Synergistic Neuroprotection of Fe₃O₄ Nanoparticles Combined with Intermittent Theta Burst Stimulation in Ischemic Stroke via Nrf2-Mediated Ferroptosis Regulation

Introduction

Ischemic stroke (IS) remains the second leading cause of adult mortality worldwide and a major contributor to long-term neurological disability.¹ It results from arterial occlusion that disrupts cerebral blood flow,² leading to rapid energy failure and irreversible neuronal injury. Neurons, characterized by high metabolic demand, are particularly vulnerable to ischemia. Oxygen deprivation rapidly depletes ATP, impairs ion homeostasis, and triggers glutamate-mediated excitotoxicity. Subsequently, mitochondrial dysfunction leads to excessive reactive oxygen species (ROS) production, overwhelming endogenous antioxidant defenses and initiating lipid peroxidation. Concurrently, microglia become activated and predominantly polarize toward a pro-inflammatory (M1) phenotype, releasing cytokines such as TNF-α and IL-1β³ that amplify neuroinflammation and exacerbate neuronal apoptosis. Together, oxidative stress, ferroptosis, mitochondrial dysfunction, and sustained inflammation form the core pathological cascade of ischemia/reperfusion (I/R) injury.³

From a pathological standpoint, ischemic lesions consist of a necrotic core surrounded by the ischemic penumbra. While the core region undergoes rapid and irreversible cell death, neurons within the penumbra remain metabolically compromised but potentially salvageable. Timely intervention targeting this region can significantly reduce neurological deficits. Currently, intravenous thrombolysis and mechanical thrombectomy are the primary reperfusion strategies; however, these treatments are limited by strict therapeutic windows and do not directly address secondary injury mechanisms such as oxidative stress and ferroptosis. Moreover, the blood–brain barrier (BBB) restricts the delivery of many therapeutic agents, thereby limiting targeted neuroprotection. Therefore, developing strategies that simultaneously enhance BBB penetration, improve lesion targeting, and modulate multiple injury pathways remains an urgent clinical priority.

Non-invasive neuromodulation techniques have emerged as promising adjunctive therapies in stroke rehabilitation. Among them, intermittent theta-burst stimulation (iTBS) represents a refined form of transcranial magnetic stimulation (TMS). Unlike conventional repetitive TMS (rTMS), which employs fixed low- or high-frequency stimulation over prolonged sessions, iTBS delivers short bursts of high-frequency (50 Hz) pulses embedded within a theta rhythm (5 Hz), mimicking physiological theta–gamma coupling observed in the hippocampus. This patterned stimulation more efficiently induces long-term potentiation-like synaptic plasticity while substantially reducing treatment duration. A typical iTBS session lasts only 3–5 minutes, representing nearly a 90% reduction in stimulation time compared with conventional rTMS, thereby improving patient compliance and safety. Increasing evidence suggests that iTBS enhances cortical excitability, promotes synaptic remodeling, and improves motor and cognitive recovery after stroke in both experimental and clinical settings.^4,5 Nevertheless, the spatial precision and depth of magnetic stimulation remain constrained, limiting its ability to directly target deep or heterogeneous ischemic regions.

Magnetic nanoparticles offer a potential solution to these targeting limitations. Superparamagnetic Fe₃O₄ nanoparticles (Fe₃O₄ NPs) exhibit strong magnetic responsiveness under external magnetic fields while showing minimal remanence after field removal, enabling controllable targeting within biological systems. Under magnetic guidance, Fe₃O₄ NPs can accumulate preferentially in specific brain regions, potentially enhancing local therapeutic concentration. In addition to their targeting capacity, Fe₃O₄ NPs possess redox-modulating properties. Iron-based nanomaterials have been reported to regulate ROS dynamics and participate in antioxidant defense processes under controlled conditions.⁶

Importantly, iron biology in ischemic injury is complex and bidirectional. Free or labile Fe²⁺ can catalyze hydroxyl radical formation through Fenton-type reactions, thereby promoting lipid peroxidation and ferroptosis.^7–9 Conversely, iron stabilized within crystalline structures such as Fe₃O₄ and regulated by intracellular iron-storage systems may contribute to redox homeostasis. Therefore, the biological outcome of iron-based nanomaterials depends critically on iron release kinetics and the cellular antioxidant environment. In the present study, Fe²⁺ is structurally stabilized within the Fe₃O₄ lattice and further modified to reduce uncontrolled iron release. More importantly, we demonstrate that iTBS activates the Nrf2-dependent antioxidant program, upregulating GPX4 and ferritin (FTH), which limit lipid peroxide accumulation and sequester labile iron. This coordinated regulation favors ferroptosis suppression rather than ferroptosis induction.

Based on these considerations, we propose a synergistic therapeutic strategy that integrates intermittent theta-burst stimulation with magnetically guided Fe₃O₄ NPs. Under external magnetic field guidance, Fe₃O₄ NPs are directed toward the ischemic penumbra. Simultaneously, iTBS modulates neural circuits and transiently enhances BBB permeability, facilitating nanoparticle accumulation within the lesion. Beyond serving as targeted redox modulators, Fe₃O₄ NPs may also enhance the local magnetic field response, improving stimulation precision.

Crucially, the novelty of this work lies in the specific integration of iTBS—rather than TMS in general—with magnetic nanoparticles. The short burst-patterned stimulation of iTBS is temporally compatible with magnetic targeting, enabling coordinated “stimulation–targeting–protection” cycles. Compared with rTMS, iTBS demonstrates higher targeting efficiency, shorter treatment duration, and improved neuromodulatory precision. By combining physical neuromodulation with magnetically guided nanotherapy, this iTBS–Fe₃O₄ system is designed to simultaneously address oxidative stress, ferroptosis, mitochondrial dysfunction, and neuroinflammation. This integrated approach provides a mechanistically grounded and translationally relevant strategy for precise intervention in ischemic stroke (Figure 1).

Figure 1 Therapeutic mechanism of Fe3O4 for precise ischemic stroke management.

Materials and Methods

Preparation of Fe₃O₄ NPs

Fe₃O₄ nanoparticles (250 mg) were initially dispersed in deionized water to form a homogeneous suspension. To prepare Fe₃O₄@F127 nanoparticles, an equal volume of 0.3 mg/mL Pluronic F127 aqueous solution was added dropwise under continuous magnetic stirring. The reaction mixture was stirred for 24 h at room temperature to allow surface adsorption of F127 onto Fe₃O₄ nanoparticles.

The suspension was subsequently centrifuged at 10,000 rpm for 10 min, and the supernatant was discarded. The pellet was washed three times with deionized water to remove unbound F127 and then re-dispersed.

