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。

1。准备插管<ol><li>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。 </li><li>对角线拉伸管生成一个斜面的顶端。 </li><li>切油管创建的5-6寸段的另一边缘。 </li><li>插入32计，½英寸皮下注射针入非斜面边缘的油管。 </li></ol> 2。注射用PBS做准备<ol><li>填写3毫升的注射器，用PBS。 </li><li>用螺丝将附加的注射器针头注射器上拉，PE10管材事先准备好的。 </li><li>弗里克注射器推注射器的活塞，以确保PBS充满了针管和有没有在注射器中的气泡。 </li><li>与PBS将准备好的注射器，注射器持有人的橡皮泥塑造出。此保持器将注射器保持稳定，而管被插入到所述导管。 </li></ol> 3。其他设置<ol><li>称取0.1克催化剂到一个小的容器， 例如 ，一个24孔板中。 </li><li> 1毫升树脂拉成一个3毫升的注射器。 注：催化剂和树脂的相对量可以调整，以改变固化时间。催化剂可降低量，然而，增加投收缩和导致一个不太理想的铸造。 注：处理树脂时，采取预防措施，避免接触树脂或树脂烟雾吸入的。一定要戴上手套和执行步骤，涉及树脂在通风良好的地方，如化学通风柜 </li><li>切下一块10.0丝线缝合约5英寸用作结扎。 </li></ol> 4。准备好鼠标<ol&gt; <li>牺牲成年小鼠。将鼠标在它的后面，打开腹腔。 </li><li>莱鼠标解剖范围平台。 15毫升锥形管的位置垂直下方的鼠标，以增加可视性和可肝脏。 </li><li>湿的棉签PBS，并用它向上翻转的肝脏，离你暴露的肝脏，与肝外门静脉或胆总管背视图。 </li><li>门静脉管型，可以防止血液凝固与室温下PBS冲洗静脉。将针3毫升注射器充满了PBS和肝外门静脉的肝，你可以插入针。 ，轻轻推PBS通过进入门静脉。让一个缺口排出血液中常见的大是大非静脉。用湿棉签肝按摩，促进血液引流。这一步是不推荐的胆管和，所有血管系统可能没有必要。 注：此步骤也可以射孔rmed代替PBS中，用4％多聚甲醛。 4％多聚甲醛的使用可能会增加血管的完整性和结果在一个更精确的铸铁，特别是较小的血管结构。 </li></ol> 5。导管插入肝外门静脉或胆总管<ol><li>用弯钳通过下面的手术缝合线结扎，约¼英寸的肝门静脉或胆管。 </li><li>扎缝合成一个松散的普通结。不要拧紧结。 </li><li>如果您使用的是一个开放式系统，它可能是有帮助的另一端系统，以配合并拧紧结扎。如果你已经打开了常见的大是大非的门静脉血液从静脉漏，领带结扎，周围增加门静脉内压力，防止树脂泄漏到中央肝静脉。 </li><li>使用春风似剪刀门静脉或胆管，约¼英寸以下的缝合打结，使一小截。这立方米t应该渗透不超过通过门静脉或胆管的直径的一半。 </li><li>使用＃5镊子，将门静脉或胆管的部位切和插入的管子坡口端进入胆管。 </li><li>前端油管推过去预先打结的绑带和对肝脏。 </li><li>注射PBS通过套管和观看，看它是否进入门静脉或胆管插管精度试验。 </li><li>拧紧结扎，插管的地方举行。 </li></ol> 6。树脂浇铸体的门静脉或肝内胆管<ol><li>加入预测1毫升的树脂预测得的0.1 g催化剂，调匀。避免产生气泡。 </li><li>绘制树脂催化剂混合物放回3毫升注射器。删除任何气泡，轻轻弹注射器和排出气泡。 注意：用于铸造小的结构，它可能是有帮助的使用的真空室，除去很小的气泡，从第Ë树脂和增强铸就品质。为了允许的时间来执行此步骤，这将是必要的，以减少从上述建议，以便减缓树脂的固化的催化剂/树脂比。 </li><li>小心地更换PBS含含树脂的注射器，注射器与留下的32号针头连接到所述套管。 </li><li>慢慢地，轻轻一推树脂进入门静脉或胆管，直到阻力增加，肝脏充满。 </li><li>用湿棉签轻轻按摩肝，鼓励甚至填补。 </li><li>删除套管的结构和迅速重新拧紧结扎，防止树脂泄漏。 </li><li>允许，鼠标平放20分钟，在室温下树脂固化，然后取出肝脏。 </li><li>继续清除或浸取的肝脏。 </li></ol> 7。清晰的可视化原位肝<ol><li>修复肝脏在4％多聚甲醛固定过夜，在4℃下，摆动。 </li><li>洗肝在PBS中，在至少四个小时，摆动。在这一点上，如果需要的话，你可以分开的肝叶。 </li><li>至少四个小时每个脱水，在50％甲醇，然后在100％甲醇，在室温下摇动。 </li><li>肝脏清除在1:2的苄醇，苯甲酸苄酯（巴布）溶液中至少24小时，摆动。可能需要较长的洗涤时间。如果肝脏没有完全明确后2-3天，回迁到100％甲醇24小时，和重复巴布结算。 </li><li>淹没在巴布肝脏原位成像的玻璃培养皿。 </li></ol> 8。浸软肝组织<ol><li>删除从小鼠肝脏，并放入50毫升锥形管与水。等待1个小时，完全固化。单独肝叶如果需要。 </li><li>倒掉水，移动肝脏玻璃瓶。淹没在15％KOH。发布于室温下过夜。不要晃动。 </li><li>小心倒掉KOH和，蒸馏水洗铸造的。注：中投版Ÿ脆弱，应特别小心。避免扰乱投当添加或删除从管中的流体。 </li><li>当树脂浇铸体是干净的，轻轻地躺在一个的Kimwipe以干燥，然后转移到一个容器存储。 </li></ol> 9。代表性的成果门静脉或胆管一个成功的演员，将显示一个连续的网络，逐步从肺门延伸到肝脏周围的小树枝。 图1示出了门静脉投作为可视化后在原位巴布结算。 图1A示出了铸造的整个左肝叶的形状和尺寸的铸造， 图1B示出了同样的铸造的特写视图，展示了渗透到最小的门静脉分支在肝脏周围的树脂。 图2示出了已经经历的KOH毫安的门静脉投细齿， 图2A示出了整个左肝叶和图2B是小周分支以特写的方式。 这种技术也被成功地应用于肝内胆管和发布使用巴布结算及KOH浸渍技术1,2,7。 常见的问题，包括不完整的填充，在树脂中，溢出到邻近的系统或组织的气泡。 图3演示如何认识到来自门静脉的一个不完整的填充（ 图3A），气泡（ 图3A），和一个溢出的树脂中央静脉（ 图3B）。 所有图像都使用Leica MZ 16 FA立体镜和QImaging RETIGA 4000R摄像头。 <img alt="图1" src="/files/ftp_upload/4272/4272fig1.jpg" /> 图1巴布清除树脂浇铸体肝左叶肝门静脉。 A.树脂施放示出了从肺门到肝脏周延伸的分层分支结构。树脂浇铸体出现在黄色蓝色调的肝叶内。 B.密切了树脂填充延伸到在肝脏周围的小树枝。通过清除肝树脂是白色的。比例尺= 1毫米。 <img alt="图2" src="/files/ftp_upload/4272/4272fig2.jpg" /> 图2。组织浸渍树脂浇铸体肝左叶肝门静脉。 A.树脂浇铸体的整个肝叶显示，许多大小不等的分支。 B.关闭的小树枝在周围的肝。羽化外观上的小树枝树脂充填的正弦空间，可能会或可能不会显示在树脂蒙上，如果存在明显的分支密度将增加。树脂是白色的。比例尺= 1毫米。 图3。通用与树脂类型转换的问题。 A.一个不完整的填充左叶门静脉部分或全部投没有延伸到肝脏边缘分支是明显的。这个演员还显示气泡，可见在连续铸钢（箭头）休息。 B.由于邻近的门静脉和中央静脉分支在一些地方，的门静脉树脂，经常进入并填写中央静脉。有两种截然不同的肺门分支（箭头）和两个分支的结构，有不同的图案和重叠时，这可以被识别。可以通过两种方式区分技术错误和真正的形态学改变。首先，将铸造结构应该比预期的结构的基础上的2维分析或其他方法。其次，重要的是执行多个复制，并且在可能的情况下，量化的结构尤尔并进行统计分析，以确定的重现性和不同的实验和控制臂之间和之内，如果有一个统计上显着的形态学改变。

