这项工作是支持的补助金BAV的克罗恩病和结肠炎基金会和加拿大卫生研究院（CIHR）加拿大（CCFC）。 GB CIHR研究生奖学金的资助。 BAV是的儿童肠和肝脏疾病（儿童）基金会主席儿科IBD的研究和加拿大研究主席在儿科胃肠病学。

1。 枸橼酸rodentium接种物的制备和小鼠灌胃<ol><li>准备和高压灭菌卢里亚Bertani肉汤（LB）。 </li><li>获得可行的C. LB琼脂平板上，用无菌接种环或移液器吸头rodentium从冷冻甘油股票和条纹上。孵育，37℃培养过夜。 ，接种3毫升无菌的LB肉汤猎鹰文化的LB平板上的菌落，用接种环或吸液管头管。应做的接种物的制备，使用无菌技术。 </li><li>孵育C. rodentium文化有氧，在37°C在200转台式培养摇床过夜。接种LB培养基过夜培养后出现混浊。 </li><li>使用一个灯泡放倒连接到1 ml注射器管饲法用100μl的隔夜C.各小鼠灌胃针rodentium文化。鼠标轻轻颈背，紧紧抓住它的脖子和背部松弛的皮肤用拇指和手指。回力动物的头，用食指固定头部和整顿灌胃针插入食道。 </li><li>保持在直立位置的鼠标和指示灌胃针沿它的嘴侧以及全球舌头灯泡小费。轻轻走过针沿着屋顶的嘴和食道推进。如果有阻力，在此过程中感受到，删除灌胃针，并重新将其插入。 </li><li>慢慢注入100μL的细菌接种灌胃针，轻轻取出。监控呼吸速率的鼠标行为后返回到它的笼子。 </li></ol>注： <ul><li>在步骤2和3中，还准备猎鹰培养管中，使用无菌的LB肉汤中培养过夜，用3毫升的无菌技术，以确保有肉汤本身是无细菌污染。 LB培养基过夜培养后出现清晰的。 </li><l&gt;轻轻地搅动文化的Falcon管内装入注射器灌胃同等剂量的细菌传递给每一个鼠标，在步骤4中。 </li><li>应该被丢弃过夜培养，如果它已被超过24小时接种。 </li><li>所有C. rodentium感染和受感染的动物应进行住房在二级生物安全设施。 </li></ul> 2。测量结肠上皮屏障通透性C. rodentium感染的小鼠<ol><li>在期望的时间点感染后，测量屏障的完整性。 </li><li>在测定的当天，准备足够的4 kDa的荧光素异硫氰酸酯（FITC） -葡聚糖溶解在磷酸盐缓冲盐水（PBS）的浓度为80毫克毫升-1灌胃每只小鼠用150μl，并制备标准曲线。 </li><li>浮渣的鼠标和管饲法用150微升的FITC-葡聚糖。在这个时候食物从笼子里取出。 </li><li>麻醉小鼠后4小时gavaging，并收集尽可能多的（450微升）的血液通过心脏穿刺。将血液添加至终浓度为3％酸 - 柠檬酸葡萄糖在微量离心管，以防止凝血。安乐死小鼠，并收集在PBS中的结肠组织细菌计数（步骤3.1 - 3.5）和10％福尔马林固定的组织，并最终免疫荧光染色（步骤4.1 - 4.8）。 </li><li>附带的血液样本在离心机中以1,000 xg离心12分钟，在4℃下收集血清，并稀释至1/10和1/100的PBS。每个样品中加入100μl在这两个稀释到96孔板中复制。 </li><li>为了制备的标准曲线，稀原来的80毫克毫升-1 FITC-葡聚糖用管饲法在PBS的小鼠的浓度如下：800，400，200，100，50，25，12.5，6.25，和0微克/毫升。将100微升各浓度添加到96孔板中，一式三份。 </li><li>使用在激发荧光wav文件，量化各样品的荧光elength表示，485 nm和535 nm的发射波长。 </li><li>分析的原始数据，通过绘制标准曲线。输入的原始数据对每个样品所产生的标准曲线，以确定在每个样品中的浓度的FITC-葡聚糖的线入方程。 </li></ol>注： <ul><li>从第4步向前，保持血液样本，冰，尽量减少暴露在光线下。 </li><li>当测量的时间点之前，或在感染后7天，上皮屏障的完整性是最优的，这一点后，会发生严重的组织损伤。测量前屏障的完整性受到严重损害发生，让您透性C.确定最大的差异小鼠品系之间rodentium感染。 </li><li>确保未感染对照组小鼠上皮屏障的完整性测试。 </li></ul> 3。 C.组织中的细菌负荷测量rodentium感染的小鼠<ol><li>加入1毫升无菌PBS一个第二蒸压一个圆形的金属珠见底2 mL离心管中，用无菌技术。从每个动物收集的单个组织将被放置在单独的PBS管。前称量试管加入组织。 </li><li>删除从鼠标盲肠和结肠。分离盲肠，结肠和推动管腔内容，使用产钳。添加到PBS管的内容。分离后0.5厘米部分，从远端结肠和盲肠的10％福尔马林固定，切开剩余的结肠和盲肠和纵向管组织，用无菌PBS洗涤前。 </li><li>称量PBS管与组织，然后使用珠的搅拌器搅拌6分钟在30 Hz均质化样品。 </li><li>使用无菌技术，180微升无菌PBS中，每孔添加到96孔板中。加入20μl的每个均质样品，每一行的第一口井。搅拌均匀，连续稀释，从每个井获得加入20μl稀释10 -1（第一我们LL）到10 -6。板10微升每种稀释液从每个样品一式三份一个方形的底部与一个网格的LB平板上。孵育过夜，在37°C。 </li><li>计数菌落形成单位（CFU），然后平均3个计数，并记录稀释每个样品计数。乘以平均CFU 50，使用原来的1毫升样品为20微升，和由该样品进行计数得到的以CFU / ml的稀释因子。最后，除以组织CFU / g的重量，以确定。 </li></ol> 4。组织学评估和感染肠组织免疫荧光染色<ol><li>在步骤3.2收集组织。商店在福尔马林隔夜组织，然后用70％的乙醇。嵌入组织中的石蜡和削减5μm切片进行染色。用苏木精 - 伊红染色组织学评估，并进行免疫荧光染色，继续进行下面的步骤。 </li><li>要deparaffinize组织染色前，地点幻灯片一个科普林氏染色缸，放在65°C水浴10分钟。接下来，将幻灯片二甲苯洗涤四次，每2分钟。相片数，然后再水化与两个5分钟的在100％乙醇洗涤，然后由一个5分钟的洗涤在每个下列：95％乙醇，75％乙醇和蒸馏水2 0。这些清洗应在通风橱内进行，以避免暴露于有害气体。 </li><li>将幻灯片中的科普林氏罐预热的柠檬酸钠缓冲液，然后入蒸笼30分钟的地方，然后坐30分钟。此过程会破坏蛋白交联形成的福尔马林固定检索潜在的抗原位点。 </li><li>叠氮化用PBS洗涤5分钟，三次。干片和一个PAP笔创建一个疏水性的障碍，使局部组织染色试剂与周围组织的标志区。 </li><li>组织为1小时，在室温下用封闭缓冲液（ 例如：α-山羊血清）中的免疫染色的湿气室内座。 </li><li>稀释PRIM进制所需浓度的抗体，抗体稀释液。倒掉封闭缓冲液和组织中添加50〜100μl的主要抗体。孵育在4℃孵育过夜或在室温下搅拌2小时。 </li><li>叠氮化用PBS洗涤5分钟，三次。加入50-100微升的抗体稀释缓冲液中稀释到组织中，在室温下孵育1小时，在黑暗中的二次抗体。洗净用dH 2 O 5分钟，三次。 </li><li>脱水幻灯片，的DAPI延长黄金的安装介质和适用的盖玻片。查看幻灯片在荧光显微镜下。 </li></ol>注： <ul><li> ，PBS中与新鲜制备的PBS中，可以被取代的磷酰基叠氮化物，作为一种替代，从步骤4开始，以避免在洗涤介质中的细菌的生长。 </li></ul>

