The Mechanism Of Remifentanil Induced Hyperalgesia Modulated By Wnt/?-catenin-NMDA Signaling Pathway

This study demonstrated that remifentanil could induce hyperalgesia in rat,characterized by decreased paw withdrawal mechanical thresholds and paw withdrawal thermal latency to mechanical and thermal stimulation.Single remifentanil could even produced more severe thermal hyperalgesia in the rats.Both of the remifentanil and surgical stimulus could enhance the expression of the c-fos.NMDA receptors,such as ketamine and magnesium sulphate could inhibit remifentanil induced hyperalgesia.Ketamine was more in inhibiting thermal hyperalgesia and mechanical allodynia in comparison with magnesium sulphate.Both of ketamine and magnesium sulphate could dose-dependently inhibit hyperalgesia.1 week later from the initial exposure of NMDA receptor antagonists,remifentanil induced hyperalgesia could not been suppressed by the previous infusion of ketamine or magnesium sulphate.NR2 B played a key role in remifentanil induced hyperalgesia.NMDA receptors could inhibit phosphorylation of NR2 B subunit of NMDA receptor.The following research have revealed that remifentanil could activate the wnt/?-catenin and enhance the expression of wnt3,wnt5 and ?-catenin.Through the inhibition of wnt activation,both of NMDA receptor NR2 B subunit antagonists Ro25-8961 and wnt scavenger iwp-2 inhibit the expression of wnts in the dorsal neuron in the lumber spinal cord,and inhibit the following pain related molecular in the wnt signaling pathway.Immunochemistry test revealed that wnt scavenger iwp-2 could inhibit more in the number of activated wnt-3 in the dorsal neuron of the lumber spinal cord,however,Ro25-8961 could have better effect in inhibiting hyperalgesia in the behavior tests,including higher paw withdrawal mechanical thresholds and longer paw withdrawal thermal latency to mechanical and thermal stimulation.Which suggested that Wnt/?-catenin took part in the progress of remifentanil induced hyperalgesia,but Wnt/?-catenin was not the sole pathway.The research explored the remifentanil induced hyperalgesia via molecular(NR2B subunit),cellular(dorsal neuron),comprehensive(sensory level)level by behavioral tests,Western-Blot?immunochemistry.Part 1: Establishment of the remifentanil induced hyperalgesia in the ratsPurpose: To explore the hyperalgesia effect by remifentanil infusion.Establishment of animal mode of remifentanil infusion induced hyperalgesia.Method: 24 SD rats were enrolled and divided into 4 groups: controlled group,single remifentanil infusion group,single surgical incision group and combined remifentanil and surgical incision.Remifentanil infusion induced hyperalgesia was observed by behavioral tests,including thermal hyperalgesia,mechanical allodynia,detecting the amount of c-fos,detecting the amount of activated NR2 B subunit in the lumber spinal cord.Result: Remifentanil administration plus surgical incision induced significant postoperative hyperalgesia,as indicated by decreased paw withdrawal mechanical thresholds and paw withdrawal thermal latency to mechanical and thermal stimulation.There was no difference in the c-fos amount in these two groups.The group of remifentanil infusion combined with surgical incision induced most severe mechanical allodynia and thermal hyperalgesia,had highest level of c-fos amount.Conclusion: The group of remifentanil infusion combined with surgical incision produced most severe hyperalgesia and highest level of c-fos.Interestingly,single remifentanil infusion induced more severe thermal hyperalgesia than single surgical incision group.There were no statistical differences in the other hyperalgesia related parameters.Part 2:The effect of the NMDA receptor antagonists,ketamine and magnesium sulphate,on the remifentanil induced hyperalgesia in the ratsPurpose: Activation of NMDA receptors play an important role in the development of remifentanil-induced