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Received Date : 19-Jun-2014 Revision Requested: 22-Jul-2014 Final Revision Received: 28-Nov-2014 Accepted Date: 02-Dec-2014 Article type : Regular Paper
Peripheral alpha4beta2 nicotinic acetylcholine receptor signaling attenuates tactile allodynia and thermal hyperalgesia after nerve injury in mice
Fumihiro Saika, Norikazu Kiguchi, Yuka Kobayashi, Shiroh Kishioka Department of Pharmacology, Wakayama Medical University, Wakayama, Japan
Short title: Neuropathic pain relief and α4β2 nAChR
Corresponding Author Shiroh Kishioka, M.D., Ph.D. Department of Pharmacology, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-0012, Japan Tel: +81 73 441 0629 Fax: +81 73 446 3806 E-mail: [email protected] This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/apha.12437 This article is protected by copyright. All rights reserved.
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Abstract Aim: Neuropathic pain is often refractory to conventional analgesics including opioids and non-steroidal anti-inflammatory drugs. Evidence suggests nicotinic acetylcholine receptor ligands regulate pain transmission. Effects of α4β2 nicotinic acetylcholine receptor activation on pain behaviors after nerve injury were studied.
Methods: Mice were subjected to partial sciatic nerve ligation. Nicotinic acetylcholine receptor α4 and β2 subunits localization in injured nerves were evaluated by immunohistochemistry. Neuropathic pain, assessed by tactile allodynia and thermal hyperalgesia, were examined by von Frey test and Hargreaves test, respectively.
Results: Nicotinic acetylcholine receptor α4 and β2 subunits were up-regulated in injured nerves and were expressed on F4/80 positive macrophages. When nicotine was perineurally administered daily for 4 days (day 7–10; maintenance phase) after nerve injury, pain behaviors were significantly alleviated. The inhibitory effects of nicotine were reversed by co-administration of mecamylamine (non-selective nicotinic acetylcholine receptor antagonist) and dihydro-β-erythroidine (selective α4β2 nicotinic acetylcholine receptor antagonist). Likewise, when α4β2 nicotinic acetylcholine receptor agonists (TC2559 or ABT418) were administered daily for 4 days (day 7–10) after nerve injury, pain behaviors
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homopentameric α7 (McGehee et al., 1995) in the nervous system. These nAChR subunits have the gene coding sequences with high homology across vertebrate species and there are many commonalities in biological mechanisms (Le Novere and Changeux, 1995). These nAChR subtypes participate in neurological disorders such as pain, and cognitive and neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease (Gotti and Clementi, 2004).
Accumulating evidence indicates that non-neuronal tissues such as the vessel and immune cells throughout the body express nAChRs. The nAChRs may affect the proliferation, adhesion and migration of the cells in the non-neuronal tissues and their actions are manifold such as wound healing and angiogenesis (Gotti and Clementi, 2004). Notably, previous reports indicate that cholinergic anti-inflammatory pathway via nAChRs signaling on macrophages has been identified, and stimulation of vagus nerve suppressed inflammatory responses in vitro and in vivo (de Jonge et al., 2005, Wang et al., 2003).
Several lines of evidence indicate that nAChR ligands regulate pain transmission, because α4β2 and α7 nAChR subtypes are located in pain inhibitory GABAergic or opioidergic neurons, and selective agonists for α4β2 or α7 nAChR subtypes suppress various types of pain through the release of these inhibitory neurotransmitters in the central nervous systems
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homopentameric α7 (McGehee et al., 1995) in the nervous system. These nAChR subunits have the gene coding sequences with high homology across vertebrate species and there are many commonalities in biological mechanisms (Le Novere and Changeux, 1995). These nAChR subtypes participate in neurological disorders such as pain, and cognitive and neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease (Gotti and Clementi, 2004).
Accumulating evidence indicates that non-neuronal tissues such as the vessel and immune cells throughout the body express nAChRs. The nAChRs may affect the proliferation, adhesion and migration of the cells in the non-neuronal tissues and their actions are manifold such as wound healing and angiogenesis (Gotti and Clementi, 2004). Notably, previous reports indicate that cholinergic anti-inflammatory pathway via nAChRs signaling on macrophages has been identified, and stimulation of vagus nerve suppressed inflammatory responses in vitro and in vivo (de Jonge et al., 2005, Wang et al., 2003).
Several lines of evidence indicate that nAChR ligands regulate pain transmission, because α4β2 and α7 nAChR subtypes are located in pain inhibitory GABAergic or opioidergic neurons, and selective agonists for α4β2 or α7 nAChR subtypes suppress various types of pain through the release of these inhibitory neurotransmitters in the central nervous systems
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Takeda, D., Nakatsuka, T., Papke, R. & Gu, J. G. 2003. Modulation of inhibitory synaptic activity by a non-alpha4beta2, non-alpha7 subtype of nicotinic receptors in the substantia gelatinosa of adult rat spinal cord. Pain 101, 13-23. Terrando, N., Eriksson, L. I., Ryu, J. K., Yang, T., Monaco, C., Feldmann, M., Jonsson Fagerlund, M., Charo, I. F., Akassoglou, K. & Maze, M. 2011. Resolving postoperative neuroinflammation and cognitive decline. Ann Neurol 70, 986-995. Tofaris, G. K., Patterson, P. H., Jessen, K. R. & Mirsky, R. 2002. Denervated Schwann cells attract macrophages by secretion of leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 in a process regulated by interleukin-6 and LIF. J Neurosci 22, 6696-6703. Tracey, K. J. 2002. The inflammatory reflex. Nature 420, 853-859. van der Zanden, E. P., Snoek, S. A., Heinsbroek, S. E., Stanisor, O. I., Verseijden, C., Boeckxstaens, G. E., Peppelenbosch, M. P., Greaves, D. R., Gordon, S. & De Jonge, W. J. 2009. Vagus nerve activity augments intestinal macrophage phagocytosis via nicotinic acetylcholine receptor alpha4beta2. Gastroenterology 137, 1029-1039, 1039 e1-4. Wang, H., Yu, M., Ochani, M., Amella, C. A., Tanovic, M., Susarla, S., Li, J. H., Wang, H., Yang, H., Ulloa, L., Al-Abed, Y., Czura, C. J. & Tracey, K. J. 2003. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature
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Surgery and drug administration For the neuropathic pain model, mice underwent PSL as described previously (Seltzer et al., 1990). Briefly, mice were anesthetized with 80 mg kg-1of sodium pentobarbital intraperitoneally (i.p.). Under the condition, the right common sciatic nerve (SCN) was exposed. The SCN was isolated from surrounding tissue and approximately one-third to one-half of the SCN thickness was tightly ligated with silk suture (No. 1; Natsume Seisakusho Co., Tokyo, Japan). Finally, the muscle and skin layers were closed with sutures. For the sham operation, the SCN was only exposed without ligation and the incision was closed. PSL-induced long-lasting allodynia and hyperalgesia mimics the neuropathic pain in human patients.
