Remifentanil-induced Hyperalgesia In Rats Research Articles

Spinal NLRP3 inflammasome activation mediates IL-1β release and contributes to remifentanil-induced postoperative hyperalgesia by regulating NMDA receptor NR1 subunit phosphorylation and GLT-1 expression in rats.

Trafficking and activation of N-methyl-D-aspartate (NMDA) receptors play an important role in initiating and maintaining postoperative remifentanil-induced hyperalgesia (RIH). Activation of the NOD-like receptor protein 3 (NLRP3) inflammasome has been linked to the development of inflammatory and neuropathic pain. We hypothesized that activation of NLRP3 inflammasome mediates IL-1β release and contributes to RIH in rats by increasing NMDA receptor NR1 (NR1) subunit phosphorylation and decreasing glutamate transporter-1 (GLT-1) expression. Acute exposure to remifentanil (1.2μg/kg/min for 60 min) was used to establish RIH in rats. Thermal and mechanical hyperalgesia were tested at baseline (24 h before remifentanil infusion) and 2, 6, 24, and 48 h after remifentanil infusion. The levels of IL-1β, GLT-1, phosphorylated NR1 (phospho-NR1), and NLRP3 inflammasome activation indicators [NLRP3, Toll-like receptor 4 (TLR4), P2X purinoceptor 7 (P2X7R), and caspase-1] were measured after the last behavioral test. A selective IL-1β inhibitor (IL-1β inhibitor antagonist; IL-1ra) or three different selective NLRP3 inflammasome activation inhibitors [(+)-naloxone (a TLR4 inhibitor), A438079 (a P2X7R inhibitor), or ac-YVADcmk (a caspase-1 inhibitor)] were intrathecally administered immediately before remifentanil infusion into rats. Remifentanil induced significant postoperative hyperalgesia, increased IL-1β and phospho-NR1 levels and activated the NLRP3 inflammasome by increasing TLR4, P2X7R, NLRP3, and caspase-1 expression, but it decreased GLT-1 expression in the L4-L6 spinal cord segments of rats, which was markedly improved by intrathecal administration of IL-1ra, (+)-naloxone, A438079, or ac-YVADcmk. NLRP3 inflammasome activation mediates IL-1β release and contributes to RIH in rats by inducing NMDA receptor NR1 subunit phosphorylation and decreasing GLT-1 expression. Inhibiting the activation of the NLRP3 inflammasome may be an effective treatment for RIH.

A Role for Transmembrane Protein 16C/Slack Impairment in Excitatory Nociceptive Synaptic Plasticity in the Pathogenesis of Remifentanil-induced Hyperalgesia in Rats

Remifentanil is widely used to control intraoperative pain. However, its analgesic effect is limited by the generation of postoperative hyperalgesia. In this study, we investigated whether the impairment of transmembrane protein 16C (TMEM16C)/Slack is required for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) activation in remifentanil-induced postoperative hyperalgesia. Remifentanil anesthesia reduced the paw withdrawal threshold from 2 h to 48 h postoperatively, with a decrease in the expression of TMEM16C and Slack in the dorsal root ganglia (DRG) and spinal cord. Knockdown of TMEM16C in the DRG reduced the expression of Slack and elevated the basal peripheral sensitivity and AMPAR expression and function. Overexpression of TMEM16C in the DRG impaired remifentanil-induced ERK1/2 phosphorylation and behavioral hyperalgesia. AMPAR-mediated current and neuronal excitability were downregulated by TMEM16C overexpression in the spinal cord. Taken together, these findings suggest that TMEM16C/Slack regulation of excitatory synaptic plasticity via GluA1-containing AMPARs is critical in the pathogenesis of remifentanil-induced postoperative hyperalgesia in rats.

<p>Wnt3a Inhibitor Attenuates Remifentanil-Induced Hyperalgesia via Downregulating Spinal NMDA Receptor in Rats</p>

PurposeThe upregulation of spinal NMDA receptor is a crucial mechanism in remifentanil-induced hyperalgesia (RIH). Wnt3a/β-catenin pathway plays an important role in neuropathic pain. We hypothesized that wnt3a inhibitor (iwp-2) could downregulate the expression of NR2B subunit in NMDA receptor, in order to relieve RIH.Materials and MethodsThe study has 2 phases. The phase 1 study is designed by different doses of iwp-2 groups to create an appropriate iwp-2 dose used in RIH alleviation. The phase 2 study is designed to prove that the wnt3a inhibitor could downregulate the activation of the NR2B to inhibit RIH in rats. Thermal hyperalgesia (PWTL) and mechanical allodynia (PWMT) were evaluated after RIH. The area under the PWTL and PWMT curves (AUC) were calculated. The amount of activated NR2B subunit, c-fos, NF-κB, β-catenin, wnt3a and p-GSK-3β (Ser9) were detected in the lumbar spinal cord.ResultsRemifentanil infusion could induce overexpression of β-catenin and wnt3a in rats. Iwp-2 (60μM, 120μM, 180μM) could dose-dependently inhibit thermal hyperalgesia and mechanical allodynia in rats. In phase 2 study, both NR2B subunit antagonist Ro25-6981 and iwp-2 decreased the amount of activated NR2B, enhanced p-GSK-3β (Ser9), reduced β-catenin, c-fos and NF-κB in the lumbar spinal cord (p < 0.001). In comparison with the group iwp-2, the group of Ro25-6981 had more benefit in reversing hyperalgesia, including higher AUC value of PWTL (p = 0.022) and PWMT (p = 0.035).ConclusionRemifentanil exposure could induce overexpression of wnt3a and enhance the production of β-catenin in the spinal dorsal horn. Inhibition of wnt3a response was capable of attenuating RIH in alleviating hyperalgesia-related behavioral parameters, as well as reducing overexpression of c-fos, NF-κB, NR2B in spinal dorsal horn.

