Physiology & Behavior 151 (2015) 55–63

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Persistent, comorbid pain and anxiety can be uncoupled in a mouse model Yan Liu a, Liu Yang a,b,⁎, Jin Yu b, Yu-Qiu Zhang a,⁎ a b

Institute of Neurobiology, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China Department of Integrative Medicine and Neurobiology, Shanghai Medical College, Fudan University, Shanghai 200032, China

H I G H L I G H T S • We compare the interaction between chronic pain and anxiety in two mice strains. • Pain and anxiety may not necessarily exacerbate one another in mouse models. • FVB mice were more resilient in behavioral responses to social stress than C57 mice.

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Article history: Received 10 April 2015 Received in revised form 3 July 2015 Accepted 5 July 2015 Available online 10 July 2015 Keywords: Anxiety-like behavior Allodynia and hyperalgesia Comorbidity Inflammatory pain Social defeat Social interaction avoidance

a b s t r a c t Clinically, pain and anxiety frequently coexist; however, these two conditions' interaction is limited and contradictory in animal studies. In this study, we combined social defeat (SD) stress with Freund's adjuvant (CFA)-induced persistent inflammatory pain to investigate the reciprocal relationship between anxiety-like and nociceptive behaviors in two mouse strains. C57BL/6J mice subjected to the 10-day period of SD stress by repeated CD-1 mice aggression exhibited significant social interaction avoidance behaviors in the social interaction (SI) test, which is believed to represent the symptoms of anxiety. These mice also displayed anxiety-like behaviors in elevated plus maze (EPM) and open field (OF) tests. Compared to C57BL/6J mice, FVB/NJNju mice showed less basal social contact, but their behavioral responses to 10-day SD stress were more resilient. CFAinflammatory mice showed robust mechanical allodynia and thermal hyperalgesia in both strains, but did not develop obvious social avoidance and anxiety-like behaviors 10 days after CFA-inflammation. Interestingly, CFAinflammatory mice exposed to SD stress were not accompanied by a worsening of pain and anxiety-like behaviors in most tests. In contrast, the SD stress-induced social avoidance was significantly antagonized by combining with CFA-inflammatory pain. These findings suggest that persistent inflammatory pain and SD stress-induced anxiety may not necessarily exacerbate one another in animal models of comorbidity. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Clinical comorbidity of pain and mood disorders (anxiety or depression) is widely recognized. Epidemiological studies have reported that chronic pain and mood disorders are reciprocally linked. The prevalence of pain in subjects with anxiety or depression, and that of anxiety or depression in subjects with pain, is higher than in the cohort with either condition alone [1–3]. In addition to the large amounts of literature on comorbidity of pain and mood disorders in human subjects, there have been many studies investigating this issue in animal models [4]. Although the general data from patient studies suggest that comorbidity

⁎ Corresponding authors at: Y.-Q. Zhang, Institute of Neurobiology, Fudan University, 1202 Mingdao Building, 131 Dong An Road, Shanghai 200032, China. L. Yang, now at Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China. E-mail addresses: [email protected] (L. Yang), [email protected] (Y.-Q. Zhang).

http://dx.doi.org/10.1016/j.physbeh.2015.07.004 0031-9384/© 2015 Elsevier Inc. All rights reserved.

provokes a general worsening in both conditions [5,6], animal studies found conflicting results, as the relationship between chronic pain and mood disorders depends on the differences in animal species/strains, models, test periods and behavioral test paradigms [4,7]. For example, a study by Mao showed that in social stress-induced anhedonic rats (Wistar), arthritic pain worsened depressive-like behaviors in forced swimming and tail suspension tests and was associated with exacerbated thermal hyperalgesia and mechanical allodynia [8]. In contrast, Bravo and colleagues showed that the combination of chronic constriction injury (CCI, a model of neuropathic pain) and unpredictable chronic mild stress (CMS) had no effect on the CCI-induced mechanical allodynia and CMS-induced anhedonia and behavioral despair in Sprague–Dawley rats [9]. Chronic social defeat (SD) stress in rodents induces social interaction avoidance and the anxious/depressive-phenotype [10,11]. This ethologically relevant stressor involves forcing a rodent to intrude on the home cage of a more aggressive rodent, which overpowers it until

