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Serotonin Plays a Key Role in the Development of Opioid-Induced Hyperalgesia in Mice

Open AccessPublished:January 16, 2021DOI:https://doi.org/10.1016/j.jpain.2020.12.008

      Highlights

      • Administering of chronic morphine induces mechanical hypersensitivity in mice.
      • Inhibition of 5-HT3 receptor attenuated opioid-induced hyperalgesia (OIH).
      • Inhibition of serotonin synthesis completely inhibited the development of OIH.
      • Astrocyte activation was observed in OIH model mice.
      • Inhibition of astrocytes activation does not ameliorate OIH.

      Abstract

      Opioid usage for pain therapy is limited by its undesirable clinical effects, including paradoxical hyperalgesia, also known as opioid-induced hyperalgesia (OIH). However, the mechanisms associated with the development and maintenance of OIH remain unclear. Here, we investigated the effect of serotonin inhibition by the 5-HT3 receptor antagonist, ondansetron (OND), as well as serotonin deprivation via its synthesis inhibitor para-chlorophenylalanine, on mouse OIH models, with particular focus on astrocyte activation. Co-administering of OND and morphine, in combination with serotonin depletion, inhibited mechanical hyperalgesia and astrocyte activation in the spinal dorsal horn of mouse OIH models. Although previous studies have suggested that activation of astrocytes in the spinal dorsal horn is essential for the development and maintenance of OIH, herein, treatment with carbenoxolone (CBX), a gap junction inhibitor that suppresses astrocyte activation, did not ameliorate mechanical hyperalgesia in mouse OIH models. These results indicate that serotonin in the spinal dorsal horn, and activation of the 5-HT3 receptor play essential roles in OIH induced by chronic morphine, while astrocyte activation in the spinal dorsal horn serves as a secondary effect of OIH. Our findings further suggest that serotonergic regulation in the spinal dorsal horn may be a therapeutic target of OIH.

      Perspective

      The current study revealed that the descending serotonergic pain-facilitatory system in the spinal dorsal horn is crucial in OIH, and that activation of astrocytes is a secondary phenotype of OIH. Our study offers new therapeutic targets for OIH and may help reduce inappropriate opioid use.

      Key words

      Opioids are essential for the management of perioperative pain as well as palliative treatment of cancer pain. However, chronic administering of opioids may cause a reduction in the analgesic effect (tolerance), and lead to paradoxical hyperalgesia (opioid-induced hyperalgesia [OIH]) and subsequent physical dependency.
      • Angst MS
      • Clark JD
      Opioid-induced hyperalgesia: a qualitative systematic review.
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      • Mao J
      Opioid-induced abnormal pain sensitivity: implications in clinical opioid therapy.
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      • Xu XJ
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      • Wiesenfeld-Hallin Z
      Opioid hyperalgesia and tolerance versus 5-HT1A receptor-mediated inverse tolerance.
      More specifically, OIH is often associated with a requirement for increased, and often long-term, administering of opioids, which worsen tolerance, resulting in physical and mental dependence. Considering that these are pressing clinical issues,
      • Ballantyne JC
      Pain medicine: repairing a fractured dream.
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      • Okie S
      A flood of opioids, a rising tide of deaths.
      elucidating mechanisms that underlie OIH may be essential for the purpose of identifying therapeutic targets that help reduce both long-term and overall opioid use.
      Several mechanisms, including internalization of the µ-opioid receptor
      • Burford NT
      • Tolbert LM
      • Sadee W
      Specific G protein activation and mu-opioid receptor internalization caused by morphine, DAMGO and endomorphin I.
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      Role of receptor internalization in opioid tolerance and dependence.
      and abnormal activation of the N-methyl-D-aspartate (NMDA) receptor, leading to central sensitization to innoxious stimuli, have been suggested to underlie OIH development.
      • Koppert W
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      The impact of opioid-induced hyperalgesia for postoperative pain.
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      • Zhao M
      • Joo DT
      Enhancement of spinal N-methyl-D-aspartate receptor function by remifentanil action at delta-opioid receptors as a mechanism for acute opioid-induced hyperalgesia or tolerance.
      Moreover, opioids are known to induce the release of serotonin (5-hydroxytryptamine [5-HT]) in the spinal dorsal horn, which contributes to opioid analgesia.
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      • Goldstein FJ
      Opioid-monoamine interactions in spinal antinociception: evidence for serotonin but not norepinephrine reciprocity.
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      • Kimura M
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      • Saito S
      Peripheral nerve injury reduces analgesic effects of systemic morphine via spinal 5-hydroxytryptamine 3 receptors.
      However, 5-HT in the spinal dorsal horn reportedly plays conflicting roles in pain sensation.
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      Dual effect of serotonin on formalin-induced nociception in the rat spinal cord.
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      Bad news from the brain: descending 5-HT pathways that control spinal pain processing.
      Furthermore, administering ondansetron (OND), a 5-HT3 receptor antagonist, alone, prevented the establishment of OIH following chronic treatment with morphine, indicating that serotonin together with activation of the 5-HT3 receptor contributed to OIH.
      • Liang DY
      • Li X
      • Clark JD
      5-hydroxytryptamine type 3 receptor modulates opioid-induced hyperalgesia and tolerance in mice.
      Furthermore, inhibition of the 5-HT3 receptor effectively suppressed the development of OIH and tolerance. However, detailed cellular mechanisms underlying the role of serotonin in the development of OIH due to the repeated administering of opioids, remains largely unknown.
      Furthermore, repeated administering of morphine also activates astrocytes in the spinal dorsal horn.
      • Mattioli TA
      • Milne B
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      Ultra-low dose naltrexone attenuates chronic morphine-induced gliosis in rats.
      ,
      • Narita M
      • Suzuki M
      • Narita M
      • Yajima Y
      • Suzuki R
      • Shioda S
      • Suzuki T
      Neuronal protein kinase C gamma-dependent proliferation and hypertrophy of spinal cord astrocytes following repeated in vivo administration of morphine.
      Recent studies have demonstrated that astrocytes become activated and proliferate in animals showing neuropathic pain.
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      • Kitano J
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      • Ji RR
      • Salter MW
      • Inoue K
      JAK-STAT3 pathway regulates spinal astrocyte proliferation and neuropathic pain maintenance in rats.
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      • Xu M
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      • Ippolito DL
      • Gendron L
      • Chavkin C
      Sciatic nerve ligation-induced proliferation of spinal cord astrocytes is mediated by kappa opioid activation of p38 mitogen-activated protein kinase.
      Such morphine-driven astrocyte activation may contribute to sensory hypersensitivity.
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      • Airavaara M
      • Tian L
      • Rauhala PV
      • Kalso EA
      Differential spinal and supraspinal activation of glia in a rat model of morphine tolerance.
      A previous study has indicated that administering morphine (1 μg/kg) subcutaneously induces hypersensitivity to thermal stimuli in mice, while inhibition of JNK, which participates in astrocyte activation, suppresses astrocyte activation and hypersensitivity.
      • Sanna MD
      • Ghelardini C
      • Galeotti N
      Activation of JNK pathway in spinal astrocytes contributes to acute ultra-low-dose morphine thermal hyperalgesia.
      However, there is little evidence that opioid-induced paradoxical hyperalgesia can be suppressed by inhibiting the activation of astrocytes.
      We hypothesized that serotonin is essential for the development of morphine-induced mechanical hyperalgesia, whereas serotonin depletion will effectively prevent development of OIH. In order to confirm that activation of serotonin, particularly via 5-HT3 receptors in the spinal dorsal horn, is essential for OIH development, we investigated whether co-administering OND with morphine, or serotonin depletion via inhibition of serotonin synthesis, can suppress OIH development. Moreover, in order to confirm whether activation of astrocytes in the spinal dorsal horn is responsible for OIH, as proposed in previous studies, we investigated whether acute or chronic inhibition of astrocyte activation by carbenoxolone (CBX), which inhibits gap junctions in astrocytes and suppresses reactive astrocytes, can effectively suppress OIH.

