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Changes in pain behavior induced by formalin, substance P, glutamate and pro-inflammatory cytokines in immobilization-induced stress mouse model
Brain Research Bulletin 71 (2006) 279–286
Changes in pain behavior induced by formalin, substance P, glutamate and pro-inflammatory cytokines in immobilization-induced stress mouse model Young-Jun Seo, Min-Soo Kwon, Eon-Jeong Shim, Soo-Hyun Park, Ok-Sun Choi, Hong-Won Suh ∗ Department of Pharmacology and Institute of Natural Medicine, College of Medicine, Hallym University, 1 Okcheon-Dong, Chuncheon, Gangwon-Do 200-702, South Korea Received 23 June 2006; received in revised form 5 September 2006; accepted 12 September 2006 Available online 4 October 2006
Abstract In the present study, we examined the change of pain behaviors induced by formalin injected subcutaneously (s.c.) into the hind paw, or substance P (SP), glutamate, and pro-inflammatory cytokines (TNF-␣, IL-1, and IFN-␥) injected intrathecally (i.t.) in the mouse immobilization stress model. The mouse was restrained either once for 1 h or five times for 5 days (once/day). In the formalin test, a single immobilization stress attenuated pain behaviors (licking, biting or scratching) in the second phase, while it had no effect on the pain behaviors revealed during the first phase. In addition, repeated immobilization stress attenuated pain behaviors revealed during the second phase but not in the first phase. A single as well as repeated immobilization stress decreased pain behaviors induced by substance P i.t. injection, but there were no significant changes in the glutamate test. In the pro-inflammatory cytokine pain model, a single immobilization stress decreased the pain behaviors induced by TNF-␣, IL-1 administered i.t. but not by IFN-␥ administered i.t. Moreover, a mouse applied with repeated immobilization stress did not show any changes in pain behaviors elicited by pro-inflammatory cytokines (TNF-␣, IL-1 and IFN-␥) compared to the control group. These results suggest that a single and repeated immobilization stress differentially affects such nociceptive processing induced by formalin, SP, glutamate and pro-inflammatory cytokines in different manners. © 2006 Elsevier Inc. All rights reserved. Keywords: Immobilization stress; Pain behavior; Stress-induced analgesia
1. Introduction Stress is defined as a state of disharmony or threatened homeostasis and results in various physiological and behavioral changes [15]. It has been characterized that stress influences brain activity and promotes long-term changes in various neural systems. Stress therefore elicits a cluster of neuronal disorders that is implicated in cognitive, endocrinal and psychiatric problems [39,47,52]. In addition, a series of studies has demonstrated that stress generally decreases the nociception referred as a stress-induced analgesia that is considered to be implicated with endogenous opioid systems [2,4,32,50,54].
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0361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2006.09.012
Immobilization is widely used stress model, which inflict potent physical and psychological stress on experimental animals to make various psychopathology [25]. Immobilization stress produces antinociceptive effects which are supported by the findings that immobilization stress increases the latency of the hot-plate response [2]. Although the exact roles underlying immobilization-induced antinociception are not fully understood, it has been suggested that the endogenous opioid system is attributed to the production of antinociceptive effects induced by immobilization, at least in part [3,6,8,42]. Recent studies have shown that single and repeated immobilization stresses induced antinociception effects differently to tail-flick latency [31]. In addition, it has also been shown that a single immobilization attenuated only the second phase of formalin induced pain behaviors in male and female rats, respectively [1]. Although many previous studies have demonstrated mainly immobilization induced analgesia or antinociception in the
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tail-flick [20,33,36,57] and the hot-plate test [26], it has not been well known that antinociceptive profiles of a single- and repeated immobilization stress occur in various pain models. Subcutaneous (s.c.) injections of 1% formalin in mouse left hind paw induce nociceptive behaviors like licking, biting, scratching forward to injected sites. Generally, nociceptive behaviors induced by formalin s.c. show a biphasic pattern. The early phase of the nociceptive response normally peaks between 0 to 5 min, and the late phase is manifested between 20 to 40 min after formalin injection, representing the direct effect on nociceptors and inflammatory nociceptive responses, respectively [27]. It has also been reported that i.t. injections of substance P (SP) or glutamate in mice induce a behavioral response similar to that caused by noxious stimulation and showed a similar response, consisting of biting, scratching and licking the lumbar and caudal parts of the body. For these reasons, i.t. SP or glutamate injection has been widely used for pain models to study the nociceptive/antinociceptive mechanism [11,16,30]. Although the exact mechanism leading to formalin-induced nociceptive response is not well known yet, several studies have been suggested that spinally located SP may play important roles in the nociceptive processing of both the first and second phase of pain behaviors, which are consisted with direct mechano- or chemo-receptor activation and inflammation, respectively, induced by formalin s.c. injection [19,28,44]. Furthermore, glutamate are mainly involved in the central sensitization which is induced by inflammatory processing in the second phase of formalin responses or neuropathic pain [9,17,37,43,48]. Recent study has shown that i.t. injections of mouse proinflammatory cytokines are a useful pain model which also evoked nociceptive behaviors [10]. Pro-inflammatory cytokines (TNF-␣, IL-1 and IFN-␥) are well known to be involved in the pathophysiology of pathological pain states that are related in hyperalgesia or allodynia [45,51,56]. As we mentioned above, the effects of a single and repeated immobilization stress on nociceptive behaviors elicited by various pain models have not been well characterized yet. In the present study, we therefore examined the effect of a single and repeated immobilization stress on nociceptive behaviors induced by formalin, SP, glutamate and pro-inflammatory cytokines. 2. Materials and methods These experiments were approved by the Hallym University Animal Care and Use Committee. All procedures were conducted in accordance with the ’Guide for Care and Use of Laboratory Animals’ published by the National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain.
2.1. Experimental animals Male ICR mice (MJ Ltd., Seoul, Korea) weighing 23–25 g were used for all the experiments. Animals were housed five per cage in a room maintained at 22 ± 0.5 ◦ C with an alternating 12 h light-dark cycle for at least 5 days before the experiments were started and food and water were available ad libitum. The animals were allowed to adapt to the experimental condition in the laboratory for at least 2 h before immobilization stress or pain testing. To reduce variation, all experiments were performed during the light phase of the cycle (10:00–17:00).
2.2. Immobilization stress procedure The mice were subjected to restraint stress as described in a previous study [50]. In brief, restraint was carried out by placing the mouse in a 50 ml corning tube, and adjusting it with an iron nail on the outside, which crossed in the caudal part of the animal. Adequate ventilation was provided by means of holes at the sides of the tubes. The mice were stressed by restraint for 1 h for daily, and for 5 days in the repeated model. In the single model there was a single exposure. The control group was submitted to the same handling at the same time except for the immobilization procedure.
2.3. Intrathecal (i.t.) injection of drugs The i.t. administration was performed in conscious mice following the previously established method [29,30] using a 30-gauge needle connected to a 25 l Hamilton syringe with polyethylene tubing. The i.t. injection volume was 5 ml and the injection site was verified by injecting a similar volume of 1% methylene blue solution and determining the distribution of the injected dye in the spinal cord. The dye injected i.t. was distributed both rostrally and caudally but with in a short distance (about 0.5 cm) and no dye was found in the brain. The success rate for the injections was consistently found to be over 95%, before the experiments were done.
