这项工作是由来自墨西哥议会科学与技术（国家科学技术委员会，207268和233395迈克尔·施诺尔）资助。 KFCO是国家科学技术委员会津贴（396260）的获得者，获得硕士学位。

所有的动物实验已通过CINVESTAV的机构动物护理和使用委员会。 1. DSS饮用水的制备及结肠炎的诱导<ol><li>制备3.5％w / v的葡聚糖硫酸钠（DSS）在高压灭菌的饮用水溶液。 DSS容易溶解并且不要求过滤最终的解决方案。 注:7天DSS处理诱发严重的结肠炎。如果诱导轻度结肠炎，3 - 推荐治疗4天。只要DSS的饮用水保持清晰，这是没有必要改变水。然而，如果它变成浑浊，它必须被更换。 </li><li>记录雄性小鼠的基线重量。确保他们8 - 年龄和范围21 12周内 - 25克体重。 注:适当浓度DSS必须在每个实验室进行优化。结果可以变化很大，这取决于外壳的条件。在2.5的浓度 - 5％通常被报道诱导精通Colitis在C57BL / 6J WT小鼠10。 DSS来自不同公司和不同批应测试，作为结果还可以与决策支持系统的不同来源的不同而不同。 </li><li>提供水自由采食 ，必要时用新鲜的3.5％DSS水代替它。 </li></ol>灌胃营养补剂2.应用<ol><li>准备在适当的溶剂中的"给送"所需的营养补充品的溶液。在这项研究中，我们使用含有200毫克/千克L-精氨酸，83毫克/公斤维生素C，46毫克/公斤维生素E，77毫克/公斤的二十碳五烯酸（EPA），和115毫克/千克二十二碳六烯酸（混合物的DHA ）以1:水和红花油1溶液。 </li><li>填充1毫升注射器连接到灌胃针（15克×50毫米）与营养补充剂混合物。擦拭灌胃针的外面以除去任何化合物的针之外，并确保适当的剂量应用时。 </li><li>通过抓住尾部与基座抑制鼠标一方面通过在颈部的用另一只手的拇指和食指背面牢固地把持皮肤褶皱。放置第三手指和保持该颈部同一只手的拇指的基部之间的尾巴。 </li><li>同时保持在直立位置的鼠标，针头插入动物的嘴的左侧，认真遵循口定位食道的屋顶。如果遇到没有抵抗力，对促进胃针。 注:验证动物继续之前正常呼吸。 </li><li>辖混合物慢慢地，慢慢拔出注射器取出试管，然后松开鼠标。结肠炎实验的过程中，每天一次申请的营养补充剂通过管饲法。 </li></ol> 3.疾病活动指数的测定注:本病活动指数是分数减肥，肛周出血，大便的一致性，这是肥胖的组合tained每天13。 <ol><li>每日测量体重并根据重量损失百分数得分的0分配到4（0:0％，1:1 - 5％，2:5 - 10％，3:10 - 20％，4:&gt; 20 ％）。 注:体重损失是由DSS结肠炎的诱导和实际重量之前计算的基底重量％之间的差来确定。 </li><li>要确定肛周出血的水平，收集新鲜粪便样品，并使用所提供的喷头就从零陵香大便隐血检测试剂盒的幻灯片薄薄涂抹。使用新鲜涂抹器对每个样品。 <ol><li>关闭的前盖并打开滑盖背面的窗口。施加显影剂溶液的两滴在样品位置的背面。 </li><li> 30 s后读取结果。蓝色的痕迹是潜血阳性。分配比分0比4（0:无，0.5 - 2.5:积极的愈创木大便潜血试验（取决于信号强度），和3 - 4:常务顾问小组还将SS出血）。 注意:如果便血是可见的，愈创木脂的粪便潜隐血测试没有被执行，和3的得分，3.5，或4分配，这取决于可见血液量。 </li></ol></li><li>通过观察一个新鲜粪便样品确定大便的一致性。得分为0分配给4（0:正常/固，0.5 - 2.5:糊状大便，3 - 4:腹泻）。 </li></ol> 4.确定肠上皮通透性在体内被伊文思蓝含量<ol><li>通过在盐水溶液（0.9％NaCl）中稀释氯胺酮（100mg / kg的体重）的混合物和甲苯噻嗪（13毫克/公斤体重）腹膜内注射它们麻醉动物，并通过监控夹送评估麻醉深度撤回反射。 </li><li>放置麻醉动物处于仰卧位置和进行剖腹，露出肠道。 </li><li>找到盲肠和作一小切口在结肠近段scendens（理想情况下，紧邻盲肠）。 </li><li>插入喂食针（G22）中，用使用公共丝线一个结扎固定。小心地冲洗管PBS丰富来冲从结肠粪便全部。灌输伊文思蓝溶液（1.5％w / v的在PBS中）进入结肠，直到它到达肛门。 </li><li>孵育Evans蓝15分钟。冲洗丰富PBS管，直到肛周冲洗明确洗出的染料。 </li><li>通过颈脱位安乐死动物。 </li><li>切除结肠和丰富PBS再冲洗。用1mL 6毫Ñ-acetylcysteine在PBS中冲洗一次，以消除任何染料粘到结肠粘液。 </li><li>纵向切开结肠，用PBS和1mL 6毫米ñ-acetylcysteine的一次冲洗。 </li><li>记录重量和长度。以提取伊文思蓝染料，过夜放置结肠在2mL的N，N-二甲基甲酰胺在室温下轻轻agitatioñ。分光光度计测量染料浓度在610纳米。 </li></ol> 5.组织收集<ol><li>通过在盐水溶液（0.9％NaCl）中稀释氯胺酮（100mg / kg的体重）的混合物和甲苯噻嗪（13毫克/公斤体重）腹膜内注射它们麻醉动物，并通过监控夹送评估麻醉深度撤回反射。 </li><li>通过颈脱位安乐死动物。 </li><li>放置动物处于仰卧位置和进行剖腹，露出腹腔。 </li><li>使用剪刀，解剖整个结肠和记录它的长度。 </li><li>用冷的PBS冲洗结肠仔细几次以除去粪便。