Arch Toxicol (2014) 88:853–855 DOI 10.1007/s00204-014-1203-0
LETTER TO THE EDITOR
The impact of social isolation on immunological parameters in rats Ute Krügel · Johannes Fischer · Katrin Bauer · Ulrich Sack · Hubertus Himmerich
Received: 4 December 2013 / Accepted: 15 January 2014 / Published online: 6 February 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract In various toxicological studies, single housing of rodents is preferred to standardize for regulatory purposes. However, housing conditions can have severe, often underestimated, impact on results in toxicological examinations. As different husbandry conditions have been shown to impose stress, we investigated their influence on plasma cytokines. Adult male Wistar rats were assigned to one group housed in cages of four and another housed singly for 28 days. Eight animals per group were tested in the forced swim test (FST) for symptoms of “behavioral despair,” and in another eight animals per group, plasma concentrations of the stress hormone ACTH, of the proinflammatory cytokines TNF-α, IFN-γ, IL-2 and IL-22, and of the anti-inflammatory cytokines IL-4 and IL-10 were analyzed. Group-housed animals had significantly lower body weight than individually housed animals. The FST
Ute Krügel and Johannes Fischer have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00204-014-1203-0) contains supplementary material, which is available to authorized users. U. Krügel (*) · J. Fischer Rudolf Boehm Institute of Pharmacology and Toxicology, Medical Faculty, University of Leipzig, Härtelstraße 16‑18, 04107 Leipzig, Germany e-mail: [email protected]‑leipzig.de K. Bauer · U. Sack Department of Clinical Immunology, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany H. Himmerich Department of Psychiatry and Psychotherapy, Medical Faculty, University of Leipzig, Semmelweisstraße 10, 04103 Leipzig, Germany
revealed symptoms of “behavioral despair” of individually housed rats accompanied by higher levels of ACTH and TNF-α but also of IL-4 and IL-10. No significant differences between housing conditions were found for IFN-γ, IL-2 and IL-22. Social isolation by husbandry conditions, apart from any other manipulation, alters the behavioral and immunological status of rats and must be considered when immunological effects are examined in various experimental protocols. Keywords Housing · Stress · Cytokines · Rats · IL-2 · IL-4 · IL-10 · IL-22 · TNF-α · IFN-γ
Housing conditions have an important influence on rodent research results (Everitt and Foster 2004). When alterations of social environment challenge the biological balance of an animal interfering with its homeostasis and if the animal is unable to maintain this homeostasis by behavioral or physiological responses, stress will develop (Koolhaas et al. 2001). In various experimental approaches, husbandry conditions interfere with the need to obtain data from individual laboratory animals by single housing. The observation, e.g., of individual food intake, metabolism or renal functions in metabolic cages or postsurgery recovery require lasting or at least intermittent individual housing. In most toxicological studies, single housing is also preferred to standardize for regulatory purposes (Verwer et al. 2007). However, the influence of housing conditions on study results is often underestimated. As rats, compared with mice, are more gregarious species (Karolewicz et al. 2001), the withholding of social contacts has been associated with stress-induced behavioral changes (Bardo et al. 2001) and negative-stressrelated physiological effects, e.g., reduced growth and adrenal hyper- or hypotrophy (Westenbroek et al. 2005).
