以光學方式觀察慢性壓迫神經小鼠痛覺神經的變化

Visualize nociceptor changes in the hind paw of neuropathic mice with chronic constriction injury

吳雅雯(Ya-Wen Wu)

指導教授 ： 嚴震東

國立臺灣大學/生命科學院/生命科學系/碩士(2018年)

https://doi.org/10.6342/NTU201802114

摘要

神經性疼痛是由感覺神經系統損傷引起的。在本研究中，我們使用顯微鏡方法來檢查坐骨神經的慢性壓迫損傷（CCI）後的皮膚下的傷害感受器的變化。我們使用標記螢光纖維Nav1.8-tdTomato的基因轉殖小鼠，並直接利用顯微觀察神經末梢的疼痛感受器並觀察神經性疼痛的發展變化過程。行為測試包括Von-Frey刺激，輻射熱板測試，分別測試其對機械和熱刺激的敏感性。在CCI手術前及3〜21天後，利用共聚焦顯微鏡和雙光子螢光顯微鏡觀察後腳掌神經叢和游離神經末梢的動態變化。我們初步的數據顯示神經纖維的數量與痛覺敏感程度並不一致。利用ATF3檢驗老鼠背根神經細胞受損的程度發現在不同腳掌神經損害組別間沒有顯著差異。由此推論證明受傷後早期的腳掌神經末梢的密度可能是CCI神經病變嚴重程度的有用生物標記。

關鍵字

神經病變性疼痛 ； 痛感受器 ； 慢性神經壓迫 ； 雙光子螢光顯微鏡

並列摘要

Neuropathic pain is caused by injury of the somatosensory nervous system. In this study we aim to use in vivo and ex vivo microscopic methods to examine the change in nociceptors under the paw skin after chronic constriction injury (CCI) of the sciatic nerve, a commonly used animal model for neuropathic pain research. Our basic hypothesis is that direct microscopic observation of free nerve endings of the nociceptors in situ and en bloc will reveal the life history of the development and maintenance of the neuropathic pain. We used genetically modified mice that intrinsically express the fluorophore tdTomato tagged to a small fiber marker, the Nav1.8 molecule.  A battery of behavior tests were applied to the hind paws of the mice to test their sensitivity to mechanical and heat stimuli, respectively. Confocal microscopic and longitudinal two-photon fluorescent microscopic observation of dynamic changes of nerve plexus and free nerve endings in the hind paw were performed first before and then 2 to 21 days after CCI of the sciatic nerve. Using behavioral tests we validated mechanical and thermal hyperalgesia of the CCI mice. Our preliminary data revealed a mismatch of the severity of mechanical hypersensitivity versus the extent of fibers degeneration. Use ATF3-IHC we found that there was no significant different of nerve injured between mild and severe groups. Thus the density of the Nav1.8-tdTomato nerve ending in the hind paw at early injury stage can be a useful biomarker for the severity of the CCI induced neuropathy.

並列關鍵字

Neuropathic pain ； Nociceptor ； chronic constriction injury ； two-photon fluorescent microscopy

參考文獻

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