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a rapidly submissive phenotype emerges [12]. Social defeat bears anxiogenic consequences, as assessed by unconditioned anxiety tests [13,14]. The previous studies from our and other laboratories showed that C57BL/6J mice subjected to a 6- or 10-day period of SD stress induced by repeated CD-1 mouse aggression displayed significant social interaction avoidance behaviors representing symptoms of anxiety [15–17]. In the present study, by combining SD stress with complete Freund's adjuvant (CFA)-induced chronic inflammatory pain models, we further investigated the reciprocal relationship between pain and anxiety-like behaviors. Because of the known variability among animal strains in their expression of either pain or anxiety/depression-like behaviors [18–20], we studied two strains (C57BL/6J and FVB/NJNju) in the present study. 2. Materials and methods 2.1. Experimental animals Adult male C57BL/6J mice (7–9 weeks) from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences and FVB/ NJNju mice (7–9 weeks) from the Model Animal Research Center of Nanjing University (Nanjing, China) were housed in groups in a temperature- and humidity-controlled room with a 12:12 light– dark cycle (lights on 07:00) and water available ad libitum. CD-1 mice that were retired breeders (male, 8–10 months) from Vital River Laboratories (Beijing, China) were used as the aggressors. The aggressors were screened every 3 months to ensure their antagonistic interactions. Six groups were tested in both strains of mice (C57BL/6J and FVB/NJNju): sham-SD control, SD stress, sham-CFA control, CFA-inflammation, CFA plus SD, and sham-CFA plus SD. To control for possible effects of time of day, mice were trained and tested at approximately the same time of day (3–6 h after lights on). All of the behavioral tests were performed by the same experimenter blinded to the group assignment to minimize the differences between-experimenters. All experimental protocols and animal handling procedures were permitted by the Shanghai Animal Care and Use Committee and Animal Ethical Committee of Fudan University, and were consistent with the policies issued by the International Association for the Study of Pain. 2.2. Social defeat stress and social interaction test Social defeat (SD) stress was conducted as previously described [16]. Briefly, the C57BL/6J or FVB/NJNju mouse was individually introduced to the home cage of an unfamiliar aggressive CD-1 resident mouse for 6–10 min and physically defeated. During the exposure, when the C57BL/6J or FVB/NJNju mouse showed signs of subordination (freezing or upright submissive postures) or when three attacks on the CD-1 mouse were observed, the defeated C57BL/6J or FVB/NJNju mouse was separated from the aggressive CD-1 mouse by introducing a perforated plastic divider into the cage to allow visual, auditory and olfactory contacts for the remainder of 24 h. If the aggressive CD-1 mouse did not initiate an attack within 10 min, a new aggressive CD-1 mouse was introduced. The next day, the experimental mouse was transferred to a new cage where another unfamiliar aggressive CD-1 mouse resided. During a period of SD stress, these mice were subjected to social defeat for 10 consecutive days (exposure to 10 different CD-1 mice). Considering that the damage by the social aggression may influence nociceptive responses, experimental mice that showed obvious visible wounds resulting from the SD stress procedure were excluded from the experiments. Control animals were kept in identical home cages in pairs (separated by the shielding board) during the 10 days. The schematic diagram of the experimental apparatus is shown in Fig. 1A. The social interaction (SI) test was performed on day 11 in a clean, open arena (42 cm × 42 cm). Each test consisted of two 2.5-min sessions, separated by an interval of 30–60 s. In the first (also called No

Fig. 1. Social defeat paradigm. (A) A schematic representation of the experimental process for social defeat (SD) stress. An experimental mouse (C57BL/6J or FVB/NJNju) was introduced to the cage of a CD-1 resident mouse (aggressor) for 6–10 min and physically defeated; then, they were housed together but separated by perforated plastic divider to allow visual, olfactory and auditory contact for the remainder of 24 h. (B) The social interaction test was performed in an open field like. The area around the cage (8 cm from the mesh) was the interaction zone. The time spent in the interaction zone was recorded.

