Brain, Behavior, and Immunity 44 (2015) 235246

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Brain, Behavior, and Immunity

journal homepage: www.elsevier.com/locate/ybrbi

Programming of formalin-induced nociception by neonatal LPS

exposure: Maintenance by peripheral and central neuroimmune activity
Ihssane Zouikr a,b,c,, Abdulrzag F. Ahmed b, Jay C. Horvat b,c, Kenneth W. Beagley d, Vicki L. Clifton e,
Allyson Ray a, Rick F. Thorne b,c, Andrew G. Jarnicki c, Philip M. Hansbro c, Deborah M. Hodgson a
a

Laboratory of Neuroimmunology, School of Psychology, University of Newcastle, Newcastle, New South Wales, Australia
School of Biomedical Sciences and Pharmacy, University of Newcastle, Newcastle, New South Wales, Australia
Hunter Medical Research Institute, Newcastle, New South Wales, Australia
d
Institute of Health Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia
e
Robinson Institute, University of Adelaide, Adelaide, South Australia, Australia
b
c

a r t i c l e

i n f o

Article history:
Received 17 July 2014
Received in revised form 10 October 2014
Accepted 23 October 2014
Available online 31 October 2014
Keywords:
LPS
Neonatal immune challenge
IL-1b
Hippocampus
Formalin test
Pain
Mast cell
Degranulation
Cytokines
Inammation

a b s t r a c t
The immune and nociceptive systems are shaped during the neonatal period where they undergo netuning and maturation. Painful experiences during this sensitive period of development are known to
produce long-lasting effects on the immune and nociceptive responses. It is less clear, however, whether
inammatory pain responses are primed by neonatal exposure to mild immunological stimuli, such as
with lipopolysaccharide (LPS). Here, we examine the impact of neonatal LPS exposure on inammatory
pain responses, peripheral and hippocampal interleukin-1b (IL-1b), as well as mast cell number and
degranulation in preadolescent and adult rats. Wistar rats were injected with LPS (0.05 mg/kg IP, Salmonella enteritidis) or saline on postnatal days (PNDs) 3 and 5 and later subjected to the formalin test at
PNDs 22 and 8097. At both time-points, and one-hour after formalin injection, blood and hippocampus
were collected for measuring circulating and central IL-1b levels using ELISA and Western blot, respectively. Paw tissue was also isolated to assess mast cell number and degree of degranulation using Toluidine Blue staining. Behavioural analyses indicate that at PND 22, LPS-challenged rats displayed enhanced
inching (p < .01) and licking (p < .01) in response to formalin injection. At PNDs 8097, LPS-challenged
rats exhibited increased inching (p < .05), an effect observed in males only. Furthermore, neonatal LPS
exposure enhanced circulating IL-1b and mast cell degranulation in preadolescent but not adult rats following formalin injection. Hippocampal IL-1b levels were increased in LPS-treated adult but not preadolescent rats in response to formalin injection. These data suggest neonatal LPS exposure produces
developmentally regulated changes in formalin-induced behavioural responses, peripheral and central
IL-1b levels, as well as mast cell degranulation following noxious stimulation later in life. These ndings
highlight the importance of immune activation during the neonatal period in shaping immune response
and pain sensitivity later in life. This is of clinical relevance given the high prevalence of bacterial infection during the neonatal period, particularly in the vulnerable population of preterm infants admitted to
neonatal intensive care units.
Crown Copyright 2014 Published by Elsevier Inc. All rights reserved.

1. Introduction
The neonatal period represents a unique developmental phase
during which both the nociceptive and immune systems are
highly plastic. The maturation and ne-tuning of pain processing
and immune responses are dependent on environmental,
Corresponding author at: Hunter Medical Research Institute (HMRI), Level 3
East. 1/Kookaburra Circuit, New Lambton Heights, New South Wales 2305,
Australia. Tel.: +61 240420812; fax: +61 249216980.
E-mail address: Ihssane.Zouikr@uon.edu.au (I. Zouikr).
http://dx.doi.org/10.1016/j.bbi.2014.10.014
0889-1591/Crown Copyright 2014 Published by Elsevier Inc. All rights reserved.

immunological and sensory stimuli encountered during this vulnerable period of development (Adkins et al., 2004; Fitzgerald,
1995, 2005; Whitelaw and Parkin, 1988). Previous studies have
demonstrated that exposure to the bacterial mimetic, lipopolysaccharide (LPS) during the neonatal period produces long-term alterations in the immunological and neuroendocrine responses later in
life (Ellis et al., 2005; Hodgson et al., 2001; Shanks et al., 1995,
2000; Spencer et al., 2006; Walker et al., 2009a, 2004). There is
now good evidence that pain behaviour can also be altered by early
pain experience (Beggs et al., 2012; Ren et al., 2004; Walker et al.,
2009b; Wang et al., 2004). Of particular interest, experimental

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administration of LPS in rodents and humans has been reported to

produce transient immune activation and pain facilitation including thermal hyperalgesia, mechanical allodynia and hyperalgesia
associated with an upregulation of IL-1b in the circulation as well
as in the inammed tissue (Hutchinson et al., 2013; Mason, 1993;
Watkins and Maier, 2000; Watkins et al., 1995; Wegner et al.,
2014). These studies assessed pain responses following LPS administration in adulthood. However, less is known about the impact of
neonatal LPS exposure on future inammatory pain responses.
Immune system to brain communication modulates a variety of
physiological responses including pain (Dantzer and Kelley, 2007;
Dantzer et al., 2008; Maier, 2003; Marchand et al., 2005; Ren and
Dubner, 2010). Pro-inammatory cytokines, particularly IL-1b,
released by immune cells in response to infection play a key role
in this bidirectional communication (Besedovsky and del Rey,
2011; Maier, 2003; Watkins et al., 2007; Watkins and Maier,
2005). Central action of pro-inammatory cytokines produces sickness behaviours such as loss of appetite, sleepiness, fever and fatigue (Dantzer, 2001; Kent et al., 1992). Importantly, hyperalgesia is
also considered an integral part of sickness behaviour aimed at
reducing activity (Watkins and Maier, 2000, 2005; Watkins et al.,
1994a,b). IL-1b, among the many pro-inammatory cytokines that
have been implicated in peripherally and centrally-mediated
hyperalgesia, is the most well characterized (Ferreira et al., 1988;
Fukuoka et al., 1994; Oka et al., 1993; Watkins et al., 1994b). Intracerebroventricular (ICV) injection of recombinant IL-1b (rIL-1b) in
rats signicantly decreases paw lick latency to thermal noxious
stimulus (Oka et al., 1993). Blocking the activation of IL-1b using
an IL-1 receptor antagonist attenuated thermal hyperalgesia (Oka
et al., 1993), and formalin-induced hyperalgesia (Watkins et al.,
1997).
In addition to its traditional role in memory formation and consolidation (Bird and Burgess, 2008), the hippocampus has recently
been implicated in pain modulation via upregulation of IL-1b
expression (del Rey et al., 2011) and is considered to be a critical
supraspinal region involved in the transition from acute to chronic
pain (Apkarian, 2008; Apkarian et al., 2011). Rats that were subjected to spare nerve injury exhibited mechanical hyperalgesia
associated with signicant increase in IL-1b mRNA levels in the
hippocampus 10 days and 24 days following injury (del Rey et al.,
2011). Neonatal LPS exposure is also associated with an elevation
of IL-1b protein expression in the hippocampus following an emotional stress in adulthood (Walker et al., 2010). Since pain is an
emotional aversive experience, we hypothesized that neonatal
LPS exposure is likely to produce increased pain sensitivity in
inammatory pain models (i.e. formalin test) via upregulation of
hippocampal IL-1b.
An increasing body of literature documents the important role
of immune cells in the initiation and maintenance of chronic pain
(Austin et al., 2012; Costigan et al., 2009; Grace et al., 2011). The
majority of documented research in this area focuses mainly on
astrocytes, microglia, and T cells. Signicantly, very few studies
have investigated the effects of mast cells in the pathogenesis of
pain. This knowledge gap is particularly surprising given the
well-characterized role of mast cells in mediating inammatory
cascades within tissue, which corresponds with the release of a
number of hyperalgesic agents, and their close proximity to nociceptors in the peripheral nervous system and dura within the
CNS (Kumar and Sharma, 2010; Silver and Curley, 2013). Upon
degranulation (i.e. following physical or chemical injury), mast
cells release algogenic substances such as histamine, bradykinin
and substance P, that contribute directly to the hyper-sensitization
of local nociceptor which results in hyperalgesia (Xanthos et al.,
2011). Nerve-resident peripheral nerve mast cells are the rst to
be activated following tissue damage and contribute to the recruitment of neutrophils and macrophages (Zuo et al., 2003). More