记录组织重量和用于下面的实验中制备，如在步骤5.6中所述 - 5.8。 </li><li>对于RNA分离和髓过氧化物酶（MPO）测定法，切断一个大小合适的组织样品（100毫克通常是足够的），将其放置一管内，和SNA在液氮中对 - 冻结它。在-80℃，以备将来使用存储的组织。 </li><li>对于免疫荧光染色和氧化应力测定（见下文），切一个小结肠样品（0.5 - 1厘米），并在填充有最佳切割温度（OCT）化合物小的铝杯浸没它。放置在干冰上的杯，并让他们慢慢冻结，以避免形成气泡。冷冻组织样品可以储存在-80℃以供将来使用。 </li><li>瑞士卷14，纵向打开结肠，小心使用镊子取出粪便。把它卷起来，与粘膜向内，用细尖镊子或薄，圆木棍。小心地将其放在包埋盒里面，然后继续步骤6.1。 注:DSS诱导的结肠损害。然而，损伤分布从近端到远端结肠而变化，与通常在远端结肠和直肠看出大多数损害。因此，我们建议在分析组织学无论是在山坳远端上或在整个结肠的瑞士角色。 </li></ol> 6.苏木伊红染色分析结肠组织学<ol><li> 组织准备 <ol><li>对于组织学分析，淹没或瑞士卷或从控制的某些结肠区域的小组织片段和处理的小鼠在5毫升10％的甲醛，在室温下（RT）下48小时，以实现适当的固定。 注:在第6步中所有其他的步骤都在室温下进行，除非另有说明。 </li><li>用自来水洗18小时。 </li><li>用15mL 70％的乙醇的脱水它们1小时，1小时96％，和1小时的无水乙醇。在进行下一浓度之前重复用新鲜的醇每一个步骤。 </li><li>继续脱水在7.5毫升无水乙醇和7.5毫升无水二甲苯中1小时的混合。传递样本以15毫升无水二甲苯中1小时，然后重复一次。用新鲜的二甲苯重复此步骤一次。 </li></OL&gt; </li><li> 在石蜡包埋结肠组织样本 <ol><li>沉浸在50mL液体石蜡的样品17小时。重复此步骤一次，但只有3小时。 </li><li>在810毫升液体石蜡:浸入立方体样品（22×22×22 mm塑料，模具方形，大小）。 </li><li>允许与结肠组织样本的石蜡固化在室温24小时。 </li></ol></li><li> 切割用切片组织的横截面 <ol><li>切使用切片机结肠样品，按制造商的说明，在其厚度为5微米。 </li><li>收集在水浴（1L）含有0.3％明胶的削减。这可以防止组织的横截面的折叠。恢复组织切片和它们安装在载玻片上。 </li></ol></li><li> 组织横截面的苏木精和曙红染色 <ol><li>在培养箱Deparaffinize 18小时的样品在60℃。为了这个目的，使用玻璃小号立德架带把手。 </li><li>脱石蜡后，浸泡在70毫升绝对二甲苯的载玻片5分钟。用新鲜的二甲苯重复此步骤一次。 </li><li>转移样品到70毫升无水乙醇的1分钟。用新鲜的酒精重复此步骤一次。 </li><li>孵育在70毫升乙醇中96％的结肠组织样品1分钟。用新鲜的酒精重复此步骤一次。 </li><li>在洗净的自来水样本进行两次2秒。 </li><li>孵育在哈里斯苏木组织7分钟。 </li><li>在洗净的自来水样本进行两次2秒。 </li><li>孵育在酸性醇的样品（70毫升乙醇的70％和700微升的1M盐酸）为7秒。 </li><li>在洗净的自来水样本进行两次2秒。 </li><li>孵育在70毫升碳酸锂（1克碳酸锂在100毫升的蒸馏水）为7秒的组织样本。 </li><li>在洗净的自来水样本进行两次2秒。 </li><li>孵育样品在70毫升乙醇80％1分钟。 </li><li>转移到样品70毫升曙红-Y为15秒。 </li><li>孵育它们在70毫升96％乙醇中1分钟。用新鲜的酒精重复此步骤一次。 </li><li>孵育在70毫升的无水乙醇（乙醇绝对无水的）组织样品1分钟。用新鲜的酒精重复此步骤一次。 </li><li>孵育在70毫升35毫升无水乙醇的35毫升无水二甲苯中1分钟的混合物的样品。 </li><li>转移样品到70毫升无水二甲苯中1分钟。在70毫升新鲜绝对二甲苯重复此步骤5分钟。 </li><li>添加80μL的合成树脂的结肠组织样品，用盖玻片覆盖它们，让它们干燥在室温24小时。最后，分析使用明场显微镜8样本。 </li></ol></li></ol>通过免疫荧光分析7结组成<ol><li>如步骤5.7所述，准备组织切成8微米厚截面使用低温恒温器。 </li><li>固定，并在-20℃下透化用96％的乙醇结肠横截面为20分钟。 注意:所有后续孵育应于RT下进行，除非另有说明，在潮湿的暗箱，防止干燥和荧光染料褪色。 </li><li>风干样品，然后冲洗一下5分钟3次，每次用1×PBS </li><li>用封闭溶液（含有的PBS 0.01％吐温和2％BSA）中2小时孵育样品。 </li><li>除去封闭溶液，并用PBS-0.01％吐温冲洗10分钟。 </li><li>在此期间，稀释的初级抗体，以在PBS-0.01％吐温所需浓度。 </li><li>除去洗涤液，应用来自前面步骤的抗体溶液中，并在加湿箱在4℃孵育过夜。 </li><li>除去抗体溶液并用PBS-0.01％吐温用去离子水洗涤3次，每次5分钟，一次10分钟，用非常温和搅拌。 </li><li>虽然洗涤，离心氟ochromeconjugated，种特异性在4℃在14,000rpm xg离心20分钟，二次抗体。 </li><li>稀释而不沉淀的二级抗体通过在PBS-0.01％吐温的提供者所指示的，应用它们，并在室温下孵育1小时。 </li><li>重复步骤7.8和完全去除清洗液成为可能。 </li><li>吸管4μL的防褪色剂在幻灯片上每个样品。 </li><li>盖上盖玻片样品。 </li><li>空气干燥1小时。 </li><li>用指甲油密封幻灯片。玻片可以储存在-20℃直至进一步分析。 </li><li>共聚焦激光显微镜分析8。 </li></ol> 8.测定氧化应激乙锭氧化<ol><li>经过7天DSS结肠炎，在厚度为5微米的低温恒温器在步骤5.7所描述和切结肠段准备组织。安装的横截面到玻片上用聚-L-赖氨酸溶液处理并孵育样品机智H 5在水中二氢乙（DHE），在37℃下在黑暗中30分钟的微米。 </li><li>用PBS（pH为7.6），用于与在RT轻轻搅动每15分钟三次洗样品。空气干燥该样品，并添加封固剂，以保护荧光的下降。观察激光扫描共聚焦显微镜氧化乙啶（放大40倍，568波长）的荧光。 </li></ol> 9. MPO含量分析白细胞募集<ol><li>经过7天DSS结肠炎，收集结肠组织和使样品均质化 - 用在冰上均化器（200 400毫克）用1mL十六烷基的（HTAB）缓冲液（在50mM 0.5％HTAB磷酸盐缓冲液，pH 6.0）。 </li><li>用1 HTAB毫升缓冲漂洗均化的尖端两次以包括溶解物所有组织和超声处理它在40％振幅五次，每次10秒。冻融在液氮中三次。 </li><li>离心在14,000rpm xg离心15分钟，回收上清。通过混合柠檬酸钠（在水中的1M），0.1毫升过氧化氢的ABTS，5毫升制备2,2&#39;-连氮基 - 双（3​​-乙基苯并噻唑-6-磺酸）（ABTS）溶液，和45毫升双向的-蒸馏水。 </li><li>混合0.1mL的含0.1 ABTS毫升溶液每种上清液在96孔平底板上。 10分钟后，测量在使用分光光度计460nm处的吸光度。 </li></ol> 10.评估促炎性细胞因子的通过PCR表达<ol><li> 组织均质 <ol><li>使用功率匀浆均质结肠组织样本50到100mg的1ml的酸性硫氰酸胍 - 苯酚 - 氯仿混合物。 </li><li>孵育，在室温下15分钟。 </li></ol></li><li> 相分离 <ol><li>加入0.2毫升氯仿，剧烈摇晃30秒。 </li><li>孵育在4℃下30分钟。 </li><li>离心机以12,000 xg离心在4℃下60分钟。 </li><li>转移的UPPER水相到一个新的管（〜500μL）。 </li></ol></li><li> RNA沉淀和再悬浮 <ol><li>加入0.5毫升异丙醇中并在4℃孵育样品60分钟。 </li><li>离心机以12,000 xg离心在4℃下30分钟。 </li><li>彻底去除上清。 </li><li>加入1毫升乙醇的75％和旋涡。 </li><li>离心机以12,000 xg离心在4℃下30分钟。 </li><li>去除上清。 </li><li>使剩余的乙醇风干3-5分钟。 注意:不要让RNA沉淀完全干燥，因为这将极大地降低其溶解度是很重要的。 </li><li>重新溶解在0.3毫升酸二乙酯的（DEPC）处理的水的颗粒。立即转录RNA样品为cDNA（更稳定，见步骤10.5），或储存在-80℃。 </li></ol></li><li> RNA定量 <ol><li>稀释的RNA 1微升99微升DEPC处理过的水。 </li><li>阅读日在260nm和使用UV / VIS分光光度计来确定样品浓度和纯度280纳米ë外径。 </li></ol></li><li> cDNA合成 <ol><li>与寡的1微升（dT）的12-18（0.5微克/微升）和DEPC处理的水（12微升的总体积）在无RNase无菌试管混合的RNA样品（1-5微克）。 </li><li>孵育在70℃下10分钟。 </li><li>加入2微升10X PCR缓冲液，50毫1微升的MgCl 2，10毫dNTP混合物1μL的，和0.1M二硫苏糖醇的2微升（DTT）。 </li><li>孵育在4℃下5分钟。 </li><li>加逆转录酶的1微升并在42℃下孵育5分钟。 </li><li>孵育在70℃下15分钟。 </li><li>孵育在4℃下15分钟。 cDNA可被储存在-20℃直至进一步使用。 </li></ol></li><li> PCR <ol><li>拌2.5微升10X PCR缓冲液中，有25毫1.5微升MgCl 2的，10毫的dNTP 0.5μL混合，0.25μ大号Taq DNA聚合酶，引物各0.25微升（10μM）和cDNA（100毫微克），以及无菌水至25微升的总体积。 </li><li>上的96孔热循环仪上运行样本。为了防止基因组DNA的扩增，正向和反向引应位于不同的外显子:IL1β-FW:GCAACTGTTCCTGAACTCAACT和IL1β-RE:TCTTTTGGGGTCCGTCAACT; IL6-FW:CCTTCCTACCCCAATTTCCAA和IL6-RE:AGATGAATTGGATGGTCTTGGTC; KC-FW:TGTCAGTGCCTGCAGACCAT和KC-RE:CCTGAGGGCAACACCTTCA;和肌动蛋白FW:TATCCACCTTCCAGCAGATGT和肌动蛋白-RE:AGCTCAGTAACAGTCCGCCTA。使用以下PCR条件:95℃5分钟，随后30个循环的95℃15秒，58℃30秒，72℃30秒，再加上在72℃最后延伸10分钟。 </li></ol></li></ol>