target) session, the experimental (C57BL/6J or FVB/NJNju) mouse was introduced to the arena with an empty mesh cage (10 cm × 6 cm). In the second (Target) session, a mesh cage with an unfamiliar CD-1 mouse replaced the empty cage. The mesh cage allowed visual and olfactory interactions (but not physical contact) between the experimental and target mice. The area (14 cm × 26 cm, meaning 8 cm around the mesh cage) was defined as the interaction zone (IZ) (Fig. 1B). The time spent in the IZ was measured. A social interaction ratio (SIR) was calculated as follows: dividing the interaction time of the second (Target) session by the first (No target). 2.3. Model of inflammatory persistent pain Complete Freund's adjuvant (CFA, 10 μl, Sigma, USA) was injected into the plantar surface of the unilateral hindpaw under brief isoflurane anesthesia. Sham control animals received 10 μl of incomplete Freund's adjuvant. Local redness and swelling were observed in CFA-injected mice but not sham-CFA mice during the experimental period. 2.4. Comorbidity of persistent inflammatory pain and SD stress One day after intraplatar injection of CFA (or sham), experimental mice received the SD stress as described above. Behavioral tests were performed on day 12. 2.5. Nociceptive behavioral tests 2.5.1. von Frey test Mechanical allodynia was assessed by measuring paw withdrawal thresholds (PWTs) in response to a calibrated series of von Frey hairs

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Fig. 2. Effects of social defeat (SD) stress and chronic inflammatory pain on mouse social interaction level. (A–D) SD stress-induced social interaction avoidance, as indicated by the time spent in the interaction zone in the absence and presence of a social target (A, C) and the social interaction ratio (SIR, the time spent in the interaction zone in the presence of a target divided by that in the absence of a target) (B, D) in both C57BL/6J (A, B) and FVB/NJNju (C, D) mice. Intraplantar (i.pl.) injection of CFA and sham groups did not produce social avoidance in either strain. **p b 0.01 versus no target; ##p b 0.01 versus sham-SD group.

(Stolting Co., Wood Dale, IL). Mice were placed individually into Plexiglas chambers on an elevated wire-mesh floor and a series of von Frey hairs were applied to the plantar surface of the hindpaw in ascending order (0.16, 0.4, 0.6, 1.0, 1.4, 2.0 and 4 g) with a sufficient force to bend the hair for 2 s or until paw withdrawal. The PWT was defined as the lowest force in grams that produced at least 3 withdrawal responses in 5 consecutive applications. 2.5.2. Hargreaves' test Thermal hyperalgesia was assessed by measuring the paw withdrawal latencies (PWLs) in response to a radiant heat source using Hargreaves' test. Mice were put in Plexiglas chambers on an elevated glass platform. Heat was delivered to the glabrous surface of paw through the glass plate using a radiant heat source (IITC Lifescience Inc., Woodland Hills, CA). The time from the onset of radiant heat application to the withdrawal of the hindpaw was defined as the PWL, and a 15-s cutoff was set to avoid tissue damage. The von Frey and Hargreaves' tests were performed in the sham-SD, SD, sham-CFA, CFA, sham-CFA + SD and CFA + SD mice; the von Frey test was followed by the Hargreaves' test with an interval of 2 h in home cages.

2.6.2. Elevated plus maze test The elevated plus maze (EPM) consisted of two equally sized open arms (without walls) crossed with two equally sized closed arms (flanked by 20-cm opaque walls). All arms communicated through a central zone, allowing animals to move freely into each arm of the maze. Light intensities in the central area, opened and closed arms were set to 15, 15 and 5 lx, respectively. Mice were placed in the center area facing the open arms, and activity was recorded for 5 min by video and analyzed using the MED-VPM-RS system (MED-Associates, USA). The number of entries into the open arms and time spent in the open arms (covering 20 cm × 20 cm) were used as the measure of anxiety. For the different behavioral tests (OF and EPM), we used separate batches of animals. 2.7. Statistical analysis Data are presented as the mean ± SEM. Student's t-test, one-way ANOVA, two-way ANOVA or repeated measures ANOVA (RM ANOVA) followed by post hoc Student–Newman–Keuls test were used to identify significant differences. In all cases, p b 0.05 was considered statistically significant.

