Electroacupuncture Pretreatment Alleviates Cerebral Ischemic Injury Through α7 Nicotinic Acetylcholine Receptor-Mediated Phenotypic Conversion of Microglia.

Zhi Ma,Tao Jiang,Chaoying Yan,Qiang Wang,Fuhai Bai,Zengli Zhang

doi:10.3389/fncel.2019.00537

Abstract

Electroacupuncture (EA) pretreatment alleviates cerebral ischemic injury through α7 nicotinic acetylcholine receptor (α7nAChR). We attempted to investigate whether the phenotypic conversion of microglia was involved in the therapeutic effect of EA pretreatment in cerebral ischemia through α7nAChR. Adult male Sprague–Dawley (SD) rats were subjected to middle cerebral artery occlusion (MCAO) after EA or α7nAChR agonist N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-furo[2,3-c]pyridine-5-carboxamide hydrochloride (PHA-543,613 hydrochloride) and antagonist α-bungarotoxin (α-BGT) pretreatment. Primary microglia were subjected to drug pretreatment and oxygen-glucose deprivation (OGD). The expressions of the classical activated phenotype (M1) microglia markers induced nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and cluster of differentiation 86 (CD86); the alternative activated phenotype (M2) microglia markers arginase-1 (Arg-1), transforming growth factor-β1 (TGF-β1), and cluster of differentiation 206 (CD206); and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and anti-inflammatory cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10) in the ischemic penumbra or in the supernatant of primary microglia were analyzed. The infarction volume and neurological scores were assessed 72 h after reperfusion. The cell viability and lactate dehydrogenase (LDH) release of neurons co-cultured with microglia were analyzed using cell counting kit-8 (CCK-8) and LDH release assays. EA pretreatment decreased the expressions of M1 markers (iNOS, IL-1β, and CD86) and pro-inflammatory cytokines (TNF-α and IL-6), whereas it increased the expressions of M2 markers (Arg-1, TGF-β1, and CD206) and anti-inflammatory cytokines (IL-4 and IL-10) by activating α7nAChR. EA pretreatment also significantly reduced the infarction volume and improved the neurological deficit. The activation of α7nAChR in microglia relieved the inflammatory response of primary microglia subjected to OGD and attenuated the injury of neurons co-cultured with microglia. In conclusion, EA pretreatment alleviates cerebral ischemic injury through α7nAChR-mediated phenotypic conversion of microglia, which may be a new mechanism for the EA pretreatment-induced neuroprotection against cerebral ischemia.

Highlights

Stroke is one of the leading causes of death worldwide (Mozaffarian et al, 2016)
We detected the expression of α7 nicotinic acetylcholine receptor (α7nAChR) in microglia by immunofluorescent double labeling of α7nAChR and Iba1. It showed that EA pretreatment, compared with the middle cerebral artery occlusion (MCAO) group, upregulated microglial α7nAChR expression in the ischemic penumbra (Figure 1F)
These results indicated that EA pretreatment exerted neuroprotective effects and reversed the effects of MCAO on the expression of α7nAChR in the ischemic penumbra of rats subjected to ischemia injury

Summary

Introduction

Stroke is one of the leading causes of death worldwide (Mozaffarian et al, 2016). High mortality and disability after stroke significantly increase the financial burden of the society and threaten public health. At later time points after ischemic stroke, M1 phenotype microglia converted to the alternative activated phenotype (M2) microglia, which release anti-inflammatory cytokines and neurotrophic substances and contribute to repair of injury and neuroprotection (Kawabori and Yenari, 2015; Zhai et al, 2017). It may be a potential therapeutic target for the treatment of ischemic stroke to facilitate the phenotypic conversion of microglia from M1 to M2

Methods

Results

Conclusion