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</ol>

作者宣称，他们没有竞争的金融利益。

枸橼酸rodentium感染传染病的小鼠结肠炎的研究提供了一个有价值的模型。这种独特的模式允许两个宿主反应，以及细菌的致病特性的表征。这种模式的成功使用该协议的细节中列出的步骤。 有几个关键的步骤，在此协议时，请记住诱导结肠炎和分析反应。首先，准备一个新鲜的过夜C. rodentium在LB肉汤接种感染小鼠的成功。由于需要的剂量为10 8 -10 9生物体感染大部分品系的小鼠，如果接种物留在振动筛上或在室温下长时间的台式，也不会有足够的活的微生物培养中剩余感染后诱发结肠炎。同样重要的是搅拌细菌接种之前加载感染的注射器，以确保每个鼠标接收等剂量的细菌。当收集在总站的感染组织，完成淹没足够的容积的10％福尔马林结肠组织中所需的适当的组织固定发生。有人建议，组织被一个5毫升的小瓶中，而不是作为固定液到组织建议的比例为10:1到微量离心管中加入福尔马林。适当的固定是必不可少的购买组织学分析，以及这些组织的免疫荧光染色。同样重要的是要记住，不同的基因敲除株都会有不同的反应，诱导时，C. rodentium结肠炎。在感染的一个新菌株第一次，小鼠应密切监测，以确定一个人性化的终点的试验，如果需要的话。可以定义基于鼠标的响应上上皮屏障完整性，细菌载量，和组织学分析的时间点为应变感兴趣的感染。 由于电镀组织匀浆的LB平板上，以确定细菌负荷是不完全选择性的方法，进行PCR为C. rodentium特定基因的细菌菌落可以做，以区分C.上盘rodentium从其他bacterialcolonies的。另一个选项是在MacConkey琼脂板组织匀浆，这种介质是用于 C更有选择性比LB培养基rodentium。另一种方法是使用链霉素抗性野生型C. rodentium的应变与链霉素抗性菌株instead.Substitution将导致在此协议中描述的相同的结肠炎模型的感应。使用链霉素耐药株的好处是更容易分析的细菌负荷，组织匀浆从这些感染可镀上，而不是单靠LB琼脂LB琼脂辅以链霉素。这避免了潜在的增长的通讯ensal微生物盘上，使CFU计票过程更容易，更准确。另一种替代方法，而测量细菌负荷，是孵育板电镀后在LB琼脂稀释组织匀浆，在任37℃孵育过夜或在室温下2-3天，导致细菌生长较慢，计数前。 该协议列出所需的步骤来产生一个强大的感染性结肠炎模型的方法，以及用来描述C.在小鼠的rodentium感染。除了使用这个模型来检验病原 - 宿主相互作用和免疫反应，它也可以被用来研究如何细菌感染可以增加患结肠癌的风险。这是可以做到通过暴露C. rodentium感染小鼠的致癌物质氧化偶氮甲烷，研究如何感染上皮细胞增殖的影响，或基因参与上皮细胞的分化或肿瘤的发生发展15。我们ING在本协议中所述的感染性结肠炎模型，细菌数量还可以进行评估，在肠外网站，如肠系膜淋巴结，脾脏和肝脏。在这些器官中的较高的数字可能表明，被测试的小鼠品系的感染是高度敏感的。使用免疫荧光染色技术，对感染的反应几乎是无止境的一些机制可以探索。一旦熟悉这里详述的技术，该协议可以被修改，以收集组织测量其他端点，例如RNA的提取和评估的基因的表达水平，以更彻底地表征到 C的反应rodentium感染。