2.4. Formalin treatment and nociceptive behavioral analysis A 10 l of 1.0% formalin solution, made up in physiologic saline (0.9% NaCl), was injected subcutaneously (s.c.) under the plantar surface of the left hind paw. For the behavioral study, animals were restrained once for 1 h or daily for 5 days prior to the behavioral study although the control group was not submitted to restraint and was injected with formalin without delay. Following the intraplantar injection of formalin, the mouse was immediately placed in an acrylic observation chamber (20 cm high, 20 cm diameter), and the time spent licking, shaking and biting the injected paw was measured with a stop-watch timer and considered as indicative of nociception [27].
2.5. Substance P or glutamate-induced nociceptive behavioral test For the behavioral study, mice were restrained once for 1 h or daily for 5 days prior to the behavioral study although the control group was not submitted to restraint. The mouse was injected i.t. with the Substance P (SP; 0.7 g/5 l) or glutamate (20 g/5 l) after immobilization stress without delay. Following the intrathecal injection of SP or glutamate, the animals were immediately placed in a glass cylinder chamber (20 cm high, 20 cm diameter) and the duration of nociceptive behavioral responses, which were manifested by licking, biting a nd scratching directed toward the lumbar and caudal regions of the spinal cord, was measured for 30 min [30]. Characteristic behaviors (biting, licking and scratching at the abdomen and hind portions of the body) induced by pharmacological effects of SP or glutamate were not observed in the vehicle-treated control group [16,30,45].
2.6. Pro-inflammatory cytokines-induced nociceptive behavioral test The intrathecally injected TNF-␣, IL-1 and IFN-␥-induced nociceptive behavioral tests were performed by the following procedures. Mice were restrained once for 1 h once or daily for 5 days prior to the behavioral study although the control group was not submitted to restraint. Mice were injected i.t. with the TNF-␣ (100 pg/5 l), IL-1 (100 pg/5 l) or IFN-␥ (100pg/5 l) after immobilization stress without delay. Immediately after the i.t. injection, each mouse was placed in an observation chamber (20 cm high, 20 cm diameter) and their behavioral responses such as licking, biting and scratching directed toward the lumbar and caudal regions of the spinal cord were recorded for 30 min. The cumulative response time(s) of scratching and biting episodes were measured with a stop-watch timer. Characteristic behaviors (biting, licking and scratching at the abdomen and hind portions of the body) induced by pharmacological effects pro-inflammatory cytokines (TNF-␣, IL-1 and IFN-␥) were not observed in the vehicle-treated control group [16,30,45].
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Fig. 1. The effect of a single and repeated immobilization stress on pain behaviors induced by intraplantar formalin injection. (A) For a single immobilization stress, mice were restrained for 1 h, and then administered subcutaneously under the plantar surface of the left hind paw with formalin solution (1%, 10 l). The cumulative response time of licking and biting the injected paw was measured in the first phase (0–5 min) and the second phase (20–40 min) of the formalin response. (B) Mice were restrained for 1 h per day for 5 days, and the pain behavior induced by formalin s.c. injection was measured immediately after the last restraint stress. The control group was submitted to same handling at the same time except immobilization procedure. The vertical bars indicate the standard error of the mean. The number of animals used for each group was 7–11. * P < 0.05, *** P < 0.001 compared with the control group.
2.7. Drugs Formalin, SP and Glutamate were purchased from Sigma Chemical Co. (St. Louis, MO). TNF-␣, IL-1 and IFN-␥ were purchased from R and D Systems Inc. (Minneapolis, MN, USA). All drugs were prepared just before use in 0.9% (w/v) of NaCl. All drug doses, which were determined as critical concentration for statistical analysis, for formalin, SP, glutamate and pro-inflammatory cytokines pain models were chosen based on previous studies [11,16,35,49].
2.8. Statistical analysis Data were presented as the mean ± S.E.M. The statistical significance of differences between groups was assessed with one-way ANOVA with Bonferroni’s post hoc test using GraphPad Prism version 4.0 for Windows XP (GraphPad Software, San Diego, CA, USA); P < 0.05 was considered significant.
3. Results 3.1. The effect of a single and repeated immobilization stress on pain behaviors induced by intraplantar formalin injection The s.c. injection of 1% formalin into the left hind paw caused an acute, immediate nociceptive response, i.e., licking, shaking and biting the injected paw, which lasted for 5 min (first phase). The second phase formalin response began about 20 min after formalin administration and lasted for about 20 min. However, s.c. injection of saline into the left hind paw did not induce any considerable nociceptive behavior (data not shown). We examined the effect of immobilization stress on nociceptive behavior induced by formalin s.c. injection. A single immobilization stress for 1 h and repeated stress restrained daily for 5 days were carried out respectively. Mice were injected with s.c. formalin into the left hind paw immediatly after immobilization stress. A single immobilization stress decreased significantly the cumulative time of intraplantar
formalin-induced nociceptive behaviors only during the second phase, but not during the first phase (Fig. 1A). Repeated immobilization stress also decreased the cumulative time of intraplantar formalin-induced nociceptive behaviors only during the second phase. 3.2. The effect of a single and repeated immobilization stress on pain behavior elicited by SP administered i.t. The i.t. injection of SP (0.7 g) caused an acute, immediate behavioral response, i.e., licking and biting, which lasted about 30 min. As shown in Fig. 2A and B, pain behaviors induced by SP i.t. injection were significantly attenuated by a single and repeated immobilization stress as well. 3.3. The effect of a single and repeated immobilization stress on pain behavior elicited by glutamate administered i.t. The i.t. injection of glutamate (20 g) caused an acute, immediate behavioral response, i.e., licking and biting, which lasted about 30 min. As shown in Fig. 3A and B, neither a single nor repeated immobilization stress decreased pain behaviors induced by glutamate i.t. injection. 3.4. The effect of a single and repeated immobilization stress on pain behavior elicited by TNF-α administered i.t. The i.t. injection of TNF-␣ (100 pg/5 l) caused an acute, immediate behavioral response, i.e., licking and biting, which lasted about 30 min. As shown in Fig. 4A, a single immobilization stress significantly attenuated pain behaviors induced by TNF-␣ i.t. injection. On the other hand, repeated immobilization stress has no effects on pain behaviors induced by TNF-␣ i.t. injection (Fig. 4B).
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Fig. 2. The effect of a single and repeated immobilization stress on pain behaviors elicited by SP administered i.t. (A) A single immobilization stress performed by restraining mice for 1 h, and then administering intrathecally with SP (0.7 g). The pain behaviors such as licking, biting and scratching responses were measured for 30 min after SP injection. (B) Mice were restrained for 1 h per day for 5 days, and the pain behavior induced by i.t. SP injection was measured immediately after the last restraint stress. The control group was submitted to same handling at the same time except immobilization procedure. The vertical bars indicate the standard error of the mean. The number of animals used for each group was 10. * P < 0.05, ** P < 0.01 compared with the control group.
