在农业和农业食品加拿大研究中心，莱斯布里奇，阿尔伯达省，加拿大的许多人没有奉献精神和努力，这项研究可能已经完成。在这方面，我们感谢：塔拉谢尔顿前和术后护理的动物，手术准备，​​麻醉科，样品分析，并与视频影像的援助;兰迪王尔德和ESTELA哥斯达黎加前和术后护理;珍妮Gusse样品分析;拜伦李套管形象;和布赖恩Egland养羊。我们也感谢提供视频设备和意见，以及其他援助（即执行生产）布伦特Selinger（莱斯布里奇大学生物科学系），和课程的再开发中心，莱斯布里奇大学录像专长。这项研究是支持部分由高级食品材料网（GDI），加拿大阿尔伯塔省肉牛业发展基金（GDI），和加拿大农业和农业食品（GDI和JPK）的赠款。

实验过程： <ol><li>所有仪器蒸压;非autoclaveable材料Germex冷至少10分钟的灭菌水溶液消毒。 </li><li>羊acepromazine（0.05毫克/公斤），格隆（0.005毫克/公斤），与布托啡诺酒石酸盐（0.2毫克/公斤）IM的前药，并且留15-20分钟。 </li><li>剃光和颈部皮肤手术擦洗。一旦颈内静脉是可视化的，在静脉注射0.1毫升利多卡因资深大律师，引入静脉导管，并固定在手术过程中使用牲畜识别标签水泥和2“医院磁带皮肤。动物10毫升Plasmalyte 148 /公斤/小时（重力流）。 </li><li>地西泮（0.2毫克/公斤）管理四，麻醉诱导硫喷妥钠（10毫克/公斤）。利多卡因气管内喷雾，适用于声带，羊气管插管，并在4 L / min的的速率（1.5-2.5％异氟醚维持在100％O 2）手术时间。 </li><li>动物被放置在一个加热垫背斜卧在手术过程中，以帮助维持体温。胃管插入瘤胃，以防止过度tympany。 </li><li>剃腹腹部和三个Prepodyne手术磨砂皮肤手术准备。腹部，然后搭着，确保保持手术部位适当的曝光（脐腹部尾鳍）。 </li><li> 15-20厘米正中切口是使用electroscapel，尾端从肚脐，并回盲部的褶皱是在位于右下腹，腹部。然后exteriorized尾回肠和盲肠。整个过程中，处理肠道的最小化和护理是采取措施，确保exteriorized肠道保持湿润，温暖（37-39 ° C）无菌磷酸盐缓冲液（PBS，pH值7.2）。 </li><li> 85厘米的回肠段（即“肠段”）是测量，然后在每个段年底东银钳（肠道夹具）和大凯利钳钳制。在肠道段中，“循环”和“空隙”之间的循环将建立。老前辈钳放在一边要加入的回肠（吻合），和凯利钳放置小肠回肠段方。 </li><li>回肠减少彼此之间东银和凯利forcep和肠道段指定的for循环是60毫升的温暖PBS轻轻冲洗两次删除ingesta。 </li><li>广谱抗生素（200毫克恩诺沙星和甲硝唑在温暖的PBS 60毫升，总量250毫克）的鸡尾酒是分布在整个肠道段和30分钟离开。 </li><li>回肠（小肠段除外）的近端和远端切端加入，形成一个连续的和功能性肠道。首先，回肠是由两个留缝线对齐，放置在肠道内的肠系膜和antimesenteric边界分别。然后，一个简单的连续缝合模式（2-0薇乔）完成对肠道双方一起加入肠道。 </li><li>暖PBS（3-5毫升）注射吻合部位，以检查是否有泄漏和缝合失败。随后，凯利forcep提示按下对肠道浆膜面，关闭网站的吻合部位，先进的吻合部位向内，以确保肠腔大和专利。 </li><li>抗生素的鸡尾酒是从肠道段删除。每个段的结束，是封闭，用一个简单的连续缝合模式（可吸收缝合线; 2-0薇乔），由一个反相缝合用2-0薇乔格局。 </li><li> 85厘米长的小肠段被分成3个15厘米的循环“，两个15厘米长的”空隙“之间的循环，和两个5厘米长的平端在小肠段的总站车厢（图1） 。循环使用2-0丝绸肠道周​​围放置一个结扎。值得注意的是主要肠系膜血管不结扎，在此过程中，因为这些都是需要供应血液肠道，保持肠道的可行性。此外，连字左边长，因为它们将被用于安全导管。 </li><li>导管（例如硅橡胶管）单独确定使用永久性标记。被置于一个硅球（5毫米直径），包围导管，从导管末端4-5厘米，以帮助防止导管滑动小肠（图1）。