importantly, mast cells can release IL-1b following infection, toxic

injury or trauma, subsequently contributing to neuropathic hyperalgesia (Sandler et al., 2007; Sommer et al., 1999).
The aim of the present study was to investigate the behavioural
and immune changes in response to neonatal LPS exposure and
subsequent formalin injection in preadolescent (i.e. PND 22) and
adult (i.e. PNDs 8097) rats, with particular focus on circulating
and hippocampal IL-1b as well as on mast cell number and degranulation in tissue following formalin injection. Our hypothesis is
that neonatal LPS exposure will produce both peripheral and central changes in mast cell and IL-1b responses that will accompany
the formalin-induced behavioural hyperalgesia.
2. Materials and methods
2.1. Experimental procedures
23 experimentally nave female Wistar rats were obtained from
the University of Newcastle Animal House and allowed one week
acclimatization prior to mating in a vivarium. Mating resulted in
184 offspring, 116 of which were used in this study; the remaining
pups were allocated to other studies. After two weeks, the male
was removed from the harem upon which dams were housed individually in custom designed polycarbonate-perspex home boxes
(43.5 cm  28 cm  12.5 cm; Mascot Wire Works, Sydney, Australia) until delivery (PND 1). Pups were left undisturbed with their
mother until PND 3 and 5 when they were briey removed from
their home boxes, weighed and injected (IP) with either LPS (Salmonella enterica, serotype enteritidis; Sigma-Aldrich Chemical Co.,
USA, dissolved in 20 lL sterile pyrogen-free saline, 50 lg/kg) or
an equivolume of sterile pyrogen-free saline (Livingstone International, Australia). All pups from the same litter were treated
identically.
Following neonatal drug administration, pups were left undisturbed with their mother until testing, when they were exposed
to the formalin test as described below. Rats were randomly
assigned testing days based on the two ages examined: PND 22
and PNDs 8097, with a maximum of three pups per litter being
assigned to each group. Following the formalin test, blood, paw,
and brain tissue samples were collected and subsequently used
for assessment of plasma IL-1b levels, histological analysis of mast
cells inltration and degranulation, and IL-1b protein levels in the
hippocampus. Animals were distributed evenly from all litters
used per treatment to avoid potential litter bias. Until their allocated testing day, rats were maintained in a temperature
(21 1 C) and humidity (45%) controlled environment, under a
12 h/12 h light-dark cycle (light on 0600 h) with food and water
available ad libitum.
All experiments were carried out in accordance with the 2013
National Health and Medical Research Council of Australian Code
for the care and use of animals for scientic purposes. All procedures were reviewed and approved by the Ethics committee of
the University of Newcastle (Ethics approval no. A-2010-127).
2.2. Formalin behavioural testing and analysis
Preadolescent rats (PND 22) were injected with 1.1% formalin
into the plantar surface of the left hindpaw and adult rats (PND
8097) were injected with 2.25% formalin into the plantar surface
of the right hindpaw. Injections were made using a 31G needle at
PND 22 and a 30 G needle at PNDs 8097. The choice of formalin
concentration, site of injection and injection volume (10 lL for
PND 22 rats and 50 lL for PNDs 8097 rats), testing apparatus as
well as behavioural scoring techniques are all described in detail
in our previous studies (Zouikr et al., 2014a,b; Zouikr et al.,

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2013). Behaviourally, no saline-injected rats were included in this

study since it has been previously shown that rats subjected to a
subcutaneous injection of saline into the plantar surface of the
hindpaw do not display inching or licking behaviour (Butkevich
and Vershinina, 2001; Guy and Abbott, 1992; Okuda et al., 2001).

2.3. Blood collection and enzyme-linked-immunosorbent assay (ELISA)

One hour following the formalin test, preadolescent and adult
rats were deeply anaesthetized with an overdose of Lethabarb
(2 mg/kg IP; Virbac, Pty. Ltd, Milperra, Australia) and blood was
collected by briey restraining the rat (<40 s) and puncturing the
saphenous vein using a 21G needle and collecting 170180 ll
of blood into EDTA-coated tubes (Livingstone International, Australia). Blood was then centrifuged at 1000g for 20 min at 4 C prior
to collecting the supernatant (plasma), which was stored at 20 C
until assayed. Circulating levels of IL-1b concentrations were
determined using a Quantikine Rat IL-1b kit (R&D Systems, USA)
according to the manifacturers instructions. The mean recovery
of IL-1b in plasma was 98% with a mean inter- and intra-assay variability of 4.7% and 5.53% respectively.

2.4. IL-1b Western blot methods

30 rats (PND 22 = 20 (8 saline; 12 LPS); PNDs 8097 = 10 rats (5
saline; 5 LPS); equally distributed between males and females)
were used to assess IL-1b protein levels in the hippocampus. 1 h
following formalin behavioural testing, animals were deeply
anaesthetized with an overdose of Pentobarbitol Sodium (Lethabarb; 2 mL/kg IP, Virbac, Pty., Ltd, Miperra, Australia). After
decapitation, brains were rapidly removed and hippocampus isolated. Samples were homogenized in ice-cold NDE lysis buffer
(1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 66 mM
EDTA, 10 mM Tris-HCl, pH 7.4) supplemented with protease and
phosphatase inhibitors (Complete protease inhibitor cocktail and
PhoSTOP; Roche Applied Science, Australia) as previously
described (Sadeqzadeh et al., 2011). Samples containing equal protein concentrations (30 lg/mL) were prepared in lithium dodecyl
sulphate sample buffer (Invitrogen) prior to electrophoresis. In
order to analyse IL-1b (37 kDa), samples were applied to commercial acrylamide gels (NuPAGE 12% Bis-Tris gels (Novex, Invitrogen) using MES SDS running Buffer. Electrophoresis was
performed in SDS running buffer before transferring proteins to
nitrocellulose membranes using a semidry blotting system (iBlot
Transfer Stack and iBlot device, Invitrogen). Membranes were
washed in TBST (50 mM Tris, 150 mM, 150 mM NaCl, 0.05% Tween
20, pH 7.6). Western blotting was performed by rst blocking the
membranes in 5% skim milk in TBST 1 h at room temperature.
Membranes were then incubated with the primary antibodies
(anti-IL1b, rabbit polyclonal, 1:200, anti-GAPDH, rabbit polyclonal,
1:5000; Santa Cruz) overnight at 4 C. Following 3  5 min washes
in TBST, membranes were incubated with the secondary antibody
(Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, Biorad).
Immunocomplexes were detected by the ECL-based detection system (Sadeqzadeh et al., 2011) with the bands visualized using a
cooled charge-coupled device camera system (Fuji-LAS-4000, Fujilm Life Science Systems). Bands were quantitated using the MultiGuage v3.0 software package (Fujilm Life Science Systems). The
optical density (OD) obtained for IL-1b was divided by the OD of
the endogenous control gene product GAPDH yielding a ratio.
The changes in IL-1b were normalized against GAPDH and
expressed as fold change relative to saline control.

237

2.5. Paw histology

One hour following formalin/saline injection, paw tissue (without bones; 35 mm from formalin-injected site) from both treatment and age groups were extracted and xed in 10% Neutral
Buffered Formalin (NBF pH 7.4; Fronine, Lomb Scientic, Taren
Point, Australia) for 48 h. Paw tissue was then embedded in parafn, sectioned (5 lm thickness), and rehydrated as follows: sections
were immersed in xylene for 2 min. After sequential immersion in
solutions of decreasing ethanol concentrations (absolute ethanol
(2  2 min), 90% ethanol (2 min), 70% ethanol (2 min)), sections
were then immersed in water for 2 min. After incubation in 1%
Toluidine Blue for 1 min, sections were blot dried then dipped
two times in absolute ethanol. Ethanol was then cleared by
immersing the sections in xylene for 2 min. Sections were nally
mounted using Ultramount (No. 4, 500 mL, Fronine Pathology,
ThermoFisher Scientic, Australia). Observations of the slides were
made under a light microscope (Carl Zeiss Axioplan 2 imaging,
Germany).
The total number of mast cells and their degree of degranulation were scored by a researcher blinded to the different treatment
groups. Total mast cell number was scored as the average total
number of mast cells observed per eld (20x magnication = 144.37 mm2) from 10 snapshots (taken from 10 random
locations in a given slide). One slide was used per treatment (formalin, non-injected, saline-injected) per rat. Degree of mast cell
degranulation was calculated using the following equation: degree
of degranulation = (no. of mast cells in scale 1  0 + no. of mast
cells in scale 2  1 + no. of mast cells in scale 3  2 + no. of mast
cells in scale 4  3)/(total no. of mast cells). Non-degranulating
mast cells were characterized as scale 1, mast cells with a low or
high level of degranulation were characterized as scale 2 or 3,
respectively, and completely degranulated mast cells were characterized as scale 4. Cell enumeration and degranulation was conducted using image analysis software Image J (version 1.4.3.67).
Two saline-injected rats were added at each time-point to
demonstrate that the physical process of intraplantar injection of
1050 lL volume of liquid per se does not affect mast cell number
or degranulation.

