Effect Of Microglia P2X4 Receptor On Central Sensitization In Chronic Migraine

PART I EFFECT OF MICROGLIA P2X4 RECEPTOR ONCENTRAL SENSITIZATION IN CHRONIC MIGRAINEObjective:The pathophysiological mechanism of chronic migraine is not clear.This part of the study was used to explore the role of microglia P2X4 receptor in mice with chronic migraine induced by repeated nitroglycerin(NTG)injection,and to provide a new direction for the treatment of chronic migraine.Methods:1.The animal model of chronic migraine was established by intraperitoneal injection of 10 mg/kg NTG every other day for 9 days,totally 5 times(i.e.,the first,third,fifth,seventh and ninth days were the administration time).The mechanical threshold of hind paw was measured by electronic von Frey filament before and after each NTG administration.Meanwhile,the expression of P2X4 receptor in the trigeminal subnucleus caudalis(TNC)and the number and morphological changes of microglia marker Iba1 were detected.2.Mice were randomly assigned to receive 5-BDBD(P2X4 receptorspecific inhibitor)or saline daily.Then the mechanical threshold of hind paw was measured.WB was used to investigate the level of p-ERK in the TNC.Immunofluorescence and immunohistochemistry were used to detect the expression of CGRP and c-Fos,respectively.3.P2X4 receptor agonist IVM(0.1mg/kg,1mg/kg)were intraperitoneally injected every other day.In the first,third,fifth,seventh and ninth days of the experiment,the mechanical and thermal threshold of hind paw were detected,and the expression of p-ERK,c-Fos and CGRP in TNC were also measured.Results:1.After repeated NTG stimulation,microglia in TNC were activated,which showed that the number of Iba1 immunofluorescent positive cells increased(p < 0.05),the area of Iba1 fluorescence increased(p < 0.05),and cell body of microglia enlarged,but still showed branching.Microglia inhibitor minocycline(MC)can inhibit the activation of microglia and the decrease of NTG-induced basic pain threshold in mice(p < 0.05),but it has no effect on acute hyperalgesia.2.After repeated NTG stimulation,the expression of P2X4 receptor in TNC increased(p < 0.05).Immunofluorescence results showed that P2X4 receptor was mainly co-localized with microglia.The administration of 5-BDBD,an inhibitor of P2X4 receptor,could improve the hyperalgesia of hind paw induced by repeated NTG stimulation(p < 0.05).Meanwhile,5-BDBD treatment inhibited the activation of p-ERK,c-Fos and reduced the release of CGRP in the TNC induced by NTG.3.Repeated activation of P2X4 receptor by IVM could induce hyperalgesia of mice hind paw,and the high dose group was significantly stronger than the low dose group(p < 0.05).In addition,repeated IVM stimulation could induce the activation of p-ERK and c-Fos and promote the release of CGRP in the TNC.Conclusion:This part of the study shows that the process of migraine chronic accompanied by microglia activation and P2X4 receptor increasing,inhibition of microglia activation or P2X4 receptor function can prevent hyperalgesia,and other migraine-related biochemical indicators.Therefore,P2X4 receptor may be involved in the chronic migraine,and regulating the function of P2X4 receptor may be a potential therapeutic strategy.PART II P2X4 RECEPTOR MEDIATES THE SYNTHESIS AND RELEASE OF BDNF IN MICROGLIA BY ACTIVATING P38MAPKObjective:To investigate the effect of P2X4 receptor on the synthesis and release of BDNF and its possible intracellular signaling pathways,and to study the potential pathophysiological mechanism of P2X4 receptor in chronic migraine.Methods:1.Microglial cell line BV2 were cultured in vitro and stimulated by50 uM ATP.At different time points of 30 min,60 min,120 min and 180 min,the content of BDNF in cell and supernatant were detected,and the maximum time point of synthesis and release of BDNF were confirmed,which served as the basis for subsequent experiments.2.Fifteen minutes before ATP stimulated BV2 cells,5-BDBD(20uM),a specific inhibitor of P2X4 receptor,was given to detect the changes of BDNF levels in cells and supernatants.Western blot,IF and ELISA methods were used.3.The changes of p-p38 and p-38 MAPK levels were detected,and the changes of BDNF contents in cell