Funding from the Australian Research Council (ARC) and National Health and Medical Research Council (NHMRC) is gratefully acknowledged.

在这个手稿中描述的研究，批准由当地动物伦理委员会，并按照澳大利亚和新西兰会动物的护理研究与教学的指导方针进行的。在此之前开始的任何动物的程序，确保适当的权限是通过当地的机构/组织获得。与所有的动物外科手术，确保手术是经过适当培训的操作人员进行的，在无菌条件和麻醉剂，止痛药和抗生素时，必须确保道德，并取得圆满成功的管理。 1.准备前一天的手术过程<ol><li>如果需要手术的前一天晚上快的老鼠。确保鼠有自由饮水。 </li><li>制备的溶液，以施用给大鼠。 <ol><li>制备补液溶液例如无菌盐水或林格溶液。 </li><li>准备作为审美要求的解决方案。在这里所描述的实验中使用的麻醉剂包括"鸡尾酒1"的制备通过组合1.9毫升氯胺酮100毫克/毫升，0.5毫升甲苯噻嗪100毫克/毫升，0.2的乙酰丙嗪10毫克/毫升毫升和2.5ml盐水和"鸡尾酒2"由1毫升氯胺酮100毫克/毫升和0.1ml乙酰丙嗪10毫克/毫升。注:吸入异氟烷或七氟醚可以是优选，因为它可以为更长的时间期间，提供麻醉的手术平面。 </li><li>任选制备含有药物的制剂中，例如，脂质乳剂。执行适当的稳定性研究，以保证制剂的稳定性在储存和给药期。 注:该制剂和药物的确切性质待施用将随着每次研究淋巴药物转运研究的目的是最经常以评价药物的淋巴运输的效率作为制剂和药物ADMINI的函数stered。 <ol><li>用在实验中的代表图4和5此处所述制剂被制备，如先前描述的43。简言之，将包含14 C-油酸（2-5微居里）和卤泛群（200微克）到40毫克油酸之前分散在5.6组成为5mM牛磺胆酸钠在磷酸盐缓冲盐水（pH值6.9）的水相的溶液中。 </li><li>对于含有2-油酸单甘油酯的配方，加入7.1毫克2-油酸单甘油酯连同其它部件，以水相的同时作为油酸含卤泛群酸相。制备含有2-油酸单甘油酯前立即施用到老鼠2-油酸单甘油酯是相对不稳定的，isomerises〜1-油酸单甘油酯的配方。 </li><li>接着乳化该制剂通过超声在室温2分钟。 注:由于先前已描述43，监视制剂用i的稳定性）乳状的外观检查在偏振光下传送给确保乳液不相分离，ⅱ）粒度分析以下使用动态光散射以提供任何相变和/或分离的指示给药制剂后即刻和，以及iii）用卤泛群浓度的分析经过验证的HPLC测定。 </li></ol></li></ol></li><li>制备在无菌水中含10mg抗凝血剂溶液/ ml的EDTA或10国际单位/毫升肝素的生理盐水冲洗淋巴插管。还制备含有2.5抗凝血剂溶液 - 10国际单位/毫升肝素的生理盐水冲洗颈动脉插管。 </li><li>制备的聚乙烯插管，用于插入肠系膜淋巴管（0.8毫米外径，0.5毫米内径），颈动脉（0.96毫米外径，0.58毫米内径0.8毫米外径，0.5毫米内径）和十二指肠（0.96毫米外径，0.58毫米内径） <ol><li>切开插管到所需的长度（通常为25 - 30厘米为颈动脉和十二指肠插管和10 - 24厘米为淋巴卡恩ULA），并放置一斜面在用无菌手术刮刀将插管的尖端（参见图1）。 注意:这增加了便于插管插入并减少套管堵塞后插入的发生率。 </li><li>通过循环插管的小部分回到它自身上，用打火机或热板加热，然后切割成尺寸放置在十二指肠内插管J形锚点（ 见图1）。 </li></ol></li><li>附加淋巴插管将25g针头和注射器含有抗凝血剂溶液（在步骤1.3中制备）。填充淋巴插管用抗凝血剂溶液中并保留在插管澳溶液/ N，减少淋巴收集期间凝块形成在淋巴插管的发生率。 </li><li>制备管淋巴和血液样品收集。预称量淋巴收集管，标号管淋巴和血液样品的收集和添加抗凝血剂溶液中。添加足够的抗凝血剂SOLUT离子达到1毫克/毫升EDTA的无菌水的最终浓度，或10 - 20国际单位/毫升肝素的收集淋巴或血液。 </li></ol> 2.准备之前立即开始手术过程<ol><li>检查所有的插管通过用无菌盐水或抗凝血剂溶液冲洗，以确保它们是专利。 </li><li>制备大鼠的手术过程。 注:肠系膜淋巴管是在吃了或已给予的油剂量为肠系膜淋巴结变成乳白色不透明具有较高的脂质负荷大鼠更为明显。一些运营商，特别是那些新的外科手术，因此可以更容易导管插入肠系膜淋巴管时大鼠预先服用一种油（大约0.1 - 1毫升）中，如橄榄油或豆油0.5 - 2小时之前开始手术。然而，给药前油影响脂质，亲脂性药物和其他因素在淋巴结的淋巴运输，使得它可能不是很理想，以预剂量油根据实验的目的。 <ol><li>整个麻醉手术和样本收集期的老鼠。 <ol><li>例如，通过皮下注射1.5毫升/千克鸡尾酒的启动麻醉朕（从步骤1.2.2），使用1ml注射器附连到地下25针插入皮肤褶皱在大鼠颈背部。保持经由腹膜内注射0.44毫升/约根据需要，使用1ml注射器附连到地下25针每小时公斤鸡尾酒2（来自步骤1.2.2）的麻醉。 </li><li>之前开始的外科手术确保麻醉深度足以通过观察呼吸速率，晶须运动，肌肉张力和响应刺激如捏脚。根据需要管理更多的麻醉。 </li></ol></li><li>剃从手术区域，这包括右侧腹部淋巴结和十二指肠插管的毛皮，以及颈部和左锁骨区域为颈动脉插管化。 </li><li>清洁无菌使用povidine碘溶液或洗必泰溶液和70％的乙醇擦洗手术区域。重复此操作3次对每个区域，用70％乙醇的最终清洗整理。 </li></ol></li><li>将背recumbence动物在丢球一个以上的手术加热垫（37°C）。 </li></ol> 3.插管的肠系膜淋巴管<ol><li>将动物用其右侧面朝向操作者。具有或不具有根据需要手术显微镜的帮助下进行手术。 </li><li>从中线（xyphoid处理）用无菌解剖刀刀片的直4厘米切口延伸到右侧约2厘米以下的肋骨（肋缘）打开腹部肌肉壁的顶层（ 见图2B）。 </li><li>从4打开腹部肌壁的其余层 - 5毫米外侧中线的右侧有一个小的对手术剪（ 见图2B）。 </li><li>收回小肠下，左腹部肌肉壁，并保持它在使用2个地方 - 3个无菌纱布，用生理盐水饱和。 </li><li>弥合老鼠在10毫升的塑料注射器在老鼠背上的右肾来缓解肠系膜淋巴管可视化水平横放。 </li><li>定位肠系膜淋巴管:一个容器大约0.5 - 1毫米直径的垂直于右肾和立即延髓并平行于肠系膜动脉（脉动暗红色血管;参见图2C）。 注意:在非禁食或油预先给药的大鼠的血管是白色的，不透明的，更容易形象化比在禁食大鼠中它是相当透明的。 </li><li>检查区域紧接尾向大肠系膜淋巴管和肠系膜动脉，以确定是否一个第二，较小的附件淋巴管是目前。如果存在的话，方框淋巴液通过导管的流动通过切割导管和强力胶阻塞，或捆扎缝线周围的风道，以确保淋巴从肠流出的整个体积被从肠系膜淋巴管收集。 </li><li>通过肾周脂肪床通过一对直镊子在右肾的下缘，并通过结缔组织层紧接腔静脉的下方，在与肠系膜淋巴管平行的方向。 </li><li>使用镊子的顶端握住淋巴插管并通过肾周脂肪床一端紧邻肠系膜淋巴管，而另一个从动物exteriorised在右肾的水平拉出。 </li><li>由钝性分离覆结缔组织和脂肪组织层隔离肠系膜淋巴管。小心不要刺破或损坏的淋巴管。 </li><li>隔离淋巴管后，让小何乐在淋巴管有两种微型剪刀，珠宝商的镊子尖端或将25g针头。小心不要完全切断血管。 </li><li>确保淋巴插管完全充满抗凝血剂溶液（使用注射器和25g针），没有空气间隙。通过小孔用小钳子的帮助4毫米肠系膜淋巴管内 - 将淋巴导管大约2。 </li><li>观察淋巴插管几分钟。观察肠淋巴结的从套管的自由端收集的逐渐流如果插管是成功的。如果插管不成功，重复步骤3.8 - 3.12。 </li><li>如果插管是成功的，通过将一小滴兽医粘合剂的比入口孔进入淋巴管固定套管。小心不要通过多余的兽医胶粘剂的应用堵塞血管。 </li><li>虽然等待兽医粘合剂设置，安装前后已经淋巴液几分钟的流动，以确保所述的插管过程是成功的。一旦某些插管的成功，取出纱布枚被小心地放置在腹腔，并把小肠在其原来的位置。 </li><li>放置含有抗凝血剂淋巴收集管（参见步骤1.6）在所述套管的端部，以收集自由流动的淋巴。 </li><li>接着，导管插入十二指肠输注补液溶液和/或制剂。 </li></ol> 4.插管十二指肠<ol><li>附上十二指肠插管到注射器（ 例如，10ml）中填充有补液溶液。 </li><li>确定十二指肠的亮粉色（有更多的血管）小肠部分，它在温和的向下拉动揭示了胃。 </li><li>通过使约2厘米以下的胃和十二指肠（幽门）的交界处十二指肠小穿刺孔无菌23政针。 </li><li>将通过穿刺孔十二指肠插管的歼形钩端。安全的地方用一滴氰基丙烯酸酯胶。 </li><li>通过将插管在输注泵中装入的补液注射器开始的大鼠的水合作用。据文献报道补液率从0.5 - 3毫升/小时15,25。 </li><li>淋巴和十二指肠插管完成后，关闭该切口在腹部肌肉壁与缝合。然后通过施加几滴组织粘合剂（腈基丙烯酸酯）的沿所述切口的侧和夹持在切口两侧的皮肤一起关闭皮肤切口。 </li></ol> 5.插管的颈动脉颈动脉插管可能之前或肠系膜淋巴管的插管之后进行。一些运营商宁愿导管插入颈动脉cannulating的淋巴管，以便减少婆之前移动的动物时，tentially撞出淋巴导管。 <ol><li>放置一个标记上的颈动脉插管从该斜面尖端2.5厘米识别管道的所述部分插入到动脉中。连接套管（ 即没有斜角）的另一端，以附着在填充有抗凝血剂溶液的注射器23或地下25针和填充套管与抗凝血剂溶液，确保没有空气间隙存在。 </li><li>为了便于插管，放置在背卧麻醉大鼠伸展颈部和头部指向操作人员。请注意，一些运营商更喜欢用老鼠的尾巴指向操作者执行该技术。 </li><li>通过皮肤层在矢状平面中的气管的左侧上方1.5厘米纵向切口 - 放置1。 </li><li>解剖使用钝头镊子以暴露底层配对颈部肌肉皮下结缔组织。 </li><li>收回颈部肌肉吨Ó揭示左颈动脉（位于约1厘米以下的皮肤表面和脉冲）。清理从动脉钝性分离上覆结缔组织。 </li><li>仔细分离使用钝组织钳，注意不要损坏相邻的迷走神经的轨道动脉的约1cm部分。通过打开和关闭所述钝头镊子垂直沿动脉的任一侧，以从周围组织和迷走神经释放它执行此。 </li><li>用细尖镊子，螺纹2丝线缝合颈动脉下方，并将其放置在分离的动脉部的每一端。摘下领带最接近的缝合操作和最远从大鼠的心脏和锁骨（ 图3A）。 </li><li>通过将一对直的细尖镊子的动脉（ 图3B）下方闭塞通过动脉的血流。 </li><li>使用细尖虹膜剪，放置一个小切口上的动脉（约1/3的顶面方式向下从分离区的顶部）。刷新任何泻血用肝素盐水动脉表面上，并用棉花提示轻轻擦拭。 </li><li>插入插管的尖端插入有一对弯曲的尖镊子的动脉。一旦插入，删除该直细尖镊子阻断血流，推进套管2.5厘米成使用两对镊子的一对保持动脉内插管动脉阻止血液泄漏背面，另一插入插管。 </li><li>用小动脉夹来保存动脉内插管，并删除被放置动脉以下步骤5.8闭塞血流直细尖镊子。 </li><li>通过注射抗凝血剂溶液进入插管和拉伸背部少量血液（ 图3C）证实插管的通畅。随后把抗凝溶液插管并使用的打火机的火焰或套管插头密封端。 </li><li>配合日Ë插管到位与两个缝合步骤5.8动脉之下。 </li><li>取出动脉夹，固定动脉插管，并围绕第三缝合。关闭伤口缝合带在脖子上。 </li></ol> 6.手术后期间和输液制剂<ol><li>继续保持在麻醉下和在加热垫的大鼠。 </li><li>补充水分经由输注补液溶液大鼠进入十二指肠为至少0.5小时以下的插管的完成。观察淋巴流率减少（〜0.1 - 以0.5ml /小时）在初始阶段之后插管并很快增加到一稳定的基线（0.4 - 2.5毫升/小时取决于复水率）。 </li><li>以下恢复期，注入感兴趣的制剂（含有脂质，药物等 ）插入通过套管十二指肠。任选地，用一个输注泵来调节制剂的流速。注:在这里描述的协议动物保持anaesthetised整个手术和淋巴收集周期和麻醉下，使得不需要额外的镇痛实施安乐死。如果该协议被修改和动物启用恢复知觉，然后适当的镇痛和术后护理是必需的。 </li><li>在完成制定输液继续补充水分大鼠在1.5-3毫升/小时用生理盐水率进入十二指肠，以防止脱水。 </li><li>通过继续向下轻柔按压，以保持大鼠在37˚C加热手术垫，以保持温度（如每步2.3），明确尿液从膀胱放在小腹的要求，并重新眼药膏每隔一小时（如每步2.2。 3）。常规体位改变建议，如果动物是在手术后恢复意识。 </li></ol> 7.集合淋巴和血液样本，以评估脂质和药物吸收<ol><li>收集淋巴不断进入管含有抗凝血剂。在规定的时间间隔改变管（ 如每小时）。 </li><li>采集血液样本在设定的时间点。 <ol><li>通过弯曲套管后面约2厘米的密封端阻塞通过套管的血流。启封插管通过切断密封端或移除套管插头。 </li><li>用空的注射器，收回肝素盐水从套管，直到血液充满整个插管。 </li><li>采用了全新的空的注射器，收回血和地点所需的体积含有抗凝血剂的管子。 </li><li>使用第一注射器，更换采样之前所采取的血液，以减少不必要的血液损失。注射前，轻弹注射器，同时保持直立，以确保无气泡进入插管。 </li><li>使用注射器，取代的血液从与抗凝血剂溶液中的鼠取出的体积。确保无残留的血液是可见的插管，任何剩余的可凝结并阻止插管。 </li><li>弯曲插管从所述未密封端约2cm到方框血流并卸下注射器。使用的打火机的火焰或套管插头，重新密封的套管。 </li></ol></li><li>在完成血液和淋巴收集从大鼠，经由&gt; 100毫克/公斤戊巴比妥钠腹膜内给药安乐死大鼠。 </li><li>通过测量淋巴在每个时间段收集的质量确定淋巴流速。 </li><li>测量药物和脂质的浓度淋巴和血液使用中，例如，高效液相色谱法，HPLC-MS，或者使用市售试剂盒，以评估脂质和药物的吸收效率。 </li><li>计算药物和脂质在淋巴质量传输从收集到的测得的浓度和淋巴的容积的乘积。 </li></ol>