      Methods

      Animals

      All animal experiments were approved by the Ethics Committee for Animal Experiments of the Niigata University Graduate School of Medicine and Dentistry (approval numbers: SA00251 and SA00678) and were performed according to the guidelines of the Science Council of Japan. Five to 6-week-old male C57BL/6N mice (Asazuma Animal Equipment Store, Niigata, Japan), weighing 20 to 30 g, were divided into groups of 4 or 5 mice per cage and housed under 12-hour light/dark cycle conditions. A total of 159 mice were used in this study, of which 5 to 15 mice per group were used for the behavioral experiments, while 4 to 6 mice per group were used for immunohistochemical and western blotting experiments. All animals were euthanized via isoflurane inhalation overdose.
      A mouse OIH model was developed according to a previous study
      • Liang DY
      • Li X
      • Clark JD
      5-hydroxytryptamine type 3 receptor modulates opioid-induced hyperalgesia and tolerance in mice.
      with specific modifications. Briefly, mice were intraperitoneally administered 20 mg/kg morphine hydrochloride (10 mg/mL, Shionogi, Osaka, Japan) twice daily (9 AM and 5 PM) for 4 days, whereas control mice were treated with the same amount of saline.

      Behavioral Assessment

      All behavioral tests were performed at 8:00 AM and administering of drugs was performed from approximately 9:00 AM to ensure that circadian rhythms were not affected.

      Von Frey Tests

      Mechanical allodynia was evaluated using von Frey filaments as previously described.
      • Amaya F
      • Samad TA
      • Barrett L
      • Broom DC
      • Woolf CJ
      Periganglionic inflammation elicits a distally radiating pain hypersensitivity by promoting COX-2 induction in the dorsal root ganglion.
      Briefly, the mice were placed in a red acrylic plate chamber with a wire mesh grid (1 cell size: 8 cm length × 10 cm width × 12 cm height, consisting of 10 cells) underneath and were acclimatized for 1 hour. Then, the plantar surface of the hindpaw was stimulated using von Frey hairs of various thicknesses (Semmes-Weinstein von Frey Anesthesiometer, Muromachi Machine Co., Ltd., Tokyo, Japan) before administering the daily drug regimen. The withdrawal threshold was defined as the filament of the lowest thickness to which the mice positively responded 2 times out of 10 stimuli. Withdrawal thresholds were logarithmically transformed to a linear scale for statistical analysis.
      • Horii Y
      • Matsuda M
      • Takemura H
      • Ishikawa D
      • Sawa T
      • Amaya F
      Spinal and peripheral mechanisms individually lead to the development of remifentanil-induced hyperalgesia.
      ,
      • Mills C
      • Leblond D
      • Joshi S
      • Zhu C
      • Hsieh G
      • Jacobson P
      • Meyer M
      • Decker M
      Estimating efficacy and drug ED50′s using von Frey thresholds: impact of weber's law and log transformation.

      Radiant Heat Tests

      Heat hypersensitivity was assessed via radiant heat stimulation using a Hargreaves Apparatus (model 7370, Ugo Basile, Comerio, Italy). Mice were placed on a red acrylic cage, similar to that described above, on a glass plate for 1 hour. The plantar surface of the hindpaw was then stimulated with radiant heat once per day before administering the daily drug regimen. The cut-off latency was set to 20 seconds to avoid damage to skin on the hindpaw. Each mouse was assessed 3 times at 5 minute intervals, and the mean of the release latency values was set as the threshold latency.

      Treatment with Ondansetron (OND), Para-Chlorophenylalanine (PCPA), and Carbenoxolone (CBX)

      OND (Tokyo Chemical Industries, Tokyo, Japan), which selectively inhibits 5-HT3 receptors, was co-administered with morphine at 0.5 mg/kg, 1.0 mg/kg, and 2.0 mg/kg doses diluted in saline (Fig 1A).
      Figure 1
      Figure 1Repetitive administering of morphine induces mechanical hyperalgesia in mice, while simultaneous administering of ondansetron (OND) with morphine prevents its development. (A) Time course for administering drugs. (B) Changes in paw-withdrawal thresholds. Symbols and colors corresponding to experimental groups are shown in graph legends. Saline (control) group: n = 9; OND 2.0 mg/kg only group: n = 9; morphine group: n = 13; morphine + OND (0.5 mg/kg) group: n = 9; morphine + OND (1.0 mg/kg) group: n = 9; morphine + OND (2.0 mg/kg) group: n = 8, respectively. (C) Paw withdrawal thresholds by von Frey hair stimulation on day 5 after starting the morphine treatment in all experimental groups were compared using two-way ANOVA followed by Bonferroni test. Application of OND at more than 1.0 mg/kg per morphine treatment significantly suppressed the reduction in paw-withdrawal threshold due to repetitive administering of morphine.
      PCPA (Sigma, St. Louis, MO), which inhibits tryptophan hydroxylase and depletes serotonin, was administered intraperitoneally at 150 mg/kg (diluted in saline) once daily. One group that received PCPA from day 1 to 4 before morphine was administered, was then started on morphine only, while the other group received PCPA from day 1 to 8 and concurrently morphine from day 5 to 8 (ie, PCPA was administered for a total of 8 days).The control group received the same amount of saline from day 1 to 4 before the administering of morphine (Fig 3A, B).
      CBX inhibits astrocyte gap junctions and suppresses reactive astrocytes. Briefly, following the measurement of baseline paw-withdrawal threshold, morphine (20 mg/kg) was administered twice per day from day 1 to 4 as described above. On day 5, paw-withdrawal thresholds were measured to verify that OIH was established, and CBX (10 and 30 nmol/5 µL) or saline was intrathecally administered through a spinal cord puncture using a 30-gauge needle between L5 and L6.
      • Hylden JL
      • Wilcox GL
      Intrathecal morphine in mice: a new technique.
      On day 5 after initiating the administering of morphine and after establishing the paw-withdrawal threshold following CBX pretreatment, thresholds were measured again at 0.5, 1, and 2 hour post CBX treatment. Further, we examined the effect of administering CBX intraperitoneally on OIH to determine whether a change in the method of administering CBX would exert an effect. For this purpose, 20 mg/kg CBX was administered intraperitoneally with morphine once daily from day 1 to 4 (Fig 7).