非抗生素软膏（Orbeseal）经高压灭菌消毒后，在无菌条件下插入导管末端（3-5毫米）;确保导管仍然专利（即它不会成为插入脱落黏膜内环路）。 </li><li>三环路颅方面被放置在一个小切口。每孔用导管导引（即“血管扩张”），并扩大6-8厘米的段的导管插入尾端成循环，直到silcone球传递到管腔（图1）。 </li><li>肠壁周围的导管与荷包缝合（2-0薇乔）担保。该导管是进一步巩固肠道配合导管结扎丝绸，划定颅循环结束。 </li><li>氨苄青霉素（5毫升100毫克/毫升在温暖的PBS）注入每个循环和空隙，是摆在腹腔和肠道。头孢唑啉（8毫升100毫克/毫升在温暖的PBS）是通过腹部切口腹腔注入。 </li><li>经腹壁正中切口附近，是一个单一的小刺切口导管通过这个网站exteriorized。 </li><li>腹腔内导管的位置和张力调整。关闭间断缝合，用一个简单的模式（可吸收缝合线，羊肠线＃3），腹部肌肉和皮肤中断的水平褥式模式（非吸收缝合线; Supramid＃1）关闭。 </li><li>一个弧形的空心不锈钢管（7毫米的ID; 10毫米外径，长度65厘米）插入下腹部切口附近的皮肤，在退出网站只是尾椎颈部皮肤和肩膀之间的隧道。导管插入管，推管的长度至少有50％的。管与导管，然后在出口部位的皮肤拉，露出导管。 </li><li>导管的张力调整刺切口部位，皮肤是封闭Supramid＃1的。 </li><li>个人发现导管内绷带“袋”，这是Supramid＃1缝合皮肤。小袋保护导管，从后续的损害。 </li><li>手术后，羊注射氟尼辛（SID）的IM初始剂量2.2毫克/公斤，其次为3天的维持剂量为1.1毫克/公斤的SID。羊也被注入Hemostam（3毫升）和恩诺沙星（2.5毫克/公斤）SID IM为3至5天，分别为。动物保持不断蠕动泵，以确保继续不分体位的流量（145毫升/小时），5％葡萄糖溶液静脉Plasmalyte 148。他们被允许喝自由采食 ，但饲料，直到恢复正常的瘤胃和肠道功能受到限制。食物摄入量，耗水量，体温，通过粪便和尿液，肠道噪音，腹部不适，言行举止，和血糖浓度密切监察（BID），必要时，在静脉注射液中的葡萄糖浓度调整，以维持血糖浓度在正常范围内。血生化和血液学（即白蛋白，碱性磷酸酶，谷丙转氨酶，淀粉酶，钙，胆固醇，肌酐，葡萄糖，无机磷，总胆红素，总蛋白，尿素，球蛋白，钠，钾，氯，氧气分压，pH值，部分压力二氧化碳，碳酸氢盐，和总二氧化碳，红，白血细胞计数，红细胞压积，血红蛋白，差白细胞计数和血小板计数）前和手术后立即监测，以后隔日。一旦开始正常的排便功能，静脉补液治疗是终止和动物转移到围场（即5-7天手术后）。个人钢笔（其他动物的视线内），从而减轻压力和导管袋（如由penmate咀嚼）遭受损害的机会，在保持动物。 </li><li>要注入个人循环的治疗，使用无菌注射器，装有一个18针，钝端针导管注入5毫升的治疗。为了确保所有进入循环的治疗的沉积，导管再注入3毫升的PBS。 </li></ol>关键点： 这种外科手术的成功至关重要的步骤包括：（1）核实，肠道本身不扭曲之前建立的吻合;（2）保证吻合口的完整性和通畅;（3）充分保证导管结扎划定循环颅结束循环墙，但确保该附件不会影响导管的通畅;（4）尽量减少肠道处理和确保exteriorized肠道保持湿润;（5）确保适当的导管紧张;（6）确保在肌肉和皮肤缝合，导管的完整性是没有影响;（7）防止羊手术后狼吞虎咽（即在手术后肠梗阻的时期），这可能会导致瘤胃嵌塞和减值瘤胃功能;（8），以确保动物得到足够的静脉输液治疗，手术后第3-5天，即血糖浓度是认真的监测，静脉注射液中的葡萄糖浓度adjus特德如果必要，以防止低血糖。 <img src="/files/ftp_upload/1301/1301fig1.jpg" alt="图1" /> 图1。插管回肠循环的一个示意图。注意三个15厘米长插管“循环”，两个15厘米长的非插管的“空隙”的循环，和两个5厘米长的码头段之间建立，所有内设立一个85厘米长的“肠的部分“一只绵羊回肠。