Spinal caspase-6 contributes to remifentanil-induced hyperalgesia via regulating CCL21/CXCR3 pathway in rats

BackgroundNeuroinflammation in the spinal cord is a pathological event in remifentanil-induced hyperalgesia (RIH), but its underlying molecular mechanisms remain unclear. Recent studies recapitulate the significance of the intracellular protease caspase-6 in the release of inflammatory mediators and synaptic plasticity in pathologic pain. Also, chemokine CCL21 is involved in microglia activation and nociceptive transduction. This study examined whether spinal caspase-6 is associated with RIH via CCL21 and its receptor CXCR3. MethodsThe acute exposure to remifentanil (1 μg kg−1 min−1for 60 min) was used to establish RIH, verified by assessment of mechanical paw withdrawal threshold and thermal paw withdrawal latency. The caspase-6 inhibitor, a neutralizing antibody against CCL21 (anti-CCL21), a selective CXCR3 antagonist NBI-74330, recombinant caspase-6 and CCL21 were used for the investigation of pathogenesis as well as the prevention of hyperalgesia. The expression of caspase-6, CCL21 and CXCR3 was also evaluated by RT-qPCR and Western blot. ResultsThis study discovered mechanical allodynia and thermal hyperalgesia along with the increase in the expression of spinal caspase-6 and CCL21/CXCR3 after remifentanil exposure. Central caspase-6 inhibition prevented behavioral RIH and spinal up-regulation of CCL21/CXCR3 level. Intrathecal anti-CCL21 injection reduced RIH and spinal expression of CXCR3. The delivery of recombinant caspase-6 facilitated acute nociceptive hypersensitivity and increased spinal CXCR3 release in naïve rats, reversing by co-application of anti-CCL21. Also, NBI-74330 attenuated RIH and exogenous CCL21-caused acute pain behaviors. ConclusionThis study highlighted that spinal caspase-6-mediated up-regulation of CCL21/CXCR3 is vital in the pathogenesis of RIH in rats.

Acid-sensing ion channel 3 expression is increased in dorsal root ganglion, hippocampus and hypothalamus in remifentanil-induced hyperalgesia in rats

BackgroundRemifentanil induces hyperalgesia, but the underlying mechanisms are not fully understood. Acid-sensing ion channel 3 (ASIC3) plays a regulatory role in the pain pathway. This study aimed to explore the effect of remifentanil administration on postoperative pain and on ASIC3 expression at the prespinal and supraspinal levels in a rat model. MethodsRats were randomly allocated to the control, incision, remifentanil, and remifentanil + incision groups. Remifentanil was given by a 1-h intravenous infusion prior to plantar incision. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured at different time points before and after incision to evaluate mechanical and thermal hyperalgesia, respectively. The dorsal root ganglion (DRG), hippocampus, and hypothalamus were obtained after sacrifice at 48 h post-incision for determination of the protein expression of ASIC3 using western blot. ResultsRemifentanil administration significantly induced mechanical and thermal hyperalgesia from 2 to 48 h after incision. In addition, remifentanil exposure remarkably stimulated ASIC3 protein expression in DRG, hippocampus, and hypothalamus of rats at 48 h after incision. ConclusionRemifentanil-induced hyperalgesia is accompanied by increased ASIC3 expression at the DRG and supraspinal levels, implying a possible involvement of ASIC3 in remifentanil-induced hyperalgesia.

TRPV1 and spinal astrocyte activation contribute to remifentanil-induced hyperalgesia in rats.

Remifentanil is an ultra-short-acting µ-opioid receptor agonist, which is widely used in general anesthesia. However, comparing with other opioids, remifentanil often induces hyperalgesia. Accumulating evidence suggests that the transient receptor potential (TRP) channels and glial cells activation were involved in the development of neuropathic pain and hyperalgesia. However, whether the TRP channels and glial cells contribute to remifentanil-induced hyperalgesia is still unknown. In this study, we used the hot-plate and Von Frey tests to evaluate the thermal and mechanical hyperalgesia. Protein expressions of TRPV1 and protein kinase C (PKC) in dorsal root ganglion were assayed by western blotting and mRNA level of Trpv1, Trpa1, Trpv4, and Trpm8 were assayed by real-time PCR. TNF-α, IL-1β, and IL-6 levels in spinal cord were measured by ELISA. Immunofluorescence assay was applied to analyze the activation of astrocyte in spinal cord. Continuing infusion of remifentanil induced thermal hyperalgesia and mechanical allodynia, which were accompanied by upregulation of TRPV1 and PKC protein in dorsal root ganglion. Moreover, remifentanil also increased the TNF-α, IL-1β, and IL-6 levels and activates the astrocyte in spinal cord. Our findings suggested that TRPV1 is involved in the TRPV1-PKC signaling pathway, which contributes to the persistence of remifentanil-induced postoperative hyperalgesia. In addition, the spinal astrocyte activation and inflammatory reaction are involved in the remifentanil-induced postoperative hyperalgesia.