肠道细菌病原体感染引发胃肠道（GI）的炎症，以及肠道病理学与病理生理学，包括腹泻和肠上皮屏障功能障碍。细菌病原体的殖民他们的主机，以及具体的抵御这种感染的宿主反应，胃肠道的致病机制了解甚少，但最近的进步已经开始在肠道细菌感染的造型，以帮助我们了解这些过程。肠道细菌也被连接到的炎症性肠病（IBDs）。 IBDs克罗恩病（CD）和UC复杂疾病的病因不明的慢性肠道炎症和组织损伤，其特征。许多模型小鼠肠道炎症的存在，从在遗传修饰的菌株，如IL-10 - / - 小鼠的自发炎症的化合物，如葡聚糖硫酸钠（DSS）和d，化学挑战initrobenzene磺酸（DNBS）1。据推测，适应不良的慢性炎症反应在IBD患者在遗传易感个体发展后肠黏膜免疫系统的异常暴露的肠道细菌，因此感染性结肠炎模型的研究也提供了显着的潜力，确定潜在的致病主机肠道细菌的反应。 枸橼酸杆菌rodentium诱导的结肠炎是罕见的模式已感染性结肠炎，以及其特征在于1,3之一，允许对分析的宿主反应，以肠溶性的细菌和潜在的IBD发病机制的进一步理解，一个重要的步骤在开发新的预防和治疗方法。 C. rodentium是革兰氏阴性安装和谦虚（A / E），小鼠的特异性细菌性病原体是密切相关的人体重要病原体肠致病性大肠杆菌大肠杆菌 （EPEC），肠出血性大肠杆菌大肠杆菌 （EHEC）3-8。的家庭的A / E病原体密切附加到根尖盲肠和结肠上皮细胞的宿主细胞的膜，形成一种非侵入性的台座状结构的宿主细胞。口腔挑战C. rodentium 10 8 -10 9生物体产生一个强大的感染性结肠炎模型，其特征在于由结肠的隐窝，单核的免疫细胞浸润和杯状细胞耗竭3,4增生或伸长。初始站点的殖民统治，几个小时后，面临的挑战，是在盲肠修补程序，然后发展到远端结肠感染后2〜3天，3。在免疫功能正常的小鼠品系中，间隙实现的病原体感染后3〜4周1,3,4。然而，许多转基因的菌株， 即基因缺失或基因敲除小鼠（ - / - ），已被发现显示增加sed的感染导致夸张的损坏和/或慢性感染和炎症9-14的易感性。这在这些基因敲除株的感染性结肠炎模型，许多人缺乏与生俱来的信号蛋白，一直是不可或缺的分辨率的肠道感染和炎症的组成部分露出几个宿主蛋白。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>卢里亚肉汤</td><td> ABM </td><td> G247 </td><td>添加LB粉25克至1L水。高压灭菌后方可使用。 </td></tr><tr><td>广场底板与电网</td><td>费舍尔</td><td> 08-757-11A </td><td></td></tr><tr><td>猎鹰文化管</td><td>萨尔斯塔特</td><td> 62.515.006 </td><td></td></tr><tr><td>灯泡放倒灌胃针</td><td>精细科学工具</td><td> 18060-20 </td><td></td></tr><tr><td> 1ml注射器</td><td> BD Biosciences公司</td><td> 309659 </td><td></td></tr><tr><td> 4 kDa的FITC-葡聚糖</td><td>西格玛</td><td> FD-4 </td><td></td></tr><tr><td>柠檬酸</td><td>西格玛</td><td> C7129 &lt;/ TD&gt; <td></td></tr><tr><td>柠檬酸钠</td><td>费舍尔</td><td> S279-500 </td><td></td></tr><tr><td>葡萄糖</td><td>费舍尔</td><td> D16.1 </td><td></td></tr><tr><td>柠檬酸盐葡萄糖</td><td></td><td></td><td>的20mM ctiric酸，110 mM柠檬酸钠，5mM的葡萄糖</td></tr><tr><td>黑色96 - 孔板</td><td>费舍尔</td><td> 07-200-762 </td><td></td></tr><tr><td>金属珠（5毫米） </td><td> Qiagen公司</td><td> 69989 </td><td></td></tr><tr><td> 10％的福尔马林</td><td>费舍尔</td><td> 5F93-4 </td><td></td></tr><tr><td> 5毫升小瓶</td><td> DiaMed </td><td> STK3205 </td><td></td></tr><tr><td>苏木</td><td>费舍尔</td><td> H345-23 </td><td></td></tr><tr><td>曙红</td><td>费舍尔</td><td> E511-100 </td><td></td></tr><tr><td>二甲苯</td><td>费舍尔</td><tD&gt; HC700-1GAL </td><td></td></tr><tr><td>吐温20 </td><td>西格玛</td><td> P5927 </td><td></td></tr><tr><td>科普林氏染色缸</td><td> VWR </td><td> 47751-792 </td><td></td></tr><tr><td>柠檬酸钠缓冲液</td><td></td><td></td><td> 10mM柠檬酸钠，0.05％吐温20，pH 6.0的</td></tr><tr><td>巴氏笔</td><td> CEDARLANE </td><td> Mu22 </td><td></td></tr><tr><td>山羊血清</td><td>西格玛</td><td> G902-3 </td><td></td></tr><tr><td>牛血清白蛋白（BSA） </td><td>费舍尔</td><td> BP1600-100 </td><td></td></tr><tr><td>的Triton X-100的</td><td>西格玛</td><td> T8532 </td><td></td></tr><tr><td>叠氮化钠</td><td>西格玛</td><td> SZ002 </td><td></td></tr><tr><td>封闭缓冲液</td><td></td><td></td><td> 2％山羊血清，1％BSA，0.1％Triton X-100，0.05％吐温20，0.05％的氢氧化钠叠氮钠，0.01 M PBS，pH值7.2，混合储存于4°C。 </td></tr><tr><td>抗体稀释液</td><td></td><td></td><td> 0.1％Triton X-100，0.1％BSA，0.05％叠氮钠，0.04％EDTA </td></tr><tr><td>阻断缓冲液和抗体稀释液TIR </td><td></td><td></td><td>与上述相同的配方，但不添加的洗涤剂（TRITON X-100和吐温20） </td></tr><tr><td>延长Gold抗淬灭试剂与DAPI </td><td> Invitrogen公司</td><td> P-36931 </td><td></td></tr><tr><td>盖玻片</td><td>费舍尔</td><td> 12.54SE </td><td></td></tr><tr><td>台式孵化振动筛</td><td> Barnstead实验室线</td><td>最大Q4000 </td><td></td></tr><tr><td>荧光计</td><td> Perkin Elmer公司</td><td> Victor2D </td><td></td></tr><tr><td>冷冻离心机</td><td> Beckman Coulter公司</td><td>微型离心22R</td><td></td></tr><tr><td>汽船</td><td> Black＆Decker公司</td><td></td><td></td></tr><tr><td>荧光显微镜</td><td>蔡司</td><td> Axio上Image.Z1 </td><td></td></tr></tbody></table>