3. Data analysis
Data analyses were conducted using the Statistical Package for
the Social Sciences, Version 20 (SPSS, Inc.). Two methods were
applied to analyze the behavioural data: during the early phase
(the rst 5 min), an independent samples t-test was used where
inching and licking at 5 min were both analyzed as outcome variables and treatment and sex were treated as xed factors. During
the late phase, the Area Under the Curve (AUC) for both inching
and licking was calculated as the sum of responses from 10 to
40 min for preadolescent rats and from 10 to 60 min for adult rats.
The AUC for inching or licking was analyzed as outcome variables
and treatment and sex were treated as xed factors. This analysis
determines the temporal pattern of the response and was analyzed
using univariate between subjects ANOVA. Plots of the mean levels
of inching and licking, as well as a histogram displaying the AUC
for both responses, were generated. male to female ratio and litter size were used as covariates and reported only when signicantly contributing to the data.
For all other data, because the variance in the data was not constant (larger variability), a Linear Mixed Model (LMM) was applied
since it can handle the failure of constant variance assumption.
Planned comparisons were performed using the Least Signicant
Differences (LSD). The signicance level was set at p = .05.

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4. Results
4.1. Neonatal LPS exposure induces long-term alterations in formalininduced nociception
4.1.1. Preadolescent rats (PND 22)
At this time-point, the two-way interaction of sex and
treatment did not reach signicance for either Flinching
[F(1, 26.58) =.036, p > .05] or Licking [F(1,42.02) = 2.74, p > .05].
A clear biphasic response was observed in both inching and
licking behaviours (Fig. 1). During the early phase, no signicant
differences were found in inching or licking the injected paw
between the two groups. However, during the late phase, analysis of the AUC in inching and licking responses revealed a significant main effect of treatment [F(1,23) = 13.88, p < .01;
F(1,23) = 13.13, p < .01; respectively] with LPS-challenged rats
displaying higher inching responses and spending more time
licking the injected paw compared to the saline-challenged group
(Fig. 1F and H).

4.1.2. Adult rats (PND 8097)

Although no signicant differences were observed between the
two groups in terms of licking behaviour [F(1,36) = .755, p > .05;
Fig. 2D and E], analysis of the AUC in inching the injected paw
during the late phase revealed a signicant main effect of treatment [F(1,36) = 5.49, p < .05] with LPS-challenged rats exhibiting
higher inching responses compared to saline-treated rats. Univariate ANOVA analysis of the AUC in inching responses also
revealed a signicant main effect of sex [F(1,36) = 5.23, p < .05].
The two-way interaction of sex and treatment did not reach significance [F(1,36) = .794, p > .05]. In males alone, LPS-treated rats displayed signicantly higher inching behaviour during the late
phase compared to saline-treated rats (p < .05; Fig. 2A). However,
females from both treatment groups displayed a similar inching
behaviour throughout the one-hour recording (Fig. 2B). The twoway interaction of sex and treatment did not reach signicance
for licking behaviour in adult rats [F(1,36) = .495, p > .05]. Additionally, no signicant differences were found during the early phase in
both inching and licking behaviours (Fig. 2).

Fig. 1. Time-course of inching (A and B) and licking (C and D) responses in neonatally saline-(male: n = 6; females: n = 6) and LPS-treated rats (male: n = 6; female: n = 7)
following an intraplantar injection of 1.1% formalin at PND 22 (mean SEM). (E and G) depict licking and inching pattern (respectively) in both males and females. (F and H)
depict the area under the curve (AUC) for licking and inching pattern (respectively) for both sexes. p < .01.

I. Zouikr et al. / Brain, Behavior, and Immunity 44 (2015) 235246

239

Fig. 2. Time-course of inching (A and B) and licking responses (D and E) following neonatal challenge with saline (males: n = 11; females: n = 9) or LPS (males: n = 13;
females: n = 9) and subsequent intraplantar injection of 2.25% formalin at PNDs 8097. (C and F) depict the area under the curve (AUC) for inching and licking pattern
(respectively) for both sexes. Data are presented as mean SEM. p < .05.

Overall, these data suggest that neonatal LPS exposure exerts

long-term effects on formalin-induced behavioural responses as
indicated by the increased susceptibility to inammatory noxious
stimuli in preadolescent and adult rats. The impact of neonatal
LPS exposure on inching behaviour becomes sex-dependent when
the rats reach adulthood.
4.2. Neonatal LPS exerts developmentally regulated effects on
circulating IL-1b
We have previously demonstrated the absence of sex dimorphism in formalin-induced behavioural responses at PND 22
(Zouikr et al., 2014b). In the current study, we were able to replicate these ndings and thus at this age (i.e. PND 22), we assessed
plasma IL-1b levels in males only.
At PND 22, LMM analysis revealed a signicant main effect of
treatment on plasma IL-1b level [F(1,13) = 11.41; p < .01] with neonatal LPS-treated male rats displaying signicantly increased IL-1b
plasma levels following formalin injection (see Fig. 3A). However,
at PNDs 8097, neonatal LPS exposure did not alter IL-1b plasma
levels [F(1,8) = 1.51; p > .05]. No sex differences were noticed in
adulthood in terms of plasma IL-1b levels as the two-way interaction of sex and treatment did not reach signicance [F(1,16) = .235,
p > .05] (Fig. 3B).
These results suggest that neonatal LPS exposure produces agedependent alterations in cytokine responses following formalin

injection as indicated by increased plasma IL-1b in preadolescent

but not adult rats.
4.3. LPS exposure during the neonatal period results in age- and sexdependent effects on hippocampal IL-1b following formalin injection
4.3.1. Preadolescent rats (PND 22)
At PND 22, LMM revealed that treatment was not a signicant
factor [F(1,7) = 1.81, p > .05] implying that both neonatal saline
and LPS-treated preadolescent rats displayed similar levels of hippocampal IL-1b following formalin injection. Additionally, no sex
differences were observed at this time-point as the two-way interaction of treatment and sex did not reach signicance
[F(1,7) = .597, p > .05; Fig. 4].
4.3.2. Adult rats (PNDs 8097)
In adulthood, LMM revealed a signicant two-way interaction
of sex and treatment [F(1,8) = 7.09, p < .05]. In males, neonatal
LPS-treated rats displayed increased IL-1b in the hippocampus
compared to saline-treated group [F(1,6) = 5.56, p = .05; Fig. 5C].
In females, there was a signicant main effect of treatment
[F(1,6) = 7.11, p < .05] with LPS-treated rats displaying decreased
levels of IL-1b in the hippocampus following the formalin test
(Fig. 5A). Collectively, these results suggest that the effects of neonatal immune challenge on hippocampal IL-1b following an
inammatory noxious stimulus are age and sex-dependent as indi-

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Fig. 3. Circulating levels of IL-1b in PND 22 (A) and PNDs 8097 (B) rats following formalin injection into the hindpaw. In preadolescence (PND 22), 14 males (7 Saline and 7
LPS) had either saline or LPS at PNDs 3 and 5 and subsequent formalin injection at PND 22. In adulthood (PNDs 8097), 9 rats (5 males and 4 females) and 13 rats (7 males and
6 females) had either saline or LPS, respectively, at PNDs 3 and 5 followed by formalin injection at PNDs 8097. n.s.: non signicant. p < .01.

Fig. 4. Hippocampal IL-1b levels in female (saline: n = 4; LPS: n = 6; A) and male (saline: n = 4; LPS: n = 6; C) rats following formalin injection at PND 22. B and D represent
IL-1b (in fold change relative to the endogenous control gene GAPDH) for neonatal saline-(white bar) and LPS (grey bar)-treated rats.

Fig. 5. Hippocampal IL-1b levels in female (saline: n = 3; LPS: n = 3; A) and male (saline: n = 2; LPS: n = 2; C) rats following formalin injection at PNDs 8097. B and D
represent IL-1b (in fold change relative to the endogenous control gene GAPDH) for neonatal saline-(white bar) and LPS-(grey bar) treated rats. p < .05. Please note that the
asterisk depicted in A and C represents non specic bands.

