Journal of Affective Disorders 274 (2020) 759–767

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Journal of Affective Disorders

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

Research paper

Social stress as a trigger for depressive-like behavior and persistent T

hyperalgesia in mice: study of the comorbidity between depression and
chronic pain
LN Piardia,1, M Pagliusia,1, IJM Bonetb, AF Brandãoa, SF Magalhãesa, FB Zanelattoa, CH Tambelia,
CA Paradaa, CR Sartoria,
⁎

a
Department of Structural and Functional Biology, University of Campinas, Rua Monteiro Lobato, 255, Cidade Universitaria Zeferino Vaz, Box 6109, Campinas, SP
13083-865, Brazil
b
Department of Oral and Maxillofacial Surgery, University of California San Francisco, 513 Parnassus Ave, Box 0440 S709, San Francisco, CA 94143, United States of
America

ARTICLE INFO ABSTRACT

Keywords: Background: There is great comorbidity and similarity between chronic pain and major depressive disorders. We
Chronic pain have recently shown that 10 days of social defeat stress (SDS) induces hyperalgesia regardless depressive-like
depression behavior in mice. Here we aimed to investigate whether social stress predisposes to chronic pain and, inversely,
social defeat stress whether chronic pain predisposes to stress-induced depression.
hyperalgesia
Methods: Firstly, we used the 10 days SDS paradigm in mice followed by a mild protocol of repetitive in-
social avoidance
flammatory stimulus to evaluate if SDS would predispose to persistent hyperalgesia development. Secondly, we
PGE2
used the intense protocol of repetitive inflammatory stimulus followed by a subthreshold SDS to evaluate if
persistent hyperalgesia would predispose to depressive-like behavior of social avoidance.
Results: Our results showed that SDS predispose to chronic pain, since stressed mice injected with PGE2 for 7
days (mild protocol), stimuli normally not sufficient to trigger chronic pain, showed persistent hyperalgesia.
Also, we showed that persistent hyperalgesia induced by repetitive inflammatory stimuli predispose to long-
lasting depressive-like behavior of social avoidance induced by subthreshold SDS.
Limitations: We did not analyze molecular mechanism associated with chronic pain and depressive-like behavior
induced by SDS. However, we hypothesized that SDS and 14 days of PGE2 would generate neuroplasticity on
brain areas shared by chronic pain and depression, predisposing to pain chronification and depressive-like be-
havior, respectively.
Conclusions: We can conclude social stress as a key and a common factor for chronic pain and depression. We can
also conclude that SDS predisposes to chronic pain and, inversely, chronic pain predisposes to depressive-like
behavior.

1. Introduction
Several studies have shown the great comorbid between chronic
pain and major depressive disorders (MDD) (Arnow et al., 2009;
Robinson et al., 2009; Goldenberg, 2010; Li, 2017). This comorbidity
between chronic pain and MDD is reflected in clinical conditions,
neural circuits, neurotransmitter systems, and neural structures that are
similarly altered in patients with these conditions (Robinson et al.,
2009; Goldenberg, 2010; Li, 2017). Understanding the relationship
between chronic pain and MDD is important for more effective clinical
interventions (Li, 2017). Recently, using a 10 days social defeat stress
(SDS) paradigm that allows the separation of susceptible and resilient
phenotypes for depressive-like behavior (Golden et al., 2011), our re-
search group showed that SDS induced mechanical and chemical hy-
peralgesia regardless depressive-like behavior in mice (Pagliusi Jr et al.,
2018). Rivat et al. (2010), using a different SDS rat model, also showed
chronic social stress triggering sensory hypersensitivity and anxiety
behavior, which is closely associated with depression (Tiller, 2013).
These results show that stress is a key factor to trigger both chronic pain
and depressive-like behavior.

⁎
Corresponding author.
E-mail address: sartoric@unicamp.br (C. Sartori).
1
Contributed equally to this article.

