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Glycogen Synthase Kinase-3β Inhibition Prevents Remifentanil-Induced Postoperative Hyperalgesia via Regulating the Expression and Function of AMPA Receptors

Li, Yi-ze, PhD; Tang, Xiao-hong, MD; Wang, Chun-yan, PhD; Hu, Nan, PhD; Xie, Ke-liang, MD, PhD; Wang, Hai-yun, MD, PhD; Yu, Yong-hao, MD, PhD; Wang, Guo-lin, MD

doi: 10.1213/ANE.0000000000000365
Pain and Analgesic Mechanisms: Research Report
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BACKGROUND: Many studies have confirmed that brief remifentanil exposure can enhance pain sensitivity. We previously reported that activation of glycogen synthase kinase-3β (GSK-3β) contributes to remifentanil-induced hyperalgesia via regulating N-methyl-D-aspartate receptor plasticity in the spinal dorsal horn. In this study, we demonstrated that GSK-3β inhibition prevented remifentanil-induced postoperative hyperalgesia via regulating α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression and function in the spinal dorsal horn.

METHODS: Using a rat model of remifentanil-induced incision hyperalgesia, mechanical and thermal pain was tested 1 day before infusion and 2 hours, 6 hours, 1 day, 2 days, 3 days, 5 days, and 7 days after infusion. Western blot analysis was used to detect AMPAR subunit (GluR1 and GluR2) trafficking, AMPAR phosphorylation status, and GSK-3β activity in the spinal dorsal horn. Furthermore, whole-cell patch-clamp recording was used to analyze the effect of GSK-3β inhibition on AMPAR-induced current in the spinal dorsal horn.

RESULTS: Membrane AMPAR subunit GluR1 was upregulated in the spinal cord in remifentanil-induced postoperative hyperalgesia rats (275 ± 36.54 [mean ± SD] vs 100 ± 9.53, P = 0.0009). Selective GSK-3β inhibitors, LiCl and TDZD, treatment ameliorates remifentanil-induced postoperative hyperalgesia, and this was associated with the downregulated GluR1 subunit in the membrane fraction (254 ± 23.51 vs 119 ± 14.74, P = 0.0027; 254 ± 23.51 vs 124 ± 9.35, P = 0.0032). Moreover, remifentanil incubation increased the amplitude and the frequency of AMPAR-induced current in dorsal horn neurons (61.09 ± 9.34 pA vs 32.56 ± 6.44 pA, P = 0.0009; 118.32 ± 20.33 milliseconds vs 643.67 ± 43.29 milliseconds, P = 0.0002), which was prevented with the application of LiCl and TDZD, respectively. Remifentanil-induced postoperative pain induced an increase in pGluR1 Ser845 and Rab5, which was prevented with the application of LiCl and TDZD.

CONCLUSIONS: These results indicate that amelioration of remifentanil-induced postoperative hyperalgesia by GSK-3β inhibition is attributed to downregulated AMPAR GluR1 expression in the membrane fraction and inhibition of AMPAR function via altering pGluR1 and Rab5 expression in the spinal dorsal horn.

Published ahead of print August 14, 2014.

From the Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin Research Institute of Anesthesiology, Tianjin, China.

Accepted for publication March 28, 2014.

Published ahead of print August 14, 2014.

Funding: This study was supported by research grants from the National Natural Science Foundation of China (81371245, 30972847, 81300960), Natural Science Foundation of Tianjin (11JCYBJC12900), Key Projects in the Tianjin Science & Technology Pillar Program (12ZCZDSY03000), and Science & Technology Foundation of Tianjin Health Bureau (2013KZ124).

The authors declare no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website.

Reprints will not be available from the authors.

Address correspondence to Guo-lin Wang, MD, Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin Research Institute of Anesthesiology, Tianjin 300052, China. Address e-mail to wangguolinemail@126.com.