Chemokine CCL7 regulates spinal phosphorylation of GluA1-containing AMPA receptor via interleukin-18 in remifentanil-induced hyperalgesia in rats

BackgroundRemifentanil-induced hyperalgesia (RIH) may be a potential component for acute confusional state and chronic pain after surgery, but specific mechanism remains elusive. Chemokines and inflammatory mediators associated neuroinflammation is involved in central nociceptive sensitization. The contribution of AMPA receptor to excitatory synaptic transmission in pathologic pain has been indicated. This study investigated whether chemokine CCL7 modulated spinal phosphorylation of AMPA receptor via interleukin (IL)-18 in a rat model of RIH. MethodsMechanical allodynia and thermal hyperalgesia after remifentanil infusion were verified by assessment of paw withdrawal threshold and paw withdrawal latency, respectively. A neutralizing antibody against CCL7, IL-18 antagonist and recombinant CCL7 were employed to identify the mechanisms. Levels of CCL7 and its receptors, expressions of IL-18 and its receptor IL-18R, and the phosphorylation of GluA1 in the spinal cord dorsal horn were evaluated. ResultsWe reported that acute exposure to remifentanil caused and maintained behavioral RIH with up-regulation of expression in CCL7/CCR2 and IL-18/IL-18R and the phosphorylation of GluA1 in the dorsal horn. Spinal inhibition of CCL7/CCR2 impaired RIH, spinal expression of IL-18/IL-18R and the phosphorylation of GluA1. Central blockage of IL-18/IL-18R reduced spinal phosphorylation of GluA1 and behavioral RIH. Also, intrathecal injection of exogenous CCL7 facilitated acute pain behaviors and spinal phosphorylation of GluA1 in naïve mice, which was suppressed by co-administration of IL-18 antagonist. ConclusionOur current findings demonstrate that spinal CCL7/CCR2 modulation of GluA1-containing AMPA receptor activation via IL-18 and IL-18R over-expression is an important pathogenesis of remifentanil-induced hyperalgesia in rats.

Spinal Protein Kinase Mζ Regulates α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor Trafficking and Dendritic Spine Plasticity via Kalirin-7 in the Pathogenesis of Remifentanil-induced Postincisional Hyperalgesia in Rats.

Intraoperative remifentanil anesthesia exaggerates postoperative pain sensitivity. Recent studies recapitulate the significance of protein kinase Mζ in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated pathologic pain. Kalirin-7, a Rho guanine nucleotide exchange factor, coordinates AMPA receptor trafficking and dendritic spine plasticity. This study examines whether protein kinase Mζ and Kalirin-7 contribute to remifentanil-induced postincisional hyperalgesia via AMPA receptor. Plantar incision was performed 10 min after the start of remifentanil infusion (1 µg · kg · min for 60 min). Paw withdrawal threshold (primary outcome), spinal protein kinase Mζ activity, Kalirin-7 expression, AMPA receptor trafficking, and spine morphology were assessed. Protein kinase Mζ inhibitor and Kalirin-7 knockdown by short hairpin RNA elucidated the mechanism and prevention of hyperalgesia. Whole-cell patch-clamp recording analyzed the role of protein kinase Mζ in spinal AMPA receptor-induced current. Remifentanil reduced postincisional paw withdrawal threshold (mean ± SD, control vs. hyperalgesia, 18.9 ± 1.6 vs. 5.3 ± 1.2 g, n = 7) at postoperative 48 h, which was accompanied by an increase in spinal protein kinase Mζ phosphorylation (97.8 ± 25.1 vs. 181.5 ± 18.3%, n = 4), Kalirin-7 production (101.9 ± 29.1 vs. 371.2 ± 59.1%, n = 4), and number of spines/10 µm (2.0 ± 0.3 vs. 13.0 ± 1.6, n = 4). Protein kinase Mζ inhibitor reduced remifentanil-induced hyperalgesia, Kalirin-7 expression, and GluA1 trafficking. Incubation with protein kinase Mζ inhibitor reversed remifentanil-enhanced AMPA receptor-induced current in dorsal horn neurons. Kalirin-7 deficiency impaired remifentanil-caused hyperalgesia, postsynaptic GluA1 insertion, and spine plasticity. Selective GluA2-lacking AMPA receptor antagonist prevented hyperalgesia in a dose-dependent manner. Spinal protein kinase Mζ regulation of GluA1-containing AMPA receptor trafficking and spine morphology via Kalirin-7 overexpression is a fundamental pathogenesis of remifentanil-induced hyperalgesia in rats.

Role of spinal CCR5 in remifentanil-induced hyperalgesia in rats with incisional pain