hyperalgesia.We hypothesized that in addition to ketamine,intrathecal Mg SO4 could also relieve thermal and mechanical hyperalgesia in rats.Methods: 40 rats were divided into control group,model group,model group plus 100 ?g Mg SO4,300?g Mg SO4 and 10?g ketamine respectively.Paw withdrawal mechanical thresholds and paw withdrawal thermal latency tests were performed at-24 h,2 h,6 h,24 h,48 h,72 h and 7 day after the surgical procedure.After behavior assessment on the 7th day,remifentanil was given again to ascertain whether or not NMDA antagonists could suppress the re-exposure of remifentanil-induced hyperalgesia.Results: In addition to ketamine,intrathecal Mg SO4(100,300 ?g)dose-dependently reduced remifentanil-induced mechanical and thermal hyperalgesia.Ketamine had less mechanical hyperalgesia in 6 h(p = 0.018),24 h(p = 0.014)and 48 h(p = 0.011)than 300 ?g Mg SO4.There was no difference in inhibiting thermal hyperalgesia between the group ketamine and group Mg SO4(300 ?g).The rats were given remifentanil again 7 days later after the first exposure of remifentanil.The hyperalgesic effect induced by re-exposure of remifentanil was not reversed in any groups of Mg SO4 or ketamine.Remifentanil infusion remarkably stimulated the expression of p NR2 B.Nevertheless,the increased amount of p NR2 B by RIH was dose-dependently suppressed by intrathecal infusion of Mg SO4 in rats.Conclusions: In addition to ketamine,intrathecal administration of Mg SO4 dose-dependently reduced remifentanil induced hyperalgesia in a surgical incision mode.Re-exposure to remifentanil 1 week later again produced hyperalgesia,and this was not altered by the prior intrathecal treatments in any 4 groups treated with Mg SO4 or ketamine.Remifentanil induced hyperalgesia/allodynia could be ameliorated by targeting the Mg-mediated,or ketamine blockade of NMDA receptors.It provides a new therapeutic choice for the management of remifentanil induced hyperalgesia.Part 3: The effect of the wnt/?-catenin-NMDA signaling pathway on remifentanil induced hyperalgesiaPurpose: To explore the effect of wnt/?-catenin signal pathway on remifentanil induced hyperalgesia.Method: SD rats were enrolled and divided into 4 groups: controlled group,remifentanil induced hyperalgesia(RIH)group,RIH plus NR2 B subunit antagonist Ro25-8961 group,and RIH plus wnt scavenger(iwp-2)group.The effect was observed by behavioral tests,including thermal hyperalgesia,mechanical allodynia,detecting the amount of activated NR2 B subunit and wnt3 in the lumber spinal cord,detecting the location of the activated wnt-3 in the dorsal spinal horn.Result: Remifentanil infusion could enhance the expression of wnt3 and wnt5 activation in the spinal cord.Both of iwp-2 and Ro25-8961 could suppress the amount of activated NR2 B and wnt3 in the lumber spinal cord,and the down-stream pain related molecular in the wnt/?-catenin signal pathway.The group of Ro25-8961 had more benefit in the behavioral tests,including higher threshold of thermal hyperalgesia and mechanical allodynia compared with group iwp-2.Immunochemistry test detect the enhancement of expression of the wnt3 in the dorsal horn of the lumber spinal cord.Both of iwp-2 and Ro25-8961 could suppress the amount of activated wnt3 in dorsal horn of the lumber spinal cord.Conclusion: Remifentanil infusion could enhance the expression of wnt3 and wnt5 activation in the dorsal neuron of spinal cord.Both of Ro25-8961 and iwp-2 could suppress the amount of activated NR2 B,wnt3 and ?-catenin in the dorsal lesion of lumber spinal cord.The group of Ro25-8961 had more benefit in the behavioral tests,including higher threshold of thermal hyperalgesia and mechanical allodynia compared with group iwp-2.Remifentanil could enhance the expression of the wnt3 in the dorsal horn of the lumber spinal cord.Both of iwp-2 and Ro25-8961 could suppress the amount of activated wnt3 in dorsal horn of the lumber spinal cord.