The authors have nothing to disclose.

大鼠肠系膜淋巴结插管模型使浓度和各种细胞和分子（例如脂质和药物）从肠进入淋巴和修改这些发生在响应的传输速率的直接量化挑战各种物质（饮食，抗原，药物，制剂等 ）10,27和疾病（癌症，病毒，大肠炎，胰岛素抗性等 ）5-7。收集在淋巴结的组件也可被进一步用于另外的实验。例如，细胞可以是培养的44，脂蛋白分馏和淋巴或它的组分，例如脂蛋白?...

从小肠通过一个单向过程，起源于所包含的每个小肠绒毛1内单乳糜淋巴流动。乳糜是相对可渗透的流体，大分子和细胞和淋巴形成从而开始与这些因素进入乳糜。在乳糜初始淋巴随后从肠经由淋巴微血管网络，收集（传入）淋巴管中，一系列的肠系膜淋巴结，并最终在后节点（传出）淋巴管流动。中的节点，淋巴通过一系列髓窦的其中交换发生在节点驻留的免疫细胞，以及材料进入从血液中的节点传递。所有淋巴从小肠流入最终收敛到传出肠系膜淋巴管，随后将乳糜池。该乳糜池还收集淋巴引流尾外周组织，INTEST伊纳勒，肝和腰部区域和一起加入胸淋巴导管与来自纵隔和身体的颅部位淋巴结。胸淋巴导管直接排空淋巴液进入静脉系统在左侧颈内静脉和锁骨下静脉的交界处。这里所描述的协议，使淋巴收集直接从肠系膜淋巴管，从而便于直接从通过肠道淋巴系统肠道全身（普通）循环过渡的各种因素进行分析。 分配到肠淋巴系统的主要生理功能是维持体液平衡，以促进脂质和亲脂性分子的吸收，以及使适当的免疫应答1。肿瘤细胞和病毒也可通过淋巴管肠道淋巴管内发生的几种炎症和代谢病症5-7 2-4，关键的变化传播。可以的肠系膜淋巴管nulation收集淋巴结内肠系膜使得能够经由肠淋巴散装流体流动分析的各种细胞和分子的浓度和输送速率，以及量化。改变浓度或这些因素的过境响应于各种挑战（ 例如，饮食，抗原，药物）和在疾病模型（ 例如，结肠炎，艾滋病，糖尿病）也可以评估。而这是不可能广泛描述其可以被分析和比较，这里的每个淋巴成分，肠系膜淋巴结简单化由含水，类脂和细胞相。的在水相中的兴趣的组件包括肽和蛋白质，例如抗原或耐受原8，免疫信使如细胞因子和肥大细胞介质9和代谢介质如肠降血糖素10。后节点肠系膜淋巴结的细胞部分包含lymphocyt的几乎完全（99％以上）上课11。各种免疫细胞（树突状细胞，肥大细胞等 ）进入预结肠系膜淋巴管，但仍然在节点12内。如果内输入淋巴细胞是感兴趣的，则可以通过去除肠系膜淋巴结的收集这些细胞中的肠系膜淋巴管12的插管前节点几天。以这种方式，传入和传出淋巴管直接连接和淋巴细胞传入淋巴直接传递到肠系膜淋巴管。各种免疫细胞通过淋巴管肠道的运输和表型可以这样进行检查。或许引用于收集肠系膜淋巴结迄今为止最常见的原因，但是，是研究肠处理，吸收和膳食脂质和亲油性分子10的运输。 以下摄取，膳食脂质被消化（例如，从甘油三酯脂肪酸和单甘油酯，phospholipid到脂肪酸和溶血磷脂，和胆固醇酯脂肪酸和胆固醇等 ），并经由加法从胆汁两亲物的肠腔成小胶束和囊泡结构中分散（磷脂，胆固醇和胆汁盐）和作用胰腺酶10,13。从这里，他们被吸收到肠。被吸收的组分的比例被重新酯化，以形成吸收细胞（肠）内的甘油三酯，磷脂和胆固醇酯。这些再酯化脂肪是由摄入外源性脂质成分和内源性脂质成分的自分泌胆汁，粘膜脂质池或肠道血液供应13的组合装配。从这里的酯化脂质要么存储肠内或组装成肠脂蛋白（乳糜微粒，极低密度脂蛋白（VLDL））连同各种脱辅基蛋白和其它亲脂性分子（ <eM&gt;如维生素）10,13。退出后肠，脂蛋白是专门从肠道通过肠系膜淋巴系统运输到全身循环作为肠乳糜更可渗透其比肠毛细血管条目。吸收的脂质组分的比例还通过毛细血管和门静脉作为单独的，非相关的脂蛋白运送从肠道到体循环，分子14。在一般情况下，然而，门静脉输送路径是唯一的一个显著球员的短链和中链长度的脂质吸收。 肠系膜淋巴结的收集由此使从肠脂蛋白和相关的部件（脂质，亲脂性分子，脱辅基蛋白）的传输的评估。脂蛋白可定量和表征的优点，即肠系膜淋巴结脂蛋白，在一般情况下，是在一个nascen吨状态，因为它们没有被广泛地通过全身酶改性，如脂蛋白脂肪酶15。而肠系膜淋巴结插管的大鼠模型已经也许历来最广泛的脂质/脂蛋白运输从肠道的分析描述的，扩大的兴趣的区域是淋巴管中的亲脂性药物，前药和其他外源物13,16的传输中的作用这是在这里描述的模型的焦点。亲脂性药物（一般是那些与log P&gt; 5和溶解度在长链甘油三酯&gt; 50毫克/克，尽管异常是明显的）17,18，前药19和其他外源物13,16可以访问肠淋巴或者被动地或通过积极融入肠道脂蛋白运输通路19。 大鼠肠系膜淋巴结插管技术因此有许多应用。博尔曼等人首先描述一个techniqUE到导管插入大鼠的肠系膜淋巴管在1948年20，从此一数量的变型的模型已经描述。例如，收集可在昏迷状态，同时抑制15或自由活动23,24发生时，老鼠被麻醉的各种麻醉剂21,22，或。大鼠可以施用不同补液溶液和其他物质如脂质和药物制剂以不同的速率进入胃，肠或肠胃外（一般0 -为5毫升/小时）25。在一些研究中胸淋巴导管，而不是肠系膜淋巴管插管经由淋巴管来估计传输从肠道尽管这可能高估中转从小肠，取决于感兴趣的因素，因为胸淋巴导管还接收来自其它淋巴区22,26。淋巴插管车型也已经在其他几个物种，包括小鼠15,27，迷你PI描述GS 12，28,29羊，猪30和狗31。然而，在大鼠模型中是最广泛和一致引用。对肠系膜淋巴管随后采集淋巴液中有意识25或麻醉22大鼠和小鼠15,27的插管的详细方案先前已公布的和有兴趣的读者是针对这些协议。这个协议是第一次证明该技术在一个可视化格式。 淋巴空心大鼠模型拥有较大的动物模型的优势在费用方面，易用性的手术和伦理方面的考虑。相比，小鼠模型中，肠系膜淋巴结插管手术也是在大鼠容易虽然小鼠模型能够在转基因动物27更详细的研究。尽管如此，也有在大鼠模型中，特别是那些在生理差异有关，该限extrapolat的一些局限离子对其他临床前和临床情况。例如，在大鼠胆汁流量是恒定的，独立的食物摄取，而在更高的物种的食物或脂质刺激胆汁流32。这产生挑战获得代表前和餐后环境在大鼠反映了什么被认为是在较大的品种和人类。用于药物递送的研究，大的物质也可以评估淋巴运输时后的现实人类剂量的给药形式25优选的。在最近的研究中，在肠系膜淋巴结脂质转运率被发现是跨越一个等效质量和脂质的类型，它提供了一些信心跨物种27外插脂质转运数据给药后物种（小鼠，大鼠，狗）媲美。然而，模型的亲脂性药物，卤泛群运输，动物的大小（ 即狗&gt;大鼠&gt;小鼠）的顺序排名。比例因子可能因此被要求前trapolate大鼠淋巴药物传送数据到其他物种。 的淋巴插管模型的限制，一般来说，是直接被动淋巴收集从淋巴管可以修改淋巴流动和传输自淋巴管的工作对一个压力梯度被改变，一旦容器被插入套管33。淋巴插管模型也可以是难以建立在实验室中是不熟悉的技术。替代模式进行了说明。举例来说，经由肠道淋巴系统的因素，如脂蛋白和亲脂性分子的转运，已经间接通过采集血液的研究。一个这样的模型涉及口服给药后，在存在和不存在抑制剂的比较脂质和/或药物的血药浓度（ 例如，秋水仙碱，普朗尼克L81，放线菌酮）肠脂蛋白生产阻断淋巴运输34。优势的经由采集血样 ​​量化淋巴运输间接模型是，它使淋巴运输的一些评估在人中作为侵入性手术，不需要35。然而，淋巴运输的抑制剂不特定且经由淋巴管输送因子稀释并修改在全身循环变得复杂这样的评估， 在体外替代物也被描述。例如，Caco-2细胞或分离的肠细胞培养物已被用于更详细地研究该进入淋巴管36-38分子的肠分泌。一种先进的体外模型是比较有代表性的人体肠道微环境也在最近描述39。在此模型中的淋巴管内皮细胞层是共培养Caco-2细胞使物质转移从肠进入淋巴管的更详细的分析。然而， 在VITRO单元系统缺乏交流流动和转移与肠腔和潜在血液和淋巴血管供应，即互联。在另一种方法，Kassis 等人建立了原位成像系统，使血管收缩，淋巴流动和肠系膜淋巴管荧光灯33血脂浓度之间的定量比较，双通道（高速明场视频和荧光）。该模型在上述的体外系统的一个优点是，它使免疫细胞通过淋巴管的通道的准确的跟踪。质量的脂质（或药物）的运输的绝对测量，但是，尚未建立使用的成像方法， 在体外和在硅片接近经由肠淋巴也已出版40-42特异性预测脂溶性药物输送的程度。例如， 离体数的c亲和力ompounds血浆乳糜微粒被发现关联相当好与它们在体内的41淋巴运输。接着，在同一组，在硅片模型建立一个预测药物亲和性基于多个物理化学性质40乳糜微粒。冬青等人还建立了在计算机芯片模型相对复杂，以彻底预测分子描述符42的基础上，亲脂性化合物的淋巴运输。这些模型可提供预测未知药物淋巴运输的程度上有用的方法。与广泛的药物和不同的实验室模型的验证将然而，需要确认其准确性和可重复性。 的肠系膜淋巴管插管因而仍然是唯一的手段来直接检查淋巴引流小肠和的因子（细胞，蛋白质的复数排列的传输速率的内容，肽在体内的情况淋巴液，脂类，药物）。这里我们描述了一个协议，用于在肠系膜淋巴管和颈动脉，使肠系膜淋巴结和从麻醉大鼠的全身血液的收集的插管。代表性的数据说明了如何在模型可用于通过肠系膜淋巴系统以检查脂质和药物输送从肠道。这之后，可以在建立模型和故障排除指南会遇到一个困难的讨论。一旦建立了模型是一个强大的工具，调查肠道淋巴运输。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Sterile saline</td>
