Skip to main content
NCBI home page
Molecular Pain logoLink to Molecular Pain
. 2023 Jun 23;19:17448069231181973. doi: 10.1177/17448069231181973

Activation of neurons and satellite glial cells in the DRG produces morphine-induced hyperalgesia

Shunsuke Yamakita 1,2, Daisuke Fujita 1,2, Kazuki Sudo 1, Daiki Ishikawa 1,2, Kohsuke Kushimoto 1,2, Yasuhiko Horii 1,2, Fumimasa Amaya 2,3,✉
PMCID: PMC10291868  PMID: 37254240

Abstract

Activation of neurons and glial cells in the dorsal root ganglion is one of the key mechanisms for the development of hyperalgesia. The aim of the present study was to examine the role of neuroglial activity in the development of opioid-induced hyperalgesia. Male rats were treated with morphine daily for 3 days. The resultant phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in the dorsal root ganglion was analyzed by immunohistochemistry and Western blotting. Pain hypersensitivity was analyzed using behavioral studies. The amount of cytokine expression in the dorsal root ganglion was also analyzed. Repeated morphine treatment induced hyperalgesia and marked induction of phosphorylated ERK1/2 in the neurons and satellite glial cells on day 3. An opioid receptor antagonist, toll like receptor-4 inhibitor, MAP/ERK kinase (MEK) inhibitor and gap junction inhibitor inhibited morphine-induced hyperalgesia and ERK1/2 phosphorylation. Morphine treatment induced alteration of cytokine expression, which was inhibited by the opioid receptor antagonist, toll like receptor-4 inhibitor, MEK inhibitor and gap junction inhibitor. Dexamethasone inhibited morphine-induced hyperalgesia and ERK1/2 phosphorylation after morphine treatment. The peripherally restricted opioid receptor antagonist, methylnaltrexone, inhibited hyperalgesia and ERK1/2 phosphorylation. Morphine activates ERK1/2 in neurons and satellite glial cells in the dorsal root ganglion via the opioid receptor and toll like receptor-4. ERK1/2 phosphorylation is gap junction-dependent and is associated with the alteration of cytokine expression. Inhibition of neuroinflammation by activation of neurons and glia might be a promising target to prevent opioid-induced hyperalgesia.

Keywords: Opioid-induced hyperalgesia, morphine, satellite glial cells, dorsal root ganglion, ERK1/2

Introduction

In several clinical situations, patients who receive opioid treatment show worsening of ongoing pain, hyperalgesia and allodynia. 1 These symptoms are called opioid-induced hyperalgesia (OIH). 2 The presence of OIH has been described in postsurgical patients 3 and those with chronic pain.4,5 OIH complicates pain treatment and sometimes aggravates refractory pain syndrome. Currently, OIH is clinically inevitable in patients on opioid therapy due to lack of an effective preventive strategy.

Previous studies demonstrated the crucial role of the opioid receptor in the peripheral sensory nerve in the development of OIH.6–8 Genetic deletion of μ-opioid receptor in nociceptors resulted in a reduced degree of OIH. 8 In addition to opioid receptors, toll like receptor-4 (TLR4) has also been characterized as a target molecule of OIH. Opioids activate TLR4 signaling in vitro, 9 and TLR4 mediates opioid-induced neuroinflammation in vivo. 10 In the peripheral nerve, opioid receptors 11 and TLR412–15 are present in the neurons of the dorsal root ganglion (DRG). However, the precise behavior of DRG cells responsible for the development of OIH is largely unknown.

Extracellular signal-regulated kinase (ERK) 1/2 signaling is one of the prominent intracellular events that leads to the sensitization of sensory neurons. 16 Phosphorylated ERK1/2 (pERK1/2) expression has been detected in DRG neurons after the direct stimulation of peripheral nerves, 17 growth factor treatment18,19 and tissue inflammation. 20 We previously demonstrated that tissue injury induced rapid phosphorylation of ERK1/2 in the neurons and satellite glial cells (SGCs) of the DRG, and that coupled activation of neurons and SGCs is mediated by the gap junction. 21 SGCs produce proinflammatory cytokines that further activate nociceptor neurons to facilitate pain hypersensitivity. 22

In the present study, cellular activity of neurons and SGCs in the DRG was investigated by analyzing ERK1/2 phosphorylation during the development of morphine-induced hyperalgesia. We also investigated the roles of opioid receptors and TLR4, as well as the gap junction, in ERK1/2 phosphorylation and behavioral hyperalgesia. Cytokine expression profiles in the DRG and their association with neuronal and glial activity were also analyzed.

Method

Animals and treatments

This study was approved by the Kyoto Prefectural University of Medicine Animal Care Committee and performed according to the guidelines of the National Institutes of Health and The International Association for the Study of Pain. Sprague-Dawley rats (male, 200–250 g; Shimizu Laboratory Supplies Co. Ltd., Kyoto, Japan) were housed in cages, with three rats per cage, and kept on a 12 h light/dark cycle. Drug injections and surgeries were performed under 2% v/v isoflurane anesthesia. We determined the sample size for each experiment based on the results obtained in our previous studies.

Experiment 1: Effect of morphine treatment on behavioral hyperalgesia and pERK1/2 expression in the DRG

Rats were randomly assigned to a vehicle or a morphine-treated group (n = 5 for each). The morphine-treated group received daily intraperitoneal injection of morphine (Daiichi Sankyo, Tokyo, Japan, 20 mg/kg) for three consecutive days. The vehicle group received the same volume of saline as a control. The treatment protocol for morphine-induced hyperalgesia was adopted from a previous study with slight modification. 23 Behavioral testing was performed 1 h before the drug administration on each day. Subsequently, the left L5 DRG was removed from rats in each group 24 h after the last drug administration for immunohistochemistry and Western blotting analyses (n = 5 for each).

Experiment 2: Effects of a TLR inhibitor, opioid receptor antagonist and MAP/ERK kinase (MEK) inhibitor on the development of morphine-induced hyperalgesia

Rats were randomly assigned to vehicle, morphine, morphine with TAK-242 (TLR4 inhibitor, Merck, Darmstadt, Germany, 3 mg/kg dissolved in 20% DMSO in 0.1 M phosphate-buffered saline (PBS)), morphine with naloxone (opioid receptor antagonist, Abcam PLC, Cambridge, UK, 2 mg/kg dissolved in water to 100 mM) or morphine with U0126 (MEK inhibitor, Promega Corp., Fitchburg, WI, USA, 0.1 mg/kg dissolved in 20 μL of 10% DMSO in 0.1 M PBS) groups (n = 5 for each). TAK-242 and naloxone were injected intraperitoneally, while U0126 was administered intrathecally. The treatment protocol and drug dose for TAK0242, 24 naloxone 25 and U0126 26 were determined based on previous studies. Rats were treated with these reagents immediately after the morphine injection for three consecutive days. Morphine injection and behavioral testing were performed as described in Experiment 1. Subsequently, the left L5 DRG was removed from rats in each group 24 h after the last drug administration for immunohistochemistry, Western blotting and RNA scope assay (n = 5 for each).

Experiment 3: Effect of a gap junction inhibitor on the development of morphine-induced hyperalgesia

Rats were randomly assigned to vehicle, morphine, or morphine with carbenoxolone (gap junction inhibitor, Sigma-Aldrich, St. Louis, MO, USA, 100 mg/kg dissolved in saline) groups (n = 5 for each). Carbenoxolone was administered intraperitoneally according to the protocol described in a previous study. 21 Morphine injections and behavioral testing were performed as described in Experiment 2. Subsequently, the left L5 DRG was removed from rats in the three groups (n = 5 for each), as described in Experiment 2.

