Repetitive transcranial magnetic stimulation ameliorates cognitive deficits in mice with radiation-induced brain injury by attenuating microglial pyroptosis and promoting neurogenesis via BDNF pathway

Abstract

Background: Radiation-induced brain injury (RIBI) is a common and severe complication during radiotherapy for head and neck tumor. Repetitive transcranial magnetic stimulation (rTMS) is a novel and non-invasive method of brain stimulation, which has been applied in various neurological diseases. rTMS has been proved to be effective for treatment of RIBI, while its mechanisms have not been well understood.

Methods: RIBI mouse model was established by cranial irradiation, K252a was daily injected intraperitoneally to block BDNF pathway. Immunofluorescence staining, immunohistochemistry and western blotting were performed to examine the microglial pyroptosis and hippocampal neurogenesis. Behavioral tests were used to assess the cognitive function and emotionality of mice. Golgi staining was applied to observe the structure of dendritic spine in hippocampus.

Results: rTMS significantly promoted hippocampal neurogenesis and mitigated neuroinflammation, with ameliorating pyroptosis in microglia, as well as downregulation of the protein expression level of NLRP3 inflammasome and key pyroptosis factor Gasdermin D (GSDMD). BDNF signaling pathway might be involved in it. After blocking BDNF pathway by K252a, a specific BDNF pathway inhibitor, the neuroprotective effect of rTMS was markedly reversed. Evaluated by behavioral tests, the cognitive dysfunction and anxiety-like behavior were found aggravated with the comparison of mice in rTMS intervention group. Moreover, the level of hippocampal neurogenesis was found to be attenuated, the pyroptosis of microglia as well as the levels of GSDMD, NLRP3 inflammasome and IL-1β were upregulated.

Conclusion: Our study indicated that rTMS notably ameliorated RIBI-induced cognitive disorders, by mitigating pyroptosis in microglia and promoting hippocampal neurogenesis via mediating BDNF pathway.

Keywords: BDNF; Neurogenesis; Neuroinflammation; RIBI; rTMS.

Fig. 1

rTMS ameoliated hippocampal neurogenesis impairment induced by cranial irradiation. (A) Schematic diagram of the experimental design, including animal grouping, radiation and rTMS intervention protocol. (B, D) Representative immunofluorescence staining images of DCX and ki67 in hippocampus. Scale bar = 100 μm. (C) The average fluorescence intesity of DCX in DG region of hippocampus. (E) The number of ki67 in DG region. n = 3 for each group. Data are presented as mean ± S.E.M, ^**P < 0.01, ^***P < 0.001 vs. Sham group; ^##P < 0.01 vs. Radiation group

Fig. 2

rTMS attenuated microglia pyroptosis induced by NLRP3 inflammasome. (A) Immunohistochemical staining of Iba-1 for indication of microglia in hippocampus. Scale bar = 100 μm, the enlarged image indicated the morphology of microglia. (B) The number of Iba-1⁺ in DG region. Data are presented as mean ± S.E.M. (C) Immunofluorescence staining of GSDMD and Iba-1, the arrows indicate co-localization of GSDMD with Iba-1. Scale bar = 50 μm. (D) The quantitative analysis of number of GSDMD⁺/Iba-1⁺ in DG region. (E-J) Western blotting representative bands and quantitative analysis. Protein expression level was normalized to β-actin. The data are presented as mean ± S.E.M, n = 3 for each group. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 vs. Sham group, ^#P < 0.05; ^###P < 0.001 vs. Radiation group

Fig. 3

rTMS activated BDNF signaling pathway in hippocampus after RIBI. (A) Representative images of western blot bands. (B-D) The relative protein expression level of BDNF, p-TrkB/TrkB and p-CREB/CREB. Data are presented as mean ± S.E.M, n = 3 for each group. ^*P < 0.05, ^**P < 0.01 vs. Sham group; ^#P < 0.05, ^##P < 0.01 vs. Radiation group

Fig. 4

Inhibiting BDNF pathway reversed the improvement of cognitive function and emotionality induced by rTMS in RIBI mice. (A) Schematic diagram of the experimental design, including animal grouping, drug administration, intervention protocol, behavioral tests and other experiments. (B) The escape latency during navigation test phase. (C) The time spent in the target quadrant during probe test phase. (D) The number of platform crossings during probe trial. (E) The locomotion total distance in open field. (F) The total time spent in central area of open field. (G) The frequency of central entries in open field. (H) The time spent in open arms of elevated plus maze. (I) The frequency of open arms entries. (J) Representative MWM training trace of mice in each group. (K) Representative movement trace of mice in the open field, the four squares in the middle represent central area. (L) Representative movement trace of mice in the elevated plus maze, open and closed arms are indicated in the diagrams. Data are presented as mean ± S.E.M, n = 11 or 12 for each group. ^*P < 0.05, ^**P < 0.01 vs. Sham group; ^#P < 0.05, ^##P < 0.01 vs. Radiation group; ^&P < 0.05, ^&&P < 0.01 vs. rTMS group; ns. no significance

Fig. 5

Inhibiting BDNF pathway attenuated the level of hippocampal neurogenesis after rTMS treatment. (A) Representative images of immunofluorescence staining of DCX (green)/BrdU (red) in hippocampal sections from Sham and cranial irradiation mice treated with rTMS and K252a. Scale bar = 100 μm. (B) Integrated density of DCX in DG region of hippocampus. (C) The number of DCX/BrdU positive cells in DG. (D) Representative images immunofluorescence staining of NeuN (green)/BrdU (red) in hippocampal sections. Scale bar = 100 μm. (E) The number of NeuN/BrdU positive cells in DG of hippocampus. All data are presented as mean ± S.E.M, n = 3 for each group. ^*P < 0.05, ^**P < 0.01 vs. Sham group; ^##P < 0.01 vs. Radiation group; ^&P < 0.05, ^&&P < 0.01 vs. rTMS group; ns. no significance

Fig. 6

Inhibiting BDNF pathway reversed the anti-pyroptosis effect of rTMS. (A) Representative immunofluorescence double staining images of Iba-1 and GSDMD, arrows indicated co-localization of GSDMD with Iba-1. Scale bar = 100 μm. (B) The number of GSDMD/Iba-1 positive cells in DG. (C) Quantitative analysis of integrated intensity of GSDMD. (D) Representative bands of western blotting. (E-I) Quantitative analysis of protein expression level by one-way ANOVA followed by Tukey’s multiple comparison test. Data are presented as mean ± S.E.M, n = 3 for each group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. Sham group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. Radiation group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001 vs. rTMS group; ns. no significance

Fig. 7

Inhibiting BDNF pathway reversed the enhancement of synaptic plasticity after rTMS treatment. (A-C) The protein levels of PSD95 and Synaptophysin, n = 3 for each group. (D) Representative images of Golgi staining and tertiary dendritic profile of granule cells in DG region of hippocampus. Scale bar = 200 μm for images of hippocampus; and scale bar = 10 μm for tertiary dendritic images. (E) The spines density of tertiary dendritic per horizons. n = 2 for each group and 6 horizons were randomly chosen for analysis. All data are presented as mean ± SEM, ^*P < 0.05, ^**P < 0.01 vs. Sham group; ^#P < 0.05, ^##P < 0.01 vs. Radiation group; ^&P < 0.05, ^&&P < 0.01 vs. rTMS group

Fig. 8

The hypothesized schematic diagram of possible relation between BDNF and NLRP3-mediated pyroptosis in RIBI