The Mechanisms Of Kalirin-7-shank3-GluA1 Membrane Insertion In Remifentanil Induced Hyperalgesia After Second Surgery

There were clinical studies found that Visual Analogue Score（VAS）after the second surgery was higher than that after the first.And the dose of postoperative analgesics was also increased after the second surgery.Remifentanil is an ultra-short-acting opioid agonist which is widely used in clinical work.It has many advantages,such as rapid onset,rapid metabolism and little accumulation in the body.But it is worth noting that remifentanil could induce hyperalgesia（RIH）after surgery.To alleviate patients’postoperative pain and promote recovery,the effect of remifentanil anesthesia on the second postoperative pain should be clarified.Recent studies have found that GluA1,the subnit of AMPA receptor,membrane trafficking is involved in RIH,but the regulation mechanism of GluA1 is blear.Shank3 protein is a kind of multidomain scaffold protein in the PDZ domain,which can bind multiple postsynaptic proteins and become the assembly platform of postsynaptic assembly.GluA1 Shank3PDZ domain binds to GluA1 carboxy-terminal PDZ binding motif to regulate GluA1memburanetraffickingandneurodevelopment.Enhancement of synaptic plasticity is a key point to central sensitization which could leading to hyperalgesia.Kalirin-7 is a multifunctional guanine nucleotide exchange factor（GEF）which located in the post excitatory synapses.It can interact with glutamate receptors and have the ability to regulate the neural cytoskeleton dynamicly.Spinal Kalirin-7 is essensial for pain activity-dependent long-term potentiation（LTP）and synapse remodeling.This study was designed to perform Brennan incision twice under remifentanil aneathesia on Sprague Dawley（SD）rats under a short period interval to observe the effect of remifentanil on postoperative pain and try to explain the spinal mechnism of the occurrence.Part 1:Comparison of postoperative pain in rats after the first and second incision and investigating the effects of remifentanil on the postoperative painObjective:To compare the postoperative pain in rats after the first and second incision and to investigate the effects of remifentanil on the postoperative pain.Method:Male SD rats underwent two set treatmens with 8 days interval.The set treatmens were NS（normal saline）:sham surgery and intravenous infusion of normal saline for 60 min at a rate of 0.1 mL/（kg·min）,I（incision）:Brennan surgery,R（remifentanil）:intravenous infusion of remifentanil at a rate of 1μg/（kg·min）for 60min,IR（incision+remifentanil）:Incision pain surgery was performed,and remifentanil was intravenously infused at a rate of 1μg/（kg·min）for 60 min.According to different treatments,the rats were divided into 6 groups（N=48,N=8）:（1）Control:NS&NS;（2）I&I;（3）IR&NS;（4）IR&I;（5）IR&R;（6）IR&IR.Two set treatments were indicated before and after the symbol“&”.Paw withdrawal threshold（PWT）and paw withdrawal latency（PWL）were measured 2 hours（h）,6h,1 day（d）,2d,3d,5d and 8d after the first treatment and 2h,6h,1d,2d,3d,7d 14d,and 18d after the second treatment.The changes of pain threshhold of rats in every group were observed after two treatments.Results:PWTs and PWLs in I&I group spent 5 days after the first surgery and 7 days after the second surgery to recover to baseline（BS）.The lowest PWT and PWL values in I&I group after the second surgery were lower than those after the first surgery（P<0.05）.Compared with I&I group,PWTs and PWLs in group IR&I,IR&R and IR&IR decreased after the first and second treatments（P<0.05）.PWT and PWL of IR&IR group were lower at 6h after the second surgery than 6h after the first surgery（P<0.05）.The lowest values of PWT and PWL after the first treatment in IR&IR group appeared on the second day after the surgery,and the lowest values of PWT and PWL after the second treatment appeared on the first day after the surgery.The lowest PWT and PWL values after the second surgery in IR&IR group were lower than those after the first surgery（P<0.05）.Conclusion:The pain was worse after the second surgery than the first.Remifentanil aggravated postoperative pain and induced second hyperalgesia.Part 2:Effects of GluA1 membrane trafficking on the neuronal plasticity in the spinal dorsal horn of rats with remifentanil induced hyperalgesia after second surgeryObjective:To observe the GluA1 expression and neuronal plasticity plasticity in the spinal dorsal horn（SDH）of rats with remifentanil induced hyperalgesia after second surgery（SRIH）.Method:Rats in this part