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cated by increased (in males) and decreased (in females) hippocampal IL-1b levels in adult rats but no changes in preadolescent
rats.
4.4. Neonatal LPS exposure induces higher mast cell degranulation in
preadolescent but not adult rats following formalin injection
At both ages, sex was not a signicant factor [F(1,48) = .704,
p > .05 at PND 22 & F(1,46) = .027, p > .05 at PNDs 8097]. Data
from both sexes were combined to produce plots of mean total
mast cells number and degree of degranulation per eld view.
4.4.1. Preadolescent rats (PND 22)
Histological analysis of the rats paw one hour following formalin injection revealed the presence of mast cells in both neonatal
treatment groups. At PND 22, LMM analysis of the degree of mast
cell degranulation revealed a signicant main effect of injection
[F(2,48) = 14.28; p < .001], and a signicant two-way interaction
of treatment and injections [F(1,48) = 6.1; p < .05]. Pair-wise comparisons revealed that the formalin-injected hindpaw had signicantly higher degree of mast cell degranulation compared to the
non-injected paw (p < .001) or saline-injected paw (p < .05). In both
neonatal saline and LPS-treated rats, formalin injection produced a
higher degree of mast cell degranulation compared to non-injected
or saline-injected paw. More importantly, neonatal LPS-challenged

241

rats displayed higher degree of mast cell degranulation following

formalin injection at PND 22 compared to saline-challenged rats
(p < .01; see Fig. 6B). No signicant differences were observed
between saline-injected paw and non-injected paw across the
treatment implying that the differences observed in the degree of
mast degranulation is not due to the injection of uid per se but
rather to the neonatal immune challenge. In regards to the total
number of mast cells, formalin injection increased the total mast
cells number compared to non-injected paw or saline-injected
but this did not reach signicance (see Fig. 6A). Additionally, the
number of mast cells was similar between LPS-treated rats and saline-treated rats. No signicant differences were observed between
saline-injected paws and non-injected paw.

4.4.2. Adult rats

In terms of degree of degranulation, LMM analysis revealed a
signicant main effect of injection [F(2,46) = 15.04, p < .001], with
the formalin-injected paw showing higher degree of degranulation
compared to the non-injected paw (p < .001). No signicant differences were observed between saline-injected and non-injected
paw. However, the two-way interactions of injections and treatment did not reach signicance [F(1,46) = .117, p > .05] implying
that neonatal immune challenge did not affect mast cells degree
of degranulation in response to formalin injection in adulthood.

Fig. 6. Average mast cell count (A) and average mast cell degranulation (B) following neonatal exposure to LPS and subsequent formalin injection in PND 22 rats. 12 rats (6
males and 6 females) neonatal saline-treated and 11 rats (5 males and 6 females) neonatal LPS-treated were used. Formalin-injected paw (Formalin) and non-injected paw
(control) was taken from the same rat. Additionally 2 rats received intraplantar injection of saline (Neosaline/saline). (C) paw sections from rats that had saline or LPS as
neonates and formalin injection at PND 22 using toluidine blue. Higher magnication (X 63) reveals higher degree of degranulation in LPS group. Arrows indicate the level of
degranulation: e.g. 1 indicates a non-degranulating mast cell. Scale = 50 lm.

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Fig. 7. Average mast cell count and average mast cell degranulation following neonatal exposure to LPS and subsequent formalin injection in PNDs 8097 rats. 10 rats (4
males and 6 females) neonatal saline-treated and 12 rats (6 males and 6 females) neonatal LPS-treated were used. Formalin-injected paw (formalin) and non-injected paw
(control) was taken from the same rat. Additionally 2 rats received intraplantar injection of saline (Neosaline/saline).

Also, the total number of mast cells did not differ between the two
groups regardless of the nature of the injection (Fig. 7).
Collectively, these results suggest neonatal LPS exposure exerts
age-dependent effects on mast cell degranulation following an
inammatory noxious stimulus encountered later in life as indicated by increased susceptibility towards higher degree of degranulation in LPS-treated rats when subjected to formalin injection at
PND 22 but not at PNDs 8097.

5. Discussion
Our study demonstrates that neonatal immune challenge plays
a crucial role in programming inammatory pain sensitivity later
in life as indicated by enhanced susceptibility to formalin-induced
pain in preadolescent rats. This hyperalgesia persists into adulthood long after the neonatal immune inammation has resolved.
These long-term behavioural changes following neonatal LPS exposure coincided with developmentally regulated changes in peripheral and central cytokine responses as indicated by increased
circulating levels of IL-1b at PND 22 but not PNDs 8097 and
increased hippocampal levels of IL-1b in adult but not preadolescent rats. Additionally LPS-treated rats displayed enhanced mast
cell degranulation following formalin injection at PND 22 but not
PNDs 8097. Overall these ndings demonstrate that a neonatal
immune challenge is accompanied by long-term and developmentally regulated changes in inammatory pain responses following a
subsequent hindpaw inammation later in life. This programming
seems to involve peripheral and hippocampal IL-1b as well as mast
cell degranulation.
5.1. LPS-induced hyperalgesia is accompanied by developmentally
regulated changes in peripheral and central IL-1b
It is now well appreciated that early life exposure to painful
experiences such as neonatal hindpaw incision or inammation
is accompanied by long-term alterations in the nociceptive pathways including increased behavioural responses to thermal and
mechanical stimuli (Beggs et al., 2012; Ren et al., 2004) as well
as altered synaptic input to the supercial dorsal horn (Li and
Baccei, 2011). In the present study, we demonstrate that an
immune challenge during the neonatal period can also alter pain

sensitivity later in life as indicated by enhanced formalin-induced

nociception during the late phase at PND 22 & PNDs 8097. This
is in agreement with a recent study demonstrating that at 4 and
8 h post LPS administration, PND 21 rats (PND 0 as birth) exhibited increased formalin-induced nociception during the late phase
(Hunter et al., 2014). Moreover, Boisse et al. have previously
reported that exposure to LPS during PND 14 is associated with
increased pain sensitivity to thermal and mechanical noxious
stimuli in adult rats (Boisse et al., 2005). We have previously
reported that neonatal LPS exposure produced a signicant
increase in inching responses and a trend towards increased
licking responses in preadolescent rats (Zouikr et al., 2014a,b).
This priming of pain sensitivity by a neonatal immune challenge
involved spinal dorsal horn neuronal responses and HPA axis
activity (Zouikr et al., 2014b). Altered formalin-induced pain in
preadolescent rats following a neonatal immune challenge also
involves supraspinal modulation (Zouikr et al., 2014a). In the current study, increased inching and licking responses were
observed in preadolescent rats neonatally challenged with LPS.
The increase in sample size and therefore increased power in
the current study contributed to identifying the differences in
licking behaviour between saline and LPS at PND 22. This altered
formalin-induced behavioural response persisted into adulthood
as indicated by increased inching response in adult males. Surprisingly, both neonatal saline and LPS animals displayed similar
licking behaviour in adulthood. Currently, we do not have an
explanation for this atypical behaviour observed in neonatal saline-injected adult rats.
The mechanisms underlying this programming of pain sensitivity by an immune challenge can also involve peripheral and central
components of the immune system given the well-established role
of the immune system in modulating pain sensitivity (Grace et al.,
2014b; Marchand et al., 2005; Ren and Dubner, 2010). The immune
system plays a crucial role in both peripheral and central processing of pain (Grace et al., 2014b; Marchand et al., 2005; Ren and
Dubner, 2010). When exposed to LPS, resident immune cells such
as mast cells and macrophages release a plethora of pro-inammatory cytokines including IL-1b, IL-6 and tumor necrosis factor
(TNF)a which are known to induce hyperalgesia (DeLeo et al.,
1996; Reeve et al., 2000; Sung et al., 2004). Among these cytokines,
IL-1b, which can be released in response to LPS administration, is a
well-established contributor of pain enhancement (Watkins et al.,