https://doi.org/10.1016/j.jad.2020.05.144
Received 4 December 2019; Received in revised form 17 April 2020; Accepted 27 May 2020
Available online 31 May 2020
0165-0327/ © 2020 Elsevier B.V. All rights reserved.
L. Piardi, et al.
A rodent model using repetitive inflammatory stimulus with pros-
taglandin E2 (PGE2) have been widely used to mimic human chronic
pain conditions (Ferreira et al., 1990; Villarreal, Sachs, et al., 2009; Dias
et al., 2015). In this model, PGE2 is daily injected subcutaneously in the
hind paw for 14 days and, as consequence, the rodent remains hyper-
algesic for at least 30 days (i.e. chronic pain) after the end of the in-
jections (Ferreira et al., 1990). An important property of this model is
that it allows us to analyze the susceptibility of the animal to develop
chronic pain. This is possible because a mild repetitive inflammatory
stimulus protocol (7 days of PGE2 injections) is not sufficient to trigger
chronic pain in healthy animals (Dias et al., 2015). Therefore, this mild
protocol plus additional manipulation (e.g. social stress, molecular
manipulations in the central nervous system) could trigger a chronic
pain condition in the individual, allowing us to study comorbidities and
molecular mechanisms associated with pain chronification. Also, using
this model we can assess the hypothesis that the social defeat stress
would induce a priming effect on nociceptor, which could become more
sensitive to PGE2.
Studying diseases of modernity, such as chronic pain and MDD,
require us to use animal models that mimic the stressful environment
where human is inserted and which, directly, is being responsible for
the epidemic indices of these diseases (Moussavi et al., 2007;
Hidaka, 2012). In this context, nowadays it seems unlikely for a human
being to be subjected to situations of physical stress, such as physical
restraint or electrical shocks, type of stressors which are being used as
animal models for depression and pain (Da Silva Torres et al., 2003;
Gameiro et al., 2006; Bali & Jaggi, 2015). Because of this we used in the
present study the SDS paradigm, considering that stress associated with
social relationships like family, work and urban conditions are the most
significative stress that affects modern humans and can trigger MDD
(Jasnow et al., 2005; Kumpulainen, 2008; Davidson & McEwen, 2012).
Even with the well-known abovementioned comorbidity between
chronic pain and MDD, there is a lack of studies in the literature aiming
the relationship between these two conditions, mainly in a social stress
context. Taking this into account, the present study assessed the re-
lationship between chronic pain and depression using a social stress
context. Based on this approach, this study aimed to investigate if the
SDS paradigm would predispose to pain chronification and, inversely, if
chronic pain induced by repetitive inflammatory stimuli would pre-
dispose to depressive-like behavior in mice.
2. Methods
Male eight weeks old C57BL/6J mice and male Swiss mice retired
breeders were used and obtained from the Multidisciplinary Center for
Biological Investigation (CEMIB) at the State University of Campinas
(UNICAMP). Mice were housed under standard laboratory conditions
(22 ± 1oC, 12-hour light/dark cycles, food and water ad libitum). All
experiments were approved by the Ethics Committee on the Use of
Animals at the Biology Institute of the University of Campinas (pro-
tocol4019-1).
In the present study we performed two sets of experiments. In the
experiment 1 we used the 10 days SDS paradigm in mice followed by a
mild protocol of repetitive inflammatory stimulus (7 days of PGE2 hind
paw injections) to evaluate if SDS would predispose to the development
of persistent hyperalgesia. In the experiment 2 we used an intense
protocol of repetitive inflammatory stimulus (14 days of PGE2 hind
paw injections) followed by a subthreshold SDS (1-day defeat protocol,
a.k.a. microdefeat) to evaluate if persistent hyperalgesia would pre-
dispose to depressive-like behavior of social avoidance. It is worth
mentioning that all behavioral tests were performed under red light
conditions and in a blinded fashion.
Experimental design is represented in the graphic diagram (figure 1):
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Journal of Affective Disorders 274 (2020) 759–767
2.1. Social defeat stress (SDS)
We used an adapted version (Pagliusi Jr. & Sartori, 2019) of the
social defeat stress protocol standardized by Golden et al. (2011).
Briefly, C57BL/6J mice were daily introduced for 10 consecutive days
in the same cage of a resident Swiss mouse (previously selected for its
aggressive behavior), each day with a different resident Swiss mouse.
Each social defeat session lasted 10 min. After this period of agonistic
body contact both mice, C57BL/6J and Swiss, remained for 24h in the
same cage but separated by a clear perforated acrylic divider allowing
only sensorial contact. These mice were designated to the “SDS” group.
Control mice were maintained in pairs (both C57BL/6J) in a cage di-
vided by a clear perforated acrylic divider allowing sensorial contact
and were handled similarly to the SDS mice during all the protocol.
These mice were designated to the “no-SDS”group. All mice were
constantly evaluated during the defeat protocol, and, as standardized
by Golden et al. (2011), mice with open wounds exceeding 1 cm were
removed from the study and immediately euthanized.
2.2. Subthreshold social defeat stress (SSDS)
For the subthreshold social defeat stress (also denominated micro-
defeat) protocol C57BL/6J mice were introduced to the cage of resident
Swiss mice (previously selected for its aggressive behavior) in three 5-
min session on a single day, with 15 min of rest, with sensorial contact,
between each session (Krishnan et al., 2007).
2.3. Persistent hyperalgesia induction model
To induce persistent hyperalgesia, we used an intense protocol of
repetitive inflammatory stimulus previously described by Ferreira et al.
(1990) and Villarreal et al. (2009). In this protocol we performed daily
injections (using a hypodermic 26-gauge needle) of prostaglandin E2
(PGE2) for 14 days in the plantar surface of the mice hind paw (90μg/
15μl/paw). After these two weeks of PGE2 injections mice shows long-
lasting mechanical hiperalgesia for, at least, 30 days.
2.4. Mild protocol of repetitive inflammatory stimulus
To evaluate predisposition to chronic pain we used a mild protocol
of repetitive inflammatory stimulus described by Dias et al. (2015),
which is a short-term version of the persistent hyperalgesia induction
model (Ferreira et al., 1990; Villarreal, Funez, et al., 2009). In this
protocol we performed daily injections (using a hypodermic 26-gauge
needle) of prostaglandin E2 (PGE2) for 7 days in the plantar surface of
the mice hind paw (90μg/15μl/paw). This protocol is used to evaluate
the susceptibility for pain chronification since it is not sufficient to
induce per se long-lasting mechanical hiperalgesia in a healthy in-
dividual.
2.5. Social interaction test
Social interaction behavior was evaluated through the social inter-
action test 24 hours after the last (S)SDS session, as previously de-
scribed by Golden et al. (2011). For this test we used an open field arena
(42cm x 42cm x 42cm) with two zones of interest: interaction zone and
corner zone, as shown in figure 2 (upper right corner). This test con-
sisted of two 150s consecutive sessions, separated from each other by
30s. The first session is called “no target” and an empty perforated
acrylic enclosure was placed in the center of the interaction zone. The
second session is called “target” and is similar to the first session,
however a Swiss mouse (selected as an aggressor but not used during
SDS) was placed into the perforated plastic enclosure. In both sessions
the C57BL/6J mouse was placed into the rear part of the open field
 L. Piardi, et al.

