P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine
Abstract Background Microglial activation contributes to the development of chronic migraine (CM). The P2Y12 receptor (P2Y12R), a metabolic purinoceptor that is expressed on microglia in the central nervous system (CNS), has been indicated to play a critical role in the pathogenesis of chronic pain....
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doaj-fc2f599ec0f342adac7859b958def23b2020-11-25T04:09:17ZengBMCJournal of Neuroinflammation1742-20942019-11-0116112010.1186/s12974-019-1603-4P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraineFeng Jing0Yixin Zhang1Ting Long2Wei He3Guangcheng Qin4Dunke Zhang5Lixue Chen6Jiying Zhou7Department of Neurology, The First Affiliated Hospital of Chongqing Medical UniversityDepartment of Neurology, The First Affiliated Hospital of Chongqing Medical UniversityDepartment of Neurology, The First Affiliated Hospital of Chongqing Medical UniversityDepartment of Neurology, The First Affiliated Hospital of Chongqing Medical UniversityLaboratory Research Center, The First Affiliated Hospital of Chongqing Medical UniversityLaboratory Research Center, The First Affiliated Hospital of Chongqing Medical UniversityLaboratory Research Center, The First Affiliated Hospital of Chongqing Medical UniversityDepartment of Neurology, The First Affiliated Hospital of Chongqing Medical UniversityAbstract Background Microglial activation contributes to the development of chronic migraine (CM). The P2Y12 receptor (P2Y12R), a metabolic purinoceptor that is expressed on microglia in the central nervous system (CNS), has been indicated to play a critical role in the pathogenesis of chronic pain. However, whether it contributes to the mechanism of CM remains unknown. Thus, the present study investigated the precise details of microglial P2Y12R involvement in CM. Methods Mice subjected to recurrent nitroglycerin (NTG) treatment were used as the CM model. Hyperalgesia were assessed by mechanical withdrawal threshold to electronic von Frey and thermal withdrawal latency to radiant heat. Western blot and immunohistochemical analyses were employed to detect the expression of P2Y12R, Iba-1, RhoA, and ROCK2 in the trigeminal nucleus caudalis (TNC). To confirm the role of P2Y12R and RhoA/ROCK in CM, we systemically administered P2Y12R antagonists (MRS2395 and clopidogrel) and a ROCK2 inhibitor (fasudil) and investigated their effects on microglial activation, c-fos, and calcitonin gene-related peptide (CGRP) expression in the TNC. To further confirm the effect of P2Y12R on microglial activation, we preincubated lipopolysaccharide (LPS)-treated BV-2 microglia with MRS2395 and clopidogrel. ELISA was used to evaluate the levels of inflammatory cytokines. Results The protein levels of P2Y12R, GTP-RhoA, ROCK2, CGRP, c-fos, and inducible nitric oxide synthase (iNOS) in the TNC were increased after recurrent NTG injection. A double labeling study showed that P2Y12R was restricted to microglia in the TNC. MRS2395 and clopidogrel attenuated the development of tactile allodynia and suppressed the expression of CGRP, c-fos, and GTP-RhoA/ROCK2 in the TNC. Furthermore, fasudil also prevented hyperalgesia and suppressed the expression of CGRP in the TNC. In addition, inhibiting P2Y12R and ROCK2 activities suppressed NTG-induced microglial morphological changes (process retraction) and iNOS production in the TNC. In vitro, a double labeling study showed that P2Y12R was colocalized with BV-2 cells, and the levels of iNOS, IL-1β, and TNF-α in LPS-stimulated BV-2 microglia were reduced by P2Y12R inhibitors. Conclusions These data demonstrate that microglial P2Y12R in the TNC plays a critical role in the pathogenesis of CM by regulating microglial activation in the TNC via RhoA/ROCK pathway.http://link.springer.com/article/10.1186/s12974-019-1603-4P2Y12 receptorMicrogliaChronic migraineTrigeminal nucleus caudalisHyperalgesiaNitroglycerin |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Feng Jing Yixin Zhang Ting Long Wei He Guangcheng Qin Dunke Zhang Lixue Chen Jiying Zhou |
spellingShingle |
Feng Jing Yixin Zhang Ting Long Wei He Guangcheng Qin Dunke Zhang Lixue Chen Jiying Zhou P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine Journal of Neuroinflammation P2Y12 receptor Microglia Chronic migraine Trigeminal nucleus caudalis Hyperalgesia Nitroglycerin |
author_facet |
Feng Jing Yixin Zhang Ting Long Wei He Guangcheng Qin Dunke Zhang Lixue Chen Jiying Zhou |
author_sort |
Feng Jing |
title |
P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine |
title_short |
P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine |
title_full |
P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine |
title_fullStr |
P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine |
title_full_unstemmed |
P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine |
title_sort |
p2y12 receptor mediates microglial activation via rhoa/rock pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine |
publisher |
BMC |
series |
Journal of Neuroinflammation |
issn |
1742-2094 |
publishDate |
2019-11-01 |
description |
Abstract Background Microglial activation contributes to the development of chronic migraine (CM). The P2Y12 receptor (P2Y12R), a metabolic purinoceptor that is expressed on microglia in the central nervous system (CNS), has been indicated to play a critical role in the pathogenesis of chronic pain. However, whether it contributes to the mechanism of CM remains unknown. Thus, the present study investigated the precise details of microglial P2Y12R involvement in CM. Methods Mice subjected to recurrent nitroglycerin (NTG) treatment were used as the CM model. Hyperalgesia were assessed by mechanical withdrawal threshold to electronic von Frey and thermal withdrawal latency to radiant heat. Western blot and immunohistochemical analyses were employed to detect the expression of P2Y12R, Iba-1, RhoA, and ROCK2 in the trigeminal nucleus caudalis (TNC). To confirm the role of P2Y12R and RhoA/ROCK in CM, we systemically administered P2Y12R antagonists (MRS2395 and clopidogrel) and a ROCK2 inhibitor (fasudil) and investigated their effects on microglial activation, c-fos, and calcitonin gene-related peptide (CGRP) expression in the TNC. To further confirm the effect of P2Y12R on microglial activation, we preincubated lipopolysaccharide (LPS)-treated BV-2 microglia with MRS2395 and clopidogrel. ELISA was used to evaluate the levels of inflammatory cytokines. Results The protein levels of P2Y12R, GTP-RhoA, ROCK2, CGRP, c-fos, and inducible nitric oxide synthase (iNOS) in the TNC were increased after recurrent NTG injection. A double labeling study showed that P2Y12R was restricted to microglia in the TNC. MRS2395 and clopidogrel attenuated the development of tactile allodynia and suppressed the expression of CGRP, c-fos, and GTP-RhoA/ROCK2 in the TNC. Furthermore, fasudil also prevented hyperalgesia and suppressed the expression of CGRP in the TNC. In addition, inhibiting P2Y12R and ROCK2 activities suppressed NTG-induced microglial morphological changes (process retraction) and iNOS production in the TNC. In vitro, a double labeling study showed that P2Y12R was colocalized with BV-2 cells, and the levels of iNOS, IL-1β, and TNF-α in LPS-stimulated BV-2 microglia were reduced by P2Y12R inhibitors. Conclusions These data demonstrate that microglial P2Y12R in the TNC plays a critical role in the pathogenesis of CM by regulating microglial activation in the TNC via RhoA/ROCK pathway. |
topic |
P2Y12 receptor Microglia Chronic migraine Trigeminal nucleus caudalis Hyperalgesia Nitroglycerin |
url |
http://link.springer.com/article/10.1186/s12974-019-1603-4 |
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