I. Zouikr et al. / Brain, Behavior, and Immunity 44 (2015) 235246

2007; Watkins and Maier, 1999, 2000; Watkins et al., 1994b). This
cytokine can act either peripherally by sensitization of nociceptors
(Fukuoka et al., 1994) or centrally by enhancing the excitability of
spinal dorsal horn neurons (Takano, 1996). In the present study, we
demonstrated that the LPS-induced enhanced inching responses
observed 1 h following formalin injection in preadolescent rats
paralleled elevated plasma levels of IL-1b. IL-1b has been previously reported to contribute to LPS-induced hyperalgesia as a local
administration of the IL-1b antagonist IL-1ra attenuated the
mechanical hyperalgesic effect of LPS injection into the hindpaw
(Cunha et al., 2000). Additionally, IL-1b can contribute to inching
responses in the formalin test as an intraplantar injection in rats of
anti-IL-1b serum but not non-immune serum prior to formalin
injection signicantly attenuated inching responses during the
late phase of the formalin test (Granados-Soto et al., 2001). It is
noteworthy that the altered cytokine responses persisted long after
the neonatal inammation had healed. This suggests that exposure
to an immune challenge during a period where the developing
immune system is highly vulnerable to environmental insult can
interfere with the normal developmental trajectory of the immune
responses, leading to hyper-reactivity of immune responses to a
second hit, notably an inammatory stimulus later in life. Surprisingly, this altered peripheral cytokine response following neonatal
LPS exposure and subsequent formalin injection does not persist
into adulthood whereby plasma levels of IL-1b were similar
between the two groups. In contrast, elevated IL-1b of central origin was observed in adult rats treated with LPS as neonates, an
effect not observed in preadolescent rats. Thus it seems that neonatal LPS exposure caused a developmental shift in cytokine
responses with peripheral changes in preadolescence and central
changes in adulthood following formalin injection.
An increasing body of evidence suggests abnormal hippocampal function in chronic pain, for review see (Liu and Chen, 2009).
Indeed given that pain is an aversive emotional experience and
considering that hippocampal amygdala reciprocal connections
are implicated in memory formation of emotional experience
(Izquierdo et al., 1997; Pitkanen et al., 2000), it is not surprising
that the hippocampus plays a crucial role in pain modulation.
Recent studies point towards an important role of hippocampal
IL-1b in neuropathic pain. In spared nerve injury (SNI) and
constriction nerve injury (CCI) models, an upregulation of
hippocampal IL-1b was correlated with mechanical allodynia
(del Rey et al., 2011). Of particular interest, exposure to an
immune stress can also affect IL-1b expression in the hippocampus. For instance, adult rats neonatally infected with Escherichia
coli displayed exaggerated levels of IL-1b in the hippocampus in
response to LPS treatment (Bilbo et al., 2005). Moreover, intraperitoneal administration of LPS induces the expression of IL-1b
mRNA and protein levels at various brain regions including the
hippocampus (Hillhouse and Mosley, 1993; Laye et al., 1994;
Quan et al., 1998).
The mechanisms underlying increased pain sensitivity via elevated levels of peripheral IL-1b are numerous. IL-1b, which can
be released from immune cells in response to a bacterial infection
(Watkins et al., 1994b), can directly activate nociceptors. IL-1b
receptor, IL-1R1 is expressed in sensory neurons (Obreja et al.,
2002). IL-1b has been shown in a nerve-skin in vitro preparation
to excite nociceptive bres (Fukuoka et al., 1994). IL-1b-induced
hyperalgesia can also be relayed to the brain via vagal afferences
as subdiaphragmatic vagotomy abolished the hyperalgesic effects
of IL-1b (Watkins et al., 1994b). Another possibility that can be
taken into account is that LPS-induced hyperalgesia will lead to
the release of IL-1b which, in turn, leads to the release of prostaglandins that are known to sensitize nociceptors (Cunha et al.,
2000). Local injection of IL-1ra inhibited the hyperalgesic response
to LPS and abolished the responses to the hyperalgesic agents

243

bradykinin, TNFa and IL-1b (Cunha et al., 2000). Finally, IL-1b can
directly act on the brain through active transport via the blood
brain barrier (BBB) (Banks et al., 1989), entry via circumventricular
structures (Blatteis et al., 1983), or binding to endothelial cells
leading to production of central prostaglandin (Dantzer, 2004).
5.2. Neonatal LPS exposure increased mast cell degranulation in
preadolescent but not adult rats following an inammatory pain
stimulus
Mast cells are granulated resident immune cells that participate
in innate immune responses and allergic reactions. They degranulate within minutes of inammatory reaction which in turn results
in the release of pro-inammatory mediators such as histamine,
bradykinin, and cytokines (Gaboury et al., 1995; Lawrence et al.,
2002; Metcalfe et al., 1997). Mast cells are found in the vicinity
of primary nociceptive neurons and vasculature and their degranulation is known to modulate the excitability of nociceptive nerve
endings (Kovacs et al., 2006). Mast cell degranulation also contributes to increased pain sensitivity. Previous studies showed that
mast cell degranulation contributes to thermal hyperalgesia via
the release of nerve growth factor (NGF) (Lewin et al., 1994). The
present study demonstrates that neonatal LPS exposure enhanced
mast cell degranulation following formalin injection in preadolescent but not adult rats. Injection of saline into the hindpaw failed
to increase mast cell degranulation, as the degree of mast cell
degranulation was similar to that in non-injected paw. These
observations suggest that the observed higher percentage of
degranulation is due to the neonatal immune challenge and not
to the injected volume per se. The higher mast cell degranulation
paralleled the LPS-induced hyperalgesia observed at PND 22. This
is in agreement with previous studies demonstrating an important
role of mast cell activity in the induction of the late phase of the
formalin test. For instance, inhibition of mast cell activity using
the mast cell stabilizer cromolyn inhibits formalin-induced nociceptive behaviour in the late phase (Parada et al., 2001). It further
follows from these ndings that mast cell-derived mediators such
as histamine are likely to be involved in the hyperalgesic response
and indeed histamine is known to sensitize nociceptors (Herbert
et al., 2001; Mizumura et al., 2000). Another possibility is the
recruitment of leukocytes induced by histamine released from
mast cells, which can also contribute to nociceptor sensitization
via the release of inammatory molecules (Yamaki et al., 1998).
It is noteworthy that circulating IL-1b levels were increased in
LPS-treated adolescent rats 1 h following formalin injection, the
same time-point as when a higher degree of mast cell degranulation was observed. Although we cannot conrm it directly in our
experiment, it is reasonable to speculate that the observed
increased IL-1b might result from mast cells during degranulation,
from the inltrating inammatory cells, or from de novo synthesis
of IL-1b. This assumption is conrmed by the fact that mast cells
have been previously shown to release IL-1b following stimulation
by peptidoglycan (McCurdy et al., 2003) or by granulocyte macrophage-colony stimulating factor (GM-CSF) (Hamilton, 2002). Furthermore, mast cell activation is known to contribute to
neuropathic hyperalgesia via the release of IL-1b (Sommer et al.,
1999). Finally, mast cells could contribute directly to nociceptor
sensitization as recent ndings suggest that mast cell degranulation requires direct contact with peripheral nerve endings via a calcium-dependent cell adhesion molecule-N-cadherin (Folgueras
et al., 2009). The absence of differences in mast cell degranulation
in LPS-treated adult rats following formalin injection is surprising.
Currently we do not have an explanation for this difference but our
results suggest a higher susceptibility of mast cell to degranulate in
response to an inammatory challenge in preadolescence. It is
probable that a second immunological hit is necessary in

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I. Zouikr et al. / Brain, Behavior, and Immunity 44 (2015) 235246

adulthood to unmask altered mast cell responses. Further studies

are necessary to explain these developmental changes in mast cell
degranulation in response to a neonatal immune challenge and
subsequent inammatory stimulus later in life.
In our experimental design formalin is setting the baseline,
since all animals had formalin injection, if there are any changes
it can only be attributed to early life intervention. Given that
peripheral IL-1b levels (hindpaw extract) did not signicantly differ between nave non-injected animals and animals that received
saline injection into their hindpaw (Saade et al., 2002) and that IL1b mRNA was not detected throughout the brain (including the
hippocampus) of rats subcutaneously injected with saline
(Yabuuchi et al., 1996), the peripheral and central immune changes
observed in our study are unlikely to be due to formalin only. However, it must be considered that there could be an effect between
formalin and LPS that goes beyond just neonatal intervention.
Future studies are required to elucidate the true causal relationship
between neonatal LPS exposure and later life exposure to formalin.
Although in the current study we did not assess the causal
effects of peripheral and central IL-1b on formalin-induced pain
following a neonatal immune challenge, our study is the rst to
show that neonatal immune challenge coincides with developmentally regulated increases in mast cell degranulation following
formalin injection as indicated by enhanced mast degranulation
in preadolescent but not adult rats following formalin injection.
This enhanced mast cell degranulation paralleled the LPS-induced
hyperalgesia and LPS-induced increased IL-1b response observed
in PND 22 rats following formalin injection.
5.3. Sex differences
Experimental and clinical studies have documented sex differences in pain (Aloisi et al., 1994; Aloisi and Ceccarelli, 2000;
Mogil, 2012) as well as cytokine expression in the hippocampus
(Walker et al., 2010). Prior studies have reported sex differences
in formalin-induced pain in pre-pubertal period (Butkevich and
Vershinina, 2001). Formalin injection at PND 25 elicited higher
inching responses in females (Butkevich et al., 2009). In the present study, we did not observe sex differences in inching responses
at PND 22. This discrepancy may be attributed to the difference in
the experimental paradigm in regards to the timing (gestational vs.
neonatal) and nature (restraint stress vs. immune challenge) of the
stressor. We have also shown that neonatal LPS exposure induces
more inching in adult males following formalin injection. The
absence of sex differences at PND 22 could be attributed to the late
onset of puberty at this time-point. Vaginal opening occurs around
PNDs 32-34 in females whereas male maturity occurs at a later
time-point around PNDs 45-48 (Lewis et al., 2002). Our laboratory
has previously demonstrated that neonatal LPS exposure induced
higher IL-1b levels in the hippocampus in both sexes following
adult exposure to restraint stress (Walker et al., 2010). In the present study, LPS-treated male rats displayed higher hippocampal IL1b compared to females, which responded to the formalin injection
by an attenuated IL-1b response in the hippocampus. Again this
discrepancy could be attributed to differences in the nature of
the stressor used (formalin injection vs. restraint stress), which
can differently affect cytokines expression in the hippocampus.
For instance, del Rey et al. did not observe sex differences in IL1b mRNA levels in the hippocampus in rats subjected to neuropathic pain (del Rey et al., 2011).
6. Conclusions
We have recently documented the importance of neonatal
exposure to immunological insults in shaping formalin-induced

