstress-related and GI disorders also display altered HPA-axis, oftenresulting in higher cortisol levels compared with healthy people[14,51].

Circulating cytokines levels were measured to investigate thepotential of this stress procedure to alter immune responses underbaseline conditions, without any immune stimulating agent (e.g.LPS or ConA stimulation), as often used in most social stress studiesto date [36,45,52]. It has been widely described that social stress canimpair immune response, in a status-dependent way, with submis-sive resident mice being the most susceptible to immune alteration;an effect which can be explained by the immuno-suppressantaction of corticosterone levels increase. However, as described byBartolomucci et al. [35] and already stated above, corticosteroneincrease itself does not seem to be systematically status-dependent,involving the influence of other physiological or genetic factors.The present study confirms the original finding that both acuteand repeated social stress are able to induce systemic release of sixdifferent pro- and anti-inflammatory cytokines reflecting a persis-tent immune response at both early and late stages. When animalswere separated according to dominance status, a pattern expres-sion similar to corticosterone was found for IL-1�, IL-6 and mKCwith both acute and submissive mice displaying higher increasesthan dominant mice (see Table 1). This supports the former find-ings that there is a link between social status and physiologicalparameters, with dominant mice being more resistant to stress ordisplaying better adaptation than submissive ones do. It shouldbe noted that increased levels of these cytokines are observed indepressed and in IBS patients [13–15]. This shows that our model isable to mimic some of the physiological alterations in these stress-related disorders. These changes appear to be at least partiallystatus-dependent, as observed in other studies [34,45]. Sheridanand colleagues in a series of elegant studies have shown that socialdisruption stress is able to increase the susceptibility to endotoxicshock [53] and induce glucocorticoid resistance [54], coupled withdownregulation of mRNA glucocorticoid receptors and increasedpro-inflammatory cytokines. Therefore, one could interpret the dif-ferential immune pattern observed between stressed mice as beingdue to a diminished HPA-axis and immune response in dominantmice as they manage to adapt and cope with the stress, comparedwith that seen in submissive mice. Together, the present proceduremay have induced a low-grade inflammation, fitting with one of themajor hypothesis of IBS aetiology [5,13].

A question to address is whether the physical contact andinjuries were at the origin of the observed immune response.Most social stress protocols involving physical interaction observewounds, independently of the stress protocol used: “Social Stress”,“Social Defeat” or “Social Disruption”, and for a 10-min or 2-hinteraction [29,32,33,37,54]. In these studies, wounds are oftenreported, although this is not common to all social stress stud-ies. When mentioned in the literature, wounds are described asminor. However, except for articles specifically assessing the effectsof wounds on immunology, concrete data on wounds are rarely

presented, making it difficult to draw conclusions about the rela-tive contribution of minor wounds to the behavioural phenotypeobserved. Interestingly, Merlot and colleagues [29], suggest thatminor wounds appearing following an acute social stress can-not trigger the rapid and exaggerated systemic immune response

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bserved in their studies. They also suggest that if injuries directlynfluenced the immune response, cytokine levels should be highern day 6 than 1, which they don’t find and which also fit withur data. In addition, it is reported that the immune response fol-owing social stress interaction is independent of injuries [37] andt is widely demonstrated that stress is linked to an exaggeratedmmune response notably in IL-6, even without any physical inter-ction, fitting with data from human depressed and IBS patients13,55].

Nonetheless, for objectivity, the potential impact of minorounds on endocrine and immune parameters was assessed in

he present study. The assessment of fur state was also conducteds another index of physical state [36,37]. Fur score was overallncreased in chronically stressed mice, but as expected, more sig-ificantly so in submissive animals, as this status has been linkedo higher stress response and likelihood to display wounds. Thisas therefore coupled to a higher number of injuries, although thisumber remained stable on the last days of stress exposure. Stress islso known to slow down the wound healing process [40], this mayxplain the differences in minor wound number observed betweeness-stressed, better coping dominant mice and more stressed sub-

issive animals. Nevertheless, no positive correlation was foundetween injuries and endocrine and immune parameters, as alsohown in a recent study by Bailey and colleagues [37]. Further,nd as expected, a correlation was found between corticosteronend cytokines levels. When data were analysed with all groups,positive correlation appeared for most cytokines. Besides, when

nalysing within the chronic group, there was also positive cor-elations between corticosterone and specific cytokines that arepecifically elevated in IBS and depressed patients [13,43]. Accord-ngly, the observed immune response in the present study is likelyo be due to the social interaction stress itself and not the physicalspects of minor wounds.