Fig. 3. The effect of a single and repeated immobilization stress on pain behaviors elicited by glutamate administered i.t. (A) A single immobilization stress performed by restraining mice for 1 h, and then administering intrathecally with glutamate (20 g). The pain behaviors such as licking, biting and scratching responses were measured for 30 min after glutamate injection. (B) Mice were restrained for 1 h per day for 5 days, and the pain behaviors induced by i.t. glutamate injection were measured immediately after the last restraint stress. The control group was submitted to same handling at the same time except immobilization procedure. The vertical bars indicate the standard error of the mean. The number of animals used for each group was 12.
3.5. The effect of a single and repeated immobilization stress on pain behavior elicited by IL-1β administered i.t. The i.t. injection of IL-1 (100 pg/5 l) caused an acute, immediate behavioral response, i.e., licking and biting, which
lasted about 30 min. As shown in Fig. 5A, a single immobilization stress significantly attenuated pain behaviors induced by IL-1 i.t. injection. On the other hand, repeated immobilization stress has no effects on pain behaviors induced by IL-1 i.t. injection (Fig. 5B).
Fig. 4. The effect of a single and repeated immobilization stress on pain behaviors elicited by TNF-␣ administered i.t. (A) A single immobilization stress performed by restraining mice for 1 h, and then administering intrathecally with TNF-␣ (100 pg per 5 l). The pain behaviors such as licking, biting and scratching responses were measured for 30 min after TNF-␣ injection. (B) Mice were restrained for 1 h per day for 5 days, and the pain behaviors induced by i.t. TNF-␣ injection were measured immediately after the last restraint stress. The vertical bars indicate the standard error of the mean. The control group was submitted to same handling at the same time except immobilization procedure. The number of animals used for each group was 10. *** P < 0.001 compared with the control group.
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Fig. 5. The effect of a single and repeated immobilization stress on pain behaviors elicited by IL-1 administered i.t. (A) A single immobilization stress performed by restraining mice for 1 h, and then administering intrathecally with IL-1 (100 pg per 5 l). The pain behaviors such as licking, biting and scratching responses were measured for 30 min after IL-1 injection. (B) Mice were restrained for 1 h per day for 5 days, and the pain behaviors induced by i.t. IL-1 injection were measured immediately after the last restraint stress. The control group was submitted to same handling at the same time except immobilization procedure. The vertical bars indicate the standard error of the mean. The number of animals used for each group was 10–12. *** P < 0.001 compared with the control group.
Fig. 6. The effect of a single and repeated immobilization stress on pain behaviors elicited by IFN-␥ administered i.t. (A) A single immobilization stress performed by restraining mice for 1 h, and then administering intrathecally with IFN-␥ (100 pg per 5 l). The pain behaviors such as licking, biting and scratching responses were measured for 30 min after IFN-␥ injection. (B) Mice were restrained for 1 h per day for 5 days, and the pain behaviors induced by i.t. IFN-␥ injection were measured immediately after the last restraint stress. The control group was submitted to same handling at the same time except immobilization procedure. The vertical bars indicate the standard error of the mean. The number of animals used for each group was 10–11.
3.6. The effect of a single and repeated immobilization stress on pain behavior elicited by IFN-γ administered i.t. The i.t. injection of IFN-␥ (100 pg/5 l) caused an acute, immediate behavioral response, i.e., licking and biting, which lasted about 30 min. As shown in Fig. 6A and B, neither a single nor repeated immobilization stress decreased pain behaviors induced by IFN-␥ i.t. injection. 4. Discussion Although we compared the relative effects of immobilization stress on various pain models because the direct comparison between formalin and other pain models which were induced by i.t. injected drugs was not appropriate for its different behavioral parameters to determine pain behaviors, in the present study, we clearly demonstrated that immobilization stress applied once as well as repeatedly attenuated pain behaviors induced by formalin s.c. and SP i.t. injection. In addition, a single and repeated immobilization stress showed significant difference between patterns of antinociception especially in the TNF-␣ and IL-1 pain models. In these results, our understanding of how immobilization
stress affected differentially on various nociceptive stimuli can be accessed by two main points, the differential modality of nociceptive processing and the nature of analgesia induced by immobilization stress. A single stress attenuated the second phase of pain behaviors induced by formalin (s.c.) injection but not the first phase (Fig. 1A). Moreover, repeated stress also attenuated the second phase not the first phase of pain behaviors elicited by s.c. formalin (Fig. 1B). On the other hand, both a single and repeated immobilization stress attenuated pain behaviors induced by i.t. SP injection (Fig. 2A and B), however, neither a single nor repeated stress has significant effects on pain behaviors induced by i.t. glutamate injection (Fig. 3A and B). It has been widely believed that various nociceptive stimulus, which have different modality, were processed by different nociceptive pathways [11,16,28]. Furthermore, series of studies have previously demonstrated that the neurochemical and neurological basis of stress-induced analgesia differs according to the type of pain and the type of stress [5,18,28,31,53]. Especially, there are wide differences between inflammatory pain and neurogenic pain in the processing of synaptic transmission and neuronal pathways [28,37,38]. It has been known that SP is mainly related with
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both acute phasic nociceptove processing in the first phase and tonic inflammatory nociceptive processing in the second phase of pain behaviors induced by formalin s.c. injection, while glutamate may be involved in the tonic chronic nociceptive processing during the second phase of pain behaviors elicited by formalin s.c. injection and neuropathic central sensitization as well [9,17,37,43,48]. Hunt and Mantyh have suggested that SP is mainly involved with the spinoparabrachial pathway which is concerned with the emotional or neuromodulatory effect of pain, while SP is hardly involved in the spinothalamic pathway which is concerned with both discrimination and affect of pain [28]. In these regards, we can speculated that the analgesia induced by a single or repeated immobilization stress in the second phase of formalin test and SP i.t. test may be involved with the SPpositive afferents pathways, but not glutamate induced neuronal alterations. Accumulating data suggested that intrathecal injection of proinflammatory cytokines like TNF-␣, IL-1 or IFN-␥ makes hyperalgesia and allodynia [45,51,56] in which it has been reported that IFN-␥ may play important roles in neuropathic pain development [46]. Pain behaviors induced by TNF-␣ and IL-1 were significantly attenuated by a single immobilization stress (Figs. 4A and 5A). However repeated immobilization stress has no effects on pain behaviors induced by TNF-␣, IL1 (Figs. 4B and 5B). Furthermore, both a single and repeated immobilization stress-induced analgesia were not observed in pain behaviors induced by IFN-␥ (Fig. 6A and B). As pointed out above, the differential effect of immobilization stress on pain behaviors induced by TNF-␣, IL-1 or IFN-␥ may due to the differential nociceptive processing in the spinal and supraspinal mechanisms between inflammatory pain and neuropathic pain, in which descending pain modulatory mechanisms induced by immobilization stress affect differently on different pathways, respectively. The striking feature in our result was that the antinociceptive effect of a single immobilization stress differs from repeated immobilization stress in pain behaviors