2.1 Animals and reagents

Male Sprague–Dawley rats (weighing 405–450 g; 12-week) were purchased from Hunan SJA Laboratory Animal Center (Changsha, Hunan, China). All rats were housed under standard conditions: 12 h light/dark cycle, room temperature 23–27°C, humidity 45%–55%, and access to standardized laboratory pellet food and water. Adaptive feeding was conducted for 1 week before the treatment. All experiments were performed in compliance with the Regulations for the Administration of Affairs Concerning Experimental Animals (China) and approved by the Animal Use and Protection Committee of University of South China. Animal suffering was minimized to the greatest extent possible. Anti-acyl-CoA synthetase long-chain family member 4 (ACSL4) antibody (Catalog no. A16848) and anti-transferrin receptor (TFRC) antibody (Catalog no. A5865) were obtained from ABclonal (Wuhan, Hubei, China), and Anti-glutathione-dependent peroxidase 4 (GPX4) antibody (Catalog no. ab125066) was purchased from Abcam (Cambridge, UK). Anti-β-actin antibody (Catalog no. 66009-1-Ig), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (Catalog no. SA00001-1), and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (Catalog no. SA00001-2) were supplied by Proteintech (Chicago, IL, USA).

2.2 MCAO (transient middle cerebral artery occlusion) rat model and grouping

With some slight modifications (Wang et al., 2008) from Zea Longa's previous method (Longa et al., 1989), the transient MCAO operation using the intraluminal filament method was performed. Briefly, a peritoneal injection of 1% sodium pentobarbital (Sigma, St. Louis, MO, USA) (0.5 mL per 100 g) was used to anesthetize the rats. Subsequently, supine rats were incised on the neck midline, and the left common carotid artery, the left external carotid artery and the left inner carotid artery (ICA) exposed. Then a monofilament nylon suture with a rounded tip (diameter of 0.43 mm) (L4300, Guangzhou Jialing Biotechnology, Guangzhou, Guangdong, China) was carefully inserted into the left ICA and occluded the middle cerebral artery. After 1.5 h of ischemia, the nylon suture was withdrawn gently to achieve reperfusion. Sham rats were treated with the same procedures without inserting the nylon suture. Throughout the procedures, the whole body temperature was maintained at 37.0 ± 0.5°C with a heating pad and monitored using a rectal temperature probe.

The rats were randomly assigned to three experimental groups: the sham group (sham group), the MCAO ischemia-reperfusion model group (MCAO group), and the rTMS treatment group (MCAO + rTMS group). All the rats, except those in sham group, were subjected to MCAO procedure, and only the rats in the MCAO + rTMS group underwent rTMS treatment.

2.3 Repetitive transcranial magnetic stimulation (rTMS) treatment

rTMS was delivered using a magnetic-electric stimulator (YRD-CCY-1, Wuhan, Hubei, China) with a round coil (Y064, 3.5 cm diameter). The treatment was initiated at 6 h after reperfusion. All procedures were based on published animal experiment protocols in previous studies (Guo et al., 2017). In brief, the coil was positioned perpendicular to the cortex in the cortical surface projection area of the left cerebral hemisphere of rats. The treatment protocol consisted of stimulation for 10 s followed by rest for 50 s (repeated 10 times) at a rate of 10 Hz. The stimulation intensity was set at 33% of the maximum stimulator output. These steps were repeated daily for 7 days.

2.4 Assessment of neurological function

The neurological deficits were assessed using the Longa scoring system (Longa et al., 1989) 7 days after reperfusion, and score of neurological function was recorded. The scoring criteria were as follows: 0, no observable deficit; 1, failure to extend right forepaw on lifting the whole body by tail; 2, circling to the contralateral side; 3, loss of walking or righting reflex; 4, no spontaneous motor activity.

2.5 Hematoxylin–eosin (HE) staining

The pathological changes of brain tissues were assessed using hematoxylin–eosin (HE) staining. Freshly obtained brain samples were fixed overnight in 4% formaldehyde and embedded in paraffin. Then HE staining was performed according to the instruction manual of the HE staining kit (G1120, Solarbio, Beijing, China). The observations were made under the microscope.

2.6 Reverse transcription quantitative PCR (RT-qPCR)

The relative mRNA expression was analyzed by reverse transcription quantitative PCR (RT-qPCR) as previously described (Hu et al., 2016). The total RNA extract was performed with TRIzol Plus RNA Purification Kit (15596026, Thermo Scientific, MA, USA) as directed by the manufacturer, and the concentration of total RNA was measured spectrophotometrically. RNA was reverse-transcribed into cDNA using a reverse transcription kit (CW2569, CWBIO, Beijing, China). Subsequently, the quantitative PCR was performed using the UltraSYBR Mixture (CW2601, CWBIO, Beijing, China) according to the manufacturer's instructions. Amplification conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. The primers used in PCR amplification and real-time RT-PCR for specific genes are listed in Table 1. The relative amount of mRNA was calculated using the comparative Ct (ΔΔCt) method. All specific genes mRNA expression were normalized against β-actin.