Experiment 4: Effect of morphine treatment on cytokine expression in the DRG

Rats were randomly assigned to vehicle or drug-treated groups (n = 5 for each). Drug-treated groups received morphine (20 mg/kg), morphine with TAK-242 (3 mg/kg), morphine with naloxone (2 mg/kg), morphine with carbenoxolone (100 mg/kg) or morphine with dexamethasone (Aspen Japan K.K., Tokyo, Japan, 1 mg/kg) intraperitoneally. Drug injections were performed as described in Experiment 2. One day after the last procedure, the left L5 DRG was removed and processed for multiple cytokine assay (n = 5 for each).

Experiment 5: Effect of dexamethasone on morphine-induced hyperalgesia

Rats were randomly assigned to vehicle or drug-treated groups (n = 5 for each). Drug-treated groups received morphine (20 mg/kg) or morphine with dexamethasone (1 mg/kg dissolved in saline). The dose of dexamethasone was determined according to a previous study. 27 Drug injections and behavioral testing were performed as described in Experiment 2. Subsequently, the left L5 DRG was removed from rats as described in Experiment 2 (n = 5 for each).

Experiment 6: Effect of methylnaltrexone on the development of morphine-induced hyperalgesia

Rats were randomly assigned to vehicle or drug-treated groups (n = 5 for each). Drug-treated groups received morphine (20 mg/kg) or morphine with methylnaltrexone (3 mg/kg dissolved in water). The treatment protocol of methylnaltrexone was determined according to a previous study. 25 Drug injections and behavioral testing were performed as described in Experiment 2. Subsequently, the left L5 DRG was removed from rats as described in Experiment 2 (n = 5 for each).

Behavioral assessment

All behavioral experiments were conducted by a single experimenter (Y.S.) who was blinded to the rats’ experimental conditions. Mechanical sensitivity was assessed using withdrawal thresholds to von Frey stimulation (Muromachi Kikai, Tokyo, Japan). Rats were placed in transparent plastic chambers with a metal wire grid floor within which they could move freely. After the rats adapted to the chamber, their withdrawal responses to mechanical stimulation were measured using a calibrated von Frey monofilament set. Stimulation by the von Frey filament was applied to the left hind paw; the minimum force at which a clear withdrawal response was obtained at least twice with 10 stimuli was set as the threshold. To avoid tissue damage, a cut-off value of 60 g was set prior to the experiment.

Heat sensitivity was determined as the withdrawal latency to radiant heat (Ugo Basile, 37370, Italy). Rats were allowed to adapt in transparent plastic cages placed on a glass floor. The left hind paw was then exposed to a focused subfloor radiant heat source. To avoid burns, a cut-off latency of 20 s was set prior to the experiment. Each rat was tested three times at 5 min intervals, and the mean of the three values was used as the withdrawal latency.

Immunohistochemistry

Animals were perfused transcardially with 0.9% NaCl and 10% neutralized formalin (Wako Pure Chemical Industries, Ltd., Osaka, Japan). L5 DRG tissues were immersed in 20% sucrose in 0.1 M phosphate-buffer (pH 7.4) at 4°C for 24 h, and then cut into 10-μm-thick sections using a cryostat (CM1850, Leica Microsystems, Wetzlar, Germany). Sections were incubated in Blocking One P (Nacalai Tesque Inc., Kyoto, Japan) for 30 min at room temperature prior to incubation with the primary antibody, followed by incubation with rabbit anti-pERK1/2 (1:4000, Merck Millipore, Billerica, MA, USA) in 0.1% Tween 20 in 0.1 M tris-buffered saline (TBS, pH 7.4) containing 1% blocking reagent (EMD Millipore, Billerica, MA, USA) at 4°C for 3 days. After the sections were washed with 0.1 M PBS, they were incubated with Cy3-conjugated anti-rabbit secondary antibody (1:1000, Merck Millipore) in 0.1 M TBS containing 1% blocking reagent at 4°C for 24 h.

For double-labeling immunohistochemistry, sections were incubated with rabbit anti-p-ERK1/2, and guinea pig anti-3-phosphoglycerate dehydrogenase (1:400, 3PGDH, Frontier Institute, Sapporo, Japan) or mouse anti-NeuN (1:100, Merck Millipore) in 0.1 M TBS containing 1% blocking reagent at 4°C for 3 days. The sections were then incubated with Cy3-conjugated anti-rabbit secondary antibody, and FITC-conjugated anti-guinea pig secondary antibody (1:1000, Merck Millipore) or FITC-conjugated anti-mouse secondary antibody (1:100 Merck Millipore).

The sections were visualized by a fluorescence microscope with digital camera system (Nikon, Tokyo, Japan).

Assessment of immunohistochemistry images

Immunohistochemistry images were analyzed by Image-J software (NIH, Bethesda, MD, USA) on a Macintosh computer system. We identified pERK1/2 or DAPI positive nuclei in the DRG neurons based on the nuclear size (>50 μm2) to investigate pERK1/2 expression. We confirmed in preliminary experiments that the signal selection based on these criteria could successfully identify pERK1/2/DAPI signals in the neurons, while excluding glial signals.

Four sections of the DRG, spaced at least 100 μm apart, were selected for each rat, and the number of pERK1/2 or DAPI positive neurons was counted and their fraction was calculated. The numbers of neurons counted in the present study are summarized in the supplemental file. To analyze the size-frequency distribution of pERK1/2 positive neurons, the surface area of the cell body of the neurons was analyzed. To examine pERK1/2 expression in the SGCs, we identified 3PGDH positive regions in the DRG section. Next, the profile of 3PGDH positive regions was applied to the pERK1/2 image of the same section to confirm pERK1/2 expression within the 3PGDH positive regions. We calculated the proportion of the area with both 3PGDH and pERK positivity relative to the area with only 3PGDH positivity to quantify pERK expression in glial cell. The regions of pERK1/2 or 3PGDH on four sections spaced at least 100 μm apart were measured and the fraction was calculated separately for each rat. For evaluation of the neurons, an experimenter who was blind to the study condition determined positive/negative reactions. In the case of glial cells, positive areas were determined by Image-J software with a signal intensity of >25 as the threshold.

In order to analyze the spatial relationship of pERK1/2 expression between neurons and SGCs, we chose five DRG sections from five rats in the morphine-treated group. We identified neurons surrounded by SGCs with pERK1/2 positivity and calculated the fraction of pERK1/2 positive neurons.

Western blotting

L5 DRGs were homogenized in a homogenate buffer (20 mM Tris-HCl (pH 8.0) containing 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1% TritonX-100, 1 mM PMSF, 10 mg/mL leupeptin and 10 mg/mL pepstatin A) with a protease/phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration of each homogenate was determined by the Bradford reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After separation of 50 μg of the cell lysate on 10% SDS-PAGE, the samples were transferred to PVDF membranes.

The membranes were incubated with rabbit anti-pERK1/2 (1:1000; Cell Signaling Technology Danvers, MA, USA) or rabbit anti-GAPDH (1:20000; Sigma-Aldrich, St. Louis, MO, USA) at 4°C for 24 h, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (Cytiva, Tokyo, Japan).