induced by TNF-␣ or IL-1. Although the repeated immobilization stress schedule in our study was less than the previous studies, consecutive immobilization stress for 5 days is enough to compare the effect of a single and repeated stress for which affect differently on the expression of various signal molecules in the descending pain regulatory regions [34]. Moreover, it has also been reported that repeated restraint stress for 7 days elicit the stress-induced hyperalgesia [18]. The differential effect of a single and repeated immobilization stress on pain behaviors induced by i.t. TNF␣ and IL-1 may imply the adaptive processing to stress or activation of descending pain facilitatory system for prolonged stress. In part, it is supported by previous studies that immobilization stress-induced antinociceptive effects were switched to pronociceptive or hyperalgesic effects as a result of habituation or hyperactivity of the pain modulation system induced by prolonged stress, in which serotonergic neurons in nucleus raphe magnus and endogenous opioid system may be modified [23,24,31]. Although we don’t know exactly that the discrepancy between the analgesic effect of a single and repeated immobilization stress was observed in TNF-␣ and IL-1 pain models,
not in formalin and SP pain models, we can speculate that the stress-induced analgesia has not only differential regulatory process to various nociceptive stimulations, but also the differential modality in a single and repeated stress. Although the exact mechanisms of analgesia induced by a single and repeated immobilization stress are currently not clear, it may be reasonable to suppose that supraspinal centers such as hippocampus, periaqueductal gray matter, nucleus raphe magnus, arcuate nucleus of hypothalamus, prefrontal cortex and some other regions are affected by stress and produce endogenous nociceptive modulation. In aspects of neurotransmittion, the stress-induced antinociception is classified as opioid or non-opioid effects. This is supported by previous studies that cross-tolerance of the analgesic effects of morphine after repeated stress [41], in addition, different mediators, such as serotonin, dopamine, histamine or excitatory amino acids also play a key role in analgesic effects after certain stress conditions [54,55]. In the previous studies, it has also been suggested that various neuronal pathways may play an important role in the adaptive process to the stress, such as the modulation of opioids, changes of monoaminergic sites, GABAergic and cholinergic function [6–8,21,22]. We have recently reported that the differential regulation of pERK and pCaMK-II induced by s.c. formalin or i.t. SP injection is shown in the CA3 regions and dentate gyrus of the mouse hippocampus [12–14]. Moreover, we found that the differential changes of nociceptive behaviors induced by formalin, SP and glutamate in the hippocampal CA3 region damaged mouse (not published data). Several lines of evidence have demonstrated that the stress-induced remodeling of dendrites in the hippocampal CA3 region may be an underlying cause of neurologic defects like the cognitive impairment in the learning of spatial and short-term memory tasks, in which adrenal steroids participate along with excitatory amino acids [39,40]. In this context, we can speculate that the differential effect of stressinduced analgesia on formalin, SP, glutamate and inflammatory cytokines-induced nociception may be involved with the neuronal remodeling elicited by immobilization stress especially in CA3 regions and dentate gyrus of the hippocampus. However, the exact role of the hippocampal change induced by immobilization stress on nociceptive processing remains to be further elucidated. Previously, we have demonstrated that the hypothalamic pERK expression was different with a single and repeated immobilization stress. Moreover, it was also observed that c-Fos and pCaMK-II expression in locus coeruleus (LC) induced by immobilization stress are significantly different in a single and repeated stress [34]. As mentioned above, LC and hypothalamus play important roles in stress-induced pain modulation. Although the exact mechanism about differential regulation of a single or repeated immobilization stress induced pain modulation was not clearly demonstrated yet, we can suppose that the various neuronal processing involved with the hypothalamus and LC may also play an important role in stress-induced pain modulation, in which antinociceptive effects of a single and repeated immobilization stress seem to be mediated by various neurotransmitter systems to be regulated by c-Fos, pERK,
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pCaMK-II or pCREB expression. However, further studies are needed to elucidate the differential processing mechanisms of a single or repeated immobilization stress on various pain models. In summary, the present study clearly showed the different effects of immobilization induced antinociception on various pain models. Our results suggest that nociceptive behaviors induced by formalin, SP, glutamate and pro-inflammatory cytokines were modulated in different way by a single and repeated immobilization stress. However, the exact mechanisms of antinociception induced by a single and repeated immobilization stress should be further investigated in future studies. Acknowledgements This research was supported by research grants from the Korean Ministry of Science and Technology under the auspices of the Brain Frontier (M103KV010014-06K2201-01410) and the MRC program of MOST/KOSEF (R13-2005-022-01001-0). References [1] A.M. Aloisi, I. Ceccarelli, C. Lupo, Behavioural and hormonal effects of restraint stress and formalin test in male and female rats, Brain Res. Bull. 1 (47) (1998) 57–62. [2] S. Amir, Z. Amit, Endogenous opioid ligands may mediate stress-induced changes in the affective properties of pain related activating a descending pain inhibitory system and releasbehavior in rats, Life Sci. 23 (1978). [3] B.D. Appelbaum, S.G. Holtzman, Restraint stress enhances morphineinduced analgesia in the rat without changing apparent affinity of receptor, Life Sci. 36 (1985) 1069–1074. [4] R.J. Bodnar, Neuropharmacological and neuroendocrine substrates of stressinduced analgesia, Ann. N.Y. Acad. Sci. 467 (1986) 345–360. [5] D.J. Calcagnetti, S.W. Fleetwood, S.G. Holtzman, Pharmacological profile of the potentiation of opioid analgesia by restraint stress, Pharmacol. Biochem. Behav. 37 (1990) 193–199. [6] L.M. Cancela, C. Bregonzio, V.A. Molina, Anxiolytic-like effect induced by chronic stress is reversed by naloxone pretreatment, Brain Res. Bull. 36 (1995) 209–213. [7] L.M. Cancela, M. Volosin, V.A. Molina, Chronic stress attenuation a2adrenoceptor reactivity is reversed by naltrexone, Pharmacol. Biochem. Behav. 31 (1988) 25–33. [8] L.M. Cancela, M. Volosin, V.A. Molina, Opioid involvement in the adaptive change of 5-HT1a receptors induced by chronic restraint, Eur. J. Pharmacol. 176 (1990) 313–319. [9] H.S. Chen, S.A. Lipton, The chemical biology of clinically tolerated NMDA receptor antagonists, J. Neurochem. 97 (2006) 1611–1626. [10] S.S. Choi, K.J. Han, J.K. Lee, H.K. Lee, E.J. Han, D.H. Kim, H.W. Suh, Antinociceptive mechanisms of orally administered decursinol in the mouse, Life Sci. 13 (73) (2003) 471–485. [11] S.S. Choi, J.K. Lee, H.W. Suh, Antinociceptive profiles of aspirin and acetaminophen in formalin, substance P and glutamate pain models, Brain Res. 7 (921) (2001) 233–239. [12] S.S. Choi, Y.J. Seo, M.S. Kwon, E.J. Shim, J.Y. Lee, Y.O. Ham, H.K. Lee, H.W. Suh, Increase of phosphorylation of calcium/calmodulin-dependent protein kinase-II in several brain regions by substance P administered intrathecally in mice, Brain Res. Bull. 65 (2005) 375–381. [13] S.S. Choi, Y.J. Seo, M.S. Kwon, E.J. Shim, J.Y. Lee, Y.O. Ham, S.H. Park, H.W. Suh, Involvement of phosphorylated extracellular signal-regulated kinase in the mouse substance P pain model, Brain Res. Mol. Brain Res. 137 (2005) 152–158. [14] S.S. Choi, Y.J. Seo, M.S. Kwon, E.J. Shim, J.Y. Lee, Y.O. Ham, H.W. Suh, Involvement of Phosphorylated Ca2+ /calmodulin-dependent protein kinase II and phosphorylated extracellular signal-regulated protein in the mouse formalin pain model, Brain Res. 1108 (2006) 28–38.