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作者什么都没有透露。

为了使用在临床研究中的营养补充品，所述的这类补充剂的安全必须小心地在动物研究中体内评价。在结肠炎的情况下，类似于IBD的临床征兆若干适当的动物模型已经建立，包括使用的DSS，TNBS，或乙酸化学模型;敲除（KO）等车型IL10-KO;和使用过继T细胞转移19-21免疫细胞介导的结肠炎。结肠炎的DSS模型是诱导结肠炎类似于人类IBD的许多疾病的症状和在研究调查结肠炎22的分子机制过多已被使用的快速和可靠的方法。此外，DDS大肠炎是可逆的，因而也允许组织愈合的研究colitogenic刺激已被移除之后。该协议提供的详细介绍已经在我们的实验室8被先前的优化DSS结肠炎模型。 <p类="jove_content"&gt;一个缺点是，DSS浓度的优化，必须在每一个实验室进行，因为环境因素大大影响疾病的发展和进展23。另外，决策支持系统必须在一个分子量范围40 - 50 kDa的诱导结肠炎。对于没有经验的研究人员，这将是重要的发展以眼的大便性状及肛周出血DAI参数的公正的评估。如果评估由雏研究者进行的，它是推荐的是有一个同事证实评估，最好在盲方式。 以分析大分子肠上皮通透性，三种主要方法已被用于在整个文献:伊文思蓝染料测定中，如这里描述4- kDa的FITC-葡聚糖在肠袢滴眼;和4- kDa的FITC-葡聚糖13,24,25的供给。在后一种测定法中，FITC-葡聚糖是通过强饲法供给，和FITC葡聚糖的横渡上皮和渗漏到血液流量从血清样品荧光测定进行测定。在该测定中，整个胃肠道的渗透性被测量，因为示踪剂传递从食道到直肠一路和大部分示踪剂将到达结肠之前通过。在循环模型中，渗透率在用于循环（通常是小肠）25密闭肠区域中测量。与此相反，伊文思蓝测定具有仅在结肠上皮通透性测量中，由于示踪剂直接灌输到整个结肠的优点。因此，使用该试验中，我们可以得出这样的结论结肠上皮是由饮食中补充保护。 它始终是重要的记录提取后使用其他检测组织前结肠重量和长度，因为一些数据（ 例如，泄露伊文思蓝的通透性ASSAy）的每克组织的表达。此外，重量与长度比率是组织损伤26的一个独立的读出。在一般情况下，组织形态是使用用苏木精和曙红染色的石蜡包埋的组织进行分析，如这里所述。石蜡包埋的优点是，该组织形态好得多保守相比其他嵌入方法。这允许不同的细胞类型的粘膜内的明确的识别（ 即，免疫细胞，上皮细胞，杯状细胞， 等 ）和组织改变的结肠炎中的分析，如水肿形成，白细胞浸润和上皮心尖糜烂。另一方面，石蜡具有的缺点是免疫荧光染色后，才能费时表位恢复执行。因而，我们总是准备嵌入在OCT化合物中冷冻不同组织活检。这样组织样本可使用低温恒温器很容易地切开并显示AUT低水平ofluorescence。我们用乙醇固定通过脱水，因为它也没有与肌动蛋白丝干扰（如甲醇那样），也不会触发自发荧光（如PFA那样）。使用这些技术，我们发现，抗炎和抗氧化的饮食补充剂可结肠炎（比较图2）期间改善组织形态和结架构。 过度中性粒细胞募集是受生产趋化因子的响应于炎症17诱导的结肠炎中的关键事件。中性粒细胞募集是一种生理事件，因为嗜中性粒细胞向去除炎性球杆的和随后的伤口愈合。但是，如果此先天免疫应答没有适当的控制和终止，在粘膜的中性粒细胞可以通过释放蛋白酶和活性氧向组织损伤。嗜中性粒细胞含有MPO，可以很容易地测量，并使用量化比色测定，其中的MPO的量成正比的中性粒细胞的数量。 图3A示出了结肠炎和饮食干预期间中性粒细胞募集的增加可以防止这种过度和不受控制的免疫反应。按照在粘膜丰富嗜中性粒细胞的存在，氧化应激结肠炎（ 图3B）中增加。 我们的饮食中补充明确保护免受氧化应激，这是最有可能的中性粒细胞募集减少的后果粘膜。如前所述，中性粒细胞趋化因子吸引和生产趋化因子是由通过各种细胞类型结肠炎中分泌的促炎性细胞因子诱导的。因此，我们假设中性粒细胞募集减少是降低细胞因子和趋化因子产生的后果。 图3C表明，促炎细胞因子IL-1β和IL-6，以及趋化因子KC，DSS诱导的结肠炎期间上调。饮食中补充反转的IL-6的水平恢复到对照水平和改善的产量增加的IL-1β和KC的。值得注意的是，KC为小鼠嗜中性粒细胞的最重要的化学引诱物。 因此，它是诱人的推测组织形态的观察保存是由于减少了中性粒细胞浸润，这一点，反过来，KC的产量减少的结果。判断的RNA生产通过RT-PCR的DSS处理过的组织中是有问题的，当残余的DSS存在于分离的RNA。这是因为DSS同时抑制逆转录酶和Taq-聚合酶。如果没有扩增可以实现的，这将是必要的LiCl介导的沉淀以进一步纯化的RNA样品，如别处27所述。 总之，我们描述在期间DSS结肠炎和如何d表示组织损伤的分析细节不同的方法豪悦国际可以通过膳食补充剂得到改善。知识从这样的动物实验获得可以证明，建立患者群的研究在人类IBD分析的补充的保护作用。然后从临床研究获得的数据可能会开辟新的途径，制定IBD的处理方案治疗策略。