The membranes were visualized using an Enhanced Chemiluminescence Select Western Blotting Detection Kit (Cytiva). The intensity of the selected band was measured and analyzed using Image-J software (NIH) on a Macintosh computer.

Double-labeling RNA scope assay with immunohistochemistry

In order to characterize TLR4 expression in the DRG, double-labeling RNA scope assay with immunohistochemistry was performed to visualize TLR4 mRNA and 3PGDH or NeuN. Animals were perfused and processed to obtain L5 DRG sections, as described in the immunohistochemistry protocol. Expression of mRNA in the DRGs was evaluated using an RNA scope multiplex fluorescent reagent kit v2 (Advanced Cell Diagnostics, Inc., Newark, CA, USA). The sections were dehydrated in EtOH, followed by hydrogen peroxide incubation at room temperature for 10 min. Protease IV was added to the sections at room temperature for 30 min. Then, the sections were washed with 0.1 M PBS, and Probe Mix (RNA scope Probe-Rn-Tlr4; Advanced Cell Diagnostics, Inc.) was added at 40°C for 2 h. Next, the samples were washed with wash buffer, and Amp1 at 40°C for 30 min, Amp2 at 40°C for 30 min and Amp3 at 40°C for 15 min were added in succession. Following washing with wash buffer between each step, HRP-C1 at 40°C for 15 min, Fluorescein-C1 at 40°C for 30 min and HRP Blocker at 40°C for 15 min were added in succession. The sections were then incubated with guinea pig anti-3PGDH or mouse anti-NeuN in 0.1 M TBS containing 1% blocking reagent at 4°C for 3 days. The sections were then incubated with FITC-conjugated anti-guinea pig secondary antibody or FITC-conjugated anti-mouse secondary antibody.

The sections were visualized using a fluorescence microscope with digital camera system (Nikon).

Cytokine assay

Protein was extracted from the L5 DRGs by the same method as for Western blotting. The amounts of cytokines from the lysate were measured using a Bio-Plex Pro rat cytokine assay kit (Bio-Rad Laboratories, Inc.), which enabled the quantification of multiple biomolecules, including GM-CSF, IFN-gamma, IL-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13 and TNF-alpha. The amounts of cytokines were standardized using the protein assay of each DRG.

Statistical analysis

Statistical analysis was performed using an unpaired t test, one-way ANOVA with the Holm-Sidak multiple comparison test, Kruskal–Wallis test with Dunn’s multiple comparison test, or two-way ANOVA with the Holm-Sidak multiple comparison test on GraphPad Prism8 software (GraphPad Software, La Jolla, CA, USA). p < 0.05 was considered statistically significant. All data are presented as the mean ± SEM.

Result

Experiment 1: Effect of morphine treatment on behavioral hyperalgesia and pERK1/2 expression in the DRG

Repeated morphine treatment induced decline in the withdrawal threshold against mechanical stimulation (Figure 1(a)) and reduction of the withdrawal latency against heat stimulation (Figure 1(b)), suggesting morphine-induced hyperalgesia on days 2 and 3. On day 3, marked induction of phosphorylated ERK1/2 (pERK1/2) (Figure 1(c)) was observed in the neurons and SGC of the DRG of rats in the morphine-treated group. Most pERK1/2 positive SGCs were in the vicinity of pERK1/2 positive neurons. In five rats in the morphine-treated group, 151 neurons were identified with surrounding SGC positivity for pERK1/2. Among them, 148 neurons (98%) were themselves positive for pERK1/2 (arrow in Figure 1(c)). Distribution of pERK1/2 was observed in the nucleus of the neurons, whereas it was observed in the cytoplasm of the SGC.

Figure 1.

Figure 1.

Open in a new tab

Behavioral testing demonstrating the effect of morphine administration on noxious thresholds (a, b). The morphine group showed a significant decline in the withdrawal threshold against mechanical stimulation (a) on days 1, 2 and 3, and withdrawal latency against thermal stimulation (b) on days 2 and 3 compared to the vehicle group. n = 5 for each group. #p < 0.05, ##p < 0.01 versus the vehicle group. (c) Expression of pERK1/2 in the DRG. Weak pERK1/2 signals were detected in the DRG of the vehicle group. Significant pERK1/2 expression was detected in the DRG after morphine administration. pERK1/2 was detected in the nuclei of the neurons (arrow) and non-neuronal cells (arrowhead). Scale bar = 50 μm. (d) Double-labeling immunohistochemistry for pERK1/2 and NeuN. Neuronal pERK1/2 expression was detected in NeuN positive cells in the DRG. The fraction of pERK1/2 positive neurons increased significantly in the morphine groups. n = 5 for each group. **p < 0.01. Scale bar = 50 μm. (e) Double-labeling immunohistochemistry for pERK1/2 and 3PGDH. Non-neuronal pERK1/2 expression was detected in 3PGDH positive SGCs in the DRG. The fraction of pERK1/2 positive areas in the 3PGDH positive area increased significantly in the morphine group. n = 5 for each group. **p < 0.01. Scale bar = 50 μm. (f) Western blotting of pERK1/2 expression in the DRG. The amount of pERK1/2 in the DRG increased significantly in the morphine group. n = 5 for each group. *p < 0.05. (g) Size-frequency distribution of pERK1/2 in the DRG. pERK1/2 expression was detected in small to large neurons. pERK1/2: phosphorylated ERK1/2; DRG: dorsal root ganglion; 3PGDH: 3-phosphoglycerate dehydrogenase; V: vehicle; M: morphine.

pERK1/2 expression was detected within NeuN positive cells, indicating their expression in neurons (Figure 1(d)). The ratio of neuronal nuclei positive for pERK1/2 expression was significantly increased in the morphine-treated group. pERK1/2 was also detected within 3PGDH positive cells, indicating their expression in SGCs (Figure 1(e)). The ratio of the pERK1/2 positive area within the 3PGDH positive area was significantly increased in the morphine group. Western blotting showed a significant increase in the amount of pERK1/2 expression in the DRG in the morphine group (Figure 1(f)). The size-frequency distribution of pERK1/2 is shown in Figure 1(g) pERK1/2 expression was detected in both large neurons and small to medium-sized neurons.

Our preliminary study showed similar pERK1/2 induction in L1-4 DRGs as well.

Experiment 2: Effects of the TLR inhibitor, opioid receptor antagonist and MEK inhibitor on the development of morphine-induced hyperalgesia

To identify the molecular target of morphine-induced hyperalgesia, TAK-242, naloxone and U0126 were used to inhibit TLR4, opioid receptors and MEK1/2 pathways, respectively. Behavioral analysis demonstrated that morphine-induced mechanical (Figure 2(a)) and thermal (Figure 2(b)) hyperalgesia were inhibited in the morphine with TLR4 inhibitor group, morphine with naloxone group and morphine with MEK inhibitor group. Western blotting analysis demonstrated significantly increased pERK1/2 levels in the morphine group. Phosphorylation of ERK1/2 was significantly inhibited in the morphine with TLR4 inhibitor, morphine with naloxone and morphine with MEK inhibitor groups (Figure 2(c)). Quantitative immunohistochemical analysis demonstrated a significant increase in the number of pERK1/2 positive neurons and pERK1/2 signal amount within 3PGDH positive cells in the morphine group. Further, the TLR4 inhibitor, naloxone and MEK inhibitor inhibited morphine-induced neuronal and glial ERK1/2 phosphorylation (Figure 3(a)). Additionally, the expression of TLR4 mRNA in the DRG was detected in NeuN positive DRG neurons, but not in 3PGDH positive non-neuronal cells (Figure 3(b)).