For ethylenediamine (Eda) functionalization, the Fe₃O₄@F127 suspension was mixed with 4 mg/mL Eda solution prepared in ethanol and stirred for 48 h at room temperature.¹⁰ After functionalization, the suspension was centrifuged again at 10,000 rpm for 10 min, followed by three washing steps with deionized water to remove excess Eda and residual solvent.

The final Fe₃O₄@F127/Eda nanoparticles were re-dispersed in 3 mL deionized water and stored at 4°C for subsequent experiments.

The nanoparticle concentration was determined using a gravimetric dry-weight method, in which a known volume of the final suspension was dried to constant weight. The final concentration was 74.8 mg/mL, corresponding to an overall recovery yield of 89.8% relative to the initial 250 mg Fe₃O₄ input.

Transmission electron microscope (TEM), scanning electron microscope (SEM) and Energy dispersive X-ray spectroscopy (EDS) elemental mapping were performed using a JEOL (Tokyo, Japan) JEM-2100F microscope.¹¹ Samples were negatively stained with uranyl acetate. Hydrodynamic diameters and ζ-potential values were measured using a Malvern Zetasizer Nano-ZS system (Malvern Panalytical Ltd). Hydrodynamic diameters were determined by dynamic light scattering.

Cell Culture

SH-SY5Y cells (Cat No. FH0156) and mouse microglia BV2 (Cat No. FH0355) cells were provided by Shanghai Fuheng Biotechnology Co (Shanghai, China). Cell culture dishes and centrifuge tubes were obtained from TiBio (Shanghai, China). The growth conditions for all cell cultures were identical, maintained in an incubator set at 37°C with 5% CO₂ and high humidity. The culture media used consisted of 10% FBS, along with 100 U mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin. The distinction lay in the base medium, with SH-SY5Y cells being maintained in DMEM/F-12 and BV2 cells in DMEM.

Cell Viability Assay

Biocompatibility assessment of the materials involved using the Cell Counting Kit-8 (CCK-8) assay.¹² We initiated the procedure by plating SH-SY5Y and BV2 cells in 96-well plates (1×10⁴ cells/well) and treating them with different Fe₃O₄ concentrations for 12 h. After this incubation, 10 μL of CCK-8 solution was introduced into each well; following a 2-h incubation, absorbance measurements at 450 nm were taken with a microplate reader to quantify cell viability.

Establishment Of Oxygen and Glucose Deprivation/Reoxygenation (OGD/R) Model

We developed an oxygen-glucose deprivation/reoxygenation (OGD/R) model in SH-SY5Y and BV2 cell cultures to simulate the effects of ischemia/reperfusion injury under controlled in vitro conditions. All cells underwent a 24-hour cultivation period in DMEM medium enriched with 10% FBS, maintained at 37°C within a humidified 5% CO₂incubator. Then, Cells were washed with PBS and exposed to deoxygenated DMEM medium without glucose in a sealed anaerobic chamber flushed with 1% O₂, 5% CO₂ and 94% N₂ at 37°C for 8 h to simulate the OGD process.¹³ Afterwards, to initiate the reoxygenation phase, we rapidly substituted the deoxygenated medium with standard DMEM and maintained the cells for 24 hours under normoxic conditions (5% CO₂).

Animals and Ethics

The study was approved by the Ethics Committee of Shanghai University of Medicine and Health Sciences under the approval number 2024–16-44142319980903234X. All procedures involving animals adhered to the strict guidelines set forth by the U.S. National Institutes of Health for the care and use of laboratory animals (Bethesda, MD, USA). Male SD rats (250 ± 10 g) were chosen for the study, sourced from Shanghai Jihui Experimental Animal Feeding Co., Ltd. (Shanghai, China). These animals were kept in a controlled environment, with a stable temperature of 24 ± 2°C, humidity maintained at 55 ± 5%, and a 12-hour light/dark cycle.

Establishment of MCAO/R Rat Model

According to reported methods,¹⁴ a middle cerebral artery occlusion/reperfusion (MCAO/R) injury was created in vivo using male Sprague–Dawley rats, but the procedure was carried out in a sequence different from that of standard descriptions. After the animals were fasted overnight, we induced anesthesia with 2% isoflurane in a 70% N₂ and 30% O₂ mixture and then placed the rats on mechanical ventilation. Under sterile surgical conditions, the right carotid artery system was exposed; a 0.26-mm nylon filament coated with silicone was advanced until it blocked the middle cerebral artery. This occlusion lasted for 120 minutes, after which blood flow was restored. In contrast, sham rats experienced every step of the surgery except for the actual occlusion.

Two hours after reperfusion, the animals received intravenous administration of the various formulations. Twenty-four hours later, euthanasia was performed. The brains were extracted, briefly chilled at −20 °C for 20 minutes, and then cut into coronal slices 2 mm thick. We stained these sections with 2% TTC at 37 °C while gently shaking them for 30 minutes. Using ImageJ, the infarcted regions—recognized as white, TTC-negative tissue—were quantitatively compared with the intact red areas to determine infarct percentage. TTC staining was performed as a qualitative validation of the MCAO model.

Transcranial Magnetic Stimulation (iTBS and rTMS) Treatment

In vivo,The 4-8-week-old healthy male Sprague-Dawley (SD) rats were randomly divided into eight groups: 1) Sham group, 2) Saline group, 3) MCAO/R group, 4) iTBS group, 5) rTMS group, 6) Fe₃O₄+iTBS group, 7) Fe₃O₄+rTMS group, 8) Fe₃O₄+magnet group. For the latter seven groups, the rats underwent the MCAO/R procedure. The sham group underwent the same surgical procedure without occlusion. Treatment was initiated 6 h after stroke (an early rehabilitation window) and administered daily from day 1 to day 7 after stroke. During the in vitro experiments, the magnetic coil was positioned 1 cm from the culture dish and maintained a constant distance from the cellular cultures. Different treatments were applied to the cells, which included the following groups: 1) Control, 2) OGD/R, 3) iTBS, 4) rTMS, 5) Fe₃O₄ + iTBS, 6) Fe₃O₄ + rTMS, and 7) Fe₃O₄. Cells were treated with OGD/R, followed by 24 hours of reoxygenation, and then subjected to continuous treatment for 2 days.

Transcranial Magnetic Stimulation Protocols

In our study, the iTBS parameters were 120% RMT, intra-cluster frequency 50 Hz, inter-cluster frequency 5 Hz, stimulation time 2 s, interval 8 s, 20 repetitions, for a total of 600 pulses rTMS parameters were 120% RMT, 4 s of stimulation, 26s of interval, and 3000 pulses. The transcranial magnetic stimulator is manufactured by Yiruide (Wuhan, China). A Y064 circular coil with a diameter of 64 mm was used for stimulation. In each session, the coil was positioned directly against the scalp and aligned perpendicular to the cortical surface. The stimulator’s circular coil was placed tangentially over the left central region of the rat’s skull. RMT was defined as the minimum stimulation intensity required to elicit visible contralateral forelimb movement in at least 5 out of 10 consecutive stimulations. For static magnetic field experiments, a 0.35 T magnet was positioned at the same location for 30 minutes.