      Immunohistochemistry

      More than 12 hours after the last drug being administered, mice were euthanized via inhalation of an overdose of the anesthetic, isoflurane. Immediately, 25 mL of saline followed by an equal amount of 4% paraformaldehyde (Mildform 10N, Fujifilm Wako Pure Chemical Company, Osaka, Japan) were injected via transcardial perfusion. Each lumbar enlargement of the spinal cord was resected and fixed in 4% paraformaldehyde for 1 hour, and the spinal cord was cryoprotected at 4°C overnight in 20% sucrose in 0.1 M phosphate buffer
      • Sasaki M
      • Hashimoto S
      • Sawa T
      • Amaya F
      Tumor necrosis factor-alpha induces expression of C/EBP-beta in primary afferent neurons following nerve injury.
      . Tissues were embedded in FSC22 frozen section medium (Leica Biosystems, Wetzlar, Germany) and cryopreserved at −70°C until sectioning. Spinal cord sections with a thickness of 10 μm, prepared via thin slicing using a frozen microtome (CM1520, Leica Biosystems, Wetzlar, Germany), were mounted on aminopropyltriethoxysilane (APS)-coated glass slides (Matsunami Glass Ind. Ltd., Osaka, Japan). These spinal cord sections were then cryopreserved at −70°C until required for immunohistochemical analysis.
      The sections were washed twice with TNT buffer (10% 1 M Tris-HCl, pH 7.5, 5% 3 M NaCl, 0.03% Tween 20) and incubated with Blocking One Histo (Nacalai tesque, Kyoto, Japan) at room temperature (23–25°C) for 1 hour. Following the removal of the blocking buffer, the sections were incubated in a humidified chamber at 4°C for 2 days with the following primary antibodies: rabbit anti-5-HT (Serotonin) IgG (1:20,000; Cat. #; 20080, Immunostar, Hudson, WI), rat anti-glial fibrially acidic protein (GFAP) IgG2a (1:1000; Cat. #; 13-0300, Thermo Fisher Scientific, Waltham, MA) or rabbit anti-phospho-p44/42 MAPK (p-ERK 1/2) (1:100, Cat. #;9101, Cell Signaling Technology, Danvers, MA). The antibodies were diluted with 0.1% Tween 20 in TNB buffer (0.1 M Tris-HCl buffered saline, pH 7.5, containing 1% blocking reagent). Sections were then rinsed twice with TNT buffer and incubated overnight in a humidified chamber at 4°C with Cy3-conjugated goat anti-rabbit IgG (1:1000; Cat. #; 111-167-003, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for anti-5-HT IgG, Alexa Fluor 546 goat anti-rat IgG (1:1000; Cat. #; A-11081, Thermo Fisher Scientific, Waltham, MA) for anti-rabbit GFAP IgG2a or Alexa Fluor 488 goat anti-rabbit IgG (1:200; Cat. #; ab150077, Thermo Fisher Scientific, Waltham, MA) for anti-phospho-p44/42 MAPK (p-ERK 1/2). The antibodies were diluted in 0.1% Tween 20 in TNB buffer. The sections were washed twice with TNT buffer, embedded using VECTASHIELD mounting medium with DAPI (4’,6-diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA), and visualized using a fluorescence microscope (BX 53, Olympus, Tokyo, Japan) equipped with a digital camera system (DP73, Olympus, Tokyo, Japan), or all-in-one fluorescence microscope (BZ-X810, Keyence, Osaka, Japan).

      Quantification of Serotonin-Positive Puncta in the Spinal Dorsal Horn

      The area of serotonin-positive puncta was quantified using a Keyence BZ-X810 microscope and BZ-H4C analyzer software (Keyence). For each mouse, 3 or 4 photographs of the lumbar spinal enlargement, taken at least 100 μm apart, were randomly selected. The number of serotonin-positive puncta in lamina I, II was quantified using the hybrid cell count module of the BZ-H4C analyzer software. The brightness of serotonin fluorescence was manually set to the maximum threshold at which the background was barely visible. The thresholds were set independently for each mouse, and mouse-specific brightness and threshold settings were applied to all measurements on a section of the individual mouse. After these parameters were set, the number of serotonin-positive puncta in all images was obtained and the average of all images was calculated for each mouse.

      Western Blotting

      Following euthanasia, the spinal dorsal horns of the lumbar enlargement were collected, homogenized with a T-PER tissue protein extraction reagent supplemented with Halt protease and phosphatase inhibitor single-use cocktail (x100; Thermo Fisher Scientific, Waltham, MA), and centrifuged at 12,000 x g for 30 minutes at 4°C. The supernatant was collected, and total protein was determined using a BCA protein assay kit (Millipore, Burlington, MA). Supernatant containing 30 μg total protein was mixed with 4x Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA) and boiled at 95°C for 5 minutes. Samples were electrophoresed using a 12% Mini-PROTEAN TGX gel (Bio-Rad Laboratories) and transferred to iBlot Gel Transfer Stacks polyvinylidene difluoride membranes (Life Technologies, Carlsbad, CA).
      The transferred membranes were incubated with 5% skimmed milk in TBS-T (1 M Tris-HCl, 30% NaCl, 0.05% Tween 20, pH 7.5) for 1 hour at room temperature. The membranes were washed with TBS-T for 5 minutes and incubated overnight at 4°C with the following primary antibodies: anti-human glial fibrillary acidic protein (GFAP; 1:300; Cat. #; M 0761, Dako A/S, Glostrup, Denmark), anti-β-actin (1:5000; Cat. #; A5441, Sigma-Aldrich, Inc., MO), anti-phospho-p44/42 MAPK (p-ERK 1/2) (1:1000, Cat. #;9101, Cell Signaling Technology, Danvers, MA), and anti-p44/42 MAPK (ERK 1/2; 1:1000, Cat. #; 4695, Cell Signaling Technology) diluted in Can Get Signal 1 (Toyobo Co., Ltd., Osaka, Japan). The membranes were washed thrice with TBS-T for 5 minute each time and incubated for 1 hour at room temperature with the following secondary antibodies: anti-mouse IgG, horseradish peroxidase-linked antibody (1:5000; Cat. #; NA935), and anti-rabbit IgG horseradish peroxidase-linked antibody (1:1000 or 5000; Cat. #; NA934, Cytiva, Marlborough, MA) diluted in Can Get Signal 2 (Toyobo Co., Ltd., Osaka, Japan). The membranes were washed thrice, 5 minutes each time, with TBS-T, and the target proteins were detected using Enhanced Chemi Luminescence (ECL) Select Western Blotting Detection Reagent (Cytiva) and visualized on Gene Gnome-5 (Syngene, Cambridge, UK) for chemiluminescence imaging. The MagicMark XP Western Protein Standard (Novex, Thermo Fisher Scientific) was used to estimate molecular weights.
      GFAP, β-actin, p-ERK, and ERK were detected using the same membranes. When detecting another primary antibody, a stripping agent was used to remove the previously detected primary antibody. After the observation of luminescence, membranes were washed with TBS-T for 5 minutes, incubated with the WB stripping solution (Nacalai tesque) for 30 minutes at room temperature, washed again with TBS-T twice, 5 minutes each time, blocked with 5% skimmed milk and incubated with the next primary antibody.