Figure 2.

Figure 2.

Open in a new tab

Behavioral testing demonstrating the effects of the drugs (a, b). The morphine group showed a significant decline in the withdrawal threshold against mechanical stimulation (a) on days 1, 2 and 3, and withdrawal latency against thermal stimulation (b) on days 2 and 3 compared to the vehicle group. Decline of the withdrawal threshold and withdrawal latency was significantly inhibited in the morphine with TAK-242, morphine with naloxone and morphine with U0126 groups compared with the morphine group. n = 5 for each group. #p < 0.05, ##p < 0.01 versus the vehicle group. *p < 0.05, **p < 0.01 versus the morphine group. (c) Western blotting of pERK1/2 expression in the DRG. The amount of pERK1/2 in the DRG, measured by Western blotting, increased significantly in the morphine group. There was a significant decrease in the amount of pERK1/2 in the morphine with TAK-242, morphine with naloxone and morphine with U0126 groups compared to the morphine group. n = 5 for each group. ##p < 0.01 versus the vehicle group. *p < 0.05, **p < 0.01. V: vehicle; M: morphine; T: TAK-242; N: Naloxone; U: U0126.

Figure 3.

Figure 3.

Open in a new tab

(a) pERK1/2 expression in the neurons and SGCs of the DRG. The fraction of pERK1/2 positive neurons increased significantly in the morphine, morphine with TAK-242 and morphine with naloxone groups compared to the vehicle group. Induction of pERK1/2 in the neurons was significantly inhibited in the morphine with TAK-242, morphine with naloxone and morphine with U0126 groups compared to the morphine group. The fraction of pERK1/2 positive areas in the 3PGDH positive area increased significantly in the morphine, morphine with TAK-242 and morphine with naloxone groups compared to the vehicle group. Induction of pERK1/2 in 3PGDH positive areas was significantly inhibited in the morphine with TAK-242, morphine with naloxone and morphine with U0126 groups compared to the morphine group. n = 5 for each group. #p < 0.05, ##p < 0.01 versus the vehicle group. **p < 0.01. Scale bar = 50 μm. (b) Double-labeling immunohistochemistry for TLR4 mRNA and 3PGDH or NeuN in the DRG. TLR4 mRNA was detected in NeuN positive DRG neurons, but not in 3PGDH positive non-neuronal cells. Scale bar = 50 μm.

Experiment 3: Effect of a gap junction inhibitor on the development of morphine-induced hyperalgesia

Next, we investigated the role of a neuron-glia interaction on the development of morphine-induced hyperalgesia. Carbenoxolone, a selective gap junction inhibitor, was used to test the involvement of signal communication between neurons and glial cells in the development of morphine-induced hyperalgesia. Mechanical (Figure 4(a)) and thermal (Figure 4(b)) hyperalgesia induced by morphine treatment was significantly inhibited in the morphine with carbenoxolone group. Western blotting showed a significant decrease in the pERK1/2 amount in the morphine with carbenoxolone group compared to the morphine group (Figure 4(c)). Immunohistochemistry demonstrated that the increases in the number of pERK1/2 positive neurons in the DRG and pERK1/2 signal amount within 3PGDH positive cells were significantly inhibited in the morphine with carbenoxolone group (Figure 4(d)). These results suggest that gap junction signaling plays a crucial role in the development of hyperalgesia and pERK1/2 expression after morphine treatment.

Figure 4.

Figure 4.

Open in a new tab

Behavioral testing demonstrating the effect of carbenoxolone (a, b). The morphine group showed a significant decline in the withdrawal threshold against mechanical stimulation (a) on days 1, 2 and 3, and withdrawal latency against thermal stimulation (b) on days 2 and 3 compared to the vehicle group. Decline in the withdrawal threshold and withdrawal latency was significantly inhibited in the morphine with carbenoxolone group compared to the morphine group. n = 5 for each group. #p < 0.05, ##p < 0.01 versus the vehicle group. *p < 0.05, **p < 0.01 versus the morphine group. (c) Western blotting of pERK1/2 expression in the DRG. The amount of pERK1/2 in the DRG measured by Western blotting increased significantly in the morphine group. There was a significant decrease in the amount of pERK1/2 in the morphine with carbenoxolone group compared to the morphine group. n = 5 for each group. ##p < 0.01 versus the vehicle group. **p < 0.01. (d) pERK1/2 expression in the neurons and SGCs of the DRG. The fraction of pERK1/2 positive neurons increased significantly in the morphine and morphine with carbenoxolone groups compared to the vehicle group. Induction of pERK1/2 in the neurons was significantly inhibited in the morphine with carbenoxolone group compared to the morphine group. The fraction of the pERK1/2 positive area in the 3PGDH positive area increased significantly in the morphine and morphine with carbenoxolone groups compared to the vehicle group. Induction of pERK1/2 in the 3PGDH positive area was significantly inhibited in the morphine with carbenoxolone group compared to the morphine group. n = 5 for each group. #p < 0.05, ##p < 0.01 versus the vehicle group. **p < 0.01. Scale bar = 50 μm. CBX: Carbenoxolone; V: vehicle; M: morphine; C: Carbenoxolone.

Experiment 4: Effect of morphine treatment on cytokine expression in the DRG

Figure 5 demonstrates the results of multiplex cytokine analysis of the DRG samples. There were significant inductions of GM-CSF, IL-1alpha, IL-2, IL-4, IL-5, IL-6, IL-12, IL-13 and TNF-alpha in the DRGs of the morphine group. TAK-242, naloxone and carbenoxolone successfully inhibited morphine-induced cytokine production in the morphine with TAK-242, morphine with naloxone and morphine with carbenoxolone groups, respectively. Dexamethasone also inhibited the production of inflammatory cytokines resulting from morphine treatment.

Figure 5.

Figure 5.

Open in a new tab

Multiplex cytokine assay. GM-CSF, IL-1alpha, IL-2, IL-4, IL-5, IL-6, IL-12, IL-13 and TNF-alpha in the DRG increased significantly in the morphine group compared to the vehicle group. Induction of cytokines were significantly inhibited in the morphine with TAK-242, morphine with naloxone, morphine with carbenoxolone and morphine with dexamethasone groups compared to the morphine group. n = 5 for each group. #p < 0.05, ##p < 0.01 versus the vehicle group. *p < 0.05, **p < 0.01. V: vehicle; M: morphine; T: TAK-242; N: naloxone; C: carbenoxolone; D: dexamethasone; GM-CSF: granulocyte-macrophage - colony-stimulating factor; IFN-g: interferon-gamma; IL: interleukin; TNF-alpha: tumor necrosis factor-alpha.

Experiment 5: Effect of dexamethasone on morphine-induced hyperalgesia

Since dexamethasone successfully inhibited morphine-induced production of inflammatory cytokines, we investigated the effect of dexamethasone on behavioral hyperalgesia and ERK1/2 phosphorylation in the DRG after morphine treatment. Dexamethasone successfully inhibited the mechanical (Figure 6(a)) and thermal (Figure 6(b)) hyperalgesia resulting from morphine treatment. Western blotting showed that the morphine-induced increase of ERK1/2 phosphorylation in the DRG (Figure 6(c)) was inhibited in the morphine with dexamethasone group. Immunohistochemical analysis demonstrated that the morphine-induced pERK1/2 expression in the neurons and glia of the DRG was inhibited in the morphine with dexamethasone group (Figure 6(d)).