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Changes in pain behavior induced by formalin, substance P, glutamate and pro-inflammatory cytokines in immobilization-induced stress mouse model Young-Jun Seo, Min-Soo Kwon, Eon-Jeong Shim, Soo-Hyun Park, Ok-Sun Choi, Hong-Won Suh ∗ Department of Pharmacology and Institute of Natural Medicine, College of Medicine, Hallym University, 1 Okcheon-Dong, Chuncheon, Gangwon-Do 200-702, South Korea Received 23 June 2006; received in revised form 5 September 2006; accepted 12 September 2006 Available online 4 October 2006
Abstract In the present study, we examined the change of pain behaviors induced by formalin injected subcutaneously (s.c.) into the hind paw, or substance P (SP), glutamate, and pro-inflammatory cytokines (TNF-␣, IL-1, and IFN-␥) injected intrathecally (i.t.) in the mouse immobilization stress model. The mouse was restrained either once for 1 h or five times for 5 days (once/day). In the formalin test, a single immobilization stress attenuated pain behaviors (licking, biting or scratching) in the second phase, while it had no effect on the pain behaviors revealed during the first phase. In addition, repeated immobilization stress attenuated pain behaviors revealed during the second phase but not in the first phase. A single as well as repeated immobilization stress decreased pain behaviors induced by substance P i.t. injection, but there were no significant changes in the glutamate test. In the pro-inflammatory cytokine pain model, a single immobilization stress decreased the pain behaviors induced by TNF-␣, IL-1 administered i.t. but not by IFN-␥ administered i.t. Moreover, a mouse applied with repeated immobilization stress did not show any changes in pain behaviors elicited by pro-inflammatory cytokines (TNF-␣, IL-1 and IFN-␥) compared to the control group. These results suggest that a single and repeated immobilization stress differentially affects such nociceptive processing induced by formalin, SP, glutamate and pro-inflammatory cytokines in different manners. © 2006 Elsevier Inc. All rights reserved. Keywords: Immobilization stress; Pain behavior; Stress-induced analgesia
1. Introduction Stress is defined as a state of disharmony or threatened homeostasis and results in various physiological and behavioral changes [15]. It has been characterized that stress influences brain activity and promotes long-term changes in various neural systems. Stress therefore elicits a cluster of neuronal disorders that is implicated in cognitive, endocrinal and psychiatric problems [39,47,52]. In addition, a series of studies has demonstrated that stress generally decreases the nociception referred as a stress-induced analgesia that is considered to be implicated with endogenous opioid systems [2,4,32,50,54].
∗
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0361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2006.09.012
Immobilization is widely used stress model, which inflict potent physical and psychological stress on experimental animals to make various psychopathology [25]. Immobilization stress produces antinociceptive effects which are supported by the findings that immobilization stress increases the latency of the hot-plate response [2]. Although the exact roles underlying immobilization-induced antinociception are not fully understood, it has been suggested that the endogenous opioid system is attributed to the production of antinociceptive effects induced by immobilization, at least in part [3,6,8,42]. Recent studies have shown that single and repeated immobilization stresses induced antinociception effects differently to tail-flick latency [31]. In addition, it has also been shown that a single immobilization attenuated only the second phase of formalin induced pain behaviors in male and female rats, respectively [1]. Although many previous studies have demonstrated mainly immobilization induced analgesia or antinociception in the
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tail-flick [20,33,36,57] and the hot-plate test [26], it has not been well known that antinociceptive profiles of a single- and repeated immobilization stress occur in various pain models. Subcutaneous (s.c.) injections of 1% formalin in mouse left hind paw induce nociceptive behaviors like licking, biting, scratching forward to injected sites. Generally, nociceptive behaviors induced by formalin s.c. show a biphasic pattern. The early phase of the nociceptive response normally peaks between 0 to 5 min, and the late phase is manifested between 20 to 40 min after formalin injection, representing the direct effect on nociceptors and inflammatory nociceptive responses, respectively [27]. It has also been reported that i.t. injections of substance P (SP) or glutamate in mice induce a behavioral response similar to that caused by noxious stimulation and showed a similar response, consisting of biting, scratching and licking the lumbar and caudal parts of the body. For these reasons, i.t. SP or glutamate injection has been widely used for pain models to study the nociceptive/antinociceptive mechanism [11,16,30]. Although the exact mechanism leading to formalin-induced nociceptive response is not well known yet, several studies have been suggested that spinally located SP may play important roles in the nociceptive processing of both the first and second phase of pain behaviors, which are consisted with direct mechano- or chemo-receptor activation and inflammation, respectively, induced by formalin s.c. injection [19,28,44]. Furthermore, glutamate are mainly involved in the central sensitization which is induced by inflammatory processing in the second phase of formalin responses or neuropathic pain [9,17,37,43,48]. Recent study has shown that i.t. injections of mouse proinflammatory cytokines are a useful pain model which also evoked nociceptive behaviors [10]. Pro-inflammatory cytokines (TNF-␣, IL-1 and IFN-␥) are well known to be involved in the pathophysiology of pathological pain states that are related in hyperalgesia or allodynia [45,51,56]. As we mentioned above, the effects of a single and repeated immobilization stress on nociceptive behaviors elicited by various pain models have not been well characterized yet. In the present study, we therefore examined the effect of a single and repeated immobilization stress on nociceptive behaviors induced by formalin, SP, glutamate and pro-inflammatory cytokines. 2. Materials and methods These experiments were approved by the Hallym University Animal Care and Use Committee. All procedures were conducted in accordance with the ’Guide for Care and Use of Laboratory Animals’ published by the National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain.
2.1. Experimental animals Male ICR mice (MJ Ltd., Seoul, Korea) weighing 23–25 g were used for all the experiments. Animals were housed five per cage in a room maintained at 22 ± 0.5 ◦ C with an alternating 12 h light-dark cycle for at least 5 days before the experiments were started and food and water were available ad libitum. The animals were allowed to adapt to the experimental condition in the laboratory for at least 2 h before immobilization stress or pain testing. To reduce variation, all experiments were performed during the light phase of the cycle (10:00–17:00).
2.2. Immobilization stress procedure The mice were subjected to restraint stress as described in a previous study [50]. In brief, restraint was carried out by placing the mouse in a 50 ml corning tube, and adjusting it with an iron nail on the outside, which crossed in the caudal part of the animal. Adequate ventilation was provided by means of holes at the sides of the tubes. The mice were stressed by restraint for 1 h for daily, and for 5 days in the repeated model. In the single model there was a single exposure. The control group was submitted to the same handling at the same time except for the immobilization procedure.
2.3. Intrathecal (i.t.) injection of drugs The i.t. administration was performed in conscious mice following the previously established method [29,30] using a 30-gauge needle connected to a 25 l Hamilton syringe with polyethylene tubing. The i.t. injection volume was 5 ml and the injection site was verified by injecting a similar volume of 1% methylene blue solution and determining the distribution of the injected dye in the spinal cord. The dye injected i.t. was distributed both rostrally and caudally but with in a short distance (about 0.5 cm) and no dye was found in the brain. The success rate for the injections was consistently found to be over 95%, before the experiments were done.