			<td>Baxter healthcare</td>
			<td>AHB 1307</td>
			<td>Any brand can be used. Example here is Baxter 100 ml saline bags, box of 50</td>
		</tr>
		<tr >
			<td >70 % ethanol in water</td>
			<td>Any</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Chlorhexidine gluconate solution (Microshield 4)</td>
			<td>Livingstone International</td>
			<td>JJ60243L</td>
			<td>Any brand can be used. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=JJ60243L</td>
		</tr>
		<tr >
			<td >Betadine solution</td>
			<td>Livingstone International</td>
			<td>BU0510</td>
			<td>Any brand can be used. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=BU0510</td>
		</tr>
		<tr >
			<td >Ilium Ketamil (Ketamine 100 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >KETA I 1</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >Ilium Xylazil (Xylazine 100 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >TRO-3828</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >ACP 10 Injection (Acepromazine 10 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >VTG-DACP010020</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >Sodium pentobarbitone</td>
			<td>PROVET VICTORIA&#160;</td>
			<td>24529</td>
			<td>Any brand can be used. Example here is Lethabarb&#174; 325 mg/ml sodium pentobarbitone, Virbac Animal Health. http://www.provet.com.au</td>
		</tr>
		<tr >
			<td >Heparin (35000I.U. in 35 mL)</td>
			<td>Sigma Pharmaceuticals</td>
			<td>337220</td>
			<td>http://sigmaco.com.au/</td>
		</tr>
		<tr >
			<td >Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate</td>
			<td >Sigma-Aldrich</td>
			<td >E1644</td>
			<td>Any brand can be used. Example here is disodium salt of EDTA from Sigma.&#160;</td>
		</tr>
		<tr >
			<td >Polyethylene (PE) cannula o.d. 0.96 mm x i.d. 0.58 mm</td>
			<td>Microtube extensions</td>
			<td>PE8050</td>
			<td>Any brand can be used. Example here is PE tubing 0.8x0.5 mm, 30 m</td>
		</tr>
		<tr >
			<td >Polyethylene (PE) cannula o.d. 0.8 mm x i.d. 0.5 mm</td>
			<td>Microtube extensions</td>
			<td>PE9658</td>
			<td>Any brand can be used. Example here is PE tubing 0.96x0.58 mm, 30 m</td>
		</tr>
		<tr >
			<td >Ruler</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Markers</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Cigarette lighter</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Cyanoacrylate glue</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >23 gauge needles</td>
			<td>Livingstone International</td>
			<td>DN23GX0.75LV</td>
			<td>Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 23GX0.75inch, 100/BOX. http://www.livingstone.com.au/?PG=search_result&#38;CAT= 
			6&#38;search=DN23GX0.75LV</td>
		</tr>
		<tr >
			<td >25 gauge needles</td>
			<td>Livingstone International</td>
			<td>DN25GX1.0LV</td>
			<td>Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 25GX1.0inch, 100/BOX. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search= 
			DN25GX1.0LV</td>
		</tr>
		<tr >
			<td >1 ml syringe</td>
			<td>Livingstone International</td>
			<td>T3SS01TA</td>