761
Figure 1. Graphic diagram of the experimental design. (A) First set of experiments to evaluate if SDS would predispose to persistent hyperalgesia, and (B) second set of experiments to evaluate if persistent hyperalgesia
would predispose depressive-like behavior of social avoidance after subthreshold SDS.
Journal of Affective Disorders 274 (2020) 759–767
L. Piardi, et al.
Figure 2. (A) Total time spent in the interaction zone during target session of the social interaction test performed 24h after the last defeat session of the SDS protocol
(B) Total time spent in the corner zone during target session of the social interaction test performed 24h after the last defeat session of the SDS protocol. (N=19-20;
***p≤0.001 different from no-SDS group). Figure on the upper right corner shows the social interaction arena (IZ = interaction zone and CZ = corners zone).
opposite the interaction zone. Both sessions were recorded by a camera
system and analyzed in specialized software (X-Plo-Rat – developed by
Dr. Silvio Morato, USP, Brazil) to obtain the total time spent in the
interaction and corners zone. The results are shown as the total time
spent in the interaction and corner zone in the target session. Less time
in the interaction zone and more time in the corner zone, when com-
pared to the control no- SDS group, are considered depressive-like be-
havior of social avoidance. In this test is also possible to segregate the
SDS mice in “susceptible” and “resilient”. Susceptible mice have social
interaction ratio (SI) – calculated by dividing the time spent in the in-
teraction zone during “target” session by the time spent in the inter-
action zone during “no target” session – less than 1 (SI < 1); and re-
silient mice have SI greater or equal than 1 (SI ≥ 1). It means that
susceptible mice show depressive-like behavior of social avoidance and
resilient mice are resistant, not showing socialavoidance.
2.6. Electronic von Frey test
The electronic von Frey test was performed to evaluate the me-
chanical nociceptive threshold, as previously described (Cunha et al.,
2004). Briefly, this test consisted of evoking a hind paw flexion with a
hand-held force transducer adapted with a 0.5mm polypropylene tip
(electronic von Frey; IITC Life Science, Woodland Hills, CA, USA). A
tilted mirror placed under the grid provided a clear view of the mouse
hind paw. The experimenter was trained to apply a pressure on the
plantar surface of the mouse hindpaw with a constant gain of strength
until the mouse withdraws the paw stereotypically in a clear flinching
response. Then the stimulus was automatically discontinued, and its
intensity was recorded when the paw was withdrawn. For habituation,
mice were placed in the von Frey apparatus 30min before starting the
test. The von Frey apparatus consists in acrylic cages measuring
12 × 20 × 17cm with a floor made of a 5mm2 mesh of non-malleable
wire with a thickness of 1mm. The results are expressed by the variation
of the nociceptive threshold in grams (gram- force) obtained by sub-
tracting the average of three values observed in the baseline (before
starting the SDS protocol or PGE2 injections) from those of the average
of three values obtained after the SDS protocol or PGE2 injections (∆
mechanical nociceptive threshold in grams). This test was used to
evaluate mechanical hyperalgesia intensity. The experimenter was
blind to all experimentaltreatments.
2.7. Capsaicin test
To evaluate the mice response to chemical nociceptive stimulus, we
performed the capsaicin test. For habituation, 30min before starting the
experiment mice were placed in the capsaicin test apparatus consisted
in an observation box measuring 30 × 30 × 30cm with of one clear
glass wall at the front, three mirrored walls, and a mirrored floor.
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Journal of Affective Disorders 274 (2020) 759–767
Following the habituation period, capsaicin was injected in the sub-
cutaneous tissue of the left hind paw (0.1µg/15µl/paw). The nocicep-
tive response was characterized by the act of lifting the paw stereo-
typically (flinching) and was quantified during a 5 min period. This test
was used to evaluate chemical hyperalgesia intensity.
2.8. Statistical analysis
Mice were randomly assigned to the different experimental condi-
tions. Statistical analysis was performed in Graph-Pad Prism version 6.0
for Windows (GraphPad Software, San Diego, CA, USA). Data were
analyzed using unpaired T test and ANOVA techniques (one-way or
two-way depending on the number of sources of variances) followed by