结肠炎是能引起腹泻和腹痛结肠的炎性病症。结肠炎可以是急性，响应于感染或压力，或者它可以发展为慢性疾病，如溃疡性结肠炎（UC），其属于下组的炎性肠疾病（IBD）的。尽管UC的临床症状得到了很好的描述，发病机制仍然知之甚少1。据专家们承认，UC是多因素疾病，与遗传突变和异常的免疫反应扮演一个重要的角色2。然而，环境因素，如生活和营养的样式，也有助于疾病的发生和发展3。 不幸的是，IBD是不可治愈的，但也有旨在缓解临床症状很多的治疗方案。目前的治疗包括抗炎药，如柳氮磺吡啶和皮质类固醇;免疫抑制剂，如azathioPRINE;单克隆抗体是捕获促炎细胞因子，如肿瘤坏死因子α（TNF-α），或嵌段粘附分子，如整，以降低白细胞募集过度;并针对触发促炎症途径，如Janus激酶（JAK）4激酶抑制剂。并非所有的患者对所有治疗，这样的治疗策略必须个体化5。此外，大多数的这些治疗药物干扰了代谢和免疫反应，常常引起严重的副作用。出于这个原因，替代治疗方案进行了研究。 替代治疗包括益生菌和营养补充剂，已在动物模型和临床试验中成功6,7-不同程度的应用。我们最近发现，不同的单营养补充剂，作为抗氧化维生素或ω3-多不饱和脂肪酸，例如应用（PUFAS），不如这种补充剂的组合在减轻结肠炎和心血管疾病的症状8,9中的应用。这些研究在小鼠中进行，因此，临床研究必须在人类中进行，以确定这些结果是否也将是适用于人类。临床研究开始之前，新的治疗方案的有效性和安全性，必须在动物模型中进行评估。 对于IBD的葡聚糖硫酸钠（DSS）的模式已被广泛用于研究疾病发展的机制以及药物和营养补充剂6,10的有益效果。在大多数研究中，只有一种急性疾病超过七天被诱导的时间段;然而，在这些动物中观察到的临床症状密切类似于那些在IBD患者中观察到（ 即，血性腹泻，体重减轻，上皮功能障碍，和免疫细胞浸润）10。 DSS诱导糜烂在粘膜，导致屏障功能障碍和增加肠道上皮通透性11。确切机制尚不清楚。然而，研究表明，DSS与中链长度的脂肪酸的相互作用形成的纳米lipocomplexes是能够进入上皮细胞和诱导炎症信号传导途径12。在本文中，我们详细描述如何结肠炎是引起并在小鼠中，补充剂如何营养通过管饲法应用到确保在每个动物的恒定剂量分析，以及如何进行检查各种结肠炎症状例如补充剂的效果。