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854
Changes in peripheral cytokines released from various cell types, e.g., macrophages, T cells and fibroblasts, were repeatedly reported in the development of stress-induced psychiatric disorders like depression and depression-like behavior in animals (Himmerich et al. 2008, 2013). Therefore, social isolation of naturally collectively living animals as rats, often a mandatory requirement in research or toxicological examination, might have an important impact on plasma cytokines and on motivated behavior; to our knowledge, however, the influence of single housing on plasma cytokines has not yet been investigated. Here, we point to the impact of 4 weeks of single housing of adult rats on biological markers of stress such as body weight, behavioral despair measured in the forced swim test (FST) and the stress hormone ACTH and on plasma cytokines compared with group housing without any other immune challenge (for methods see supplementary online material). In the present study, the final body weight of grouphoused animals was significantly lower than that of individually housed animals (P = 0.021, Supplementary Table 1). This observation agrees with multiple reports showing that social isolation, as another quality of stress, could increase weight gain, obviously not only because of boredom, reduced motor activity and/or frustration, but also because of lack of feeding competition (Fischer et al. 2012; Nyuyki et al. 2012). In this experiment, the enduring lack of social contacts, e.g., olfactory, tactile or thermal stimuli by conspecifics, not only induced a prolonged immobility in the FST, which has to be interpreted as increased helplessness and despair (Porsolt et al. 1977), but also reduced the time spent in escape attempts (P = 0.001 and P = 0.004, Supplementary Table 1). Forced swimming of naïve grouphoused rats elicited acute responses in the peripheral immune system (Himmerich et al. 2013). To prevent interference of the FST with effects of single housing, plasma concentrations were analyzed in separate rats not exposed to FST. In agreement with behavioral deficits, the circulating stress hormone ACTH was higher in isolated animals (P = 0.029, Supplementary Table 1). Wistar rats exposed to chronic mild stress develop adrenal hypertrophy mediated by elevated ACTH and higher corticosterone, both indicators for activation of the hypothalamic–pituitary– adrenal (HPA) axis (Garcia et al. 2009). This study also revealed higher levels of the pro-inflammatory cytokines TNF-α (P = 0.002) as well as of anti-inflammatory IL-4 (P = 0.025) and IL-10 (P = 0.003) in singly housed rats, but no significant changes in IL-2 (P = 0.093), IFN-γ (P = 0.881) and IL-22 (P = 0.381) all together suggesting that social isolation favors a generalized immune response (supplementary figure 1). TNF-α is released from monocytes and macrophages contributing to host defense, fever, sickness behavior and
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Arch Toxicol (2014) 88:853–855
depressive mood. Its hyperproduction is induced by various stress paradigms (Himmerich et al. 2013). TNF-α modulates HPA axis activity and increases ACTH production (Straub et al. 2011). Therefore, increase in ACTH could be a consequence of stimulated TNF-α production. Anti-inflammatory IL-4 and IL-10 produced by T2 helper (Th2) cells are rarely investigated in animal stress paradigms (Himmerich et al. 2013). Single housing elevated both cytokines. IL-10 is suggested to inhibit the (over)expression of pro-inflammatory cytokines (IL-1, INF-γ and TNF-α) and may serve to supersede pro-inflammatory cytokine signaling cascade but also modulates mice depressive-like behavior without detectable variations in INF-γ and TNF-α (Mesquita et al. 2008). IL-4 suppresses the production of Th1 cells and macrophages and therefore contributes to immune homeostasis. It is further discussed to influence higher brain function (Gadani et al. 2012), possibly mediating a higher susceptibility to live stressors, e.g., of asthma patients. This is not proved in rodent experimental approaches but should be considered in respective investigations. Besides the housing conditions, various additional parameters influence the concentrations of plasma cytokines and advise caution in their interpretation of snapshots in cytokine analysis. Plasma cytokine concentrations are influenced by onset, duration, sequence and impact of certain stimuli including, e.g., the treatment with control solutions (Krügel et al. 2013; Möller et al. 2013) or type of anesthesia (Hofstetter et al. 2005). Furthermore, reviewing the literature, species, strain, sex and age are often sources for divergent results. The immune response to bacterial lipopolysaccharide (LPS) is markedly influenced by social environment and gender of Wistar rats (Yee and Pendergast 2010). In Sprague–Dawley rats, another outbreed strain, social isolation beginning at weaning and vehicle treatment caused a reduction in the anti-inflammatory IL-4 and IL-6 (Möller et al. 2013). In opposite to rats, wild mice tend to be behaviorally isolated and form strong dominant hierarchies in groups resulting in social defeat of subordinates, which might be immunologically relevant and can interfere with results of further treatment (Karolewicz et al. 2001). In summary, single housing protocols for rats apart from other treatments provoke social-isolation-induced stress symptoms of behavioral despair accompanied by profound alterations in pro- and anti-inflammatory plasma cytokines. This has to be considered, in particular when behavioral or immunological data are consulted to evaluate animal models in basic research and toxicity studies. Acknowledgments This study was supported by the ClaussenSimon-Foundation and the Deutsche Forschungsgemeinschaft (KR 3614/2-1). The funding had no role in study design, collection, analysis or interpretation of data or in submission of the manuscript. Prof. Himmerich received speaker honoraria from AstraZeneca, Lilly and
Arch Toxicol (2014) 88:853–855 Servier, consulting fees from Bristol-Myers Squibb and chemical substances for study support from Lundbeck, AstraZeneca, Novartis and Wyeth. All other authors reported no biomedical financial interests or potential conflicts of interest. The authors would like to acknowledge the excellent technical assistance from Mrs. A.K. Krause and Mr. L. Feige.