Objective To evaluate the role of spinal C-C motif chemokine receptor 5 (CCR5) in remifentanil-induced hyperalgesia in rats with incisional pain. Methods Thirty-two male Sprague-Dawley rats, weighing 240-260 g, aged 2-3 months, in which intrathecal and caudal catheters were successfully implanted, were divided into 4 groups (n=8 each) using a random number table: control group (group C), CCR5 antagonist maraviroc group (group M), remifentanil plus incisional pain group (group R+ I) and maraviroc plus remifentanil plus incisional pain group (group M+ R+ I). Phosphate buffer solution (PBS) 10 μl was intrathecally injected and normal saline was infused for 60 min at 1 μg·kg-1·min-1 via the caudal vein in group C. Maraviroc 100 pmol (in 10 μl of PBS) was intrathecally injected and normal saline was infused for 60 min at 1 μg·kg-1·min-1 via the caudal vein in group M. PBS 10 μl was intrathecally injected, then the model of incisional pain was established, and remifentanil 1 μg·kg-1·min-1 was infused for 60 min via the caudal vein in group R+ I.Maraviroc 100 pmol (in 10 μl of PBS) was intrathecally injected, then the model of incisional pain was established, and remifentanil 1 μg·kg-1·min-1 was infused for 60 min via the caudal vein in group M+ R+ I.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before infusion of remifentanil or normal saline (T0) and 2, 6, 24 and 48 h after stopping infusion (T1-4). The rats were sacrificed after the last measurement of pain threshold, and L4-6 segments of the spinal cord were removed for determination of the expression of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule-1 (Iba-1) by Western blot. Results Compared with group C, the MWT was significantly decreased and TWL was shortened at T1-4, and the expression of GFAP and Iba-1 in the spinal cord was up-regulated in R+ I and M+ R+ I groups (P 0.05). Compared with group R+ I, the MWT was significantly increased and TWL was prolonged at T1-4, and the expression of GFAP and Iba-1 in the spinal cord was down-regulated in group M+ R+ I (P<0.05). Conclusion Spinal CCR5 is involved in remifentanil-induced hyperalgesia in the rats with incisional pain, and the mechanism may be related to activating astrocytes and microglias. Key words: Piperidines; Hyperalgesia; Pain, postoperative; Receptors, CCR5; Spinal cord

Role of autophagy in remifentanil-induced hyperalgesia in rats with incisional pain

Objective To evaluate the role of autophagy in remifentanil-induced hyperalgesia in the rats with incisional pain. Methods Forty pathogen-free healthy male Sprague-Dawley rats, aged 2-3 months, weighing 250-280 g, were divided into 5 groups(n=8 each)using a random number table: control group(group C), remifentanil group(group R), incisional pain group(group I), incisional pain plus remifentanil group(group IR)and incisional pain plus remifentanil plus autophagy inhibitor 3-methyladenine(3-MA)group(group MA). In group R, remifentanil was infused for 60 min at a rate of 1 μg·kg-1·min-1 through the caudal vein.In group IR, a 1-cm longitudinal incision was made through skin, fascia and muscle of the plantar aspect of both hindpaws in anesthetized rats at 10 min of remifentanil infusion.In group MA, 3-MA 15 mg/kg was injected intraperitoneally at 1 h before establishment of the model, and the other treatments were similar to those previously described in group IR, while the equal volume of normal saline was given in the other groups.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured at 24 h before infusion and 2, 6, 24 and 48 h after infusion(T0-4). The rats were sacrificed after the last measurement of pain threshold at T4, and the L4-6 segments of the spinal cord were removed for determination of the expression of microtubule-associated protein 1 light chain 3 Ⅱ(LC3 Ⅱ), Beclin-1 and P62 by Western blot. Results Compared with group C, the MWT was significantly decreased and the TWL was shortened at T1-4 in R, I, IR and MA groups, the expression of LC3Ⅱ and Beclin-1 was up-regulated, and P62 expression was down-regulated in R and IR groups, the expression of LC3Ⅱ was up-regulated at T4 in group I, and the expression of LC3Ⅱ and Beclin-1 was up-regulated at T4 in group MA(P<0.05). Compared with group R, the MWT was significantly decreased and the TWL was shortened at T1-4, and the expression of LC3Ⅱ was up-regulated at T4 in group IR(P<0.05). Compared with group I, the MWT was significantly decreased and the TWL was shortened at T1-4, and the expression of LC3Ⅱ and Beclin-1 was up-regulated and P62 expression was down-regulated at T4 in group IR(P<0.05). Compared with group IR, the MWT was significantly decreased and the TWL was shortened at T1-4, and the expression of LC3Ⅱ and Beclin-1 was down-regulated and P62 expression was up-regulated at T4 in group MA(P<0.05). Conclusion Autophagy is involved in the development and maintenance of remifentanil-induced hyperalgesia in the rats with incisional pain. Key words: Autophagy; Piperidines; Pain, postoperative; Hyperalgesia

Role of spinal kinesin superfamily motor protein 17 in remifentanil-induced hyperalgesia in rats with incisional pain

Objective To evaluate the role of spinal kinesin superfamily motor protein 17(KIF17)in remifentanil-induced hyperalgesia in rats with incisional pain. Methods Forty male Sprague-Dawley rats, aged 2-3 months, weighing 240-260 g, were divided into 5 groups(n=8 each)using a random number table: control group(group C), remifentanil group(group R), incisional pain group(group I), remifentanil plus incisional pain group(group R+ I)and remifentanil plus incisional pain plus KIF17 inhibitor group(group R+ I+ M). Normal saline 1.5 ml was intravenously infused for 60 min in group C. Remifentanil 1 μg·kg-1·min-1 was intravenously infused for 60 min in group R. In group I, incisional pain model was established, and normal saline 1.5 ml was intravenously infused for 60 min at the same time.In group R+ I, incisional pain model was established, and remifentanil 1.5 ml was intravenously infused for 60 min at the same time.In group R+ I+ M, KIF17 inhibitor Myr-Rc-13 10 μg(in 10 μl dimethyl sulfoxide)was injected intrathecally, and remifentanil 1 μg·kg-1·min-1 was intravenously infused for 60 min while incisional pain model was established.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured at 24 h before remifentanil or normal saline infusion and at 2, 6, 24 and 48 h after the end of infusion.The rats were sacrificed after the last measurement of pain threshold, and the lumbar segment of the spinal cord was removed for determination of the expression of KIF17 and phosphorylated KIF17(pKIF17)by Western blot. Results Compared with group C, MWT was significantly decreased, TWL was shortened, and the expression of KIF17 and pKIF17 was up-regulated in R, I and R+ I groups(P<0.05). Compared with R and I groups, MWT was significantly decreased, TWL was shortened, and the expression of KIF17 and pKIF17 was up-regulated in group R+ I(P<0.05). Compared with group I+ R, MWT was significantly increased, TWL was prolonged, and the expression of KIF17 and pKIF17 was down-regulated in group R+ I+ M(P<0.05). Conclusion Increased activity of KIF17 is involved in the development and maintenance of remifentanil-induced hyperalgesia in rats with incisional pain. Key words: Piperidines; Pain, postoperative; Hyperalgesia; Kinesin; Spinal cord