<table><tbody><tr><td>0.3% of gelatin&amp;nbsp;</td><td>Bioxon</td><td>158</td><td></td></tr><tr><td>10% formaldehyde</td><td>J.T. Baker</td><td>2106-03</td><td></td></tr><tr><td>96-well plate with flat bottom</td><td>Corning</td><td>3368</td><td></td></tr><tr><td>Absolute ethanol&amp;nbsp;</td><td>J.T. Baker</td><td>9000-03</td><td></td></tr><tr><td>Absolute xylene</td><td>J.T. Baker</td><td>9490-03</td><td></td></tr><tr><td>ABTS</td><td>Sigma Aldrich</td><td>H5882</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>9048-46-8</td><td></td></tr><tr><td>ColoScreen</td><td>Helena Laboratories</td><td>5072</td><td></td></tr><tr><td>Corabion (Kindly provided by Merck, Naucalpan, Mexico)</td><td>Merck</td><td></td><td></td></tr><tr><td>Dextran Sulfate Sodium Salt</td><td>Affymetrix</td><td>9011-18-1</td><td>(M.W. 40,000-50,000)</td></tr><tr><td>Dihydroethidium</td><td>Life Technology</td><td>D11347</td><td></td></tr><tr><td>Eosin-Y&amp;nbsp;</td><td>J.T. Baker</td><td>L087-03</td><td></td></tr><tr><td>Evans blue</td><td>Sigma-Aldrich</td><td>314-13-6</td><td></td></tr><tr><td>Feeding&amp;nbsp; needle</td><td>Cadence Science Inc.</td><td>9921</td><td></td></tr><tr><td>Glass slide rack with handle</td><td>Electron Microscopy</td><td>70312-16</td><td></td></tr><tr><td>Glass Slides</td><td>Corning</td><td>2947</td><td></td></tr><tr><td>Harris hematoxylin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H3136</td><td></td></tr><tr><td>Hexadecyltrimethyammonium (HTAB)</td><td>Sigma Aldrich</td><td>H6269</td><td></td></tr><tr><td>Histosette (Embedding cassette)</td><td>Simport</td><td>M498.2</td><td></td></tr><tr><td>Hydrochloric acid 1 M</td><td>J.T. Baker</td><td>9535-62</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma Aldrich</td><td>H3410</td><td></td></tr><tr><td>Ketamine</td><td>PiSA Agropecuaria</td><td>Q-7833-028</td><td></td></tr><tr><td>Liquid paraffin&amp;nbsp;</td><td>Paraplast</td><td>39501-006</td><td></td></tr><tr><td>Lithium carbonate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>2362</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-dimethylformamide</td><td>J.T. Baker</td><td>68-12-2</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-acetylcysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td></td></tr><tr><td>Plastic cubes</td><td>Electron Microscopy</td><td>70181</td><td></td></tr><tr><td>Poly-L-Lysine Solution</td><td>Sigma-Aldrich</td><td>25988-63-0</td><td></td></tr><tr><td>Prism 5 statistical software</td><td>GraphPad Software</td><td>Prism 5</td><td></td></tr><tr><td>Saline Solution 0.9% NaCl</td><td>CS PiSA</td><td>Q-7833-009</td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma-Aldrich</td><td>W302600</td><td></td></tr><tr><td>Synthetic resin&amp;nbsp;</td><td>Poly Mont</td><td>7987</td><td></td></tr><tr><td>Taq DNA polymerase</td><td>Invitrogen</td><td>11615-010</td><td></td></tr><tr><td>Tissue-tek. O.C.T Compound</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>Tuberculine Syringe</td><td>BD Plastipak</td><td>305945</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>9005-64-5</td><td></td></tr><tr><td>VECTASHIELD Antifade Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>Xylazine</td><td>PiSA Agropecuaria</td><td>Q-7833-099</td><td></td></tr></tbody></table>

analyzing beneficial effects of nutritional supplements on intestinal epithelial barrier functions during experimental colitis

炎症性肠疾病（IBD），包括克罗恩病和溃疡性结肠炎，是肠内的慢性复发病症。他们造成严重的问题，如腹部绞痛，血性腹泻，体重减轻，在受影响的个人。不幸的是，目前还没有治愈的是，和治疗仅旨在减轻症状。目前的治疗包括抗炎和免疫抑制药物，可能导致严重的副作用。这是值得的替代治疗方案，如营养补充剂，不引起副作用的搜索。他们在临床研究中的应用之前，这种化合物必须进行严格测试在动物模型的有效性和安全性。一个可靠的实验模型是在小鼠葡聚糖硫酸钠（DSS）结肠炎模型，其再现许多的溃疡性结肠炎在人体中的临床症状。我们最近应用该模型以测试含营养补充剂的有益效果维生素C和E，L-精氨酸，和ω3多不饱和脂肪酸（PUFA）。我们分析了各种疾病参数，发现这补充能够改善水肿形成，组织损伤，白细胞浸润，氧化应激和促炎性细胞因子，导致在疾病活动指数的整体改善。在这篇文章中，我们详细使用C57BL / 6小鼠的DSS结肠炎模型的营养补充品的正确使用，以及如何疾病参数，如组织学，氧化应激和炎症进行评估解释。分析不同饮食补充剂的有益效果可接着最终打开的替代治疗策略，减轻IBD的症状和/或延长缓解期的相位，而不会引起严重的副作用的发展新的途径。