Opioids have been considered the most effective treatment for acute, chronic, and cancer pain.1 However, prolonged opioid exposure has been associated with an increase in pain sensitivity, known as opioid-induced hyperalgesia.2 Remifentanil, an ultrashort-acting μ-opioid receptor (MOR) agonist, has been widely used for the management of operative pain, due to its reliability, rapid onset, and small risk of respiratory depression or delayed postoperative recovery.3 However, many studies demonstrate that remifentanil-induced postoperative pain is more rapid and frequent than that induced by other opioids.4

In the spinal cord dorsal horn, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated excitatory postsynaptic potentials are supposed to play a pivotal role in the transmission of both acute and chronic pain.5–8 AMPARs are ligand-gated ion channels composed of 4 subunits (GluR1-4) that are encoded by 4 separate genes, gria1-4. The composition of AMPAR subunits is thought to determine its functional properties, such as conduction and trafficking behavior.9 In the rat spinal dorsal horn, synaptic AMPARs are mainly composed of GluR1 and GluR2 subunits.10 GluR1-containing AMPARs are permeable to Ca2+, while GluR2-containing AMPARs have little Ca2+ permeability. The ratio of GluR1/GluR2 determines the electrophysiology function of AMPARs in the spinal cord.11,12 Persistent inflammatory pain increases AMPAR Ca2+ permeability and alters AMPAR subunit compositions in the spinal cord dorsal horn.13,14 Drdla-Schutting et al.15 suggested that acute opioid application modified subcellular distribution of the AMPA-GluR1 receptor subunit in dendrites of neurons in mice. Although modulation by AMPARs of spinal nociception is a potential mechanism of opioid-induced hyperalgesia, the trafficking and function of AMPARs during remifentanil-induced hyperalgesia are still not well studied.

Our previous study found that the activity of glycogen synthase kinase-3β (GSK-3β) was increased with the application of remifentanil in the spinal cord dorsal horn. GSK-3, a multifunctional serine/threonine protein kinase, has been shown to be a key regulator of synaptic plasticity.16 To date, 2 highly homologous isoforms of GSK-3, GSK-3α and GSK-3β, have been discovered.17 Only GSK-3β is highly expressed in the central nervous system, where it has been implicated in several central nervous system dysfunctions, such as schizophrenia, Alzheimer disease, and bipolar disorders.17–22 Studies have found that constitutively active endogenous GSK-3 plays an important role in maintaining AMPARs at the synaptic membrane.23 Our previous study has found that activation of GSK-3β contributes to remifentanil-induced postoperative hyperalgesia via regulating N-methyl-D-aspartate receptor (NMDAR) trafficking and function in the spinal cord.24 However, the role of GSK-3β in regulating AMPAR expression and function in remifentanil-induced hyperalgesia and its maintenance has not been studied.

We hypothesized that GSK-3β inhibition could prevent the development and maintenance of remifentanil-induced postoperative hyperalgesia via regulating the AMPAR expression and function in the spinal cord.

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METHODS

Ethics Statements

All protocols and procedures were approved by the Committee on the Ethics of Experiments of Tianjin Medical University General Hospital, Tianjin, China (Permit Number: 2012-X11-10) and the Institutional Animal Care Committee of Tianjin Medical University. All surgeries and drug deliveries were performed under sevoflurane anesthesia, and all efforts were made to use the minimum number of animals and to minimize their suffering.

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Animals

Experiments were performed on adult (weighing 240–260 g) and newborn (14–21 days old) male Sprague-Dawley rats. Rats were provided by the Laboratory Animal Center of the Military Medical Sciences Academy of the Chinese PLA and housed in cages with a 12-hour light/12-hour dark cycle at a temperature of 22°C ± 2°C. Rats had access to water and food ad libitum.