2.6. Anxiety-like behavioral tests

3. Results

2.6.1. Open field test The open field (OF) test was performed in a Plexiglas quadratic box (40 cm × 40 cm × 40 cm), which was evenly illuminated to 15 lx. Mice were individually placed in the field and allowed to explore for 5 min. Activity was recorded by video and analyzed using the MED-VOF-RS system (MED-Associates, USA). Time spent and distance traveled in the center zone (covering 20 cm × 20 cm) was used as the measure of anxiety.

3.1. Effects of social defeat stress and chronic inflammatory pain on mouse social interaction behavior Performance on the social interaction test is thought to be a measure of anxiety. Mice were exposed to a different aggressor (CD1) daily for 10 days, and then, social contact was measured using a two-trial interaction paradigm (Fig. 1) with an empty social target trial (no target) followed by a social target trial (target). As shown in Fig. 2A, undefeated

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Fig. 3. Effects of social defeat stress and chronic inflammatory pain on nociceptive behaviors. (A–D) Intraplantar (i.pl.) injection of CFA induced robust mechanical allodynia (A, C) and thermal hyperalgesia (B, D) at 6 and 11 days after injection in both C57BL/6J (A, B) and FVB/NJNju (C, D) mice. SD stress did not affect the paw withdrawal thresholds (PWTs) to von Frey hairs and paw withdrawal latencies (PWLs) to radiant heat stimulation in both strains. *p b 0.05, **p b 0.01 versus sham-CFA group.

C57BL/6J mice spent more time in the interaction zone when a social target was introduced. The mice exposed to social defeated (SD) stress spent less time in close proximity to the target mouse. Complete Freund's adjuvant (CFA)-induced inflammatory pain did not affect the social behavior of C57BL/6J mice. When presented with a target mouse, the CFA-inflammatory mice spent a similar time period as sham controls in the interaction zone, which significantly differed from SD mice (One-way ANOVA, F3,31 = 40.939, p b 0.001). The social interaction ratio (SIR, the time spent in the interaction zone in the presence of a target divided by that in the absence of a target) of SD mice was significantly lower than that in controls and inflammatory mice (One-way ANOVA, F3,31 = 4.673, p = 0.009) (Fig. 2B). Interestingly, although the FVB/NJNju mice also spent more time in the interaction zone in the presence of a target than that in the absence of a target, this strain showed a decreased social approach compared with C57BL/6J (Fig. 2C). Two-way ANOVA analysis revealed significant effects of strain (C57BL/6J verse FVB/NJNju) when presented with a target mouse (F1,79 = 26.723, p b 0.001). Neither SD stress nor inflammatory pain markedly influenced the interaction behavior of FVB/NJNju mice. There was no significant difference in the SIR among the treated groups (One-way ANOVA, F3,46 = 0.131, p = 0.941) (Fig. 2D). 3.2. Effects of social defeat stress and chronic inflammatory pain on nociceptive behaviors The von Frey and Hargreaves' tests were used to measure the mechanical and thermal response threshold. In the absence of SD stress and CFA-inflammation, paw withdrawal thresholds (PWTs) to von

Frey hairs and paw withdrawal latencies (PWLs) to radiant heat stimulation were stable for 10 days in both strains. In the C57BL/6J and FVB/ NJNju groups, intraplantar (i.pl.) injection of CFA induced robust mechanical allodynia and thermal hyperalgesia (Fig. 3A–D). Two-way RM ANOVA analysis revealed a significant effect of CFA treatment (PWTC57: F3,48 = 20, p b 0.001; PWTFVB: F3,42 = 29.408, p b 0.001; PWLC57: F3,48 = 7.139, p b 0.001; PWFVB: F3,42 = 9.563, p b 0.001). SD stress did not significantly change the mechanical and thermal response thresholds in both strains, although SD stress caused a decreasing trend in PWLs on day 10. No differences in PWTs and PWLs were found between the C57BL/6j and FVB/NJNju mice. 3.3. Effects of social defeat stress and chronic inflammatory pain on anxietylike behaviors In the open-field (OF) test, mice naturally prefer to be near a protective wall rather than exposed to danger in the open, but a competing foraging instinct will motivate them to explore. Mice spent less time in the center of the open field, which is interpreted as anxiety-like behavior [21]. C57BL/6J mice exposed to SD stress spent less time (Oneway ANOVA, F3, 36 = 9.616, p b 0.001) and traveled shorter distances (One-way ANOVA, F3, 36 = 17.103, p b 0.001) in the central zone of the open field (Fig. 4A–D). Because the OF test can also be used as a measure of overall activity, we measured the total distance traveled in the open-field and calculated the percentage of central distance (Fig. 4D). Unexpectedly, CFA-inflammatory mice only showed slightly decreased central time and central distance with a similar percentage of central distance to sham controls. However, we found little difference between FVB/NJNju mice with and without SD stress or CFA-inflammation