饮食补充剂可以从DSS结肠炎保护抗炎和抗氧化的饮食补充剂，例如维生素C，维生素E，所述一氧化氮（NO）-source L-精氨酸，和ω3多不饱和脂肪酸（ω3多不饱和脂肪酸），已被用于在动物不同的成功模型以减轻炎性疾病3,6,15的迹象。营养不良，氧化应激和促炎细胞因子的可触发的急性和慢性结肠炎3。在先前的研究中，我们证明了微量营养素的组合显示出对体内 DSS诱导的结肠炎的保护作用。在这里，我们提供这些效果如何测量8详细的协议。首先，我们通过确定疾病活动指数（DAI），包括减肥的三个参数的分析对疾病进展的营养补充品的效果，大便性状及肛周出血。代表性的结果表明，DSS处理导致不断增加的DAI，符合市场预期，达到对天七（ 图1A）最多8.2±0.46。抗炎饮食补充剂延迟结肠炎的发病，但在稍后的时间点，当结肠炎是非常强的保护作用微乎其微，不再具有统计学显著，这表明当结肠炎还不太强，饮食补充剂才有效。重要的是，膳食干预可以防止在肠上皮通透性的DSS诱导的增加，这是结肠炎的（ 图1B）的一个重要特征。 DSS结肠炎导致减少结肠的长度，并且这缩短也由抗炎和抗氧化饮食补充物（ 图1C）改善。因此，结肠炎的临床症状的确可以通过饮食成分的改变缓解。 分析为什么结肠炎进展是接收饮食中补充动物不太严重，组织形态，苏木/伊红（H / E）石蜡包埋远端结肠组织染色后查处。对照样品表现出正常的形态（ 图2A，上图），而用3.5％DSS处理的动物表现出结肠炎的典型体征（ 即，白细胞浸润，水肿形成，隐窝发育不良，和溃疡; 图2A，中图）。附注，饮食补充剂明显缓解结肠炎引起的形态学变化并行处理，与整体形态，这是更具有可比性对照组（ 图2A，下图）。这些观察结果非常相似，已经报道用于益生菌13和清楚共治疗表明，饮食习惯的改变可以抵消结肠炎的发展。 我们已经知道，结肠炎引起的肠道上皮通透性可以通过饮食补充抵消。通透性增加是IBD的一个标志，它是在所造成的稳定粘着结（AJ）和紧密连接（TJ）的拆卸很大一部分。由连接蛋白的内化上皮细胞接触不稳定是由促炎性细胞因子，其结肠炎16中大量产生的诱导。我们发现，通过远端结肠组织的免疫荧光染色，该TJ分子紧密连接-1（ZO-1）和AJ分子E-钙粘蛋白的DSS诱导的结肠炎中被内化和这些蛋白质的共定位部分是丢失在隐窝上皮细胞接触（ 图2B）。一世mportantly，E-钙粘蛋白和当DSS处理的小鼠共同处理与抗炎和抗氧化饮食补充物（ 图2B）ZO-1减少的内化。整体，隐窝和结结构更媲美当施加饮食补充控制，这表明所观察到的保护作用至少部分是由于组织结构的保存。 饮食干预可以抵消炎症中性粒细胞募集，氧化应激和促炎细胞因子的生产 IBD的另一个特点是不受控制的中性粒细胞浸润。招募嗜中性粒细胞通过产生和释放的活性氧（ROS）的和蛋白酶17向组织损伤。使用MPO活性分析，我们发现强大的中性粒细胞募集在结肠响应DSS，这是抵消饮食补充剂（ 图3A）。 氧化应激是结肠炎的另一重要特征，引起活性氧从招募嗜中性粒细胞的释放。因此，我们分析了结肠截面ROS的产生。 图3B说明了DSS处理强烈氧化应激增加。值得注意的是，饮食习惯的改变完全阻止DSS诱导的氧化应激（ 图3B），这是最有可能的中性粒细胞募集（ 图3A）减少的结果。 促炎性细胞因子和趋化因子的强烈由各种细胞类型结肠炎18过程中产生的。这样的炎症介质有助于我们已经知道可以通过改变饮食衰减中性粒细胞募集和连接蛋白内化的特点。通过RT-PCR表达分析表明，DSS增加IL1β的表达，IL-6，和角化细胞衍生的趋化因子（KC），如预期的（ 图3C），并且消炎饮食补充减少此DSS诱导的增加（ 图3C）。这些数据意味着，补充营养，的确可以起到抵消IBD的临床症状。 <img alt="图1" src="/files/ftp_upload/55095/55095fig1.jpg" /> 图1.营养补充剂可以缓解疾病活动指数，肠上皮通透性和大肠缩短。 （A）中的疾病活动指数（DAI，值从0 - 12）包括体重减轻，粪便稠度，和肛周出血的三个参数，并在控制（CTRL），结肠炎，和结肠炎的实验组每天记录+营养补充品（结肠炎+增刊）。对照组没有显示肠炎的迹象，导致0 throughou的戴t为试验期。 N =每组5只。 （B）的肠上皮通透性通过伊文思蓝泄漏在上述实验组测量，并表示为每克组织的620纳米的吸收。 N =每组8只。所有数据表示为平均值±平均值（SDM）的标准偏差。 * P &lt;0.05; ** P &lt;0.01; *** P &lt;0.001。统计通过单向ANOVA计算的。 （C）治疗7天后的各实验组的整个结肠的代表性图像。在左边的刻度是厘米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55095/55095fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55095/55095fig2.jpg" /> 图2.组织形态和结架构可在结肠炎更好地保存营养申请时，苏pplements。 （A）中从各实验组的远端结肠组织活检的5微米石蜡截面用苏木精/伊红染色，并针对结肠炎的典型症状，如水肿形成（箭头），白细胞浸润（箭头）分析和溃疡（*）。结肠炎+增刊的总体组织形态。更有一批类似于较结肠炎组的控制，对证明DSS结肠炎的整体保护作用。图像代表每组3个独立的组织筹备工作。图像用一个10X物镜拍摄。栏= 100微米。 （B）的各组的8微米结肠冷冻切片用抗ZO-1抗体（绿色）和E-钙粘蛋白（红色）染色。共同定位（在合并后的图像黄色;箭头）E-cadherin和ZO-1在隐窝上皮细胞的接触，这是结肠炎过程中丢失的，大多保存在结肠炎+增刊。组。图像代表3独立的组织筹备工作。栏= 50微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55095/55095fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55095/55095fig3.jpg" /> 图3.中性粒细胞募集，氧化应激和促炎细胞因子的生产可以通过营养补充剂减少。 （A）髓过氧化物酶（MPO）活性测定白细胞募集的金额将分析结肠组织实验组:对照组（CTRL），结肠炎，和结肠炎+营养补充（结肠炎+增刊）。 N =每组5只; * P &lt;0.05。数据显示为平均值±SDM。统计通过单向ANOVA计算的。 （B）中的氧化乙锭的荧光，在共同的8微米的冷冻切片中的红色描绘为氧化应激的量度LON组织，激光共聚焦显微镜记录。图像代表了3个独立的组织筹备工作。栏= 100微米。 （C）中生产的促炎性细胞因子IL1β和IL-6和KC通过半定量RT-PCR在1.5％琼脂糖凝胶中确定的趋化因子（黑色和白色已还原为清楚起见）。 β肌动蛋白担任管家基因。的三个独立的cDNA制备的代表图像被示出。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55095/55095fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis at JoVE.com