CXCL13 regulates the trafficking of GluN2B-containing NMDA receptor via IL-17 in the development of remifentanil-induced hyperalgesia in rats

BackgroundThis study aimed to investigate whether CXCL13 modulated the trafficking of NMDA receptor via interleukin (IL)-17 in a rat model of remifentanil-induced hyperalgesia (RIH).Although chemokines are crucial regulators of neuroinflammation, spinal N-methyl-d-aspartate (NMDA) receptor activation, and development of hypernociceptive process, little is known about specific pathogenesis and effective treatment. Inflammatory mediators are required for excitatory synaptic transmission in pathologic pain. MethodsA neutralizing antibody against CXCL13 (anti-CXCL13), antiserum against IL-17 (anti-IL-17), and recombinant CXCL13 and IL-17 were administered intrathecally to explore the roles of CXCL13, IL-17, and NMDA receptor, as well as the prevention of hyperalgesia. Paw withdrawal threshold and paw withdrawal latency were employed to record mechanical allodynia and thermal hyperalgesia. Reverse transcriptase quantitative polymerase chain reaction was used to evaluate the levels of CXCL13/CXCR5 and IL-17/IL-17RA in the spinal dorsal horn. The trafficking of spinal GluN2B-containing NMDA receptor was assessed by Western blot after nociceptive testing. ResultsThis study found mechanical allodynia and thermal hyperalgesia with a remarkable increase in the expression of spinal CXCL13/CXCR5 and IL-17/IL-17RA and trafficking of GluN2B-containing NMDA receptor after remifentanil exposure. Behavioral RIH and elevated GluN2B trafficking were dampened by intrathecal anti-CXCL13 and anti-IL-17, respectively. The delivery of exogenous CXCL13 dose-dependently generated a rapid nociceptive hypersensitivity in naïve rats, which was prevented by coadministering anti-IL-17. CXCL13-induced IL-17RA overproduction and GluN2B trafficking were reversed by anti-IL-17 treatment. GluN2B antagonist also blocked CXCL13, and IL-17 directly induced hyperalgesia. ConclusionThis study highlighted the contribution of IL-17 pathway in the trafficking of CXCL13-induced GluN2B-containing NMDA receptor in the pathogenesis of RIH.

Changes in expression of TRPV1 in dorsal root ganglions during remifentanil-induced hyperalgesia in rats with incisional pain

Objective To evaluate the changes in the expression of transient receptor potential vanilloid l (TRPV1) in dorsal root ganglions (DRGs) during remifentanil-induced hyperalgesia in the rats with incisional pain. Methods Thirty-two SPF healthy male Sprague-Dawley rats, weighing 240-260 g, aged 2-3 months, in which caudal catheters were successfully implanted, were divided into 4 groups (n=8 each) using a random number table: control group (group C), incisional pain group (group I), remifentanil group (group R), and incisional pain + remifentanil group (group I+ R). A 1 cm longitudinal incision was made through skin, fascia and muscle of the plantar aspect of the left hindpaw to establish the model of incisional pain.In group R, remifentanil was intravenously infused for 60 min at a rate of 1.2 μg·kg-1·min-1.In group I, the model of incisional pain was established, and the equal volume of normal saline was intravenously infused for 60 min at the same time.In group I+ R, the model of incisional pain was established, and remifentanil was intravenously infused for 60 min at a rate of 1.2 μg·kg-1·min-1 at the same time.In group C, the equal volume of normal saline was intravenously infused for 60 min.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawl latency (TWL) were measured at 24 h before normal saline or remifentanil infusion (T0) and 2, 6, 24 and 48 h after the end of infusion (T1-4). The rats were sacrificed after the last measurement of pain threshold, and the DRGs of the lumbar segment (L4-6) were removed for determination of the expression of TRPV1 protein and mRNA by Western blot and real-time polymerase chain reaction, respectively. Results Compared with group C, the MWT was significantly decreased, and the TWL was shortened at T1-4, and the expression of TRPV1 protein and mRNA was up-regulated in R, I and I+ R groups (P<0.05). Compared with group R or group I, the MWT was significantly decreased, and the TWL was shortened at T1-4, and the expression of TRPV1 protein and mRNA was up-regulated in group I+ R (P<0.05) Conclusion Up-regulated expression of TRPV1 in DRGs may be involved in the mechanism underlying remifentanil-induced hyperalgesia in the rats with incisional pain. Key words: TRPV cation channels; Piperidines; Hyperalgesia; Ganglia, spinal