      Statistical Analysis

      Statistical analysis was performed using an unpaired Student's t-test, 1-way ANOVA with Bonferroni's multiple comparison test or a 2-way ANOVA, followed by Bonferroni's multiple comparison test using the GraphPad Prism8 software (GraphPad Software, La Jolla, CA). Statistical significance was set at P < .05. Since the result of the paw withdrawal threshold was discrete data, the final data of the behavioral experiment session was presented as a boxplot, where the bottom and the top of the box were the first and third quartiles, respectively, and the whiskers above and below the box indicated the maximum and minimum values, respectively. The median (horizontal line in the box) was indicated.

      Results

      Effect of OND Treatment on Morphine-Induced Hypersensitivity

      All behavioral assessments were performed prior to the administering of morphine, in order to avoid the antinociceptive effect of morphine. Morphine treatment induced reduction in paw-withdrawal threshold against mechanical stimulation (n = 9 for saline group, n = 13 for morphine group, respectively; F [20, 204] = 3.876, P< .0001 between treatments and time; 2-way ANOVA). Thus, OIH was successfully established by administering morphine. Experimental time course is shown (Fig 1A).
      To investigate whether OND inhibits development of morphine-induced hypersensitivity, OND was simultaneously administered with morphine. Compared with the morphine group, paw-withdrawal threshold for mechanical stimulation was significantly increased in the morphine + OND group at day 3 (Fig 1). The increases in paw-withdrawal threshold at day 5 were significant for OND doses between 0.5 mg/kg and 2.0 mg/kg (morphine group vs morphine + OND, .5 mg/kg, P = .015; morphine group vs. morphine + OND, 1.0 mg/kg, P = 0.002; morphine group vs morphine + OND, 2.0 mg/kg, P = .005; Fig. 1B and C). OND alone did not influence mechanical paw-withdrawal threshold (saline vs OND, 2.0 mg/kg, P = 1.0: saline vs morphine, P < .0001; Fig. 1B and 1C). Withdrawal latency for thermal stimulation was not significantly shortened in the morphine group compared to that in the control group, as was shown previously,
      • Liang D
      • Li X
      • Lighthall G
      • Clark JD
      Heme oxygenase type 2 modulates behavioral and molecular changes during chronic exposure to morphine.
      and it was not significantly changed in the morphine + OND 2.0 mg/kg groups compared to the morphine group (n = 5 for saline group, n = 7 for morphine group and n = 7 for morphine + OND 2.0 mg/kg, F [8, 64] = 2.006, P= .06 between treatment and time; 2-way ANOVA; Fig. 2A and 2B).
      Figure 2
      Figure 2Repetitive administering of morphine did not affect thermal hypersensitivity. Time course for drug administering was the same as that described for A. (B) Changes in paw-withdrawal latencies. Symbols and colors corresponding to experimental groups shown in the graph legends are similar to that in . Saline (control) group: n = 5; morphine group: n = 7; morphine + OND (2.0 mg/kg) group: n = 7, respectively. (C) Paw withdrawal latencies against radiant heat. The development of heat hypersensitivity due to repetitive administering of morphine was weak, and the difference in paw-withdrawal latencies was not significant (P = 0.28; 1-way ANOVA followed by Bonferroni test).

      Effect of PCPA Pretreatment on Spinal Dorsal Horn Serotonin Expression

      Experimental time course is shown (Fig. 3A and 3B). Continuous intraperitoneal administering of PCPA once daily resulted in a near complete decrease in serotonin expression in the dorsal horn of the mouse spinal cord on day 5 of treatment, and the decrease in serotonin was maintained on day 8 after continued administering (Fig. 3C–3E). Next, we investigated the changes in mechanical withdrawal threshold induced by the administering of morphine. PCPA was then administered for 4 days starting from day 1, following which PCPA was discontinued and morphine treatment was initiated on day 5. Compared to mouse OIH models, PCPA pretreatment delayed the onset of hypersensitivity on day 5 of administering morphine. Interestingly, consecutive administering of PCPA from day 1 to day 8, followed by morphine treatment from day 5 to day 8 resulted in complete suppression of OIH development (day 9; morphine vs PCPA pretreatment, P = 1.0; morphine vs PCPA co-administration with morphine, P < .0001; F [4, 47] = 17.27; 1-way ANOVA; Fig. 3F and 3G). PCPA administered alone from day 1 to day 8 did not exert an effect on paw withdrawal threshold on day 9, which was when measurements were started, compared with saline group (saline vs PCPA for 8 day group, P = 1.0; saline vs morphine, P < .0001; PCPA for 8 day group vs morphine, P < .0001; Fig. 3F and 3G).
      Figure 3
      Figure 3Opioid-induced mechanical hyperalgesia (OIH) does not develop in serotonin-deprived animals. Effect of administering PCPA on serotonin expression in the spinal dorsal horn. (A) Time course showing that PCPA was administered for 4 days prior to the administering of morphine. (B) Time course showing that PCPA was administered for 4 days before and during the administering of morphine (ie, a total of 8 days). Serotonin-positive puncta (C) are restricted to laminae I and II in the naive mice, (D) but were not observed in the 4-day PCPA group. (E) Serotonin depletion is maintained in the 8-day PCPA group; scale bar: 50 µm. (F) Mechanical hypersensitivity. Symbols and colors corresponding to experimental groups in the graph legends are as in . Saline (control): n = 9; PCPA (150 mg/kg) for 8 days: n = 9; morphine: n = 15; PCPA (150 mg/kg) for 8 days + morphine: n = 9; PCPA (150 mg/kg) for 4 days + morphine group: n = 9, respectively. (F, G) Mechanical paw-withdrawal threshold on day 5 after administering morphine. Administering PCPA influenced paw-withdrawal threshold by mechanical stimulation. The degree of opioid-induced hyperalgesia in the group pretreated with PCPA was the same as that in the morphine group, although its onset was delayed. Meanwhile, opioid-induced hyperalgesia was completely suppressed when PCPA was co-administered with morphine.

      Changes in Serotonin Levels in OIH Model Mice

      Serotonin expression was confirmed in laminae I and II of the spinal dorsal horn of naive mice. Serotonin expression in the morphine group was significantly increased compared with that of naive mice (number of serotonin-positive puncta number in spinal dorsal horn: naive vs morphine group, F [4, 21] = 45.47, P = .0016; 1-way ANOVA followed by Bonferroni test; Fig. 4A, 4B, and 4F). However, co-administering OND with morphine did not reduce serotonin expression, even though it significantly suppressed mechanical hypersensitivity in OIH (P = .17; Fig. 4C and 4F).
      Figure 4
      Figure 4Serotonin levels in the spinal dorsal horn of (A) naive mice. (B) Repetitive administering of morphine significantly increased serotonin levels in the superficial layer (laminae I and outer layer of II). (C) Co-administering of OND did not influence serotonin levels, whereas (D) PCPA pretreatment enhanced serotonin levels. (E) Co-administering of PCPA and morphine strongly suppressed serotonin levels; Scale bar: 50 µm. (F) Quantification of serotonin-positive puncta in the spinal dorsal horn. Saline (control) group: n = 5; morphine group: n = 6; morphine + OND (2.0 mg/kg) group: n = 5; PCPA (150 mg/kg) for 4 days + morphine group: n = 4; PCPA (150 mg/kg) for 8 days + morphine group: n = 6, respectively. One-way ANOVA followed by Bonferroni test was used for multiple comparisons.
      PCPA was administered for 4 days, and on the day 5 complete suppression of serotonin expression was observed in the spinal dorsal horn. However, administering morphine after PCPA pretreatment dramatically increased serotonin expression in a manner similar to that seen in the mouse OIH model (morphine only vs morphine + OND, 2.0 mg/kg, P = .17; morphine only vs PCPA pretreatment + morphine, P = 1.0; morphine only vs PCPA co-administration with morphine, P < .0001; Fig 4D and 4F). These results suggested that instead of influencing serotonin expression, OND inhibited signals via the 5-HT3 receptor and suppressed mechanical hyperalgesia. In addition, morphine-induced tryptophan hydroxylase activation remained strong even after tryptophan hydroxylase was suppressed by PCPA pre-treatment (Fig 3E). By contrast, serotonin expression was significantly suppressed after tryptophan hydroxylase was inhibited by co-administering PCPA with morphine (Fig. 4E and 4F). These data suggest that serotonin expression in the spinal dorsal horn is a key factor in OIH development.