Nicotine tartrate, mecamylamine hydrochloride (non-specific nAChR antagonist; Mec), dihydro-β-erythroidine
(α4β2
nAChR
antagonist;
DHβE),
and
ABT418
[(S)-3-methyl-5-(1-methyl-2-pyrrolidinyl) isoxazole] (α4β2 nAChR agonist) were purchased from
Sigma
Aldrich
(Tokyo,
Japan).
[4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine
TC2559 difumarate]
difumarate (α4β2
nAChR
agonist) was purchased from R&D Systems (Minneapolis, MN, USA). All drugs were dissolved in sterile phosphate-buffered saline (PBS). Under sodium pentobarbital anesthesia (80 mg kg-1, i.p.), these agents (10 µL) were injected into a region surrounding the SCN
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without a skin incision using 30-gauge needle attached to a glass microsyringe in accordance with previous reports (Ma and Quirion, 2006, Maeda et al., 2009). According to the preventive effects of local nicotine (1-20 nmol) administration against tactile allodynia and thermal hyperalgesia after PSL on initial phase (Kiguchi et al., 2012b), we applied the dose of nicotine (20 nmol) into the injured SCN.
Immunohistochemistry Immunohistochemistry was performed according to the method of our previous papers (Saika et al., 2012, Kiguchi et al., 2012a). Frozen sections were cut (10 μm thick) at -20°C by cryostat and transferred to silane-coated glass slides. They were immersed and shaken in PBS containing 0.1% Triton X-100 (30 min). Then, the sections were blocked with 4% bovine serum albumin (BSA) and PBS containing 0.1% Triton X-100 for six hours at room temperature. The sections were incubated overnight with specific primary antibodies against F4/80 (1:200, Cedarlane Laboratories, Burlington, Ontario, Canada) and nAChR α4 and β2 subunit (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C. The sections were washed with PBS containing 0.1% Triton X-100 (5 min×5) and incubated with secondary antibodies solution (Alexa Fluor-488 or AlexaFluor-594 -conjugated IgG, 1:200; Invitrogen, Carlsbad, CA, USA) for 2 hour at room temperature in the dark. The section was washed with PBS containing 0.1% Triton X-100 (30 min), followed by nuclear staining using
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Hoechst 33342 (1:1000; Invitrogen) for 10 min at room temperature in the dark. Finally, the sections were washed with PBS (10 min) and mounted by a cover slip with Perma Fluor (Thermo Fisher Scientific, Waltham, MA, USA). Immunoreactivity was captured using a confocal laser scanning microscope. For quantitative analysis, F4/80 positive macrophages were counted in the 200 × 200 μm square area.
Assessment of neuropathic pain Mice were habituated to the environment for the assessments for at least two days before baseline test. Tactile allodynia was evaluated by the von Frey test (Kiguchi et al., 2010) using von Frey filaments (Neuroscience, Tokyo, Japan). Mice were placed on a wire mesh (5 × 5 mm) grid floor, and covered with an individual opaque container to avoid visual stimulation. The mice were adapted for 1–2 hour before the test. The von Frey filaments (0.07 or 0.16 g) were applied ten times from underneath the mesh grid floor to the central surface of each hind paw. The escape responses such as withdrawal lifting or flinching of the paw against the filament stimulation were counted for each hind paw. Tactile allodynia was presented as the percentage of the withdrawal responses against filament stimuli.
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Thermal hyperalgesia was evaluated by the Hargreaves test (Hargreaves et al., 1988) using the IITC 390 Plantar Test Analgesia Meter (Neuroscience, Tokyo, Japan). Mice were placed in a clear plastic cage on an elevated glass sheet and allowed to adapt for 1–2 hour. The radiant heat source positioned under the glass sheet was applied to the central surface of each hind paw. The latencies to escape the hind paw against radiant heat were measured ten times for each hind paw at a 5-min interval. Data are expressed as the mean latency of ten stimulations. The heat intensity was calibrated to give a control latency of approximately 10 s.
Statistical analysis All data are expressed as mean ± S.E.M. Statistical analysis was performed by a two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons test, and one-way ANOVA followed by Tukey’s multiple comparisons test. Values of P < 0.05 were considered statistically significant. Results Localization of α4β2 nAChRs in accumulating macrophages Immunohistochemical analysis demonstrated F4/80 positive macrophages were increased in the vicinity of the injured SCN on day 7 after PSL, whereas immune cells were rarely observed in the SCN in the sham group. When we evaluated the number of F4/80 positive
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cells in SCN, there was a marked increase in the number of F4/80 positive cells in the PSL mice, while rare F4/80 positive cells in sham mice. In addition, nAChR α4 and/or β2 subunits were co-localized with F4/80 positive cells. Moreover, nAChR α4 and β2 subunits were
prominently
up-regulated
on
F4/80 positive
macrophages
(Fig.
1).
The
immunoreactivity of nAChR subunits and macrophage did not detect in the SCN on day 7 after PSL, when their primary antibodies were not applied on the sections (Supplemental fig. 1).
Effects of peripheral administration of nAChR agonists on tactile allodynia and thermal hyperalgesia after PSL in the maintenance phase Tactile allodynia and thermal hyperalgesia were observed from day 7 to day 21 after PSL but not after sham. When nicotine 20 nmol, based on the report of Kiguchi et al. (2012), was perineurally administered once a day for 4 days (day 7–10; maintenance phase) after PSL, PSL-induced tactile allodynia and thermal hyperalgesia were significantly attenuated (Fig. 2A, B). The inhibitory effects of nicotine were antagonized by co-administration with not only mecamylamine, a non-selective nAChR antagonist, but also DHβE, a selective α4β2 nAChR antagonist on day 14 after PSL (Fig. 2C, D). These studies were performed by blind assessment. Perineural administration of a selective α4β2 nAChR agonist, TC2559, administered once a day for 4 days (20 nmol, day 7–10), also suppressed tactile allodynia and
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thermal hyperalgesia after PSL (Fig. 3A, B). On day 14, tactile allodynia and thermal hyperalgesia were attenuated by treatment with TC2559 (day 7–10) in a dose-dependent manner (Fig. 3C, D). Similarly, perineural administration of another α4β2 nAChR agonist, ABT418, administered once a day for 4 days (1–20 nmol, day 7–10), reduced tactile allodynia and thermal hyperalgesia on day 14 after PSL (Fig. 4), indicating tactile allodynia and thermal hyperalgesia after PSL in the maintenance phase was alleviated by treatment with the α4β2 nAChR agonists in this phase. Effects of peripheral administration of nAChR agonists on tactile allodynia and thermal hyperalgesia after PSL in the initiation phase Nicotine was perineurally administered once a day for 4 days (day 0–3; initiation phase) after PSL. On day 7 after PSL, tactile allodynia and thermal hyperalgesia could be observed. Nicotine significantly suppressed tactile allodynia and thermal hyperalgesia on day 7 after PSL, and the inhibitory effects of nicotine were reversed by co-administration of DHβE (Fig. 5A, B). TC2559, administered once a day for 4 days (20 nmol, day 0–3), significantly attenuated tactile allodynia and thermal hyperalgesia on day 7 after PSL (Fig. 5C, D), indicating that tactile allodynia and thermal hyperalgesia after PSL in the initiation phase was alleviated by α4β2 nAChR agonists. Moreover, TC2559, administered to male C57BL/6 mice and female ICR mice once a day for 4 days (20 nmol, day 0–3), also significantly attenuated tactile allodynia on day 7 after PSL (Fig. 5E, F).
