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Protective Effects of Repetitive Transcranial Magnetic Stimulation Against Streptozotocin-Induced Alzheimer's Disease - PubMed

Protective Effects of Repetitive Transcranial Magnetic Stimulation Against Streptozotocin-Induced Alzheimer's Disease

Seul-Ki Kim et al. Mol Neurobiol. 2024 Mar.

Abstract

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation under investigation for treatment of a wide range of neurological disorders. In particular, the therapeutic application of rTMS for neurodegenerative diseases such as Alzheimer's disease (AD) is attracting attention. However, the mechanisms underlying the therapeutic efficacy of rTMS have not yet been elucidated, and few studies have systematically analyzed the stimulation parameters. In this study, we found that treatment with rTMS contributed to restoration of memory deficits by activating genes involved in synaptic plasticity and long-term memory. We evaluated changes in several intracellular signaling pathways in response to rTMS stimulation; rTMS treatment activated STAT, MAPK, Akt/p70S6K, and CREB signaling. We also systematically investigated the influence of rTMS parameters. We found an effective range of applications for rTMS and determined the optimal combination to achieve the highest efficiency. Moreover, application of rTMS inhibited the increase in cell death induced by hydrogen peroxide. These results suggest that rTMS treatment exerts a neuroprotective effect on cellular damage induced by oxidative stress, which plays an important role in the pathogenesis of neurological disorders. rTMS treatment attenuated streptozotocin (STZ)-mediated cell death and AD-like pathology in neuronal cells. In an animal model of sporadic AD caused by intracerebroventricular STZ injection, rTMS application improved cognitive decline and showed neuroprotective effects on hippocampal histology. Overall, this study will help in the design of stimulation protocols for rTMS application and presents a novel mechanism that may explain the therapeutic effects of rTMS in neurodegenerative diseases, including AD.

Keywords: Alzheimer’s disease; Cognitive impairment improvement; Neuroprotective effects; Phosphorylation of intracellular signaling pathways; Repetitive transcranial magnetic stimulation (rTMS).

© 2023. The Author(s).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1

rTMS treatment increases the expression of genes related to synaptic plasticity and long-term memory. SH-SY5Y cells were treated with low frequency (1 Hz) or high frequency (10 Hz) rTMS at 1.75 T for 30 min. mRNA levels of c-Fos, c-Jun, JunB, Arc, Egr-1, NR4A1, NPAS4, and Homer1a were analyzed by quantitative real-time PCR. GAPDH was used as a standard. Data are shown as mean ± SD (n = 3). NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.005, determined by Student’s t-test

Fig. 2
Fig. 2

Treatment with rTMS induces activation of STAT, MAPK, Akt/p70S6K, and CREB signaling pathways. A SH-SY5Y cells were treated with 10 Hz rTMS for 30 min at the indicated intensity. B SH-SY5Y cells were treated with low frequency (1 Hz) or high frequency (10 Hz) rTMS for 30 min at 1.75 T. C HT22 cells were treated with 10 Hz rTMS for 30 min at the indicated intensity. D HT22 cells were treated with low frequency (1 Hz) or high frequency (10 Hz) rTMS for 30 min at 1.75 T. Cell lysates were subjected to immunoblotting with the indicated antibodies. α-tubulin was used as a standard

Fig. 3
Fig. 3

Induction of protein phosphorylation by rTMS treatment is faster and longer lasting at high frequencies. A SH-SY5Y cells were treated with low frequency (1 Hz) rTMS at 1.75 T for the indicated times. B SH-SY5Y cells were treated with high frequency (10 Hz) rTMS at 1.75 T for the indicated times. C Schematic overview of experimental design. Image created with BioRender.com. D SH-SY5Y cells were treated with low frequency (1 Hz) rTMS at 1.75 T for 30 min and then incubated for the indicated time period without stimulation. E SH-SY5Y cells were treated with high frequency (10 Hz) rTMS at 1.75 T for 30 min and then incubated for the indicated time period without stimulation. A–E Cell lysates were subjected to immunoblotting with the indicated antibodies. α-tubulin was used as a standard

Fig. 4
Fig. 4

Excessive rTMS treatment causes neuronal cell damage and death. SH-SY5Y cells were treated with 10 Hz rTMS at 2 T for the indicated times. A Morphology was observed using a phase contrast inverted microscope. Scale bar, 100 µM. B Cell viability was determined by crystal violet staining. Representative images are shown on the left, and quantification is shown on the right. C, D Cell lysates were subjected to immunoblotting with the indicated antibodies. α-tubulin was used as a standard. Values are shown as mean ± SD of three replicates. *P < 0.05 vs. untreated control by Student’s t-test

Fig. 5
Fig. 5

rTMS treatment reduces H2O2-induced neuronal cell death. SH-SY5Y cells were treated with 10 Hz rTMS at 1.75 T for 30 min, three times a day for 2 days for a total of six times. Then, 48 h after the initial rTMS stimulation, cells were treated with 400 µM H2O2 for an additional 12 or 24 h. A Cell viability was determined by MTT assay. B Dead cells were measured by staining with IncuCyte CytoTox Green Reagent. C, D Cell lysates were subjected to immunoblotting with the indicated antibodies. α-tubulin was used as a standard. E, F Apoptotic cells were detected using annexin V-FITC/PI staining and analyzed by flow cytometry. Annexin V-positive cells were considered apoptotic. All data are expressed as mean ± standard deviation of three replicates, and representative data are shown. *P < 0.05, **P < 0.01, ***P < 0.005, determined by Student’s t-test

Fig. 6
Fig. 6

rTMS treatment alleviates STZ-induced neuronal cell death and AD-like pathology. SH-SY5Y cells were treated with 10 Hz rTMS at 1.75 T for 30 min, three times a day for 2 days for a total of six times. Then, 48 h after the initial rTMS stimulation, cells were treated with 5 mM STZ for an additional 12 or 24 h. A Cell viability was determined by MTT assay. B Dead cells were measured by staining with IncuCyte CytoTox Green Reagent. C, D, G, H Cell lysates were subjected to immunoblotting with the indicated antibodies. α-tubulin was used as a standard. E, F Apoptotic cells were detected using annexin V-FITC/PI staining and analyzed by flow cytometry. Annexin V-positive cells were considered apoptotic. All data are expressed as mean ± standard deviation of three replicates, and representative data are shown. *P < 0.05, **P < 0.01, ***P < 0.005, determined by Student’s t-test

Fig. 7
Fig. 7

rTMS ameliorates memory deficit behavioral disorders and neuronal cell death in ICV-STZ rats. Rats were injected ICV with STZ (1.5 mg/kg, saline 5 µl). After 7 days of STZ administration, the rTMS application group was treated with high frequency (10 Hz) rTMS for 20 min a day, 5 days a week, for a total of 4 weeks. A Experimental design for animal experiments. Image created with BioRender.com. B Photograph of immobilized rats for rTMS application in the awake state. C Behavioral experiment on spontaneous alternation using the Y-maze. D Hepatic injury was assessed by measuring serum levels of albumin. E Representative images of Nissl staining in the CA1, CA2, CA3, and DG regions of the hippocampus. F Quantification of Nissl stain mean intensity in the CA1, CA2, CA3, and DG regions of the hippocampus. All data are presented as mean ± standard deviation (n = 4). *P < 0.05, analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test

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