a pos hoc Bonferroni multiple comparison tests to compare the groups.
Data are presented as the mean ± standard error of mean (SEM).
Statistical significance was defined as p ≤ 0.05.
3. Results
3.1. Experiment 1 – social stress-induced predisposition for chronic pain
Social defeat stress (SDS) induced depressive-like behavior of
social avoidance
After 10 days of social defeat stress we performed the social inter-
action test where mice from SDS group spent less time in interaction
zone and more time in corner zone during the target session when
compared to mice from no-SDS group (figure 2). As previously de-
scribed (Golden et al., 2011), this is a typical depressive-like behavior of
social avoidance and demonstrates that the model standardized by
Golden et al. (2011) was replicated in the present study. Figure 2A
shows the total time spent in the interaction zone by mice from each
group during target session (no-SDS: 78.26 ± 5.264s; SDS:
51.65 ± 5.028s). Unpaired T test revealed statistical difference be-
tween groups (p=0.0008; t=3.658; df=37). Figure 2B shows the total
time spent in the corner zone by mice from each group during target
session (no-SDS: 17.58 ± 1.972s; SDS: 41.05 ± 4.343s). Unpaired T test
revealed statistical difference between groups (p≤0.0001; t=4.834;
df=37).
3.2. Social defeat stress (SDS)-induced mechanical hyperalgesia was long-
lasting
After the end of the SDS protocol we performed weekly assessment
of the mechanical nociceptive threshold to evaluate how long SDS-in-
duced hyperalgesia last. As shown in figure 3, on week 0 (48h after the
last defeat session) we replicate our previously results (Pagliusi Jr.
et al., 2018) showing hyperalgesia induced by SDS (no-SDS:
-0.0118 ± 0.1896g; SDS: 1.84 ± 0.2503g). One week after the last
L. Piardi, et al.
Figure 3. Variation of the mechanical nociceptive threshold. Figure shows
thevariation of the mechanical nociceptive threshold 0, 1, 2 and 3 weeks after
the last defeat session of the SDS protocol (N=17-20; **p≤0.01 different from
no-SDS group; ***p≤0.001 different from no-SDS group; week 0 means 48h
after the last defeat session).
defeat session (on week 1) mice from SDS group were still showing
mechanical hyperalgesia when compared to mice from no-SDS group
(control: -0.1176 ± 0.1654g; SDS: 1.765 ± 0.2643g). Same occurred
two weeks after the last defeat session (on week 2), when mice from
SDS group was still showing mechanical hyperalgesia when compared
to mice from no-SDS group (control: -0.0294 ± 0.1548g; SDS:
1.045 ± 0.3169g). However, three weeks after the last defeat session
(on week 3) the mechanical nociceptive threshold of mice from SDS
group returned to the baseline (control: 0.1 ± 0.1906g; SDS:
0.235 ± 0.1907g) and became similar to the no-SDS group. Two-way
analyses of variance (ANOVA) showed significant differences for all
effects (Interaction: F3,105= 13.54 p≤0.0001; Time: F3,105= 9.157
p≤0.0001; Column Factor: F1,35= 22.41 p≤0.0001; Subjects: F35,105=
5.440 p≤0.0001). The post hoc Bonferroni's multiple comparison test
revealed statistical differences between no-SDS and SDS groups on
week 0 (p≤0.001), week 1 (p≤0.001) and week 2 (p=0.0048).
3.3. Social defeat stress (SDS) predisposed to persistent hyperalgesia in a
mild protocol of repetitive inflammatory stimulus
After SDS-induced mechanical hiperalgesia returns to the baseline (3
weeks, see figure 4), we perform a mild protocol of repetitive inflammatory
stimulus consisted by only 7 days of PGE2 (or saline as control for PGE2)
injections subcutaneously in the hind paw. As shown in figure 4A, 24h after
the last PGE2 injection (day 1) mice from both no-SDS and SDS groups
showed mechanical hyperalgesia (i.e. are statistical different from mice of
no-SDS Saline group). SDS mice injected with saline (SDS Saline group) did
not show mechanical hyperalgesia on day 1, although it has increased their
mechanical nociceptive threshold when compared to the day before starting
saline injections (no-SDS Saline: 0.1143 ± 0.2355g; no-SDS PGE2:
2.57 ± 0.2608g; SDS Saline: 1.38 ± 0.3723g; SDS PGE2: 3.49 ± 0.2892g).
After 8 days of the last injections (day 8) only SDS groups (Saline and PGE2)
showed long-lasting mechanical hyperalgesia. As expected in a mild per-
sistent hyperalgesia induction protocol, mice from no-SDS PGE2 group did
not show long-lasting mechanical hyperalgesia (no-SDS Saline: -0.3429
± 0.2608g; no-SDS PGE2: -0.34 ± 0.4064g; SDS Saline: 1.34 ± 0.3925g;
SDS PGE2: 1.58 ± 0.4555g). Two-way ANOVA showed significant differ-
ences for all effects (Interaction: F6,66=7.286 p≤ 0.0001; Time:
F2,66=34.07 p≤0.0001; Column Factor: F3,33=8.207 p=0.0003; Subjects:
F33,66=2.180 p=0.0036). The post hoc Bonferroni's multiple comparison
test revealed statistical differences on day 1 between no-SDS Saline and
both PGE2 groups (p≤0.001) and between SDS Saline and SDS PGE2
(p<0.001). Still observing the data from day 1, it's important to note that
there is no statistical difference between SDS saline and no-SDS saline
groups. On day 8 the post hoc Bonferroni's multiple comparison test re-
vealed statistical differences between no-SDS and SDS groups (p≤0.05
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Journal of Affective Disorders 274 (2020) 759–767
between no-SDS Saline and SDS Saline and p≤0.01 between no-SDS Saline
and SDS PGE2, and between no-SDS PGE2 and both SDS).
Figure 4B shows mechanical hyperalgesia 8 days after the last PGE2
injection but, at this moment, we separated the phenotypes generated
by SDS protocol (resilient and susceptible mice) evaluated in the social
interaction test performed on day -28 of the experimental design. We
can see that susceptible mice, but not resilient, shows persistent me-
chanical hyperalgesia (no-SDS PGE2: 0.0222 ± 0.2060g; resilient PGE2:
0.5200 ± 0.3121g; susceptible PGE2: 2.640 ± 0.5240g). One-way
ANOVA showed significant differences between groups (F2,16=17.33;
p≤0.0001). The post hoc Bonferroni's multiple comparison test re-
vealed statistical differences between susceptible PGE2 and no-SDS
PGE2 (p≤0.001) and resilient PGE2 (p≤0.01).
After 10 days of the last injections (day 10) we performed the
capsaicin test (figure 4C) and only SDS PGE2 group showed long-lasting
chemical hyperalgesia (no-SDS saline: 48.75 ± 3.400; no-SDS PGE2:
56.30 ± 2.028; SDS saline: 61.70 ± 3.986; SDS PGE2: 64.90 ± 4.040).
One-way ANOVA showed significant differences between groups
(F3,34=3.821; p=0.0184). The post hoc Bonferroni's multiple com-
parison test revealed statistical differences between SDS PGE2 and no-
SDS saline (p=0.0161)
3.4. Social defeat stress (SDS)-induced depressive-like behavior of social
avoidance was long-lasting regardless saline or PGE2 injection in the hind
paw
After 9 days after the last injection (PGE2 or saline), i.e. 38 days after the
SDS protocol, we performed another social interaction test and both, saline
and PGE2, SDS groups spent less time in interaction zone (figure 5A) and
more time in corner zone (figure 5B) during the target session when
compared to no-SDS groups, which indicate that they are still showing
depressive-like behavior of social avoidance (Time in interaction zone –
no-SDS Saline: 70.50 ± 6.150s; no-SDS PGE2: 70.00 ± 6.396s; SDS Saline:
33.00 ± 4.240s; SDS PGE2: 31.20 ± 4.950s. Time in corner zone – no-SDS
Saline: 19.75 ± 3.115s; no-SDS PGE2: 22.30 ± 6.289s; SDS Saline:
54.80 ± 7.212s; SDS PGE2: 46.70 ± 7.149s). One-way ANOVA showed
significant differences between groups in the time in interaction zone (Time
in interaction zone – F3,34=16.23; p≤0.0001. Time in corner zone –
F3,34=7.322; p=0.0006). The post hoc Bonferroni's multiple comparison
test revealed statistical differences between SDS groups (saline and PGE2)
and both no-SDS groups (saline and PGE2) (Time in interaction zone –
p≤0.001; Time in corner zone – p≤0.01 between SDS saline and control
groups and p≤0.05 between SDS PGE2 and control groups).
3.5. Experiment 2 – chronic pain-induced predisposition for depressive-like
behavior of social avoidance
14 days of PGE2 injections in the hind paw induced persistent
hyperalgesia
After 1 day and 15 days of the last PGE2 (or saline) injection we
performed an electronic von Frey test to assess the mechanical noci-
ceptive threshold (figure 6). As previously described (Ferreira et al.,
1990; Villarreal, Funez, et al., 2009), all mice injected with PGE2 for 14
days showed persistent mechanical hyperalgesia (Day 1 – saline:
0.3824 ± 0.1102g; PGE2: 3.4263 ± 0.2405g. Day 15 – saline:
0.1618 ± 0.225g; PGE2: 2.5526 ± 0.2065g). Two-way ANOVA showed
significant differences for time, column factor and subjects (Interaction:
F1,34=3.415 p=0.0733; Time: F1,34=9.597 p=0.0039; Column Factor:
F1,34=120.6 p≤0.0001; Subjects: F34,34=1.961 p=0.0267). The post
hoc Bonferroni's multiple comparison test revealed statistical differ-
ences on day 1 and 15 between saline and PGE2 groups (p ≤0.001).
3.6. Persistent hyperalgesia predisposes to depressive-like behavior in a
subthreshold social defeat stressparadigm
After 14 days of PGE2 injections we performed a subthreshold social
L. Piardi, et al. Journal of Affective Disorders 274 (2020) 759–767