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Discussion In the present study, we demonstrated that F4/80 positive macrophages were observed in the vicinity of the injured SCN on day 7 after PSL, and that nAChR α4 and β2 subunits were expressed on the infiltrating macrophages as assessed by immunohistochemistry. The perineural injection of nicotine for 4 days (day 7–10; maintenance phase) attenuated tactile allodynia and thermal hyperalgesia after PSL, indicating the inhibitory effects of nicotine on PSL-induced pain behaviors in the maintenance phase. It is noteworthy that nicotine attenuated pain behaviors in the maintenance phase, because medical treatment usually begins after the development of neuropathic pain (maintenance phase) in most patients. Similarly, PSL-induced pain behaviors were also inhibited by selective α4β2 nAChR agonists (TC2559; in the both initial and maintenance phase, ABT418; in the maintenance phase), and the nicotine-induced attenuation of pain behaviors was antagonized by a selective α4β2 nAChR antagonist, DHβE, suggesting that α4β2 nAChR may play an important role in the alleviation of PSL-induced pain behaviors in both initial and maintenance phases. We showed that nicotine and TC2559, which were perineurally injected for 4 days (day 7 –10; maintenance phase), elicited the attenuation of pain behaviors until day 21. On the other hand, the pain inhibitory effects of ABT418 were evaluated on day 14. The pain inhibitory effects of ABT418 on PSL-induced pain behaviors could be observed on 4 day after cessation of repeated administration (day 7 –10), indicating that the inhibitory effects are prolonged after
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the cessation of administration. ABT418 is the α4β2 nAChR agonist as well as TC2559. Therefore, we have an assumption that ABT418 would also show the pain inhibitory effects over day 14 as well as the effects of nicotine and TC2559.
Accumulating evidence indicates that interactions among several types of inflammatory cells in the injured nerve are implicated in the pathogenesis of neuropathic pain (Calvo et al., 2012, Moalem and Tracey, 2006). First, tissue resident macrophages and schwann cells are activated by peripheral nerve injury and release inflammatory cytokines, including IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) (Mueller et al., 2001, Tofaris et al., 2002). Thereafter, circulating neutrophils, macrophages, and lymphocytes are recruited to the injured nerve (Kim and Moalem-Taylor, 2011), and cause chronic inflammation, resulting in neuropathic pain (Scholz and Woolf, 2007). Notably, it has been considered that macrophages play a critical role in neuroinflammation after peripheral nerve injury (Moalem and Tracey, 2006). Indeed, depletion of macrophages by clodronate improved neuropathic pain (Liu et al., 2000). Moreover, we also reported that macrophage-derived cytokines and chemokines, such as IL-1β and macrophage inflammatory proteins (CCL3 and CCL4), are essential factors for the development of neuropathic pain (Kiguchi et al., 2010, Saika et al., 2012). Therefore, macrophages may be important therapeutic targets for the treatment of neuropathic pain.
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Recently, the anti-inflammatory effects of nAChRs ligands acting on macrophages have been clarified using various experimental models (de Jonge et al., 2005, Wang et al., 2003). For example, activation of nAChRs expressed on macrophages by nicotine suppressed lipopolysaccharide-induced expression of proinflammatory cytokines (IL-1β and IL-6, TNF-α) (Wang et al., 2003). Nicotine ameliorated surgery-induced inflammation through the suppression of macrophages (de Jonge et al., 2005). In this study, we showed that macrophages infiltrated into the injured SCN on day 7 after PSL and that neuropathic pain was observed at least, for 7–21 days, suggesting that infiltrating macrophages might play an important role in the maintenance phase of neuropathic pain. Furthermore, it was reported that nAChR α4 subunit positive cells were located on not only macrophage, but also other types of cells such as neutrophiles and lymphocytes by immunohistochemistry (Kiguchi et al., 2012b). These nAChR α4 subunit positive cells may be also involved in neuropathic pain. Actually, lymphocytes also participate in the maintenance phase of neuropathic pain (Moalem et al., 2004). Additionally, T lymphocytes express choline acetyltransferase, an enzyme of acetylcholine synthesis (Kawashima et al., 2012). These reports suggested the presence of non-neuronal cholinergic system in immune cells and its involvement in the regulation of immune function.
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Our previous report (Kiguchi et al., 2012b) shows that expression of nAChR α4 subunit of SCN after nerve-injury was measured by Western blotting. The peak of its expression was observed on initial phase after PSL, and the up-regulation had been sustained until at least day 7. Typically, chronic exposure to ligand results in surface receptor down-regulation to attenuate its intracellular signaling and desensitizes receptors, and tolerance is developed subsequently (Giniatullin et al., 2005). However, in the case of nAChR, chronic exposure to nAChR agonist results in up-regulation (Akaike et al., 2010, Ke et al., 1998, Sekhon et al., 1999), which has been proposed to be a compensatory mechanism to replace desensitized receptors at the cell surface (Darsow et al., 2005). Moreover, we showed that nAChR located on macrophage may play an important role in the relief of neuropathic pain in this experiment. On the other hand, there is no report that nicotine desensitizes nAChRs on macrophages. Thus, the nAChRs between neuron and non-neuronal cells might be different in response to nicotine. In other words, we thought that repeated administration of nicotine and α4β2 nAChR agonists might be effective in this mouse model.