<ol>
	<li>Sparks, E. E., Perrien, D. S., Huppert, K. A., Peterson, T. E., Huppert, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defects+in+hepatic+Notch+signaling+result+in+disruption+of+the+communicating+intrahepatic+bile+duct+network+in+mice.">Defects in hepatic Notch signaling result in disruption of the communicating intrahepatic bile duct network in mice.</a> Dis. Model Mech. 4, 359-367 (2011).</li><li>Vanderpool, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+interactions+between+hepatocyte+nuclear+factor-6+and+notch+signaling+regulate+mouse+intrahepatic+bile+duct+development+in+vivo.">Genetic interactions between hepatocyte nuclear factor-6 and notch signaling regulate mouse intrahepatic bile duct development in vivo.</a> Hepatology. 55, 233-243 (2012).</li><li>Meyer, E. P., Ulmann-Schuler, A., Staufenbiel, M., Krucker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+morphology+and+3D+architecture+of+brain+vasculature+in+a+mouse+model+for+Alzheimer's+disease.">Altered morphology and 3D architecture of brain vasculature in a mouse model for Alzheimer's disease.</a> Proceedings of the National Academy of Sciences. 105, 3587-3592 (2008).</li><li>Van Steenkiste, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+corrosion+casting:+analyzing+wall+shear+stress+in+the+portal+vein+and+vascular+abnormalities+in+portal+hypertensive+and+cirrhotic+rodents.">Vascular corrosion casting: analyzing wall shear stress in the portal vein and vascular abnormalities in portal hypertensive and cirrhotic rodents.</a> Lab Invest. 90, 1558-1572 (2010).</li><li>Dickie, R., Cormack, M., Semmler-Behnke, M., Kreyling, W. G., Tsuda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+pulmonary+lymphatics+in+immature+lungs.">Deep pulmonary lymphatics in immature lungs.</a> Journal of Applied Physiology. 107, 859-863 (2009).</li><li>Kelly, D. M., McEntee, G. P., McGeenery, K. F., Fitzpatrick, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvasculature+of+the+pancreas,+liver,+and+kidney+in+cerulein-induced+pancreatitis.">Microvasculature of the pancreas, liver, and kidney in cerulein-induced pancreatitis.</a> Archives of Surgery. 128, 293-295 (1993).</li><li>Sparks, E. E., Huppert, K. A., Brown, M. A., Washington, M. K., Huppert, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+regulates+formation+of+the+three-dimensional+architecture+of+intrahepatic+bile+ducts+in+mice.">Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice.</a> Hepatology. 51, 1391-1400 (2010).</li><li>Carlson, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+expression+of+constitutively+active+Notch4+elicits+reversible+arteriovenous+malformations+in+adult+mice.">Endothelial expression of constitutively active Notch4 elicits reversible arteriovenous malformations in adult mice.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 9884-9889 (2005).</li><li>Hemmeryckx, B., Emmerechts, J., Bovill, E. G., Hoylaerts, M. F., Lijnen, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ageing+on+the+murine+venous+circulation.">Effect of ageing on the murine venous circulation.</a> Histochem. Cell Biol. , (2012).</li><li>Hossler, F. E., Olson, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvasculature+of+the+nasal+salt+gland+of+the+duckling,+Anas+platyrhynchos:+quantitative+responses+to+osmotic+adaptation+and+deadaptation+studied+with+vascular+corrosion+casting.">Microvasculature of the nasal salt gland of the duckling, Anas platyrhynchos: quantitative responses to osmotic adaptation and deadaptation studied with vascular corrosion casting.</a> J. Exp. Zool. 254, 237-247 (1990).</li><li>Hossler, F. E., West, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venous+valve+anatomy+and+morphometry:+studies+on+the+duckling+using+vascular+corrosion+casting.">Venous valve anatomy and morphometry: studies on the duckling using vascular corrosion casting.</a> Am. J. Anat. 181, 425-432 (1988).</li><li>Hossler, F. E., Monson, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+blood+supply+of+intrinsic+lymph+nodes+in+the+wall+of+the+rabbit+urinary+bladder--studies+with+light+microscopy,+electron+microscopy,+and+vascular+corrosion+casting.">Structure and blood supply of intrinsic lymph nodes in the wall of the rabbit urinary bladder--studies with light microscopy, electron microscopy, and vascular corrosion casting.</a> Anat. Rec. 252, 477-484 (1998).</li><li>Hossler, F. E., Monson, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+unique+elastic+sheath+surrounding+the+vesicular+arteries+of+the+rabbit+urinary+bladder--studies+of+the+microvasculature+with+microscopy+and+vascular+corrosion+casting.">Evidence for a unique elastic sheath surrounding the vesicular arteries of the rabbit urinary bladder--studies of the microvasculature with microscopy and vascular corrosion casting.</a> Anat. Rec. 252, 472-476 (1998).</li><li>Hossler, F. E., Kao, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvasculature+of+the+urinary+bladder+of+the+dog:+a+study+using+vascular+corrosion+casting.">Microvasculature of the urinary bladder of the dog: a study using vascular corrosion casting.</a> Microsc. Microanal. 13, 220-227 (2007).</li><li>Wischgoll, T., Choy, J. S., Kassab, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+morphometry+and+branching+angles+of+porcine+coronary+arterial+tree+from+CT+images.">Extraction of morphometry and branching angles of porcine coronary arterial tree from CT images.</a> Am J Physiol. Heart. Circ. Physiol. 297, H1949-H1955 (2009).</li><li>Hossler, F. E., Douglas, J. E., Douglas, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+morphometry+of+myocardial+capillaries+studied+with+vascular+corrosion+casting+and+scanning+electron+microscopy:+a+method+for+rat+heart.">Anatomy and morphometry of myocardial capillaries studied with vascular corrosion casting and scanning electron microscopy: a method for rat heart.</a> Scan Electron Microsc. , 1469-1475 (1986).</li><li>Hossler, F. E., Douglas, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Corrosion+Casting:+Review+of+Advantages+and+Limitations+in+the+Application+of+Some+Simple+Quantitative+Methods.">Vascular Corrosion Casting: Review of Advantages and Limitations in the Application of Some Simple Quantitative Methods.</a> Microsc. Microanal. 7, 253-264 (2001).</li><li>Mondy, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-CT+of+corrosion+casts+for+use+in+the+computer-aided+design+of+microvasculature.">Micro-CT of corrosion casts for use in the computer-aided design of microvasculature.</a> Tissue Eng. Part C Methods. 15, 729-738 (2009).</li><li>Masyuk, T. V., Ritman, E. L., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Assessment+of+the+Rat+Intrahepatic+Biliary+System+by+Three-Dimensional+Reconstruction.">Quantitative Assessment of the Rat Intrahepatic Biliary System by Three-Dimensional Reconstruction.</a> The American Journal of Pathology. 158, 2079-2088 (2001).</li><li>Masyuk, T. V., Ritman, E. L., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+Artery+and+Portal+Vein+Remodeling+in+Rat+Liver:+Vascular+Response+to+Selective+Cholangiocyte+Proliferation.">Hepatic Artery and Portal Vein Remodeling in Rat Liver: Vascular Response to Selective Cholangiocyte Proliferation.</a> The American Journal of Pathology. 162, 1175-1182 (2003).</li></ol>

