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Vagus nerve stimulation as a promising neuroprotection for ischemic stroke via α7nAchR-dependent inactivation of microglial NLRP3 inflammasome

Abstract

Ischemic stroke is a major cause of disability and death worldwide, and its management requires urgent attention. Previous studies have shown that vagus nerve stimulation (VNS) exerts neuroprotection in ischemic stroke by inhibiting neuroinflammation and apoptosis. In this study, we evaluated the timing for VNS intervention in ischemic stroke, and the underlying mechanisms  of VNS-induced neuroprotection. Mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 60 min. The left vagus nerve at cervical level was exposed and attached to an electrode connected to a low-frequency electrical stimulator. Vagus nerve stimulation (VNS) was given for 60 min before, during and after tMCAO (Pre-VNS, Dur-VNS, Post-VNS). Neurological function was assessed 24 h after reperfusion. We found that all the three VNS significantly protected against the tMCAO-induced injury evidenced by improved neurological function and reduced infarct volume. Moreover, the Pre-VNS was the most effective against the ischemic injury. We found that tMCAO activated microglia in the ischemic core and penumbra regions of the brain, followed by the NLRP3 inflammasome activation-induced neuroinflammation, which finally triggered neuronal death. VNS treatment preserved α7nAChR expression in the penumbra regions, inhibited NLRP3 inflammasome activation and ensuing neuroinflammation, rescuing cerebral neurons. The role of α7nAChR in microglial NLRP3 inflammasome activation in ischemic stroke was further validated using genetic manipulations, including Chrna7 knockout mice and microglial Chrna7 overexpression mice, as well as pharmacological interventions using the α7nAChR inhibitor methyllycaconitine and agonist PNU-282987. Collectively, this study demonstrates the potential of VNS as a safe and effective strategy to treat ischemic stroke, and presents a new approach targeting microglial NLRP3 inflammasome, which might be therapeutic for other inflammation-related diseases.

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Fig. 1: VNS treatment improves neurological function and reduces infarct volume in the tMCAO mouse model.
Fig. 2: VNS treatment rescues neuronal loss via inhibiting microglia activation-induced neuroinflammation.
Fig. 3: VNS treatment inhibits microglial NLRP3 inflammasome activation in the penumbra region of tMCAO mice.
Fig. 4: VNS treatment preserves α7nAChR expression in the penumbra area of tMCAO mice.
Fig. 5: Pharmacological inhibition of α7nAChR abrogates the protective effect of VNS treatment on ischemic injury.
Fig. 6: Genetic deletion of Chrna7 abolishes the therapeutic effect of VNS treatment on ischemic injury.
Fig. 7: Overexpression of microglial Chrna7 reinstates the therapeutic efficacy of VNS intervention on ischemic injury.
Fig. 8: Activation of α7nAChR by PNU treatment inhibits NLRP3 expression through NF-κB signaling.
Fig. 9: Activation of α7nAChR by PNU treatment inhibits microglial NLRP3 inflammasome-induced neuronal death.
Fig. 10: Schematic illustration of α7nAChR-dependent inactivation of microglial NLRP3 inflammasome.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (82204357, 82173797), the National Key R&D Program of China (2021ZD0202903), the Natural Science Fund of the Basic Research Program of the Science and Technology Department of Jiangsu Province (BK20231267) and the Medical Research Project of Jiangsu Provincial Health Commission (M2022071).

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ML, XL, and LC conceived and designed this study. XMX, YD, YPW, SYJ, and YFD acquired, analyzed, and interpreted the data. YZ, RXH, and CQ contributed to the methodologies. LC wrote the original manuscript draft. ML and XL revised the manuscript and supervised the project. All authors have confirmed the submission of this manuscript.

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Correspondence to Lei Cao, Xiao Lu or Ming Lu.

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Xia, Xm., Duan, Y., Wang, Yp. et al. Vagus nerve stimulation as a promising neuroprotection for ischemic stroke via α7nAchR-dependent inactivation of microglial NLRP3 inflammasome. Acta Pharmacol Sin 45, 1349–1365 (2024). https://doi.org/10.1038/s41401-024-01245-4

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