Cathepsin S in the spinal microglia contributes to remifentanil-induced hyperalgesia in rats

Cysteine protease Cathepsin S (CatS) expressed by spinal microglia has been shown to play a critical role in nerve injury and inflammation-induced chronic pain. However, whether microglial CatS contributes to remifentanil-induced acute hyperalgesia remains unstudied. In the present study, intravenous remifentanil infusion induced a significant increase in the expression of premature and mature form of CatS in the activated microglia in the spinal cord. Spinal delivery of irreversible CatS inhibitor LHVS reduced hyperalgesia, attenuated activation of spinal microglia and blocked phosphorylation of NMDA receptor NR1 subunit induced by remifentanil. Furthermore, inhibition of microglia by minocycline effectively suppressed remifentanil-induced hyperalgesia, as well as CatS upregulation. In addition, remifentanil infusion also induced an increase in reactive oxygen species (ROS) levels in spinal neurons. Systemic administration of ROS scavenger PBN was sufficient to suppress remifentanil-induced painful hypersensitivity. Removal of ROS by PBN prevented upregulation of mature CatS in spinal microglia. However, increased protein level of premature form of CatS was not affected by PBN. Altogether, our findings demonstrate that neuronal ROS promote maturation of microglial CatS which facilitates activation of NMDA in the spinal dorsal horn. Therefore, such mechanism is involved in neuron-microglia positive feedback and contributes to remifentanil-induced hyperalgesia.

Effect of dexmedetomidine on trafficking of N-methyl-D-aspartate receptors in spinal dorsal horn neurons during remifentanil-induced hyperalgesia in rats with incisional pain

Objective To investigate the effect of dexmedetomidine on trafficking of N-methyl-D-aspartate(NMDA)receptors in spinal dorsal horn neurons during remifentanil-induced hyperalgesia in the rats with incisional pain. Methods Twenty-four male Sprague-Dawley rats, aged 2-3 months, weighing 240-260 g, were randomly divided into 3 groups(n=8 each)using a random number table: control group(group C), remifentanil + incisional pain group(group R+ I), and dexmedetomidine + remifentanil + incisional pain group(group D+ R+ I). A 1 cm longitudinal incision was made in the plantar surface of the left hindpaw to establish the model of incisional pain in anesthetized rats.In group D+ R+ I, dexmedetomidine 50 μg/kg was injected subcutaneously at 30 min before establishment of the model.In R+ I and D+ R+ I groups, remifentanil was infused for 90 min at a rate of 1.2 μg·kg-1·min-1 at the same time as the model was established.In group C, normal saline was infused for 90 min at a rate of 0.12 ml·kg-1·min-1.The thermal paw withdrawal latency(TWL)and mechanical paw withdrawal threshold(MWT)were measured before administration of dexmedetomidine(T0), and at 2, 6, 24 and 48 h after stopping infusion of remifentanil(T1-4). The rats were sacrificed after the last behavioral testing, and the L4-6 segments of the spinal cord were removed for determination of the expression of NMDA receptor NR1, NR2A and NR2B subunits in membrane(m)and total(t)proteins in the spinal dorsal horn by Western blot analysis.The ratios of mNR1/tNR1, mNR2A/tNR2A and mNR2B/tNR2B were calculated. Results Compared with group C, the TWL was significantly shortened, the MWT was significantly decreased, the expression of mNR1, tNR1, mNR2B and tNR2B was significantly up-regulated, the ratios of mNR1/tNR1 and mNR2B/tNR2B were significantly increased(P 0.05). Compared with group R+ I, the TWL was significantly prolonged, the MWT was significantly increased, the expression of mNR1, tNR1, mNR2B and tNR2B was significantly down-regulated, the ratios of mNR1/tNR1 and mNR2B/tNR2B were significantly decreased(P 0.05). Conclusion The mechanism by which dexmedetomidine prevents remifentanil-induced hyperalgesia is related to reduction of trafficking of NR1 and NR2B subunits-containing NMDA receptors in spinal dorsal horn neurons of the rats with incisional pain. Key words: Dexmedetomidine; Piperidines; Receptors, N-methyl-D-aspartate; Pain; Postoperative complications; Hyperalgesia; Spinal cord

Involvement of CCL3/CCR5 Signaling in Dorsal Root Ganglion in Remifentanil-induced Hyperalgesia in Rats.

