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Aberrant neuronal excitation promotes neuroinflammation in the primary motor cortex of ischemic stroke mice

Acta Pharmacologica Sinica volume 46, pages 2105–2119 (2025)Cite this article

Abstract

Current treatments for ischemic stroke aim to achieve rapid reperfusion with intravenous thrombolysis and/or endovascular thrombectomy, which have proven to attenuate disability. Despite the significant progress in reperfusion therapies, functional recovery remains inconsistent, primarily due to ongoing neuronal excitotoxicity and neuroinflammation. In this study we investigated the relationship between neuronal activity and neuroinflammation in an ischemic mouse model using chemogenetic techniques. MCAO cerebral ischemia model was established in mice; in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) was established in PC12 neurons. By measuring c-Fos expression, we showed that MCAO caused the activation of both excitatory and inhibitory neurons within the M1 primary motor cortex, which subsequently induced reactive activation of local microglia through the secretion of unique neuronal extracellular vesicles (EVs). Chemogenetic inhibition of abnormal neuronal activity in stroke-affected cortical neurons reversed microglia activation and reduced neuronal apoptosis. By analyzing the miRNAs in EVs from the ischemic M1 cortex, we found that miR-128-3p was significantly downregulated in ischemia-challenged neurons and their EVs, leading to neuronal injury and proinflammatory polarization of microglia. Intravenous injection of miR-128-3p mimics significantly improved neuronal survival, reduced neuroinflammation accompanied by better functional recovery after ischemic stroke. In summary, stroke-induced abnormal neuronal activity reduces miR-128-3p levels in ischemic neurons and EVs, leading to increased microglia activation and neuronal injury after a stroke. The study highlights that inhibiting abnormal neuronal activity or delivering miR-128-3p-enriched EVs as novel methods for stroke treatment.

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Fig. 1: Inhibition the activity of the excitatory neurons in M1 cortex improved brain injury following stroke.
Fig. 2: The down-regulated miR-128-3p levels in M1 cortex-EVs were negatively correlated with neuronal activation.
Fig. 3: MiR‐128‐3p in EVs improved microglial activation and neuronal apoptosis.
Fig. 4: MiR‐128‐3p targeted Klf4 signaling to polarize microglia.
Fig. 5: Klf4 activated Pdcd4 transcription.
Fig. 6: Engineered RVG-miR-128-EVs selectively targeted neurons.
Fig. 7: RVG-EVs delivery of miR-128-3p enhanced functional recovery and reduced microglial activation in MCAO mice.

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Acknowledgements

Research funding support for this work was from the National Natural Science Foundation of China (82271327, 82072535 and 81873768) to Zhen Wang and Natural Science Foundation of Shandong Provine (ZR2024MH038) to Zhen Wang. We are grateful with the National Natural Science Foundation of China under Grant (No. U23A6011) to Dr. Wen-qiang Chen for providing us with financial support. We are grateful to Dr. Yan Li (the First Affiliated Hospital of Shandong First Medical University and Shandong Provincial Qianfoshan Hospital) for the action potential assay. We thank the Translational Medicine Center of Shandong University for the advice and instruments provided for our research. The graphical abstract was created with BioRender.com.

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  1. These authors contributed equally: Ting-ting Li, Xiao-fan Guo

Authors and Affiliations

  1. Department of Physiology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Ji-nan, 250012, China

    Ting-ting Li, Yi-jing Zhao, Ya-hong Cheng, Dan-qing Xin, Yan Song & Zhen Wang

  2. Department of Neurology, Loma Linda University Health, Loma Linda, CA, 92354, USA

    Xiao-fan Guo

  3. Department of Medical Psychology and Ethics, School of Basic Medicine Sciences, Cheeloo College of Medicine, Shandong University, Ji-nan, 250012, China

    Dan-qing Xin & De-xiang Liu

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Contributions

ZW led the project, such as, in concepts, designing, supervising, funding and writing and revising of the manuscript; TTL made substantial contributions to laboratory work and the analysis of data; XFG provided the experiment ideas and revised the manuscript; YJZ performed cell culture; DQX performed Ca2+ image and electrophysiology; YHC and YS took part in behavioral testing; DXL supervised this project and revised the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Zhen Wang.

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Li, Tt., Guo, Xf., Zhao, Yj. et al. Aberrant neuronal excitation promotes neuroinflammation in the primary motor cortex of ischemic stroke mice. Acta Pharmacol Sin 46, 2105–2119 (2025). https://doi.org/10.1038/s41401-025-01518-6

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