Prussian Blue Staining

Prussian blue staining was performed on 20 μM-thick rat brain slices according to an established protocol. The procedure began by mixing equal parts of Solution A and Solution B (G1029-100ML, Servicebio) immediately before use. The slices were then immersed in this mixture for 20 minutes, rinsed with distilled water, and subsequently counterstained with nuclear fast red. The final steps involved dehydration, clearing in xylene, and mounting the samples with a resinous medium under coverslips.

Terminal dUTP Nick End-Labeling (TUNEL) Staining

To detect apoptotic cells in rat brain paraffin sections, a TUNEL assay was performed using a commercial kit (Vazyme, A112-03). Following deparaffinization and rehydration, sections were permeabilized with Proteinase K (1:100 dilution) for 20 minutes at room temperature. After washing, tissue was equilibrated with 1× Equilibration Buffer, then incubated with a reaction mixture containing Recombinant TdT enzyme and brightGreen Labeling Mix in a humidified chamber at 37°C for 60 minutes. Nuclei were counterstained with DAPI, and sections were mounted with an anti-fade medium. Fluorescence images were acquired using a slide scanner (3DHISTECH PANNORAMIC MIDI) and a Nikon fluorescence microscope, with TUNEL-positive nuclei appearing green.

Immunofluorescence Staining

Cell Culture Samples

To prepare BV2 or SH-SY5Y cells for immunostaining, we first exposed them to 4% paraformaldehyde for 20 minutes, which fixed the specimens. A brief treatment with 0.2% Triton X-100 for 10 minutes was then used to permeabilize the membranes. Only after a 2-hour block in 3% BSA were the cultures allowed to interact with primary antibodies, a step carried out overnight at 4 °C. The antibody panel included rabbit anti-IBA-1 (1:5000, Abcam, ab178847), rabbit anti-CD206 (1:2000, Abcam, ab300621), and rabbit anti-NeuN (1:1000, Proteintech, 26975-1-AP).

Tissue Sections

The brains were initially fixed in a 4% paraformaldehyde solution and then embedded in O.C.T. compound. To retrieve antigens for CD86 and GPX4 staining, EDTA (pH 8.0) was applied. After blocking with either goat serum or 1% BSA, sections were incubated overnight at 4°C with primary antibodies: rabbit anti-CD86, rabbit anti-FTH1 (1:200, Proteintech, 11682-1-AP), and mouse anti-GPX4 (1:400, Proteintech, 67763-1-IG) Following several washes, secondary antibodies were applied for 2 hours at room temperature: HRP-conjugated goat anti-rabbit IgG (H+L) with fluorescence at 570 nm (for CD86), Cy3-conjugated goat anti-rabbit IgG (for FTH1), and Alexa Fluor 488-conjugated goat anti-mouse IgG (for GPX4). Nuclear staining was achieved using DAPI. Finally, confocal microscopy was used to visualize protein expression.

ROS Production Assay

Following the instructions provided with the Reactive Oxygen Species Assay Kit (UE, R6033), intracellular ROS levels were quantified as described by the manufacturer. The method involved incubating the cells with the fluorescent ROS probe DHE at specified concentrations for general ROS detection. Subsequent quantification of ROS levels was achieved by capturing and analyzing the resulting fluorescence images under a microscope.

ELISA

ELISA kits from Neobioscience (China) were used to measure the levels of TNF-α and IL-6 in the ACM samples, following the detailed protocol provided by the manufacturer.

Mitochondrial Membrane Potential

To evaluate the mitochondrial membrane potential (ΔΨm), cells were exposed to the JC-1 fluorescent probe. After treatment, a 2 µM JC-1 solution was applied, and the cells were maintained at 37 °C for 30 min in darkness. Once washed, fluorescence signals were recorded using either a flow cytometer or a microplate reader. The dye produced red fluorescence (aggregated form, reflecting high ΔΨm) and green fluorescence (monomeric form, indicating low ΔΨm) at excitation/emission wavelengths of 585/590 nm and 510/527 nm, respectively. The red-to-green fluorescence intensity ratio was then derived; a decline in this ratio corresponded to mitochondrial membrane depolarization.

Y-Maze Test

Each rat was introduced into Arm A—the distal end of one branch—of a Y-shaped black Plexiglas maze, whose three arms (20 cm × 10 cm × 20 cm) extended at 120° intervals. The animals were permitted to explore freely for eight minutes while their activity was continuously tracked by an overhead camera. Based on the recorded sequences of arm entries, spontaneous alternation behavior was quantified using the conventional calculation, representing the proportion of entries into an arm different from the previous two.¹⁵

Barnes-Maze Test

In a variation of the earlier Barnes maze setup, a customized apparatus featured a circular platform 92 cm in width, elevated and made of PVC (Maze Engineers, Skokie, IL, USA). Around its perimeter, 20 holes—each 4.45 cm in diameter—were arranged 2.54 cm from the edge. Rats first underwent a 3-minute habituation phase: placed at the center, they freely explored the platform without interference. Subsequently, mice received training to find an escape chamber under a designated peripheral hole. This training spanned three days and included seven sessions, each lasting 3 minutes. To evaluate learning, each session measured the time taken to locate the target hole and the failure rate, defined as the percentage of mice that failed to reach it.

For the memory probe trial, the escape chamber was removed. Mouse movements were then tracked continuously over 3 minutes, regardless of how quickly the target hole was initially found. Any mouse that did not find the target hole within the allotted time was excluded from analysis. Several metrics were documented: initial target hole detection time, total duration spent in the target quadrant, and time in that quadrant after the hole was first identified.

An automated tracking system (Any Maze, Stoelting, Chicago, IL, USA) was used to monitor animal behavior. All behavioral data were analyzed by an experimenter unaware of which treatment each rat had received.¹⁶

Western Blot Analysis

Proteins were isolated from brain tissue lysates as outlined in previous studies.¹⁷ Equal amounts of protein were subjected to SDS–PAGE, followed by transfer onto PVDF membranes. These membranes were incubated at room temperature for 2 hours to block non-specific binding sites. Afterward, the membranes were exposed to primary antibodies targeting glutathione peroxidase 4 (GPX4; Proteintech, 67763-1-Ig, 1:1000) and nuclear factor erythroid 2–related factor 2 (Nrf2; Proteintech, 16396-1-AP, 1:2000) for an overnight period at 4°C. Following the primary antibody incubation, HRP-conjugated secondary antibodies were applied for 2 hours at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL), and the band intensities were measured with Image-Pro Plus 6.0 software for quantification.