Figure 6.

Figure 6.

Open in a new tab

Behavioral testing demonstrating the effect of dexamethasone (a, b). The morphine group showed a significant decline in the withdrawal threshold against mechanical stimulation (a) on days 1, 2 and 3, and withdrawal latency against thermal stimulation (b) on days 2 and 3 compared to the vehicle group. Decline of the withdrawal threshold and withdrawal latency was significantly inhibited in the morphine with dexamethasone group compared to the morphine group. n = 5 for each group. ##p < 0.01 versus the vehicle group. *p < 0.05, **p < 0.01 versus the morphine group. (c) Western blotting of pERK1/2 expression in the DRG. The amount of pERK1/2 in the DRG measured by Western blotting increased significantly in the morphine group. There was a significant decrease in the amount of pERK1/2 in the morphine with dexamethasone group compared to the morphine group. n = 5 for each group. ##p < 0.01 versus the vehicle group. *p < 0.05. (d) pERK1/2 expression in the neurons and SGCs of the DRG. The fraction of pERK1/2 positive neurons increased significantly in the morphine and morphine with dexamethasone groups compared to the vehicle group. Induction of pERK1/2 in the neurons was significantly inhibited in the morphine with dexamethasone group compared to the morphine group. The fraction of pERK1/2 positive area in the 3PGDH positive area increased significantly in the morphine and morphine with dexamethasone groups compared to the vehicle group. Induction of pERK1/2 in the 3PGDH positive area was significantly inhibited in the morphine with dexamethasone group compared to the morphine group. n = 5 for each group. ##p < 0.01 versus the vehicle group. **p < 0.01. Scale bar = 50 μm. V: vehicle; M: morphine; D: Dexamethasone.

Experiment 6: Effect of methylnaltrexone on the development of morphine-induced hyperalgesia

We investigated the effect of methylnaltrexone on behavioral hyperalgesia and ERK1/2 phosphorylation in the DRG after morphine treatment. Methylnaltrexone inhibited the mechanical (Figure 7(a)) and thermal (Figure 7(b)) hyperalgesia resulting from morphine treatment. Western blotting showed that the morphine-induced increase of ERK1/2 phosphorylation in the DRG (Figure 7(c)) was inhibited in the morphine with methylnaltrexone group. Immunohistochemical analysis demonstrated that the morphine-induced pERK1/2 expression in the neurons and glia of the DRG was inhibited in the morphine with methylnaltrexone group (Figure 7(d)).

Figure 7.

Figure 7.

Open in a new tab

Behavioral testing demonstrating the effect of methylnaltrexone (a, b). The morphine group showed a significant decline in the withdrawal threshold against mechanical stimulation (a) on days 2 and 3, and withdrawal latency against thermal stimulation (b) on day 3 compared to the vehicle group. Decline in the withdrawal threshold and withdrawal latency was significantly inhibited in the morphine with methylnaltrexone group compared to the morphine group. n = 5 for each group. ##p < 0.01 versus the vehicle group. *p < 0.05, **p < 0.01 versus the morphine group. (c) Western blotting of pERK1/2 expression in the DRG. The amount of pERK1/2 in the DRG measured by Western blotting increased significantly in the morphine group. There was a significant decrease in the amount of pERK1/2 in the morphine with methylnaltrexone group compared to the morphine group. n = 5 for each group. #p < 0.05 versus the vehicle group. *p < 0.05. (d) pERK1/2 expression in the neurons and SGCs of the DRG. The fraction of pERK1/2 positive neurons increased significantly in the morphine group compared to the vehicle group. Induction of pERK1/2 in the neurons was significantly inhibited in the morphine with methylnaltrexone group compared to the morphine group. The fraction of the pERK1/2 positive area in the 3PGDH positive area increased significantly in the morphine group compared to the vehicle group. Induction of pERK1/2 in the 3PGDH positive area was significantly inhibited in the morphine with methylnaltrexone group compared to the morphine group. n = 5 for each group. ##p < 0.01 versus the vehicle group. **p < 0.01. Scale bar = 50 μm. V: vehicle; M: morphine; MNX: Methylnaltrexone.

Discussion

In this study, we explored the molecular mechanism of OIH induced by acute morphine treatment. Morphine treatment induced ERK1/2 phosphorylation in neurons and SGCs of the DRG. The gap junction inhibitor inhibited morphine-induced hyperalgesia, as well as phosphorylation of ERK1/2 and cytokine expression. These lines of evidence indicate that activation of neurons and SGCs and subsequent neuroinflammation in the DRG play a crucial role in morphine-induced hyperalgesia.

The ERK1/2 cascade is one of the central signaling pathways that regulates many neuronal and non-neuronal cellular activities, including nociceptive processing. 16 Intrathecal administration of an MEK inhibitor and methylnaltrexone, a peripherally restricted opioid receptor antagonist, successfully inhibited both pERK1/2 phosphorylation in the DRG and behavioral hyperalgesia, suggesting that activation of ERK1/2 signaling in the DRG is likely to play a crucial role in the development of hyperalgesia after morphine treatment. Various functions of ERK1/2 in the modulation of the nociceptive system have been identified, including transcriptional and post-transcriptional regulation. 16 In this study, most of the neuronal pERK1/2 was detected in the nucleus, whereas pERK1/2 in the SGC was observed in the cytoplasm, suggesting different downstream effectors between neurons and SGCs.

Previous investigations testing the involvement of opioid receptors or TLR4 in the pathophysiology of OIH showed conflicting results. 28 Opioid receptors and TLR4 agonists are capable of inducing phosphorylation of ERK1/2.12,29 In this study, we tested their relative contributions to the development of hyperalgesia after morphine treatment. Opioid receptor and TLR4 antagonists individually inhibited the development of morphine-induced hyperalgesia. Consistently, inhibition of opioid receptors or TLR4 signals inhibited morphine-induced ERK1/2 phosphorylation. These results demonstrated that both opioid receptors and TLR4 are required for morphine-induced activation of DRG cells and the development of behavioral hyperalgesia. Previous studies suggested that there is functional crosstalk between opioid receptors and TLR4 in neurons and immune cells. 30 Other previous studies suggested an intracellular interaction between g-protein coupled receptor and TLRs by signaling molecules such as regulator of G-protein signaling (RGS) in immune cells.31,32 A similar interaction in the DRG neurons might contribute to the development of OIH. Further analysis to determine the interaction between opioid receptors and TLR4 might help to explain the mechanisms of morphine-induced hyperalgesia in greater detail.

Opioid receptors 11 and TLR412–15 exist exclusively in small DRG neurons. We confirmed the expression of TLR4 in DRG neurons using RNA scope in situ hybridization. In contrast, ERK1/2 phosphorylation was observed in SGCs or neurons with large cell bodies. This suggests that ERK1/2 is probably phosphorylated in opioid receptors or TLR4 deficient cells. On the other hand, inhibition of these receptors blocked ERK1/2 phosphorylation, as well as signals arising from TLR4 or opioid receptor expressing neurons that are required for the activation of ERK1/2 in the SGCs. Carbenoxolone, a gap junction inhibitor, inhibited ERK1/2 phosphorylation in the DRG and alleviated behavioral hyperalgesia. The gap junction in the DRG has been shown to facilitate activation of DRG neurons and SGCs in several pain models.33–36 Taken together, the evidence suggests that SGCs might receive direct signals from activated neurons possibly through gap junctions.