Several mechanisms of remifentanil-induced hyperalgesia in spinal cord mainly have been explained such as N-methyl-D-aspartate receptors activation, but the mechanism in dorsal root ganglion (DRG) is poorly understood. It has been reported that CCL3 may be a regulator in both inflammatory pain and hyperalgesia. In this paper we explored whether CCL3 and CCR5, the mainly receptor of CCL3, play a role in the remifentanil-induced hyperalgesia in DRG by using a rat model with remifentanil administration. The von Frey test and hot plate test were performed to measure the different threshold to evaluate mechanical and thermal hyperalgesia. Real-time polymerase chain reaction and Western blot analysis were used to evaluate time course of CCL3 and CCR5 expression in DRG after remifentanil infusion. The changes of glial cells and the expression of CCL3 and CCR5 were detected by immunofluorescence assay. Finally, intrathecal injection of CCL3-neutralizing antibody and maraviroc, the CCR5 antagonists, were used sevoflurane to confirm the role of CCL3/CCR5 signaling in the rat model of remifentanil-induced hyperalgesia. Remifentanil infusion profoundly increased thermal and mechanical hyperalgesia from 2 to 48 hours according to paw withdrawal latency (PWL) and paw withdrawal thresholds (PWT) (mean±SD, hyperalgesia vs. control, 17.4±0.91 vs. 8.5±1.46 s; 20.1±0.32 vs. 9.6±0.5 g, n=8). Moreover, the expression of mRNAs and proteins of CCL3 and its receptor CCR5 in DRG were dramatically increased after remifentanil infusion as compared with the normal saline group. We also found that CCL3 and CCR5 were colocalized with glial cells or neurons. Furthermore, intrathecal injection of CCL3-neutralizing antibody (mean±SD, hyperalgesia vs. control, 17.4±0.91 vs. 8.5±1.46 s; 20.1±0.32 vs. 9.6±0.5 g, n=5) and maraviroc(mean±SD, hyperalgesia vs. control, 17.4±0.91 vs. 8.5±1.46 s; 20.1±0.32 vs. 9.6±0.5 g, n=5) were able to suppress remifentanil-related thermal and mechanical hyperalgesia according to behavioral test. The results highlighted the fact that CCL3 and its receptor CCR5 in DRG might contribute to remifentanil-induced hyperalgesia. Thus CCL3/CCR5 signaling may be further considered in the development of new therapeutic strategies.

Effect of dexmedetomidine on spinal p38MAPK expression during remifentanil-induced postoperative hyperalgesia in rats with incisional pain

Objective To evaluate the effect of dexmedetomidine on spinal p38 mitogen-activated protein kinase (p38MAPK) expression during remifentanil-induced hyperalgesia in rats with incisional pain. Methods Forty-eight healthy male Sprague-Dawley rats, aged 6 weeks, weighing 220-250 g, were randomly divided into 4 groups (n= 12 each) using a random number table: control group (group C), incision pain group (group IP), incision pain + remifentanil group (group IP+ R), and incision pain + remifentanil + dexmedetomidine group (group IP+ R+ D). After successful establishment of the model of incisionsal pian, remifentanil 1.0 μg/kg was infused for 4 h via the tail vein in group IP+ R; remifentanil 1.0 μg/kg was infused for 4 h via the tail vein, and dexmedetomidine 10 μg/kg was simultaneously infused for 4 h via the jugular vein in group IP+ R+ D; the equal volume of normal saline was infused for 4 h via the tail and jugular veins in C and IP groups.The mechanical paw withdrawal threshold (MWT) was measured at 24 h before operation (T0), and at 4, 6, 24 and 48 h after the end of drug infusion (T1-4). After measurement of MWT at T4, the expression of p38MAPK was determined using immuno-histochemistry. Results Compared with group C, the MWT was significantly decreased at T1-4, and the expression of p38MAPK was up-regulated at T4 in IP and IP+ R groups (P 0.05). Compared with group IP, the MWT was significantly decreased at T1-4, and the expression of p38MAPK was up-regulated at T4 in group P+ R, and the MWT was significantly increased at T1-4, and the expression of p38MAPK was down-regulated at T4 in group IP+ R+ D (P<0.05). Compared with group IP+ R, the MWT was significantly increased at T1-4, and the expression of p38MAPK was down-regulated at T4 in group IP+ R+ D (P<0.05). Conclusion The mechanism by which dexmedetomidine reduces hyperalgesia induced by remifentanil is related to down-regulation of spinal p38MAPK expression in the rats with incisional pain. Key words: Dexmedetomidine; Pain, postoperative; Hyperalgesia; p38 mitogen-activated protein kinases; Spinal cord

Role of spinal protein kinase C in remifentanil-induced hyperalgesia in rats with incisional pain: the relationship with phosphorylation of NR1

Objective To evaluate the role of spinal protein kinase C(PKC)in remifentanil-induced hyperalgesia in rats with incisional pain and the relationship with phosphorylation of R1 subunit-containing NMDA receptor(NR1). Methods Sixty SPF adult male Sprague-Dawley rats, weighing 220–250 g, were randomly divided into 5 groups(n=12 each)using a random number table: control group(group C), incisional pain group(group I), remifentanil group(group R), Go7874 + incisional pain group(group G), and Go7874 + remifentanil group(group G+ R). Incisional pain was induced by an incision made into the plantar surface of the right hindpaw of anesthetized rats in I, R, G and G+ R groups except group C. In addition, remifentanil(0.04 mg/kg, remifentanil 1 mg in 40 ml of normal saline)was infused subcutaneously over 30 min during operation in R and G+ R groups, PKC inhibitor Go7874 10 μl(12.5 nmol, in 10 μl of 10% dimethyl sulfoxide)were intrathecally injected at 30 min before operation in G and G+ R groups, and 10% dimethyl sulfoxide 10 μl was intrathecally injected in the other groups.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured at 24 h before operation and 2, 6, 24 and 48 h after operation(T0-4). At 24 h after operation, 3 rats in each group were sacrificed, and the L3-5 lumbar segment of the spinal cord was removed for determination of the phosphorylated NR1(p-NR1)expression in the dorsal horn on the right side using Western blot. Results Compared with group C, the MWT was significantly decreased, and TWL was shortened at T1-4 in I, R and G+ R groups, and at T3, 4 in group G, and the expression of p-NR1 was up-regulated at T3 in I, R, G and G+ R groups(P<0.05). Compared with group I, the MWT was significantly decreased, and TWL was shortened at T1-4 in group R, the MWT was increased, and TWL was prolonged at T1-4 in group G, the MWT was increased at T3, 4, and TWL was prolonged at T1-4 in group G+ R, the expression of p-NR1 was up-regulated at T3 in group R, and the expression of p-NR1 was down-regulated at T3 in G and G+ R groups(P<0.05). Compared with group R, the MWT was significantly increased, and TWL was prolonged at T1-4, and the expression of p-NR1 was down-regulated at T3 in group G+ R(P<0.05). Conclusion Spinal PKC is involved in remifentanil-induced hyperalgesia in rats with incisional pain, which is related to decrease in the phosphorylation of NR1. Key words: Protein kinase C; Spinal cord; Piperidines; Pain, postoperative; Hyperalgesia; Receptors, N-methyl-D-aspartate