of the Control group,IR&NS group,IR&I group,IR&R group and IR&IR group were treated as the first part.Section1:1)The L₄-L₆ SDHs of IR&IR rats were taken（n=8 at each time point）at 2h,6h,1d,3d,7d,14d,and 18d after the second intravenous administration to explore the expression of GluA1.2)At the first day after second intravenous administration,the SDHs of L₄-L₆ in C group,IR&NS group,IR&I group,IR&R group,and IR&IR group were obtained for Western Blot（WB）to observe the expression of GluA1.3)At the first day after the second intravenous administration,the C fiber evoked potential was tested on the rat in C group and IR&IR group.The L₄-L₆ were also obtain for Golgi staining and recording the AMPA receptor-mediated microexcitatory postsynaptic currents（mEPSC）.Section2:Male SD rats were randomly divided into 4 groups.（1）C group;（2）IR&IR group;（3）NASPM group:Rats in this group were given 100μg AMPA receptor antagonist NASPM by intrathecal administration;（4）IR&IR+NASPM group:Rats experienced the same treatment as the rats in IR&IR group,but 100μg NASPM was given intrathecally before the second intravenous administration.1)PWTs and PWLs were measured before and after the treatment.2)At the fist day after second intravenous administration,the SDHs of L₄-L₆ in group IR&IR and IR&IR+NASPM were obtained for WB to observe the expression and membrane trafficking of GluA1.3)At the first day after the second intravenous administration,the C fiber evoked potential was tested on the rats in IR&IR group and IR&IR+NASPM group.The L₄-L₆were also obtain for Golgi staining and recording the AMPA receptor-mediated mEPSC.Results:In IR&IR rats GluA1 membrane trafficking was the most at the first day after second intravenous administration.Compared with C group,IR&NS group,IR&I group,and IR&R group,GluA1 membran trafficking in SDH was increased in group IR&IR（P<0.05）.Compared with C group,in IR&IR group the primary dendritic branches,dendritic spine density and length of SDH neurons increased,the C fiber evoked potential increased,and the frequency and amplitude of AMPA receptor-mediated mEPSC increased（P<0.05）.Compared with IR&IR group,in group IR&IR+NASPM group the PWTs and PWLs increased after the second intravenous administration,the pain threshold returned to baseline time ealier,the primary dendritic branches,dendritic spine density and length of SDH neurons decreased,the C fiber evoked potential of decreased,and the frequency and amplitude of AMPA receptor-mediated mEPSC decreased（P<0.05）.Conclusion:GluA1 membrane trafficking in the SDH of SRIH rats were increased,which enhanced the structural and functional plasticity of neurons and promoted the occurrence of SRIH.Part 3:Effects of Shank3 protein on the neuronal plasticity in SDH of rats with SRIH and the regulation of GluA1 membrane trffickingObjective:To observe the expression of Shank3 in SDH of rats with SRIH,and to explore the effect of Shank3 on SRIH.Method:Rats in this part of the Control group,IR&NS group,IR&I group,IR&R group and IR&IR group were treated as the first part.Section1:1)The L₄-L₆ spinal dorsal horns（n=8 at each time point）of IR&IR rats were taken at 2h,6h,1d,3d,7d,14d,and 18d after the second intravenous administration to explore the expression of Shank3.2)At the first day after second intravenous administration,the SDHs of L₄-L₆ in C group,IR&NS group,IR&I group,IR&R group,and IR&IR group were obtained for WB to observe the expression of Shank3.3)The co-expression of Shank3 and GluA1 in group C and IR&IR were observed by immunofluorescence.Section2:Male SD rats were randomly divided into 5 groups.（1）C group.（2）IR&IR group.（3）siRsh3 group:Rats in this group were intrathecally given 10μg siRNA which targeted to Shank3 mRNA（siRsh3）.（4）IR&IR+siRsh3 group:Rats experienced the same treatment as the rats in IR&IR group,but 10μg siRsh3 was intrathecally given before the second intravenous administration.