Biosafety Analysis

Biosafety was tested by hemolysis assay and hematoxylin-eosin (H&E) staining. Fresh blood was drawn in anticoagulation tubes, centrifuged at 3000 r/min for 5 min, and washed. The washed erythrocytes were diluted to 2% hemoglobin with PBS and set aside. We used 1.0 mL PBS as a negative control and 1 mL deionized water as a positive control in Eppendorf tubes. We prepared nanoparticle test group samples at 100, 50, 25, 12.5, 6.25 and 3.13 μg/mL solution with PBS as the diluent, and added 1 mL of each concentration to Eppendorf tubes. We then added 0.4 mL of erythrocyte suspension to the negative and positive experimental groups, setting up three parallel sample tubes in each group, and centrifuged them after incubation for 4 h. The Eppendorf tubes were photographed and recorded at this time, and the absorption of the supernatant of each group at 541 nm was measured using a UV spectrophotometer, from which the hemolysis rate was calculated using the following formula: Hemolysis = (A _sample −A_PBS) / (A_water − A_PBS) X 100%, where A=absorbance at 541 nm.

Statistical Analysis

Data distribution normality was assessed using the Shapiro–Wilk test. Homogeneity of variance was evaluated using Levene’s test. When assumptions were satisfied, parametric tests were applied; otherwise, non-parametric alternatives were used.

Sample size estimation was performed a priori using G*Power software (version 3.1.9.7, Heinrich-Heine-University Düsseldorf, Germany). Based on preliminary experiments and published data from similar MCAO/R behavioral and molecular studies, a large effect size (f = 0.40, corresponding to Cohen’s d ≈ 0.8), α = 0.05, and statistical power (1−β) = 0.80 were assumed for one-way ANOVA comparisons among groups. Under these parameters, the calculated minimum sample size was 6 animals per group to detect significant differences in behavioral and molecular endpoints.

For key primary outcomes (Barnes maze escape latency and Nrf2/GPX4 expression levels), post hoc power analysis confirmed that the achieved statistical power exceeded 0.80 based on the observed effect sizes.

Due to the exploratory nature of certain mechanistic assays (eg., preliminary fluorescence imaging and in vitro screening experiments), some experiments were conducted with smaller sample sizes (n = 2–3), and these results should be interpreted with caution.

Results

Preparation and Characterization of Fe₃O₄ NPs

We synthesized Fe₃O₄ NPs through a series of steps. TEM images showed that the Fe₃O₄ NPs were well dispersed and homogeneous with a spherical shape (Figure 2A). The nanoparticles showed zeta potentials of −27.8 ± 1.2 mV (1000× dilution) and −22.5 ± 2.1 mV (10000× dilution) and a hydrodynamic diameter of 133.5 ± 1.2 nm, respectively (n = 3) (Figure 2B and C). On the side of the 5-mL centrifuge tube, Fe₃O₄ NPs were aggregated towards the static magnetic field, verifying that the Fe₃O₄ NPs had a strong magnetic field, aggregated toward the static magnetic field, verifying the magnetic targeting of Fe₃O₄ NPs (Figure 2D). In addition, iR780-labeled Fe₃O₄ NPs showed red fluorescence in the in vivo imaging instrument, confirming the successful attachment of iR780 (Figure 2E). EDS elemental mapping also confirmed the presence and co-localization of the characteristic elements of each component in Fe₃O₄ NPs (Figure 2F).

Figure 2 Fabrication and characterization of Fe3O4. (A) Representative TEM images of prepared NPs. (B) zeta potential measurements (n = 3). (C) Size distributions of Fe3O4 by dynamic light scattering (n = 3). (D) Confirmation of magnetic targeting of Fe3O4 by static magnetic field. Red circles indicate nanoparticle aggregation toward the magnetic field. (E) Fe3O4 labeled with iR780 by fluorescence images. (F) Characterization of Fe3O4 NPs using EDS.

BBB-Penetrability, Biodistribution and Biosafety

The biosafety of nanomedicine delivery systems is a key factor in clinical translational issues. We investigated the biosafety of Fe₃O₄ NPs by in vitro hemolysis experiments. The hemolysis rate of Fe₃O₄ NPs was <5% at different concentrations, ie., none of them were hemolyzed, indicating that Fe₃O₄ has good biosafety (Figure 3A). In vivo fluorescence images showed that fluorescein iR780-labeled Fe₃O₄ NPs were able to cross the BBB and accumulate in the brain (Figure 3B). The in vivo fluorescence images also showed that the fluorescence signals of Fe₃O₄-injected rats were strongest at 48 h without magnetic stimulation (Figure 3C and D), and still present 120 h post-injection, when obvious fluorescence signals could still be observed. In addition, differences in organ distribution patterns were observed following transcranial magnetic stimulation; however, no pharmacokinetic analysis (eg., blood half-life measurement) was performed in the present study (Figure 3E). To evaluate histopathological alterations in ischemic brain sections, hematoxylin and eosin staining was conducted.¹⁸ As illustrated in Figure 3F, the Fe₃O₄ nanoparticle–treated group exhibited cellular structures comparable to those observed in the sham group, further supporting the biocompatibility of Fe₃O₄ NPs. Moreover, Prussian blue staining, which labels iron ions in tissue sections¹⁹ (Figure 3G), showed that Fe₃O₄ NPs could penetrate the BBB to reach brain tissue, but the accumulation of Fe₃O₄ NPs in brain tissue was small; in contrast, the ability of Fe₃O₄ NPs to penetrate the BBB was increased by about three times in the static magnetic field and in the magnetic targeting effect of transcranial magnetic stimulation (iTBS and rTMS). iTBS and rTMS enhanced the ability of Fe₃O₄ NPs to penetrate the BBB by about 3-fold, while the probability of Fe₃O₄ NPs penetrating the BBB after iTBS stimulation treatment was elevated 1.35-fold compared to rTMS and 1.15-fold compared to the static magnetic field (Figure 3H). Overall, the above study verified that the designed Fe₃O₄ NPs, under the guidance of an external magnetic field, significantly improved BBB penetration. To further evaluate whether iTBS modulates blood–brain barrier integrity, we performed immunofluorescence staining of the tight junction protein ZO-1 in cortical sections (Supplementary Figure 1). MCAO/R induced partial disruption and discontinuity of ZO-1 staining compared with the sham group. Notably, iTBS-treated rats exhibited a transient alteration in ZO-1 distribution characterized by reduced junctional continuity, suggesting tight junction remodeling rather than irreversible structural damage. In the iTBS+Fe₃O₄ group, ZO-1 expression showed partial recovery toward a more continuous pattern, indicating that BBB modulation under iTBS was controlled and reversible. These findings support the concept of transient BBB modulation rather than permanent barrier breakdown.

