9日龄小鼠新生儿肝外胆管和胆囊清扫术
Summary
为了观察小鼠新生儿胆管疾病，需要完整的胆管和有效的准备。因此，在保持胆管完整性的同时，成功研制出分离小鼠新生儿整个肝外胆管系统的新方法。
Abstract
小鼠新生儿胆管的清扫被描述为困难。所述标准操作程序的主要目的是在制备过程中在不损坏胆管的情况下分离小鼠新生儿的肝外胆管（EBD）。由于与胆管细胞系和整个肝外胆管系统（EBDS）的收获相比，其制备异常紧密，因此所描述的方法在研究新生儿胆管疾病（例如胆道闭锁）的动物模型时非常有用。安乐死后，进入腹膜腔，并通过独特的 En-bloc-切除术 （EbR） 提取胆管系统、十二指肠和肝脏。将提取的样品放在泡沫垫上，并在没有必要接触的情况下从无创伤性细胞中解剖EBD。解剖整个EBDS是这种方法的一个显着优势。由于胆管组织的体积和数量小，必须小心。使用所描述的技术，对胆管细胞没有损伤。此外，该技术的纯度是可重复的（n = 10）。因此，可以收获最佳可比的样品。此外，没有胆管组织受到伤害，因为在制备过程中可以避免与胆管系统的任何接触，将胆汁留在胆囊内。最重要的是，在进行最后的胆囊和胆管清扫时，无创伤的微器械只在胆管的稍微外侧使用，而不会挤压胆管。这是获得干净完整样本的关键，对于进一步的组织学检查或胆管细胞分离至关重要。总而言之，所描述的创新解剖技术使特别没有经验的操作人员能够使用必要的设备尽可能干净地隔离EBDS。
Introduction
胆管病（如胆道闭锁、原发性硬化性胆管炎 （PSC） 和原发性胆汁性胆管炎 （PBC））的起源和进展未知或不完全1，2。对这些疾病的起源和进展的了解有限，导致缺乏治疗选择3。研究新生儿胆管疾病的最困难的障碍是获得对病理生理学的分子理解。更好地了解分子病理学的重要关键之一是对受影响组织进行最佳观察。为了避免降低研究之间的可比性和差异，例如观察胆道闭锁4的潜在病毒性病因，需要尽可能最好地准备和共享所执行的解剖技术。靶组织的纯制备对于以后的显微镜研究或育种细胞和3D类器官培养物是必要的。然而，在小鼠新生儿疾病中，组织样本很少见，并且由于体积非常小，仅少量出现。关于胆管疾病，已经描述了小鼠新生儿胆管清洁制备的困难5。由于新生儿发育阶段，组织分化并不过分，与成人样品的制备相比，这会使制备复杂化并增加难度。因此，操作工作组研究了在新生小鼠模型中制备EBDS的新策略。在本研究中，该技术允许对每个样品进行有效解剖。
胆管系统腹膜内放置在右上腹部，起源于肝脏。胆囊位于肝脏右叶的内脏表面下方。胆管与门静脉和肝动脉一起嵌入肝十二指肠韧带中。它直接连接肝脏和十二指肠，并将胆汁排入十二指肠6。在解剖学上，胆管分为左右肝管、肝总管、胆囊管和胆总管，胆总管和胆总管7汇合而成。这个最终通过Vater壶腹 将 胆汁和唾液从胰管排空到十二指肠。
胆管细胞在肝内和肝外排列胆管，栖息在复杂的解剖壁龛中，在那里它们有助于胆汁的产生和稳态8。胆汁每天以高浓度通过这些专门的上皮细胞。特别是，HCO3-伞的维护对于防止胆汁酸毒性非常重要9。胆管细胞是肝胆系统中针对例如管腔微生物的第一道防线10。胆管细胞对毒性攻击的防御能力可能会因遗传易感性而减弱。毒性超负荷会导致损伤和破坏，因此可导致胆管病。此外，发育中的胆管不能完全具有所有自我保护机制，导致新生儿胆管对环境毒素的敏感性更高11。
Protocol
Representative Results
Discussion
本文报道并讨论了一种新的手术方法的创建和验证，用于去除安乐死新生小鼠的EBDS。显微镜和组织学发现表明，该方法可快速检测EBD并在导管边缘附近解剖它们，即使在新生小鼠中也是如此。所述方案只需要手术器械和20倍放大倍率的显微镜。此外，该方法允许隔离整个EBDS。该技术高效、简单且易于复制。
为了研究胆管疾病，如胆道闭锁，PSC和PBC，经常需要机械提取整个胆?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢Johanna Hagens，Pauline Schuppert，Clara Philippi，PD Dr. Medicine Christian Tomuschat，Svenja Warnke，PD Dr. Diana Lindner，Dirk Westermann教授，Miriam Tomczak，Nicole Lüder，Nadine Kurzawa，Dr. rer nat. Laia Pagerols Raluy，Birgit Appl和Magdalena Trochimiuk的贡献。汉斯·克里斯蒂安·施密特（Hans Christian Schmidt）得到了汉堡UKE的Else Kröner-Fresenius-Stiftung iPRIME奖学金（2021_EKPK.10）的资助。
Materials
| 2-Propanol | CHEMSOLUTE | 11365000 | used as a dehydrating agent |
| 30 G canula | B Braun/Sterican, Melsungen Germany | 4656300 | canula for hydration of the sample |
| Air vent | C + P Möbelsysteme GmbH & Co. KG, Breidenbach, Germany | Tec-Ononmic AZ 1200 | the use of an air vent helps to avoid inhalation of formalin-containing fixatives |
| Aqua ad injectabilia Braun | B Braun, Melsungen, Germany | 2351744 | saline; Container: Mini-Plasco connect, 20 x 10 mL, sterile |
| Bigger microsurgical Forceps | DIADUST von Aesculap, Trossingen Deutschland | FD253R | straight, 180 mm (7"), platform tip, round handle, width: 0,800 mm, diamond dust coated, non-sterile, reusable optional tool for observation and every step of preparation except very final preparation; Dividing skin of the peritoneum |
| Camera “SmartCAM 5” | Basler and Vision Engineering, Send, United Kingdom | EVC131A | optional Lynx Exo camera modul: sensortype: CMOS, resolution 2560 x 1920 pixels, sensor size: 1/2"; Used for videoproduction and technical evaluation |
| Dehydration machine/Citadel 2000 Tissue Processor | Fisher Scientific GmbH, Schwerte, Germany | 12612613 | used for automatic dehydration, short program (approx. 4.8 h) |