Figure 4. (A) Shows the variation of the mechanical nociceptive threshold 1 day before (experimental day -6), 1 and 8 days after PGE2 (or saline) injections (N=7-
10; *p≤0.05 no-SDS Saline vs. SDS Saline; **p≤0.01 no-SDS Saline vs. SDS PGE2; no-SDS PGE2 vs. SDS Saline; no-SDS PGE2 vs. SDS PGE2; ***p≤0.001 no-SDS
Saline vs. no-SDS PGE2; no-SDS Saline vs. SDS PGE2; ###p≤0.001 SDS Saline vs. SDS PGE2); (B) shows variation of the mechanical nociceptive threshold 8 days after
PGE2 injections, dividing SDS group in susceptible (SI ratio ≥ 1) and resilient (SI ratio < 1) (N=5-9; **p≤0.01; ***p≤0.001) and (C) shows the number of flinches
for each experimental group in the capsaicin test performed 10 days after the last saline/PGE2 injection (N=7-10; **p≤0.01 different from no-SDS Saline group).

Figure 5. (A) Total time spent in the interaction zone during target session 9 days after the last saline/PGE2 injection. (B) Total time spent in the corner zone during
target session 9 days after the last saline/PGE2 injection (N=8-10; *p≤0.05 different from no-SDS group; **p≤0.01 different from no-SDS group; ***p≤0.001
different from no-SDS group). Figure on the upper right corner shows the social interaction arena (IZ = interaction zone and CZ = corners zone).

