| Opioids have been regarded as the most effective analgesics for management of acute or chronic pain and cancer pain. However, they also produce a lot of adverse effects, including hyperalgesia, which limits their usefulness. Remifentanil is a potent short-acting μ-opioid receptor agonist, as it is a rapid-onset opioid and has short duration, remifentanil have been widely used as anagesic in clinical practice. Many researches show that the incidence rate of remifentanil-induced hyperalgesia is significantly higher than other opioids. There is increasing evidence that N-methyl-d-aspartate(NMDA) receptor play a critical role in the development of opioid-induced hyperalgesia. However, the change of NMDA receptor subunits trafficking in remifentanil-induced hyperalgesia has not been reported yet.Glycogen synthase kinase3(GSK-3) is a multifunctional serine/threonine protein kinase, ubiquitous in eukaryotes. GSK-3contained two subtypes GSK-3a and GSK-3β in mammals. The main function of this enzyme includes regulating the synthesis of glycogen metabolism, participate in cell differentiation and proliferation. Recent studies have found that, GSK-3affects synaptic plasticity, and has an important role in mediating NMDA receptor’s trafficking that the application of GSK-3inhibitors can inhibit the NMDA receptor expression in the postsynaptic membrane. But in remifentanil-induced hyperalgesia model, the change of GSK-3has not been studied systematically.In this study, using a rat model of postoperative pain, we investigated the changes of GSK-3(3expression and NMDA receptor subunits (NR1, NR2A, and NR2B) trafficking in rats’spinal cord when remifentanil-induced hyperalgesia developed. We also examined the impact of TDZD-8, a GSK-3(3inhibitor, on NMDA receptor subunits trafficking, and the impact of naltrindole, a DOR inhibitor, on GSK-3β expression in rats’spinal cord when remifentanil-induced hyperalgesia developed.Part1Changes of glycogen synthase kinase-3β mRNA in spinal cord neurons in rats with incisional pain (IP)-remifentanil-induced hyperalgesia Objective To investigate the changes in GSK-3β mRNA in spinal cord neurons in rats with IP-remifentanil-induced hyperalgesia.Methods32SD male rats(240-260g) were randomly divided into4groups (n=8each):group R, remifentanil (1.2μg·kg-1·min-1); group I, IP+the same volume of saline infused; group R+I, IP+remifentanil; group C, the same volume of saline. IP was established as Brennan’s description. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured24h before anesthesia and2h,6h,24h,48h after anesthesia. The rats were sacrificed after the last threshold measurement. The expressions of GSK-3β mRNA in rats’spinal cord neurons were determined by real-time PCR.Results Remifentanil-induced hyperalgesia developed in group R, I and R+I. The expression of GSK-3(3mRNA in rats’spinal cord neurons was highest in group R+I.Conclusion These data indicate that the increased GSK-3β mRNA in rats spinal cord neurons is involved in remifentanil-induced hyperalgesia.Part2Changes in N-methyl-d-aspartate receptor subunits trafficking in spinal cord neurons in rats with IP-remifentanil-induced hyperalgesiaObjective To investigate the changes in N-methyl-d-aspartate receptor (NMDAR) subunits trafficking in spinal cord neurons in rats with IP-remifentanil-induced hyperalgesia.Methods32SD male rats(240-260g) were randomly divided into4groups (n=8each):group R, remifentanil (1.2μg·kg-1·min-1); group I, IP+the same volume of saline; group R+I, IP+remifentanil (1.2μg·kg-1·min-1); group C, the same volume of saline. IP was established as Brennan’s description. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured24h before anesthesia and2h,6h,24h,48h after anesthesia. The rats were sacrificed after the last threshold measurement. The surface and intracellular NMDAR subunits (NR1, NR2A, and NR2B) expressions in rats’spinal cord neurons were determined by western blot.The ratio of sNRl/iNRl, sNR2A/iNR2A and sNR2B/iNR2B were calculated.Results Remifentanil-induced hyperalgesia developed in group R, I and R+I. The amount of surface NR1and NR2B protein increased in group R, I and R+I when compared with group C (P<0.05). As opposed to this increase in the surface pool, the amounts of intracellular NR1and NR2B in group R, I and R+I decreased when compared with group C (P<0.05). The ratio of sNRl/iNRl and sNR2B/iNR2B markedly increased in group R, I and R+I when compared with group C (P<0.05). The amount of surface NR1and NR2B protein increased, the amounts of intracellular NR1and NR2B decreased, the ratio of sNRl/iNRl and sNR2B/iNR2B markedly increased in group R+I when compared with group R and group I, respectively (P<0.05). NR2A showed no significant changes in either surface or intracellular pool (P>0.05).As a result, the ratio of sNR2A/iNR2A remained unchanged (P>0.05).Conclusion These data indicate that the increased trafficking of NMDA receptors NR1and NR2B subunits from intracellular pool to surface pool in rats spinal cord neurons is involved in remifentanil-induced hyperalgesia.Part3Effect of TDZD-8on NMDA receptor subunits (NRl and NR2B) trafficking in spinal cord in IP-remifentanil-induced hyperalgesiaObjective To investigate the regulation of Glycogen synthase kinase-3β on N-methyl-d-aspartate receptor NR1and NR2B subunits trafficking in spinal cord in a rat model of IP-remifentanil-induced hyperalgesia.Methods Twenty-four male SD rats(240-260g,2-3months old) in which caudal vein catheter was successfully placed were randomly divided into3groups (n=8each):group C:DMSO+the same volume of saline; group R+I:DMSO+remifentanil+IP; group TDZD-8:TDZD-8+remifentanil+IP. TDZD-8(1mg/kg)/DMSO (2ml/kg) was infused intravenously just before remifentanil(1.2μg·kg-1·min-160min) infusion, IP was established as Brennan’s description. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured24h before and2,6,24,48h after the infusion of intravenous remifentanil. The rats were sacrificed after the last threshold measurement. The surface and intracellular NMDAR NR1and NR2B subunits expressions in rats’spinal cord were determined by western blot. The ratio of sNR1/iNR1and sNR2B/iNR2B were calculated.Results Compared with group C, PWT and PWL decreased, the amounts of surface NR1and NR2B proteins increased, the amounts of intracellular NR1and NR2B proteins decreased, the ratio of sNR1/iNR1and sNR2B/iNR2B increased in group R+I and group TDZD-8(P<0.05). Compared with groupR+I, PWT and PWL increased, the amounts of surface NR1and NR2B proteins decreased, the amounts of intracellular NR1and NR2B proteins increased, the ratio of sNR1/iNR1and sNR2B/iNR2B decreased in group TDZD-8(P<0.05)Conclusion Glycogen synthase kinase-3β was involved in the regulation of N-methyl-d-aspartate receptor NR1and NR2B subunits trafficking in spinal cord in a rat model of IP-remifentanil-induced hyperalgesia.Part4Effects of naltrindole on the changed expression of GSK-3β mRNA in spinal cord in IP-remifentanil-induced hyperalgesiaObjective To investigate the regulation of delta-opioid receptor (DOR) on the changed expression of GSK-3β mRNA in spinal cord in IP-remifentanil-induced hyperalgesia.Methods Twenty-four male SD rats(240-260g,2-3months old) in which caudal vein catheter was successfully placed were randomly divided into3groups (n=8each):group C:saline+the same volume of saline; group R+I:saline+remifentanil+IP; group naltrindole:naltrindole+remifentanil+IP. Naltrindole (0.1mg/kg)/saline was administered intraperitoneally just before remifentanil(1.2μg·kg-1·min-160min) infusion, IP was established as Brennan’s description. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured24h before and2,6,24,48h after the infusion of intravenous remifentanil. The rats were sacrificed after the last threshold measurement. The expressions of GSK-3β mRNA in rats’ spinal cord neurons were determined by real-time PCR.Results Compared with group C, PWT and PWL decreased, the expressions of GSK-3β mRNA increased in group R+I and group naltrindole (P<0.05). Compared with groupR+I, PWT and PWL increased, the expressions of GSK-3β mRNA decreased in group naltridole (P<0.05)Conclusion Delta-opioid receptor was involved in the regulation of the expression of GSK-3β mRNA in spinal cord in a rat model of IP-remifentanil-induced hyperalgesia. |
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