the citrobacter rodentium mouse model: studying pathogen and host contributions to infectious colitis

该协议列出的步骤需要产生一个强大的感染性疾病和肠炎的模型，以及用来描述枸橼酸rodentium感染的小鼠的方法。C. rodentium是一种革兰氏染色阴性，小鼠特异性细菌性病原体，临床上重要的人类病原体肠致病性大肠杆菌是密切相关的大肠杆菌和肠出血性大肠杆菌大肠杆菌 。一旦感染C. rodentium，免疫小鼠患上温和和短暂的体重减轻和腹泻。组织学观察，肠隐窝伸长率，免疫细胞浸润，杯状细胞耗竭。实现间隙感染后3〜4周。肠上皮屏障的完整性，细菌量，以及组织损伤，感染后的不同时间点的测量，让易受感染的小鼠品系的特征。 致病机制s的细菌病原体定植于肠道他们的主机，以及具体的抵御这种感染的宿主反应，却知之甚少。因此，C. rodentium作为一个有价值的工具，以帮助我们了解这些过程模型肠道细菌感染。肠道细菌也被连接到的炎症性肠病（IBDs）。据推测，在肠黏膜免疫系统的异常暴露的肠道细菌基因易感个体适应不良的慢性炎症反应的IBD患者中发展。因此，感染性结肠炎模型的研究提供了重要C.确定潜在的致病性肠道细菌的宿主反应的潜力。 rodentium诱导的结肠炎就是这样一个难得的模式，让肠道细菌的宿主反应的分析，我们进一步了解鸡传染性法氏囊病发病的潜在机制;新的预防和治疗方法的发展至关重要。

