| Project/Area Number |
15591827
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Otorhinolaryngology
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| Research Institution | Kyoto Prefectural University of Medicine |
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Principal Investigator |
UNO Toshiyuki KPUM, Associate professor, 医学研究科, 助教授 (70254349)
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| Co-Investigator(Kenkyū-buntansha) |
HISA Yasuo KPUM, Professor, 医学研究科, 教授 (50181087)
YAOI Takeshi KPUM, Assistant, 医学研究科, 助手 (40311914)
SUZUKI Toshihiko KPUM, Assistant, 医学研究科, 助手 (20275225)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | recurrent laryngeal nerve injury / nucleus ambiguus / single-cell RT-PCR / Stat3 / Reg-2 / Bax / Bcl-2 / 喉頭 / 反回神経 / 神経再生関連因子 / 単一細胞PCR / 神経再生関連遺伝子 |
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| Research Abstract |
To elucidate molecular changes of motoneurons which innervate laryngeal muscles after transection of rat recurrent laryngeal nerve, we have devised methods ; nucleus ambiguus(NA) neurons projecting to intrinsic laryngeal muscles were labeled retrogradely with DiI by injecting into bilateral thyroarytenoid muscles, then either one of recurrent laryngeal nerves was transected, and fluorescence-bright single neurons were microdissected, followed by a single-cell reverse transcription (RT)-PCR. Immunohistochemically, many DiI-labeled motoneurons in NA were immunoreactive to phosphorylated Stat3 in the nucleus on 1, 7, 10 and 14 days after axotomy. Thirty m-thick frozen sections of brain stem were mounted onto membrane slide, and DiI-labeled single neurons in NA were microdissected under a fluorescence microscope. By real-time quantitative RT-PCR for single motoneurons, the expression levels of GAP-43 and nNOS genes were up-regulated at 7 days and 1 day after axotomy, respectively. The expression levels of Stat3, Reg-2, and Bcl-2 genes were up-regulated at 7 days, whereas that of Bax was down-regulated at 1 and 7 days after axotomy as compared with control levels. We could demonstrate a part of molecular cascades involving Stat3 in neurons projecting to laryngeal muscles after axotomy with this novel method in which single-cell RT-PCR was combined with neurotracer DiI labeling.
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