| Remifentanil is an ultra-short potent ?- opioid receptor agonist and widely used in clinical anesthesia, as it rapid onset, rapid clearance and no accumulation. But it induces rapider and commoner hyperalgesia than other opioid analgesics, which limits the clinical application of remifentanil. Activation of N-methyl-D-aspartate(NMDA) receptors takes a pivotal part in remifentanil-induced hyperalgesia. Like NMDARs, the ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors(AMPARs) are excitatory ion glutamate receptors in postsynaptic membrane which are involved in transmission of both acute and chronic pain. Protein interacting with C kinase 1(PICK1) plays an important role in NMDA receptor-mediated internalization of GluR2-containing AMPARs, and contributes to the induction and maintenance of inflammation-induced pain. The present study aimed to test the hypothesis that PICK1 contributes to remifentanil-induced hyperalgesia by regulating AMPARs expression and trafficking in the spinal cord.Part 1 Changes of PICK1 mRNA and protein in spinal cord neurons in rats with remifentanil-induced hyperalgesiaObjective To investigate the changes of PICK1 mRNA and protein in spinal cord neurons in rats with remifentanil-induced hyperalgesia and disscuss the possible mechanism of PICK1 in remifentanil-induced hyperalgesia.Methods 16 SD male rats(240-260 g, 42 ~ 49 days old) with caudal vein catheter were randomly divided into 2 groups(n=8): Remifentanil group(group R),infused with remifentanil at the rate of 1.2 ?g· kg-1·min-1 for 60min; Control group(group C), infused with saline at the rate of 0.12 ml·kg-1·min-1 for 60 min. The paw withdrawal threshold(PWT) and paw withdrawal latency(PWL) were measured at24 h before saline or remifentanil infusion, and at 2, 6, 24, and 48 h after the end of saline or remifentanil infusion by Von Frey filaments test and Hot Plate test.The rats were sacrificed after the last measurement of pain threshold.The lumbar segment(L4~6) of the spinal cord was removed for determination of the expression of PICKl mRNA( by quantitative real-time reverse transcriptase-polymerase chain reaction)and PICKl protein(by Western blot).Results Compared with group C,the PWT was significantly decreased,the PWL was shortened(P <0.05), and the expression of PICKI mRNA and protein was up-regulated in group R(P <0.05).Conclusion Remifentanil infusion induced distinct hyperalgesia, which may be related to up-regulation of PICK1 expression in the spinal cord neutons of rats.Part 2 Changes of AMPA receptor subunits expression and trafficking in spinal cord neurons in rats with remifentanil-induced hyperalgesiaObjective To investigate the changes of AMPA receptor subunits expression and trafficking in spinal cord neurons in rats with remifentanil-induced hyperalgesia and disscuss the possible mechanism of AMPA receptor in remifentanil-induced hyperalgesia.Methods 16 SD male rats(240-260 g, 42 ~ 49 days old) with caudal vein catheter were randomly divided into 2 groups(n=8): Remifentanil group(group R),infused with remifentanil at the rate of 1.2 ?g· kg-1·min-1 for 60min; Control group(group C), infused with saline at the rate of 0.12 ml·kg-1·min-1 for 60 min. The paw withdrawal threshold(PWT) and paw withdrawal latency(PWL) were measured at24 h before saline or remifentanil infusion, and at 2, 6, 24, and 48 h after the end of saline or remifentanil infusion by Von Frey filaments test and Hot Plate test.The rats were sacrificed after the last measurement of pain threshold.The lumbar segment(L4~6) of the spinal cord was removed for determination of the expression of AMPA receptor subunits expression(by Immunofluorescence staining) and trafficking(by Western blot).Results Compared with group C,the PWT was significantly decreased,the PWL was shortened(P <0.05),the expression of GluR1 and GluR3 subunits positive cells in rats' spinal cord dorsal horn were up-regulated while GluR2 was down-regulated, and the expression in cell membrane of GluR1 and GluR3 were significantly increased while GluR2 was decreased in group R(P<0.05).Conclusion Remifentanil infusion induced distinct hyperalgesia, which may be related to up-regulation of AMPA receptor subunits GluR1 and GluR3 positive cells and down-regulation of GluR2 positive cells and the increased trafficking of GluR1 and GluR3 to membrane and GluR2 to cytoplasm in the spinal cord of rats.Part 3 the effect of PICK1 antisense(AS) oligodeoxynucleotide(ODN) on the expression and trafficking of AMPA receptor subunits in spinal cord neurons in rats with remifentanil-induced hyperalgesiaObjective To investigate the effect of PICK1 on the expression and trafficking of AMPA receptor subunits in spinal cord neurons in rats with remifentanil-induced hyperalgesia by intrathecal administration of PICK1 antisense(AS)oligodeoxynucleotide(ODN).Methods Part A 96 SD male rats(240-260 g, 42~49 days old) with caudal vein catheter were randomly divided into 3 groups(n=32): group NS, group MS and group AS. The intrathecal polyethylene(PE-10) catheter was inserted into the subarachnoid space in all three groups. The rats were allowed to recover for a week before being used experimentally. Rats showing any neurological deficits postoperatively were discarded. Every 24 h for 4 days, group NS was injected intrathecally with 10?l saline while group MS and group AS were separately injected intrathecally with 10?g / 10?l PICK1 missense nucleotide acid(MSODN) and antisense nucleotide acid(ASODN), followed by an injection of 10 ?l of saline to flush the catheter. On the fifth till eighth day, to verify the effect of PICK1 AS ODN,we randomly euthanize 4 rats from each group every day and collected the spinal cord segment L4-6 for western blot. Part B The rest rats in group NS and group AS were randomly divided into 4 groups(n=8): group NS+NS, group NS+R, group AS+NS and group AS+R. group NS+R and group AS+R were infused intravenously remifentanil 1.2 ?g ? kg-1 ? min-1 60 min while group NS+NS and group AS+NS were infused intravenously saline 0.12 ml·kg-1·min-1 for 60 min. Paw withdrawal threshold(PWT) and paw withdrawal latency(PWL) were measured 24 h before and 2, 6, 24,48 h after the infusion of intravenous infusion. The rats were sacrificed after the last threshold measurement. The AMPAR GluR1, GluR2 and GluR3 subunits expressions in rats' spinal cord dorsal horn were determined by immunofluorescence staining.And the cell membrane and intracellular AMPAR GluR1, GluR2 and GluR3subunits' expression in rats' spinal cord were determined by western blot.Results Compared with group NS+NS, PWT was significantly decreased and PWL was significantly shortened, the expression of GluR1 and GluR3 wereup-regulated while GluR2 was decreased, and the amounts of memebane GluR1 and GluR3 increased but memebane GluR2 was decreased(P<0.05)while the amounts of intracellular GluR1,GluR2 and GluR3 have no significant changes in group NS+R and group AS+R. Compared with group NS+R, PWT was decreased and PWL was shortened(P<0.05), the expression of GluR2 was up-regulated while GluR3 was down-regulated(P<0.05)while GluR1 has no significant change and the amounts of surface GluR2 proteins increased but GluR3 decreased(P<0.05)while the amounts of surface GluR1, intracellular GluR1, GluR2 and GluR3 have no significant changes in group AS+R.Conclusion PICK1 deficiency might partly reverse remifentanil-induced hyperalgesia, through regulating AMPA receptor subunits GluR2 and GluR3 expression and trafficking in spinal cord in a rat model of remifentanil-induced hyperalgesia, but has no effect on GluR1 subunit. |
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