2.4. Formalin treatment and nociceptive behavioral analysis A 10 l of 1.0% formalin solution, made up in physiologic saline (0.9% NaCl), was injected subcutaneously (s.c.) under the plantar surface of the left hind paw. For the behavioral study, animals were restrained once for 1 h or daily for 5 days prior to the behavioral study although the control group was not submitted to restraint and was injected with formalin without delay. Following the intraplantar injection of formalin, the mouse was immediately placed in an acrylic observation chamber (20 cm high, 20 cm diameter), and the time spent licking, shaking and biting the injected paw was measured with a stop-watch timer and considered as indicative of nociception [27].
2.5. Substance P or glutamate-induced nociceptive behavioral test For the behavioral study, mice were restrained once for 1 h or daily for 5 days prior to the behavioral study although the control group was not submitted to restraint. The mouse was injected i.t. with the Substance P (SP; 0.7 g/5 l) or glutamate (20 g/5 l) after immobilization stress without delay. Following the intrathecal injection of SP or glutamate, the animals were immediately placed in a glass cylinder chamber (20 cm high, 20 cm diameter) and the duration of nociceptive behavioral responses, which were manifested by licking, biting a nd scratching directed toward the lumbar and caudal regions of the spinal cord, was measured for 30 min [30]. Characteristic behaviors (biting, licking and scratching at the abdomen and hind portions of the body) induced by pharmacological effects of SP or glutamate were not observed in the vehicle-treated control group [16,30,45].
2.6. Pro-inflammatory cytokines-induced nociceptive behavioral test The intrathecally injected TNF-␣, IL-1 and IFN-␥-induced nociceptive behavioral tests were performed by the following procedures. Mice were restrained once for 1 h once or daily for 5 days prior to the behavioral study although the control group was not submitted to restraint. Mice were injected i.t. with the TNF-␣ (100 pg/5 l), IL-1 (100 pg/5 l) or IFN-␥ (100pg/5 l) after immobilization stress without delay. Immediately after the i.t. injection, each mouse was placed in an observation chamber (20 cm high, 20 cm diameter) and their behavioral responses such as licking, biting and scratching directed toward the lumbar and caudal regions of the spinal cord were recorded for 30 min. The cumulative response time(s) of scratching and biting episodes were measured with a stop-watch timer. Characteristic behaviors (biting, licking and scratching at the abdomen and hind portions of the body) induced by pharmacological effects pro-inflammatory cytokines (TNF-␣, IL-1 and IFN-␥) were not observed in the vehicle-treated control group [16,30,45].
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Fig. 1. The effect of a single and repeated immobilization stress on pain behaviors induced by intraplantar formalin injection. (A) For a single immobilization stress, mice were restrained for 1 h, and then administered subcutaneously under the plantar surface of the left hind paw with formalin solution (1%, 10 l). The cumulative response time of licking and biting the injected paw was measured in the first phase (0–5 min) and the second phase (20–40 min) of the formalin response. (B) Mice were restrained for 1 h per day for 5 days, and the pain behavior induced by formalin s.c. injection was measured immediately after the last restraint stress. The control group was submitted to same handling at the same time except immobilization procedure. The vertical bars indicate the standard error of the mean. The number of animals used for each group was 7–11. * P < 0.05, *** P < 0.001 compared with the control group.
2.7. Drugs Formalin, SP and Glutamate were purchased from Sigma Chemical Co. (St. Louis, MO). TNF-␣, IL-1 and IFN-␥ were purchased from R and D Systems Inc. (Minneapolis, MN, USA). All drugs were prepared just before use in 0.9% (w/v) of NaCl. All drug doses, which were determined as critical concentration for statistical analysis, for formalin, SP, glutamate and pro-inflammatory cytokines pain models were chosen based on previous studies [11,16,35,49].
2.8. Statistical analysis Data were presented as the mean ± S.E.M. The statistical significance of differences between groups was assessed with one-way ANOVA with Bonferroni’s post hoc test using GraphPad Prism version 4.0 for Windows XP (GraphPad Software, San Diego, CA, USA); P < 0.05 was considered significant.
3. Results 3.1. The effect of a single and repeated immobilization stress on pain behaviors induced by intraplantar formalin injection The s.c. injection of 1% formalin into the left hind paw caused an acute, immediate nociceptive response, i.e., licking, shaking and biting the injected paw, which lasted for 5 min (first phase). The second phase formalin response began about 20 min after formalin administration and lasted for about 20 min. However, s.c. injection of saline into the left hind paw did not induce any considerable nociceptive behavior (data not shown). We examined the effect of immobilization stress on nociceptive behavior induced by formalin s.c. injection. A single immobilization stress for 1 h and repeated stress restrained daily for 5 days were carried out respectively. Mice were injected with s.c. formalin into the left hind paw immediatly after immobilization stress. A single immobilization stress decreased significantly the cumulative time of intraplantar
formalin-induced nociceptive behaviors only during the second phase, but not during the first phase (Fig. 1A). Repeated immobilization stress also decreased the cumulative time of intraplantar formalin-induced nociceptive behaviors only during the second phase. 3.2. The effect of a single and repeated immobilization stress on pain behavior elicited by SP administered i.t. The i.t. injection of SP (0.7 g) caused an acute, immediate behavioral response, i.e., licking and biting, which lasted about 30 min. As shown in Fig. 2A and B, pain behaviors induced by SP i.t. injection were significantly attenuated by a single and repeated immobilization stress as well. 3.3. The effect of a single and repeated immobilization stress on pain behavior elicited by glutamate administered i.t. The i.t. injection of glutamate (20 g) caused an acute, immediate behavioral response, i.e., licking and biting, which lasted about 30 min. As shown in Fig. 3A and B, neither a single nor repeated immobilization stress decreased pain behaviors induced by glutamate i.t. injection. 3.4. The effect of a single and repeated immobilization stress on pain behavior elicited by TNF-α administered i.t. The i.t. injection of TNF-␣ (100 pg/5 l) caused an acute, immediate behavioral response, i.e., licking and biting, which lasted about 30 min. As shown in Fig. 4A, a single immobilization stress significantly attenuated pain behaviors induced by TNF-␣ i.t. injection. On the other hand, repeated immobilization stress has no effects on pain behaviors induced by TNF-␣ i.t. injection (Fig. 4B).
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Fig. 2. The effect of a single and repeated immobilization stress on pain behaviors elicited by SP administered i.t. (A) A single immobilization stress performed by restraining mice for 1 h, and then administering intrathecally with SP (0.7 g). The pain behaviors such as licking, biting and scratching responses were measured for 30 min after SP injection. (B) Mice were restrained for 1 h per day for 5 days, and the pain behavior induced by i.t. SP injection was measured immediately after the last restraint stress. The control group was submitted to same handling at the same time except immobilization procedure. The vertical bars indicate the standard error of the mean. The number of animals used for each group was 10. * P < 0.05, ** P < 0.01 compared with the control group.
Fig. 3. The effect of a single and repeated immobilization stress on pain behaviors elicited by glutamate administered i.t. (A) A single immobilization stress performed by restraining mice for 1 h, and then administering intrathecally with glutamate (20 g). The pain behaviors such as licking, biting and scratching responses were measured for 30 min after glutamate injection. (B) Mice were restrained for 1 h per day for 5 days, and the pain behaviors induced by i.t. glutamate injection were measured immediately after the last restraint stress. The control group was submitted to same handling at the same time except immobilization procedure. The vertical bars indicate the standard error of the mean. The number of animals used for each group was 12.