Mounting evidence has shown that inflammatory cytokines modulate neuronal function and lead to the development of pain hypersensitivity.37–40 Activated SGCs are capable of synthesizing inflammatory cytokines and releasing them in a paracrine manner.41–43 In this study, most of the pERK1/2 positive neurons were surrounded by pERK1/2 expressing SGCs, suggesting a special relationship between neurons and SGCs. Previous studies showed that opioids induce activation of astrocytes 44 or macrophages 45 that leads to the production of proinflammatory cytokines 46 and chemokines 47 in the central nervous system. Compared to the central nervous system, less is known about the neuroinflammatory response in the peripheral nervous system. In the DRG, morphine activates the production of IL-1β 48 and SDF1. 49 Our study demonstrated that morphine alters cytokine expression profiles in the DRG, including activation of several proinflammatory (IL-1α, TNF-α) cytokines. Moreover, inhibition of the gap junction successfully inhibited cytokine production, suggesting the involvement of neuroglial communication. Dexamethasone, one of the synthetic glucocorticoids widely used to reduce the immune reaction in clinical settings, inhibited ERK1/2 phosphorylation and the development of morphine-induced hyperalgesia. These results demonstrated that cytokines in the DRG contribute to the development of morphine-induced hyperalgesia.

Methylnaltrexone does not cross the blood–brain barrier due to its high polarity and low lipid solubility. 50 In our study, methylnaltrexone prevented morphine-induced hyperalgesia without affecting its antinociceptive effect. This is inconsistent with the observation of the genetic deletion of μ-opioid receptors in DRG nociceptors. 8 Methylnaltrexone is widely used to treat opioid-induced constipation in clinical settings. 51 The ability of this drug to inhibit OIH should be tested in future clinical studies.

Our study has several limitations that should be noted. Intrathecal drug injection, as used in our protocol, has been shown to selectively deliver the drug to the L5 DRG, 52 although we cannot totally exclude the possibility that the MEK inhibitor might have affected the spinal cord and/or central nervous system.53,54 Similarly, systemic treatment with carbenoxolone, dexamethasone and TLR4 antagonists showed inhibitory effects on DRG cells. However, their effect on the central nervous system cannot be totally denied. Additionally, TAK242, naloxone, U0126, carbenoxolone, dexamethasone and methylnaltrexone were dissolved in different solutions (10–20% DMSO in 0.1 M PBS, saline and water). Although our preliminary study showed that these solutions do not have any effect on neuronal function, we cannot completely exclude a possible effect, since we did not assess a control group receiving only the vehicle. Finally, in this study, we did not examine the effect of the MEK inhibitor and methylnaltrexone on cytokine expression in the DRG.

In conclusion, morphine induces activation of ERK1/2 signaling in neurons and SGCs that leads to neuroinflammation in the DRG. Activation of neuroglial components and neuroinflammation in the DRG might be a promising target for preventing OIH.

Supplemental Material

Supplemental Material - Activation of neurons and satellite glial cells in the DRG produces morphine-induced hyperalgesia
Click here for additional data file. (233.6KB, pdf)

Supplemental Material for Activation of neurons and satellite glial cells in the DRG produces morphine-induced hyperalgesia by Shunsuke Yamakita, Daisuke Fujita, Kazuki Sudo, Daiki Ishikawa, Kousuke Kushimoto, Yasuhiko Horii and Fumimasa Amaya in Molecular Pain.

Author contributions: SY designed the study protocol, conducted the experiments and wrote the article. DF, KS, DI, KK, and YH conducted the experiments. FA designed the study protocol and wrote the article.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: SY was supported by Grants-in-Aid for Scientific Research from the Japan Society for Promotion of Science (19K18250 and 22K16593). FA was supported by Grants-in-Aid for Scientific Research from the Japan Society for Promotion of Science (20K21635 and 21H03026). YH was supported by a Grant-in-Aid for Scientific Research from the Japan Society for Promotion of Science (22K16614).

Supplemental Material: Supplemental material for this article is available online.