			<td>Any brand can be used. Example here is Terumo syringe 1 ml Slip Tuberculin 100/Box. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=T3SS01TA</td>
		</tr>
		<tr >
			<td >10 ml syringe</td>
			<td>Livingstone International</td>
			<td>T3SS10SA</td>
			<td>Any brand can be used. Example here is Terumo syringe 10 ml Slip 100/Box. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=T3SS10SA</td>
		</tr>
		<tr >
			<td >Gauze swabs</td>
			<td>Livingstone International</td>
			<td>GSC075</td>
			<td>Any brand can be used and cut to required size. Example here is gauze swabs cotton filled 7.5x7.5 cm, 8 ply. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=GSC075</td>
		</tr>
		<tr >
			<td >Cotton buds</td>
			<td>Livingstone International</td>
			<td>CTAST075DP</td>
			<td>Any brand can be used. Example here is Livingstone cotton applicator plastic double tipped. 75MM. 100/PK. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=CTAST075DP</td>
		</tr>
		<tr >
			<td >Heating pad</td>
			<td>Ratek</td>
			<td>WT1</td>
			<td>Any brand that keeps temperature at 37C can be used. Example here is Ratek warming tray.</td>
		</tr>
		<tr >
			<td >Surgical light</td>
			<td>Harvard Apparatus</td>
			<td>72-0215 with 72-0267</td>
			<td>Any brand can be used. Example here is Harvard apparatus V-Lux 1000 Cold Light Source with Bifurcated Gooseneck Light Guide, Black, 4.7 mm fiber diameter (each arm). http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_50601_ 
			-1_HAI_ProductDetail and&#160; http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_35487_ 
			-1_HAI_ProductDetail___</td>
		</tr>
		<tr >
			<td >Surgical microscope</td>
			<td>Zeiss</td>
			<td>495005-0014-000</td>
			<td>Any brand can be used. Example here is Zeiss Stereomicroscope Stemi 2000-C with Stand S Double Spot and KL 300 LED. https://www.micro-shop.zeiss.com/?l=en&#38;p=us&#38;f=e&#38;i=10143</td>
		</tr>
		<tr >
			<td >Silk suture</td>
			<td>Livingstone International</td>
			<td>DTSK163019F4</td>
			<td>Any brand can be used. Example here is *&#160; 
			Email this item to my friend 
			3/8 Circle Reverse Cut Silk Suture 3/0 Thread 19mm. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=DTSK163019F4</td>
		</tr>
		<tr >
			<td >Scalpel blades</td>
			<td >Fine Science Tools (FST)</td>
			<td >10020-00</td>
			<td>Any brand can be used. Example here is FST Scalpel Blade #20. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=191</td>
		</tr>
		<tr >
			<td >Scalpel handle</td>
			<td >Fine Science Tools (FST)</td>
			<td>10004-13</td>
			<td>Any brand can be used. Example here is FST Scalpel Handle #4. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=298&#38;CategoryId=51</td>
		</tr>
		<tr >
			<td >1 x Small surgical scissors</td>
			<td >Fine Science Tools (FST)</td>
			<td>14060-09</td>
			<td>Any brand can be used. Example here is FST Fine Scissors, 9 cm with 21 mm cutting edge, sharp, straight. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=40&#38;CategoryId=17</td>
		</tr>
		<tr >
			<td >2 x Forceps with serrated curved tip</td>
			<td >Fine Science Tools (FST)</td>
			<td >11001-13</td>
			<td>Any brand can be used. Example here is FST 13 cm standard pattern forceps with curved 2.8x1.4 mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=405&#38;CategoryId=32</td>
		</tr>
		<tr >
			<td >1 x Iridectomy scissors</td>
			<td >Fine Science Tools (FST)</td>
			<td>15000-08</td>
			<td>Any brand can be used. Example here is FST Vannas Spring Scissors - 2.5mm Cutting Edge, Straight. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=17&#38;CategoryId=16&#160;</td>