nociception throughout the rst three weeks of postnatal life

(Zouikr et al., 2014b). Such priming of pain responsiveness
involved increased spinal dorsal horn neuronal responses as well
as heightened HPA axis activity (Zouikr et al., 2014b). We have
recently shown that such priming of pain sensitivity also involves
supra-spinal modulation as indicated by decreased Fos-protein
expression in LPS-treated preadolescent rats (Zouikr et al.,
2014a). In the current study, we expanded these ndings to show
that neonatal LPS exposure can program formalin-induced pain
with alterations in inching responses persisting into adulthood.
Neonatal LPS exposure was also accompanied by developmentally
regulated changes in immune responses with higher mast cell
degranulation and enhanced circulating levels of IL-1b observed
in preadolescent rats and an upregulation of hippocampal IL-1b
in adult rats following a neonatal immune challenge. It is now well
appreciated that the immune system plays a crucial role in modulating pain responsiveness (Grace et al., 2014a; Marchand et al.,
2005; Ren and Dubner, 2010; Watkins and Maier, 1999, 2000).
Pharmacological treatments aimed at blocking proinammatory
cytokine release or mast cell degranulation constitute a promising
therapeutic approach to treat chronic pain. Additionally, understanding the impact of neonatal bacterial infection on pain sensitivity later in life is of clinical importance given the high risk of
having a bacterial infection during the neonatal period (Williams,
1988) and given the failure of current therapy to treat chronic pain.
Role of the funding source
This work was supported by a grant from the Australian
Research Council (ARC DP 09787599). ARC had no further role in
the study design; in collection, analysis, and interpretation of data;
in the writing of the manuscript, or in the decision to submit the
manuscript.
Acknowledgments
The authors would like to thank all conjoint BSAF staff for their
assistance in animal husbandry. We also would like to thank Mrs.
Debbie-Gai Papperall for her assistance with mast cell histology.
References
Adkins, B., Leclerc, C., Marshall-Clarke, S., 2004. Neonatal adaptive immunity comes
of age. Nat. Rev. Immunol. 4, 553564.
Aloisi, A.M., Ceccarelli, I., 2000. Role of gonadal hormones in formalin-induced pain
responses of male rats: modulation by estradiol and naloxone administration.
Neuroscience 95, 559566.
Aloisi, A.M., Albonetti, M.E., Carli, G., 1994. Sex differences in the behavioural
response to persistent pain in rats. Neurosci. Lett. 179, 7982.
Apkarian, A.V., 2008. Pain perception in relation to emotional learning. Curr. Opin.
Neurobiol. 18, 464468.
Apkarian, A.V., Hashmi, J.A., Baliki, M.N., 2011. Pain and the brain: specicity and
plasticity of the brain in clinical chronic pain. Pain 152, S4964.
Austin, P.J., Kim, C.F., Perera, C.J., Moalem-Taylor, G., 2012. Regulatory T cells
attenuate neuropathic pain following peripheral nerve injury and experimental
autoimmune neuritis. Pain 153, 19161931.
Banks, W.A., Kastin, A.J., Durham, D.A., 1989. Bidirectional transport of interleukin-1
alpha across the blood-brain barrier. Brain Res. Bull. 23, 433437.
Beggs, S., Currie, G., Salter, M.W., Fitzgerald, M., Walker, S.M., 2012. Priming of adult
pain responses by neonatal pain experience: maintenance by central
neuroimmune activity. Brain: J. Neurol. 135, 404417.
Besedovsky, H.O., del Rey, A., 2011. Central and peripheral cytokines mediate
immune-brain connectivity. Neurochem. Res. 36, 16.
Bilbo, S.D., Biedenkapp, J.C., Der-Avakian, A., Watkins, L.R., Rudy, J.W., Maier, S.F.,
2005. Neonatal infection-induced memory impairment after lipopolysaccharide
in adulthood is prevented via caspase-1 inhibition. J. Neurosci.: Ofcial J. Soc.
Neurosci. 25, 80008009.
Bird, C.M., Burgess, N., 2008. The hippocampus and memory: insights from spatial
processing. Nat. Rev. Neurosci. 9, 182194.
Blatteis, C.M., Bealer, S.L., Hunter, W.S., Llanos, Q.J., Ahokas, R.A., Mashburn Jr., T.A.,
1983. Suppression of fever after lesions of the anteroventral third ventricle in
guinea pigs. Brain Res. Bull. 11, 519526.

I. Zouikr et al. / Brain, Behavior, and Immunity 44 (2015) 235246

Boisse, L., Spencer, S.J., Mouihate, A., Vergnolle, N., Pittman, Q.J., 2005. Neonatal
immune challenge alters nociception in the adult rat. Pain 119, 133141.
Butkevich, I.P., Vershinina, E.A., 2001. Prenatal stress alters time characteristics and
intensity of formalin-induced pain responses in juvenile rats. Brain Res. 915,
8893.
Butkevich, I.P., Mikhailenko, V.A., Bagaeva, T.R., Makukhina, G.V., 2009. Persistent
pain responses in inammation and corticosterone levels in juvenile rats born
to adrenalectomized dams. Neurosci. Behav. Physiol. 39, 297300.
Costigan, M., Moss, A., Latremoliere, A., Johnston, C., Verma-Gandhu, M., Herbert,
T.A., Barrett, L., Brenner, G.J., Vardeh, D., Woolf, C.J., Fitzgerald, M., 2009. T-cell
inltration and signaling in the adult dorsal spinal cord is a major contributor to
neuropathic pain-like hypersensitivity. J. Neurosci.: Ofcial J. Soc. Neurosci. 29,
1441514422.
Cunha, J.M., Cunha, F.Q., Poole, S., Ferreira, S.H., 2000. Cytokine-mediated
inammatory hyperalgesia limited by interleukin-1 receptor antagonist. Br. J.
Pharmacol. 130, 14181424.
Dantzer, R., 2001. Cytokine-induced sickness behavior: mechanisms and
implications. Ann. N. Y. Acad. Sci. 933, 222234.
Dantzer, R., 2004. Cytokine-induced sickness behaviour: a neuroimmune response
to activation of innate immunity. Eur. J. Pharmacol. 500, 399411.
Dantzer, R., Kelley, K.W., 2007. Twenty years of research on cytokine-induced
sickness behavior. Brain Behav. Immun. 21, 153160.
Dantzer, R., OConnor, J.C., Freund, G.G., Johnson, R.W., Kelley, K.W., 2008. From
inammation to sickness and depression: when the immune system subjugates
the brain. Nat. Rev. Neurosci. 9, 4656.
del Rey, A., Yau, H.J., Randolf, A., Centeno, M.V., Wildmann, J., Martina, M.,
Besedovsky, H.O., Apkarian, A.V., 2011. Chronic neuropathic pain-like behavior
correlates with IL-1beta expression and disrupts cytokine interactions in the
hippocampus. Pain 152, 28272835.
DeLeo, J.A., Colburn, R.W., Nichols, M., Malhotra, A., 1996. Interleukin-6-mediated
hyperalgesia/allodynia and increased spinal IL-6 expression in a rat
mononeuropathy model. J. Interferon Cytokine Res. 16, 695700.
Ellis, S., Mouihate, A., Pittman, Q.J., 2005. Early life immune challenge alters innate
immune responses to lipopolysaccharide: implications for host defense as
adults. FASEB J.: Ofcial Publ. Fed. Am. Soc. Exp. Biol. 19, 15191521.
Ferreira, S.H., Lorenzetti, B.B., Bristow, A.F., Poole, S., 1988. Interleukin-1 beta as a
potent hyperalgesic agent antagonized by a tripeptide analogue. Nature 334,
698700.
Fitzgerald, M., 1995. Developmental biology of inammatory pain. Br. J. Anaesth. 75,
177185.
Fitzgerald, M., 2005. The development of nociceptive circuits. Nat. Rev. Neurosci. 6,
507520.
Folgueras, A.R., Valdes-Sanchez, T., Llano, E., Menendez, L., Baamonde, A., Denlinger,
B.L., Belmonte, C., Juarez, L., Lastra, A., Garcia-Suarez, O., Astudillo, A., Kirstein,
M., Pendas, A.M., Farinas, I., Lopez-Otin, C., 2009. Metalloproteinase MT5-MMP
is an essential modulator of neuro-immune interactions in thermal pain
stimulation. Proc. Natl. Acad. Sci. U.S.A. 106, 1645116456.
Fukuoka, H., Kawatani, M., Hisamitsu, T., Takeshige, C., 1994. Cutaneous
hyperalgesia induced by peripheral injection of interleukin-1 beta in the rat.
Brain Res. 657, 133140.
Gaboury, J.P., Johnston, B., Niu, X.F., Kubes, P., 1995. Mechanisms underlying acute
mast cell-induced leukocyte rolling and adhesion in vivo. J. Immunol. 154, 804
813.
Grace, P.M., Rolan, P.E., Hutchinson, M.R., 2011. Peripheral immune contributions to
the maintenance of central glial activation underlying neuropathic pain. Brain
Behav. Immun. 25, 13221332.
Grace, P.M., Hutchinson, M.R., Maier, S.F., Watkins, L.R., 2014a. Pathological pain
and the neuroimmune interface. Nat. Rev. Immunol. 14, 217231.
Grace, P.M., Hutchinson, M.R., Maier, S.F., Watkins, L.R., 2014b. Pathological pain
and the neuroimmune interface. Nat. Rev. Immunol.
Granados-Soto, V., Alonso-Lopez, R., Asomoza-Espinosa, R., Runo, M.O., GomesLopes, L.D., Ferreira, S.H., 2001. Participation of COX, IL-1 beta and TNF alpha in
formalin-induced inammatory pain. Proc. West. Pharmacol. Soc. 44, 1517.
Guy, E.R., Abbott, F.V., 1992. The behavioral response to formalin in preweanling
rats. Pain 51, 8190.
Hamilton, J.A., 2002. GM-CSF in inammation and autoimmunity. Trends Immunol.
23, 403408.
Herbert, M.K., Just, H., Schmidt, R.F., 2001. Histamine excites groups III and IV
afferents from the cat knee joint depending on their resting activity. Neurosci.
Lett. 305, 9598.
Hillhouse, E.W., Mosley, K., 1993. Peripheral endotoxin induces hypothalamic
immunoreactive interleukin-1 beta in the rat. Br. J. Pharmacol. 109,
289290.
Hodgson, D.M., Knott, B., Walker, F.R., 2001. Neonatal endotoxin exposure
inuences HPA responsivity and impairs tumor immunity in Fischer 344 rats
in adulthood. Pediatr. Res. 50, 750755.
Hunter, D., Chai, C., Barr, G.A., 2014. Effects of COX inhibition and LPS on formalin
induced pain in the infant rat. Dev. Neurobiol. http://dx.doi.org/10.1002/
dneu.22230, in press.
Hutchinson, M.R., Buijs, M., Tuke, J., Kwok, Y.H., Gentgall, M., Williams, D., Rolan, P.,
2013. Low-dose endotoxin potentiates capsaicin-induced pain in man: evidence
for a pain neuroimmune connection. Brain Behav. Immun. 30, 311.
Izquierdo, I., Quillfeldt, J.A., Zanatta, M.S., Quevedo, J., Schaeffer, E., Schmitz, P.K.,
Medina, J.H., 1997. Sequential role of hippocampus and amygdala, entorhinal
cortex and parietal cortex in formation and retrieval of memory for inhibitory
avoidance in rats. Eur. J. Neurosci. 9, 786793.