3.5. The effect of a single and repeated immobilization stress on pain behavior elicited by IL-1β administered i.t. The i.t. injection of IL-1 (100 pg/5 l) caused an acute, immediate behavioral response, i.e., licking and biting, which
lasted about 30 min. As shown in Fig. 5A, a single immobilization stress significantly attenuated pain behaviors induced by IL-1 i.t. injection. On the other hand, repeated immobilization stress has no effects on pain behaviors induced by IL-1 i.t. injection (Fig. 5B).
Fig. 4. The effect of a single and repeated immobilization stress on pain behaviors elicited by TNF-␣ administered i.t. (A) A single immobilization stress performed by restraining mice for 1 h, and then administering intrathecally with TNF-␣ (100 pg per 5 l). The pain behaviors such as licking, biting and scratching responses were measured for 30 min after TNF-␣ injection. (B) Mice were restrained for 1 h per day for 5 days, and the pain behaviors induced by i.t. TNF-␣ injection were measured immediately after the last restraint stress. The vertical bars indicate the standard error of the mean. The control group was submitted to same handling at the same time except immobilization procedure. The number of animals used for each group was 10. *** P < 0.001 compared with the control group.
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Fig. 5. The effect of a single and repeated immobilization stress on pain behaviors elicited by IL-1 administered i.t. (A) A single immobilization stress performed by restraining mice for 1 h, and then administering intrathecally with IL-1 (100 pg per 5 l). The pain behaviors such as licking, biting and scratching responses were measured for 30 min after IL-1 injection. (B) Mice were restrained for 1 h per day for 5 days, and the pain behaviors induced by i.t. IL-1 injection were measured immediately after the last restraint stress. The control group was submitted to same handling at the same time except immobilization procedure. The vertical bars indicate the standard error of the mean. The number of animals used for each group was 10–12. *** P < 0.001 compared with the control group.
Fig. 6. The effect of a single and repeated immobilization stress on pain behaviors elicited by IFN-␥ administered i.t. (A) A single immobilization stress performed by restraining mice for 1 h, and then administering intrathecally with IFN-␥ (100 pg per 5 l). The pain behaviors such as licking, biting and scratching responses were measured for 30 min after IFN-␥ injection. (B) Mice were restrained for 1 h per day for 5 days, and the pain behaviors induced by i.t. IFN-␥ injection were measured immediately after the last restraint stress. The control group was submitted to same handling at the same time except immobilization procedure. The vertical bars indicate the standard error of the mean. The number of animals used for each group was 10–11.
3.6. The effect of a single and repeated immobilization stress on pain behavior elicited by IFN-γ administered i.t. The i.t. injection of IFN-␥ (100 pg/5 l) caused an acute, immediate behavioral response, i.e., licking and biting, which lasted about 30 min. As shown in Fig. 6A and B, neither a single nor repeated immobilization stress decreased pain behaviors induced by IFN-␥ i.t. injection. 4. Discussion Although we compared the relative effects of immobilization stress on various pain models because the direct comparison between formalin and other pain models which were induced by i.t. injected drugs was not appropriate for its different behavioral parameters to determine pain behaviors, in the present study, we clearly demonstrated that immobilization stress applied once as well as repeatedly attenuated pain behaviors induced by formalin s.c. and SP i.t. injection. In addition, a single and repeated immobilization stress showed significant difference between patterns of antinociception especially in the TNF-␣ and IL-1 pain models. In these results, our understanding of how immobilization
stress affected differentially on various nociceptive stimuli can be accessed by two main points, the differential modality of nociceptive processing and the nature of analgesia induced by immobilization stress. A single stress attenuated the second phase of pain behaviors induced by formalin (s.c.) injection but not the first phase (Fig. 1A). Moreover, repeated stress also attenuated the second phase not the first phase of pain behaviors elicited by s.c. formalin (Fig. 1B). On the other hand, both a single and repeated immobilization stress attenuated pain behaviors induced by i.t. SP injection (Fig. 2A and B), however, neither a single nor repeated stress has significant effects on pain behaviors induced by i.t. glutamate injection (Fig. 3A and B). It has been widely believed that various nociceptive stimulus, which have different modality, were processed by different nociceptive pathways [11,16,28]. Furthermore, series of studies have previously demonstrated that the neurochemical and neurological basis of stress-induced analgesia differs according to the type of pain and the type of stress [5,18,28,31,53]. Especially, there are wide differences between inflammatory pain and neurogenic pain in the processing of synaptic transmission and neuronal pathways [28,37,38]. It has been known that SP is mainly related with
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both acute phasic nociceptove processing in the first phase and tonic inflammatory nociceptive processing in the second phase of pain behaviors induced by formalin s.c. injection, while glutamate may be involved in the tonic chronic nociceptive processing during the second phase of pain behaviors elicited by formalin s.c. injection and neuropathic central sensitization as well [9,17,37,43,48]. Hunt and Mantyh have suggested that SP is mainly involved with the spinoparabrachial pathway which is concerned with the emotional or neuromodulatory effect of pain, while SP is hardly involved in the spinothalamic pathway which is concerned with both discrimination and affect of pain [28]. In these regards, we can speculated that the analgesia induced by a single or repeated immobilization stress in the second phase of formalin test and SP i.t. test may be involved with the SPpositive afferents pathways, but not glutamate induced neuronal alterations. Accumulating data suggested that intrathecal injection of proinflammatory cytokines like TNF-␣, IL-1 or IFN-␥ makes hyperalgesia and allodynia [45,51,56] in which it has been reported that IFN-␥ may play important roles in neuropathic pain development [46]. Pain behaviors induced by TNF-␣ and IL-1 were significantly attenuated by a single immobilization stress (Figs. 4A and 5A). However repeated immobilization stress has no effects on pain behaviors induced by TNF-␣, IL1 (Figs. 4B and 5B). Furthermore, both a single and repeated immobilization stress-induced analgesia were not observed in pain behaviors induced by IFN-␥ (Fig. 6A and B). As pointed out above, the differential effect of immobilization stress on pain behaviors induced by TNF-␣, IL-1 or IFN-␥ may due to the differential nociceptive processing in the spinal and supraspinal mechanisms between inflammatory pain and neuropathic pain, in which descending pain modulatory mechanisms induced by immobilization stress affect differently on different pathways, respectively. The striking feature in our result was that the antinociceptive effect of a single immobilization stress differs from repeated immobilization stress in pain behaviors induced by TNF-␣ or IL-1. Although the repeated immobilization stress schedule in our study was less than the previous studies, consecutive immobilization stress for 5 days is enough to compare the effect of a single and repeated stress for which affect differently on the expression of various signal molecules in the descending pain regulatory regions [34]. Moreover, it has also been reported that repeated restraint stress for 7 days elicit the stress-induced hyperalgesia [18]. The differential effect of a single and repeated immobilization stress on pain behaviors induced by i.t. TNF␣ and IL-1 may imply the adaptive processing to stress or activation of descending pain facilitatory system for prolonged stress. In part, it is supported by previous studies that immobilization stress-induced antinociceptive effects were switched to pronociceptive or hyperalgesic effects as a result of habituation or hyperactivity of the pain modulation system induced by prolonged stress, in which serotonergic neurons in nucleus raphe magnus and endogenous opioid system may be modified [23,24,31]. Although we don’t know exactly that the discrepancy between the analgesic effect of a single and repeated immobilization stress was observed in TNF-␣ and IL-1 pain models,
