Research On The Role And Mechanism Of Microglial P2X7R In Central Sensitization Of Chronic Migraine

PART 1 P2X7R-MEDIATIED MICROGLIA ACTIVATION CONTRIBUTES TO CENTRAL SENSITIZATION OF CHRONIC MIGRAINEObjectiveCentral sensitization is a crucial mechanism of chronic migraine（CM）.Studies have indicated that the microglia activation and subsequent inflammation conduce to central sensitization.P2X7R could mediate inflammation by activating microglia.At present,no research explored P2X7R in CM model.Therefore,this part of the experiment was designed to achieve the following purposes:1.To analyzed the expression of P2X7R in the trigeminal nucleus caudalis（TNC）in CM model;2.To explore the effect of P2X7R in the TNC on central sensitization of CM;3.To explore the effect of P2X7R on the activation of microglia.Methods1.The CM model was established by repeated intraperitoneal injection of NTG.Western blotting（WB）was used to analyze the time course of P2X7R expression on the 1^st,3^rd,5^thand 9^thday of modeling.Real-time RT-PCR（RT-q PCR）and immunofluorescence assays were preformed to compare the expression profile of P2X7R in TNC between SHAM and NTG group.Immunofluorescence double-labeling was used to co-localize the P2X7R with microglia,astrocytes and neurons.2.P2X7R antagonist,brilliant blue G（BBG）,and the same volume of solvent were given.Randomly divided the Mice into five groups:SHAM group,SHAM+BBG group,NTG group,NTG+VEH group and NTG+BBG group.To assess the changes of hyperalgesia,we detected the mechanical and thermal threshold by electronic Von Frey and radiant heat.WB and immunofluorescence were used to determine the effect of BBG on expression profile of CGRP and c-fos.3.In vivo,we performed immunofluorescence to determine the effect of BBG on the number and morphology of microglia.WB were conducted to detect the changes of NLRP3,IL-1βand IL-18 proteins.4.In vitro,0.8m M Bz ATP was used to stimulate BV-2 cells to imitate the P2X7R activation in the TNC area in CM.WB was performed to determine the P2X7R expression.BV-2 cells were stimulated by BBG and the same volume of solvent.The cells were divided into four groups:VEH group,BBG group,Bz ATP group and Bz ATP+BBG group.The cell morphology and i NOS expression were assessed by immunofluorescence.WB and ELSA were combined to assess the change of inflammation.Results1.WB showed that,during the modeling,the P2X7R in the TNC was gradually up-regulated with a significant increase on the 5^thday（p<0.001）and continuing the whole modeling process.RT-q PCR and immunofluorescence revealed that the expression of P2X7R m RNA and P2X7R positive cells as well as immunofluorescence intensity in the model group were significantly higher than those in the SHAM group.Immunofluorescence double-labeling revealed that P2X7R was mainly co-localized with microglial marker,Iba-1,in the TNC area.2.The data of behavioral assessment indicated that recurrent NTG injection induced hyperalgesia which featured as decreased mechanical and thermal threshold.BBG intervention significantly improved the hyperalgesia of modeling mice.Additionally,protein level of CGRP and c-fos in the TNC area were decreased by BBG treatment.3.In vivo,NTG treatment markedly increased the Iba-1 positive cells with enhanced immunoreactivity.Morphologically,the microglia presented with hypertrophied cell bodies and shorter and fewer processes.The quantitative analysis showed that the total and mean length of processes decreased.After intervention with BBG,the activation of microglia was inhibited in the TNC area.In addition,BBG significantly decreased NTG induced expression of NLRP3,IL-1βand IL-18（p<0.01）.4.In vitro,the protein level of P2X7R was augmented after treatment with Bz ATP in BV-2 cells which peaked at 120min,maintained for 180min.Compared with the SHAM group,the BV-2 cells became round and enlarged with shorter processes after Bz ATP treatment.The i NOS expression was also increased after Bz ATP treatment.BBG inhibited the BV-2 cells activation and decreased the i NOS expression.WB and ELISA showed that BBG significantly decreased the Bz ATP induced expression of NLRP3 protein,IL-1βprotein and IL-18 protein.ConclusionsIn the TNC area of CM,the expression of microglia P2X7R was up-regulated,accompanied by activation of microglia and NLRP3inflammasome.P2X7R antagonist inhibited the microglia activation and subsequent inflammation,then improved central sensitization.These results indicates that P2X7R participates in the central sensitization of CM by mediating the microglia activation.The intervention against P2X7R might benefits to CM prevention.PART2 P2X7R CONTRIBUTES TO CENTRAL SENSITIZATION OF CHRONIC MIGRAINE BY ACTIVATING MICROGLIA THROUGH AUTOPHAGYObjectiveAutophagy regulates the microglia activation and subsequent inflammation.Studies have suggested that P2X7R can regulate the process of autophagy.In CM model,whether autophagy is implicated in mediating the effect of P2X7R on microglia activation has not been reported.Additionally,no research explored autophagy in CM.Therefore,this part of the experiment was designed to achieve the following purposes:1.To explore the changes of autophagy in the TNC area of CM model;2.To study the effect of autophagy on central sensitization of CM;3.To explore whether autophagy is implicated in the effect of P2X7R on the microglia activation.Methods1.The CM model was established by repeated intraperitoneal injection of NTG.WB was conducted to analyze the expression of autophagy-related proteins,while transmission electron microscope（TEM）was performed to detect the autophagosome in microglia in TNC area.Finally,chloroquine（CQ）was treated to analyze the autophagic flux.2.Autophagy inducer rapamycin（RAPA）and the same volume solvent were given.Four groups were randomly created:SHAM-VEH group,SHAM-RAPA group,NTG-VEH group and NTG-RAPA group.To assess the behavior change,we tested both mechanical and thermal threshold.Change of CGRP and c-fos expression profile were also detected.3.WB was performed to analyze the expression profile of P2X7R and autophagy-related protein after RAPA or BBG administration.4.We performed immunofluorescence to evaluate the effect of RAPA on the number and morphology of microglia.WB were conducted to detect the expression of NLRP3,IL-1βand IL-18 after RAPA treatment.Results1.Recurrent NTG treatment significantly up-regulated the expression of LC3-II and p62,and did not alter the level of beclin1.Compared with mice receiving NTG only,the combination treatment of CQ and NTG did not further increase LC3-II proteins,which indicated the impaired autophagic flux in the TNC of the CM model.TEM showed the number of autophagosomes in the microglia increased at the TNC area of CM model which was consistent with the augmented LC3-II by WB analysis.2.The data of behavioral assessment showed that recurrent NTG injection induced hyperalgesia which featured as decreased mechanical and thermal threshold.The autophagy inducer,RAPA,significantly improved basal rather than acute hyperalgesia.Additionally,RAPA treatment markedly suppressed CGRP and c-fos expression.3.WB showed decreased LC3-II and p62 after BBG treatment,while there was no change in P2X7R after RAPA administration.4.Immunofluorescence showed that RAPA pretreatment inhibited microgliosis,reduced Iba-1 immunoreactivity,and changed the microglia into a ramified shape,exhibiting increased total and mean length of processes.Additionally,mice receiving RAPA treatment exhibited decreased NLRP3,IL-1βand IL-18（p<0.05）.ConclusionsOur results reveals the autophagic impairment in CM.Enhanced autophagy improved central sensitization by inhibiting microglia activation.P2X7R promotes central sensitization by mediating microglia activation through autophagic impairment.The involvement of autophagy in CM introduces new information for investigation of the mechanism of CM and provides potential therapeutic targets for CM prevention.