245

Kent, S., Bluthe, R.M., Kelley, K.W., Dantzer, R., 1992. Sickness behavior as a new
target for drug development. Trends Pharmacol. Sci. 13, 2428.
Kovacs, P., Hernadi, I., Wilhelm, M., 2006. Mast cells modulate maintained neuronal
activity in the thalamus in vivo. J. Neuroimmunol. 171, 17.
Kumar, V., Sharma, A., 2010. Mast cells: emerging sentinel innate immune cells with
diverse role in immunity. Mol. Immunol. 48, 1425.
Lawrence, T., Willoughby, D.A., Gilroy, D.W., 2002. Anti-inammatory lipid
mediators and insights into the resolution of inammation. Nat. Rev.
Immunol. 2, 787795.
Laye, S., Parnet, P., Goujon, E., Dantzer, R., 1994. Peripheral administration of
lipopolysaccharide induces the expression of cytokine transcripts in the brain
and pituitary of mice. Brain Res. Mol. Brain Res. 27, 157162.
Lewin, G.R., Rueff, A., Mendell, L.M., 1994. Peripheral and central mechanisms of
NGF-induced hyperalgesia. Eur. J. Neurosci. 6, 19031912.
Lewis, E.M., Barnett Jr., J.F., Freshwater, L., Hoberman, A.M., Christian, M.S., 2002.
Sexual maturation data for Crl Sprague-Dawley rats: criteria and confounding
factors. Drug Chem. Toxicol. 25, 437458.
Li, J., Baccei, M.L., 2011. Neonatal tissue damage facilitates nociceptive synaptic
input to the developing supercial dorsal horn via NGF-dependent
mechanisms. Pain 152, 18461855.
Liu, M.G., Chen, J., 2009. Roles of the hippocampal formation in pain information
processing. Neurosci. Bull. 25, 237266.
Maier, S.F., 2003. Bi-directional immune-brain communication: Implications for
understanding stress, pain, and cognition. Brain Behav. Immun. 17, 6985.
Marchand, F., Perretti, M., McMahon, S.B., 2005. Role of the immune system in
chronic pain. Nat. Rev. Neurosci. 6, 521532.
Mason, P., 1993. Lipopolysaccharide induces fever and decreases tail ick latency in
awake rats. Neurosci. Lett. 154, 134136.
McCurdy, J.D., Olynych, T.J., Maher, L.H., Marshall, J.S., 2003. Cutting edge: distinct
Toll-like receptor 2 activators selectively induce different classes of mediator
production from human mast cells. J. Immunol. 170, 16251629.
Metcalfe, D.D., Baram, D., Mekori, Y.A., 1997. Mast cells. Physiol. Rev. 77, 1033
1079.
Mizumura, K., Koda, H., Kumazawa, T., 2000. Possible contribution of protein kinase
C in the effects of histamine on the visceral nociceptor activities in vitro.
Neurosci. Res. 37, 183190.
Mogil, J.S., 2012. Sex differences in pain and pain inhibition: multiple explanations
of a controversial phenomenon. Nat. Rev. Neurosci. 13, 859866.
Obreja, O., Rathee, P.K., Lips, K.S., Distler, C., Kress, M., 2002. IL-1 beta potentiates
heat-activated currents in rat sensory neurons: involvement of IL-1RI, tyrosine
kinase, and protein kinase C. FASEB J.: Ofcial Publ. Fed. Am. Soc. Exp. Biol. 16,
14971503.
Oka, T., Aou, S., Hori, T., 1993. Intracerebroventricular injection of interleukin-1 beta
induces hyperalgesia in rats. Brain Res. 624, 6168.
Okuda, K., Sakurada, C., Takahashi, M., Yamada, T., Sakurada, T., 2001.
Characterization of nociceptive responses and spinal releases of nitric oxide
metabolites and glutamate evoked by different concentrations of formalin in
rats. Pain 92, 107115.
Parada, C.A., Tambeli, C.H., Cunha, F.Q., Ferreira, S.H., 2001. The major role of
peripheral release of histamine and 5-hydroxytryptamine in formalin-induced
nociception. Neuroscience 102, 937944.
Pitkanen, A., Pikkarainen, M., Nurminen, N., Ylinen, A., 2000. Reciprocal connections
between the amygdala and the hippocampal formation, perirhinal cortex, and
postrhinal cortex in rat: A review. Ann. N. Y. Acad. Sci. 911, 369391.
Quan, N., Whiteside, M., Herkenham, M., 1998. Time course and localization
patterns of interleukin-1beta messenger RNA expression in brain and pituitary
after peripheral administration of lipopolysaccharide. Neuroscience 83, 281
293.
Reeve, A.J., Patel, S., Fox, A., Walker, K., Urban, L., 2000. Intrathecally administered
endotoxin or cytokines produce allodynia, hyperalgesia and changes in spinal
cord neuronal responses to nociceptive stimuli in the rat. Eur. J. Pain 4, 247
257.
Ren, K., Dubner, R., 2010. Interactions between the immune and nervous systems in
pain. Nat. Med. 16, 12671276.
Ren, K., Anseloni, V., Zou, S.P., Wade, E.B., Novikova, S.I., Ennis, M., Traub, R.J., Gold,
M.S., Dubner, R., Lidow, M.S., 2004. Characterization of basal and reinammation-associated long-term alteration in pain responsivity following
short-lasting neonatal local inammatory insult. Pain 110, 588596.
Saade, N.E., Massaad, C.A., Ochoa-Chaar, C.I., Jabbur, S.J., Saeh-Garabedian, B.,
Atweh, S.F., 2002. Upregulation of proinammatory cytokines and nerve growth
factor by intraplantar injection of capsaicin in rats. J. Physiol. 545, 241253.
Sadeqzadeh, E., de Bock, C.E., Zhang, X.D., Shipman, K.L., Scott, N.M., Song, C.,
Yeadon, T., Oliveira, C.S., Jin, B., Hersey, P., Boyd, A.W., Burns, G.F., Thorne, R.F.,
2011. Dual processing of FAT1 cadherin protein by human melanoma cells
generates distinct protein products. J. Biol. Chem. 286, 2818128191.
Sandler, C., Lindstedt, K.A., Joutsiniemi, S., Lappalainen, J., Juutilainen, T., Kolah, J.,
Kovanen, P.T., Eklund, K.K., 2007. Selective activation of mast cells in
rheumatoid synovial tissue results in production of TNF-alpha, IL-1beta and
IL-1Ra. Inammation Res.: Ofcial J. Eur. Histamine Res. Soc. 56, 230239.
Shanks, N., Larocque, S., Meaney, M.J., 1995. Neonatal endotoxin exposure alters the
development of the hypothalamic-pituitary-adrenal axis: early illness and later
responsivity to stress. J. Neurosci.: Ofcial J. Soc. Neurosci. 15, 376384.
Shanks, N., Windle, R.J., Perks, P.A., Harbuz, M.S., Jessop, D.S., Ingram, C.D., Lightman,
S.L., 2000. Early-life exposure to endotoxin alters hypothalamic-pituitaryadrenal function and predisposition to inammation. Proc. Natl. Acad. Sci. U.S.A.
97, 56455650.