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(Fig. 4A–D). A two-way ANOVA analysis revealed significant effects of strains (C57BL/6J and FVB/NJNju) in central time and central distance (Central time: F1, 72 = 9.345, p = 0.003; Central distances: F1,72 = 4.216, p = 0.044). We used the elevated-plus maze (EPM) test as an additional test of anxiety-like behavior. In the elevated-plus maze, the time spent in the open arms is interpreted as a measure of reduced anxiety-like behavior [21]. C57BL/6J mice exposed to SD stress showed significantly lower entries (One-way ANOVA, F3, 36 = 21.823, p b 0.001) and less time (Oneway ANOVA, F3, 36 = 18.777, p b 0.001) in the open arms than those without SD stress. Although C57BL/6J mice with CFA-inflammatory pain also showed decreases in open-arm entry, no differences in the percentage of open-arm entry, open-arm time and percentage of open-arm time were found between inflammatory and sham mice (Fig. 5A–D). In FVB/NJNju mice, either SD stress or CFA-inflammation produced a decrease in open-arm entries, but there was no statistical difference in the percentage of open-arm entry between SD-stressexposed and sham-SD mice (Fig. 5A and B). Moreover, neither openarm time nor percentage of open-arm time was different among all treatment groups (One-way ANOVA, F3, 35 = 0.574, p = 0.636) (Fig. 5C and D). Taken together, although there were no consistent changes in the two related measures (OFT and EPM), the behavioral responses of FVB/NJNju mice seem to be more resilient. 3.4. Effects of comorbid social defeat stress and chronic inflammatory pain on anxiety-like and nociceptive behaviors As mentioned above, following SD stress, C57BL/6J mice spent less time in the interaction zone when presented with a target mouse (Fig. 2A and B). Similarly, the mice who received combined sham-CFA

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with SD stress (sham-CFA + SD) treatment showed a lower SIR (Fig. 6A). Unexpectedly, with the combination of CFA-inflammation and SD stress (CFA + SD), mice showed significantly higher SIR compared with sham-CFA + SD treated mice (Student's t-test, p b 0.001) (Fig. 6A). In FVB/NJNju mice, the SIR was similar between groups of sham-CFA + SD and CFA + SD treatment (Fig. 6A). In the OF and EPM tests, neither C57BL/6J nor FVB/NJNju mice showed significant differences between groups of sham-CFA + SD and CFA + SD treatment (Fig. 6B–D). Similar to CFA-inflammatory C57BL/6J mice, severe mechanical allodynia and thermal hyperalgesia of the ipsilateral hind paw were evident in the CFA + SD mice on days 6 and 10. Interestingly, there was no significant worsening of allodynia and hyperalgesia in CFA + SD mice compared with CFA alone in this strain (Fig. 6E and F). In FVB/NJNju mice subjected to CFA + SD, although both allodynic and hyperalgesic effects were more intense than that observed in CFA alone, only worse hyperalgesia reached statistical significance (Fig. 6G and H). 4. Discussion Chronic pain is clinically associated with the development of affective disorders. A reciprocal relationship between allodynia and/or hyperalgesia and anxiety/depression-like behaviors has also been present in rats with combined sciatic nerve injury and/or temporomandibular joint inflammation and acute/chronic stress [8,18,20]. Using the mouse models of combined CFA-inflammation (a model of chronic inflammatory pain) and social-defeat stress and both alone, the present study demonstrated that nociceptive hypersensitivity and anxiety-like behaviors were not exacerbated by comorbidity of pain and anxiety. Clinically, humans with chronic pain often have higher scores of anxiety and depression [3,22–24]. However, experimental animal studies