      Changes in the Phosphorylation Ratio of ERK in the Spinal Dorsal Horn

      Mitogen-activated protein kinases (MAPKs) are known to play a crucial role in pain sensation in the spinal dorsal horn. Specifically, extracellular signal-regulated kinases (ERK1: p44-MAPK and ERK2: p42-MAPK), found in the neurons of the spinal cord, are known to be phosphorylated during neuropathic pain and inflammatory pain. Therefore, we speculated that, in a manner similar to that in other animal pain models, ERKs phosphorylation in the spinal dorsal horn of mouse OIH models is increased, resulting in the proportion of phosphorylated ERKs (p-ERKs) to total ERKs expressed in the spinal dorsal horn being increased.
      Small numbers of pERK-positive nuclei were originally found in the spinal dorsal horn of naive mice (Fig 5A). pERK-positive nuclei in the superficial layer of spinal dorsal horn were increased after morphine was administered (Fig 5B). Immunohistochemical analysis indicated that both co-administering of OND and PCPA suppressed the increase in pERK-positive nuclei induced by morphine treatment (Fig. 5C and 5D). The expression of p-ERK was not co-localized with GFAP in the spinal dorsal horn (Fig. 5A–5D; inset). As Immunohistochemical analysis does not distinguish between variant forms of ERKs (ERK1 and ERK2), western blot analysis was performed to quantify the phosphorylation ratio of ERK1 and ERK2 in the spinal dorsal horn. Although the phosphorylation ratio of ERK1 was slightly elevated following treatment with morphine, it did not reach statistical significance (Fig 5F), whereas that of ERK2 was significantly increased (Fig 5G). Although co-administering of OND tended to reduce the phosphorylated ERK2/total ERK2 ratio, this reduction was not statistically significant, whereas PCPA treatment significantly decreased ERK2 phosphorylation ratio (ERK1: F [3, 28] = 1.178, P = 0.338; ERK2: F [3, 28] = 5.55, P = .004; 1-way ANOVA followed by Bonferroni test; Fig 5). Since ERK2 has been observed in neurons and astrocytes, but not in microglia of the spinal dorsal horn,
      • Otsubo Y
      • Satoh Y
      • Kodama M
      • Araki Y
      • Satomoto M
      • Sakamoto E
      • Pages G
      • Pouyssegur J
      • Endo S
      • Kazama T
      Mechanical allodynia but not thermal hyperalgesia is impaired in mice deficient for ERK2 in the central nervous system.
      these results indicate that repetitive administering of morphine enhances neuronal and astrocyte activities in the spinal dorsal horn.
      Figure 5
      Figure 5Phosphorylation of ERK2 (p42-MAPK) in the spinal dorsal horn is enhanced following repetitive administering of morphine and is reversed by serotonin deprivation via the administering of PCPA. (A–D) Representative immunohistochemistry against pERK and GFAP in the spinal dorsal horns of experimental group mice. (A) Naive group, (B) morphine group, (C) morphine + OND 2.0 mg/kg group and (D) PCPA 150 mg/kg for 8 days + morphine group. White arrows indicate the expression of p-ERK in the spinal dorsal horn. Scale bar: 50 µm in large figures and 20 µm in insets. (E) Western blot analysis. Relative phosphorylation ratios of (F) ERK1 (p44-MAPK) and (G) ERK2 (p42-MAPK). OND (2.0 mg/kg) tended to reduce the phosphorylation ratio of ERK2, although not to a significant level. Neither OND nor serotonin deprivation affected the phosphorylation ratio of ERK1. Saline (control) group: n = 11; morphine group: n = 11; morphine + OND (2.0 mg/kg) group: n = 6; PCPA (150 mg/kg) for 8 days + morphine group: n = 7, respectively.

      Changes in GFAP in the Spinal Dorsal Horn

      Following peripheral nerve injury, astrocytes in the dorsal horn of the spinal cord undergo a series of phenotypic and functional changes referred to as reactive astrogliosis.
      • Tsuda M
      • Kohro Y
      • Yano T
      • Tsujikawa T
      • Kitano J
      • Tozaki-Saitoh H
      • Koyanagi S
      • Ohdo S
      • Ji RR
      • Salter MW
      • Inoue K
      JAK-STAT3 pathway regulates spinal astrocyte proliferation and neuropathic pain maintenance in rats.
      ,
      • Zhuang ZY
      • Gerner P
      • Woolf CJ
      • Ji RR
      ERK is sequentially activated in neurons, microglia, and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model.
      During this process, naive astrocytes exhibit enlarged projections, hypertrophy of the cell body and increased GFAP expression, all of which characterize the reactive astrocyte phenotype.
      • Li T
      • Chen X
      • Zhang C
      • Zhang Y
      • Yao W
      An update on reactive astrocytes in chronic pain.
      ,
      • Ohmichi M
      • Ohmichi Y
      • Ohishi H
      • Yoshimoto T
      • Morimoto A
      • Li Y
      • Sakurai H
      • Nakano T
      • Sato J
      Activated spinal astrocytes are involved in the maintenance of chronic widespread mechanical hyperalgesia after cast immobilization.
      ,
      • Tsuda M
      • Kohro Y
      • Yano T
      • Tsujikawa T
      • Kitano J
      • Tozaki-Saitoh H
      • Koyanagi S
      • Ohdo S
      • Ji RR
      • Salter MW
      • Inoue K
      JAK-STAT3 pathway regulates spinal astrocyte proliferation and neuropathic pain maintenance in rats.
      GFAP immunoreactivity was increased in the inner layer of the spinal dorsal horn (laminae II and III) of the mouse OIH model. The shape of GFAP-positive cells became thicker, while the projections became thick and short (Fig. 6A and 6B). Meanwhile, both co-administering OND and PCPA pretreatment suppressed GFAP immunoreactivity and morphological changes in GFAP-positive astrocytes (Fig. 6C and 6D). In addition, GFAP levels in mouse OIH models were significantly increased. However, when treated concurrently with OND and PCPA for 8 days, GFAP levels were reduced when compared with those of morphine treated mice, but not significantly so (F [3, 30] = 4.82, P = 0.007; 1-way ANOVA followed by Bonferroni test; Fig. 6E and 6F). Furthermore, there was a discrepancy between morphological changes in astrocytes and changes in GFAP levels due to treatment with OND and PCPA in mouse OIH models. Morphological changes seen in activated astrocytes did not always correspond to GFAP expression levels.
      • Safavi-Abbasi S
      • Wolff JR
      • Missler M
      Rapid morphological changes in astrocytes are accompanied by redistribution but not by quantitative changes of cytoskeletal proteins.
      Thus, we investigated whether the OIH behavioral phenotype could be reversed by CBX, an inhibitor of astrocytic gap junctions.
      Figure 6
      Figure 6Glial fibrillary acidic protein (GFAP) levels in the spinal dorsal horn. (A) Immunohistochemistry of naive mice. (B) Repetitive administering of morphine strengthened GFAP immunoreactivity, and the shape of GFAP-positive astrocytes was swollen in lamina II, III, and V. (C) OND (2.0 mg/kg) co-administered with morphine suppressed the increased GFAP immunoreactivity induced by repetitive administering of morphine (D) PCPA (150 mg/kg) administered for 8 days reduced GFAP immunoreactivity, and the morphology of the astrocytes was thinner than that in mice repetitively administered morphine. (A–D) Magnified micrographs of lamina II are shown in insets. Scale bar: 50 µm in large figures and 20 µm in insets. (E) Western blot analysis and (F) GFAP quantification using western blot. Repetitive administering of morphine increased GFAP expression. In contrast to immunohistochemistry, GFAP levels induced by the administering of morphine were neither significantly suppressed by co-administering OND (2.0 mg/kg), nor deprivation of serotonin synthesis by PCPA. Saline control group: n = 9; morphine group: n = 12; morphine + OND (2.0 mg/kg) group: n = 7; PCPA (150 mg/kg) for 8 days + morphine group: n = 7, respectively.

