| 研究課題/領域番号 |
22K09248
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| 研究種目 |
基盤研究(C)
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| 配分区分 | 基金 |
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| 応募区分 | 一般 |
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| 審査区分 |
小区分56010:脳神経外科学関連
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| 研究機関 | 国立研究開発法人国立循環器病研究センター |
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研究代表者 |
大西 諭一郎 国立研究開発法人国立循環器病研究センター, 研究所, 非常勤研究員 (00533811)
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| 研究分担者 |
山本 正道 国立研究開発法人国立循環器病研究センター, 研究所, 特任部長 (70423150)
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| 研究期間 (年度) |
2022-04-01 – 2025-03-31
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| 研究課題ステータス |
交付 (2023年度)
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| 配分額 *注記 |
4,160千円 (直接経費: 3,200千円、間接経費: 960千円)
2024年度: 1,040千円 (直接経費: 800千円、間接経費: 240千円)
2023年度: 1,560千円 (直接経費: 1,200千円、間接経費: 360千円)
2022年度: 1,560千円 (直接経費: 1,200千円、間接経費: 360千円)
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| キーワード | spinal cord injury / metabolic stress / ATP / mitochondria / 脊髄損傷 / ミトコンドリア / 末梢神経損傷 / ワーラー変性 / 神経変性 / エネルギー / 代謝 |
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| 研究開始時の研究の概要 |
１．脊髄離断モデルの作成、および組織学的評価
２．ニューロン特異的ATP可視化マウスの脊髄急性スライス標本の観察
３．脊髄損傷後慢性期の損傷尾側での阻害剤投与下でのニューロン・軸索変性の評価
４．脊髄損傷後急性期と慢性期の損傷尾側でのメタボローム解析
５．脊髄損傷後急性期と慢性期の損傷尾側でのトランスクリプトーム解析
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| 研究実績の概要 |
Spinal cord injury (SCI) presents the axonal degeneration in the caudal site of lesion. The caudal axonal degeneration prevents the functional recovery due to the axonal regenerative sprouting and sprouting at the injury site. The axonal mitochondrial transport to the distal site of lesion is impaired in SCI, leading to the metabolic stress. Previously, we revealed that axons activated the glycolysis system to maintain ATP levels, and inactivated tricarboxylic acid cycle because of mitochondrial degeneration. A gradual decrease in ATP levels was observed before the progression of axonal degeneration. Furthermore, glycolysis activation increased ATP levels and delayed axonal degeneration. In this study, we investigated the mitochondria transfer to SCI to prevent the caudal axonal degeneration.
T8 partial and complete transection were performed in GO-ATeam1 and GO-ATeam2 mice, which expressed a fluorescence resonance energy transfer-based ATP biosensor with orange fluorescent protein and green fluorescent protein in the mitochondria and cytosol, respectively. Mitochondrial isolation was proceeded by differential centrifugation from littermate liver and new born liver. Isolated mitochondria transferred to T9 spinal cord with stereotactic apparatus after cord transection. The mitochondrial and intracellular ATP level was assessed in GO-ATeam1 and GO-ATeam2 mice, respectively. Ultrathin distal spinal cord cross-sections were analyzed for axon diameter, G-ratio and number of mitochondria in axon by electron microscopy.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
JC-1 dye indicated the preservation of membrane potential in isolated mitochondria. FRET analysis also presented the ATP production in isolated mitochondria. In vivo imaging presented that the transfer of adult liver derived mitochondria elevated ATP level in the distal cord, and improved locomotor function. However, control buffer, ATP solution, and new born liver derived non-function mitochondria had no ATP elevation, and no locomotor improvement. Immunohistochemical analysis revealed that adult liver derived mitochondria were distally spread, and incorporated into neuron. The transfer of adult liver derived mitochondria increased the number of mitochondria in axon and thickness of myelin sheath.
These results indicated that the transfer of mitochondria into the distal site of spinal cord injury prevented the axonal degeneration, and improved locomotor function.
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| 今後の研究の推進方策 |
We investigate how transfferd mitochondria contribute the improvement of locomotor function.
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