- 學位論文
以活化方式增強標記小鼠皮下特定神經
Activation-Enhanced Labeling of Type Specific Cutaneous Nerve Fibers
摘要
感覺神經負責將個體從環境中接收到的刺激轉化為電訊號，然後將它們傳送到中樞神經系統。在我們的皮膚中，有多種類型的感覺神經來偵測多種不同類型的環境變化，這些不同類型的神經上具有各種受體分子、離子通道等等不同組合，而每一種構造都有其負責的感覺功能。舉例來說，TRPV1 和 MrgD 都屬於皮下神經的 C 纖維。 TRPV1 是負責偵測有害熱和痛的神經，其活化劑是辣椒素。 MrgD是偵測瘙癢和疼痛的神經，它的活化劑是β-丙氨酸。識別特定類型的感覺神經可以幫助我們進行臨床和科學研究，但目前缺乏有效的方法來識別它們。若有一種方法可以幫助我們在活體內觀察不同功能類型的感覺神經，並可隨時在體內追蹤這些神經纖維，將會是非常有趣且實用的，因此我們的目標是開發一種顯微神經測定法來選擇性地標記特定類型的神經纖維。 在本研究中，我們利用上述特定感覺具有特定受體分子或離子通道的特性，並使用小分子染料亞甲藍來標記以活化劑活化的特定感覺神經來標記活體中的皮下神經。此外，我們使用 TRPV1-tdTomato 和 MrgD-GFP 轉基因小鼠來測試我們的概念。 我們的結果分為兩個部分。第一部分是我們利用共軛焦顯微鏡觀察 TRPV1-tdTomato 和 MrgD-GFP 小鼠後腳掌的皮下神經纖維形態。第二部分是亞甲藍注射到小鼠腳掌的結果。我們的結果表明，活化劑與亞甲藍的組合增加了亞甲藍標記特定種類神經的比例。也就是說，同時注射β-丙氨酸和亞甲藍會使亞甲藍更傾向標記MrgD神經，而同時注射辣椒素和亞甲藍會使亞甲藍傾向標記TRPV1神經。 綜合上述研究，我們發現通過共軛交顯微鏡來活體掃描小鼠可以幫助我們觀察和操縱生物體內的神經纖維。此外，共同注射 MB 和活化劑可以增強亞甲藍標記特定種類的神經纖維。這些方法使我們能夠在體內觀察小鼠的皮下特定神經。
並列摘要
Sensory nerve fibers are responsible for converting stimuli the individual received from the environment into electrical signals and then transmitting them to the central nervous system. In the skin, there are multiple types of nerve fibers for detecting many different kinds of environmental changes. These different fiber types are equipped with specific combinations of receptor molecules, ion channels and signal transduction machinery, each of these structures has its own function in sensing. Recognizing specific type of sensory nerve fibers can help us conduct clinical and scientific research, but there is no efficient way to identify them intravitally. For examples, both TRPV1 and MrgD belong to the C-fiber group of the subcutaneous nerves. TRPV1 is the nerve type that detects noxious heat and pain, and its agonist is capsaicin. MrgD is the nerve type that detects itching and pain, and its agonist is β-alanine. It would be interest to have a method to visualize different functional types of sensory fibers in vivo, in situ, and capable of following them in disease states, so we aim to develop a microscopic nerve assay to selectively label specific type of nerve fibers. In the present study, we took advantage the specific sensory fibers have specific receptor molecules described above, and used a small molecule dye, methylene blue, to label agonist-activated specific sensory fibers for intravital visualization. Furthermore, we use TRPV1-tdTomato and MrgD-GFP transgenic mice to test our concept. There are two parts in our results. First, we revealed the subcutaneous nerve fibers morphology of TRPV1-tdTomato and MrgD-GFP mice hindpaws under the confocal microscope. And the second part is the results of the methylene blue injection to the mice hindpaws. Our results show that combining agonist with methylene blue increased the proportion of methylene blue staining of agonist matching specific type of fibers. That is, combined β-alanine and methylene blue subcutaneous injection caused preferential labeling of MrgD fibers, while combined capsaicin and methylene blue injection labeled TRPV1 fibers in the mice skin. In conclusion, by intravital scanning of mice can help us observe and manipulate nerve fibers in living bodies. Furthermore, co-injecting MB and ligand can enhance MB labeling specific type of fibers. These methods make us possible to observe nerve fibers of mice in vivo.
參考文獻
延伸閱讀
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