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Plantar Incision

Plantar incisional pain (nociception) was induced as described previously.25 Briefly, rats (weighing 240–260 g) were anesthetized with sevoflurane (3.0% for induction, 1.0% for maintenance) via a nose mask. A number 11 blade was used to make a 1-cm longitudinal incision through the skin, fascia, and muscle of the right hindpaw, starting 0.5 cm from the proximal edge. The skin was sutured with 4-0 silk sutures after the underlying flexor muscle was divided. The erythromycin ointment was then used to smear the incision.

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Drug Treatments

Rats were anesthetized with sevoflurane (3.0% for induction, 1.0% for maintenance) via a nose mask. A 24-gauge over-the-needle Teflon catheter was inserted into the caudal vein and flushed with heparinized saline. The caudal vein catheter was used to infuse saline and remifentanil. Control group (C) rats underwent a sham operation with saline infusion (0.1 mL·kg−1·min−1, 60 minutes); remifentanil group (R) rats underwent a sham operation and remifentanil infusion (1.0 μg·kg−1·min−1, 60 minutes); incision group (I) rats underwent a surgical incision without remifentanil infusion; remifentanil plus incision group (RI) rats underwent a surgical incision with remifentanil infusion (1.0 μg·kg−1·min−1, 60 minutes); remifentanil incision and GSK-3β inhibitor LiCl group (LiCl) rats underwent a surgical incision, remifentanil, and LiCl infusion (1.0 μg·kg−1·min−1 and 100 mg·kg−1, 60 minutes); and remifentanil, incision, and TDZD group (TDZD) rats underwent a surgical incision, remifentanil, and TDZD infusion (1.0 μg·kg−1·min−1 and 1.0 μg·kg−1, 60 minutes).

In patch-clamp recording, spinal cord slices from the control group (C) were incubated in artificial cerebrospinal fluid (ACSF) for 60 minutes; spinal cord slices from the remifentanil group (R) were incubated in ACSF with 4 nM remifentanil; spinal cord slices from the remifentanil and LiCl group (LiCl) were incubated in ACSF with 4 nM remifentanil and 20 mM LiCl; and spinal cord slices from the remifentanil and TDZD group (TDZD) were incubated in ACSF with 4 nM remifentanil and 10 μM TDZD.

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Mechanical and Thermal Hyperalgesia Testing

Paw withdrawal threshold (PWT) was determined by electronic von Frey filaments (BSEVF3, Harvard Apparatus, Edenbridge, UK) to evaluate mechanical hyperalgesia. Adult rats were placed in individual cages (20 cm × 20 cm × 20 cm) with a wire grid bottom. von Frey filaments were applied vertically to the plantar side of the right hindpaw. Flinching or lifting of the hindpaw off the cage surface was considered a positive response. A cutoff value of 60 g was used to prevent tissue damage. Each trial was repeated 5 times at 15-minute intervals. A 55°C hotplate (YLS-6B, Zheng Hua Biological Instrument Co., Anhui, China) was used to evaluate thermal hyperalgesia and was expressed as paw withdrawal latency (PWL). Each trial was repeated 5 times at 15-minute intervals, and a cutoff time of 40 seconds was used to avoid tissue damage.