		</tr>
		<tr >
			<td >1 x Forceps with straight serated tip</td>
			<td >Fine Science Tools (FST)</td>
			<td>11650-10</td>
			<td>Any brand can be used. Example here is FST Graefe 10 cm straight with serrated 1 x 0.99 mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=390&#38;CategoryId=32</td>
		</tr>
		<tr >
			<td >1 x Forceps with smooth sharp straight fine tip</td>
			<td >Fine Science Tools (FST)</td>
			<td>11251-10</td>
			<td>Any brand can be used. Example here is FST Dumont #5 forceps straight 11cm with 0.08 x 0.04mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=335&#38;CategoryId=29</td>
		</tr>
		<tr >
			<td >1 x Forceps with smooth fine curved forceps</td>
			<td >Fine Science Tools (FST)</td>
			<td>11063-07</td>
			<td>Any brand can be used. Example here is FST Delicate Forceps 9 cm with smooth 0.4 x 0.3mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=360</td>
		</tr>
		<tr >
			<td >2 x Hemostats</td>
			<td >Fine Science Tools (FST)</td>
			<td >13010-12</td>
			<td>Any brand can be used. Not all operators use the hemostats. Example here is FST 12 cm Micro-Mosquito Hemostats with 20 mm length x 1.3 mm width serrated, straight tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=377&#38;CategoryId=33</td>
		</tr>
		<tr >
			<td >1 x Suture needle holder</td>
			<td >Fine Science Tools (FST)</td>
			<td>12001-13</td>
			<td>Any brand can be used. Example here is FST 13cm Hasley Needle Holder with 16 mm length x 1.9 mm width tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=254&#38;CategoryId=70</td>
		</tr>
		<tr >
			<td >1 x Artery clamp</td>
			<td >Fine Science Tools (FST)</td>
			<td>18050-28</td>
			<td>Any brand can be used. Example here is FST Bulldog Serrefines straight, 28 mm long, 9x1.6 mm jaw dimension with medium clamp press. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=270&#38;CategoryId=82</td>
		</tr>
		<tr >
			<td >Oleic acid</td>
			<td >Sigma Aldrich</td>
			<td >O1008</td>
			<td>When required, any brand can be used. Example here is 99% pure oleic acid. http://www.sigmaaldrich.com/catalog/product/sial/o1008?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >14C-oleic acid</td>
			<td >Perkin&#160;</td>
			<td>NEC317050UC&#160;</td>
			<td>Any brand can be used. Example here is Oleic Acid, [1-14C]-, 50&#181;Ci (1.85MBq). http://www.perkinelmer.com/Catalog/Product/ID/NEC317050UC</td>
		</tr>
		<tr >
			<td >Sodium taurocholate</td>
			<td >Sigma Aldrich</td>
			<td >T4009</td>
			<td>Any brand can be used. Example here is taurocholic acid sodium salt hydrate &#8805;95% (TLC) . http://www.sigmaaldrich.com/catalog/product/sigma/t4009?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >Halofantrine</td>
			<td >Glaxo Smith Kline</td>
			<td ></td>
			<td>Halofantrine was kindly provided as a gift from Glaxo Smith Kline</td>
		</tr>
		<tr >
			<td >Sodium phosphate monobasic</td>
			<td >Sigma Aldrich</td>
			<td >71507</td>
			<td >Any brand can be used. Example here is sodium phosphate monobasic monohydrate, BioXtra, for molecular biology, &#62;99.5%. http://www.sigmaaldrich.com/catalog/product/sigma/71643?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >Sodium phosphate dibasic</td>
			<td >Sigma Aldrich</td>
			<td >71643</td>
			<td >Any brand can be used. Example here is sodium phosphate dibasic dihydrate, BioUltra, for molecular biology, &#62;99%. http://www.sigmaaldrich.com/catalog/product/sigma/71507?lang=en&#38;region=AU</td>
		</tr>
	</tbody>
</table>