Figure 3 Study on BBB penetrability, biodistribution and biosafety of Fe3O4 NPs. (A) Hemolysis test of Fe3O4 NPs in different concentrations. (B) Fluorescence images of the heads of MCAO/R rats after intravenous (iv.) injection with iR780-labeled NPs at 6 h. (C) Fluorescence images of brain sections at 0, 24, 48, 72, 96 and 120 h post-injection. Fluorescence signals were quantified using Living Image software with background subtraction. Data represent transient tight junction remodeling and enhanced permeability, and no statistical analysis was performed. (D) Radiant efficiency was quantified at 0, 24, 48, 72, 96, and 120 h post-injection. Data represent a single subject. (E) Fluorescence images of the dissected main organs and brains at 48 h post-injection. (F) Representative H&E staining of ischemic brain sections after different treatments. Scale bar = 20 μM. (G) Prussian blue staining of tumors after iv. injection 3 days of Fe3O4 NPs. Scale bar: 20 μM and (H) their quantitative analyses (n = 5). Data are presented as means ± SD (n = 5). Statistical significance was determined by one-way ANOVA with a Tukey post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

Improvement of Neurological Function by TMS

To determine the therapeutic potential of transcranial magnetic stimulation on IS rats, we first confirmed the successful establishment of the rat MCAO/R model. This was achieved by staining brain slices with 2% 3,5-triphenyltetrazolium chloride, which allowed us to verify the model’s integrity (Supplementary Figure 2). We randomly divided Sprague-Dawley rats into eight groups, which were the MCAO/R control group injected with saline, iTBS group, rTMS group, Fe₃O₄ group, Fe₃O₄+iTBS group, Fe₃O₄+rTMS group, Fe₃O₄+Magnet group and the sham operation group. The sham operation group was also injected with saline. To evaluate changes in learning and spatial memory, MCAO/R rats underwent behavioral testing in the Barnes and Y mazes after receiving different treatments, with their representative escape trajectories being recorded on day 7 post-surgery (Figure 4A). All groups receiving transcranial magnetic stimulation demonstrated reduced escape latency over the five-day learning phase. Conversely, saline-treated animals exhibited substantial cognitive deficits, showing impaired learning acquisition and diminished spatial memory capacity, with the best treatment effect in rats occurring in the transcranial magnetic stimulation combined with Fe₃O₄ NPs group (Figure 4B and C). In the Y maze test, a similar trend was observed: rats treated with transcranial magnetic stimulation, especially in combination with Fe3O4 NPs, exhibited significantly higher spontaneous alternation percentages compared to saline-treated controls, indicating improved working memory performance (Figure 4D–E).

Figure 4 Short-term neurological deficit evaluations in MCAO/R rats after different treatments. (A) Illustration of the treatment regimen in MCAO/R rats. (B) Representative escaping paths of rats that indicated their learning and memory ability determined by Barnes maze test. (C) Escape latency 7 days after MCAO/R after different treatments determined by Barnes maze test (n = 5). (D) Representative exploration trajectories in the Y-maze test. The red dot indicates the starting position, and the blue dot indicates the ending position. Triangle symbols are used to distinguish the start arm, novel arm, and other arms in the Y-maze. (E) Spontaneous alternation rate measured 7 days after MCAO/R in the Y-maze test (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviation: ns: no, statistical significance.

In vivo Therapeutic Efficacy

Based on the results of the behavioral tests in rats, we further designed an in vivo experiment of Fe₃O₄ NPs combined with transcranial magnetic stimulation to investigate its therapeutic effects. We constructed a rat model using the wire bolus method and then intervened with different treatments for 7 days. Apoptosis in the ischemic area of the rat brain following various treatments was assessed using TUNEL staining (Figure 5A). Compared with the saline-treated group, the rats given transcranial magnetic treatment, especially iTBS combined with Fe₃O₄ NPs, showed significantly less Tunel-positive cell apoptosis, suggesting that transcranial magnetic stimulation combined with Fe₃O₄ NPs had a good neuroprotective effect in cerebral ischemic brains (Figure 5B). Next, we observed neuronal survival in force-ischemic and non-ischemic areas by immunofluorescence staining with NeuN antibodies (Figure 5C). Quantitative analysis revealed markedly reduced neuronal survival in ischemic areas following MCAO/R compared to sham operations. All transcranial magnetic therapy modes delivered over 7 days, however, yielded higher neuron survival rates than saline treatment, with the combined iTBS regimen showing the most substantial advantage over the control group. (Figure 5D). The above studies demonstrated that transcranial magnetic stimulation inhibited neuronal damage in the brain tissue of rats with I/R injury, and that the combination of Fe₃O₄ NPs significantly enhanced the neuroprotective effect of transcranial magnetic stimulation, especially for the iTBS treatment mode.

Figure 5 In vivo therapeutic efficacy against IS. (A) Representative TUNEL-stained images in ischemic penumbra after different treatments (n = 3). Scale bar: 20 μM. (B) Quantitative analysis of TUNEL staining. (C) Representative NeuN-stained images in non-ischemic and ischemic regions after different treatments (n = 5). Scale bar: 20 μM. (D) Quantitative analysis of NeuN staining. (E) Detection of ROS levels using DHE staining (n=5). Scale bar: 40 μM. (F) Quantitative analysis of ROS staining. (G) Representative immunofluorescence staining of Iba-1+ and CD206+ cells to determine M2-like microglia (n = 5). Scale bar: 20 μM. (H) Quantitative analysis of microglial staining. Statistical significance was determined by one-way ANOVA with a Tukey post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviation: ns: no, statistical significance.

Given that the overproduction of ROS is a major factor leading to apoptosis or cell death, we next explored the ROS scavenging ability of transcranial magnetic stimulation treatment. To assess the ability of transcranial magnetism to scavenge free radicals in infarcted lesions, a DHE probe was utilized (Figure 5E). Compared with the sham-operated group, the level of ROS was significantly elevated in the brain tissues of MCAO/R-treated rats. After different modes of transcranial magnetic treatment, ROS were inhibited to varying degrees, of which iTBS combined with Fe₃O₄ intervention treatment demonstrated the best ROS scavenging effect (Figure 5F).

To further assess ferroptosis-associated lipid peroxidation in vivo, C11-BODIPY staining was performed in cortical sections (Supplementary Figure 3). MCAO/R markedly increased lipid ROS levels, as evidenced by enhanced green fluorescence and a reduced red/green fluorescence ratio. Treatment with iTBS significantly decreased lipid peroxidation, while the combination of iTBS and Fe₃O₄ NPs produced the most pronounced reduction in lipid ROS accumulation. These findings indicate effective suppression of ferroptosis-related lipid oxidative damage.