      Effect of CBX

      Next, we investigated whether the role of reactive astrocytes in the spinal dorsal horn was crucial role for OIH development. We found no beneficial effect in the amelioration of mechanical hyperalgesia (F [2, 15] = .293, P= .75; 1-way ANOVA followed by Bonferroni test; Fig. 7A and 7B).
      Figure 7
      Figure 7Carbenoxolone (CBX) does not improve morphine-induced mechanical hyperalgesia in mice. (A) Time course of mechanical hypersensitivity from 0 hour to 2 hours after administering CBX. Symbols and colors corresponding to experimental groups shown in the graph legends are as in . Saline (control): n = 9; CBX (10 nmol): n = 5; CBX (30 nmol): n = 4, respectively. (B) Mechanical paw-withdrawal threshold 2 h after intrathecal CBX administration. Intrathecal administering of CBX does not prevent OIH development. (C) Time course of mechanical hypersensitivity from day 1 to day 5 following the administering of CBX. CBX (20 mg/kg): n = 6; morphine: n = 10; morphine + CBX: n = 10, respectively. (D) Mechanical paw-withdrawal threshold - CBX does not prevent mechanical hypersensitivity in established OIH mouse model induced by the repetitive administering of morphine.
      We then assessed whether the inhibition of astrocyte proliferation suppresses the development of OIH. Although CBX itself reduced the paw-withdrawal threshold. this reduction was not statistically significant (day 1 vs day 5; P = .582; 2-way NOVA followed by Bonferroni test; Fig 7C). Morphine-induced reduction in the paw-withdrawal threshold was not markedly prevented by the co-administering of CBX, thereby contradicting our hypothesis (F [2, 23] = 6.15, P = .007, morphine group vs morphine + CBX 20 mg/kg: P= .24 by 2-way ANOVA followed by Bonferroni test; Fig 7D).