References Bardo MT, Klebaur JE, Valone JM, Deaton C (2001) Environmental enrichment decreases intravenous self-administration of amphetamine in female and male rats. Psychopharmacology 155:278–284 Everitt JI, Foster PMD (2004) Laboratory animal science issues in the design and conduct of studies with endocrine-active compounds. ILAR J Natl Res Counc Inst Lab Anim Resour 45:417–424 Fischer CW, Liebenberg N, Elfving B, Lund S, Wegener G (2012) Isolation-induced behavioural changes in a genetic animal model of depression. Behav Brain Res 2230:85–91 Gadani SP, Cronk JC, Norris GT, Kipnis J (2012) IL-4 in the brain: a cytokine to remember. J Immunol 189:4213–4219 Garcia LS, Comim CM, Valvassori SS, Reus GZ, Stertz L, Kapczinski F, Gavioli EC, Quevedo J (2009) Ketamine treatment reverses behavioral and physiological alterations induced by chronic mild stress in rats. Prog Neuropsychopharmacol Biol Psychiatry 33:450–455 Himmerich H, Fulda S, Linseisen J, Seiler H, Wolfram G, Himmerich S, Gedrich K, Kloiber S, Lucae S, Ising M, Uhr M, Holsboer F, Pollmächer T (2008) Depression, comorbidities and the TNFalpha system. Eur Psychiatr 23:421–429 Himmerich H, Fischer J, Bauer K, Kirkby KC, Sack U, Krügel U (2013) Stress-induced cytokine changes in rats. Eur Cytokine Netw 24:97–103 Hofstetter C, Flondor M, Boost KA, Koehler P, Bosmann M, Pfeilschifter J, Zwissler B, Mühl H (2005) A brief exposure to isoflurane (50 s) significantly impacts on plasma cytokine levels in endotoxemic rats. Int Immunopharmacol 5:1519–1522 Karolewicz B, Paul IA (2001) Group housing of mice increases immobility and antidepressant sensitivity in the forced swim and tail suspension tests. Eur J Pharmacol 415:197–201
855 Koolhaas JM, Baumans V, Blom HJM, von Holst D, Timmermans PJA, Wiepkema PR (2001) Behaviour, stress and well-being. Principles of laboratory animal science. Elsevier, Amsterdam, pp 75–99 Krügel U, Fischer J, Radicke S, Sack U, Himmerich H (2013) Antidepressant effects of TNF-α blockade in an animal model of depression. J Psychiatr Res 47:611–616 Mesquita AR, Correia-Neves M, Roque S, Castro AG, Vieira P, Pedrosa J, Palha JA, Sousa N (2008) IL-10 modulates depressivelike behavior. J Psychiatr Res 43:89–97 Möller M, Du Preez JL, Viljoen FP, Berk M, Emsley R, Harvey BH (2013) Social isolation rearing induces mitochondrial, immunological, neurochemical and behavioural deficits in rats, and is reversed by clozapine or N-acetyl cysteine. Brain Behav Immun 30:156–167 Nyuyki KD, Beiderbeck DI, Lukas M, Neumann ID, Reber SO (2012) Chronic subordinate colony housing (CSC) as a model of chronic psychosocial stress in male rats. PLoS ONE 7(12):e52371. doi:10.1371/journal.pone.0052371 Porsolt RD, Bertin A, Jalfre M (1977) Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 229:327–336 Straub RH, Buttgereit F, Cutolo M (2011) Alterations of the hypothalamic-pituitary-adrenal axis in systemic immune diseases: a role for misguided energy regulation. Clin Exp Rheumatol 29(Suppl 68):23–31 Verwer CM, van der Ven LTM, van den Bos R, Hendriksen CFM (2007) Effects of housing condition on experimental outcome in a reproduction toxicity study. Regul Toxicol Pharmacol RTP 48:184–193 Westenbroek C, Snijders TAB, den Boer JA, Gerrits M, Fokkema DS, Ter Horst GJ (2005) Pair-housing of male and female rats during chronic stress exposure results in gender-specific behavioral responses. Horm Behav 47:620–628 Yee JR, Prendergast BJ (2010) Sex-specific social regulation of inflammatory responses and sickness behaviors. Brain Behav Immun 24:942–951
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LETTER TO THE EDITOR
