The Mechanisms Of PKMζ-Kalirin-7 Mediated Synaptic Plasticity In The Pathogenesis Of Remifentanil-induced Hyperalgesia In Rats

Opioids have been widely engaged in controlling acute and chronic pain and cancer pain.Although opioids exposure initially offers analgesia,they may cause hyperalgesia,which limits their use in clinic settings.Remifentanil,as a potent short-actingμ-opioid,is a clinical balanced anesthesia component.However,numerous literature report that the incidence of remifentanil-induced hyperalgesia（RIH）seems higher than other opioids,likely due to its rapid offset.Therefore,it is of importance to clarify the specific molecular mechanism of RIH and explore the effective management for RIH.Protein kinase Mζ（PKMζ）,constitutively active form of the atypical PKC,can sustain the synaptic potentiation.PKMζhas been demonstrated to be involved in the pathogenesis of neuropathic pain and inflammatory pain,via modulating synaptic structural plasticity and functional plasticity induced and maintained by AMPA receptor activity.Kalirin,a family of Rho guanine nucleotide exchange factors in excitatory synapse,plays an important role in dendritic spine morphology,synaptic development and learns and memory.Kalirin-7 is strongly associated with AMPA receptor pathway.Recent study has found that Kalirin-7 is also implicated in nociception-related excitatory synaptic plasticity,which is indispensable to the generation and maintenance of pain hypersensitivity.Thus,we hypothesize that PKMζcontributes to RIH via Kalirin-7 mediated spine plasticity and AMPA receptor function.In this study,we evaluate how PKMζand Kalirin-7 regulate spinal dorsal horn excitatory synaptic structural and functional plasticity in the development of RIH using incision-RIH model in vivo and in vitro.This study may expedite the development of novel therapies for the treatment of RIH in clinics.Objective To investigate changes of PKMζactivity、Kalirin-7 expression and AMPAR trafficking in spinal dorsal horn in RIH and discuss the possible mechanism of postoperative RIH;To investigate effects of PKMζon Kalirin-7 expression and AMPAR trafficking in spinal dorsal horn in RIH after intrathecal PKMζinhibitor ZIP injection;To investigate effects of Kalirin-7 on behavioral hyperalgesia and spinal AMPAR trafficking in RIH through central kalirin-7 inhibition with intrathecal kalirin-7-shRNA injection;To investigate effects of GluA1 on behavioral RIH after intrathecal GluA1 selective antagonist NASPM injection;To investigate effects of PKMζand Kalirin-7 on dendritic spine morphology of spinal dorsal horn in RIH after central PKMζand Kalirin-7 inhibition;To investigate effects of PKMζand Kalirin-7on AMPA receptor-mediated mEPSCs after remifentanil incubation.MethodsⅠ44 SD male rats（240²60 g）were randomly divided into 4 groups（n=11each）:C group（control group）:underwent the infusion of normal saline 0.1ml·kg^-1·min^-1,60min;I group（incision group）:underwent incision surgery;R group（remifentanil group）:underwent the infusion of remifentanil 1.0μg·kg^-1·min^-1,60min;IR group（incision+remifentanil）:underwent incision surgery and infusion of remifentanil 1.0μg·kg^-1·min^-1,60min.PWT and PWL were measured at 1d before and2 h、6 h、1 d、2 d、3 d、5 d and 7 d after infusion（n=7 each）.The L₄-₆ segments of dorsal horn（n=4 each）were collected on 2d after remifentanil infusion for determining PKMζactivity,Kalirin-7 expression and AMPAR trafficking using IHC and Western blot.Ⅱ66 SD male rats（240²60 g）were randomly divided into 6 groups（n=11each）:C group（control group）:underwent the infusion of normal saline 0.1ml·kg^-1·min^-1,60min;IR group（incision+remifentanil）:underwent incision surgery and infusion of remifentanil 1.0μg·kg^-1·min^-1,60min;C+Z₃ group:intrathecal ZIP 100nmol before the infusion of normal saline;Z₁,Z₂,Z₃ group:intrathecal ZIP 1nmol,10nmol,100nmol before remifentanil anesthesia and incision.PWT and PWL were measured at 1d before and 2 h、6 h、1 d、2 d、3 d、5 d and 7 d after infusion（n=7 each）.The L₄-₆ segments of dorsal horn（n=4 each）were collected on 2d after remifentanil infusion for determining Kalirin-7 expression and AMPAR trafficking using Western blot.Ⅲ44 SD male rats（240²60 g）were randomly divided into 4 groups（n=11each）:C group（control group）:underwent the infusion of normal saline 0.1ml·kg^-1·min^-1,60min;IR group（incision+remifentanil）:underwent incision surgery and infusion of remifentanil 1.0μg·kg^-1·min^-1,60min;C+kalirin-7-shRNA group:the infusion of normal saline at 2 weeks after intrathecal kalirin-7-shRNA（10μL;3×10⁸transducing units[TU]/mL）;IR+kalirin-7-shRNA group:remifentanil and incision exposure at 2 weeks after intrathecal kalirin-7-shRNA（10μL;3×10⁸ transducing units[TU]/mL）.PWT and PWL were measured at 1d before and 2 h、6 h、1 d、2 d、3 d、5 d and 7 d after infusion（n=7 each）.The L₄-₆ segments of dorsal horn（n=4 each）were collected on 2d after remifentanil infusion for determining PKMζactivity and AMPAR trafficking using Western blot.