Treatment with nicotine and TC2559 for 4 days (day 0–3; initiation phase) also suppressed PSL-induced tactile allodynia and thermal hyperalgesia, indicating that the activation of α4β2 nAChRs inhibited neuropathic pain in the initiation phase. These results are consistent with reports that TC2559 or ABT594 attenuated chronic constriction injury- and spinal nerve
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ligation-induced neuropathic pain (Cheng et al., 2011, Kesingland et al., 2000). In this phase, several inflammatory cytokines (IL-1β, IL-6, and TNF) and chemokines (CCL2, CCL3) derived from infiltrating macrophages and neutrophils largely contribute to the development of neuropathic pain (Scholz and Woolf, 2007). Indeed, we reported that inhibition of IL-1β and chemokine (CCL3, CCL4) release derived from macrophages or neutrophils by local administration of neutralizing antibodies prevented the initiation of neuropathic pain (Kiguchi et al., 2012b, Saika et al., 2012). It was also reported that nicotine suppressed the production of cytokines (IL-6, IL-12, and TNF-α) in alveolar macrophages, which expressed α4β2 nAChR subtypes, but not α7 (Matsunaga et al., 2001). Activation of α4β2 nAChR located in isolated intestinal or peritoneal macrophages suppressed NF-κB activation of macrophages and reduced proinflammatory cytokine (TNF-α) release (van der Zanden et al., 2009). Thus, α4β2 nAChR agonists might reduce the triggers of neuropathic pain by suppressing inflammatory mediators, owing to the inhibition of macrophages and neutrophils. In addition, there are several reports that α4β2 nAChR agonists increase anti-inflammatory mediators and attenuate pain behavior (Terrando et al., 2011, Han et al., 2014).
We showed that α4β2 nAChR agonist, TC2559, significantly suppressed the PSL-induced tactile allodynia in the initial phase regardless of mouse strain and/or sex (Fig. 5 E and F). In the species difference, there are several reports showing that stimulation of human and/or
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murine macrophage by nicotine reduces expression of inflammatory mediators induced by endotoxin and nerve-injury, and these reports support to have high commonality in terms of nAChR subunit types between humans and mice (de Jonge et al., 2005, Wang et al., 2003, Matsunaga et al., 2001, Blanchet et al., 2004). And the α4, α7 and β2 nAChR subunits have the gene coding sequences with high homology across vertebrate species (Le Novere and Changeux, 1995).
Taken together, we propose that perineural administration of α4β2 nAChR agonists around injured nerves might attenuate neuroinflammation-related tactile allodynia and thermal hyperalgesia. This study provides a rationale for the possible utility of selective agonists of α4β2 nAChRs for the medical treatment of neuropathic pain.
Acknowledgments This study was supported by a grant from the Smoking Research Foundation.
Conflict of interest The authors declare no conflicts of interest.
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neuroinflammation. Neurochem Int 61, 1212-1219. Kiguchi, N., Maeda, T., Kobayashi, Y., Fukazawa, Y. & Kishioka, S. 2010. Macrophage inflammatory protein-1alpha mediates the development of neuropathic pain following peripheral nerve injury through interleukin-1beta up-regulation. Pain 149, 305-315. Kim, C. F. & Moalem-Taylor, G. 2011. Detailed characterization of neuro-immune responses following neuropathic injury in mice. Brain Res 1405, 95-108. Kiyosawa, A., Katsurabayashi, S., Akaike, N., Pang, Z. P. & Akaike, N. 2001. Nicotine facilitates glycine release in the rat spinal dorsal horn. J Physiol 536, 101-110. Le Novere, N. & Changeux, J. P. 1995. Molecular evolution of the nicotinic acetylcholine receptor: an example of multigene family in excitable cells. J Mol Evol 40, 155-172. Lindstrom, J. 1997. Nicotinic acetylcholine receptors in health and disease. Mol Neurobiol 15, 193-222. Liu, T., van Rooijen, N. & Tracey, D. J. 2000. Depletion of macrophages reduces axonal degeneration and hyperalgesia following nerve injury. Pain 86, 25-32. Loram, L. C., Taylor, F. R., Strand, K. A., Maier, S. F., Speake, J. D., Jordan, K. G., James, J. W., Wene, S. P., Pritchard, R. C., Green, H., Van Dyke, K., Mazarov, A., Letchworth, S. R. & Watkins, L. R. 2012. Systemic administration of an alpha-7 nicotinic acetylcholine agonist reverses neuropathic pain in male Sprague Dawley rats. J Pain 13, 1162-1171.
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Ma, W. & Quirion, R. 2006. Increased calcitonin gene-related peptide in neuroma and invading macrophages is involved in the up-regulation of interleukin-6 and thermal hyperalgesia in a rat model of mononeuropathy. J Neurochem 98, 180-192. Maeda, T., Kiguchi, N., Kobayashi, Y., Ikuta, T., Ozaki, M. & Kishioka, S. 2009. Leptin derived from adipocytes in injured peripheral nerves facilitates development of neuropathic pain via macrophage stimulation. Proc Natl Acad Sci U S A 106, 13076-13081. Matsunaga, K., Klein, T. W., Friedman, H. & Yamamoto, Y. 2001. Involvement of nicotinic acetylcholine receptors in suppression of antimicrobial activity and cytokine responses of alveolar macrophages to Legionella pneumophila infection by nicotine. J Immunol 167, 6518-6524. McGehee, D. S., Heath, M. J., Gelber, S., Devay, P. & Role, L. W. 1995. Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors. Science 269, 1692-1696. Miwa, J. M., Freedman, R. & Lester, H. A. 2011. Neural systems governed by nicotinic acetylcholine receptors: emerging hypotheses. Neuron 70, 20-33. Moalem, G. & Tracey, D. J. 2006. Immune and inflammatory mechanisms in neuropathic pain. Brain Res Rev 51, 240-264. Moalem, G., Xu, K. & Yu, L. 2004. T lymphocytes play a role in neuropathic pain following
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peripheral nerve injury in rats. Neuroscience 129, 767-777. Mueller, M., Wacker, K., Ringelstein, E. B., Hickey, W. F., Imai, Y. & Kiefer, R. 2001. Rapid response of identified resident endoneurial macrophages to nerve injury. Am J Pathol 159, 2187-2197. Rashid, M. H., Furue, H., Yoshimura, M. & Ueda, H. 2006. Tonic inhibitory role of alpha4beta2 subtype of nicotinic acetylcholine receptors on nociceptive transmission in the spinal cord in mice. Pain 125, 125-135. Saika, F., Kiguchi, N., Kobayashi, Y., Fukazawa, Y. & Kishioka, S. 2012. CC-chemokine ligand 4/macrophage inflammatory protein-1beta participates in the induction of neuropathic pain after peripheral nerve injury. Eur J Pain 16, 1271-1280. Scholz, J. & Woolf, C. J. 2007. The neuropathic pain triad: neurons, immune cells and glia. Nat Neurosci 10, 1361-1368. Sekhon, H. S., Jia, Y., Raab, R., Kuryatov, A., Pankow, J. F., Whitsett, J. A., Lindstrom, J. & Spindel, E. R. 1999. Prenatal nicotine increases pulmonary alpha7 nicotinic receptor expression and alters fetal lung development in monkeys. J Clin Invest 103, 637-647. Seltzer, Z., Dubner, R. & Shir, Y. 1990. A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 43, 205-218. Sindrup, S. H. & Jensen, T. S. 1999. Efficacy of pharmacological treatments of neuropathic pain: an update and effect related to mechanism of drug action. Pain 83, 389-400.
