Name: extrahepatic bile duct and gall bladder dissection in nine-day-old mouse neonates
Uploaded: 2022-08-23T15:00:00
Description: Watch this Scientific Journal Video about Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates at JoVE.com

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。

在伦理批准（N045/2021）后，观察雄性和雌性C57BL / 6小鼠新生儿至9天大。这些动物由德国汉堡汉堡-埃彭多夫大学医学中心的动物设施出生并用于实验目的。新生儿与亲本动物一起被关在笼子里。环境条件控制在温度（20-24 °C）、12：12 h明暗循环、相对湿度40%-70%。
1. 实验准备
<ol><li>准备外科手术所需的设备，包括剪刀，镊子等（见 材料表）。</li>
	<li...

<ol>
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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Propanol</td>
			<td>CHEMSOLUTE</td>
			<td>11365000</td>
			<td>used as a dehydrating agent</td>
		</tr>
		<tr>
			<td>30 G canula</td>
			<td>B Braun/Sterican, Melsungen Germany</td>
			<td>4656300</td>
			<td>canula for hydration of the sample</td>
		</tr>
		<tr>
			<td>Air vent</td>
			<td>C + P M&#246;belsysteme GmbH &#38; Co. KG, Breidenbach, Germany</td>
			<td>Tec-Ononmic AZ 1200</td>
			<td>the use of an air vent helps to avoid inhalation of formalin-containing fixatives</td>
		</tr>
		<tr>
			<td>Aqua ad injectabilia Braun</td>
			<td>B Braun, Melsungen, Germany</td>
			<td>2351744</td>
			<td>saline; Container: Mini-Plasco connect, 20 x 10 mL, sterile</td>
		</tr>
		<tr>
			<td>Bigger microsurgical Forceps</td>
			<td>DIADUST von Aesculap, Trossingen Deutschland</td>
			<td>FD253R</td>
			<td>straight, 180 mm (7&#34;), 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</td>
		</tr>
		<tr>
			<td>Camera &#8220;SmartCAM 5&#8221;&#160;</td>
			<td>Basler and Vision Engineering, Send, United Kingdom</td>
			<td>EVC131A</td>
			<td>optional Lynx Exo camera modul: sensortype: CMOS, resolution 2560 x 1920 pixels, sensor size: 1/2&#34;; Used for videoproduction and technical evaluation</td>
		</tr>
		<tr>
			<td>Dehydration machine/Citadel 2000 Tissue Processor</td>
			<td>Fisher Scientific GmbH, Schwerte, Germany</td>
			<td>12612613</td>
			<td>used for automatic dehydration, short program (approx. 4.8 h)</td>
		</tr>
		<tr>
			<td>Dehydration sponge&#160;</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>TT56.1</td>
			<td>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&#160; 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</td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Phosphat Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Doesn&#180;t contain Calzium or Magnesium, 500 mL</td>
		</tr>
		<tr>
			<td>Embedding cassettes</td>
			<td>Engelbrecht GmbH, Ederm&#252;nde, Germany</td>
			<td>17990</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>MEDITE Medical GmbH, Burgdorf, Germany</td>
			<td>41-6660-00</td>
			<td>staining solution, ready to use</td>
		</tr>
		<tr>
			<td>Fine Scissors CeramaCut</td>
			<td>FST, Heidelberg Germany</td>
			<td>14959-09</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>FST, Heidelberg Germany</td>
			<td>11051-10</td>
			<td>Length: 10 cm, Tip Shape: curved, serrated, Tip width: 0.8 mm, Tip Dimensions: 0.8 x 0.7 mm, Alloy /Material: Stainless Steel</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>MEDITE Medical GmbH, Burgdorf, Germany</td>
			<td>41-5130-00</td>
			<td>staining solution, ready to use</td>
		</tr>
		<tr>
			<td>Highresolotion microscope</td>
			<td>Vision Engineering, Send United Kingdom</td>
			<td>EVO503&#160;</td>
			<td>Capable of enlargement up to 60x magnification, only 6x to 20x magnification were used&#160;</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus Optical CO, Ltd., Hamburg, Germany</td>
			<td>BX60F5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glases</td>
			<td>Marienfeld, Lauda-K&#246;nigshofen, Germany</td>
			<td>101244</td>
			<td>60 mm broad, made of SCHOTT D 263 glass</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>R. Langenbrinck GmbH, Emmendingen, Germany</td>
			<td>03-0060</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica, Nu&#223;loch, Germany</td>
			<td>SM2010R</td>
			<td>Tool for sectioning (2 &#181;m-slices)&#160;</td>
		</tr>
		<tr>
			<td>Omnifix-F 1 mL syringe</td>
			<td>B Braun, Melsungen, Germany</td>
			<td>9161406V</td>
			<td>syringe without canula</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sakura Finetec, Torrance, USA</td>
			<td>4511</td>
			<td>Tissue-Tek Paraffin Wax Tek III, without DMSO</td>
		</tr>
		<tr>
			<td>Paraffin embedding machine</td>
			<td>MEDITE Medical GmbH, Burgdorf, Germany</td>
			<td>TES 99</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Morphisto</td>
			<td>1176201000</td>
			<td>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&#160;</td>
		</tr>
		<tr>
			<td>Small Microsurgical Forceps&#160;</td>
			<td>EPM (Erich Pfitzer Medizintechnik), B&#252;tthard, Bayern, Germany</td>
			<td>(00)165</td>
			<td>Round handle, straight, 0.3 mm tip, tool for observation and every step of preparation, especially useful in final preparation</td>
		</tr>
		<tr>
			<td>Stainless Steel Ruler</td>
			<td>Agntho&#39;s AB, Liding&#246;, Sweden</td>
			<td>30085-15</td>
			<td>150mm With Metric &#38; Inch Graduations</td>
		</tr>
		<tr>
			<td>Surgical Scissors &#8211; Sharp-blunt for decapitation</td>
			<td>FST, Heidelberg Germany</td>
			<td>14001-14</td>
			<td>Device for decapitation</td>
		</tr>
		<tr>
			<td>Warming cabinet</td>
			<td>Haraeus, Hanau, Germany</td>
			<td>T 6060</td>
			<td>the sliced samples should be kept in the warming cabinet to ensure the attachement of the sample on the microscope slides</td>
		</tr>
	</tbody>
</table>

extrahepatic bile duct and gall bladder dissection in nine-day-old mouse neonates