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Western Blot

After finishing behavioral tests (1 day before infusion and 2 hours, 6 hours, 12 hours, 1 day, 2 days, 5 days, and 7 days after infusion), rats were decapitated under sevoflurane anesthesia and spinal cords (L4-L6) were extruded. To prepare a total lysate, the tissue was homogenized in ice-cold lysis buffer (pH 7.5, 2% Triton X-100, 150 mM NaCl, 50 mM Tris, 1 g·mL−1 aprotinin, 100 g·mL−1 phenylmethylsulfonyl fluoride, and phosphatase inhibitors). The lysate was centrifuged at 12,000g for 30 minutes at 4°C, and the supernatant was used for Western blot analysis. To prepare membrane and cytosolic fractions, the tissue was separated into membrane, cytosolic, and nuclear fractions by a membrane, cytosolic, and nuclear compartment protein extraction kit (Biochain Institute, Inc., Hayward, CA). The inner references of the cytosolic and membrane protein were β-actin antibody (1:5000; Sigma-Aldrich, St. Louis, MO) and N-Cad antibody (1:2000; MBL, Naka-ku, Nagoya, Japan), respectively. Ten percent sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate samples (20 μg protein), and then the proteins were transferred onto nitrocellulose membranes. All membranes were blocked with 5% nonfat milk in Tris-TBST for 1 hour (TBST, 50 mM Tris-HCl, 154 mM NaCl, and 0.05% Tween 20, pH 7.4) and subsequently incubated overnight at 4°C with antibodies against GluR1, GluR2, phosphorylated (Ser845) GluR1, phosphorylated (Ser880) GluR2 (all 1:500, Chemicon, Temecula, CA), GSK-3β, phosphorylated (Try216 and Ser9) GSK-3β antibodies, PSD-95, and Rab5 (all 1:1000, Cell Signaling Technology). Immunoreactivity was detected by incubation with horseradish peroxidase–conjugated secondary antibodies (1:2000 in 5% nonfat milk in TBST, Jackson Immuno Research, Danvers, MA) for 1 hour. The secondary antibodies were visualized using a chemiluminescence imaging system (Syngene, Cambridge, UK) and measured using an imaging analysis system (Gene Tools Match software; Syngene).

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Spinal Cord Slice Preparation and Whole-Cell Patch-Clamp Recording

The method used for obtaining rat spinal cord slices was described previously.26,27 Rats (14–21 days old) were anesthetized with sevoflurane (3.0% for induction, 1.0% for maintenance). The anterior approach was used to separate spinal cords (L4-L5), and the spinal cord was sliced into transverse slices (350 μm) with a vibratome (VT1000S, Leica, Wetzlar, Germany). The slices were then incubated in ACSF at room temperature (22°C–25°C). ACSF was aerated with 5% CO2 and 95% O2 at pH 7.4 for 1 hour before incubation. ASCF is composed of (in mM) 126 NaCl, 2 MgCl2, 1.25 NaH2PO4, 3.5 KCl, 26 NaHCO3, 10 D-glucose, and 2 CaCl2. Each slice was transferred into a recording chamber. The chamber was placed on an upright microscope equipped (BX51W1, Olympus, Tokyo, Japan) and continuously perfused with oxygenated ACSF. Individual neurons were identified through a television monitor connected to a low light-sensitive CCD camera (710M, DVC, New York, NY). Borosilicate glass patch microelectrodes were produced by the vertical electrode puller (PIP5, HEKA, Lambrecht, Germany) and used for whole-cell patch-clamp recording with tip openings of 1 to 2 μm and a series resistance of 3 to 5 MΩ. Microelectrodes were filled with an intracellular solution containing (in mM) 130 KCl, 10 HEPES, 0.5 CaCl2, 10 EGTA, 2 MgCl2, 2 Mg-ATP, and 0.3 Na-GTP, pH 7.3. To isolate the AMPAR miniature excitatory postsynaptic currents (mEPSC), (2R)-amino-5-phosphonovaleric acid (40 μM), tetrodotoxin (0.5 μM), strychnine (2 μM), and bicuculline (5 μM) were added into the perfusion solution. The AMPAR-mediated mEPSC was analyzed by Clampfit 9.0 (Axon Instruments, Foster City, CA).

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Statistical Analysis

Statistical analysis was performed with SPSS 19.0 (IBM, Chicago, IL). Values are expressed as mean ± SD. To determine the sample size, a power analysis was performed based on a preliminary experiment. PWL in the control group was 18.6 seconds, and in the remifentanil group it was 7.2 seconds. Assuming an α error of 0.05 with a power of 0.90, we calculated a necessary sample size of 6 to show a significant effect. Based on this calculation and to ensure reasonable data, we increased the sample size to 8 and the samples were distributed normally. Interaction between time and group factors in a 2-way analysis of variance with repeated measurements was used to analyze the difference in PWT and PWL in different groups. Multiple comparisons in PWT and PWL were adjusted using the Tukey–Kramer method. Western blot results, amplitude, and interevent intervals of AMPAR-mediated mEPSC were analyzed by 1-way analysis of variance followed by post hoc comparisons using Tukey-Kramer test. P < 0.01 value was considered statistically significant.

