Magnetic brain stimulation using iron oxide nanoparticle-mediated selective treatment of the left prelimbic cortex as a novel strategy to rapidly improve depressive-like symptoms in mice
磁刺激极大地促进了神经科学的进步，其中高频（10 Hz）重复经颅磁刺激（rTMS）刺激左背外侧前额叶皮层（DLPFC）已于2008年获得美国食品药品监督管理局（FDA）的批准，用于治疗成人难治性抑郁。但目前rTMS仍然存在技术局限性，包括穿透力差，磁刺激聚焦精度不足，刺激深度有限等。特别是在小动物精神疾病模型中，即使最小的通用线圈也会刺激整个半球甚至整个大脑，无法做到精准定位，难以研究rTMS的神经调控机制等脑科学相关的重大科学问题。针对此问题，我们提出了利用获得中国FDA批准的超顺磁性纳米氧化铁（SPIO）药物，结合轻度磁脉冲序列，以提高局部皮层敏感性的磁性脑刺激的新策略，即联合磁性刺激系统（c-MSS）。小鼠左前额叶皮层中与人类DLPFC同源的脑区为PrL，磁共振成像证明SPIO可稳定存在于PrL脑区至少11天。从细胞、动物和体外模型多方面证明了c-MSS的安全性和物理性特征。建立慢性不可预测的轻度应激（CUMS）抑郁模型小鼠，使用0.1T c-MSS（每次5分钟，每天两次）进行磁干预，结果表明10 Hz磁刺激5天可快速改善CUMS小鼠的抑郁样症状，这可能与增加脑源性神经营养因子（BDNF）和改善下丘脑-垂体-肾上腺（HPA）轴有密切关系。c-MSS是一种可精准刺激小动物任意皮层靶标的新方法，这一项研究成果将极大地丰富磁刺激的潜在应用领域，促进基于磁性纳米药物的磁刺激技术临床转化。Abstract:
Magnetic brain stimulation has greatly contributed to the advancement of neuroscience. However, challenges remain in the power of penetration and precision of magnetic stimulation, especially in small animals. Here, a novel combined magnetic stimulation system (c-MSS) was established for brain stimulation in mice. The c-MSS uses a mild magnetic pulse sequence and injection of superparamagnetic iron oxide (SPIO) nanodrugs to elevate local cortical susceptibility. After imaging of the SPIO nanoparticles in the left prelimbic (PrL) cortex in mice, we determined their safety and physical characteristics. Depressive-like behavior was established in mice using a chronic unpredictable mild stress (CUMS) model. SPIO nanodrugs were then delivered precisely to the left PrL cortex using in situ injection. A 0.1 T magnetic field (adjustable frequency) was used for magnetic stimulation (5 min/session, two sessions daily). Biomarkers representing therapeutic effects were measured before and after c-MSS intervention. Results showed that c-MSS rapidly improved depressive-like symptoms in CUMS mice after stimulation with a 10 Hz field for 5 d, combined with increased brain-derived neurotrophic factor (BDNF) and inactivation of hypothalamic-pituitary-adrenal (HPA) axis function, which enhanced neuronal activity due to SPIO nanoparticle-mediated effects. The c-MSS was safe and effective, representing a novel approach in the selective stimulation of arbitrary cortical targets in small animals, playing a bioelectric role in neural circuit regulation, including antidepressant effects in CUMS mice. This expands the potential applications of magnetic stimulation and progresses brain research towards clinical application.
Figure 1. Construction of combined magnetic stimulation system (c-MSS) and microinjection of mice
A: TEM image of PSC-capped γ-Fe2O3 nanoparticles and external view of injection (Inset). B: Schematic of injection process in left PrL cortex of mice. C: Measurement of PSC-capped γ-Fe2O3 nanoparticle size using Dynamic Light Scattering. Mean diameter was approximately 30-35 nm. D: Schematic of imposed magnetic field. E: Simulation of field intensity and magnetic flux of cone-shaped magnets. F: Magnetic hysteresis of SPIO nanoparticles. G: Output wave-profile of magnetic field. c-MSS: Combined magnetic stimulation system; TEM: Transmission electronic microscopy; PSC: Polyglucose sorbitol carboxymethylether; PrL: Prelimbic; SPIO: Superparamagnetic iron oxide.