      Discussion

      The findings of the current study revealed that co-administering 5-HT3 receptor antagonist, OND, and morphine suppressed OIH development induced by the repeated administering of morphine, and that the inhibition of serotonin-synthetic enzyme by PCPA prevented the development of morphine-induced OIH completely. In addition, substantiating the findings of other studies, we demonstrated that in the spinal dorsal horn of mouse OIH models, the proportion of phosphorylated ERK (pain-related MAP kinase) was increased; astrocytes were morphologically activated; and GFAP abundance (the cytoskeleton protein of astrocytes) was increased.
      • Mattioli TA
      • Milne B
      • Cahill CM
      Ultra-low dose naltrexone attenuates chronic morphine-induced gliosis in rats.
      ,
      • Narita M
      • Suzuki M
      • Narita M
      • Yajima Y
      • Suzuki R
      • Shioda S
      • Suzuki T
      Neuronal protein kinase C gamma-dependent proliferation and hypertrophy of spinal cord astrocytes following repeated in vivo administration of morphine.
      Furthermore, morphological changes in the astrocytes of mouse OIH-induced models tended to be suppressed after OND- and PCPA-treatment, despite a non-significant reduction in GFAP abundance. To further evaluate the contribution of astrocytic activity to behavioral changes in mice, we used CBX, an astrocytic gap junction hemichannel inhibitor that suppresses activation of astrocytes in the spinal cord. Contrary to that seen in the mouse neuropathic-pain model,
      • Chen G
      • Park CK
      • Xie RG
      • Berta T
      • Nedergaard M
      • Ji RR
      Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice.
      intrathecally administered CBX (10 and 30 nmol/5 µL) failed to suppress mechanical hypersensitivity. Moreover, the simultaneous administering of CBX (20 mg/kg) and morphine failed to suppress the development of morphine-induced OIH. Overall, these results suggested that serotonin and activation of the 5-HT3 receptor play a crucial role in OIH induced by the repetitive administering of morphine, and that the activation of astrocytes did not have a direct causal relationship with OIH.
      In addition to the direct activation of opioid receptors expressed in the sensory pathway, the descending pain modulatory pathway serves as another mechanism of opioid antinociception which comprises a noradrenergic pathway projecting from the locus coeruleus and a serotonergic pathway initiating from the rostral ventromedial medulla (RVM). Since opioids increase serotonin contents in the spinal cord, the serotonergic pathway appears to play a crucial role in opioid analgesia.
      • Kellstein DE
      • Malseed RT
      • Goldstein FJ
      Opioid-monoamine interactions in spinal antinociception: evidence for serotonin but not norepinephrine reciprocity.
      ,
      • Kimura M
      • Obata H
      • Saito S
      Peripheral nerve injury reduces analgesic effects of systemic morphine via spinal 5-hydroxytryptamine 3 receptors.
      Serotonin secretion in the spinal cord exerts an analgesic effect, and thus intrathecal injection of serotonin promotes antinociception,
      • Yaksh TL
      • Wilson PR
      Spinal serotonin terminal system mediates antinociception.
      whereas the descending pathway from the rostral depletion of serotonin reduces the antinociceptive effect of morphine.
      • Sawynok J
      • Reid A
      Lesions to ascending noradrenergic and serotonergic pathways modify antinociception produced by intracerebroventricular administration of morphine.
      By contrast, the depletion of spinal 5-HT reduces formalin-induced nociception,
      • Svensson CI
      • Tran TK
      • Fitzsimmons B
      • Yaksh TL
      • Hua XY
      Descending serotonergic facilitation of spinal ERK activation and pain behavior.
      ,
      • Wei F
      • Dubner R
      • Zou S
      • Ren K
      • Bai G
      • Wei D
      • Guo W
      Molecular depletion of descending serotonin unmasks its novel facilitatory role in the development of persistent pain.
      thereby indicating a facilitatory role. Previous studies have indicated that inhibition of 5-HT3 receptors ameliorates nociception by several stimuli
      • Green GM
      • Scarth J
      • Dickenson A
      An excitatory role for 5-HT in spinal inflammatory nociceptive transmission; state-dependent actions via dorsal horn 5-HT(3) receptors in the anaesthetized rat.
      ,
      • Greenwood-Van Meerveld B
      • Mohammadi E
      • Tyler K
      • Pietra C
      • Bee LA
      • Dickenson A
      Synergistic effect of 5-hydroxytryptamine 3 and neurokinin 1 receptor antagonism in rodent models of somatic and visceral pain.
      and hypersensitivity in animal neuropathic pain models.
      • Kimura M
      • Obata H
      • Saito S
      Peripheral nerve injury reduces analgesic effects of systemic morphine via spinal 5-hydroxytryptamine 3 receptors.
      ,
      • Patel R
      • Dickenson AH
      Modality selective roles of pro-nociceptive spinal 5-HT2A and 5-HT3 receptors in normal and neuropathic states.
      As stated above, the descending serotonergic pathway has a bidirectional role in pain sensation, while the administering of opioids facilitates serotonin secretion in the spinal cord. Our results clearly demonstrated that upregulation of serotonin contents in the spinal dorsal horn is associated with pain facilitation following chronic morphine treatment.
      A previous study has indicated that inhibition of the 5-HT3 receptor effectively suppresses hypersensitivity to mechanical stimuli in mice that were chronically administered with morphine.
      • Bannister K
      • Sikandar S
      • Bauer CS
      • Dolphin AC
      • Porreca F
      • Dickenson AH
      Pregabalin suppresses spinal neuronal hyperexcitability and visceral hypersensitivity in the absence of peripheral pathophysiology.
      ,
      • Liang DY
      • Li X
      • Clark JD
      5-hydroxytryptamine type 3 receptor modulates opioid-induced hyperalgesia and tolerance in mice.
      Using in vivo electrophysiology, Bannister et al. showed that inhibition of 5-HT3 receptor by OND (100 µM) superfusion suppressed the hyperactivity of wide dynamic range neurons in chronic morphine-treated rats. Liang et al. revealed that OND 1.0 mg/kg, systemically administered for 4 days, ameliorated mechanical hypersensitivity and tolerance induced by chronic morphine treatment. However, intraplantar administering of OND (up to 10 µg) did not affect the modulation of OIH. Furthermore, Liang et al. revealed that the systemic administering of OND prevents overexpression of pain-related genes, which encode calcitonin gene-related peptide, preprotachykinin-A, NDMA receptor subunit 1, transient receptor potential vanilloid 1 and the 5-HT3 receptor in the dorsal root ganglia. These results indicated that serotonin and the 5-HT3 receptor expressed in the dorsal root ganglia play a critical role in the development of OIH.
      However, 4 major receptors of serotonin, 5-HT1, 5-HT2, 5-HT3, and 5-HT7, are expressed in the spinal dorsal horn. Both excitation and inhibition of cells are receptor dependent as follows: inhibition; 5-HT1 and 5-HT2 receptors: excitation; 5-HT3 and 5-HT7 receptors.
      • Bardoni R
      Serotonergic modulation of nociceptive circuits in spinal cord dorsal horn.
      The physiological phenotype resulting from the elevation of serotonin in the spinal cord by morphine, may be complicated. In the current study, we depleted serotonin using PCPA pretreatment followed by co-administering with morphine, which produced the following results: 1) repetitive administering of morphine strongly induced serotonin expression in the spinal dorsal horn, as indicated by the correlation between serotonin concentrations in the spinal dorsal horn and reduced paw-withdrawal thresholds; 2) complete suppression of serotonin expression by PCPA prevented OIH development. Our findings indicate that OIH development depends on serotonin content in the spinal dorsal horn, which substantiated those of previous studies showing that the depletion of serotonin suppressed development of persistent pain.
      • Wei F
      • Dubner R
      • Zou S
      • Ren K
      • Bai G
      • Wei D
      • Guo W
      Molecular depletion of descending serotonin unmasks its novel facilitatory role in the development of persistent pain.
      Furthermore, the contribution of serotonin to nociception in OIH may be primarily mediated by 5-HT3 receptor activation, as 5-HT3 receptor inhibition improved the OIH even though spinal serotonin was upregulated by repeated administration of morphine. By contrast, inhibition of the 5-HT3 receptor did not suppress serotonin expression in the spinal dorsal horn, suggesting that its inhibition does not exert a positive feedback effect on serotonin secretion.
      In chronic pain, including OIH, phosphorylation of ERK has been observed in neurons, microglia, and astrocytes of the spinal dorsal horn.
      • Berta T
      • Liu YC
      • Xu ZZ
      • Ji RR
      Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1beta and ERK signaling in the spinal cord of mice.
      ,
      • Ji RR
      • RWt Gereau
      • Malcangio M
      • Strichartz GR
      MAP kinase and pain.
      ,
      • Sanna MD
      • Borgonetti V
      • Galeotti N
      Mu opioid receptor-triggered notch-1 activation contributes to morphine tolerance: role of neuron-glia communication.
      ,
      • Zhuang ZY
      • Gerner P
      • Woolf CJ
      • Ji RR
      ERK is sequentially activated in neurons, microglia, and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model.