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Takeda, D., Nakatsuka, T., Papke, R. & Gu, J. G. 2003. Modulation of inhibitory synaptic activity by a non-alpha4beta2, non-alpha7 subtype of nicotinic receptors in the substantia gelatinosa of adult rat spinal cord. Pain 101, 13-23. Terrando, N., Eriksson, L. I., Ryu, J. K., Yang, T., Monaco, C., Feldmann, M., Jonsson Fagerlund, M., Charo, I. F., Akassoglou, K. & Maze, M. 2011. Resolving postoperative neuroinflammation and cognitive decline. Ann Neurol 70, 986-995. Tofaris, G. K., Patterson, P. H., Jessen, K. R. & Mirsky, R. 2002. Denervated Schwann cells attract macrophages by secretion of leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 in a process regulated by interleukin-6 and LIF. J Neurosci 22, 6696-6703. Tracey, K. J. 2002. The inflammatory reflex. Nature 420, 853-859. van der Zanden, E. P., Snoek, S. A., Heinsbroek, S. E., Stanisor, O. I., Verseijden, C., Boeckxstaens, G. E., Peppelenbosch, M. P., Greaves, D. R., Gordon, S. & De Jonge, W. J. 2009. Vagus nerve activity augments intestinal macrophage phagocytosis via nicotinic acetylcholine receptor alpha4beta2. Gastroenterology 137, 1029-1039, 1039 e1-4. Wang, H., Yu, M., Ochani, M., Amella, C. A., Tanovic, M., Susarla, S., Li, J. H., Wang, H., Yang, H., Ulloa, L., Al-Abed, Y., Czura, C. J. & Tracey, K. J. 2003. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature
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421, 384-388. Woolf, C. J. & Mannion, R. J. 1999. Neuropathic pain: aetiology, symptoms, mechanisms, and management. Lancet 353, 1959-1964.
Figure Legends Fig. 1. Localization of nAChRs α4 or β2 subunits on infiltrating macrophages. Mice were subjected to sham or PSL, and the sciatic nerve was dissected on day 7 after
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operation. Localization of nAChR α4 or β2 subunits on F4/80 positive macrophages in the sciatic nerves was evaluated by immunohistochemistry. Representative micrographs of longitudinal sciatic nerve are shown. Green; nAChR α4 or β2 subunit, magenta; F4/80. By quantitative analysis, the cell count of F4/80 positive cells in the injured SCN (200 × 200 μm) was presented as the mean ± S.E.M. of three to five samples. nAChR α4 and/or β2 subunits were co-localized with F4/80 positive cells. N.D.; not detected. d7; day 7.
Fig. 2. Inhibition of PSL-induced tactile allodynia and thermal hyperalgesia by perineural nicotine in the maintenance phase. Mice were subjected to PSL, and nicotine was perineurally administered once a day for 4 days (day 7–10) after PSL. PSL-induced tactile allodynia and thermal hyperalgesia were evaluated by the von Frey test (A, C) and Hargreaves test (B, D), respectively. The time courses of nicotine (20 nmol) effects (A, B). Reversal of nicotine effects on day 14 after PSL by co-administration of nAChR antagonists (DHβE 20 nmol and Mec 20 nmol) with nicotine (20 nmol) (C, D). Veh; vehicle, Nic; nicotine, DHβE; dihydro-β-erythroidine, Mec; mecamylamine. Data are presented as the mean ± S.E.M. of 5–26 mice. Control is the response of the contralateral side of PSL+Veh. ***P
Received Date : 19-Jun-2014 Revision Requested: 22-Jul-2014 Final Revision Received: 28-Nov-2014 Accepted Date: 02-Dec-2014 Article type : Regular Paper
Peripheral alpha4beta2 nicotinic acetylcholine receptor signaling attenuates tactile allodynia and thermal hyperalgesia after nerve injury in mice
Fumihiro Saika, Norikazu Kiguchi, Yuka Kobayashi, Shiroh Kishioka Department of Pharmacology, Wakayama Medical University, Wakayama, Japan
Short title: Neuropathic pain relief and α4β2 nAChR
Corresponding Author Shiroh Kishioka, M.D., Ph.D. Department of Pharmacology, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-0012, Japan Tel: +81 73 441 0629 Fax: +81 73 446 3806 E-mail: [email protected] This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/apha.12437 This article is protected by copyright. All rights reserved.
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Abstract Aim: Neuropathic pain is often refractory to conventional analgesics including opioids and non-steroidal anti-inflammatory drugs. Evidence suggests nicotinic acetylcholine receptor ligands regulate pain transmission. Effects of α4β2 nicotinic acetylcholine receptor activation on pain behaviors after nerve injury were studied.
Methods: Mice were subjected to partial sciatic nerve ligation. Nicotinic acetylcholine receptor α4 and β2 subunits localization in injured nerves were evaluated by immunohistochemistry. Neuropathic pain, assessed by tactile allodynia and thermal hyperalgesia, were examined by von Frey test and Hargreaves test, respectively.
Results: Nicotinic acetylcholine receptor α4 and β2 subunits were up-regulated in injured nerves and were expressed on F4/80 positive macrophages. When nicotine was perineurally administered daily for 4 days (day 7–10; maintenance phase) after nerve injury, pain behaviors were significantly alleviated. The inhibitory effects of nicotine were reversed by co-administration of mecamylamine (non-selective nicotinic acetylcholine receptor antagonist) and dihydro-β-erythroidine (selective α4β2 nicotinic acetylcholine receptor antagonist). Likewise, when α4β2 nicotinic acetylcholine receptor agonists (TC2559 or ABT418) were administered daily for 4 days (day 7–10) after nerve injury, pain behaviors
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homopentameric α7 (McGehee et al., 1995) in the nervous system. These nAChR subunits have the gene coding sequences with high homology across vertebrate species and there are many commonalities in biological mechanisms (Le Novere and Changeux, 1995). These nAChR subtypes participate in neurological disorders such as pain, and cognitive and neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease (Gotti and Clementi, 2004).
Accumulating evidence indicates that non-neuronal tissues such as the vessel and immune cells throughout the body express nAChRs. The nAChRs may affect the proliferation, adhesion and migration of the cells in the non-neuronal tissues and their actions are manifold such as wound healing and angiogenesis (Gotti and Clementi, 2004). Notably, previous reports indicate that cholinergic anti-inflammatory pathway via nAChRs signaling on macrophages has been identified, and stimulation of vagus nerve suppressed inflammatory responses in vitro and in vivo (de Jonge et al., 2005, Wang et al., 2003).
Several lines of evidence indicate that nAChR ligands regulate pain transmission, because α4β2 and α7 nAChR subtypes are located in pain inhibitory GABAergic or opioidergic neurons, and selective agonists for α4β2 or α7 nAChR subtypes suppress various types of pain through the release of these inhibitory neurotransmitters in the central nervous systems
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homopentameric α7 (McGehee et al., 1995) in the nervous system. These nAChR subunits have the gene coding sequences with high homology across vertebrate species and there are many commonalities in biological mechanisms (Le Novere and Changeux, 1995). These nAChR subtypes participate in neurological disorders such as pain, and cognitive and neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease (Gotti and Clementi, 2004).