（5）IR&IR+mismatched RNA group:rats experienced the same treatment as the rats in IR&IR group,but 10μg mismatched RNA was intrathecally given before the second intravenous administration.1)PWTs and PWLs were measured before and after the treatments.2)At the fist day after second intravenous administration,the SDHs of L₄-L₆ in IR&IR group and IR&IR+siRsh3 group were obtained for WB to observe the expression and membrane trafficking of GluA1.3)At the fist day after second intravenous administration,the C fiber evoked potential was tested on the rat in IR&IR group and IR&IR+siRsh3 group.The L₄-L₆ were also obtained for Golgi staining and recording the AMPA receptor-mediated mEPSC.Results:In group IR&IR the expression of Shank3 was the most at the first day after second intravenous administration.Compared with C group,IR&NS group,IR&I group,and IR&R group,Shank3 in SDH was increased in IR&IR group（P<0.05）.Compared with C group,in IR&IR group the co-expression of Shank3 and GluA1increased（P<0.05）.Compared with IR&IR group,in IR&IR+siRsh3 group the PWTs and PWLs increased increased after the second intravenous administration,the pain threshold returned to baseline time ealier,the membrane trafficking of GluA1decreased,the primay dendritic branches dendritic spine density and length of SDH neurons decreased,the C fiber evoked potential decreased,and the frequency and amplitude of AMPA receptor-mediated mEPSC decreased（P<0.05）.Conclusion:The expression of Shank3 in the SDH of SRIH rats were increased,and positively regulated GluA1 membrane trafficking to enhance the neural structural and functional plasticity.Part 4:Effects of Kalirin-7 protein on the neuronal synaptic plasticity in SDH of rats with SRIH and the regulation of Shank3-GluA1 membrane trffickingObjective:To observe the expression of Kalirin-7 in SDH of rats with SRIH,and to explore the effect of Kalirin-7 on SRIH.Method:Rats in this part of the Control group,IR&NS group,IR&I group,IR&R group and IR&IR group were treated as the first part.Section 1:1)The L₄-L₆ spinal dorsal horns（n=8 at each time point）of IR&IR rats were taken at 2h,6h,1d,3d,7d,14d,and 18d after the second intravenous administration to explore the expression of Kalirin-7.2)At the first day after second intravenous administration,the SDHs of L₄-L₆ in C group,IR&NS group,IR&I group,IR&R group,and IR&IR group were obtained for Western Blot to observe the expression of Kalirin-7.Section 2:Male SD rats were randomly divided into 6 groups.（1）C group.（2）IR&IR group.（3）shRkal7 group:Rats in this group were intrathecally given shRNA-lentivirus which targeted to Kalirin-7 mRNA（shRkal7）.（4）Negative-LV group:Rats in this group were intrathecally given negative control shRNA-expressing lentivirus.（5）IR&IR+shRkal7 group:Rats received shRkal7 intrathecally;and 2 weeks later,underwent IR&IR treatment.（6）IR&IR+Negative-LV group:Rats received Negative-LV intrathecally;and 2 weeks later,underwent IR&IR treatment.1)Two weeks,three weeks and four weeks after intrathecal treatment,Western Blot was used to explore the expression of Kalirin-7 in SDH.2)PWTs and PWLs were measured before and after the treatment.3)At the first day after the second intravenous administration,the C fiber evoked potential was tested on the rat in IR&IR group and IR&IR+shRkal7 group.The L₄-L₆ were also obtain for Golgi staining and recording the AMPA receptor-mediated mEPSC.4)At the first day after the second intravenous administration,the expression of Shank3 and GluA1,co-locotion of Shank3 and GluA1,and co-expression of Shank3and GluA1 were observed by WB,immunofluorescence,and co-immunoprecipitation respectively.Results:The expression of Kalirin-7 in IR&IR rats was the most at first day after second intravenous administration.Compared with C group,IR&NS group,IR&I group,and IR&R group,Kalirin-7 in IR&IR group was increased（P<0.05）.Compared IR&IR+shRkal7 group,Kalirin-7 decreased in IR&IR+shRkal7 group at 2 weeks,3weeks,and 4 weeks（P<0.05）.Compared with the IR&IR group,the PWTs and PWLs in IR&IR+shRkal7 group increased,the pain threshold returned to baseline ealier,the expression of Kalirin-7 decreased,the member trafficking of GluA1 decreased,the co-location and co-expression of Shank3 and GluA1 decreased,the dendritic spine density and length of of spinal neurons decreased,the C fiber evoked potential decreased,and the frequency and amplitude of AMPA receptor-mediated mEPSC decreased（P<0.05）.Conclusion:The expression of Kalirin-7 in the SDH of SRIH rats were increased,then it positively regulated the interaction of Shank3 and GluA1,and GluA1 membran trafficking to enhance the neural structural and functional plasticity.General conclusion:The second postoperative pain was more severe than the first.The use of remifentanil analgesia in two surgeries will further aggravate second postoperative pain and induce second hyperalgesia.The mechnism may be explain below.The increase of Kalinin-7 in SDH which could promote Shank3 regulating GluA1 postsynaptic membrane trafficking.Then it leading to excitatory glutamatergic neuronal structure and function remodeling which contributing to central sensitization.