<ol>
	<li>Vidal, J. E., McClane, B. A., Saputo, J., Parker, J., Uzal, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Clostridium+perfringens+beta-toxin+on+the+rabbit+small+intestine+and+colon.">Effects of Clostridium perfringens beta-toxin on the rabbit small intestine and colon.</a> Infect. Immun. 76, 4396-4404 (2008).</li><li>Santos, R. L., Zhang, S., Tsolis, R. M., Baumler, A. J., Adams, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+molecular+characterization+of+Salmonella+typhimurium+infection+in+neonatal+calves.">Morphologic and molecular characterization of Salmonella typhimurium infection in neonatal calves.</a> Vet. Pathol. 39, 200-215 (2002).</li><li>Uzzau, S., Leori, G. S., Petruzzi, V., Watson, P. R., Schianchi, G., Bacciu, D., Mazzarello, V., Wallis, T. S., Rubino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+enterica+serovar-host+specificity+does+not+correlate+with+the+magnitude+of+intestinal+invasion+in+sheep.">Salmonella enterica serovar-host specificity does not correlate with the magnitude of intestinal invasion in sheep.</a> Infect. Immun. 69, 3092-3099 (2001).</li><li>Gerdts, V., Uwiera, R. R. E., Mutwiri, G. K., Wilson, D. J., Bowersock, T., Kindane, A., Babiuk, L. A., Griebel, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+intestinal+'loops'+provide+an+in+vivo+model+to+analyse+multiple+mucosal+immune+responses.">Multiple intestinal 'loops' provide an in vivo model to analyse multiple mucosal immune responses.</a> J. Immunol. Methods. 256, 19-33 (2001).</li><li>Greibel, P. J., Davis, W. C., Reynolds, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+the+growth+and+differentiation+of+b+cells+isolated+from+follicles+from+ileal+Peyer's+patch+of+sheep.">An analysis of the growth and differentiation of b cells isolated from follicles from ileal Peyer's patch of sheep.</a> Immunology. 75, 601-607 (1992).</li><li>Motyka, G., Greibel, P. J., Reynolds, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agents+that+activate+protein+kinase+C+resue+sheep+ileal+Peyer's+patch+B+cells+from+apoptosis.">Agents that activate protein kinase C resue sheep ileal Peyer's patch B cells from apoptosis.</a> Eur. J. Immunol. 23, 1314-1321 (1993).</li></ol>