Accumulating evidence indicates that non-neuronal tissues such as the vessel and immune cells throughout the body express nAChRs. The nAChRs may affect the proliferation, adhesion and migration of the cells in the non-neuronal tissues and their actions are manifold such as wound healing and angiogenesis (Gotti and Clementi, 2004). Notably, previous reports indicate that cholinergic anti-inflammatory pathway via nAChRs signaling on macrophages has been identified, and stimulation of vagus nerve suppressed inflammatory responses in vitro and in vivo (de Jonge et al., 2005, Wang et al., 2003).
Several lines of evidence indicate that nAChR ligands regulate pain transmission, because α4β2 and α7 nAChR subtypes are located in pain inhibitory GABAergic or opioidergic neurons, and selective agonists for α4β2 or α7 nAChR subtypes suppress various types of pain through the release of these inhibitory neurotransmitters in the central nervous systems
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Takeda, D., Nakatsuka, T., Papke, R. & Gu, J. G. 2003. Modulation of inhibitory synaptic activity by a non-alpha4beta2, non-alpha7 subtype of nicotinic receptors in the substantia gelatinosa of adult rat spinal cord. Pain 101, 13-23. Terrando, N., Eriksson, L. I., Ryu, J. K., Yang, T., Monaco, C., Feldmann, M., Jonsson Fagerlund, M., Charo, I. F., Akassoglou, K. & Maze, M. 2011. Resolving postoperative neuroinflammation and cognitive decline. Ann Neurol 70, 986-995. Tofaris, G. K., Patterson, P. H., Jessen, K. R. & Mirsky, R. 2002. Denervated Schwann cells attract macrophages by secretion of leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 in a process regulated by interleukin-6 and LIF. J Neurosci 22, 6696-6703. Tracey, K. J. 2002. The inflammatory reflex. Nature 420, 853-859. van der Zanden, E. P., Snoek, S. A., Heinsbroek, S. E., Stanisor, O. I., Verseijden, C., Boeckxstaens, G. E., Peppelenbosch, M. P., Greaves, D. R., Gordon, S. & De Jonge, W. J. 2009. Vagus nerve activity augments intestinal macrophage phagocytosis via nicotinic acetylcholine receptor alpha4beta2. Gastroenterology 137, 1029-1039, 1039 e1-4. Wang, H., Yu, M., Ochani, M., Amella, C. A., Tanovic, M., Susarla, S., Li, J. H., Wang, H., Yang, H., Ulloa, L., Al-Abed, Y., Czura, C. J. & Tracey, K. J. 2003. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature
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Surgery and drug administration For the neuropathic pain model, mice underwent PSL as described previously (Seltzer et al., 1990). Briefly, mice were anesthetized with 80 mg kg-1of sodium pentobarbital intraperitoneally (i.p.). Under the condition, the right common sciatic nerve (SCN) was exposed. The SCN was isolated from surrounding tissue and approximately one-third to one-half of the SCN thickness was tightly ligated with silk suture (No. 1; Natsume Seisakusho Co., Tokyo, Japan). Finally, the muscle and skin layers were closed with sutures. For the sham operation, the SCN was only exposed without ligation and the incision was closed. PSL-induced long-lasting allodynia and hyperalgesia mimics the neuropathic pain in human patients.
Nicotine tartrate, mecamylamine hydrochloride (non-specific nAChR antagonist; Mec), dihydro-β-erythroidine
(α4β2
nAChR
antagonist;
DHβE),
and
ABT418
[(S)-3-methyl-5-(1-methyl-2-pyrrolidinyl) isoxazole] (α4β2 nAChR agonist) were purchased from
Sigma
Aldrich
(Tokyo,
Japan).
[4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine
TC2559 difumarate]
difumarate (α4β2
nAChR
agonist) was purchased from R&D Systems (Minneapolis, MN, USA). All drugs were dissolved in sterile phosphate-buffered saline (PBS). Under sodium pentobarbital anesthesia (80 mg kg-1, i.p.), these agents (10 µL) were injected into a region surrounding the SCN
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without a skin incision using 30-gauge needle attached to a glass microsyringe in accordance with previous reports (Ma and Quirion, 2006, Maeda et al., 2009). According to the preventive effects of local nicotine (1-20 nmol) administration against tactile allodynia and thermal hyperalgesia after PSL on initial phase (Kiguchi et al., 2012b), we applied the dose of nicotine (20 nmol) into the injured SCN.
Immunohistochemistry Immunohistochemistry was performed according to the method of our previous papers (Saika et al., 2012, Kiguchi et al., 2012a). Frozen sections were cut (10 μm thick) at -20°C by cryostat and transferred to silane-coated glass slides. They were immersed and shaken in PBS containing 0.1% Triton X-100 (30 min). Then, the sections were blocked with 4% bovine serum albumin (BSA) and PBS containing 0.1% Triton X-100 for six hours at room temperature. The sections were incubated overnight with specific primary antibodies against F4/80 (1:200, Cedarlane Laboratories, Burlington, Ontario, Canada) and nAChR α4 and β2 subunit (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C. The sections were washed with PBS containing 0.1% Triton X-100 (5 min×5) and incubated with secondary antibodies solution (Alexa Fluor-488 or AlexaFluor-594 -conjugated IgG, 1:200; Invitrogen, Carlsbad, CA, USA) for 2 hour at room temperature in the dark. The section was washed with PBS containing 0.1% Triton X-100 (30 min), followed by nuclear staining using
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Hoechst 33342 (1:1000; Invitrogen) for 10 min at room temperature in the dark. Finally, the sections were washed with PBS (10 min) and mounted by a cover slip with Perma Fluor (Thermo Fisher Scientific, Waltham, MA, USA). Immunoreactivity was captured using a confocal laser scanning microscope. For quantitative analysis, F4/80 positive macrophages were counted in the 200 × 200 μm square area.
Assessment of neuropathic pain Mice were habituated to the environment for the assessments for at least two days before baseline test. Tactile allodynia was evaluated by the von Frey test (Kiguchi et al., 2010) using von Frey filaments (Neuroscience, Tokyo, Japan). Mice were placed on a wire mesh (5 × 5 mm) grid floor, and covered with an individual opaque container to avoid visual stimulation. The mice were adapted for 1–2 hour before the test. The von Frey filaments (0.07 or 0.16 g) were applied ten times from underneath the mesh grid floor to the central surface of each hind paw. The escape responses such as withdrawal lifting or flinching of the paw against the filament stimulation were counted for each hind paw. Tactile allodynia was presented as the percentage of the withdrawal responses against filament stimuli.