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RESULTS

Remifentanil-Induced Postoperative Mechanical and Thermal Hyperalgesia

Compared with the saline group, remifentanil infusion at 1 μg·kg−1·min−1 for 60 minutes caused a significant decrease of PWT and PWL from 2 to 48 hours. Those results suggest that opioid-induced thermal and mechanical hyperalgesia can be increased by remifentanil infusion at the rate of 1 μg·kg−1·min−1 (Fig. S1, Supplemental Digital Content 1, http://links.lww.com/AA/A927). Remifentanil infusion or incision increased thermal and mechanical pain hypersensitivity from 2 hours to 5 days after remifentanil infusion and/or incision. Compared with baseline and the control group, PWL of the incised paw and PWT in response to von Frey filaments applied to the incised paw were significantly decreased (Fig. 1B). Thermal and mechanical hyperalgesia appeared at 2 hours, reached a peak level on day 2, and persisted for 5 days after remifentanil infusion and incision (all Tukey P < 0.007) (Fig. 1, A and B).

Figure 1

Figure 1

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Remifentanil-Induced Postoperative Hyperalgesia Increased the Expression of GluR1 Subunit in the Plasma Membrane, but Not GluR2 Subunit in the Spinal Cord

Spinal cord (L4-L6) segments were collected at −1 day and 2 hours, 6 hours, 1 day, 2 days, 3 days, 5 days, and 7 days after infusion from rats treated with remifentanil infusion and incision. Histograms illustrate that remifentanil infusion and incision increased the expression of GluR1 in membrane fraction from 2 hours, reached a peak value on day 2, and persisted for 3 days (all Tukey P < 0.009, Fig. 2A). Remifentanil infusion and incision had no effect on the expression of cytoplasmic GluR1 (all P > 0.473, Fig. 2B). These results suggest that GluR1 moves into the plasma membrane after remifentanil infusion and incision. Surprisingly, neither cytoplasmic nor membrane protein expression showed a remifentanil- and incisioninduced change in GluR2 expression (all P > 0.582, Fig. S2, Supplemental Digital Content 2, http://links.lww.com/AA/A928).

Figure 2

Figure 2

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Expression of GluR1 in the Membrane Fraction Was Increased in Spinal Dorsal Horn with Remifentanil-Induced Postoperative Hyperalgesia

Expression of GluR1 in the membrane fraction reached a peak level on day 2 after remifentanil infusion and incision. Accordingly, L4-L6 spinal cords were harvested at 2 days after remifentanil infusion. Remifentanil infusion and incision induced increased GluR1 expression in the membrane fraction on day 2 (P = 0.004 and P = 0.002). However, neither remifentanil infusion nor incision had an effect on the expression of cytoplasmic GluR1 (all P > 0.523, Fig. 3).

Figure 3

Figure 3

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The Activity of GSK-3β Was Increased in the Spinal Dorsal Horn in Remifentanil-Induced Postoperative Hyperalgesia Rats

Phosphorylation of N-terminal serine 9 for GSK-3β has an inhibitory effect on and plays an important role in regulation of GSK-3β function.28 Phosphorylation of tyrosine in the C terminus at 216 activates GSK-3β, and its mutation decreases the activity of the enzyme.29,30 L4-L6 spinal cords were harvested at 2 days after remifentanil infusion. Remifentanil infusion and incision resulted in an increase of pGSK-3β (Tyr216) and a decrease of pGSK-3β (ser9) but had no impact on the total protein level of GSK-3β (all Tukey P < 0.005, Fig. 4). Together, these results show that remifentanil infusion and incision could augment GSK-3β activity by increasing phosphorylation of tyrosine 216 and dephosphorylation of serine 9.