没有利益冲突的声明。

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...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td> PE10聚乙烯管</td><td> Fisher Scientific则</td><td> 1417012P </td><td></td></tr><tr><td> 5-0手术黑色编织丝</td><td> Roboz手术</td><td> SUT-15-1 </td><td></td></tr><tr><td> Steriject 32 G x 13毫米针</td><td>黑云母空</td><td> TSK3213 </td><td></td></tr><tr><td>春风似剪刀</td><td>精细科学工具</td><td> 15000-08 </td><td></td></tr><tr><td> 5号钳</td><td>精细科学工具</td><td> 91150-20 </td><td></td></tr><tr><td> Mercox II试剂盒</td><td>拉德研究</td><td> 21247 </td><td></td></tr><tr><td>苯甲醇</td><td> Fisher Scientific则</td><td> 1816年至1804年</td><td> ØNLY所需的巴布结算</td></tr><tr><td>苯甲酸苄酯</td><td> MP Biomedicals，LLC </td><td> 154839 </td><td>只巴布结算需要</td></tr><tr><td>磷酸盐缓冲盐水（PBS） </td><td></td><td></td><td></td></tr><tr><td>橡皮泥</td><td></td><td></td><td></td></tr><tr><td>规模</td><td></td><td></td><td></td></tr><tr><td>实验室剪刀</td><td></td><td></td><td></td></tr><tr><td> 15毫升帽聚丙烯管</td><td></td><td></td><td></td></tr><tr><td> 4％paraformaldahyde </td><td></td><td></td><td></td></tr><tr><td> 15％的氢氧化钾（KOH）的</td><td></td><td></td><td>只需要浸渍</td></tr><tr><td>刀片</td><td></td><td></td><td></td></tr><tr><td> 100％甲醇</td><td></td><td></td><td></td></tr><tr><td> 3毫升Luer锁紧接口注射器</td><td></td><td></td><td></td></tr></tbody></table>

