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P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine

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...

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Autores principales: Jing, Feng, Zhang, Yixin, Long, Ting, He, Wei, Qin, Guangcheng, Zhang, Dunke, Chen, Lixue, Zhou, Jiying
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854723/
https://www.ncbi.nlm.nih.gov/pubmed/31722730
http://dx.doi.org/10.1186/s12974-019-1603-4
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author Jing, Feng
Zhang, Yixin
Long, Ting
He, Wei
Qin, Guangcheng
Zhang, Dunke
Chen, Lixue
Zhou, Jiying
author_facet Jing, Feng
Zhang, Yixin
Long, Ting
He, Wei
Qin, Guangcheng
Zhang, Dunke
Chen, Lixue
Zhou, Jiying
author_sort Jing, Feng
collection PubMed
description 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.
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spelling pubmed-68547232019-11-21 P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine Jing, Feng Zhang, Yixin Long, Ting He, Wei Qin, Guangcheng Zhang, Dunke Chen, Lixue Zhou, Jiying J Neuroinflammation Research 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. BioMed Central 2019-11-13 /pmc/articles/PMC6854723/ /pubmed/31722730 http://dx.doi.org/10.1186/s12974-019-1603-4 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Jing, Feng
Zhang, Yixin
Long, Ting
He, Wei
Qin, Guangcheng
Zhang, Dunke
Chen, Lixue
Zhou, Jiying
P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine
title 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_short 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
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854723/
https://www.ncbi.nlm.nih.gov/pubmed/31722730
http://dx.doi.org/10.1186/s12974-019-1603-4
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