Fig. 4. Effects of social defeat stress and chronic inflammatory pain on anxiety-like behavior in the open field test. (A) The illustrative examples of mice travel traces in the open field test. (B–D) Time spent in the central area (B), distance traveled in the central area (C) and the percentage of the central distance (D) were significantly decreased following SD stress in C57BL/6J mice but not in FVB/NJNju mice. **p b 0.01 versus sham-SD.

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Fig. 5. Effects of social defeat stress and chronic inflammatory pain on anxiety-like behaviors in the elevated plus maze test. (A, B) The number of entries into the open arms was significantly decreased by SD stress and CFA-inflammation in both C57BL/6J and FVB/ NJNju mice (A), whereas the decrease in percentage of open arm entries only occurred in C57BL/6J mice with SD stress and FVB/NJNju mice with CFA-inflammatory pain (B). (C, D) Time spent in open arms and the percentage of open arm time were similarly decreased in C57BL/6J mice with SD stress. Neither SD stress nor CFA-inflammatory pain affect the time spent in open arms and the percentage of time spent in the open arms in FVB/ NJNju mice. *p b 0.05, **p b 0.01 versus sham-SD or sham-CFA.

using persistent pain models found conflicting results as to whether injured mice show changes in anxiety-like behaviors [9,19,25–28]. For example, Hasnie et al. reported that nerve injury did not alter OF or EPM behavior at 1, 2 or 4 weeks after injury in C57BL/6J mice [26]. Additionally, a study by Basbaum showed that mice in the SNI (spared nerve injury)-, CCI (chronic constriction injury)- and intraplantar CFA-chronic pain groups did not differ in measures of anxiety during 1–7 weeks [19]. Similar to these previous studies in mice, our current study further confirmed that CFA-induced inflammatory pain did not alter OF and

social interaction behaviors at 10 days after inflammation in both C57BL/6J and FVB/NJNju strains. In contrast, there is a large body of work on rats showing changes in anxiety and depression-like behaviors occurring in neuropathic and inflammatory pain status [25,26,28–31]. Notably, in the present study both C57BL/6J and FVB/NJNju mice with CFA-inflammatory pain showed decreased open-arm entry in the EPM test, although there were no differences in the OF test. Given that the EPM test explores the conflict between the natural behavior of rodents to explore new spaces and avoid open spaces, it is considered to be a more sensitive test of anxiety [32–34]. Our results may also suggest that mice with persistent pain develop slight anxiety. These apparent discrepancies are regarded as being related to different sources, such as differences in chronic pain models, differences in behavioral test paradigms, especially differences in animal species/strains. Indeed, the studies by Mao showed that neuropathic and inflammatory pain induced anxiety- and depression-like behaviors only in Wistar–Kyoto (WKY) but not in Wistar rats [18,20]. An important finding of the present study is that SD stress and CFAinflammation comorbid mice did not demonstrate worsening anxietylike behaviors. In contrast, with the combination of CFA-inflammation and SD stress, mice exhibited a higher social interaction level compared with SD stress treatment alone, suggesting a resiliency of inflammatory pain to SD-induced social avoidance. Theoretically and empirically, there may be a positive association between pain and anxiety [8,18,20, 35,36]. However, persistent pain is an environment in which the source of pain cannot be removed, and attention will automatically be focused on the pain, as it is the most threatening aspect of the environment [37, 38]. In the present study, we observed that in the EPM test CFAinflammatory mice had a decreased open-arm and close-arm entry alternation and spent more time licking and favoring the affected paw. It has been reported that mice with chronic pain spend less time exploring novel objects in the novel-object recognition test [39,40]. Thus, attention toward the source or location of the pain may interrupt or ignore the aggressive behavior from aggressors in the SD stress process. In addition to the attention directed toward pain, the other alternative explanations may be that various neurotransmitters and neuromodulators play roles in both pain and antidepressant effects. For example, inflammatory pain produces increased levels of 5-HT and its receptors in the brain [41,42], and an increased 5-HT signal has an antidepressant effect [43]. In regard to the influence of an anxiety-like state on nociception, we found no difference in PWTs to von Frey and PWLs to radiant heat stimulation between SD stress and sham control mice in both strains (C57BL/6J and FVB/NJNju), indicating that the basal response thresholds of mechanical and thermal stimuli were unaffected by the induction of an anxiety-like state. However, CFA-induced thermal hyperalgesia was significantly exacerbated by the comorbid disorder in FVB/NJNju mice with CFA + SD on day 10. A similar trend was also observed in C57BL/ 6J mice although there was no statistically significant result. This finding suggested that a 10-day SD stress period may facilitate CFA-induced thermal hyperalgesia. Mechanical allodynia was not worsened by the combination of SD stress with CFA-inflammatory pain in both strains. In support of this finding, it was reported that CCI-treated rats with chronic unpredictable mild stress (CMS) exhibited similar mechanical and cold allodynia compared to rats without CMS [9]. Clinically, either hypoalgesia or facilitation of experimentally evoked pain has been reported in depression patients [44–47]. Animal research also incorporated pain-increasing and pain-inhibiting influences of anxiety or depression-like states. Rats subjected to CMS exhibited increased nociceptive thresholds in normal, CFA-inflammation and spinal nerve ligated (SNL)-neuropathic pain conditions [48–50]. Additionally, food deprivation and social inequality stress decreased the pain perception in formalin-inflammatory pain [51]. The contrary reports included that formalin-inflammatory rats with CMS showed greater nociceptive responses than those without CMS [50]. Rats exposed to chronic restraint stress [52] or SD stress [8,53] developed allodynia or hyperalgesia.