ORCID iD

Fumimasa Amaya https://orcid.org/0000-0003-2934-0845

References

  • 1.Angst MS, Clark JD. Opioid-induced Hyperalgesia. Anesthesiol 2006; 104: 570–587. DOI: 10.1097/00000542-200603000-00025 [DOI] [PubMed] [Google Scholar]
  • 2.Velayudhan A, Bellingham G, Morley-Forster P. Opioid-induced hyperalgesia. Cont Educ Anaesth Crit Care & Pain 2014; 14: 125–129. DOI: 10.1093/bjaceaccp/mkt045 [DOI] [Google Scholar]
  • 3.Fletcher D, Martinez V. Opioid-induced hyperalgesia in patients after surgery: a systematic review and a meta-analysis. British J Anaesth 2014; 112: 991–1004. DOI: 10.1093/bja/aeu137 [DOI] [PubMed] [Google Scholar]
  • 4.Chu LF, Clark DJ, Angst MS. Opioid tolerance and hyperalgesia in chronic pain patients after one month of oral morphine therapy: a preliminary prospective study. J Pain 2006; 7: 43–48. DOI: 10.1016/j.jpain.2005.08.001 [DOI] [PubMed] [Google Scholar]
  • 5.Crofford LJ. Adverse effects of chronic opioid therapy for chronic musculoskeletal pain. Nature Rev Rheumatol 2010; 6: 191–197. DOI: 10.1038/nrrheum.2010.24 [DOI] [PubMed] [Google Scholar]
  • 6.Zhou HY, Chen SR, Chen H, Pan HL. Opioid-induced long-term potentiation in the spinal cord is a presynaptic event. J Neurosci 2010; 30: 4460–4466. DOI: 10.1523/JNEUROSCI.5857-09.2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Sun J, Chen SR, Chen H, Pan HL. μ‐Opioid receptors in primary sensory neurons are essential for opioid analgesic effect on acute and inflammatory pain and opioid‐induced hyperalgesia. J Physiol 2019; 597: 1661–1675. DOI: 10.1113/JP277428 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Corder G, Tawfik VL, Wang D, Sypek EI, Low SA, Dickinson JR, Sotoudeh C, Clark JD, Barres BA, Bohlen CJ, Scherrer G. Loss of μ opioid receptor signaling in nociceptors, but not microglia, abrogates morphine tolerance without disrupting analgesia. Nat Med 2017; 23: 164–173. DOI: 10.1038/nm.4262 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Hutchinson MR, Zhang Y, Shridhar M, Evans JH, Buchanan MM, Zhao TX, Slivka PF, Coats BD, Rezvani N, Wieseler J, Hughes TS, Landgraf KE, Chan S, Fong S, Phipps S, Falke JJ, Leinwand LA, Maier SF, Yin H, Rice KC, Watkins LR. Evidence that opioids may have toll-like receptor 4 and MD-2 effects. Brain, Behavior, and Immunity 2010; 24: 83–95. DOI: 10.1016/j.bbi.2009.08.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Bai L, Zhai C, Han K, Li Z, Qian J, Jing Y, Zhang W, Xu J-T. Toll-like receptor 4-mediated nuclear factor-κB activation in spinal cord contributes to chronic morphine-induced analgesic tolerance and hyperalgesia in rats. Neurosci Bull 2014; 30: 936–948. DOI: 10.1007/s12264-014-1483-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ma W, Sapio MR, Manalo AP, Maric D, Dougherty MK, Goto T, Mannes AJ, Iadarola MJ. Anatomical analysis of transient potential vanilloid receptor 1 (Trpv1+) and Mu-opioid receptor (Oprm1+) Co-expression in rat dorsal root ganglion neurons. Front Molec Neurosci 2022; 15: 926596–20220707. DOI: 10.3389/fnmol.2022.926596 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Tse KH, Chow KBS, Leung WK, Wong YH, Wise H. Lipopolysaccharide differentially modulates expression of cytokines and cyclooxygenases in dorsal root ganglion cells via Toll-like receptor-4 dependent pathways. Neurosci 2014; 267: 241–251, DOI: 10.1016/j.neuroscience.2014.02.041 [DOI] [PubMed] [Google Scholar]
  • 13.Chen T, Li H, Yin Y, Zhang Y, Liu Z, Liu H. Interactions of Notch1 and TLR4 signaling pathways in DRG neurons of in vivo and in vitro models of diabetic neuropathy. Scienti Rep 2017; 7: 14923. DOI: 10.1038/s41598-017-15053-w [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Wu Y, Wang Y, Wang J, Fan Q, Zhu J, Yang L, Rong W. TLR4 mediates upregulation and sensitization of TRPV1 in primary afferent neurons in 2,4,6-trinitrobenzene sulfate-induced colitis. Molecular Pain 2019; 15: 174480691983001. DOI: 10.1177/1744806919830018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhang J, Harada Y, Hayashi Y. A TLR-CXCL1 pathway in DRG neurons induces neutrophil accumulation in the DRG and mechanical allodynia in EAE mice. Scientific Reports 2019; 9: 12003. DOI: 10.1038/s41598-019-48558-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ji RR, RWt Gereau, Malcangio M, Strichartz GR. MAP kinase and pain. Brain Res Rev 2008; 60(1). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Dai Y, Iwata K, Fukuoka T, Kondo E, Tokunaga A, Yamanaka H, Tachibana T, Liu Y, Noguchi K. Phosphorylation of extracellular signal-regulated kinase in primary afferent neurons by noxious stimuli and its involvement in peripheral sensitization. J Neurosci 2002; 22: 7737–7745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Andres C, Hasenauer J, Ahn HS, Joseph EK, Isensee J, Theis FJ, Allgöwer F, Levine JD, Dib-Hajj SD, Waxman SG, Hucho T. Wound-healing growth factor, basic FGF, induces Erk1/2-dependent mechanical hyperalgesia. Pain 2013; 154: 2216–2226. [DOI] [PubMed] [Google Scholar]
  • 19.Delcroix JD, Valletta JS, Wu C, Hunt SJ, Kowal AS, Mobley WC. NGF signaling in sensory neurons. Neuron 2003; 39: 69–84. [DOI] [PubMed] [Google Scholar]
  • 20.Yang F, Sun W, Yang Y, Wang Y, Li CL, Fu H, Wang XL, Yang F, He T, Chen J. SDF1-CXCR4 signaling contributes to persistent pain and hypersensitivity via regulating excitability of primary nociceptive neurons: involvement of ERK-dependent Nav1.8 up-regulation. J Neuroinfl 2015; 12: 219. DOI: 10.1186/s12974-015-0441-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Yamakita S, Horii Y, Takemura H, Matsuoka Y, Yamashita A, Yamaguchi Y, Matsuda M, Sawa T, Amaya F. Synergistic activation of ERK1/2 between A-fiber neurons and glial cells in the DRG contributes to pain hypersensitivity after tissue injury. Molecular Pain 2018; 14, 1744806918767508. DOI: 10.1177/1744806918767508 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Hanani M, Spray DC. Emerging importance of satellite glia in nervous system function and dysfunction. Nat Rev Neurosci 2020; 21: 485–498. DOI: 10.1038/s41583-020-0333-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Roeckel LA, Utard V, Reiss D, Mouheiche J, Maurin H, Robé A, Audouard E, Wood JN, Goumon Y, Simonin F, Gaveriaux-Ruff C. Morphine-induced hyperalgesia involves mu opioid receptors and the metabolite morphine-3-glucuronide. Scienti Rep 2017; 7: 10406. DOI: 10.1038/s41598-017-11120-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Yamada M, Ichikawa T, Ii M, Sunamoto M, Itoh K, Tamura N, Kitazaki T. Discovery of novel and potent small-molecule inhibitors of NO and cytokine production as antisepsis agents: synthesis and biological activity of alkyl 6-(N-substituted sulfamoyl)cyclohex-1-ene-1-carboxylate. J Med Chem 2005; 48: 7457–7467. DOI: 10.1021/jm050623t [DOI] [PubMed] [Google Scholar]
  • 25.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. Neurosci 2020; 446: 28–42. DOI: 10.1016/j.neuroscience.2020.08.014 [DOI] [PubMed] [Google Scholar]
  • 26.Nag S, Mokha SS. Activation of a Gq-coupled membrane estrogen receptor rapidly attenuates α2-adrenoceptor-induced antinociception via an ERK I/II-dependent, non-genomic mechanism in the female rat. Neurosci 2014; 267: 122–134. DOI: 10.1016/j.neuroscience.2014.02.040 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Roka R, Ait-Belgnaoui A, Salvador-Cartier C, Garcia-Villar R, Fioramonti J, Eutamene H, Bueno L. Dexamethasone prevents visceral hyperalgesia but not colonic permeability increase induced by luminal protease-activated receptor-2 agonist in rats. Gut 2007; 56: 1072–1078. DOI: 10.1136/gut.2006.115352 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Roeckel LA, Le Coz GM, Gavériaux-Ruff C, Simonin F. Opioid-induced hyperalgesia: cellular and molecular mechanisms. Neurosci 2016; 338: 160–182. DOI: 10.1016/j.neuroscience.2016.06.029 [DOI] [PubMed] [Google Scholar]
