在皮肤中的单神经末梢体内双光子显微镜
Summary
该协议的动态纵向成像和神经末梢记者在转基因小鼠中的选择性激光损伤呈现。
Abstract
在皮肤神经末梢参与生理过程，如感测1以及在病理过程，如神经性疼痛2。他们贴近地表的定位有利于皮肤神经末梢的生活完整的动物显微成像。使用多光子显微术，它能够获得精细图像克服的皮肤组织的强烈的光散射问题。表达的THY-1启动子在神经元（包括周感觉神经元）的控制下EYFP记者转基因小鼠是适合于个别的神经末梢在延长的时间周期的纵向研究长达数月甚至终生。此外，使用相同的飞秒激光作为用于成像，所以能够产生神经纤维的高选择性病变的神经纤维重组的研究。在这里，我们提出了一个简单而可靠的协议进行纵向多光子活体成像和基于激光显微对小鼠皮肤的神经末梢。
Introduction
皮肤的神经末梢发生在不同的病理生理状态的动态变化。神经纤维可以通过变性和再生或重组过程中这种疾病的过程中如周围神经病变2莫顿神经瘤3。跌打损伤后，神经末梢动态皮肤的一个重要组成部分是受损区域的神经再支配。然而，对于神经末梢调查的通常的方法是在体外组织学切片，缺乏对正在进行的流程4的实时信息。用遗传编码的荧光标记物，所以能够掌握其神经末梢活体动物的皮肤，从而获得关于结构变化丰富和显著更多相关信息。皮神经末梢的调查能够使用常规荧光显微镜，然而，皮肤组织的强烈的光散射强烈破坏了质量数据的获取5。多光子显微镜能够取得在强烈散射组织中的高清晰度图像，由于只从物镜的焦点导致荧光发射的激发光的光子能量的非线性总和。这个效果导致了强劲增长的穿透深度和改善信号对噪声比的测量在皮肤组织6。使用相同的激光作为成像，所以能够产生神经纤维7的选择性清扫。在下面的协议，我们将展示在体内的皮肤神经末梢纵向成像的记者转基因小鼠结合使用市售的多光子显微镜系统选择性激光损伤的方法。
Protocol
Representative Results
Discussion
在这个视频协议，我们证明了该方法的单神经末梢的非侵入性的纵向双光子成像。
皮肤神经支配的动力学在这些疾病如牛皮癣和外周神经病变2，并且在创伤性损伤9受到影响。双光子成像允许神经纤维结构中的胶原基质的详细分析。利用转基因小鼠的报道中，有助于避免与神经纤维的染色的问题。 THY1-YFP-H菌株似乎是形态学数据分析10足够坚固，而大?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
作者要感谢Neurotar有限公司提供技术援助，CIMO基金会和FGSN提供财政支持。
Materials
| Round cover glass | Electron Microscopy Science | 72296-05 | 5 mm diameter #1.5 thickness |
| Eye drops Viscotears | Novartis | 2mg/g | |
| Superglue Loctite 401 | Henkel | 135429 | |
| Ketaminol | Intervet | Ketamine 50 mg/ml, working solution 10 mg/ml (use it 80 µg per gramm of animal weight) | |
| Rompun | Bayer Healthcare | Xylazine 20 mg/ml, working solution 1.25 mg/ml (use it 10 µg per gramm of animal weight) | |
| Working solution is a mixture of Ketamine 10 mg/ml and Xylazine 1.25 mg/ml in PBS | |||
| Ethanol 70% | |||
| Distilled water or Milli-Q water | |||
| Syringes 1ml | BD | 300013 | |
| 30G 1/2" needles | BD | 304000 | |
| Plastic packaging material | Could be purchaized from general hardware store | ||
| FV-1000MPE microscope | Olympus | FV-1000MPE | Microscope with motorized stage and rigid metal bar for fixation |
| 25X XLPlan objective | Olympus | XLPLN 25XWMP | Water imersion objective optimized for multiphoton imaging |
| Mai-Tai DeepSee laser (2W) | SpectraPhysics | ||
| Heating pad | Supertech | TMP-5b | Heating pad with a temperature controller |
| Metal ring fixator | Neurotar Ltd. | ||
| ImageJ | NIH | Open source software for image processing and analysis, http://rsbweb.nih.gov/ij/ | |
| Thy1-YFPH mice strain | JaxLab | 003782 |
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