Histological study of the colon reveals mild impairments in bothhronically stressed animals, although no colon length differenceas observed. Such findings outline that the stressor employed was

trong enough to induce physiological changes which may reflectnflammatory dysfunction by non-adaptive cytokine signalling ory brain-gut miscommunication via vagal efferents following aobust stress state [3,5,56]. Cytokines are known to stimulate cen-ral mechanisms, autonomic nervous system and trigger immunend endocrine changes and thus are poised to contribute to thehanges observed [1,5,13,57,58]. It should be noted that there iso overt tissue damage observed in IBS [13,59], whereas it is onef the hallmarks of other GI tract disorders such as inflamma-ory bowel disease, and is reproducibly observed in animal modelslbeit to a much greater extent than that observed here [60,61].owever, IBS pathophysiology is also hypothesised to be due tolow-grade inflammation, therefore the present results show thebserved increase in the levels of pro-inflammatory cytokines mayave induced gut epithelial barrier dysfunction and lead to thebserved mild colonic damage measured, featuring potential low-rade inflammation [21,62].

Spleen weight was also measured, as splenomegaly and alteredplenocyte function were reported following social stress [36,37].bsence of significant weight difference in our study suggeststressed animals may still have been in the acute phase of stressesponse to social conflict.

Finally, all stressed animal groups displayed a decrease inodyweight gain, which was more pronounced in the chronicallytressed dominant mice. The weight loss in animals that were

cutely stressed may be due to decreased food or water intaker increased faecal output. In terms of chronically stressed ani-als our data are in agreement with other studies of social stresshich also demonstrate a deficit in bodyweight gain, which is moreronounced in animals with a dominant status [34,63]. [

n Research 216 (2011) 576–584 583

BALB/c mice are known to be more anxious than other mousestrains such as C57BL/6, which are amongst the strains most com-monly used in social stress protocols. A wealth of data existsshowing altered neurochemical alterations both under baselineconditions and following stress [31]. Moreover, we have recentlyshown a distinct brain activation pattern to an acute restraint stressin BALB/c mice compared with C57BL/6 mice [64]. Further straincomparisons using a similar social stress protocol to that employedhere are now warranted. Moreover, our data highlight the utility ofthe BALB/c strain as an excellent strain for social stress assays.

5. Conclusions

The repeated short and intense social stress used in the presentstudy induced two types of behavioural and physiological modi-fications. Dominant mice would develop a better coping strategyto stress, that may be due to enhanced sympathetic activity andfewer physiological and behavioural changes. Submissive mice,prone to defeat, seem unable to cope with stress, somatise, andas a result display enhanced inflammatory activation, eventuallypriming depression and severe physiological damage. These twodifferent subtypes of stress coping in this innately anxious mousestrain may underlie the mechanisms at the origin of diseases sus-ceptibility, as described as well in C57BL/6 or CD1 mice [32,33,45].Together the model used in this study was able to induce com-plex behavioural and physiological impairments across the entirebrain-gut axis similar to those observed in some GI and psychi-atric pathologies, warranting further behavioural characterisation.This model may also prove a useful paradigm for assessing noveltherapeutic strategies for stress-related GIT disorders such as IBS.

Acknowledgements

The authors would like to thank Caroline Browne, PatrickFitzgerald, Cliona O’Mahony, Dr. Romain-Daniel Gosselin, Dr.Declan McKernan and Dr. Dervla O’Malley for technical assis-tance. We valued the assistance of Dr. Silvia Melgar for analysisof colon morphology. The Alimentary Pharmabiotic Centre is aresearch centre funded by Science Foundation Ireland (SFI), throughthe Irish Government’s National Development Plan. The authorsand their work were supported by SFI (grant nos. 02/CE/B124and 07/CE/B1368). JFC is also funded by European Community’sSeventh Framework Programme; grant Number: FP7/2007–2013,Grant Agreement 201714.

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