The impact of social isolation on immunological parameters in rats Ute Krügel · Johannes Fischer · Katrin Bauer · Ulrich Sack · Hubertus Himmerich
Received: 4 December 2013 / Accepted: 15 January 2014 / Published online: 6 February 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract In various toxicological studies, single housing of rodents is preferred to standardize for regulatory purposes. However, housing conditions can have severe, often underestimated, impact on results in toxicological examinations. As different husbandry conditions have been shown to impose stress, we investigated their influence on plasma cytokines. Adult male Wistar rats were assigned to one group housed in cages of four and another housed singly for 28 days. Eight animals per group were tested in the forced swim test (FST) for symptoms of “behavioral despair,” and in another eight animals per group, plasma concentrations of the stress hormone ACTH, of the proinflammatory cytokines TNF-α, IFN-γ, IL-2 and IL-22, and of the anti-inflammatory cytokines IL-4 and IL-10 were analyzed. Group-housed animals had significantly lower body weight than individually housed animals. The FST
Ute Krügel and Johannes Fischer have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00204-014-1203-0) contains supplementary material, which is available to authorized users. U. Krügel (*) · J. Fischer Rudolf Boehm Institute of Pharmacology and Toxicology, Medical Faculty, University of Leipzig, Härtelstraße 16‑18, 04107 Leipzig, Germany e-mail: [email protected]‑leipzig.de K. Bauer · U. Sack Department of Clinical Immunology, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany H. Himmerich Department of Psychiatry and Psychotherapy, Medical Faculty, University of Leipzig, Semmelweisstraße 10, 04103 Leipzig, Germany
revealed symptoms of “behavioral despair” of individually housed rats accompanied by higher levels of ACTH and TNF-α but also of IL-4 and IL-10. No significant differences between housing conditions were found for IFN-γ, IL-2 and IL-22. Social isolation by husbandry conditions, apart from any other manipulation, alters the behavioral and immunological status of rats and must be considered when immunological effects are examined in various experimental protocols. Keywords Housing · Stress · Cytokines · Rats · IL-2 · IL-4 · IL-10 · IL-22 · TNF-α · IFN-γ
Housing conditions have an important influence on rodent research results (Everitt and Foster 2004). When alterations of social environment challenge the biological balance of an animal interfering with its homeostasis and if the animal is unable to maintain this homeostasis by behavioral or physiological responses, stress will develop (Koolhaas et al. 2001). In various experimental approaches, husbandry conditions interfere with the need to obtain data from individual laboratory animals by single housing. The observation, e.g., of individual food intake, metabolism or renal functions in metabolic cages or postsurgery recovery require lasting or at least intermittent individual housing. In most toxicological studies, single housing is also preferred to standardize for regulatory purposes (Verwer et al. 2007). However, the influence of housing conditions on study results is often underestimated. As rats, compared with mice, are more gregarious species (Karolewicz et al. 2001), the withholding of social contacts has been associated with stress-induced behavioral changes (Bardo et al. 2001) and negative-stressrelated physiological effects, e.g., reduced growth and adrenal hyper- or hypotrophy (Westenbroek et al. 2005).
