Name: the mesenteric lymph duct cannulated rat model: application to the assessment of intestinal lymphatic drug transport
Uploaded: 2015-03-06T13:00:00.000+00:00
Duration: 13 min 17 s
Description: 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

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><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&amp;amp;CAT=6&amp;amp;search&lt;br/&gt; =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&amp;amp;CAT=6&amp;amp;search&lt;br/&gt; =BU0510</td></tr><tr><td>Ilium Ketamil (Ketamine 100 mg/ml)</td><td>PROVET VICTORIA&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>VTG-DACP010020</td><td>http://www.provet.com.au/</td></tr><tr><td>Sodium pentobarbitone</td><td>PROVET VICTORIA&amp;nbsp;</td><td>24529</td><td>Any brand can be used. Example here is Lethabarb&amp;reg; 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.&amp;nbsp;</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 0.96 x0.58 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 0.8 x0.5 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>Veterinary adhesive</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&amp;amp;CAT=&lt;br/&gt; 6&amp;amp;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&amp;amp;CAT=6&amp;amp;search=&lt;br/&gt; 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&amp;amp;CAT=6&amp;amp;search&lt;br/&gt; =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&amp;amp;CAT=6&amp;amp;search&lt;br/&gt; =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&amp;amp;CAT=6&amp;amp;search&lt;br/&gt; =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&amp;amp;CAT=6&amp;amp;search&lt;br/&gt; =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_&lt;br/&gt; -1_HAI_ProductDetail and&amp;nbsp; http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_35487_&lt;br/&gt; -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&amp;amp;p=us&amp;amp;f=e&amp;amp;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 &amp;nbsp;&lt;br/&gt;&lt;br/&gt; 3/8 Circle Reverse Cut Silk Suture 3/0 Thread 19mm. http://www.livingstone.com.au/?PG=search_result&amp;amp;CAT=6&amp;amp;search&lt;br/&gt; =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&amp;amp;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&amp;amp;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&amp;amp;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&amp;amp;CategoryId=16&amp;nbsp;</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&amp;amp;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&amp;amp;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 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&amp;amp;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&amp;amp;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&amp;amp;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&amp;amp;region=AU</td></tr><tr><td>14C-oleic acid</td><td>Perkin&amp;nbsp;</td><td>NEC317050UC&amp;nbsp;</td><td>Any brand can be used. Example here is Oleic Acid, [1-14C]-, 50&amp;micro;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 &amp;ge;95% (TLC) . http://www.sigmaaldrich.com/catalog/product/sigma/t4009?lang=en&amp;amp;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, &gt;99.5%. http://www.sigmaaldrich.com/catalog/product/sigma/71643?lang=en&amp;amp;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, &gt;99%. http://www.sigmaaldrich.com/catalog/product/sigma/71507?lang=en&amp;amp;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

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 ...

科研

免疫与感染

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

体外肠囊评估黏膜通透性在胃肠道疾病模型

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

医学

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

在新生儿猪淋巴的肠淋巴躯干和收集的长期导尿

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

大鼠肠系膜血管生成法

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

生物学

Targeting the Rat's Small Bowel: Long-Term Infusion into the Superior Mesenteric Artery

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA cannulation is more difficult in rats due to small anatomical dimensions. The aim of the study is to describe a surgical technique for cannulation of the SMA in rats to perform long-term infusion of drugs into the SMA vascular bed in unrestricted animals, which will result in a high rate of catheter patency after the post surgical recovery for 24 hours. 
To avoid the risk of SMA thrombosis or bleeding from direct access, a proximal branch of the SMA is isolated, ligated distally and cannulated with a 0.25 mm polyurethane capillary tube whose tip is advanced close to the origin of the SMA from the aorta. The cannula is then tunnelled subcutaneously to the back of the animal's neck and through the skin via an artificial valve. The external portion of the cannula is inserted in a semi-rigid support system and connected to the continuous infusion pump outside the cage where the rat is free to move. 
Correct positioning of the cannula was demonstrated by post-surgical angiography and autopsy findings. Catheter patency after 24 hours of saline infusion into the SMA region was assured in most rats by the total discharge of the pump and recognition of a functional cannula for blood sampling or saline infusion.

Access for long-term infusion in the superior mesenteric artery (SMA) of rats is a surgical procedure that consists of cannulation of a proximal branch of the SMA. The cannula exits from the abdominal wound and is tunneled through the subcutaneous space back to the interscapular fold.

靶向大鼠的小肠:长期输注到肠系膜上动脉

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA ...

生物工程

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

鼠佩耶斑块中淋巴细胞动力学的微观观察

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的 体内 动力学

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...

Mesenteric Lymph Duct Cannulation in a Rat Model to Harvest Lymph Fluid

Source: Trevaskis, N. L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52389">The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport.</a> J. Vis. Exp. (2015).
This video demonstrates a technique to cannulate the mesenteric lymph duct in rats. This method enables direct access to the entire volume of components present in the lymph fluid.

大鼠模型中的肠系膜淋巴管插管以收集淋巴液

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Assay and Analysis

Intraduodenal Cannulation in a Rat Model for Immunopathology Studies

Source: Trevaskis, N. L. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52389">The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates intraduodenal cannulation in a rat model. After anesthetizing the rat, the duodenum is located and a small puncture is created. A J-shaped cannula with a beveled tip is carefully inserted and secured, and the incision is closed. The cannulated rat model enables intra-intestinal infusion for immunopathology studies.

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

非侵入性光学成像淋巴血管的鼠标

体外肠囊评估黏膜通透性在胃肠道疾病模型

在新生儿猪淋巴的肠淋巴躯干和收集的长期导尿

大鼠肠系膜血管生成法

靶向大鼠的小肠:长期输注到肠系膜上动脉

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

鼠佩耶斑块中淋巴细胞动力学的微观观察

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的体内动力学

大鼠模型中的肠系膜淋巴管插管以收集淋巴液

用于免疫病理学研究的大鼠模型中的十二指肠内插管

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

非侵入性光学成像淋巴血管的鼠标

体外肠囊评估黏膜通透性在胃肠道疾病模型

在新生儿猪淋巴的肠淋巴躯干和收集的长期导尿

大鼠肠系膜血管生成法

靶向大鼠的小肠:长期输注到肠系膜上动脉

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

鼠佩耶斑块中淋巴细胞动力学的微观观察

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的 体内 动力学

大鼠模型中的肠系膜淋巴管插管以收集淋巴液

用于免疫病理学研究的大鼠模型中的十二指肠内插管

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

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的体内动力学