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Thermal hyperalgesia was evaluated by the Hargreaves test (Hargreaves et al., 1988) using the IITC 390 Plantar Test Analgesia Meter (Neuroscience, Tokyo, Japan). Mice were placed in a clear plastic cage on an elevated glass sheet and allowed to adapt for 1–2 hour. The radiant heat source positioned under the glass sheet was applied to the central surface of each hind paw. The latencies to escape the hind paw against radiant heat were measured ten times for each hind paw at a 5-min interval. Data are expressed as the mean latency of ten stimulations. The heat intensity was calibrated to give a control latency of approximately 10 s.
Statistical analysis All data are expressed as mean ± S.E.M. Statistical analysis was performed by a two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons test, and one-way ANOVA followed by Tukey’s multiple comparisons test. Values of P < 0.05 were considered statistically significant. Results Localization of α4β2 nAChRs in accumulating macrophages Immunohistochemical analysis demonstrated F4/80 positive macrophages were increased in the vicinity of the injured SCN on day 7 after PSL, whereas immune cells were rarely observed in the SCN in the sham group. When we evaluated the number of F4/80 positive
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cells in SCN, there was a marked increase in the number of F4/80 positive cells in the PSL mice, while rare F4/80 positive cells in sham mice. In addition, nAChR α4 and/or β2 subunits were co-localized with F4/80 positive cells. Moreover, nAChR α4 and β2 subunits were
prominently
up-regulated
on
F4/80 positive
macrophages
(Fig.
1).
The
immunoreactivity of nAChR subunits and macrophage did not detect in the SCN on day 7 after PSL, when their primary antibodies were not applied on the sections (Supplemental fig. 1).
Effects of peripheral administration of nAChR agonists on tactile allodynia and thermal hyperalgesia after PSL in the maintenance phase Tactile allodynia and thermal hyperalgesia were observed from day 7 to day 21 after PSL but not after sham. When nicotine 20 nmol, based on the report of Kiguchi et al. (2012), was perineurally administered once a day for 4 days (day 7–10; maintenance phase) after PSL, PSL-induced tactile allodynia and thermal hyperalgesia were significantly attenuated (Fig. 2A, B). The inhibitory effects of nicotine were antagonized by co-administration with not only mecamylamine, a non-selective nAChR antagonist, but also DHβE, a selective α4β2 nAChR antagonist on day 14 after PSL (Fig. 2C, D). These studies were performed by blind assessment. Perineural administration of a selective α4β2 nAChR agonist, TC2559, administered once a day for 4 days (20 nmol, day 7–10), also suppressed tactile allodynia and
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thermal hyperalgesia after PSL (Fig. 3A, B). On day 14, tactile allodynia and thermal hyperalgesia were attenuated by treatment with TC2559 (day 7–10) in a dose-dependent manner (Fig. 3C, D). Similarly, perineural administration of another α4β2 nAChR agonist, ABT418, administered once a day for 4 days (1–20 nmol, day 7–10), reduced tactile allodynia and thermal hyperalgesia on day 14 after PSL (Fig. 4), indicating tactile allodynia and thermal hyperalgesia after PSL in the maintenance phase was alleviated by treatment with the α4β2 nAChR agonists in this phase. Effects of peripheral administration of nAChR agonists on tactile allodynia and thermal hyperalgesia after PSL in the initiation phase Nicotine was perineurally administered once a day for 4 days (day 0–3; initiation phase) after PSL. On day 7 after PSL, tactile allodynia and thermal hyperalgesia could be observed. Nicotine significantly suppressed tactile allodynia and thermal hyperalgesia on day 7 after PSL, and the inhibitory effects of nicotine were reversed by co-administration of DHβE (Fig. 5A, B). TC2559, administered once a day for 4 days (20 nmol, day 0–3), significantly attenuated tactile allodynia and thermal hyperalgesia on day 7 after PSL (Fig. 5C, D), indicating that tactile allodynia and thermal hyperalgesia after PSL in the initiation phase was alleviated by α4β2 nAChR agonists. Moreover, TC2559, administered to male C57BL/6 mice and female ICR mice once a day for 4 days (20 nmol, day 0–3), also significantly attenuated tactile allodynia on day 7 after PSL (Fig. 5E, F).
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Discussion In the present study, we demonstrated that F4/80 positive macrophages were observed in the vicinity of the injured SCN on day 7 after PSL, and that nAChR α4 and β2 subunits were expressed on the infiltrating macrophages as assessed by immunohistochemistry. The perineural injection of nicotine for 4 days (day 7–10; maintenance phase) attenuated tactile allodynia and thermal hyperalgesia after PSL, indicating the inhibitory effects of nicotine on PSL-induced pain behaviors in the maintenance phase. It is noteworthy that nicotine attenuated pain behaviors in the maintenance phase, because medical treatment usually begins after the development of neuropathic pain (maintenance phase) in most patients. Similarly, PSL-induced pain behaviors were also inhibited by selective α4β2 nAChR agonists (TC2559; in the both initial and maintenance phase, ABT418; in the maintenance phase), and the nicotine-induced attenuation of pain behaviors was antagonized by a selective α4β2 nAChR antagonist, DHβE, suggesting that α4β2 nAChR may play an important role in the alleviation of PSL-induced pain behaviors in both initial and maintenance phases. We showed that nicotine and TC2559, which were perineurally injected for 4 days (day 7 –10; maintenance phase), elicited the attenuation of pain behaviors until day 21. On the other hand, the pain inhibitory effects of ABT418 were evaluated on day 14. The pain inhibitory effects of ABT418 on PSL-induced pain behaviors could be observed on 4 day after cessation of repeated administration (day 7 –10), indicating that the inhibitory effects are prolonged after
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the cessation of administration. ABT418 is the α4β2 nAChR agonist as well as TC2559. Therefore, we have an assumption that ABT418 would also show the pain inhibitory effects over day 14 as well as the effects of nicotine and TC2559.
Accumulating evidence indicates that interactions among several types of inflammatory cells in the injured nerve are implicated in the pathogenesis of neuropathic pain (Calvo et al., 2012, Moalem and Tracey, 2006). First, tissue resident macrophages and schwann cells are activated by peripheral nerve injury and release inflammatory cytokines, including IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) (Mueller et al., 2001, Tofaris et al., 2002). Thereafter, circulating neutrophils, macrophages, and lymphocytes are recruited to the injured nerve (Kim and Moalem-Taylor, 2011), and cause chronic inflammation, resulting in neuropathic pain (Scholz and Woolf, 2007). Notably, it has been considered that macrophages play a critical role in neuroinflammation after peripheral nerve injury (Moalem and Tracey, 2006). Indeed, depletion of macrophages by clodronate improved neuropathic pain (Liu et al., 2000). Moreover, we also reported that macrophage-derived cytokines and chemokines, such as IL-1β and macrophage inflammatory proteins (CCL3 and CCL4), are essential factors for the development of neuropathic pain (Kiguchi et al., 2010, Saika et al., 2012). Therefore, macrophages may be important therapeutic targets for the treatment of neuropathic pain.