2.7 Western blotting analysis

Protein expressions of GPX4, ASCL4, and TFRC were assessed by western blotting analysis according to the standard protocol. After euthanizing the rats via anesthetic overdose, left cerebral cortical tissues were immediately harvested to homogenize with RIPA buffer on the ice. Proteins were extracted from the supernatant and quantified with BCA Protein Assay Kit (Beyotime, Shanghai, China). Then total proteins were separated by 10%–12% sodium dodecyl sulfate-polyacrylamide gel under denaturing conditions and transferred to polyvinylidene difluoride membranes (Merck Millipore Ltd., USA). After being blocked with 5% nonfat milk, the membranes were incubated with relevant primary antibodies (rabbit anti-GPX4 (1:5000), rabbit anti-ASCL4 (1:2000), mouse anti-TFRC (1:2000), mouse anti-β-tubulin (1:5000)) overnight at 4°C. On the following day, the membranes were washed followed by incubation with secondary antibodies (HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (1:5000) and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (1:5000)) for 2 h (room temperature). Target proteins were visualized on the membranes using an enhanced chemiluminescence kit (Millipore Corporation, Billerica, MA, USA). Intensities of target protein bands, normalized to β-actin, were quantified using ImageJ software (NIH, Bethesda, MD, USA).

2.8 Enzyme-linked immunosorbent assay (ELISA)

The concentrations of IL-1β, IL-6, and TNF-α were detected using the following commercially available enzyme-linked immunosorbent assay (ELISA) kits: IL-1β, Rat Interleukin 1β (IL-1β) ELISA Kit (CUSABIO, Wuhan, Hubei, China); IL-6, Rat Interleukin 6 (IL-6) ELISA Kit (CUSABIO); and TNF-α, Rat Tumor necrosis factor α ELISA Kit (CUSABIO). The concentration of each inflammatory cytokine in the cerebrospinal fluid (CSF) of rats was determined by ELISA according to the manufacturer's instructions.

2.9 Statistical analysis

Statistical analysis was performed using SPSS 26.0 (SPSS Inc., Chicago, IL, USA). Data are presented as mean ± S.E.M., except for the neurological scores. Data of neurological scores were analyzed by nonparametric tests (Kruskal–Wallis test) followed by a Dunn's post hoc test. For other data that met normal distribution assumptions and equal variances, one-way analyses of variance were used with LSD post hoc tests. p Values <.05 were considered to indicate statistical significance in all statistical tests.

3.1 High-frequency rTMS attenuates cerebral I/R injury in MCAO-treated rats

To investigate whether high-frequency rTMS alleviates the neurological impairment, we used the Longa scoring system to assess neurological function of rats after MCAO. The results revealed that administration of high-frequency rTMS attenuated MCAO-induced increase in the neurological score (Figure 1A). In general, a higher score indicates decreased neurological function. So these data indicated that high-frequency rTMS attenuates the neurological deficits of MCAO-treated rats.

To further confirm the neuroprotective effect of rTMS, brain slices were stained with HE to assess the morphological changes. As shown in Figure 1B, HE staining evidenced a disorderly arrangement with cytoplasmic looseness, abnormal neuronal swelling, karyopyknosis, and cellular vacuolation in the MCAO group as compared with the sham group. Of note, such histological damage in the cortical ischemic zone could be significantly reversed following treatment with rTMS, implicating that high-frequency rTMS improves morphological damage in the cortical ischemic area of MCAO-treated rats. Taken together, these data demonstrated that high-frequency rTMS alleviates the cerebral I/R injury in MCAO-treated rats.

3.2 High-frequency rTMS attenuates ferroptosis in the ischemic cortex of MCAO-treated rats

It has been reported that excessive ferroptosis has been linked to cerebral I/R injury (Li et al., 2021). To determine the involvement of ferroptosis in rats treated with MCAO, and whether rTMS-induced rescuing effects are associated with alleviating ferroptosis, the mRNA and protein expression of genes related to ferroptosis were assessed. The RT-qPCR results showed that MCAO significantly increased the mRNA expression levels of ACSL4 and TFRC in the cortex but decreased the mRNA expression level of GPX4, which were reversed by rTMS treatment (Figure 2A). Consistently, MCAO-treated rats exhibited significant increases in the protein expression levels of ASCL4 and TFRC as well as decrease in the protein expression level of GPX4, which were also significantly abolished by treatment with rTMS (Figure 2B). These data demonstrated that high-frequency rTMS alleviates in the ischemic cortex of MCAO-treated rats.

3.3 High-frequency rTMS inhibits inflammation in the CSF of MCAO-treated rats

To investigate whether rTMS-exerted neuroprotective effects in MCAO-exposed rats are associated with inhibiting inflammation, inflammatory cytokines, including IL-1β, IL-6, and TNF-α in CSF measured by ELISA test. As shown in Figure 3, the concentrations of IL-1β (Figure 3A), IL-6 (Figure 3B), and TNF-α (Figure 3C) in the CSF of MCAO-exposed rats were markedly upregulated, which significantly reduced by treatment with high-frequency rTMS. These results implicated that high-frequency rTMS attenuates the inflammation in the CSF of MCAO-treated rats.