the mesenteric lymph duct cannulated rat model: application to the assessment of intestinal lymphatic drug transport

肠道淋巴系统在流体输送，脂质的吸收及免疫功能键的作用。淋巴流直接从小肠通过一系列的淋巴管和节点会聚在肠系膜淋巴管。的肠系膜淋巴管插管从而使肠系膜淋巴结从肠流出的集合。肠系膜淋巴结由免疫细胞（99％淋巴细胞），水相（流体，肽和蛋白质，例如细胞因子和胃肠激素）和脂蛋白组分（脂质，亲脂性分子和apo-蛋白）的细胞级分。的肠系膜淋巴管插管模型因此可用于测量通过淋巴系统的浓度和一系列从肠道因素的输送速率。改变这些因素以响应不同的挑战（ 例如，饮食，抗原，药物）和在疾病（ 例如，炎性肠病，艾滋病，糖尿病）也BË确定。扩大感兴趣的一个领域是淋巴运输中的口服亲脂性药物和药物前体，与肠脂质吸收途径关联的吸收的作用。这里，我们描述，详细地说，一个肠系膜淋巴管空心​​大鼠模型使通过淋巴系统的速率和脂质和药物输送的程度的评价为以下的肠释放数小时。该方法是很容易适应在淋巴其它参数的测量。我们提供了建立这种复杂的外科手术方法时可能遇到的困难的详细描述，以及从实验失败和成功的有代表性的数据，以提供指令，关于如何确认实验的成功和解释所获得的数据。