Figure 4

Figure 4

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Inhibition of GSK-3β Attenuates Remifentanil-Induced Postoperative Thermal and Mechanical Hyperalgesia

To investigate whether GSK-3β participates in opioid-induced postoperative hyperalgesia, 2 selective GSK-3β inhibitors LiCl (100 mg·kg−1) and TDZD (1 μg·kg−1) were infused IV before remifentanil infusion. Latencies to noxious heat and mechanical paw stimulation were recorded at −1 day and 2 hours, 6 hours, 1 days, 2 days, 3 days, 5 days, and 7 days after infusion. We found that LiCl and TDZD significantly improved PWT and PWL in rats exposed to remifentanil infusion and incision (all Tukey P < 0.005, Fig. 5). The antihyperalgesic effect of LiCl and TDZD was shown in rats with remifentanil-induced postoperative hyperalgesia. These results suggest that remifentanil-induced postoperative thermal and mechanical hyperalgesia was attenuated by inhibition of GSK-3β.

Figure 5

Figure 5

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GSK-3β Inhibition Downregulates Membrane GluR1 Subunit Expression in the Spinal Dorsal Horn

The membrane GluR1 subunit was increased in spinal cord neurons of rats exposed to remifentanil and incision (P = 0.0009). LiCl or TDZD treatment reduced the increase of GluR1 expression in these rats (P = 0.0027, P = 0.0032). However, there was no change in the cytoplasmic protein level of GluR1 expression in all groups (P = 0.832, Fig. 6). These results suggest that inhibition of GSK-3β could modulate remifentanil-induced GluR1 expression and membrane trafficking in dorsal horn neurons.

Figure 6

Figure 6

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GSK-3β Inhibition Decreases Remifentanil-Induced AMPAR Function Enhancement in Dorsal Horn Neurons

Whole-cell patch-clamp recordings were made to detect changes in remifentanil-induced AMPAR-mediated mEPSC in dorsal horn neurons. Representative traces of AMPAR-mediated mEPSC in different groups are shown in Figure 7. The amplitude of AMPAR current was increased, while the interevent interval of AMPAR current was decreased after remifentanil incubation (P = 0.0009, P = 0.0002, Fig. 7). These results indicate that AMPAR function was enhanced after the exposure of spinal slices to 4 nM remifentanil. To further confirm the role of GSK-3β on AMPAR-mediated mEPSC in the spinal cord, we examined AMPAR-mediated mEPSC with the application of remifentanil (4 nM) and LiCl (20 mM) or TDZD (10 μM). LiCl and TDZD decreased the amplitude and the frequency of AMPAR current when compared with the remifentanil (R) group (all Tukey P < 0.0009, Fig. 7). These results show that GSK-3β inhibition decreased the remifentanil-induced AMPAR function increase in the spinal cord.

Figure 7

Figure 7

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GSK-3β Downregulates GluR1 Phosphorylation at Ser845 in the Spinal Cord in Rats with Remifentanil-Induced Postoperative Hyperalgesia

L4-L6 spinal cords were collected at 2 days after remifentanil infusion. Remifentanil infusion and incision increased the phosphorylation of the GluR1 subunit at Ser845 but had no effect on the phosphorylation of GluR2 (Ser880) (P = 0.0007, P = 0.255). This increase was inhibited with the application of LiCl or TDZD, indicating that GluR1 phosphorylation was dependent on GSK-3β activity in rats with remifentanil-induced postoperative hyperalgesia (P = 0.0036, P = 0.0051, Fig. 8).

Figure 8

Figure 8

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GSK-3β Inhibitors Upregulate Rab5 Expression in the Spinal Cord of Rats with Remifentanil-Induced Postoperative Hyperalgesia

Spinal cords were collected 2 days after remifentanil infusion. Remifentanil infusion and incision decreased Rab5 expression (P = 0.0004) but had no effect on Rab4 and PSD-95 expression (P = 0.645, P = 0.732). The decrease in Rab5 expression was reversed with the application of LiCl or TDZD-8, indicating that GSK-3β modulates GluR1 trafficking via Rab5 in rats with remifentanil-induced postoperative hyperalgesia (P = 0.0003, P = 0.0001, Fig. 9).