Changes in expression of spinal p-PKMζ during remifentanil-induced hyperalgesia in rats with incisional pain

Objective To evaluate the changes in the expression of spinal phosphorylated protein kinase Mζ (p-PKMζ) during remifentanil-induced hyperalgesia in rats with incisional pain. Methods Thirty-two heathy male Sprague-Dawley rats, aged 2-3 months, weighing 240-260 g, were randomly dvided into 4 groups (n= 8 each) using a random number table: control group (group C), remifentanil group (group R), incisional pain group (group I), and incisional pain + remifentanil group (group I+ R). In group R, a catheter was inserted into the caudal vein, and remifentanil was infused for 60 min at a rate of 1 μg·kg-1·min-1 via the catheter.A 1 cm longitudinal incision was made in the plantar surface of the left hindpaw to establish the model of incisional pain in anesthetized rats.In group I, incisional pain was produced, and the normal saline equal to the volume of remifentanil was infused via the catheter.In group I+ R, incisional pain was produced, and remifentanil was infused for 60 min at a rate of 1 μg·kg-1·min-1 via the catheter.While the equal volume of normal saline was given in group C. At 24 h before remifentanil infusion, and 2, 6, 24 and 48 h after the end of remifentanil infusion (T0-4), the mechanical paw withdrawal threshold (MWT) and thermal paw withdraw latency (TWL) were measured.The rats were sacrificed after the last measurement of pain threshold, and the lumbar segments (L4-6) of the spinal cord were removed for determination of the expression of PKMζ and phosphorylated PKMζ (p-PKMζ) by Western blot.The ratio of p-PKMζ/PKMζ was calculated. Results Compared with group C, the MWT was significantly decreased, and the TWL was shortened at T1-4, the expression of PKMζ and p-PKMζ was up-regulated at T4, and the ratio of p-PKMζ/PKMζ was increased in R, I and I+ R groups (P<0.05). Compared with I and R groups, the MWT was significantly decreased, and the TWL was shortened at T1-4, the expression of PKMζ and p-PKMζ was up-regulated at T4, and the ratio of p-PKMζ/PKMζ was increased in group I+ R (P<0.05). Conclusion Up-regulated expression of spinal p-PKMζ is involved in the development of remifentanil-induced hyperalgesia in rats with incisional pain. Key words: Piperidines; Pain, postoperative; Hyperalgesia; Protein kinase C; Spinal cord

Changes in expression of PICK1 in spinal cord and dorsal root ganglion neurons during remifentanil-induced hyperalgesia in rats with incisional pain

Objective To evaluate the changes in the expression of protein interacting with Cα kinase 1 (PICK1) in the spinal cord and dorsal root ganglion (DRG) neurons during remifentanil-induced hyperalgesia in rats with incisional pain. Methods Thirty-two male Sprague-Dawley rats, weighing 240-260 g, aged 42-49 days, were randomly divided into 4 groups (n=8 each) using a random number table: control group (group C), incisional pain group (group I), remifentanil group (group R), and remifentanil + incisional pain group (group R + I). In R and R+ I groups, remifentanil was infused intravenously for 60 min at the rate of 1.2 μg· kg-1·min-1.In C and I groups, normal saline was infused intravenously for 60 min at the rate of 0.12 ml·kg-1·min-1.In I and R+ I groups, the model of incisional pain was established, and remifentanil and normal saline were infused intravenously, respectively, at the same time.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before normal saline or remifentanil infusion, and at 2, 6, 24, and 48 h after the end of normal saline or remifentanil infusion (T1-4). The rats were sacrificed after the last measurement of pain threshold.The lumbar segment (L4-6) of the spinal cord and left DRGs were removed for determination of the expression of PICK1 mRNA (by quantitative real-time reverse transcriptase-polymerase chain reaction) and PICK1 protein (by Western blot). Results Compared with group C, the MWT was significantly decreased, the TWL was shortened, and the expression of PICK1 protein and mRNA was up-regulated in R and R+ I groups, and the MWT was significantly decreased, the TWL was shortened (P 0.05). Compared with group I, the MWT was significantly decreased, the TWL was shortened, and the expression of PICK1 protein and mRNA was up-regulated in group R+ I (P 0.05). Conclusion The mechanism by which remifentanil induces hyperalgesia may be related to up-regulation of PICK1 expression in the spinal cord and DRG neurons of rats with incisional pain. Key words: Protein kinase C-alpha; Piperidines; Pain, postoperative; Hyperalgesia; Spinal cord; Ganglia, spinal; Neurons