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L. Piardi, et al.
Figure 6. Variation of the mechanical nociceptive threshold. Figure shows the
variation of the mechanical nociceptive threshold 1 and 15 days after saline/
PGE2 injections (N=17-19; ***p≤0.001 different from saline group).
defeat stress (SSDS) session as previous described (Krishnan et al.,
2007) and assessed the social interaction behavior three times: 24h (0
weeks), 2 weeks and 3 weeks after SSDS session. Figure 7 shows the
time spent in the interaction zone (A) and corner zone (B) during target
session for all three social interaction tests performed. We can see that
in both, saline and PGE2, SSDS groups spent less time in the interaction
zone and more time in the corner zone when compared to the control
no-SDS groups 24h after SSDS session, that is, showed depressive-like
behavior of social avoidance (Time in interaction zone – no-SSDS
saline: 46.10 ± 5.815s; no-SSDS PGE2: 54.20 ± 6.352s; SSDS saline:
20.67 ± 5.605s; SSDS PGE2: 13.30 ± 2.103s. Time in corner zone –
no-SSDS saline: 38.60 ± 6.791s; no-SSDS PGE2: 29.80 ± 6.354s; SSDS
saline: 72.90 ± 9.885s; SSDS PGE2: 80.30 ± 7.733s). One-way ANOVA
Figure 7. (A) Total time spent in the interaction zone during target session 0, 2
and 3 weeks after SSDS (B) Total time spent in the corner zone during target
session 0, 2 and 3 weeks after SSDS (N=7-10; *p≤0.05; **p≤0.01 and
***p≤0.001; &&p≤0.01 different from no-SSDS saline group; &&&p≤0.001
different from no-SSDS saline group; ##p≤0.01 different from no-SSDS PGE2
group; ###p≤0.001 different from no-SSDS PGE2 group; Ѳp≤0.05 different
from both saline groups; week 0 means 24h after SSDS).
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Journal of Affective Disorders 274 (2020) 759–767
showed significant differences between groups (Time in interaction
zone – F3,35=14.28; p≤0.0001. Time in corner zone – F3,36=10.18;
p≤0.0001). The post hoc Bonferroni's multiple comparison test re-
vealed statistical differences between SSDS groups (saline and PGE2)
and both no-SSDS groups (saline and PGE2) (p≤0.01 between no-SSDS
saline and SSDS saline; and p≤0.001 between no-SSDS groups and
SSDS PGE2 and between control PGE2 and SSDS saline).
In the second social interaction test, performed 14 days after SSDS
session, only SSDS PGE2 showed depressive-like behavior of social
avoidance, spending less time in the interaction zone and more time in
the corner zone when compared to the no-SSDS groups (Time in in-
teraction zone – no-SSDS saline: 64.63 ± 4.799s; no-SSDS PGE2:
70.56 ± 7.474s; SSDS saline: 53.11 ± 9.621s; SSDS PGE2:
32.10 ± 8.127s. Time in corner zone – no-SSDS saline:
25.89 ± 6.603s; – no-SSDS PGE2: 23.56 ± 4.882s; SSDS saline:
43.11 ± 9.102s; SSDS PGE2: 71.30 ± 10.73s). One-way ANOVA
showed significant differences between groups (Time in interaction
zone – F3,32=4.864; p=0.0067. Time in corner zone – F3,33=7.266;
p=0.0007). The post hoc Bonferroni's multiple comparison test re-
vealed statistical differences between SSDS PGE2 and both no SSDS
groups (saline and PGE2) (Time in interaction zone – p≤0.05 between
control saline and SSDS PGE2 and p≤0.01 between control PGE2 and
SSDS PGE2. Time in corner zone – p≤0.01 between no-SSDS groups
and SSDS PGE2).
In the third and last social interaction test, performed 21 days after
SSDS session, again only SSDS PGE2 showed depressive-like behavior of
social avoidance, spending less time in the interaction zone and more
time in the corner zone when compared to the control no-defeated
groups (Time in interaction zone – no-SSDS saline: 60.10 ± 5.767s;
no-SSDS PGE2: 65.11 ± 5.591s; SSDS saline: 59.13 ± 8.812s; SSDS
PGE2: 31.70 ± 7.448s. Time in corner zone – no-SSDS saline:
25.56 ± 4.571s; no-SSDS PGE2: 20.25 ± 2.313s; SSDS saline:
41.50 ± 10.61s; SSDS PGE2: 74.80 ± 11.92s). One-way ANOVA
showed significant differences between groups (Time in interaction
zone – F3,33=5.019; p=0.0056. Time in corner zone – F3,31=8.400;
p=0.0003). The post hoc Bonferroni's multiple comparison test re-
vealed statistical differences between SSDS PGE2 and all other groups
(Time in interaction zone – p≤0.05 between no-SSDS saline and SSDS
PGE2 and p≤0.01 between SSDS PGE2 and other groups. Time in
corner zone – p≤0.05 between SSDS PGE2 and SSDS saline and
p≤0.01 between SSDS PGE2 and no-SSDS groups).
4. Discussion
The present investigation was designed to evaluate whether social
defeat stress – that induces depressive-like behavior of social avoidance
– would predispose to persistent hyperalgesia and, inversely, weather
persistent hyperalgesia would predispose to depressive-like behavior of
social avoidance in mice. This approach was taken because there is, in
humans, great comorbidity between chronic pain and depression
(Goldenberg, 2010; Robinson et al., 2009) and few studies have in-
vestigated this relationship in animal models. We used the social defeat
stress paradigm because the stress associated with social relationships
like family, work and urban conditions are, generally, the most
common stress that affects modern humans and can trigger MDD
(Jasnow et al., 2005; Kumpulainen, 2008; Davidson & McEwen, 2012).
Also, we used the repetitive inflammatory stimulus (PGE2 injections)
paradigm to study pain chronification because it is an established
model (Ferreira et al., 1990; Villarreal, Funez, et al., 2009) and, among
many causes, chronic pain commonly results from an inflammatory
episode (Woolf, 2011) and PGE2 is a well-known inflammatory med-
iator (Ferreira et al., 1990).
Our results showed that social defeat stress induces mechanical
hyperalgesia, as we had previously described (Pagliusi et al., 2018), and
that this hyperalgesia is chronic, lasting for at least 2 weeks. Also, our
results showed that social defeat stress (SDS) predisposes to persistent
L. Piardi, et al.
hyperalgesia in a mild protocol of repetitive inflammatory stimulus
and, inversely, persistent hyperalgesia predisposes to depressive-like
behavior of social avoidance in a subthreshold social defeat stress
(SSDS) model. These results strongly corroborate clinical studies
showing the abovementioned comorbidity between chronic pain and
depression (Bair et al., 2008; Maletic & Raison, 2009; Robinson et al.,
2009; Goldenberg, 2010). To the best of our knowledge, no studies
investigated the causality relationship between inflammatory agents-
induced chronic pain and depressive-like behavior in a social stress
mouse model approach.
Social defeat stress-induced hyperalgesia has already been reported
in a mild social defeat stress model for rats (Rivatet al., 2010). In this
study, Rivat et al. (2010) showed that SDS-induced hyperalgesia was
long-lasting for at least 2 weeks, same observed for mice in the present
study. In fact, it is well established chronic stress triggering hyper-
algesia (Satoh et al., 1992; Da Silva Torres et al., 2003; Bradesi et al.,
2005; Khasar et al., 2005; Gameiro et al., 2006; Suarez-Roca et al., 2006;
Rivat et al., 2007) and depressive-like behavior (Conti et al., 2002;
Mineur et al., 2006; Bondi et al., 2008; Pollak et al., 2010). However,