3-dimensional resin casting and imaging of mouse portal vein or intrahepatic bile duct system

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。

Watch this Scientific Journal Video about 3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System at JoVE.com

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

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&gt;，系统的完整的通信体系结构的可视化或定量。

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  ...

3-dimensional-resin-casting-imaging-mouse-portal-vein-or-intrahepatic

Research

JoVE Journal

Medicine

17.1K 次观看.  Vanderbilt University. 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

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

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

科研

医学

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

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 as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

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 ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

使用多氟化胆汁酸和<em&gt;在体内</em&gt;磁共振成像来衡量胆汁酸运输

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer that often results in severe short bowel syndrome-like adverse effects including bile acid diarrhea, dehydration, electrolyte disturbances, and nutrient malabsorption. Here we introduce a murine ileal resection model, termed ileectomy, to evaluate tissue communication and the maintenance of systemic homeostasis. After ileal resection, circulating blood is permanently devoid of the ileum-specific endocrine hormone fibroblast growth factor 15 (FGF15), which releases its endocrinal inhibition of bile acid synthesis in the liver. In combination with the increased production and abolished reabsorption of bile acids after removing the ileum, mice that underwent surgery suffer from bile salt overaccumulation in the intestine and associated diarrhea, morbidity, and mortality. Novel usage of the surgery model introduced in this study may provide mechanistic and functional insights into ileal control of systemic metabolic regulation in physiology and disease.

Small intestine-dependent bile acid reabsorption and feedback inhibition of hepatic bile acid synthesis is important for systemic homeostasis and health. In this study, we describe a mouse model for ileal resection to evaluate ileectomy-induced bile malabsorption, overaccumulation, and toxicity in mouse intestine.

回肠致胆汁积滞在小鼠肠道中

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

肝分型与门静脉结扎大鼠分期肝切除 (ALPPS) 手术模型的建立

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

激光多普勒灌注成像在鼠标欣德利姆

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

通过胆管 TNBS 输液治疗慢性胰腺炎的鼠标模型

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

尽量减少小鼠肝内胰岛移植期间输注后门静脉出血

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

DUCT：双树脂铸造，然后进行微型计算机断层扫描，用于3D肝脏分析

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

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

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation of the extrahepatic bile duct (EBD) in mouse neonates without damaging the bile duct during preparation. Because of its exceptionally close preparation compared to the cholangiocytes cell line and harvesting of the entire extrahepatic bile duct system (EBDS), the described approach is extremely useful in researching animal models of newborn bile duct disorders, such as biliary atresia. After euthanasia, the peritoneal cavity was accessed, and the bile duct system, duodenum, and liver were extracted with the unique En-bloc-Resection (EbR). The extracted sample is placed on a foam mat, and the EBD is dissected from contaminating cells atraumatically without necessary touch. The dissection of the entire EBDS is a significant advantage of this method. Caution must be taken due to the small size and amount of bile duct tissue. Using the described technique, there is no damage to the cholangiocytes. Further, the purity of the technique is reproducible (n = 10). Therefore, optimally comparable samples can be harvested. Furthermore, no bile duct tissue is harmed, because any contact with the bile duct system can be avoided during preparation, leaving the bile fluid inside the gall bladder. Most importantly, while performing the final gall bladder and bile duct dissection, atraumatic microinstruments were used only slightly lateral of the bile duct without squeezing it. This is the key to a clean and intact sample, and essential for further histological investigation or the isolation of cholangiocytes. To summarize, the described innovative dissection technique enables especially inexperienced operators with the necessary equipment to isolate the EBDS as cleanly as possible.

For the observation of murine neonatal bile duct disorders, an intact bile duct and efficient preparation are required. Therefore, a new approach for isolating the entire extrahepatic bile duct system in murine neonates was successfully developed while maintaining the integrity of the bile duct.

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 ...