Stroke is a leading cause of death and long-term disability worldwide, characterized by high mortality rates, high incidence, and high recurrence rates. Ischemic stroke accounts for approximately 60–70 % of all strokes and is caused by a sudden blockage of a brain artery (Hilkens et al., 2024; Yoshimura et al., 2022). This blockage causes interrupted blood flow and oxygen supply, leading to neuronal damage or death. The progression includes irreversible death in the ischemic core, followed by damage in the penumbra (Hazelton et al., 2022; Kumari et al., 2024; Liu et al., 2024a, Liu et al., 2024b). In the treatment of stroke, acute-phase therapy primarily focuses on neuroprotection by inhibiting cell death (Qiao et al., 2023). However, due to the narrow treatment time-window, only a small number of patients benefit from these interventions. In the subacute and chronic phases, neurorehabilitation methods aimed at enhancing neural plasticity are mainly employed (Kumar and Kitago, 2019; Ward, 2017). The majority of patients improve their neurological function through rehabilitation during this stage, although the clinical effectiveness of rehabilitation therapy remains limited.

Stroke can lead to both local and global brain inflammation, and immune function plays a crucial role in regulating neuroinflammation (Shi et al., 2019). Microglia, as the brain's only resident immune cells, are the first line of defense in the central nervous system (CNS) against pathogen invasion (Gerganova et al., 2022). After stroke, the activation, polarization, and phagocytosis of microglia are essential for modulating the neuroinflammatory microenvironment and enhancing neuroplasticity (Hu et al., 2015; Marinelli et al., 2019). As the stroke progresses, in the acute phase, microglia are primarily located at the infarct core and the surrounding penumbra, predominantly exhibiting anti-inflammatory characteristics. In the subacute and chronic phases, pro-inflammatory microglial phenotypes rapidly increase and become predominant, with microglia spreading to surrounding and distant brain areas (Hu et al., 2012; Wang et al., 2022a, Wang et al., 2022b). The subacute and chronic phases after stroke are also the periods of most active spontaneous neural plasticity, which can promote functional recovery post-stroke. Our previous research has shown that the development of this neural plasticity temporally and spatially overlaps with pro-inflammatory microglial activation, which we refer to as spatiotemporal co-localization (Yu et al., 2021). This suggests that microglia may have a profound impact on neuroplasticity after stroke and could be a key therapeutic target for stroke rehabilitation.

Exogenous therapies targeting microglia become of the significance and can promote recovery after stroke. Several studies have shown that various interventions can effectively regulate the polarization of microglia to an anti-inflammatory phenotype after stroke. The common approaches include pharmacological treatments (Ran et al., 2021) or drug-contained biomaterials (Liu et al., 2023), exogenous cell therapy (Orczykowski et al., 2019), physical exercise (Svensson et al., 2016), and non-invasive brain stimulation methods which mainly include transcranial electrical stimulation (Walter et al., 2022), transcranial magnetic stimulation, transcranial focused ultrasound (Wang et al., 2021), and transcranial low-level laser therapy (Vogel et al., 2021). Among these, repetitive transcranial magnetic stimulation (rTMS) is a widely used physical therapy method with multiple targets. Previous research has indicated that it can modulate brain excitability, improve blood-brain barrier permeability, regulate neurotransmitter and cytokine levels, and reconstruct brain networks (Kemps et al., 2022). Additionally, some studies suggest that rTMS can target microglia to promote post-stroke recovery, although the underlying mechanisms have not yet been fully elucidated (Ferreira et al., 2024).

rTMS can induce both electrical currents and magnetic fields simultaneously affecting the CNS. Previous research on the mechanisms of rTMS modulation of neural function post-stroke mostly focuses on neurons. The magnetic fields produced by rTMS can induce electrical currents in the cortex, leading to neuronal depolarization. This enhances synaptic plasticity through long-term potentiation (LTP) and long-term depression (LTD). However, the effects of the magnetic field itself are still not well understood (Chervyakov et al., 2015; Monday et al., 2018). A recent study has used low-field strength transcranial magnetic stimulation on Purkinje fibers in the cerebellum. They found that the ability of rTMS to promote neural regeneration was significantly reduced in Cry1 and Cry2 knockout mice with left cerebellar lesions, indicating that Cry proteins may act as magnetic-responsive proteins and are primary candidates for biochemical magnetic receptors (Dufor et al., 2019). Cryptochromes (Cry) are flavoproteins closely related to photolyase, with Cry1 and Cry2 being the most studied. These proteins are expressed in various cells and play roles in circadian rhythm regulation, immune responses, and carbohydrate metabolism. The Cry protein family is also considered the only candidate for magnetic receptors in animals due to their ability to sense magnetic fields through quantum spin dynamics involving light-induced “free radical pairs” (Dodson et al., 2013; Karki et al., 2021; Qin et al., 2016). Additionally, the research has shown that in myeloid-derived macrophages, Cry knockdown leads to increased expression of pro-inflammatory cytokines like IL-6, chemokines, and iNOS (Narasimamurthy et al., 2012). In atherosclerosis models, Cry knockout in macrophages resulted in upregulation of pro-inflammatory factors and downregulation of anti-inflammatory factors, suggesting that Cry plays a crucial role in regulating inflammation in macrophages (Yang et al., 2015). It remains unclear whether microglia can also respond to magnetic stimulation through Cry proteins and subsequently undergo biological regulatory functions.

Therefore, our study employed a special pattern of rTMS: the FDA-approved intermittent theta-burst stimulation (iTBS) paradigm to focus on the direct effects of the magnetic field produced by iTBS on microglia. The aim is to investigate how iTBS modulates microglial polarization, thereby enhancing neuroplasticity and promoting stroke recovery, and to explore the downstream signaling pathways. This research aims to provide new theoretical insights for the clinical application of iTBS.