在活體下利用亞甲藍標記神經來觀察小鼠和人類皮膚的神經纖維

Intra-vital methylene blue labeling of nerve fibers in the skin of mice and human subjects

游少瑋(Shao-Wei Yu)

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(2020年)

https://doi.org/10.6342/NTU202003807

摘要

神經痛是臨床上難以根治的神經疾病，是由體感覺神經受損而引發的慢性疼痛，而自發性疼痛、觸覺異常與痛覺敏感化是神經痛的三大徵狀。臨床上，醫院常藉由理學檢查、電學檢查和組織切片以檢查診斷周圍神經病變的方式，但理學和電學檢查易受外部因素的影響，而組織切片有傷害的風險。因此我們想建立一種溫和、可逆且可觀察的神經測定法作為輔助診斷工具。
在以前的研究中，我們知道亞甲藍（Methylene Blue, MB）帶有藍色且對神經有親和性能將神經染色，而氧化下的藍色MB具有近紅外光 (Near Infrared, NIR)的螢光利於觀察。在本實驗中，我們將不同濃度的10μl MB注入小鼠後肢第三趾中，並在雙光子激發顯微鏡或共軛焦顯微鏡下觀察MB的螢光，試圖了解MB施打的適宜的濃度、觀察時間，和觀察位置以及MB對神經的親和性，之後在人體上測試結合MB與三倍頻影像術（third-harmonic generation imaging，THG）對皮下神經纖維標記的可行性。
我們注射MB後發現，小鼠被注射位置會染出類似神經纖維的條狀螢光，其中有部分與轉殖基因小鼠（NaV1.8-cre :: tdTomato）的神經纖維熒光重疊，表明我們可以利用MB染色小直徑的神經纖維，也有另外一部份不重疊的條狀神經螢光。而若施打部位不具有神經，如坐骨神經部分切除手術(spared nerve injury, SNI)後的第三足趾，則完全不會有條狀螢光的產生。

進行不同MB濃度的測試時，發先隨著注入不同濃度的10μl MB之後，MB所染的神經會隨著濃度增加而增多，在雙光子激發顯微鏡下，我們使用自行從固體粉末(購自sigma)調配的MB，MB在接近0.3% ( 0.3 mg/ml) 時，染色區域增加幅度最大；而在共軛焦顯微鏡下，我們改以注射用純水來稀釋醫療用的1% MB溶液(購自“安星”)，MB在稀釋至接近0.03% ( 0.03 mg/ml)時有最佳的增加幅度。小鼠在注入MB的15分鐘之後就可以觀察到螢光，最佳觀測時間為注入MB之後的30分鐘至90分鐘，超過時間之後就由條狀的類似神經纖維的螢光，變成點狀的螢光，點狀螢光最久會一直持續到注射MB後的18小時，在24小時之後MB的螢光就完全消失，而降低MB濃度的時候，同時也會縮減螢光持續時間。
在人體實驗部分，在腳踝上10公分的小腿處和手腕後10公分的前臂處以皮內注射的方式，分別注射了10μl 的液體稀釋的0.03%MB和固體調配的0.3%MB，最佳觀測時間約在注射後60分鐘至90分鐘，可以看到許多類似神經纖維的條狀螢光出現在真皮層中，並與三倍頻影像互相對比，可以有效確認為神經。
我們還進行了MB的毒性測試，發現轉殖基因小鼠（NaV1.8-cre :: tdTomato）的神經纖維螢光在注射固態MB後2~4天或注射液體MB後1~3天，都會有NaV1.8的神經纖維螢光減少的現象，但是兩者都會在MB注射後第5~7天恢復正常，表示該神經纖維的螢光減少具有可逆性，而人體試驗部分，我們依照問卷進行調查，目前受試者注射之後皆無異狀。
綜合上述研究，我們發現利用MB在皮內微注射可以染出許多真皮層中的神經纖維，其中一部分的MB條狀螢光為小直徑神經纖維。另外，結合三倍頻影像則能可以有助於定量人體皮膚的小直徑神經纖維。

關鍵字

亞甲藍 ； 近紅外光 ； 活體影像 ； 小神經纖維密度 ； 三倍頻 ； 小鼠 ； 人

並列摘要

Neuropathic pain is a neurological disease caused by injury or pathology of the somatosensory system. Over two-thirds of neuropathic pain patients suffer pain symptoms for decades. Clinically, biopsy and compound action potential are common examinations for the diagnosis of peripheral neuropathy, but the electrical tests are for large diameter nerve fibers and biopsy causes prolonged nerve damage. We aim to develop a mild, reversible and microscopic nerve assay as an auxiliary diagnostic tool.
In previous studies, we know methylene blue (MB) stains nerves, and oxidized MB has near-infrared (NIR) fluorescence for fluorescent observation. In the mice, we injected 10 μl MB of different concentrations into the third toe of the hind limb, and observed the fluorescence of MB under a confocal microscope or a two-photon fluorescent microscope. We tested the optimal concentration, observation time and the suitable observation location of MB, and then tested the feasibility of using MB on human skin and combining MB and third-harmonic generation imaging (THG) to label cutaneous nerve fibers in humans.
After we injected MB, we found that mice skin showed nerve-like fiber stripes, and some of fluorescent stripes overlapped with the fluorescence of nerve fibers in transgenic mice (NaV1.8-cre :: tdTomato), indicating MB stained small nerve fibers. In addition, if the site did not have nerves fibers, such as the third toe after spared nerve surgery (SNI) of the sciatic nerve, there was no fluorescence stripe at all.
When testing different concentrations of 10 μl MB, we found labeled area by the dye increased with increasing concentrations. Under the two-photon fluorescent microscope, MB (purchased from sigma) label increase was the greatest at 0.3% (0.3 mg/ml); while under confocal microscope, MB (purchased from "Anxing") label increase was the best at 0.03% (0.03 mg/ml). In addition, fluorescence can be observed after 15 minutes of injection, and the best observation time was 30 minutes to 90 minutes after injection. After this period, fluorescence changed from stripe to dot shape. All MB fluorescence disappeared within 24 hours, and higher concentrations of MB extended the duration of fluorescence in the skin.
In human experiments, 10 μl of 0.3% sigma MB or 0.03% Anxing MB were also injected intradermally at the lower leg 10 cm above the ankle or the forearm area 10 cm above the wrist. Many fluorescent stripes resembling nerve fibers appeared in the dermis within 30 to 90 mins. When combining with the third-harmonic generation imaging many fibers had both MB and THG fluorescence.
We also tested MB toxicity and found that the nerve fiber fluorescence of transgenic mice (NaV1.8-cre::tdTomato) decreased in 2~4 days after injection of solid 0.3% MB or 1~3 days after injection of liquid 0.03% MB. Both MB returned to baseline 5~7 days after the injection, indicating that the fluorescence decreases in the Nav1.8 nerve fiber were reversible. In the human test part, we conducted a survey according to the questionnaire, and all subjects showed no abnormalities after injection.
In summary, we found intradermal MB injection labeled many nerve fibers in the skin. Some of the MB fluorescent stripes were small-diameter nerve fibers. In addition, combining MB and third-harmonic generation imaging may help quantify small diameter nerve fibers in the human skin.

並列關鍵字

methylene blue (MB) ； near infrared fluorescence (NIR) ； Intra-vital imaging ； Small-fiber neuropathy ； third-harmonic generation imaging ； mice ； human

參考文獻

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