13
854
Changes in peripheral cytokines released from various cell types, e.g., macrophages, T cells and fibroblasts, were repeatedly reported in the development of stress-induced psychiatric disorders like depression and depression-like behavior in animals (Himmerich et al. 2008, 2013). Therefore, social isolation of naturally collectively living animals as rats, often a mandatory requirement in research or toxicological examination, might have an important impact on plasma cytokines and on motivated behavior; to our knowledge, however, the influence of single housing on plasma cytokines has not yet been investigated. Here, we point to the impact of 4 weeks of single housing of adult rats on biological markers of stress such as body weight, behavioral despair measured in the forced swim test (FST) and the stress hormone ACTH and on plasma cytokines compared with group housing without any other immune challenge (for methods see supplementary online material). In the present study, the final body weight of grouphoused animals was significantly lower than that of individually housed animals (P = 0.021, Supplementary Table 1). This observation agrees with multiple reports showing that social isolation, as another quality of stress, could increase weight gain, obviously not only because of boredom, reduced motor activity and/or frustration, but also because of lack of feeding competition (Fischer et al. 2012; Nyuyki et al. 2012). In this experiment, the enduring lack of social contacts, e.g., olfactory, tactile or thermal stimuli by conspecifics, not only induced a prolonged immobility in the FST, which has to be interpreted as increased helplessness and despair (Porsolt et al. 1977), but also reduced the time spent in escape attempts (P = 0.001 and P = 0.004, Supplementary Table 1). Forced swimming of naïve grouphoused rats elicited acute responses in the peripheral immune system (Himmerich et al. 2013). To prevent interference of the FST with effects of single housing, plasma concentrations were analyzed in separate rats not exposed to FST. In agreement with behavioral deficits, the circulating stress hormone ACTH was higher in isolated animals (P = 0.029, Supplementary Table 1). Wistar rats exposed to chronic mild stress develop adrenal hypertrophy mediated by elevated ACTH and higher corticosterone, both indicators for activation of the hypothalamic–pituitary– adrenal (HPA) axis (Garcia et al. 2009). This study also revealed higher levels of the pro-inflammatory cytokines TNF-α (P = 0.002) as well as of anti-inflammatory IL-4 (P = 0.025) and IL-10 (P = 0.003) in singly housed rats, but no significant changes in IL-2 (P = 0.093), IFN-γ (P = 0.881) and IL-22 (P = 0.381) all together suggesting that social isolation favors a generalized immune response (supplementary figure 1). TNF-α is released from monocytes and macrophages contributing to host defense, fever, sickness behavior and
13
Arch Toxicol (2014) 88:853–855
depressive mood. Its hyperproduction is induced by various stress paradigms (Himmerich et al. 2013). TNF-α modulates HPA axis activity and increases ACTH production (Straub et al. 2011). Therefore, increase in ACTH could be a consequence of stimulated TNF-α production. Anti-inflammatory IL-4 and IL-10 produced by T2 helper (Th2) cells are rarely investigated in animal stress paradigms (Himmerich et al. 2013). Single housing elevated both cytokines. IL-10 is suggested to inhibit the (over)expression of pro-inflammatory cytokines (IL-1, INF-γ and TNF-α) and may serve to supersede pro-inflammatory cytokine signaling cascade but also modulates mice depressive-like behavior without detectable variations in INF-γ and TNF-α (Mesquita et al. 2008). IL-4 suppresses the production of Th1 cells and macrophages and therefore contributes to immune homeostasis. It is further discussed to influence higher brain function (Gadani et al. 2012), possibly mediating a higher susceptibility to live stressors, e.g., of asthma patients. This is not proved in rodent experimental approaches but should be considered in respective investigations. Besides the housing conditions, various additional parameters influence the concentrations of plasma cytokines and advise caution in their interpretation of snapshots in cytokine analysis. Plasma cytokine concentrations are influenced by onset, duration, sequence and impact of certain stimuli including, e.g., the treatment with control solutions (Krügel et al. 2013; Möller et al. 2013) or type of anesthesia (Hofstetter et al. 2005). Furthermore, reviewing the literature, species, strain, sex and age are often sources for divergent results. The immune response to bacterial lipopolysaccharide (LPS) is markedly influenced by social environment and gender of Wistar rats (Yee and Pendergast 2010). In Sprague–Dawley rats, another outbreed strain, social isolation beginning at weaning and vehicle treatment caused a reduction in the anti-inflammatory IL-4 and IL-6 (Möller et al. 2013). In opposite to rats, wild mice tend to be behaviorally isolated and form strong dominant hierarchies in groups resulting in social defeat of subordinates, which might be immunologically relevant and can interfere with results of further treatment (Karolewicz et al. 2001). In summary, single housing protocols for rats apart from other treatments provoke social-isolation-induced stress symptoms of behavioral despair accompanied by profound alterations in pro- and anti-inflammatory plasma cytokines. This has to be considered, in particular when behavioral or immunological data are consulted to evaluate animal models in basic research and toxicity studies. Acknowledgments This study was supported by the ClaussenSimon-Foundation and the Deutsche Forschungsgemeinschaft (KR 3614/2-1). The funding had no role in study design, collection, analysis or interpretation of data or in submission of the manuscript. Prof. Himmerich received speaker honoraria from AstraZeneca, Lilly and
Arch Toxicol (2014) 88:853–855 Servier, consulting fees from Bristol-Myers Squibb and chemical substances for study support from Lundbeck, AstraZeneca, Novartis and Wyeth. All other authors reported no biomedical financial interests or potential conflicts of interest. The authors would like to acknowledge the excellent technical assistance from Mrs. A.K. Krause and Mr. L. Feige.