there is a lack of studies evaluating chronic stress, mainly social stress,
triggering these both conditions simultaneously.
It's important to mention that we waited the mechanical nociceptive
threshold of the stressed mice returned to baseline values (evaluated
before the SDS); this way, when beginning the PGE2 injections, all
groups would depart under equal pain sensibility conditions.
Interestingly, mechanical hyperalgesia disappears (figure 4) even
stressed mice still showing social avoidance (figure 5). We hypothe-
sized that social avoidance is a more complex behavior than pain re-
sponse elicited by mechanical stimulus, recruiting more neural net-
works and requiring greater integration in the central nervous system.
This idea is supported by the fact that resilient mice showed mechanical
hyperalgesia even not showing depressive-like behavior of social
avoidance (Pagliusi Jr. et al., 2018). That is, because it is a less complex
behavior and has arisen earlier in vertebrate phylogeny, hyperalgesia
can be previously induced by social stress and independently of social
avoidance behavior expression.
In another context, Brüning et al., (2015) showed that partial sciatic
nerve ligation, commonly reported as a comorbid pain and depression
model, can induce depressive-like behavior in mice evaluated in the
forced swimming and tail suspension tests 4 weeks after experimental
surgery. It's important to mention that 14 days of PGE2 injections
cannot induce depressive-like behavior of social avoidance by itself, as
shown in the no SSDS PGE2 group in figure 7. In fact, our results
showed that, to induce depressive-like behavior of social avoidance, 14
days of PGE2 needs to be followed by a subthreshold social defeat stress
stimulus that, in naïve mice, is not sufficient to induce social avoidance.
This way, SDS – in the experiment 1 – and 14 days of PGE2 injec-
tions – in the experiment 2 – would be generating neuroplasticity on
specifics brain areas, probably shared by chronic pain and depression,
and, as consequence, predisposing to pain chronification and depres-
sive-like behavior of social avoidance, respectively. Corroborating this
idea, Norman, Karelina and Zhang (2010a) showed that spared nerve
injury, an animal model of persistent peripheral neuropathic pain, also
induces depressive-like behavior evaluated in the forced swim test.
They also showed that 2 weeks of chronic restraint stress prior to spared
nerve injury exacerbated the hyperalgesia and depressive-likebehavior,
resulting in an increase in IL-1b gene expression in the prefrontal cortex
and brain derived neurotrophic factor (BDNF) gene expression in
periaqueductal gray matter (PAG) (Norman, Karelina, Zhang, et al.,
2010a). It's important to mention that both, prefrontal cortex and PAG,
are of great importance in the pathophysiology of both depression and
chronic pain (Heinricher, 2016; Li, 2017). Moreover, although the
present study did not investigate mechanisms behind the causality be-
tween pain and depressive-like behaviors, it is plausible to consider that
the SDS would be stimulating neuroplasticity on specifics brain areas,
such as prefrontal cortex ad PAG, probably shared by chronic pain and
766
Journal of Affective Disorders 274 (2020) 759–767
depression, and, as consequence, predisposing to both pain chron-
ification and depressive-like behavior of social avoidance.
Considering peripheral outcomes of SDS, it is worth mentioning a
possible priming effect on nociceptor induced by chronic stress. It is
well known that chronic stress, including SDS, results in chronic acti-
vation of the HPA axis, which, in turns, induces an increase of circu-
lating inflammatory cytokines (Tian et al, 2014; Nasef et al, 2017). This
inflammatory environment induced by chronic stress can switch the
nociceptor, making it more susceptible to PGE2 in a process that we call
hyperalgesic priming (Reichling and Levine, 2009). In this context,
Khasar et al (2008) showed in ratsthat repeated sound stress enhances
the susceptibility of the nociceptor to PGE2, also discussing a possible
hyperalgesic priming induced by repeated stress. Aurora et al (2018)
performed a study also suggesting this effect. In this study, authors
showed that chronic social stress slowed the recovery of the nociceptive
threshold after a post-operative pain model in rats, indicating that local
inflammatory cytokines along with stress-released cytokines would be
triggering hyperresponsiveness of the nociceptor (Aurora et al, 2018).
It is noteworthy that many studies linking stress with pain and de-
pressive-like behavior employ physical stress protocols, such as physical
restraint and forced swim (DeSantana et al., 2013; Li et al., 2017;
Nishiyori et al., 2011; Scheich et al., 2017; Sousa et al., 2018). How-
ever, nowadays, it is unlikely humans being forcibly exposed to these
forms of stressors. Therefore, in the current study, we employed a social
stress protocol model that better reflects the modern human stressful
condition. This approach has great relevance because social environ-
ment significatively influences depressive-like and pain behaviors in
both human and rodents. Norman, Karelina, Morris, et al. (2010b), for
example, showed that social interaction during two weeks prior to the
spared nerve injury surgery prevents depressive-like behavior induced
by the injury. Also, Bates et al. (2016) showed in adolescent mice that
social environment (group-housed) mitigates the development of
opioid-induced hyperalgesia and antinociceptive tolerance.
It's to be noted that the current study did not investigate mechan-
isms behind the causality between pain and depressive-like behaviors,
although this mechanistic approach is open for future studies. However,
we advance in the understanding of the interrelationships between
social stress and pain chronification, showing that social stress predis-
poses to chronic pain and, inversely, chronic pain predisposes to de-
pressive-like behavior induced by social stress. In addition, we vali-
dated the SDS paradigm used in the present study as a chronic pain
model.
Data Accessibility
All data presented in the current manuscript can be obtained from
the corresponding author.
Author Contributions
L.P.N. and M.P.Jr. contributed equally and designed the study,
performed the experiments, analyzed the data, and drafted the manu-
script. I.J.M.B. performed behavioral experiments. S.F.M. performed
behavioral experiments. F.B.Z. performed behavioral experiments.
A.F.B. performed behavioral experiments. C.H.T. analyzed the data and
drafted the manuscript. C.A.P. designed the study, analyzed the data,
and drafted the manuscript. C.R.S. designed the study, analyzed the
data, and drafted the manuscript.
Declaration of interest statement
The authors report no conflicts of interest.
L. Piardi, et al.
Acknowledgement
This study was supported by the Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP, Grant number: 2015/26777-7) and
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil
(CAPES) – Finance Code 001.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.jad.2020.05.144.
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