对实验动物的所有程序批准的机构动物保健委员会（动物使用的协议＃0609）和加拿大动物保健委员会设置的指引。 我们什么都没有透露。

在此过程中，我们能够成功地建立在羊回肠内建立的肠袢的导管。坚持严格的术后护理，动物迅速恢复从程序和手术后恢复正常活动5-7天。我们决定建立循环内导管不影响肠黏膜免疫功能（数据未显示）和动物可以生活至少8周后手术。此过程允许我们引入不同的免疫车厢多次治疗，在延长的时间间隔，一旦动物有从手术中恢复，并进行实证衡量这些治疗的生理和免疫效果。 为今后的实验中的一个重要的考虑因素是肠道内的小肠段的位置。在此过程中，我们手术创造了肠道内往往回肠（连续）Peyer氏斑（5,6）定义的“免疫功能回肠”部分。这个区域可以延长颅可达2米，超出回盲倍。尽管这个地区被认为是解剖学空肠在免疫功能方面，它是回肠，突出考虑功能描述肠道地区时的重要性。

<table border="1"><tr><td> 分类/编号 </td><td> 公司 </td><td> 目录 </td></tr><tr><td colspan="3"> 外科用品 </td></tr><tr><td>外科手套</td><td> WDDC Å </td><td> 103091 </td></tr><tr><td>外科口罩</td><td> WDDC </td><td> MAX53013 </td></tr><tr><td>手术衣</td><td> CDMV b </td><td> 3365 </td></tr><tr><td> OB的围裙</td><td> WDDC </td><td> 900062 </td></tr><tr><td>手术帽</td><td> WDDC </td><td> 903271 </td></tr><tr><td> EZ刷子刷洗</td><td> WDDC </td><td> BDC377479 </td></tr><tr><td colspan="3"> 麻醉和药物 </td></tr><tr><td>麻醉机</td><td>博伊尔</td><td> </td></tr><tr><td> Sodasorb </td><td> WDDC </td><td> 108638 </td></tr><tr><td>喉镜</td><td> WDDC </td><td> </td></tr><tr><td>喉镜片（米勒刀片＃4） </td><td> WDDC </td><td> 902667 </td></tr><tr><td>气管插管（7.0 - 8.5毫米） </td><td> WDDC </td><td> </td></tr><tr><td>利多卡因气管内喷雾</td><td> WDDC </td><td> 103364 </td></tr><tr><td> Acepromazine </td><td> WDDC </td><td> AUS01020 </td></tr><tr><td>格隆</td><td> WDDC </td><td> SAB2880 </td></tr><tr><td>布托啡诺酒石酸</td><td> WDDC </td><td> AYE304541 </td></tr><tr><td>地西泮</td><td> WDDC </td><td> sab790 </td></tr><tr><td>硫喷妥钠</td><td> WDDC </td><td> 103508 </td></tr><tr><td>盐酸肾上腺素</td><td> WDDC </td><td> 103856 </td></tr><tr><td>多沙普仑</td><td>斯特拉思科纳药房</td><td> </td></tr><tr><td>异氟醚</td><td> WDDC </td><td> 100339 </td></tr><tr><td> Hemostam </td><td> WDDC </td><td> 103857 </td></tr><tr><td>氟尼辛</td><td> WDDC </td><td> sch140100 </td></tr><tr><td>恩诺沙星</td><td> WDDC </td><td> hll0049 </td></tr><tr><td>甲硝唑</td><td> WDDC </td><td> 101653 </td></tr><tr><td>氨苄青霉素</td><td> WDDC </td><td> NOV67185 - 6 </td></tr><tr><td>头孢唑啉</td><td> WDDC </td><td> nov69397 </td></tr><tr><td colspan="3"> 手术设备和用品 </td></tr><tr><td> Electroscapel </td><td>马坎</td><td> MV8 </td></tr><tr><td>加热垫</td><td>凯恩</td><td> PHMD - 48 </td></tr><tr><td>胃管（NALGENE管5 / 16“编号） </td><td> Fisher Scientific则</td><td> </td></tr><tr><td>针驱动程序</td><td> WDDC </td><td> 108338 </td></tr><tr><td>艾迪棕色forcep </td><td> WDDC </td><td> 911075 </td></tr><tr><td>大鼠牙齿forcep </td><td> CDMV </td><td> 5275 </td></tr><tr><td>凯利forcep </td><td> WDDC </td><td> 901133 </td></tr><tr><td>蚊子止血</td><td> WDDC </td><td> 108471 </td></tr><tr><td> Carmalt forcep </td><td> WDDC </td><td> 901225 </td></tr><tr><td>手术刀处理</td><td> WDDC </td><td> 900285 </td></tr><tr><td>手术刀刀片＃22 </td><td> WDDC </td><td> 900511 </td></tr><tr><td>梅岑鲍姆剪刀</td><td> WDDC </td><td> 901166 </td></tr><tr><td>夏普锋利的剪刀</td><td> WDDC </td><td> 108264 </td></tr><tr><td>巾钳</td><td> WDDC </td><td> 901213 </td></tr><tr><td>阿里斯组织forcep </td><td> WDDC </td><td> 