代表性实验的结果以定量通过以下使用肠系膜淋巴结插管模型肠递送淋巴系统的累积程度及脂质和药物转运的速率示于图4和图5，在该实验中，200微克的模型的亲脂性的药物卤泛群施用至大鼠的十二指肠在2小时中含有40mg油酸（包括2 - 5微居里14 C-油酸）的制剂和7.1毫克2-油酸单甘油酯的磷酸分散在毫升5mM的5.6牛磺胆酸钠缓冲生理盐水pH值6.9。下列制剂给药的大鼠再?...

Watch this Scientific Journal Video about The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport at JoVE.com

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

在肠系膜淋巴管空心​​大鼠模型:应用肠淋巴药物运输的评估

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

the-mesenteric-lymph-duct-cannulated-rat-model-application-to

Research

JoVE Journal

Immunology and Infection

28.4K 次观看.  Monash University (Parkville Campus). Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

视频: The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

腹股沟淋巴结的活体显微镜

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

科研

免疫与感染

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

小鼠腘窝淋巴结的活体成像

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

淋巴结脂肪垫对嵌合体的淋巴结基质细胞起源研究的一代

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

对于检测磁力搅拌器方法<em&gt;旋毛虫</em&gt;幼虫肌肉样品

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

口服抗生素治疗小鼠肠道微生物群的定量聚合酶链反应分析

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

慢性沙门氏菌感染诱发肠纤维化

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

通过在小鼠中吸液性淋巴血管阻止淋巴流

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to ...

在肠系膜淋巴管空心大鼠模型:应用肠淋巴药物运输的评估

摘要

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此视频中的章节

腹股沟淋巴结的活体显微镜

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

小鼠腘窝淋巴结的活体成像

非侵入性光学成像淋巴血管的鼠标

淋巴结脂肪垫对嵌合体的淋巴结基质细胞起源研究的一代

对于检测磁力搅拌器方法

人类的过表达和纯化

口服抗生素治疗小鼠肠道微生物群的定量聚合酶链反应分析

慢性沙门氏菌感染诱发肠纤维化

通过在小鼠中吸液性淋巴血管阻止淋巴流

在肠系膜淋巴管空心​​大鼠模型:应用肠淋巴药物运输的评估

摘要

探索更多视频

此视频中的章节

腹股沟淋巴结的活体显微镜

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

小鼠腘窝淋巴结的活体成像

非侵入性光学成像淋巴血管的鼠标

淋巴结脂肪垫对嵌合体的淋巴结基质细胞起源研究的一代

对于检测磁力搅拌器方法

人类的过表达和纯化

口服抗生素治疗小鼠肠道微生物群的定量聚合酶链反应分析

慢性沙门氏菌感染诱发肠纤维化

通过在小鼠中吸液性淋巴血管阻止淋巴流

在肠系膜淋巴管空心大鼠模型:应用肠淋巴药物运输的评估