| Dehydration sponge | Carl Roth, Karlsruhe, Germany | TT56.1 | sponge for final dissection step, other sponges/foam pads with a minimum pore size of 60 pores per inch are also suitable, the use of two foam pads per embedding cassette is recomended to cover the sample from below and above to prevent sliding through the perforation of the embedding cassettes |
| Dulbecco´s Phosphat Buffered Saline (PBS) | Gibco | 14190-144 | Doesn´t contain Calzium or Magnesium, 500 mL |
| Embedding cassettes | Engelbrecht GmbH, Edermünde, Germany | 17990 | |
| Eosin | MEDITE Medical GmbH, Burgdorf, Germany | 41-6660-00 | staining solution, ready to use |
| Fine Scissors CeramaCut | FST, Heidelberg Germany | 14959-09 | Tips: Sharp-Sharp, Alloy / Material: Ceramic Coated Stainless Steel, Serrated:, Yes; Feature: CeramaCut, Tip Shape: Straight, Cutting Edge: 22 mm, Length: 9 cm; Skin incision, incision of the peritoneal window |
| Graefe Forceps | FST, Heidelberg Germany | 11051-10 | Length: 10 cm, Tip Shape: curved, serrated, Tip width: 0.8 mm, Tip Dimensions: 0.8 x 0.7 mm, Alloy /Material: Stainless Steel |
| Hematoxylin | MEDITE Medical GmbH, Burgdorf, Germany | 41-5130-00 | staining solution, ready to use |
| Highresolotion microscope | Vision Engineering, Send United Kingdom | EVO503 | Capable of enlargement up to 60x magnification, only 6x to 20x magnification were used |
| Microscope | Olympus Optical CO, Ltd., Hamburg, Germany | BX60F5 | |
| Microscope Cover Glases | Marienfeld, Lauda-Königshofen, Germany | 101244 | 60 mm broad, made of SCHOTT D 263 glass |
| Microscope Slides | R. Langenbrinck GmbH, Emmendingen, Germany | 03-0060 | |
| Microtome | Leica, Nußloch, Germany | SM2010R | Tool for sectioning (2 µm-slices) |
| Omnifix-F 1 mL syringe | B Braun, Melsungen, Germany | 9161406V | syringe without canula |
| Paraffin | Sakura Finetec, Torrance, USA | 4511 | Tissue-Tek Paraffin Wax Tek III, without DMSO |
| Paraffin embedding machine | MEDITE Medical GmbH, Burgdorf, Germany | TES 99 | The embedding machine used in this study contained the following three individual modules: TES 99.420, TES 99.250, TES 99.600. The sample should be embedded in Paraffin directly after the dehydration, no interim storage in a fridge should be performed due to possible shrinking and moisture in the fridge |
| Paraformaldehyde (PFA) | Morphisto | 1176201000 | Prepare 1 mL Aliquots in 2 mL Eppendorf conical Tubes for liver samples and 0.5 mL Aliquots in 1 mL Eppendorf conical Tubes for extrahepatic bile duct samples, 4% in PBS ph 7.4 |
| Small Microsurgical Forceps | EPM (Erich Pfitzer Medizintechnik), Bütthard, Bayern, Germany | (00)165 | Round handle, straight, 0.3 mm tip, tool for observation and every step of preparation, especially useful in final preparation |
| Stainless Steel Ruler | Agntho's AB, Lidingö, Sweden | 30085-15 | 150mm With Metric & Inch Graduations |
| Surgical Scissors – Sharp-blunt for decapitation | FST, Heidelberg Germany | 14001-14 | Device for decapitation |
| Warming cabinet | Haraeus, Hanau, Germany | T 6060 | the sliced samples should be kept in the warming cabinet to ensure the attachement of the sample on the microscope slides |
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