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Fig. 6. Effects of comorbid social defeat stress and chronic inflammatory pain on anxiety-like and nociceptive behaviors. (A) C57BL/6J mice subjected to CFA-inflammatory pain and SD stress exhibited resiliency to SD stress-induced social avoidance. The social interaction ratio (SIR) was significantly higher in CFA-inflammation + SD stress group than those of shamCFA + SD stress group. *p b 0.05. (B–D) Neither strain exposed to SD stress with nor without CFA-inflammation changed the anxiety-like behaviors in OF and EPM tests. (E, F) There was no difference in PWLs (E) and PWTs (F) between groups of SD stress with and without CFA-inflammation in C57BL/6J mice. (G, H) Combination of SD stress with CFA-inflammation significantly exacerbated thermal hyperalgesia (G) rather than mechanical allodynia (H) in FVB/NJNju mice.

Moreover, CFA-induced mechanical allodynia and thermal hyperalgesia were significantly exacerbated when combined with SD stress in rats [8]. Despite a large amount of animal and clinical studies, these findings have not demonstrated a clear association between anxiety and alterations of nociceptive sensation. Finally, it is worth noting that compared with C57BL/6J mice, FVB/ NJNju mice subjected to SD stress showed less anxiety-like behaviors, suggesting a strain difference. The similar studies using C57BL/6J and Balb/c mice [19] or WKY and Wistar rats [18] also revealed the differences derived from species and strains. Compared with C57BL/6J mice, FVB/NJNju mice expressed lower levels of inflammation-related chemokines [54,55], supporting that FVB/NJNju mice may have

resiliency to social defeat. Similarly, FVB/NJNju mice were reported to show less immobility than C57BL/6J mice in the forced swim test [56]. Additionally, FVB/NJNju mice have stronger cardiac pump function [57] and a higher resting metabolic rate [58] than C57BL/6J mice, and whether these affect animal behaviors remains to be studied further. In conclusion, C57BL/6J and FVB mice showed different responses to social defeat stress and CFA-induced inflammatory pain. In animal models of comorbidity, persistent pain and SD stress-induced anxiety do not necessarily exacerbate one another. In particular, combined stress caused the behavior that was more affected by inflammatory pain and less by SD stress-induced defects.

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