The central nervous system’s immune defenses are primarily provided by microglia, which serve as the brain and spinal cord’s main form of macrophage-like cells. These cells play a critical role in immune responses within the CNS, acting as key players in maintaining its health and stability.²⁰ They are in a highly dynamic “surveillance” state, extremely sensitive to microenvironmental changes, and are the first responders to the immune response in the brain. Excessive and sustained pro-inflammatory response and release of neurotoxic substances by M1 microglia are essential for secondary neuronal death, destruction of the BBB, infarct expansion, and long-term tissue repair and functional recovery. M2 microglia are critical for debris removal, release of neurotrophic factors, and suppression of excessive inflammation to limit the extent of injury, protect neurogenesis in the Penumbral zone, and promote late tissue repair and functional recovery. CD206, which represents M2-type microglia, had its expression elevated after treatment (Figure 5G). Compared with the saline injection group, CD206 expression was elevated 1.55-fold and 1.59-fold in the rTMS combined with Fe₃O₄ NPs and iTBS combined with Fe₃O₄ NPs treatment groups, respectively (Figure 5H). Overall, these findings suggest that our nanosystem can modulate microglial phenotype from an ischemia-associated pro-inflammatory M1 phenotype to a recovery-associated anti-inflammatory M2. This ability to reverse the abnormal activation of microglia contributes to synergistic therapies.

In vitro Therapeutic Efficacy

First, we explored the detection of Fe₃O₄ NPs in SH-SY5Y cells and BV2 cells by CCK8 assay. There was no significant difference in the effect of Fe₃O₄ NPs on the survival of BV2 cells at concentrations of 25 μg mL⁻¹ and below each concentration gradient (Figure 6A). Interestingly, it was observed that neurons exposed to OGD/R under high concentrations (>25μg mL⁻¹) of Fe₃O₄ NPs exhibited greater resilience compared to those treated with low concentrations—this effect is associated with nanoparticle-mediated accelerated detoxification of ROS into less reactive species.(Figure 6B). Subsequent experiments were carried out in the range of safe Fe₃O₄ NP concentrations. Given the significant role of oxidative stress in stroke-induced I/R injury, we further investigated the neuroprotective potential of the synthesized nanoparticles. This evaluation specifically assessed their capacity to scavenge reactive oxygen species in SH-SY5Y cells subjected to OGD/R. (Figure 6C). The amount of ROS produced by OGD/R-treated SH-SY5Y cells was significantly increased compared with normal SH-SY5Y cells. After transcranial magnetic stimulation, the intracellular ROS level showed a significant decreasing trend, confirming its strong neuroprotective effect in cells in vitro (Figure 6D). To assess the impact on microglial phenotype, we performed immunofluorescence labeling of microglia using CD86 (a marker of M1 microglia) (Figure 6E). Results showed significantly elevated CD86 expression in the group without OGD/R treatment. Notably, CD86 expression decreased following rTMS combined with Fe₃O₄ NPs treatment and iTBS combined with Fe₃O₄ NPs treatment (Figure 6F). Observation revealed a sharp decline in mitochondrial membrane potential (Δψm) following OGD/R compared to normal SH-SY5Y cells. To assess whether TMS could alleviate OGD/R-induced damage, we employed the JC-1 probe to track Δψm dynamics across treatment groups. After 24-hour interventions, fluorescence patterns shifted significantly: the green signal indicating damaged mitochondria weakened, while the red signal representing healthy mitochondria intensified. (Figure 6G). It was observed statistically that iTBS together with Fe₃O₄ NPs markedly restored mitochondrial membrane potential (ΔΨm) in SH-SY5Y cells damaged by OGD/R. This implies that the developed nanoplatform could effectively alleviate oxidative stress, thereby potentially preventing subsequent neuronal injury (Figure 6H). These results further confirmed that transcranial magnetic stimulation promoted reprogramming of MI-type microglia to the M2-type microglia. In addition, we measured the levels of inflammation-related cytokines in brain tissues using ELISA (Figure 6I and J). ELISA results confirmed that pro-inflammatory cytokines secreted by M1-type microglia were significantly elevated after OGD/R, including IL-β and TNF-α, and these pro-inflammatory factors were comparable to the control levels after treatment with Fe₃O₄ NPs combined with iTBS. Additionally, after treatment with Fe₃O₄ NPs combined with iTBS, there was a significant reduction in the level of malondialdehyde (Figure 6K).

Figure 6 Neuroprotective effects and modulation of microglia phenotypes in vitro. (A) and (B) The cell viability of SH-SY5Y cells and BV2 cells after OGD/R or incubation with Fe3O4 of different concentration for 12 h (n=2). (C) Fluorescence images of ROS in different groups after OGD/R (n = 3). Scale bar: 40 μM. (D) Immunofluorescence co-staining images of BV2 cells showing M1 phenotype marker CD86 (green) (n = 3). Scale bar: 20 μM, corresponding quantitative fluorescence of ROS (E) and CD86 (F). (G) The changes of Δψm in SH-SY5Y cells undergoing OGD/R after different treatments for 48 h and (H) their quantitative analyses (n = 3). Relative expression of pro-inflammatory IL-6 (I) and TNF-α (J), and MDA (K) in DMEN from BV2 cell culture. Data are presented as means ± SD (n = 3). ***P < 0.001, **P < 0.01, *P < 0.05. Abbreviation: ns: no, statistical significance.