在标准的感染实验，小鼠灌胃100μL过夜C.感染者约2.5×10 8 CFU通过rodentium文化。 与 C的C57BL / 6小鼠的感染rodentium结果，在温和和短暂的体重减轻和腹泻。虽然很少发生C57BL / 6小鼠，动物可能生病和需要euthanization。因此，鼠标应该减肥的程度和症状的困扰，如piloerect毛皮和驼背姿势进行监控，以确定不同菌株在何种程度上受到感染。 结果呈现在图1至图4是代表从冷冻甘油库存中制备使用的过夜培养完成感染。在感染后7天，小鼠麻醉，通过心脏穿刺收集血液。 图1示出了测定的血清中的FITC-葡聚糖C57BL / 6小鼠，以及Toll样受体2（TLR2）基因敲除小鼠，先前已被发现具有上皮屏障受损，完整性，在感染过程中9。在一个完整的肠上皮屏障的存在下，4 kDa的FITC-葡聚糖是通过这一层可渗透差。因此，FITC-葡聚糖水平的提高血清中的建议在感染肠上皮屏障的完整性，使这种分子泄露跨越减值。作为对照，在未感染的小鼠的血清，FITC-葡聚糖灌胃也被提出。 感染后一个星期，已经发现，在结肠中的细菌负荷峰值约10 9 CFU / g的3,4。在所需的时间点后感染细菌载量，可以测量以确认感染，以及评估，如果不同的基因敲除小鼠遭受从增加或减少细菌的负担。由于C。 rodentium的管腔致病菌，可密切ADH趁着肠上皮细胞，腔内容，盲肠，结肠组织中的细菌负荷可以被测量。 图2示出了典型的细菌负荷，在第7天测量盲肠和结肠组织感染后的C57BL / 6的肠腔内以及小鼠。 大部分的病理过程中观察到C。 rodentium在C57BL / 6小鼠的感染发生在前端2厘米的colon11。宏观在感染后的第7天，观察到结肠粘膜以及增稠的结肠长度缩短，在C57BL / 6小鼠中看到很少发生公开的损害。通常在感染过程中，C57BL / 6小鼠患有中度的炎症反应和病理组织学特点，免疫细胞浸润，结肠隐窝伸长率和杯状细胞耗竭（ 图3）。 在H＆E部分在图3中可以看出，多个宿主反应的安装过程中ING感染C. rodentium。为了进一步确定这些变化，免疫荧光染色法可用于研究蛋白质的变化，如标记细胞增殖，细胞死亡，或先天免疫和适应性免疫反应。免疫荧光染色的细菌反应的研究方面，如它的定位在受感染的组织也是有价值的。染色技术的一个例子在此，检查主机蛋白ki67的（红），一个标记的细胞增殖和C. rodentium蛋白质TIR，绿，定位在12天检查细菌感染后， 如图4所示 。 <img src="/files/ftp_upload/50222/50222fig1.jpg" alt="图1" fo:content-width="3.5in" fo:src="/files/ftp_upload/50222/50222fig1highres.jpg" /> 图1。测量血清的FITC-葡聚糖，以评估上皮屏障完整性小鼠，灌胃4 kDa的FITC-葡聚糖的未受感染的条件下，并在感染后7天，后血清的FITC-dextran水平测定。 C57BL / 6（WT）小鼠表现出增加，FITC-葡聚糖的血清水平在感染后第7天到未受感染的条件相比可以忽略不计。相反，TLR2 - / -小鼠显着增加基线建议严重受损的屏障的完整性，在该菌株相比，在血清中的水平的FTIC-葡聚糖期间C。 rodentium感染，如先前已被证明。 <img src="/files/ftp_upload/50222/50222fig2.jpg" alt="图2" fo:content-width="4in" fo:src="/files/ftp_upload/50222/50222fig2highres.jpg"/> 图2 C.收集rodentium载荷管腔，盲肠，结肠感染的C57BL / 6小鼠在感染后7天。流明，盲肠，结肠组织，匀浆，并镀在串行稀释在LB琼脂。每个Circle代表一个单独的从动物个体收集的样品。实线表示的几何平均值，而垂直误差棒表示SEM。 <img src="/files/ftp_upload/50222/50222fig3.jpg" alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/50222/50222fig3highres.jpg"/> 图3。组织学分析造成的损失由 C rodentium感染后7天感染温和的免疫细胞浸润，以及延伸的隐窝，杯状细胞耗竭和轻度水肿。相比较，观察控制未受感染的组织。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50222/50222fig3large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/50222/50222fig4.jpg" alt="图4"/> 图4。感染和第12天，感染后的免疫荧光染色C57BL / 6小鼠。TION组织中的宿主蛋白ki67的（红色）和C.远端结肠组织染色rodentium蛋白质TIR（绿色）。

