The Effect And Mechanism Of Oxytocin On Central Sensitization In Chronic Migraine

PART Ⅰ ENDOGENOUS OXYTOCIN AND OXYTOCIN RECEPTOR ARE INVOLVED IN CENTRAL SENSITIZATION OF CHRONIC MIGRAINEObjective: The pathophysiological mechanism of chronic migraine(CM)is unclear.Current studies suggest that the hypothalamus may be involved in the pathophysiology of CM,and that Oxytocin(OT),secreted from the hypothalamus,is involved in the regulation of pain.It is unclear whether endogenous OT and Oxytocin receptor(OTR)are involved in the central sensitization in CM.This part of the study was intended to investigate the expression levels of endogenous OT and OTR in the hypothalamus and Trigeminal nucleus caudalis(TNC)of CM mouse.Methods: CM mouse model was established by repeated intraperitoneal injection of Nitroglycerin(NTG)for 5 times on days 1,3,5,7 and 9.The changes of orbital and paw mechanical pain and paw thermal threshold were measured by Von Frey filaments and radiant heat equitment at 1,3,5,7,9 days before injection and on 11 days.The protein expression of c-fos and calcitonin gene-related peptide(CGRP)in TNC was detected by Western Blot(WB)to evaluate the central sensitization.WB,Quantitative real-time polymerase chain reaction(QRT-PCR)and immunofluorescence were used to compare the expression changes of OT and OTR between the sham and CM group.The subcellular localization of OTR in TNC was determined by dual immunofluorescence method.Results: After repeated NTG stimulation,the mechanical pain and thermal pain threshold of paw and periorbit was showed a gradually decreasing trend,and the pain threshold was still significantly lower than that of control group on day 11 without NTG stimulation(p<0.05).WB results showed that the expressions of c-fos and CGRP of TNC in CM group were significantly higher than those in sham group.Immunofluorescence analysis was showed that the expression of OT-positive neurons in hypothalamus and OTergic nerve fibers in TNC was decreased significantly in CM group compared with that in the control group.In addition,the immunofluorescence result showed the expression of OTR in TNC was mainly located on the cell membrane and postsynaptic membrane of neurons.WB and PCR results showed that OTR expression increased significantly after NTG stimulation compared with control group(p<0.05).Conclusion: Repeated stimulation of NTG can establish stable CM animal model and induce central sensitization.The dysfunction of endogenous OT/OTR is observed in CM model,suggesting that OT and OTR are involved in the central sensitization of CM.PART Ⅱ EFFECT OF OXYTOCIN ON HYPERALGESIA AND SYNAPTIC PLASTICITY IN CHRONIC MIGRAINE MOUSE MODELObjective: OT participates in pain regulation.Previous studies have shown abnormal synaptic plasticity after central sensitization in CM.Whether exogenous OT can regulate central sensitization of CM has not been reported.The purpose of this study was to investigate the effect of intranasal exogenous OT on central sensitization and synaptic plasticity in CM mouse,and to explore the role of OTR.Methods: The CM mouse model was established by NTG.Repeated intranasal OT and intraperitoneal L368,899,an oxytocin receptor(OTR)antagonist,were administered to investigate the effect of OT and the role of OTR.The mechanical pain and thermal pain threshold and the c-fos,CGRP expressions were measured to evaluate the effect of OT and the role of OTR.The phosphorylation level of Nmethyl D-Aspartate receptor subtype 2B subunit(NR2B)and synapse associated proteins level,including postsynaptic Density Protein 95(PSD-95),Synaptophysin-1(Syt-1)and Synaptosomal associated rotein25(Snap-25)were detected by WB to evaluate NMDA receptor function.The synaptic ultrastructure and dendrite spine of TNC neurons in CM mouse were explored by Transmission electron microscopy(TEM)and Golgi staining.Results: Repeated intranasal administration of OT(20μg/kg)improved mechanical and thermal pain sensitivity induced by NTG,and decreased the expression of CGRP and c-fos in TNC.In addition,WB results also suggested that OT treatment could terminate the phosphorylation of NR2 B and decrease the expression of synaptic related protein expression induced by NTG stimulation.Electron microscopy results and Golgi staining also showed that OT treatment improved the abnormal synaptic plasticity and dendritic spine in TNC region after NTG stimulation.The protective effect of OT was prevented by L368,899.Conclusion: Repeated intranasal OT treatment could improve hyperalgesia and central sensitization of CM mice through OTR,affect NMDA receptor function in TNC by inhibiting NR2 B phosphorylation,and result in the improvement of synaptic plasticity.Repeated intranasal OT therapy may be a potential treatment for the prevention of CM.PART Ⅲ THE AC1/PKA/PCREB PATHWAY IS INVOLVED N THE REGULATION OF OXYTOCIN IN CENTRAL ENSITIZATION OF CMObjective: Current studies show that the Adenylyl cyclase 1(AC1)/Protein kinase A(PKA)/ phosphorylated c AMP responsive element-binding protein(p CREB)pathway plays an important role in activating NMDA receptors and maintaining central sensitization and synaptic plasticity.Whether OT regulates central sensitization of CM by inhibiting AC1/PKA/p CREB pathway has not been reported.This study intends to verify the effect of OT on AC1/PKA/p CREB pathway through in vivo and in vitro experiments.Methods: 1.After OT and L368,899 intervention in CM mouse,the protein expression of AC1,PKA,CREB and p CREB in TNC was detected by WB;2.Intraperitoneal injection of NMDA receptor agonist NMDA0.5mg/kg or 5mg/kg every other day was used to simulate the administration of NTG.At 1,3,5,7,9 and 11 day before drug injection,the mechanical pain threshold and thermal pain threshold of paw were detected.The number of c-fos positive cells in TNC were detected by immunofluorescence.The protein expression of p NR2 B,PKA,CGRP and p CREB were evaluated by WB.3.PC12 cells were cultured in vitro and stimulated by different doses of NMDA,and the appropriate intervention dose was evaluated by MTT test to provide a basis for subsequent experiments.The expression levels of neuronal Nitric Oxide synthase(n NOS)and OTR in control group and NMDA group were determined to evaluate the feasibility of the cell model.4.According to the experimental purpose,the expressions of phosphorylation of NR2 B,n NOS,PKA and p CREB protein were detected by WB after OT(10μM)intervention.Results: Repeated NTG injection could significantly increase the proteins expression of AC1,PKA and p CREB in TNC,while OT treatment could inhibit the expression of these proteins.Repeated NMDA stimulation can simulate the effect of NTG to induce hyperalgesia in mouse,and induce the increase of c-fos positive cells in TNC,leading to central sensitization,and promote the expression levels of p NR2 B,PKA and p CREB protein.However,OT treatment could improve hyperalgesia and inhibit the expression of these proteins.NMDA stimulation promoted the activation of PC12 cells,resulting in the increase of n NOS and OTR in vitro experiments.OT treatment affected the activation of PC12 cells by inhibiting NR2 B phosphorylation and protein expression of n NOS,PKA and p CREB.Conclusion: The AC1/PKA/p CREB pathway is activated in CM model.OT can affect NMDA receptor function and improve hyperalgesia and central sensitization by inhibiting the activation of AC1/PKA/p CREB pathway.