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 hous-
ing 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 pro-
inflammatory 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: ute.kruegel@medizin.uni‑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 differ-
ences 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 behavio-
ral and immunological status of rats and must be consid-
ered 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 physio-
logical responses, stress will develop (Koolhaas et al. 2001).
In various experimental approaches, husbandry condi-
tions 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 toxicologi-
cal 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 gre-
garious species (Karolewicz et al. 2001), the withholding
of social contacts has been associated with stress-induced
behavioral changes (Bardo et al. 2001) and negative-stress-
related 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). There-
fore, social isolation of naturally collectively living animals
as rats, often a mandatory requirement in research or toxi-
cological examination, might have an important impact on
plasma cytokines and on motivated behavior; to our knowl-
edge, 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 hous-
ing 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 supplemen-
tary online material).
In the present study, the final body weight of group-
housed animals was significantly lower than that of individ-
ually 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 con-
specifics, 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, Sup-
plementary Table 1). Forced swimming of naïve group-
housed rats elicited acute responses in the peripheral
immune system (Himmerich et al. 2013). To prevent inter-
ference 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 circulat-
ing stress hormone ACTH was higher in isolated animals
(P  = 0.029, Supplementary Table 1). Wistar rats exposed
to chronic mild stress develop adrenal hypertrophy medi-
ated 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 modu-
lates 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 ele-
vated 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-inflam-
matory 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 mac-
rophages and therefore contributes to immune homeosta-
sis. It is further discussed to influence higher brain function
(Gadani et al. 2012), possibly mediating a higher suscepti-
bility 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 snap-
shots 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 hierar-
chies 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 mod-
els in basic research and toxicity studies.
Acknowledgments  This study was supported by the Claussen-
Simon-Foundation and the Deutsche Forschungsgemeinschaft (KR
3614/2-1). The funding had no role in study design, collection, analy-
sis or interpretation of data or in submission of the manuscript. Prof.
Himmerich received speaker honoraria from AstraZeneca, Lilly and
Arch Toxicol (2014) 88:853–855 855
Servier, consulting fees from Bristol-Myers Squibb and chemical sub-
stances 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 ampheta-
mine 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, Kapczin-
ski 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, Himmer-
ich S, Gedrich K, Kloiber S, Lucae S, Ising M, Uhr M, Holsboer
F, Pollmächer T (2008) Depression, comorbidities and the TNF-
alpha 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, Pfeils-
chifter J, Zwissler B, Mühl H (2005) A brief exposure to isoflu-
rane (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
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) Antide-
pressant effects of TNF-α blockade in an animal model of depres-
sion. 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 depressive-
like 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, immu-
nological, 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 hypotha-
lamic-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 dur-
ing 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
13