| 研究課題/領域番号 |
20K11551
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| 研究機関 | 国際基督教大学 |
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研究代表者 |
グホ サビン 国際基督教大学, 教養学部, 准教授 (30453179)
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| 研究分担者 |
宮本 泰則 お茶の水女子大学, ヒューマンライフイノベーション研究所, 教授 (50272737)
和気 秀文 順天堂大学, スポーツ健康科学部, 教授 (50274957)
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| 研究期間 (年度) |
2020-04-01 – 2024-03-31
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| キーワード | Nanoplastic / Oral exposure / Heart rate / Urine osmolality / Area postrema / NTS / Microglia reactivity / Gene expression |
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| 研究実績の概要 |
This study investigates whether long-term oral exposure to nanoplastic alters cellular and molecular characteristics in blood-brain-free areas of the brain and their physiological functions. Five weeks-old male Wistar rats (n=6 per group) were fed daily with positively or negatively charged polystyrene nanoparticles (NPs) (50 nm diameter, 12.5 mg/kg body weight) for two months. To confirm the migration of circulating NPs to AP, we injected yellow-green fluorescent cationic polystyrene nanoparticles PSNPs (F-NP) in the jugular vein of six Wistar male rats (7 weeks old, 5 and 10 gr/kg body weight) and use confocal microscopy LSM 710 with spectral imaging mode to detect their presence in AP. Our analysis of the AP confirmed the presence of F-NPs in this area.
Our previous findings indicate that a two-month NPs exposure affects heart rate and osmoregulation and increases the reactivity of microglial cells in the area postrema (AP), a blood-brain barrier (BBB)-free cardiovascular center. This year, we focused on the microglia reactivity in the Nucleus Tractus Solitarius (NTS), an area directly adjacent to the AP and protected by a BBB. NTS is a pivotal central area involved in the monitoring and regulation of blood pressure levels. The results revealed that the reactivity of microglia in NPs groups was more robust in the NTS than in the AP, indicating that the impact of circulating NPs is more severe on NTS microglia than on AP microglia.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
4: 遅れている
理由
During FY2021, the microglia morphology study on rats exposed to NPs was performed only on the AP (the experiment was time-consuming), therefore, we pursued this study during FY2022 by focusing on the NTS of those rats, an area protected by a BBB. Interestingly, the result showed that the reactivity of microglia to NP exposure was more robust in the NTS than in AP.
Another goal of this project, which was previously delayed, was to investigate the distribution of NPs in AP after intravenous injection of Fluorescent NPs (F-NP) in rats by using confocal microscopy spectral imaging mode. The experiments were performed accordingly to our plan this year and the presence of F-NPs in the AP area was observed.
Our plan for FY2022 was to investigate the inflammatory status of the AP and NTS at a transcript level by using RT-qPCR on tissues collected from the brain of rats chronically orally exposed to NPs in FY2021. Our experiences were delayed due to various issues (technical issues with our RT-qPCR equipment, the relocation of our laboratories to another building on our campus, and the fact that performing the experiments that were previously delayed was time-consuming). The total RNAs of AP and NTS were extracted, and preliminary RT-qPCR experiments to investigate the expression of a few genes coding for inflammation-related molecules were performed. The genes investigated so far did not show any clear expression differences between groups. The experience will be repeated during FY 2023 and additional genes of interest will be tested.
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| 今後の研究の推進方策 |
Since we could not perform enough experiments during FY2022, the inflammatory status of the AP and NTS will be further investigated at the molecular level during FY2023. The expression of inflammatory markers and receptors, and neuronal response-related genes will be investigated at the transcript level by using RT-qPCR in the AP and NTS collected from the brain of rats chronically orally exposed to PSNPs in FY2021.
Candidate molecules or their ligand showing a differential gene expression in the AP of rats exposed to NPs will be microinjected in the AP of anesthetized rats and their role in regulating cardiovascular parameters will be assessed acutely with Prof Waki. In the case we obtain a successful result, siRNA will be introduced to chronically silence the expression of the targeted molecule(s) in the AP to identify their functional roles in cardiovascular regulation in freely moving animals with Prof Waki.
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| 次年度使用額が生じた理由 |
-The reason for the carry-over in FY2023 stands on the fact that we did not have the chance to perform many RT-qPCR experiments in FY2022 and therefore, we did not purchase the associated consumable. The budget carried over from FY2022 to FY2023 will be used to purchase consumables such as primers/RT-qPCR kits/RNAse free tubes and tips (mainly) to perform RT-qPCR experiments and identify gene expression affected by NP exposure.
-The rest of the budget allocated to FY2023 will be used to purchase peptides, siRNA, and other necessary materials necessary for testing the functional role of candidate molecules identified by RT-qPCR experiments.
-Another part of this budget will be used to cover the cost of a trip to Korea to attend The 10th Federation of the Asian and Oceanian Physiological Societies Congress (FAOPS2023) and present the latest data of this study.
-The remaining part of this budget should be used to cover the fee for the publication of those data.
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