Intermittent theta-burst stimulation improves motor function by inhibiting neuronal pyroptosis and regulating microglial polarization via TLR4/NFκB/NLRP3 signaling pathway in cerebral ischemic mice

Background: Neuronal Pyroptosis and neuroinflammation with excess microglial activation are widely involved in the early pathological process of ischemic stroke. Repetitive transcranial magnetic stimulation (rTMS), as a non-invasive neuromodulatory technique, has recently been reported to be anti-inflammatory and regulate microglial function. However, few studies have elucidated the role and mechanism of rTMS underlying regulating neuronal Pyroptosis and microglial polarization.

Methods: We evaluated the motor function in middle cerebral artery occlusion/reperfusion (MCAO/r) injury mice after 1-week intermittent theta-burst rTMS (iTBS) treatment in the early phase with or without depletion of microglia by colony-stimulating factor 1 receptor (CSF1R) inhibitor treatment, respectively. We further explored the morphological and molecular biological alterations associated with neuronal Pyroptosis and microglial polarization via Nissl, EdU, TTC, TUNEL staining, electron microscopy, multiplex cytokine bioassays, western blot assays, immunofluorescence staining and RNA Sequencing.

Results: ITBS significantly protected against cerebral ischemia/reperfusion (I/R) injury-induced locomotor deficits and neuronal damage, which probably relied on the regulation of innate immune and inflammatory responses, as evidenced by RNA Sequencing analysis. The peak of Pyroptosis was confirmed to be later than that of Apoptosis during the early phase of stroke, and Pyroptosis was mainly located and more severe in the peri-infarcted area compared with Apoptosis. Multiplex cytokine bioassays showed that iTBS significantly ameliorated the high levels of IL-1β, IL-17A, TNF-α, IFN-γ in MCAO/r group and elevated the level of IL-10. ITBS inhibited the expression of neuronal pyroptosis-associated proteins (i.e., Caspase1, IL-1β, IL-18, ASC, GSDMD, NLRP1) in the peri-infarcted area rather than at the border of infarcted core. KEGG enrichment analysis and further studies demonstrated that iTBS significantly shifted the microglial M1/M2 phenotype balance by curbing proinflammatory M1 activation (Iba1⁺/CD86⁺) and enhancing the anti-inflammatory M2 activation (Iba1⁺/CD206⁺) in peri-infarcted area via inhibiting TLR4/NFκB/NLRP3 signaling pathway. Depletion of microglia using CSF1R inhibitor (PLX3397) eliminated the motor functional improvements after iTBS treatment.

Conclusions: rTMS could alleviate cerebral I/R injury induced locomotor deficits and neuronal Pyroptosis by modulating the microglial polarization. It is expected that these data will provide novel insights into the mechanisms of rTMS protecting against cerebral I/R injury and potential targets underlying neuronal Pyroptosis in the early phase of stroke.

Ischemic stroke; Magnetic stimulation; Microglia; Neuroinflammation; Pyroptosis.