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Recently, the anti-inflammatory effects of nAChRs ligands acting on macrophages have been clarified using various experimental models (de Jonge et al., 2005, Wang et al., 2003). For example, activation of nAChRs expressed on macrophages by nicotine suppressed lipopolysaccharide-induced expression of proinflammatory cytokines (IL-1β and IL-6, TNF-α) (Wang et al., 2003). Nicotine ameliorated surgery-induced inflammation through the suppression of macrophages (de Jonge et al., 2005). In this study, we showed that macrophages infiltrated into the injured SCN on day 7 after PSL and that neuropathic pain was observed at least, for 7–21 days, suggesting that infiltrating macrophages might play an important role in the maintenance phase of neuropathic pain. Furthermore, it was reported that nAChR α4 subunit positive cells were located on not only macrophage, but also other types of cells such as neutrophiles and lymphocytes by immunohistochemistry (Kiguchi et al., 2012b). These nAChR α4 subunit positive cells may be also involved in neuropathic pain. Actually, lymphocytes also participate in the maintenance phase of neuropathic pain (Moalem et al., 2004). Additionally, T lymphocytes express choline acetyltransferase, an enzyme of acetylcholine synthesis (Kawashima et al., 2012). These reports suggested the presence of non-neuronal cholinergic system in immune cells and its involvement in the regulation of immune function.
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Our previous report (Kiguchi et al., 2012b) shows that expression of nAChR α4 subunit of SCN after nerve-injury was measured by Western blotting. The peak of its expression was observed on initial phase after PSL, and the up-regulation had been sustained until at least day 7. Typically, chronic exposure to ligand results in surface receptor down-regulation to attenuate its intracellular signaling and desensitizes receptors, and tolerance is developed subsequently (Giniatullin et al., 2005). However, in the case of nAChR, chronic exposure to nAChR agonist results in up-regulation (Akaike et al., 2010, Ke et al., 1998, Sekhon et al., 1999), which has been proposed to be a compensatory mechanism to replace desensitized receptors at the cell surface (Darsow et al., 2005). Moreover, we showed that nAChR located on macrophage may play an important role in the relief of neuropathic pain in this experiment. On the other hand, there is no report that nicotine desensitizes nAChRs on macrophages. Thus, the nAChRs between neuron and non-neuronal cells might be different in response to nicotine. In other words, we thought that repeated administration of nicotine and α4β2 nAChR agonists might be effective in this mouse model.
Treatment with nicotine and TC2559 for 4 days (day 0–3; initiation phase) also suppressed PSL-induced tactile allodynia and thermal hyperalgesia, indicating that the activation of α4β2 nAChRs inhibited neuropathic pain in the initiation phase. These results are consistent with reports that TC2559 or ABT594 attenuated chronic constriction injury- and spinal nerve
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ligation-induced neuropathic pain (Cheng et al., 2011, Kesingland et al., 2000). In this phase, several inflammatory cytokines (IL-1β, IL-6, and TNF) and chemokines (CCL2, CCL3) derived from infiltrating macrophages and neutrophils largely contribute to the development of neuropathic pain (Scholz and Woolf, 2007). Indeed, we reported that inhibition of IL-1β and chemokine (CCL3, CCL4) release derived from macrophages or neutrophils by local administration of neutralizing antibodies prevented the initiation of neuropathic pain (Kiguchi et al., 2012b, Saika et al., 2012). It was also reported that nicotine suppressed the production of cytokines (IL-6, IL-12, and TNF-α) in alveolar macrophages, which expressed α4β2 nAChR subtypes, but not α7 (Matsunaga et al., 2001). Activation of α4β2 nAChR located in isolated intestinal or peritoneal macrophages suppressed NF-κB activation of macrophages and reduced proinflammatory cytokine (TNF-α) release (van der Zanden et al., 2009). Thus, α4β2 nAChR agonists might reduce the triggers of neuropathic pain by suppressing inflammatory mediators, owing to the inhibition of macrophages and neutrophils. In addition, there are several reports that α4β2 nAChR agonists increase anti-inflammatory mediators and attenuate pain behavior (Terrando et al., 2011, Han et al., 2014).
We showed that α4β2 nAChR agonist, TC2559, significantly suppressed the PSL-induced tactile allodynia in the initial phase regardless of mouse strain and/or sex (Fig. 5 E and F). In the species difference, there are several reports showing that stimulation of human and/or
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murine macrophage by nicotine reduces expression of inflammatory mediators induced by endotoxin and nerve-injury, and these reports support to have high commonality in terms of nAChR subunit types between humans and mice (de Jonge et al., 2005, Wang et al., 2003, Matsunaga et al., 2001, Blanchet et al., 2004). And the α4, α7 and β2 nAChR subunits have the gene coding sequences with high homology across vertebrate species (Le Novere and Changeux, 1995).
Taken together, we propose that perineural administration of α4β2 nAChR agonists around injured nerves might attenuate neuroinflammation-related tactile allodynia and thermal hyperalgesia. This study provides a rationale for the possible utility of selective agonists of α4β2 nAChRs for the medical treatment of neuropathic pain.
Acknowledgments This study was supported by a grant from the Smoking Research Foundation.
Conflict of interest The authors declare no conflicts of interest.
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Figure Legends Fig. 1. Localization of nAChRs α4 or β2 subunits on infiltrating macrophages. Mice were subjected to sham or PSL, and the sciatic nerve was dissected on day 7 after
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operation. Localization of nAChR α4 or β2 subunits on F4/80 positive macrophages in the sciatic nerves was evaluated by immunohistochemistry. Representative micrographs of longitudinal sciatic nerve are shown. Green; nAChR α4 or β2 subunit, magenta; F4/80. By quantitative analysis, the cell count of F4/80 positive cells in the injured SCN (200 × 200 μm) was presented as the mean ± S.E.M. of three to five samples. nAChR α4 and/or β2 subunits were co-localized with F4/80 positive cells. N.D.; not detected. d7; day 7.
Fig. 2. Inhibition of PSL-induced tactile allodynia and thermal hyperalgesia by perineural nicotine in the maintenance phase. Mice were subjected to PSL, and nicotine was perineurally administered once a day for 4 days (day 7–10) after PSL. PSL-induced tactile allodynia and thermal hyperalgesia were evaluated by the von Frey test (A, C) and Hargreaves test (B, D), respectively. The time courses of nicotine (20 nmol) effects (A, B). Reversal of nicotine effects on day 14 after PSL by co-administration of nAChR antagonists (DHβE 20 nmol and Mec 20 nmol) with nicotine (20 nmol) (C, D). Veh; vehicle, Nic; nicotine, DHβE; dihydro-β-erythroidine, Mec; mecamylamine. Data are presented as the mean ± S.E.M. of 5–26 mice. Control is the response of the contralateral side of PSL+Veh. ***P