Figure 2. MRI tracing and cytotoxicity of nanodrugs
A–D: in vivo MRI images of injected nanodrugs in left PrL cortex of mice after 1, 3, 7, and 11 days, respectively. n=3 in each group. E, F: CCK-8 and LDH assay of primary cortical neurons after treatment for 24 h with various doses of SPIO nanoparticles. G, H: CCK-8 and LDH assay of primary cortical neurons treated with 5 μg/mL SPIO nanoparticles for 6, 12, 24, and 48 h (n=8). I: Representative images of in situ TUNEL assay using confocal microscopy (magnification,×400). J: Percentage of TUNEL-positive cells. Scale bar: 50 μm. n=8 slices from eight mice in each group. **: P<0.01 compared with control group (one-way ANOVA followed by Bonferroni’s multiple comparison post hoc test). MRI: Magnetic resonance imaging; PrL: Prelimbic; CCK-8: Cell counting kit-8; LDH: Lactate dehydrogenase; SPIO: Superparamagnetic iron oxide; TUNEL: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling.
Figure 3. Measurement of magneto-electric induction and physical effects in a hydrogel phantom
A: Hydrogel phantom containing injected SPIO nanoparticles. B: Heating of phantom within a 10 Hz (300 r/min) magnetic field. C: Magneto-electric induction in different field frequencies. D: Schematic of measurement of magneto-electric induction. E: Acoustic signals of phantoms with SPIO nanoparticles in presence of a 5, 10, and 15 Hz magnetic field. F: Induced voltages in presence or absence of SPIO nanoparticles. Result was confirmed in triplicate. SPIO: Superparamagnetic iron oxide.
Figure 5. Treatment of CUMS mice and confirmation of effects of c-MSS through behavioral experiments
A: Experimental procedures for selection of optimal field frequency and duration of treatment. B, C: SPT and FST results in CUMS mice. n=15 mice for each group. Values are mean±SEM. ***: P<0.001 vs. before CUMS modelling (control mice) (paired Student’s t-test). D: Monitoring of body weight of mice compared with age-matched control mice. n=15 mice for each group. Values are mean±SEM. *: P<0.05 vs. Control; **: P<0.01 vs. Control; ***: P<0.001 vs. Control. E, F: SPT and FST results in CUMS mice after c-MSS treatment using different frequencies of magnetic field. G, H: SPT and FST results in CUMS mice after treatment with a 10 Hz c-MSS for a variety of durations. Values are mean±SEM, n=8-10 mice. *: P<0.05 vs. Control; **: P<0.01 vs. Control; ***: P<0.001 vs. Control. #: P<0.05 vs. CUMS; ##: P<0.01 vs. CUMS; ###: P<0.001 vs. CUMS (one-way ANOVA followed by Bonferroni’s multiple comparison post hoc test). I,J: SPT and FST results. Sham groups were administered saline and 10 Hz magnetic field, SPIO nanoparticles without a magnetic field, and Au nanoparticles in a 10 Hz magnetic field. All treatments lasted 5 days (two 5 min sessions per day). Data were calculated from six mice for each group. Values are mean±SEM. ***: P<0.001 vs. Control. ###: P<0.001 vs. CUMS (one-way ANOVA followed by Bonferroni’s multiple comparison post hoc test). c-MSS: Combined magnetic stimulation system; FST: Forced swim test; SPT: Sucrose preference test; CUMS: Chronic unpredictable mild stress; SPIO: Superparamagnetic iron oxide; PrL: Prelimbic.
Figure 6. Comparison of biomarker levels for effective anti-depression in mice
A: Representative immunohistochemistry micrographs of c-fos expression in left PrL cortex after c-MSS treatment for 5 days in mice. B: Schematic displaying region of left PrL cortex. C: c-fos-positive cells in left PrL cortex. Scale bar: 50 μm. n=8 slices from eight mice in each group. Values represent mean±SEM. ***: P<0.001 vs. Control (one-way ANOVA followed by Bonferroni’s multiple comparison post hoc test). D: BDNF protein levels in PrL whole tissue in various groups. E: Serum levels of CORT in various groups. F: Serum levels of ACTH in various groups. Data were calculated from eight mice for each group. Values are mean±SEM. **: P<0.01 vs. Control; ***: P<0.001 vs. Control. #: P<0.05 vs. CUMS+Saline+10 Hz MF; ###: P<0.001 vs. CUMS+Saline+10 Hz MF (one-way ANOVA followed by Bonferroni’s multiple comparison post hoc test). PrL: Prelimbic; CUMS: Chronic unpredictable mild stress; BDNF: Brain-derived neurotrophic factor; CORT: Corticosterone; ACTH: Adrenocorticotropic hormone.
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