Role of microglial Gria1 down-regulation in neuropathic pain

Abstract

Microglia is the resident immune cell in the central nervous system, and the activation of microglia is known to induce neuropathic pain. Previously, single cell microarray for spinal microglia in L4 spinal nerve transection (SNT) model revealed down-regulation of Gria1 mRNA in ipsilateral microglia at post-operation day 7. Gria1 gene encodes glutamate receptor 1 (GluR1) which is an AMPA receptor family. While GluR including GluR1 is known to be functionally expressed in cortical microglia, it is still unclear if Gria1 in spinal microglia is involved in the development of neuropathic pain. Therefore, this study is aimed to determine whether the Gria1 expression indeed decreases following microglial activation, and downregulation of Gria1 contributes to neuropathic pain. Primary cultured microglia from mice cerebral cortex were treated with ATP (50uM, 1hr) to induce microglial activation in vitro condition. Expression level of Gria1 and GluR1 in ATP-activated microglia wasexamined with real time PCR and immunostaining, respectively. Cytokine array (mouse cytokine array kit, panel A, R&D systems) for various cytokines was performed with ATP-treated microglia, and the change in cytokines from Gria1 knockdown microglia was determined with ELISA. The level of phosphorylation of p38 MAPK was also confirmed in Gria1 knockdown microglia with western blot. In addition, functional significance of Gria1 down-regulation in microglia was confirmed in vivo. Behavior test was performed to check the pain hypersensitivity with von Frey filament by easuring paw withdrawal threshold following intrathecal injection of Gria1 knockdown microglia to naive mice. The expression of Gria1 mRNA and GluR1 protein encoded by Gria1 were significantly attenuated in the ATP-treated microglia, compared to the control, indicating the down-regulation of Gria1 in activated microglia. Cytokine array revealed the increased level of TNF-a in activated microglia, and increased TNF-a was also mimicked in Gria1 knockdown microglia. However, the level of phosphorylation of p38 MAPK was not changed in Gria1 knockdown microglia. From the behavior study, it was found that paw withdrawal threshold significantly decreased after intrathecal injection of Gria1 knockdown microglia. Taken together, these results provide strong evidence that Gria1 expression is decreased in activated microglia, which contributes to the neuropathic pain following peripheral nerve injury. Downregulation of Gria1 in activated microglia is likely to be involved in the release of TNF-a which leads pain hypersensitivity in the neuropathic pain condition.