References Bardo MT, Klebaur JE, Valone JM, Deaton C (2001) Environmental enrichment decreases intravenous self-administration of amphetamine in female and male rats. Psychopharmacology 155:278–284 Everitt JI, Foster PMD (2004) Laboratory animal science issues in the design and conduct of studies with endocrine-active compounds. ILAR J Natl Res Counc Inst Lab Anim Resour 45:417–424 Fischer CW, Liebenberg N, Elfving B, Lund S, Wegener G (2012) Isolation-induced behavioural changes in a genetic animal model of depression. Behav Brain Res 2230:85–91 Gadani SP, Cronk JC, Norris GT, Kipnis J (2012) IL-4 in the brain: a cytokine to remember. J Immunol 189:4213–4219 Garcia LS, Comim CM, Valvassori SS, Reus GZ, Stertz L, Kapczinski F, Gavioli EC, Quevedo J (2009) Ketamine treatment reverses behavioral and physiological alterations induced by chronic mild stress in rats. Prog Neuropsychopharmacol Biol Psychiatry 33:450–455 Himmerich H, Fulda S, Linseisen J, Seiler H, Wolfram G, Himmerich S, Gedrich K, Kloiber S, Lucae S, Ising M, Uhr M, Holsboer F, Pollmächer T (2008) Depression, comorbidities and the TNFalpha system. Eur Psychiatr 23:421–429 Himmerich H, Fischer J, Bauer K, Kirkby KC, Sack U, Krügel U (2013) Stress-induced cytokine changes in rats. Eur Cytokine Netw 24:97–103 Hofstetter C, Flondor M, Boost KA, Koehler P, Bosmann M, Pfeilschifter J, Zwissler B, Mühl H (2005) A brief exposure to isoflurane (50 s) significantly impacts on plasma cytokine levels in endotoxemic rats. Int Immunopharmacol 5:1519–1522 Karolewicz B, Paul IA (2001) Group housing of mice increases immobility and antidepressant sensitivity in the forced swim and tail suspension tests. Eur J Pharmacol 415:197–201
855 Koolhaas JM, Baumans V, Blom HJM, von Holst D, Timmermans PJA, Wiepkema PR (2001) Behaviour, stress and well-being. Principles of laboratory animal science. Elsevier, Amsterdam, pp 75–99 Krügel U, Fischer J, Radicke S, Sack U, Himmerich H (2013) Antidepressant effects of TNF-α blockade in an animal model of depression. J Psychiatr Res 47:611–616 Mesquita AR, Correia-Neves M, Roque S, Castro AG, Vieira P, Pedrosa J, Palha JA, Sousa N (2008) IL-10 modulates depressivelike behavior. J Psychiatr Res 43:89–97 Möller M, Du Preez JL, Viljoen FP, Berk M, Emsley R, Harvey BH (2013) Social isolation rearing induces mitochondrial, immunological, neurochemical and behavioural deficits in rats, and is reversed by clozapine or N-acetyl cysteine. Brain Behav Immun 30:156–167 Nyuyki KD, Beiderbeck DI, Lukas M, Neumann ID, Reber SO (2012) Chronic subordinate colony housing (CSC) as a model of chronic psychosocial stress in male rats. PLoS ONE 7(12):e52371. doi:10.1371/journal.pone.0052371 Porsolt RD, Bertin A, Jalfre M (1977) Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 229:327–336 Straub RH, Buttgereit F, Cutolo M (2011) Alterations of the hypothalamic-pituitary-adrenal axis in systemic immune diseases: a role for misguided energy regulation. Clin Exp Rheumatol 29(Suppl 68):23–31 Verwer CM, van der Ven LTM, van den Bos R, Hendriksen CFM (2007) Effects of housing condition on experimental outcome in a reproduction toxicity study. Regul Toxicol Pharmacol RTP 48:184–193 Westenbroek C, Snijders TAB, den Boer JA, Gerrits M, Fokkema DS, Ter Horst GJ (2005) Pair-housing of male and female rats during chronic stress exposure results in gender-specific behavioral responses. Horm Behav 47:620–628 Yee JR, Prendergast BJ (2010) Sex-specific social regulation of inflammatory responses and sickness behaviors. Brain Behav Immun 24:942–951
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