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。

图1A 显示了用所述技术解剖的小鼠新生儿的EBDS。显微镜下，看不到进一步的肝组织。在方案的最后分离步骤中，肝组织已被移除，并且在颜色和稠度方面可以很容易地与胆管组织区分开来。 图1B 显示了分离的样品与毫米尺度的比较。EBD的长度（从胆囊到十二指肠测量）小于10毫米。非常脆弱的胆总管的直径从0.05-0.2毫米不等。 图2</strong...

Watch this Scientific Journal Video about Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates at JoVE.com

Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates

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。

9日龄小鼠新生儿肝外胆管和胆囊清扫术

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation ...

许多评估肝外胆道系统的报告报告说，他们获得了样本，但没有获得样本的方式，这引发了可比性问题。该技术的主要优点是可以解剖整个肝外胆道系统，并且可以无创伤地去除污染细胞。我们的解剖方案通常可以应用于小鼠新生儿胆管疾病的实验方法，旨在解剖肝外胆道系统。该方案的一些特征，例如，将胃附着在十二指肠上，可能有助于十二指肠闭锁的研究，以便通过胃表达的部分消化的乳汁正确定向。一开始，该技术可能需要一些时间进行培训。练习并遵循循序渐进的方法将提高速度和结果。首先，将消毒和高压灭菌的器械放在手术台旁边的无菌表面上。在腹膜上切开后，沿着左额腋线将切口扩大到斩首的位置。用无创伤钳从左侧到右侧去除皮肤。在显微镜下，握住脾脏周围的腹膜。轻轻地将其提起，直到腹膜类似于帐篷状结构，并在中心切一个一毫米的孔。等待腹膜帐篷充满空气。在由下肋骨、腹部外侧区域和下膀胱区域构成的窗口中切掉腹膜。确保完全进入肝脏、胆管系统、胃、小肠和结肠。通过小心地将剑突拉到颅腹位置来检查胆囊和胆管。避免镰状韧带撕裂，这可能导致胆囊从胆管系统撕裂。然后，释放剑突的拉力。轻轻拉下十二指肠以释放胆管系统。然后，通过识别十二指肠进行下部 En-bloc 动员，十二指肠将胆管系统连接到十二指肠。切开十二指肠，向右侧约两厘米处。然后，切开幽门区域。确保胃内容物存在于切割位置和十二指肠之间的幽门区域。要进行上部整体动员，请轻轻拉动剑突并进入镰状韧带。切开胆囊之间的镰状韧带一厘米，靠近剑突。切开肝脏和胸部之间的连接结构，例如食道、下腔静脉、胸主动脉以及围绕肝脏裸露区域的所有韧带。此外，切断所有背侧剩余的组织连接。在20倍显微镜放大倍率下，将整体样品放在泡沫垫上。通过在正确的解剖位置重新组织样本来组装样本。使用轻柔的运动，使十二指肠的口腔和口腔部分变平。使用无创伤镊子，在十二指肠处开始扁平运动，并继续到切削刃。然后，在十二指肠的口腔部分抚平胃的白色浆状内容物。识别十二指肠的口腔和腹侧部分，以排除可能的胆管旋转。在解剖胆管之前切掉大块残留的肝组织。经过几次刮擦动作后，将样品转移到泡沫垫上的清洁位置。使用背景中较少挤压的肝组织来优化EBDS和不需要的细胞之间的区分视图。处理肝十二指肠韧带，直到孤立的EBDS仍然存在。观察肝动脉和门静脉以白色和非常脆弱的细丝形式出现，在十二指肠上口服约三到五毫米。小心地，用轻轻拉动左侧来取下这根细丝。确保刮擦运动从胆管系统开始并通向肝脏边界。使用该协议，从9天大的小鼠新生儿中解剖EBDS。从胆囊到十二指肠的孤立EBDS长度小于10毫米，脆弱胆总管的直径从0.05到0.2毫米不等。苏木精伊红染色在具有开放腔的EBDS的纵向切片上进行。在管腔周围发现胆管细胞为单层，并染得更深。研究人员应共享和标准化解剖和注射方案等操作技术，以提高其研究和结果的可比性和快速可用性。先前的解剖对于组织学和单细胞方法是有利的，例如分离胆管内和胆管外细胞，因为它可以减少细胞培养污染。

Research

JoVE Journal

Medicine

Mouse Bladder Wall Injection

鼠标膀胱壁注射液

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

科研

医学

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

沃顿商学院的导管插管通过小鼠颌下腺唾液腺

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

树脂浇铸和3维成像的小鼠门静脉或肝内胆管系统

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

膀胱胀诱发内脏运动反应为膀胱疼痛小鼠模型

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

State of the Art Cranial Ultrasound Imaging in Neonates

艺术颅超声成像在新生儿的状态

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

胆管结扎小鼠：感应炎性肝损伤和纤维化的阻塞性胆汁淤积

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

通过胰管结扎手术损伤的小鼠胰腺作为模型内分泌和外重新规划和扩散

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

经皮微循环成像在早产儿

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

分离和剖析人力胆汁含外来体的MicroRNA-

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...