not in formalin and SP pain models, we can speculate that the stress-induced analgesia has not only differential regulatory process to various nociceptive stimulations, but also the differential modality in a single and repeated stress. Although the exact mechanisms of analgesia induced by a single and repeated immobilization stress are currently not clear, it may be reasonable to suppose that supraspinal centers such as hippocampus, periaqueductal gray matter, nucleus raphe magnus, arcuate nucleus of hypothalamus, prefrontal cortex and some other regions are affected by stress and produce endogenous nociceptive modulation. In aspects of neurotransmittion, the stress-induced antinociception is classified as opioid or non-opioid effects. This is supported by previous studies that cross-tolerance of the analgesic effects of morphine after repeated stress [41], in addition, different mediators, such as serotonin, dopamine, histamine or excitatory amino acids also play a key role in analgesic effects after certain stress conditions [54,55]. In the previous studies, it has also been suggested that various neuronal pathways may play an important role in the adaptive process to the stress, such as the modulation of opioids, changes of monoaminergic sites, GABAergic and cholinergic function [6–8,21,22]. We have recently reported that the differential regulation of pERK and pCaMK-II induced by s.c. formalin or i.t. SP injection is shown in the CA3 regions and dentate gyrus of the mouse hippocampus [12–14]. Moreover, we found that the differential changes of nociceptive behaviors induced by formalin, SP and glutamate in the hippocampal CA3 region damaged mouse (not published data). Several lines of evidence have demonstrated that the stress-induced remodeling of dendrites in the hippocampal CA3 region may be an underlying cause of neurologic defects like the cognitive impairment in the learning of spatial and short-term memory tasks, in which adrenal steroids participate along with excitatory amino acids [39,40]. In this context, we can speculate that the differential effect of stressinduced analgesia on formalin, SP, glutamate and inflammatory cytokines-induced nociception may be involved with the neuronal remodeling elicited by immobilization stress especially in CA3 regions and dentate gyrus of the hippocampus. However, the exact role of the hippocampal change induced by immobilization stress on nociceptive processing remains to be further elucidated. Previously, we have demonstrated that the hypothalamic pERK expression was different with a single and repeated immobilization stress. Moreover, it was also observed that c-Fos and pCaMK-II expression in locus coeruleus (LC) induced by immobilization stress are significantly different in a single and repeated stress [34]. As mentioned above, LC and hypothalamus play important roles in stress-induced pain modulation. Although the exact mechanism about differential regulation of a single or repeated immobilization stress induced pain modulation was not clearly demonstrated yet, we can suppose that the various neuronal processing involved with the hypothalamus and LC may also play an important role in stress-induced pain modulation, in which antinociceptive effects of a single and repeated immobilization stress seem to be mediated by various neurotransmitter systems to be regulated by c-Fos, pERK,
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pCaMK-II or pCREB expression. However, further studies are needed to elucidate the differential processing mechanisms of a single or repeated immobilization stress on various pain models. In summary, the present study clearly showed the different effects of immobilization induced antinociception on various pain models. Our results suggest that nociceptive behaviors induced by formalin, SP, glutamate and pro-inflammatory cytokines were modulated in different way by a single and repeated immobilization stress. However, the exact mechanisms of antinociception induced by a single and repeated immobilization stress should be further investigated in future studies. Acknowledgements This research was supported by research grants from the Korean Ministry of Science and Technology under the auspices of the Brain Frontier (M103KV010014-06K2201-01410) and the MRC program of MOST/KOSEF (R13-2005-022-01001-0). References [1] A.M. Aloisi, I. Ceccarelli, C. Lupo, Behavioural and hormonal effects of restraint stress and formalin test in male and female rats, Brain Res. Bull. 1 (47) (1998) 57–62. [2] S. Amir, Z. Amit, Endogenous opioid ligands may mediate stress-induced changes in the affective properties of pain related activating a descending pain inhibitory system and releasbehavior in rats, Life Sci. 23 (1978). [3] B.D. Appelbaum, S.G. Holtzman, Restraint stress enhances morphineinduced analgesia in the rat without changing apparent affinity of receptor, Life Sci. 36 (1985) 1069–1074. [4] R.J. Bodnar, Neuropharmacological and neuroendocrine substrates of stressinduced analgesia, Ann. N.Y. Acad. Sci. 467 (1986) 345–360. [5] D.J. Calcagnetti, S.W. Fleetwood, S.G. Holtzman, Pharmacological profile of the potentiation of opioid analgesia by restraint stress, Pharmacol. Biochem. Behav. 37 (1990) 193–199. [6] L.M. Cancela, C. Bregonzio, V.A. Molina, Anxiolytic-like effect induced by chronic stress is reversed by naloxone pretreatment, Brain Res. Bull. 36 (1995) 209–213. [7] L.M. Cancela, M. Volosin, V.A. Molina, Chronic stress attenuation a2adrenoceptor reactivity is reversed by naltrexone, Pharmacol. Biochem. Behav. 31 (1988) 25–33. [8] L.M. Cancela, M. Volosin, V.A. Molina, Opioid involvement in the adaptive change of 5-HT1a receptors induced by chronic restraint, Eur. J. Pharmacol. 176 (1990) 313–319. [9] H.S. Chen, S.A. Lipton, The chemical biology of clinically tolerated NMDA receptor antagonists, J. Neurochem. 97 (2006) 1611–1626. [10] S.S. Choi, K.J. Han, J.K. Lee, H.K. Lee, E.J. Han, D.H. Kim, H.W. Suh, Antinociceptive mechanisms of orally administered decursinol in the mouse, Life Sci. 13 (73) (2003) 471–485. [11] S.S. Choi, J.K. Lee, H.W. Suh, Antinociceptive profiles of aspirin and acetaminophen in formalin, substance P and glutamate pain models, Brain Res. 7 (921) (2001) 233–239. [12] S.S. Choi, Y.J. Seo, M.S. Kwon, E.J. Shim, J.Y. Lee, Y.O. Ham, H.K. Lee, H.W. Suh, Increase of phosphorylation of calcium/calmodulin-dependent protein kinase-II in several brain regions by substance P administered intrathecally in mice, Brain Res. Bull. 65 (2005) 375–381. [13] S.S. Choi, Y.J. Seo, M.S. Kwon, E.J. Shim, J.Y. Lee, Y.O. Ham, S.H. Park, H.W. Suh, Involvement of phosphorylated extracellular signal-regulated kinase in the mouse substance P pain model, Brain Res. Mol. Brain Res. 137 (2005) 152–158. [14] S.S. Choi, Y.J. Seo, M.S. Kwon, E.J. Shim, J.Y. Lee, Y.O. Ham, H.W. Suh, Involvement of Phosphorylated Ca2+ /calmodulin-dependent protein kinase II and phosphorylated extracellular signal-regulated protein in the mouse formalin pain model, Brain Res. 1108 (2006) 28–38.
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