supernatant were detected after the p-38 MAPK inhibitor SB203580(10 mu M)was given.Results:1.Western blot analysis showed that the synthesis of BDNF in BV2 cells increased after 60 minutes of ATP treatment,reached the peak at 120 minutes.ELISA results showed that the release of BDNF from BV2 cells increased significantly at 60 min of ATP stimulation and reached the peak at 120 min(p < 0.05),which was consistent with the trend of intracellular synthesis of BDNF.2.Western blot results showed that 5-BDBD could inhibit the synthesis of BDNF induced by ATP in BV2 cells(p < 0.05),and also inhibit the release of BDNF induced by ATP(p < 0.05).5-BDBD alone did not affect the synthesis and release of intracellular BDNF.3.Western blot results showed that ATP 50 uM could activate p38-MAPK,resulting in a significant increase in phosphorylated protein(p-p38MAPK)(p < 0.05),but the expression of total p38-MAPK remained unchanged.The phosphorylation of p38-MAPK was inhibited by the specific inhibitor of P2X4 receptor(p < 0.05).Using 5-BDBD alone did not affect the phosphorylation of p38-MAPK in cells.SB203580,an inhibitor of p-38 MAPK,could inhibit the release of BDNF in BV2(p <0.05),and also reduce the synthesis of BDNF in cells.Conclusion:ATP-stimulated P2X4 receptor induces BDNF release and synthesis,which dependent on p38-MAPK activation.BDNF may be a key moleculemediating cross-talk between microglia and neurons.P2X4R-p38 MAPKBDNF signaling pathway may contribute to the development of new therapies for chronic migraine.PART III THE EFFECT OF BDNF ON CENTRAL SENSITIZATION IN CHRONIC MIGRAINEObjective : In vivo experiments were conducted to investigate the effect of BDNF on central sensitization of chronic migraine induced by repeated NTG injection,and to further verify the role of P2X4R-p38MAPK-BDNF pathway in chronic migraine.Methods:1.The chronic migraine model was established by intraperitoneal injection of NTG every other day.The protein levels of p-38 MAPK,p-p38 and BDNF in TNC were detected on the 3rd,5th,7th and 9th day of the experiment.In addition,immunofluorescence was used to observe the cellular localization of BDNF.2.P2X4 receptor inhibitor 5-BDBD was given to detect the expression change of BDNF,p38 and p-p38 MAPK.Meanwhile,IVM(0.1mg/kg,1mg/kg)were given intermittently every other day to activate the P2X4 receptor repeatedly.The expression changes of P2X4 receptor,p38,p-p38 and BDNF were detected by WB.3.BDNF receptor TrkB specific inhibitor ANA-12,1mg/kg,was given for 11 consecutive days.The mechanical pain threshold and thermal pain threshold of hind paw,the activation of p-ERK and c-Fos at TNC and the release of CGRP in the TNC were also detected.Results:1.Repeated NTG stimulation could significantly increase the expression of BDNF protein.This upward trend lasted from day 5 to the last dose(p < 0.05).In addition,repeated NTG stimulation did not increase the expression of p38 MAPK protein in TNC,but increased the expression of p-p38MAPK(p < 0.05).This trend began on the fifth day and remained evident on the ninth day.2.Immunofluorescence resuls indicated that most of BDNF were co-localized with Iba1,while a small amount of BDNF was co-localized with neurons.3.Blocking P2X4 receptor by 5-BDBD inhibits the increase of p-p38 MAPK and BDNF induced by repeated NTG stimulation.Repeated activation of P2X4 receptor through IVM not only increased the expression of P2X4 receptor,but also increased the expression of BDNF and p-p38MAPK(P < 0.05),which had no effect on total p38 MAPK.4.ANA-12 treatment could inhibit the decrease of PWT and PWL induced by NTG in mice(p < 0.05),and inhibit the hyperalgesiof hind paw.However,ANA-12 alone did not affect the pain threshold in mice.Immunofluorescence results showed that ANA-12 inhibited NTG-induced CGRP release and c-Fos activation(p < 0.05).WB results showed that ANA-12 treatment inhibited NTG-induced p-ERK activation(p < 0.05).Conclusion:P2X4 receptor affect the synthesis and release of BDNF in microglia.BDNF acts on TrkB receptor in neurons to mediate Neuron-microglia signal.BDNF can affect the generation of central sensitization in chronic migraine by inhibiting CGRP release,ERK phosphorylation and c-Fos activation.