Ⅳ66 SD male rats（240²60 g）were randomly divided into 6 groups（n=11each）:C group（control group）:underwent the infusion of normal saline 0.1ml·kg^-1·min^-1,60min;IR group（incision+remifentanil）:underwent incision surgery and infusion of remifentanil 1.0μg·kg^-1·min^-1,60min;C+N₃ group:intrathecal NASPM 100μg before the infusion of normal saline;N₁,N₂,N₃ group:intrathecal NASPM 1μg,10μg,100μg before remifentanil anesthesia and incision.PWT and PWL were measured at 1d before and 2 h、6 h、1 d、2 d、3 d、5 d and 7 d after infusion（n=7 each）.The L₄-₆ segments of dorsal horn（n=4 each）were collected on 2d after remifentanil infusion for determining AMPAR trafficking using Western blot.Ⅴ16 SD male rats（240²60 g）were randomly divided into 4 groups（n=4each）:C group（control group）:Normal saline 0.1 ml·kg^-1·min^-1,1h,i.v.;IR group（incision+remifentanil）:underwent incision surgery and infusion of remifentanil1.0μg·kg^-1·min^-1,60min;IR+ZIP group:intrathecal ZIP 100nmol before remifentanil anesthesia and incision.IR+kalirin-7-shRNA group:remifentanil and incision exposure at 2 weeks after intrathecal kalirin-7-shRNA（10μL;3×10⁸ transducing units[TU]/mL）.The L₄-₆ segments of dorsal horn were collected on 2d after remifentanil infusion for determining spine length and number using Golgi staining.Ⅵ16 SD male rats（5⁷weeks）were randomly divided into 4 groups（n=4each）:C group:Artificial cerebrospinal fluid（ACSF）was employed to incubate spinal cord slices（L₄-₅）;R group:ACSF containing remifentanil 4nM was employed to incubate spinal cord slices;ZIP group:ACSF containing remifentanil 4nM and ZIP5μM was employed to incubate spinal cord slices;kalirin-7-shRNA group:ACSF containing remifentanil 4nM was employed to incubate spinal cord slices at 2 weeks after intrathecal kalirin-7-shRNA（10μL;3×10⁸ transducing units[TU]/mL）.We employed the whole cell patch clamp technique to assess AMPAR electrophisological function.ResultsⅠCompared with C group,rats in I,R and IR group showed a significant decrease in PWT and PWL;increase in PKMζphosphorylation and Kalirin-7 expression;increase in GluA1 expression and trafficking（P<0.05）.Compared with I group,rats in IR group showed a significant decrease in PWT and PWL;increase in PKMζphosphorylation and Kalirin-7 expression;increase in GluA1 expression and trafficking（P<0.05）.There are no significant changes in the levels of GluA2 expression and trafficking in all groups（P>0.05）.ⅡCompared with C group,rats in IR,Z₁,Z₂ and Z₃ group showed a significant decrease in PWT and PWL;increase in PKMζphosphorylation and Kalirin-7 expression;increase in GluA1 expression and trafficking（P<0.05）.Compared with IR group,rats in Z₂ and Z₃ group showed a dose-dependently increase in PWT and PWL;decrease in PKMζphosphorylation and Kalirin-7 expression;decrease in GluA1 expression and trafficking（P<0.05）.ⅢCompared with C group,rats in IR and IR+kalirin-7-shRNA group showed a significant decrease in PWT and PWL;increase in PKMζphosphorylation;increase in GluA1 expression and trafficking（P<0.05）.Compared with IR group,rats in IR+kalirin-7-shRNA group showed an increase in PWT and PWL;decrease in GluA1expression and trafficking（P<0.05）;but not change in PKMζphosphorylation.ⅣCompared with C group,rats in IR,N₁,N₂ and N₃ group showed a significant decrease in PWT and PWL;increase in GluA1 expression and trafficking（P<0.05）.Compared with IR group,rats in N₂ and N₃ group showed a dose-dependently increase in PWT and PWL;decrease in GluA1 trafficking（P<0.05）.ⅤCompared with C group,rats in IR,IR+ZIP and IR+kalirin-7-shRNA group showed a significant increase in the number and length of spine（P<0.05）.Compared with IR group,rats in IR+ZIP and IR+kalirin-7-shRNA group showed a decrease in the number and length of spine（P<0.05）.ⅥCompared with C group,rats in R,ZIP and kalirin-7-shRNA group showed a significant increase in the amplitude and frequency of mEPSCs（P<0.05）.Compared with R group,rats in ZIP and kalirin-7-shRNA group showed a decrease in the amplitude and frequency of mEPSCs（P<0.05）.Conclusion Remifentanil anesthesia may aggravate incision-induced postoperative hypernociception;Upregulation of PKMζactivity,Kalirin-7 expression and AMPAR trafficking from cytoplasm to post-synaptic membrane surface may be involved in the development of RIH;Pharmacologic suppression of central PKMζimpairs RIH in rats,via down-regulating Kalirin-7 expression and AMPAR trafficking;Spinal kalirin-7 knock-down impairs RIH in rats,via down-regulating AMPAR trafficking;Inhibition of spinal GluA1 trafficking impairs remifentanil-induced mechanical and thermal hyperalgesia in rats;Remifentanil anesthesia activates spinal PKMζand Kalirin-7 to regulate dendritic spine structure plasticity,which involves the development of RIH;Remifentanil anesthesia up-regulates the activity of spinal PKMζand Kalirin-7 to mediate excitatory glutaminergic synaptic functional plasticity,which is central for the development of RIH.