Figure 9

Figure 9

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DISCUSSION

In the present study, we found that remifentanil infusion and incision enhanced GSK-3β activity in the spinal cord by increasing dephosphorylation of Ser9 and phosphorylation of Tyr216. Remifentanil-induced postoperative hyperalgesia increased GluR1 trafficking into membranes, pGluR1 (Ser845) and Rab5, but had no effect on pGluR2, Rab4, or PSD-95 in the dorsal spinal cord. These changes can be prevented by GSK-3β inhibitors, LiCl and TDZD. In the whole-cell patch-clamp study, remifentanil enhanced the amplitude and the frequency of AMPAR-mediated mEPSC, which were also attenuated by GSK-3β inhibitors. These results suggest that GSK-3β inhibition can prevent remifentanil-induced hyperalgesia via regulating AMPAR subunit GluR1 expression and AMPAR function by altering pGluR1 and Rab5 expression in dorsal horn neurons.

In the current study, LiCl and TDZD, 2 selective GSK-3β inhibitors, reduced remifentanil-induced postoperative hyperalgesia, indicating that GSK-3β activity plays an important role in pain signal transmission and responses. Previous studies indicated that the inhibition of GSK-3β prevented the development of opioid-induced tolerance and morphine increased GSK-3β activity in mice.31,32 Another study showed that neuropathic pain-induced thermal and mechanical hyperalgesia was prevented by AR-A014418, a selective GSK-3β inhibitor, indicating that GSK-3β inhibitor has an antihyperalgesic effect in mice.33,34 These antinociceptive effects of GSK-3β inhibitors were mediated by excitatory glutamatergic receptors and metabotropic receptors.34

GSK-3β plays a pivotal role in AMPAR trafficking and function. GSK-3 inhibitors produce a significant reduction of AMPAR synaptic responses, which was accompanied by the increase of AMPAR internalization and the decrease of AMPAR surface expression.23 Trafficking of postsynaptic AMPARs is required for controlling fast synaptic transmission and excitatory synaptic efficacy.35,36 Clathrin-mediated endocytosis plays a pivotal role in AMPAR trafficking. Insulin can increase clathrin-mediated endocytosis, decrease the level of membrane AMPARs, and then induce the formation of long-term depression in hippocampal CA1 neurons.36 Insulin inactivates the enzyme GSK-3 via PKB/Akt pathway and inhibits mEPSC amplitude similar to GSK-3 inhibitors in cultured cortical neurons.23,37,38 This may explain how GSK-3β inhibitors (LiCl and TDZD) modulate trafficking and function of AMPARs.

Functional AMPARs are homomeric or heteromeric tetramers of AMPAR subunits. Homomeric channels are composed of GluR1, GluR3, and GluR4 that are Ca2+ permeable, while homomeric GluR2 channels have little Ca2+ permeability.39 GluR1 and GluR2 subunits of the AMPARs are expressed at high levels on postsynaptic neuronal membranes in the superficial dorsal horn.40 In this study, membrane GluR1 was increased in the spinal cord of rats with remifentanil-induced postoperative hyperalgesia without substantial changes in GluR2. The increased Ca2+-permeable AMPARs contribute to spinal sensitization and pain behavior. Under basal conditions, membrane insertion of GluR1-containing complexes is slow and balanced by an efflux out of the membrane; however, the insertion rate increases after increased neural activity.41 Thus, it is possible that AMPAR-mediated neural activity was increased in rats with remifentanil-induced postoperative hyperalgesia.