  • 29.Zheng H, Loh HH, Law P-Y. β-Arrestin-Dependent μ-Opioid Receptor-Activated Extracellular Signal-Regulated Kinases (ERKs) Translocate to Nucleus in Contrast to G Protein-Dependent ERK Activation. Mole Pharmacol 2008; 73: 178–190. DOI: 10.1124/mol.107.039842 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Zhang P, Yang M, Chen C, Liu L, Wei X, Zeng S. Toll-Like Receptor 4 (TLR4)/Opioid receptor pathway crosstalk and impact on opioid analgesia, immune function, and gastrointestinal motility. Front Immunol 2020; 11: 1455. DOI: 10.3389/fimmu.2020.01455 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Shi GX, Harrison K, Han SB, Moratz C, Kehrl JH. Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling. J Immunol 2004; 172: 5175–5184. DOI: 10.4049/jimmunol.172.9.5175 [DOI] [PubMed] [Google Scholar]
  • 32.Lattin J, Zidar DA, Schroder K, Kellie S, Hume DA, Sweet MJ. G-protein-coupled receptor expression, function, and signaling in macrophages. J Leuk Biol 2007; 82: 16–32. DOI: 10.1189/jlb.0107051.20070424 [DOI] [PubMed] [Google Scholar]
  • 33.Ledda M, Blum E, De Palo S, Hanani M. Augmentation in gap junction-mediated cell coupling in dorsal root ganglia following sciatic nerve neuritis in the mouse. Neurosci 2009; 164: 1538–1545. DOI: 10.1016/j.neuroscience.2009.09.038 [DOI] [PubMed] [Google Scholar]
  • 34.Warwick RA, Hanani M. The contribution of satellite glial cells to chemotherapy-induced neuropathic pain. Eur J Pain 2013; 17: 571–580. DOI: 10.1002/j.1532-2149.2012.00219.x [DOI] [PubMed] [Google Scholar]
  • 35.Kim YS, Anderson M, Park K, Zheng Q, Agarwal A, Gong C, Saijilafu Young L, Young S, He PC, LaVinka F, Zhou D, Bergles M, Hanani Y, Guan DC, Spray X, Dong X.Coupled activation of primary sensory neurons contributes to chronic pain. Neuron 2016; 91: 1085–1096. DOI: 10.1016/j.neuron.2016.07.044 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Feldman-Goriachnik R, Belzer V, Hanani M. Systemic inflammation activates satellite glial cells in the mouse nodose ganglion and alters their functions. Glia 2015; 63: 2121–2132. DOI: 10.1002/glia.22881 [DOI] [PubMed] [Google Scholar]
  • 37.Andratsch M, Mair N, Constantin CE, Scherbakov N, Benetti C, Quarta S, Vogl C, Sailer CA, Üceyler N, Brockhaus J, Martini R, Sommer C, Ulrich Zeilhofer HU, Müller W, Kuner R, Davis JB, Rose-John S, Kress M. A key role for gp130 expressed on peripheral sensory nerves in pathological pain. The Journal of Neuroscience 2009; 29: 13473–13483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Binshtok AM, Wang H, Zimmermann K, Amaya F, Vardeh D, Shi L, Brenner GJ, Ji RR, Bean BP, Woolf CJ, Samad TA. Nociceptors are Interleukin-1β Sensors. J Neurosci 2008; 28: 14062–14073. DOI: 10.1523/JNEUROSCI.3795-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Cunha TM, Verri WA, Silva JS, Poole S, Cunha FQ, Ferreira SH. A cascade of cytokines mediates mechanical inflammatory hypernociception in mice. Proc Nati Acad Sci 2005; 102: 1755–1760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Gold MS, Gebhart GF. Nociceptor sensitization in pain pathogenesis. Nat Med 2010; 16: 1248–1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Takeda M, Tanimoto T, Kadoi J, Nasu M, Takahashi M, Kitagawa J, Matsumoto S. Enhanced excitability of nociceptive trigeminal ganglion neurons by satellite glial cytokine following peripheral inflammation. Pain 2007; 129: 155–166, DOI: 10.1016/j.pain.2006.10.007 [DOI] [PubMed] [Google Scholar]
  • 42.Song DD, Li Y, Tang D, Huang LY, Yuan YZ. Neuron-glial communication mediated by TNF-α and glial activation in dorsal root ganglia in visceral inflammatory hypersensitivity. Amer J Physiol-Gastro Liv Physiol 2014; 306: G788–G795. DOI: 10.1152/ajpgi.00318 [DOI] [PubMed] [Google Scholar]
  • 43.Dubový P, Klusáková I, Svíženská I, Brázda V. Satellite glial cells express IL-6 and corresponding signal-transducing receptors in the dorsal root ganglia of rat neuropathic pain model. Neuron Glia Biol 2010; 6: 73–83. DOI: 10.1017/S1740925X10000074 [DOI] [PubMed] [Google Scholar]
  • 44.Raghavendra V, Tanga FY, DeLeo JA. Attenuation of morphine tolerance, withdrawal-induced hyperalgesia, and associated spinal inflammatory immune responses by propentofylline in rats. Neuropsychopharmacology 2004; 29: 327–334. DOI: 10.1038/sj.npp.1300315 [DOI] [PubMed] [Google Scholar]
  • 45.Agostini S, Eutamene H, Cartier C, Broccardo M, Improta G, Houdeau E, Petrella C, Ferrier L, Theodorou V, Bueno L. Evidence of central and peripheral sensitization in a rat model of narcotic bowel-like syndrome. Gastroenterol 2010; 139: 553–563. DOI: 10.1053/j.gastro.2010.03.046 [DOI] [PubMed] [Google Scholar]
  • 46.Johnston IN, Milligan ED, Wieseler-Frank J, Frank MG, Zapata V, Campisi J, Langer S, Martin D, Green P, Fleshner M, Leinwand L, Maier SF, Watkins LR. A role for proinflammatory cytokines and fractalkine in analgesia, tolerance, and subsequent pain facilitation induced by chronic intrathecal morphine. J Neurosci 2004; 24: 7353–7365. DOI: 10.1523/JNEUROSCI.1850-04 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.White F, Wilson N. Opiate-induced hypernociception and chemokine receptors. Neuropharmacol 2010; 58: 35–37. DOI: 10.1016/j.neuropharm.2009.07.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Berta T, Liu T, Liu YC, Xu ZZ, Ji RR. Acute morphine activates satellite glial cells and Up-Regulates IL-1β in Dorsal Root Ganglia in Mice via Matrix Metalloprotease-9. Molec Pain 2012; 8: 1744–8069. DOI: 10.1186/1744-8069-8-18 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Wilson NM, Jung H, Ripsch MS, Miller RJ, White FA. CXCR4 signaling mediates morphine-induced tactile hyperalgesia. Brain, Behavior, and Immunity 2011; 25: 565–573. DOI: 10.1016/j.bbi.2010.12.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Shaiova L, Rim F, Friedman D, Jahdi M. A review of methylnaltrexone, a peripheral opioid receptor antagonist, and its role in opioid-induced constipation. Palli Suppor Care 2007; 5: 161–166. DOI: 10.1017/s147895150707023x [DOI] [PubMed] [Google Scholar]
  • 51.Yuan CS, Israel RJ. Methylnaltrexone, a novel peripheral opioid receptor antagonist for the treatment of opioid side effects. Expert Opinion on Investigational Drugs 2006; 15: 541–552. DOI: 10.1517/13543784.15.5.541 [DOI] [PubMed] [Google Scholar]
  • 52.Matsuoka Y, Yamashita A, Matsuda M, Kawai K, Sawa T, Amaya F. NLRP2 inflammasome in dorsal root ganglion as a novel molecular platform that produces inflammatory pain hypersensitivity. Pain 2019; 160: 2149–2160. DOI: 10.1097/j.pain.0000000000001611 [DOI] [PubMed] [Google Scholar]
  • 53.Sanna MD, Ghelardini C, Galeotti N. Regionally selective activation of ERK and JNK in morphine paradoxical hyperalgesia: a step toward improving opioid pain therapy. Neuropharmacol 2014; 86: 67–77. DOI: 10.1016/j.neuropharm.2014.06.007 [DOI] [PubMed] [Google Scholar]
  • 54.Berta T, Liu Y-C, Xu Z-Z, Ji R-R. Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1β and ERK signaling in the spinal cord of mice. Neurosci 2013; 247: 376–385. DOI: 10.1016/j.neuroscience.2013.05.018 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material - Activation of neurons and satellite glial cells in the DRG produces morphine-induced hyperalgesia
Click here for additional data file. (233.6KB, pdf)

Supplemental Material for Activation of neurons and satellite glial cells in the DRG produces morphine-induced hyperalgesia by Shunsuke Yamakita, Daisuke Fujita, Kazuki Sudo, Daiki Ishikawa, Kousuke Kushimoto, Yasuhiko Horii and Fumimasa Amaya in Molecular Pain.

Articles from Molecular Pain are provided here courtesy of SAGE Publications

RESOURCES