901201 </td></tr><tr><td>老前辈的forcep </td><td> WDDC </td><td> 904226 </td></tr><tr><td>不锈钢碗</td><td> WDDC </td><td> 903601 </td></tr><tr><td>不锈钢套管（弯） </td><td>本地供应商</td><td> </td></tr><tr><td>导管导引</td><td> WDDC </td><td> 11440 </td></tr><tr><td>手术巾</td><td> CDMV </td><td> 7972 </td></tr><tr><td>一次性悬垂</td><td> WDDC </td><td> 900035 </td></tr><tr><td>无菌悬垂</td><td> CDMV </td><td> 2323 </td></tr><tr><td>披风（40 × 40厘米） </td><td> CDMV </td><td> 8358 </td></tr><tr><td>手术悬垂（7“× 11”开放） </td><td> CDMV </td><td> 269​​4 </td></tr><tr><td>薇乔（2-0） </td><td> WDDC </td><td> JJMJ333H </td></tr><tr><td>丝绸2-0 </td><td> WDDC </td><td> 105248 </td></tr><tr><td>羊肠线＃3 </td><td> WDDC </td><td> CSL547 - 2070 </td></tr><tr><td> Supramid＃1 </td><td> WDDC </td><td> CSL547 - 3040 </td></tr><tr><td>针（切割和非切割） </td><td> WDDC </td><td> </td></tr><tr><td colspan="3"> 导管 </td></tr><tr><td>硅橡胶管（0.040“的ID，0.085”外径） </td><td> Fisher Scientific则</td><td> 11 - 189 - 15D </td></tr><tr><td> Orbeseal软膏</td><td> WDDC </td><td> 106344 </td></tr><tr><td>清除硅（732多用途密封胶） </td><td>本地供应商</td><td> </td></tr><tr><td>钝年底针</td><td> WDDC </td><td> 905035 </td></tr><tr><td>标记（永久性的工业） </td><td>本地供应商</td><td> </td></tr><tr><td>导管袋（建造Elastoplast 7.5厘米） </td><td> WDDC </td><td> 104094 </td></tr><tr><td colspan="3"> 绷带 </td></tr><tr><td>医院磁带（2“） </td><td> WDDC </td><td> 103842 </td></tr><tr><td>医院磁带（1“） </td><td> WDDC </td><td> VLMRENF20 </td></tr><tr><td>纱布的4x4 </td><td> WDDC </td><td> 107551 </td></tr><tr><td>纱布抱住</td><td> WDDC </td><td> 107633 </td></tr><tr><td> Elastoplast7.5厘米</td><td> WDDC </td><td> 104094 </td></tr><tr><td> Elastoplast rippable绷带</td><td> WDDC </td><td> 104093 </td></tr><tr><td colspan="3"> 氖edles和注射器</td></tr><tr><td>注射器（60毫升） </td><td> WDDC </td><td> 104004 </td></tr><tr><td>注射器（10毫升） </td><td> WDDC </td><td> bdc301604 </td></tr><tr><td> 25G针</td><td> WDDC </td><td> BDC305122 </td></tr><tr><td> 20G针</td><td> WDDC </td><td> BDC305175 </td></tr><tr><td> 18G针</td><td> WDDC </td><td> 903353 </td></tr><tr><td colspan="3"> 静脉内 </td></tr><tr><td> Intracath静脉导管（16G 8“） </td><td> WDDC </td><td> bdc3831621 </td></tr><tr><td>静脉注射组（10滴） </td><td> WDDC </td><td> 101797 </td></tr><tr><td> 0.9％氯化钠溶液</td><td> WDDC </td><td> BAXJB1322P </td></tr><tr><td> Plasmalyte 148解决方案</td><td> WDDC </td><td> BAXJB2534 </td></tr><tr><td>葡萄糖</td><td> WDDC </td><td> MCT60190 </td></tr><tr><td>蠕动泵</td><td>科尔帕默</td><td> 7524-10 </td></tr><tr><td>牲畜识别标签水泥</td><td>本地供应商</td><td> </td></tr><tr><td colspan="3"> Sanatizers /消毒剂 </td></tr><tr><td> Germex解决方案</td><td> WDDC </td><td> mtc60270 </td></tr><tr><td>异丙醇</td><td> WDDC </td><td> 104590 </td></tr><tr><td> Stanhexidine洗面奶</td><td> WDDC </td><td> 109248 </td></tr><tr><td> Prepodyne解决方案</td><td> WDDC </td><td> 105710 </td></tr><tr><td> Prepodyne擦洗</td><td> WDDC </td><td> 105711 </td></tr></table> 西方的毒品分销中心有限公司，埃德蒙顿，AB 。 b的兽药经销中心公司，圣Hyacinthe，量化宽松政策。

