树脂浇铸和3维成像的小鼠门静脉或肝内胆管系统
Summary
小鼠肝门静脉或肝内胆管的3维结构的可视化和量化的方法进行说明。该树脂铸造技术也可以被应用到其他的导管或血管系统，并允许<em在原位</em>，系统的完整的通信体系结构的可视化或定量。
Abstract
在器官的血管和导管结构，正确的架构是适当的生理功能不可缺少的，这些结构的形成和维持是一个高度管制的过程。这些复杂的，3维结构的分析，极大地依赖于任一的2 – 维的检查部或对染料注入研究。然而，这些技术无法提供一个完整的，可量化的表示他们的目的是阐明导管或血管结构。或者，3 – 维的塑料树脂石膏的性质产生一个永久的系统快照是一种新颖的和有用的技术，广泛的3维结构和网络的可视化和量化。
树脂浇注系统的一个关键优势的能力，以确定完整的连接，沟通，血管或胆管结构。血管的结构和公爵河谷网络是至关重要的器官功能，该技术具有的潜力，以帮助在几个方面研究血管和导管网络。分析正常形态和功能结构的腔结构，确定发育的形态发生变化，并发现正常和疾病状态的形态在组织体系结构之间的差异，也可以使用树脂浇铸。以前的工作利用了树脂浇铸研究，例如，在小鼠肝内胆管系统的架构和功能上的缺陷，并没有反映在二维分析的结构1,2，改变脑血管的一个阿尔茨海默氏症小鼠模型3 ，门静脉异常，门脉高压症和肝硬化小鼠4，在大鼠淋巴成熟不成熟和成人之间的肺的发育步骤5，即时反应，化学性损伤大鼠肝脏，胰腺和肾脏微血管的变化6。
在这里，我们提出一种方法，产生一个3维树脂浇铸体的小鼠血管或导管的网络中，特别注重门静脉和肝内胆管。这些强制转换可以通过清零或浸软组织可视化，然后进行分析。这种技术可以应用于几乎任何血管或导管的系统，将直接适用询问到一个3 – 维的导管或血管结构的发展，功能，维护，或损伤的任何研究。
Protocol
1。准备插管随着你的指尖温暖了1英寸长的路段，PE10管材和伸展，使管变细。 注：该船只或导管，以将插管的大小将决定所需的拉伸程度。套管必须得到很好的拉伸为胆管蒙上，但可能只需要适度拉伸以适应较大的门静脉内。 对角线拉伸管生成一个斜面的顶端。 切油管创建的5-6寸段的另一边缘。 插入32计，½英寸皮下注射针入非斜面边缘的油管。 …
Discussion
我们已经介绍了如何可以转换的肝门静脉肝内胆管系统的具体例子，但这种技术可以应用到几乎任何其他的导管或血管系统有轻微的修改。先前的研究已经证明了这种技术的可行性，在多个物种，包括鼠标的2,7 ~~ 9，小鸭10,11，兔，狗14，12,13和猪15，并在许多器官，包括鼻粘膜腺体14，心脏，膀胱12,13，16和肝脏1,2,7,8。这些石膏可用于比较多…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院（NIH）的资助SSH（R01-DK078640），霍华德·休斯医学研究所（HHMI）通过在分子医学TJW HHMI /范德比尔特大学的证书课程（GRDOT56006779），范德比尔特糖尿病研究和培训中心（P30-DK020593）和范德比尔特消化疾病研究中心（P30-DK058404）提供的核心服务。
Materials
| Name of reagent | Company | Catalog number | Comments |
| PE10 polyethylene tubing | Fisher Scientific | 1417012P | |
| 5-0 surgical black braided silk | Roboz Surgical | SUT-15-1 | |
| Steriject 32 G x 13 mm needle | Air-tite | TSK3213 | |
| Spring scissors | Fine Science Tools | 15000-08 | |
| Number 5 forceps | Fine Science Tools | 91150-20 | |
| Mercox II kit | Ladd Research | 21247 | |
| Benzyl alcohol | Fisher Scientific | 1816-04 | Only required for BABB-clearing |
| Benzyl benzoate | MP Biomedicals, LLC | 154839 | Only required for BABB-clearing |
| Phosphate-buffered saline (PBS) | |||
| Modeling clay | |||
| Scale | |||
| Laboratory scissors | |||
| 15 ml cap polypropylene tubes | |||
| 4% paraformaldahyde | |||
| 15% potassium hydroxide (KOH) | Only required for maceration | ||
| Razor blade | |||
| 100% methanol | |||
| 3 ml luer lock syringe |
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Tags
3-DimensionalResin CastingImagingMouse Portal VeinIntrahepatic Bile Duct SystemArchitectureVascular StructuresDuctal StructuresPhysiological FunctionFormationMaintenanceHighly Regulated ProcessAnalysis2-dimensional ExaminationSectionDye Injection StudiesComplete RepresentationQuantifiable RepresentationNovel TechniqueVisualizing StructuresQuantifying StructuresPlastic Resin CastsPermanent SnapshotConnected StructureCommunicating StructureBlood VesselDuct NetworkOrgan FunctionMorphological ChangesDisease States
Cite This Article
Walter, T. J., Sparks, E. E., Huppert, S. S. 3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System. J. Vis. Exp. (68), e4272, doi:10.3791/4272 (2012).