In this study, we found that phosphorylation of the GluR1 subunit of AMPARs at Ser845 was increased in rats with remifentanil-induced postoperative pain. Phosphorylation of GluR1 at Ser845 by protein kinase A was required by AMPAR insertion.42,43 Acute inflammatory pain is characterized by increased thermal and mechanical pain sensitization in the spinal cord dorsal horn and increased phosphorylation of the GluR1 subunit of AMPARs at Ser845, and these effects are mediated by the activation of protein kinase A.44,45 GluR2 internalization requires NMDAR-triggered spinal cord protein kinase C α activation and protein kinase C α–mediated phosphorylation of GluR2 at Ser880.46 In the current study, there was no change in the internalization or the phosphorylation of GluR2 at Ser880, indicating that the internalization or the phosphorylation of GluR2 may not be necessary for remifentanil-induced postoperative hyperalgesia.

The essential regulators of intracellular membrane sorting are mediated by members of the Rab family in eukaryotic cells.47,48 Rab5, a member of the Rab family, can be activated by GSK-3 inhibitors in hippocampal neurons. Rab5 regulates AMPAR trafficking from plasma membrane to early endosomes, thereby decreasing the level of AMPARs on the plasma membrane. Knockdown of Rab5 eliminates the effect of GSK-3 inhibitors on the amplitude of AMPAR-mediated mEPSC.23 Also, Rab5 is necessary to clathrin-dependent AMPAR internalization, and the activity of Rab5 is required for long-term depression.49 Rab4, a small GTPase, modulates the recycling of receptors from early endosomes to the plasma membrane.50 In one study, complete Freund’s adjuvant–induced inflammatory pain was mediated by the SGK1/GRASP-1/Rab4 signaling pathway via increasing GluR1 subunit expression on the plasma membrane at the spinal cord.51 In this study, we found that inhibition of GSK-3β increases Rab5 expression but had no effect on Rab4. This suggests that GSK-3β inhibitors may reduce CluR1 expression by enhancing Rab5-mediated AMPAR internalization.

Chronic opioid exposure induces opioid receptor desensitization, which may play a crucial role in remifentanil-induced hyperalgesia. Opioid receptor desensitization involves the classical model of G protein–coupled receptor regulation, including their phosphorylation, and uncoupling of receptors from their G proteins via arrestins.52 One study examined remifentanil-induced MOR internalization in lamina II neurons of spinal dorsal horn in rats.53 Remifentanil promoted a rapid and strong desensitization of MOR by promoting Ser375 phosphorylation.54

In conclusion, our study suggests that remifentanil-induced postoperative hyperalgesia may be mediated by GSK-3β activation via increased AMPAR subunit GluR1 expression and AMPAR function regulated by altering spinal pGluR1 and Rab5 expression. The antihyperalgesic effect of GSK-3β may provide a new target for preventing and treating remifentanil-induced postoperative hyperalgesia.

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DISCLOSURES

Name: Yi-ze Li, PhD.

Contribution: This author helped design the study and write the manuscript.

Attestation: Yi-ze Li has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Xiao-hong Tang, MD.

Contribution: This author helped analyze the data.

Attestation: Xiao-hong Tang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Chun-yan Wang, PhD.

Contribution: This author helped conduct the study.

Attestation: Chun-yan Wang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Nan Hu, PhD.

Contribution: This author helped conduct the study.

Attestation: Nan Hu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Ke-liang Xie, MD, PhD.

Contribution: This author helped design the study and write the manuscript.

Attestation: Ke-liang Xie has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Hai-yun Wang, MD, PhD.

Contribution: This author helped design the study and conduct the study.

Attestation: Hai-yun Wang has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Yong-hao Yu, MD, PhD.

Contribution: This author helped design the study and analyze the data.

Attestation: Yong-hao Yu has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Guo-lin Wang, MD.

Contribution: This author helped design the study, conduct the study, and write the manuscript.

Attestation: Guo-lin Wang has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

This manuscript was handled by: Jianren Mao, MD, PhD.

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REFERENCES

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