246

I. Zouikr et al. / Brain, Behavior, and Immunity 44 (2015) 235246

Silver, R., Curley, J.P., 2013. Mast cells on the mind: new insights and opportunities.
Trends Neurosci. 36, 513521.
Sommer, C., Petrausch, S., Lindenlaub, T., Toyka, K.V., 1999. Neutralizing antibodies
to interleukin 1-receptor reduce pain associated behavior in mice with
experimental neuropathy. Neurosci. Lett. 270, 2528.
Spencer, S.J., Martin, S., Mouihate, A., Pittman, Q.J., 2006. Early-life immune
challenge: dening a critical window for effects on adult responses to
immune challenge. Neuropsychopharmacol.: Ofcial Publ. Am. College
Neuropsychopharmacol. 31, 19101918.
Sung, C.S., Wen, Z.H., Chang, W.K., Ho, S.T., Tsai, S.K., Chang, Y.C., Wong, C.S., 2004.
Intrathecal interleukin-1beta administration induces thermal hyperalgesia by
activating inducible nitric oxide synthase expression in the rat spinal cord.
Brain Res. 1015, 145153.
Takano, Y., 1996. Hyperalgesic Effects of Intrathecally Administered Interleukin-1b
in Rats. 8th World Congress on Pain, Vancouver.
Walker, F.R., Brogan, A., Smith, R., Hodgson, D.M., 2004. A prole of the immediate
endocrine, metabolic and behavioural responses following a dual exposure to
endotoxin in early life. Physiol. Behav. 83, 495504.
Walker, A.K., Nakamura, T., Byrne, R.J., Naicker, S., Tynan, R.J., Hunter, M., Hodgson,
D.M., 2009a. Neonatal lipopolysaccharide and adult stress exposure predisposes
rats to anxiety-like behaviour and blunted corticosterone responses:
implications for the double-hit hypothesis. Psychoneuroendocrinology 34,
15151525.
Walker, S.M., Tochiki, K.K., Fitzgerald, M., 2009b. Hindpaw incision in early life
increases the hyperalgesic response to repeat surgical injury: critical period and
dependence on initial afferent activity. Pain 147, 99106.
Walker, A.K., Nakamura, T., Hodgson, D.M., 2010. Neonatal lipopolysaccharide
exposure alters central cytokine responses to stress in adulthood in Wistar rats.
Stress 13, 506515.
Wang, G., Ji, Y., Lidow, M.S., Traub, R.J., 2004. Neonatal hind paw injury alters
processing of visceral and somatic nociceptive stimuli in the adult rat. J. Pain:
Ofcial J. Am. Pain Soc. 5, 440449.
Watkins, L.R., Maier, S.F., 1999. Implications of immune-to-brain communication
for sickness and pain. Proc. Natl. Acad. Sci. U.S.A. 96, 77107713.
Watkins, L.R., Maier, S.F., 2000. The pain of being sick: implications of immune-tobrain communication for understanding pain. Annu. Rev. Psychol. 51, 2957.
Watkins, L.R., Maier, S.F., 2005. Immune regulation of central nervous system
functions: from sickness responses to pathological pain. J. Intern. Med. 257,
139155.
Watkins, L.R., Wiertelak, E.P., Goehler, L.E., Mooney-Heiberger, K., Martinez, J.,
Furness, L., Smith, K.P., Maier, S.F., 1994a. Neurocircuitry of illness-induced
hyperalgesia. Brain Res. 639, 283299.

Watkins, L.R., Wiertelak, E.P., Goehler, L.E., Smith, K.P., Martin, D., Maier, S.F., 1994b.
Characterization of cytokine-induced hyperalgesia. Brain Res. 654, 1526.
Watkins, L.R., Maier, S.F., Goehler, L.E., 1995. Immune activation: the role of proinammatory cytokines in inammation, illness responses and pathological
pain states. Pain 63, 289302.
Watkins, L.R., Martin, D., Ulrich, P., Tracey, K.J., Maier, S.F., 1997. Evidence for the
involvement of spinal cord glia in subcutaneous formalin induced hyperalgesia
in the rat. Pain 71, 225235.
Watkins, L.R., Hutchinson, M.R., Milligan, E.D., Maier, S.F., 2007. Listening and
talking to neurons: implications of immune activation for pain control and
increasing the efcacy of opioids. Brain Res. Rev. 56, 148169.
Wegner, A., Elsenbruch, S., Maluck, J., Grigoleit, J.S., Engler, H., Jager, M., Spreitzer, I.,
Schedlowski, M., Benson, S., 2014. Inammation-induced hyperalgesia: effects
of timing, dosage, and negative affect on somatic pain sensitivity in human
experimental endotoxemia. Brain, Behav., Immunity, 12.
Whitelaw, A., Parkin, J., 1988. Development of immunity. Br. Med. Bull. 44, 1037
1051.
Williams, R.F., 1988. Colonization of the developing body by bacteria. In: Davis, J.A.,
Dobbing, J. (Eds.), Scientic Foundations of Paediatrics. Heinemann, London, pp.
10451065.
Xanthos, D.N., Gaderer, S., Drdla, R., Nuro, E., Abramova, A., Ellmeier, W., Sandkuhler,
J., 2011. Central nervous system mast cells in peripheral inammatory
nociception. Mol. Pain 7, 42.
Yabuuchi, K., Maruta, E., Minami, M., Satoh, M., 1996. Induction of interleukin-1
beta mRNA in the hypothalamus following subcutaneous injections of formalin
into the rat hind paws. Neurosci. Lett. 207, 109112.
Yamaki, K., Thorlacius, H., Xie, X., Lindbom, L., Hedqvist, P., Raud, J., 1998.
Characteristics of histamine-induced leukocyte rolling in the undisturbed
microcirculation of the rat mesentery. Br. J. Pharmacol. 123, 390399.
Zouikr, I., Tadros, M.A., Clifton, V.L., Beagley, K.W., Hodgson, D.M., 2013. Low
formalin concentrations induce ne-tuned responses that are sex and agedependent: a developmental study. PLoS ONE 8, e53384.
Zouikr, I., James, M.H., Campbell, E.J., Clifton, V.L., Beagley, K.W., Dayas, C.V.,
Hodgson, D.M., 2014a. Altered formalin-induced pain and Fos induction in the
periaqueductal grey of preadolescent rats following neonatal LPS exposure.
PLoS ONE 9, e98382.
Zouikr, I., Tadros, M.A., Barouei, J., Beagley, K.W., Clifton, V.L., Callister, R.J., Hodgson,
D.M., 2014b. Altered nociceptive, endocrine, and dorsal horn neuron responses
in rats following a neonatal immune challenge. Psychoneuroendocrinology 41,
112.
Zuo, Y., Perkins, N.M., Tracey, D.J., Geczy, C.L., 2003. Inammation and hyperalgesia
induced by nerve injury in the rat: a key role of mast cells. Pain 105, 467479.