Study of Molecular Mechanisms

Evidence from single-cell sequencing indicated that neuronal ferroptosis triggered by MCAO/R was suppressed following iTBS administration, primarily through modulation of Fth1, transferrin, ferritin light chain 1 (Ftl1), GPX4, and spermidine/spermine N1-acetyltransferase 1 expression. Moreover, numerous reports have demonstrated that excessive reactive oxygen species (ROS) generated during cerebral ischemia–reperfusion activate the Nrf2-mediated antioxidant defense system in vivo.²¹ Normally, Nrf2 remains inactive in the cytoplasm through its association with the repressor protein Keap1, which functions as a molecular sensor detecting oxidative stress signals such as ROS.²² When reactive oxygen species (ROS) levels rise, they oxidize the sulfhydryl (-SH) groups on Keap1, which alters its structure. This structural change weakens Keap1’s grip on Nrf2, allowing Nrf2 to detach and move into the nucleus. Once inside, Nrf2 attaches to the antioxidant response element (ARE) and activates the transcription of related protective genes. To reduce the accumulation of lipid peroxides and thereby prevent ferroptosis, Nrf2 activation is essential. The genes that are regulated downstream by Nrf2 and contribute to the suppression of lipid peroxidation and ferroptosis include Fth1, Ftl, and GPX4. These genes play a vital role in limiting oxidative damage (Figure 7A).⁸ To further explore the mechanism of action of TMS combined with Fe₃O₄ NPs in IS, Nrf2 expression was analyzed by Western blotting experiments (Figure 7B). Consistent with the Mi et al²³ and Lu et al²⁴ studies, Nrf2 is known to enhance the expression of Fth1 and Ftl,²⁵ which are key components in iron storage. Ferritin, the primary protein for storing intracellular iron, plays a crucial role in sequestering free iron in a form that is non-toxic. By doing so, it prevents the participation of iron ions in the Fenton reaction, thereby reducing the generation of free radicals. This process is vital for maintaining cellular iron balance and protecting against oxidative damage. In addition, GPX4 is important for regulating ferroptosis, and Nrf2 can inhibit ferroptosis by regulating GPX4. Immunoblotting analysis revealed that OGD/R injury significantly suppressed GPX4 expression (Figure 7C). This suppression was effectively reversed by transcranial magnetic stimulation, particularly through the combined application of Fe₃O₄ NPs with iTBS, which notably restored GPX4 protein levels (Figure 7D). Next, we further verified this with fluorescence staining (Figure 7E), and consistent with the results of GPX4 protein expression, fluorescence staining showed that Fe₃O₄ NPs in combination with iTBS resulted in a significant increase in GPX4 expression (Figure 7F). In contrast, the Nrf2 inhibitor ML385 (2 μM) significantly abolished the inhibitory effect of Fe₃O₄ NPs combined with iTBS on GPX4 in OGD/R-treated neurons. Fe₃O₄ NPs combined with iTBS reversed the inhibitory effect of ML385 on Fth1 in OGD/R-treated neurons (Figure 7G). These results support that Fe₃O₄ NPs combined with iTBS are associated with inhibition of ferroptosis via activation of the Nrf2–GPX4 axis (Figure 7H).

Figure 7 Nrf2–GPX4-mediated ferroptosis regulation in vitro. (A) Differentially expressed ferroptosis-related genes in different comparisons. (B) Representative Western blotting analysis of the protein expressions of GPX4 and Nrf2 in ischemic brain tissues after different treatments. (C) Fluorescence images of GPX4 in SH-SY5Y cells after OGD/R (n = 3). Scale bar: 20 μM. (D) Quantitative analysis of GPX4 fluorescence intensity. (E) Fluorescence expression plots of GPX4 and FTH1 in different treatment groups after addition of ML385 (n = 3). (F) Quantitative analysis of GPX4 expression. (G) Quantitative analysis of FTH1 expression. Data are presented as means ± SD (n = 3). Scale bar: 20 μM. (H) Specific mechanisms chart. **P < 0.01, *P < 0.05. Abbreviation: ns: no, statistical significance.

Consistent with the molecular findings, C11-BODIPY staining confirmed reduced lipid peroxidation in the combined treatment group (Supplementary Figure 3), further validating that activation of the Nrf2–GPX4 axis functionally translated into suppression of ferroptotic lipid damage in vivo.

Discussion

Post-ischemic reperfusion injury involves complex pathological processes, including oxidative stress, inflammatory responses and neuronal apoptosis. In this study, enhanced magnetic targeting and increased nanoparticle accumulation in the ischemic penumbra represent the primary physical effect of iTBS intervention. However, the relative quantitative contribution of magnetic guidance versus neuromodulatory activation was not independently dissected in the present study. The downstream modulation of ferroptosis, mitochondrial function, and microglial polarization should be interpreted as secondary biological consequences resulting from improved nanoparticle delivery and neuromodulatory activation rather than independent parallel mechanisms. Traditional therapies struggle to effectively counteract the cascade of secondary damage due to limitations such as difficulty penetrating the BBB and low targeting efficiency. The present study employed iTBS as the core therapeutic modality, combined with the magnetic targeting properties of Fe₃O₄ NPs, to construct an innovative treatment system integrating neuromodulation, BBB penetration and multi-pathway protection, offering a novel approach for precise intervention in IS.

As a non-invasive neuromodulation technique, iTBS significantly modulates cerebral vascular physiology through high-frequency pulse stimulation. Guided by an external magnetic field, it enhances Fe₃O₄ NPs accumulation in the ischemic penumbra. Research confirms iTBS optimizes nanoparticle delivery through the following mechanisms: 1) Vasodilation and BBB permeability regulation. iTBS stimulation promotes functional remodeling of cerebral vascular endothelial cells, transiently opening BBB tight junctions to facilitate Fe₃O₄ NPs trans-barrier transport; and 2) neuromodulation and targeted synergy. iTBS activates local neural circuits, amplifying the ischemic region’s biomechanical response to magnetically targeted nanoparticles. This increases Fe₃O₄ accumulation under magnetic guidance by approximately threefold compared to magnetic targeting alone, highlighting iTBS’s pivotal role in the targeting strategy.

Mitochondrial dysfunction and ferroptosis are key mechanisms in neuronal injury following I/R.^7,8,26 iTBS emerges as a core protective factor in this therapeutic system through multi-pathway regulation. First, it exerts dual ferroptosis inhibition by activating the Nrf2 signaling axis. iTBS significantly enhances Nrf2 transcriptional activity, synergizing with the antioxidant properties of Fe₃O₄ to block ferroptosis via two pathways. Notably, our data suggest that iTBS acts as the upstream regulatory trigger of the Nrf2 pathway, whereas Fe₃O₄ mainly enhances redox buffering capacity. The maximal ferroptosis inhibition observed in the combined group indicates a synergistic rather than additive interaction: 1) GPX4-dependent clearance of lipid peroxides. Nrf2 binds to antioxidant response elements, promoting GPX4 expression to catalyze the reduction of lipid peroxides and halt ferroptosis cascades; and 2) iron ion chelation regulation iTBS synergistically with Fe₃O₄ upregulates Fth1, chelating intracellular free iron and reducing lipid peroxidation substrate generation. The second pathway by which iTBS acts is through mitochondrial morphology and functional restoration. iTBS directly promotes mitochondrial homeostasis, while the antioxidant and redox-regulatory properties of Fe₃O₄ further stabilize mitochondrial membrane potential. This mechanistic interpretation is further supported by in vivo lipid peroxidation analysis using C11-BODIPY (Supplementary Figure 3), which demonstrated a significant reduction in lipid ROS levels following combined iTBS–Fe₃O₄ treatment. The decrease in lipid oxidative burden provides functional evidence that Nrf2 activation and GPX4 restoration effectively limit ferroptosis progression in ischemic cortex.

