| PART I MICROGLIAL P2Y12 RECEPTOR MEDIATES CENTRAL SENSITIZATION IN A MOUSE MODEL OF CHRONIC MIGRAINEObjective The pathophysiology of chronic migraine(CM)is unclear,however,central sensitization plays an important role in the occurrence and development of this disease.Microglial activation contributes to central sensitization and participates in the development of CM.The P2Y12 receptor as a specific receptor on the surface of microglia is involved in the occurrence and maintenance of chronic pain,however,whether it mediates the pathogenesis of CM remains unknown.Thus,this study analyzed the expression of microglial P2Y12 receptor in the trigeminal nucleus caudalis(TNC)in a mice model of CM,and explored the effect of this receptor in the central sensitization in CM.Methods1.Mice were injected intraperitoneally with 10mg/kg Nitroglycerin(NTG)on days 1,3,5,7 and 9 respectively to establish CM animal model,and then the protein level of P2Y12 receptor in the TNC in different groups was analyzed by Western blot(WB).The morphological characteristics and the expression of P2Y12 receptor in the TNC in control group and NTG 9d group were studied by immunofluorescence staining.Colocalization of P2Y12 receptor with neurons,microlia and astrocytes were detected by double-labeling immunofluorescence.2.CM mice were injected intraperitoneally with P2Y12 receptor antagonists MRS2395(1.5 mg/kg)and clopidogrel(15,45 and 100 mg/kg),and then the periorbital and paw mechanical thresholds were measured by electronic von Frey,paw thermal withdrawal latency was determined by radiant heat.Besides,the expression of calcitonin gene-related peptide(CGRP)and c-fos in the TNC were analyzed by WB and immunofluorescence.Results1.WB data showed that the protein level of P2Y12 receptor in the TNC was increased gradually following NTG injection,and was significantly increased on day 5 and 9 after NTG injection(p<0.01).Both the staining intensity of P2Y12 R and the number of P2Y12R-positive cells in the TNC were markedly increased after recurrent NTG administration compared with the values in the control group.The double labeling immunofluorescence results showed that P2Y12 receptor in the TNC was only co-localized with microglia.2.After NTG administration,we found that the periorbital and paw mechanical thresholds and paw withdrawal latencies were significantly decreased compared with the data in control group(p<0.05),the mechanical and thermal thresholds were markedly increased after chronic treatment with MRS2395 and clopidogrel(p<0.05).WB analysis results showed that the expression of CGRP in the TNC was markedly increased on day 5 and 9 after NTG injection compared with the value in control group(p<0.05),however,the upregulation of CGRP protein level in the NTG group was significantly decreased following MRS2395 and clopidogrel treatment(p<0.05).The results of immunofluorescence showed that the fluorescence intensity of CGRP in the NTG group was significantly increased compared with the value in control group(p <0.05),the CGRP intensity was reduced after MRS2395 and clopidogrel administration(p<0.05).Similarly,WB and immunofluorescence data showed that the expression of c-fos protein and the number of positive cells in the TNC in NTG group were significantly increased compared with those in control group(p<0.05),and the c-fos protein level and positive cells were significantly decreased after MRS2395 and clopidogrel injection(p<0.05).Conclusion The study found that the expression of microglial P2Y12 receptor in the TNC in CM mice was up-regulated,inhibiting the function of P2Y12 receptor with its antagonists MRS2395 and clopidogrel attenuated hyperalgesia,reduced the expression of CGRP and c-fos,these results suggest that P2Y12 receptor is involved in the central sensitization in CM.This study provides new ideas for the clinical treatment of CM,microglial P2Y12 receptor may be a potential target.Clopidogrel as a commonly used P2Y12 receptor antagonist may be effective in the treatment of CM.PART? P2Y12 RECEPTOR MEDIATES MICROGLIAL MORPHOLOGICAL ALTERATIONS AND INFLAMMATORY MEDIATORS PRODUCTIONObjective Activated microglia exhibit morphological changes,release a variety of pro-inflammatory factors,and enhance their ability to phagocytose and migrate.Microglial activation participates in central sensitization,and involes in the development of pain.P2Y12 receptor,a specific receptor on the surface of microglia,can mediate microglial activation,however,whether its effect on central sensitization of CM is related to the regulation of microglial function remains unknown.In this study,we analyzed the effect of P2Y12 receptor on microglial activation in the TNC in a mouse model of CM,and verified the effect of P2Y12 receptor antagonists on microglial morphological characteristics and proinflammatory factor production in BV-2 cells,to determine whether P2Y12 receptors are involved in the central sensitization in CM by mediating microglial activation.Methods1.Immunofluorescence staining was used to analyze the changes in the number and morphology of microglia in the TNC.WB was used to study the protein levels of interleukin-1?(IL-1?),tumor necrosis factor-?