Watch this Scientific Journal Video about The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis at JoVE.com

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

枸橼酸rodentium感染提供了一个有价值的模型来研究肠道细菌感染以及宿主的免疫反应和结肠炎的小鼠。该协议概述了屏障的完整性测量，病原体负荷和病理损害，从而为深入鉴定病原体和宿主小鼠的感染性肠炎的贡献。

“<em&gt;枸橼酸rodentium</em&gt;鼠标产品型号：研究感染性结肠炎的病原体和宿主的贡献

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

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Immunology and Infection

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

26.4K 次观看.  BC Children's Hospital. 枸橼酸rodentium感染提供了一个有价值的模型来研究肠道细菌感染以及宿主的免疫反应和结肠炎的小鼠。该协议概述了屏障的完整性测量，病原体负荷和病理损害，从而为深入鉴定病原体和宿主小鼠的感染性肠炎的贡献。

视频: The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

科研

免疫与感染

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

鼠标型号病原体引起的慢性炎症局部及全身站点

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

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 ...

医学

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Chronic Salmonella Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Chronic Salmonella Infected Mouse Model

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

慢性沙门氏菌感染小鼠模型

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

生物学

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

头孢哌酮治疗小鼠模型的临床相关的<em&gt;艰难梭菌</em&gt;应变R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

建立和UTI和CAUTI表征在小鼠模型

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

小鼠远端结肠造口术, 一种新的 C57BL/6 老鼠转移性结肠炎模型

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,000,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis. Although anti-inflammatory therapies may help control inflammation, they have no efficacy on fibrosis and stenosis in CD. The pathogenesis of CD is not well understood. Current studies focus mainly on delineating dysregulated gut immune response mechanisms. While CD-associated transmural inflammation, intestinal fibrosis, and luminal stenosis all represent mechanical stress to the gut wall, the role of mechanical stress in CD is not well defined. To determine if mechanical stress plays an independent pathogenic role in CD, a protocol of TNBS-induced CD-like colitis model in rodents has been developed. This TNBS-induced transmural inflammation and fibrosis model resembles pathological hallmarks of CD in the colon. It is induced by intracolonic instillation of TNBS into the distal colon of adult Sprague-Dawley rats. In this model, transmural inflammation leads to stenosis at the TNBS instillation site (Site I). Mechanical distention is observed in the portion proximal to the instillation site (Site P), representing mechanical stress but not visible inflammation. Colonic portion distal to inflammation (Site D) presents neither inflammation nor mechanical stress. Distinctive changes of gene expression, immune response, fibrosis, and smooth muscle growth at different sites (P, I, and D) were observed, highlighting a profound impact of mechanical stress. Therefore, this model of CD-like colitis will help us better understand CD&#39;s pathogenic mechanisms, particularly the role of mechanical stress and mechanical stress-induced gene expression in immune dysregulation, intestinal fibrosis, and tissue remodeling in CD.

The present protocol describes the development of a Crohn's-like colitis model in rodents. Transmural inflammation leads to stenosis at the TNBS instillation site, and mechanical enlargement is observed in the segment proximal to the stenosis. These changes allow studying mechanical stress in colitis.

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摘要

探索更多视频

4D多模态成像

鼠标型号病原体引起的慢性炎症局部及全身站点

Neuropathogenic的非侵入型

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

诱导小鼠肠道炎症由效应CD4的过继转移

慢性沙门氏菌感染小鼠模型

头孢哌酮治疗小鼠模型的临床相关的

建立和UTI和CAUTI表征在小鼠模型

小鼠远端结肠造口术, 一种新的 C57BL/6 老鼠转移性结肠炎模型

TNBS诱导的啮齿动物模型研究机械应力在克罗恩病中的致病作用