      Therefore, we examined whether activated (phosphorylated) ERK in the spinal dorsal horn was increased in mouse OIH models as also whether the inhibition of 5-HT3 receptor and depletion of serotonin synthesis suppressed phosphorylation of ERK. The p-ERK/ERK ratio in our established mouse OIH model was significantly elevated, which was consistent with the results of a previous study involving mouse chronic morphine tolerance models which found that ERK phosphorylation was enhanced without an increase in ERK 1/2 protein levels.
      • Sanna MD
      • Borgonetti V
      • Galeotti N
      Mu opioid receptor-triggered notch-1 activation contributes to morphine tolerance: role of neuron-glia communication.
      Recent studies have indicated that glial activation in the spinal cord plays a key role in the exacerbation and maintenance of chronic pain,
      • Tsuda M
      • Kohro Y
      • Yano T
      • Tsujikawa T
      • Kitano J
      • Tozaki-Saitoh H
      • Koyanagi S
      • Ohdo S
      • Ji RR
      • Salter MW
      • Inoue K
      JAK-STAT3 pathway regulates spinal astrocyte proliferation and neuropathic pain maintenance in rats.
      ,
      • Xu M
      • Bruchas MR
      • Ippolito DL
      • Gendron L
      • Chavkin C
      Sciatic nerve ligation-induced proliferation of spinal cord astrocytes is mediated by kappa opioid activation of p38 mitogen-activated protein kinase.
      and that ERK phosphorylation occurs upstream of the activation of transcription factor STAT1/3, which triggers astrocyte activation.
      • Wang Z
      • Ma W
      • Chabot JG
      • Quirion R
      Calcitonin gene-related peptide as a regulator of neuronal CaMKII-CREB, microglial p38-NFkappaB and astroglial ERK-Stat1/3 cascades mediating the development of tolerance to morphine-induced analgesia.
      We found that astroglia in the spinal dorsal horn were morphologically activated, and that the GFAP abundance was increased in comparison to that in naive mice. Moreover, both OND treatment and serotonin deprivation reversed this morphological change. However, there was no significant reduction in the level of GFAP. A previous study showed that morphological changes in astrocytes occurred without any quantitative changes in cytoskeletal proteins under culture conditions.
      • Safavi-Abbasi S
      • Wolff JR
      • Missler M
      Rapid morphological changes in astrocytes are accompanied by redistribution but not by quantitative changes of cytoskeletal proteins.
      Thus, our results suggest that repetitive administering of morphine induces astrocyte activation in the spinal dorsal horn, whereas OND treatment and serotonin deprivation suppresses its activation.
      Furthermore, activation of glial cells in the spinal dorsal horn are reportedly involved in hypersensitivity to tactile stimuli caused by inflammation,
      • Qin M
      • Wang JJ
      • Cao R
      • Zhang H
      • Duan L
      • Gao B
      • Xiong YF
      • Chen LW
      • Rao ZR
      The lumbar spinal cord glial cells actively modulate subcutaneous formalin induced hyperalgesia in the rat.
      ,
      • Sweitzer SM
      • Colburn RW
      • Rutkowski M
      • DeLeo JA
      Acute peripheral inflammation induces moderate glial activation and spinal IL-1beta expression that correlates with pain behavior in the rat.
      nerve injury,
      • Guo W
      • Miyoshi K
      • Dubner R
      • Gu M
      • Li M
      • Liu J
      • Yang J
      • Zou S
      • Ren K
      • Noguchi K
      • Wei F
      Spinal 5-HT3 receptors mediate descending facilitation and contribute to behavioral hypersensitivity via a reciprocal neuron-glial signaling cascade.
      paw injury,
      • Obata H
      • Eisenach JC
      • Hussain H
      • Bynum T
      • Vincler M
      Spinal glial activation contributes to postoperative mechanical hypersensitivity in the rat.
      and repetitive opioid administration.
      • Jokinen V
      • Sidorova Y
      • Viisanen H
      • Suleymanova I
      • Tiilikainen H
      • Li Z
      • Lilius TO
      • Matlik K
      • Anttila JE
      • Airavaara M
      • Tian L
      • Rauhala PV
      • Kalso EA
      Differential spinal and supraspinal activation of glia in a rat model of morphine tolerance.
      Moreover, suppression of astrocyte activation effectively suppressed pain behavior in several pain models.
      • Obata H
      • Eisenach JC
      • Hussain H
      • Bynum T
      • Vincler M
      Spinal glial activation contributes to postoperative mechanical hypersensitivity in the rat.
      ,
      • Ohmichi M
      • Ohmichi Y
      • Ohishi H
      • Yoshimoto T
      • Morimoto A
      • Li Y
      • Sakurai H
      • Nakano T
      • Sato J
      Activated spinal astrocytes are involved in the maintenance of chronic widespread mechanical hyperalgesia after cast immobilization.
      ,
      • Tsuda M
      • Kohro Y
      • Yano T
      • Tsujikawa T
      • Kitano J
      • Tozaki-Saitoh H
      • Koyanagi S
      • Ohdo S
      • Ji RR
      • Salter MW
      • Inoue K
      JAK-STAT3 pathway regulates spinal astrocyte proliferation and neuropathic pain maintenance in rats.
      Connexin 43 (Cx43) is a hemichannel that forms gap junctions between astrocytes as well as between neurons and astrocytes, whereas CBX inhibits Cx43 and suppresses chronic pain.
      • Chen G
      • Park CK
      • Xie RG
      • Berta T
      • Nedergaard M
      • Ji RR
      Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice.
      ,
      • Falk S
      Carbenoxolone as a novel therapy for attenuation of cancer-induced bone pain.
      Thus, we hypothesized that inhibition of astrocytes using CBX may suppress OIH development. However, CBX did not suppress tactile hypersensitivity. Moreover, its chronic administering failed to suppress the development of mechanical hyperalgesia in mouse OIH models. Notably, the doses used for intrathecal and systemic administering of CBX were sufficient to suppress tactile hypersensitivity in animal models of neuropathic pain
      • Chen G
      • Park CK
      • Xie RG
      • Berta T
      • Nedergaard M
      • Ji RR
      Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice.
      and cancer pain.
      • Falk S
      Carbenoxolone as a novel therapy for attenuation of cancer-induced bone pain.
      This indicates that unlike neuropathic and cancer pain, OIH induced by chronic morphine is triggered by upregulation of serotonin in the spinal dorsal horn, and that serotonin itself induces the excitation of sensory neurons. Although astrocyte activation was observed in the spinal dorsal horn as previously reported,
      • Li T
      • Chen X
      • Zhang C
      • Zhang Y
      • Yao W
      An update on reactive astrocytes in chronic pain.
      it may be a secondary effect of chronic pain.
      In addition, descending serotonin-mediated pain facilitation at the spinal level has been demonstrated to be involved in persistent pain development following nerve injury.
      • Guo W
      • Miyoshi K
      • Dubner R
      • Gu M
      • Li M
      • Liu J
      • Yang J
      • Zou S
      • Ren K
      • Noguchi K
      • Wei F
      Spinal 5-HT3 receptors mediate descending facilitation and contribute to behavioral hypersensitivity via a reciprocal neuron-glial signaling cascade.
      ,
      • Ossipov MH
      • Morimura K
      • Porreca F
      Descending pain modulation and chronification of pain.
      Elevation of serotonin in the spinal cord also induced astrocyte activation via chemokine (fractalkine) release from neurons and facilitated the release of the inflammatory cytokine, IL-1β, from astrocytes, which causes phosphorylation of the GluN1 subunit of NMDA receptor, subsequently inducing neuronal hyperactivity in the spinal dorsal horn.
      • Guo W
      • Miyoshi K
      • Dubner R
      • Gu M
      • Li M
      • Liu J
      • Yang J
      • Zou S
      • Ren K
      • Noguchi K
      • Wei F
      Spinal 5-HT3 receptors mediate descending facilitation and contribute to behavioral hypersensitivity via a reciprocal neuron-glial signaling cascade.
      In this study, Guo et al. showed that serotonin activated fractalkine-positive neurons in the spinal dorsal horn via the 5-HT3 receptor, where a selective 5-HT3 receptor antagonist ameliorated mechanical allodynia in developed neuropathic pain.
      The current study was beset by certain limitations. First, this study focused on the histological and biochemical changes occurring in the spinal cord of mouse OIH models. However, although the descending pain facilitatory serotonin system is located in the medulla (dorsal raphe nuclei and rostroventral medulla), we did not evaluate changes in the supraspinal neuronal circuit in our model. Second, the pain-related cerebral circuit was not evaluated. An evaluation of changes in the supraspinal neuronal circuits is required for a better understanding of all mechanisms associated with OIH.
      In conclusion, our findings suggest that the descending serotonergic facilitatory pathway, which plays a crucial role in the development and maintenance of neuropathic pain, is involved in OIH development. Our study further provides new insights into therapeutic targets for OIH. Considering that OIH is associated with inappropriate opioid use during the perioperative period, its prevention and treatment may reduce opioid usage for pain management as well as its inappropriate use.

      Acknowledgments

      We would like to thank Editage (www.editage.jp) for English language editing.

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