(TNF-?)and inducible nitric oxide synthase(i NOS)in the TNC.2.BV-2 cells changed to M1-type after lipopolysaccharide(LPS)(1?g/ml)stimulation,mimics the activation of microglia in the TNC in CM.WB was used to analyze the expression of P2Y12 receptor and i NOS in BV-2 cells during LPS stimulation.Enzyme linked immunosorbent assay(ELISA)was used to detect the release of IL-1? and TNF-? from BV-2cells treated by LPS.Immunofluorescence double-labeling to analyze the co-localization of P2Y12 receptor with BV-2 microglia.After MRS2395(20 ?M)and clopidogrel(0.18 ?M,1.8 ?M)injection,the morphological changes of BV-2 cells were analyzed by immunofluorescence,the expression of i NOS was analyzed by WB and immunofluorescence and the production of IL-1? and TNF-? was detected by ELISA.Results1.The results of immunofluorescence showed that the number and the immunoreactivity of microglia in the TNC were significantly increased(p<0.05)after NTG injection.Microglia had hypertrophic somata and shortened processes after NTG stimulation,the total length and average length of the processes were significantly shorter than those in the control group(p<0.05),MRS2395 and clopidogrel treatment did not significantly change the number and the immunoreactivity of microglia,but the total and average length of microglial processes were significantly longer than those of the NTG group(p<0.05).After NTG stimulation,the protein levels of i NOS,IL-1? and TNF-? in the TNC were significantly increased(p<0.05).The upregulation of i NOS,IL-1? and TNF-? protein levels were markedly decreased after MRS2395 and clopidogrel administration(p<0.05).2.The protein levels of P2Y12 receptor and i NOS were increased after8 h of LPS stimulation(p<0.05)and lasted for 24 h.ELISA results showed that IL-1? production was up-regulated after 24 h of LPS stimulation(p<0.05),TNF-? levels were increased after 8h of LPS stimulation(p<0.05)and lasted for 24 h.Immunofluorescence confirmed that P2Y12 receptor was co-localized with BV-2 cells.After LPS stimulation,BV-2 cells manifested as hypertrophic somata,the immunoreactivity of i NOS was increased compared with those in the control group.The upregulation of the immunoreactivity of i NOS was decreased(p<0.05)after MRS2395 and clopidogrel injection.ELISA data showed that the production of IL-1? and TNF-? were significantly reduced following MRS2395 and clopidogrel treatment compared with the LPS group(p<0.05).MRS2395 mediated microglial morphological alteration,however,clopidogrel had no significant effect on the morphological changes of BV-2 microglia.Conclusion This study found that microglia in the TNC in CM model were activated,which performed as the upregulation of the cell number,hypertrophic somata and shortened processes,and the production of pro-inflammatory factors.P2Y12 receptor inhibitors mediated microglial activation by prolonging microglial processes and reducing the release of pro-inflammatory factors.These results suggest that P2Y12 receptor may be involved in the central sensitization in CM by mediating microglial activation.PART? P2Y12 RECEPTOR MEDIATES MICROGLIAL ACTIVATION VIA RHOA/ROCK PATHWAY CONTRIBUTES TO CENTRAL SENSITIZATION IN CHRONIC MIGRAINEObjective The Rho A/Rho-associated coiled coil-containing kinase(ROCK)pathway is involved in the development of pain,P2Y12 receptor mediates the alteration of cell morphology and the release of inflammatory factors through this pathway.This study analyzed the expression of Rho A and ROCK2 in the TNC in CM model,and explored the effects of Rho A/ROCK pathway on hyperalgesia and CGRP release to clarify whether the Rho A/ROCK pathway is involved in central sensitization in CM.To clarify whether P2Y12 receptor mediates microglial activation via Rho A/ROCK pathway in CM,we analyzed the expression of Rho A and ROCK2 and explored the changes of microglial activation after inhibiting P2Y12 receptor.Methods1.WB was used to analyze the changes of GTP-Rho A and ROCK2 protein levels in the TNC after NTG injection.The periorbital and paw mechanical thresholds were measured by electronic von Frey,and paw thermal withdrawal latency was determined by radiant heat after intraperitoneal injection of the ROCK inhibitor fasudil(30mg/kg).Besides,the expression of CGRP in the TNC was analyzed by WB and immunofluorescence.2.WB was used to analyze the changes of the protein levels of GTP-Rho A and ROCK2 after P2Y12 receptor antagonists MRS2395 and clopidogrel administration,and to detect the changes of P2Y12 receptor,GTP-Rho A and ROCK2 expression after fasudil administration.3.Immunofluorescence was used to determine the alterations in the number and morphological characteristics of microglia after fasudil treatment.WB was used to explore the protein levels of IL-1?,TNF-? and i NOS in each group.Results1.WB analysis results showed that GTP-Rho A and ROCK2 expressions were up-regulated after NTG injection(p<0.05).The mechanical and thermal thresholds were increased after fasudil treatment compared with those in NTG group(p<0.05).The results of WB and immunofluorescence showed that the protein levels and immunoreactivity of CGRP in the TNC were significantly decreased after fasudil administration compared with those in NTG group(p<0.05).2.The protein levels of GTP-Rho A and ROCK2 were significantly decreased after MRS2395 and clopidogrel administration(p<0.05).The expression of GTP-Rho A and ROCK2 were decreased after fasudil intervention(p<0.05),however,fasudil had no significant effect on P2Y12 receptor protein expression.3.Fasudil had no significant effect on the number and immunoreactivity of microglia in the TNC,however,the total and mean length of microglial processes were markedly prolonged after fasudil treatment compared with those in the NTG group(p<0.05).WB data showed that the expression levels of IL-1?,TNF-? and i NOS in the TNC were significantly decreased after fasudil administration compared with those in the NTG group(p<0.05).Conclusion This study found that inhibition of the activity of Rho A/ROCK pathway can alleviate hyperalgesia and reduce the expression of CGRP in the TNC in CM model,suggesting that this pathway is involved in central sensitization in CM.Rho A/ROCK pathway acts as a downstream pathway of the P2Y12 receptor,inhibiting the activity of this pathway can affect the microglial morphological characteristics and the release of proinflammatory factors,indicating that P2Y12 receptor mediates microglial activation through Rho A/ROCK pathway contributes to the development of central sensitization in CM. |
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