Although iron is widely recognized as a key driver of ferroptosis, our findings suggest that the iTBS–Fe₃O₄ system shifts the cellular response toward iron detoxification and lipid peroxide clearance. Specifically, activation of Nrf2 not only enhances GPX4-mediated reduction of lipid peroxides but also increases ferritin expression, thereby lowering the pool of labile iron available for Fenton chemistry.^8,26 This coordinated regulation provides a mechanistic explanation for why Fe₃O₄ nanoparticles, when combined with iTBS, exert net neuroprotection rather than promoting ferroptosis.

In neuroinflammation, iTBS regulation of microglial phenotype switching is a key mechanism for mitigating I/R injury. Analysis revealed that iTBS combined with Fe₃O₄ NPs significantly suppressed microglial activation toward the pro-inflammatory M1 phenotype (reduced CD86 expression, decreased TNF-α and IL-1β expression) while promoting anti-inflammatory M2 polarization. (increased CD206 expression). Construction of a neuroprotective microenvironment: iTBS reduces neurotoxic substance release by modulating microglial phagocytic function and cytokine secretion profiles. This synergizes with Nrf2-mediated ferroptosis inhibition pathways to collectively mitigate neuronal injury severity.

Compared to traditional IS therapies, the combined treatment system centered on iTBS demonstrate notable advantages over conventional approaches: iTBS’s non-invasive nature and spatiotemporal controllability overcome the limitations of passive targeting by nanomedicines; its combination with Fe₃O₄ enables simultaneous intervention in three key pathological pathways—oxidative stress, inflammation and ferroptosis—while precisely activating the Nrf2 pathway to enhance treatment specificity. Behavioral studies indicate this approach shortens the Barnes escape latency and improves motor coordination in stroke animal models. The behavioral improvements were consistent with the molecular findings of reduced oxidative stress and enhanced Nrf2/GPX4 signaling. Within the field of IS therapy, optimizing neuromodulation techniques remains a key direction for overcoming the limitations of traditional treatments. Compared to conventional rTMS, iTBS demonstrates significant research value due to its unique stimulation pattern, high neural modulation efficiency and clinical translation potential. Its advantages are further amplified in the innovative therapeutic system combining Fe₃O₄ nanoparticles. The short pulse stimulation of iTBS (total treatment time ≤5 minutes) can synchronize with the magnetic targeting process of Fe₃O₄ nanoparticles. Through real-time coordination of “stimulation-targeting-repair,” it enhances nanoparticle accumulation efficiency in the ischemic penumbra (35% higher than the rTMS combination group). In contrast, the prolonged stimulation duration of rTMS may disrupt the magnetic field-guided trajectory of nanoparticles, reducing targeting precision. Research indicates that iTBS, by mimicking the physiological theta rhythm bursts in the hippocampal region, can more efficiently induce long-term potentiation effects, promoting synaptic remodeling and neural regeneration. Combined with its brief action duration, iTBS holds profound clinical significance. This combinatorial strategy may provide a promising framework for integrating neuromodulation with nanomedicine in ischemic stroke therapy.

Although enhanced BBB penetration, Nrf2 activation, and ferroptosis inhibition were observed, the complete causal chain linking these events was not fully dissected in a stepwise manner. Therefore, the proposed pathway should be interpreted as a mechanistic framework supported by associative evidence.

This study still has some limitations. Although enhanced nanoparticle accumulation was observed under iTBS, the present study did not include direct quantitative BBB permeability assays (eg., Evans blue extravasation or tracer-based permeability measurements). Therefore, whether iTBS directly transient tight junction remodeling and enhanced permeability or instead alters cerebral hemodynamics or magnetic targeting efficiency requires further investigation. Consistently, ZO-1 immunofluorescence analysis (Supplementary Figure 2) demonstrated altered tight junction continuity following iTBS stimulation, supporting the presence of transient tight junction remodeling. Importantly, no extensive structural disruption was observed, indicating that iTBS-induced BBB modulation may be reversible and spatially controlled. This observation aligns with the improved nanoparticle accumulation observed under iTBS guidance. Similarly, while Nrf2 signaling was significantly upregulated, the precise upstream regulatory interactions between iTBS stimulation and antioxidant transcriptional control remain to be fully elucidated. Therefore, the term “BBB modulation” may more accurately reflect our findings than definitive “BBB opening.” The present study focused on early-stage (7-day) functional and molecular outcomes following ischemic injury. While this timeframe captures acute neuroprotection and early recovery dynamics, it does not address long-term efficacy, durability of functional improvement, or delayed safety concerns such as chronic inflammation or iron accumulation. Future studies will extend observation periods to evaluate sustained behavioral recovery, long-term ferroptosis regulation, and potential late-phase adverse effects. Particular attention should be given to potential cumulative iron deposition after repeated dosing, which was not assessed in the current short-term study. From a translational perspective, the clinical feasibility of the proposed iTBS–Fe₃O₄ strategy requires consideration of treatment timing, compatibility with standard reperfusion therapies, and safety associated with transient BBB modulation. In our in vivo experiments, treatment was initiated at 6 h after reperfusion and continued daily during the acute phase (Day 1–7), which corresponds to a clinically relevant early rehabilitation window. Importantly, this approach may be compatible with intravenous thrombolysis and mechanical thrombectomy, as the primary objective is not to replace reperfusion but to mitigate secondary injury and improve functional recovery. This timing was selected to approximate an early post-reperfusion rehabilitation window in clinical practice. Nevertheless, transient tight junction remodeling and enhanced permeability may theoretically increase the risk of vasogenic edema or hemorrhagic transformation. In the present study, no obvious hemorrhagic changes were observed in histological evaluation, and Fe₃O₄ NPs exhibited favorable biosafety profiles.²⁷ Future studies will further optimize iTBS parameters, evaluate long-term vascular safety, and assess the therapeutic window in combination with thrombolysis or thrombectomy.

In addition, local temperature changes were not measured during stimulation, and thus potential magnetothermal contributions were not evaluated in the present study.

Given iTBS’s central role in neuromodulation, future research will focus on 1) Parameter optimization and mechanism refinement: Decoding the dynamic regulation of the Nrf2-GPX4/Fth1 axis by iTBS stimulation patterns to minimize off-target effects; 2) Combined therapy development: Exploring time-window matching strategies for iTBS combined with Fe₃O₄ and thrombolytic therapy to simultaneously address vascular reperfusion and secondary injury; and 3) Cross-disease application: Extending iTBS’s neuromodulatory advantages to central nervous system diseases characterized by oxidative stress/ferroptosis, such as Alzheimer’s disease and Parkinson’s disease, achieving precision treatment through customized parameter design.