catheterization of intestinal loops in ruminants

肠道是一个复杂的结构，不仅在营养物质的吸收，而且还充当之间的个人和外界的障碍。因此，肠道内起着举足轻重的作用，从肠道致病菌的免疫监视和保护。调查肠道生理学和免疫学通常采用“肠袢”作为实验模型。这些循环模型大部分都是非恢复手术过程中，研究短期（&lt;24小时）在肠道内的变化（1-3）。我们先前创建的恢复肠袢模型来具体衡量长期（&lt;6个月）羊暴露后疫苗，佐剂和病毒（4）肠道免疫变化。此过程的本地化处理，以具体的“循环”，让我们品尝这一领域的肠道。这种方法的一个显着的缺点是管理治疗的机会，即在手术时的单一窗口。此外，肠道黏膜和管腔内容的样本，只能采取在项目终止。上述模型中的其他显着的局限性，手术操作和必要的手术后的措施（如抗生素和止痛药的管理），可直接影响治疗本身和/或改变免疫功能，从而混淆的结果。因此，我们修改了我们的肠袢模型，通过长期导管插入循环。羊完全恢复的程序，并exteriorized导管的影响。值得注意的是，建立在循环导管，使我们能够引进在延长的时间间隔多次治疗后，手术和清理手术期间服用过的药物和手术后的时期的恢复。

Watch this Scientific Journal Video about Catheterization of Intestinal Loops in Ruminants at JoVE.com

Catheterization of Intestinal Loops in Ruminants

我们描述了一种新的手术方法catheterizing在羊回肠“肠袢”。一旦动物有从手术中恢复，并已清除抗生素和止痛药，多治疗，可以直接存入通过导管循环。

导尿在反刍动物的肠袢

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the ...

catheterization-of-intestinal-loops-in-ruminants

Nanyang Technological University

Research

JoVE Journal

Biology

13.6K 次观看.  University of Alberta. 我们描述了一种新的手术方法catheterizing在羊回肠“肠袢”。一旦动物有从手术中恢复，并已清除抗生素和止痛药，多治疗，可以直接存入通过导管循环。

视频: Catheterization of Intestinal Loops in Ruminants

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

调查在DSS诱导的鸡传染性法氏囊病模型的肠道炎症

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

科研

生物学

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

逆转录病毒感染的原发性肠化培养的视频协议

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

测量压力容量环中的鼠标

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

肠上皮细胞通透性的体外及体内测定方法

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

小肠部分梗阻的小鼠模型

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

海参肠细胞原培养的凋亡诱导与检测

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

肠道维利表皮的器官3D培养进行分化

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

来自肠道密码的穆林原结肠上皮单层的生成

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...