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

炎症性肠病（IBD）的当前治疗的目的是减轻疾病的症状，但可能会导致严重的副作用。因此，替代战略被动物结肠炎模型研究。在这里，我们介绍如何补充营养的临床体征IBD的有利影响在这样的肠炎模型进行了分析。

分析营养补充剂的有益作用于肠上皮屏障功能在实验性结肠炎

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

analyzing-beneficial-effects-nutritional-supplements-on-intestinal

Nanyang Technological University

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Medicine

Unit 1

Drugs for Chronic Bowel Disorders

SCIENTISTS IN ACTION

12.2K 次观看.  Center for Research and Advanced Studies of the National Polytechnic Institute. 炎症性肠病（IBD）的当前治疗的目的是减轻疾病的症状，但可能会导致严重的副作用。因此，替代战略被动物结肠炎模型研究。在这里，我们介绍如何补充营养的临床体征IBD的有利影响在这样的肠炎模型进行了分析。 

视频: Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

二硝基苯磺酸/ TNBS结肠炎模型：提供洞察炎症性肠病和膳食脂肪的影响

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

科研

医学

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 ...

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

基于内显微镜的方法，用于评估肠道渗透性和上皮细胞脱落性能

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

免疫与感染

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 ...

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

研究活体器官的肠道屏障分解

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

生物学

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Gut microbiota dysbiosis is thought to exert a role in the progression of colitis. However, the precise standards for probiotic administration in alleviating colitis remain undefined. Most analysis methods rely on limited diversity and abundance of gut microorganisms. Therefore, observational studies cannot establish causation. In this study, we applied antibiotic-induced pseudo-germ-free mice to investigate the role of gut microbiota in regulating the probiotic effects of Bacillus&#160;cereus (B. cereus)&#160;on dextran sulfate sodium (DSS)-induced colitis in mice. This process allows for evaluating the bidirectional regulating effect of B. cereus&#160;supplementation on health and provides stable and reproducible results. Here, the detailed protocols for B. cereus cultivation, gavage operation, stool collection, and antibiotic clearance treatment on colitis mice are provided. The optimization methods are also applicable for other chronic inflammatory-associated disorders. The results showed that B. cereus administration decreased body weight loss, colon length shortening, disease activity index, and histopathological scores. However, treatment with antibiotics suppressed the positive effect of B. cereus on colitis. These results indicate that gut microbiota are needed for the alleviating effects of B. cereus on colitis. Therefore, exploring the beneficial effects of probiotics in this research is a promising approach for developing novel treatment strategies for alleviating the symptoms of chronic inflammatory-associated disorders.

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Here, we present a protocol to establish the antibiotic-induced pseudo-germ-free dextran sulfate sodium-induced colitis mouse model to investigate the role of gut microbiota in regulating the positive effects of Bacillus cereus on colitis.

Gut microbiota dysbiosis is thought to exert a role in the progression of colitis. However, the precise standards for probiotic administration in ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

使用荧光素-异硫氰酸酯标记的葡聚糖评估小鼠肠道屏障完整性

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

肠道炎症对已建立的小鼠结肠样的上皮影响

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

作者聚焦:研究体外肠道炎症对已建立的小鼠结肠样蛋白的上皮效应  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

研究体 外 肠道炎症对已建立的小鼠结肠样的上皮影响

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

分析营养补充剂的有益作用于肠上皮屏障功能在实验性结肠炎

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此视频中的章节

二硝基苯磺酸/ TNBS结肠炎模型：提供洞察炎症性肠病和膳食脂肪的影响

体外肠囊评估黏膜通透性在胃肠道疾病模型

基于内显微镜的方法，用于评估肠道渗透性和上皮细胞脱落性能

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

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

研究活体器官的肠道屏障分解

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

使用荧光素-异硫氰酸酯标记的葡聚糖评估小鼠肠道屏障完整性

研究体外肠道炎症对已建立的小鼠结肠样的上皮影响

分析营养补充剂的有益作用于肠上皮屏障功能在实验性结肠炎

摘要

探索更多视频

此视频中的章节

二硝基苯磺酸/ TNBS结肠炎模型：提供洞察炎症性肠病和膳食脂肪的影响

体外肠囊评估黏膜通透性在胃肠道疾病模型

基于内显微镜的方法，用于评估肠道渗透性和上皮细胞脱落性能

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

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

研究活体器官的肠道屏障分解

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

使用荧光素-异硫氰酸酯标记的葡聚糖评估小鼠肠道屏障完整性

研究体 外 肠道炎症对已建立的小鼠结肠样的上皮影响

研究体外肠道炎症对已建立的小鼠结肠样的上皮影响