这项工作部分通过国家卫生研究院（DK097335和T32AI007260）和爱荷华大学斯蒂德家庭儿科系得到支持。

所有动物程序均由爱荷华大学机构动物护理和使用委员会（协议#8041401）批准。所有动物都安置在爱荷华大学批准的实验室动物护理评估和认证协会（AALAC）中。所有小鼠都是野生型菌株C57Bl/6J。
1. 在怀孕小鼠中建立FEMI
<ol><li>LPS 准备<ol>
<li>使用从 大肠杆菌 O55:B5（库存浓度 2 毫克/毫升）提取的 LPS。</li>
		<li>稀释 LPS 库存浓度 1:100 与无菌盐水的工作浓度为 20μg/mL。</li>
	</ol>
</li>
	<li>母体 LPS 注射<ol>
<li>在妊娠日e15注入怀孕的水坝。这个时间点是大约75%，通过阴郁的怀孕，使这个模型发展类似于人类怀孕的早期三个月，这是当大多数早产时，由于胆囊炎发生。</li>
		<li>注射前立即称量怀孕小鼠，以确定适当的LPS配给。</li>
		<li>使用以下公式计算工作浓度的剂量:5 μL x 克体重 （gbw），总剂量 LPS 为 100 μg/kg。对于对照动物，使用等量的正常盐水注射。</li>
		<li>每次注射前，涡流 LPS 解决方案在高位 15 秒内三次。</li>
		<li>将LPS音量绘制成1毫升注射器。</li>
		<li>用擦伤技术约束怀孕的老鼠。保持在后部重新任职的位置，并执行注射。<ol>
<li>插入一个30米8毫米针斜面在腹部的右下象限（以避免膀胱和腹部血管）在30\u201240°的角度。插入约1/4至1/2的针的长度。</li>
			<li>在注射前拉回注射器柱塞以确保负压。如果存在负压，则继续注射。</li>
			<li>注射后，监测小鼠约30分钟，然后返回笼子，在怀孕的剩余时间。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 分娩和照顾后代，以及肠道收获
<ol><li>通常通过e20阴道分娩交付幼崽。 注意:此模型确实具有预期的剂量依赖胎儿损失率，可在 图 1 中看到，并在以下结果中讨论。</li>
	<li>允许幼崽与母亲呆在一起，并接受脂肪饲料。</li>
	<li>在收获日，通常是产后第14天（P14），按照机构动物护理和使用委员会的议定书，通过子宫颈脱位对幼崽实施安乐死。</li>
	<li>使用剪刀和钳子，通过皮肤和腹膜，在腹部的中线下做一个垂直切口，整个腹部长度。用剪刀将小肠从胃切除到粪便，用钳子切除肠子。</li>
	<li>分离并保留小肠的1/3（人类肠的分节代表），丢弃近端小肠、粪便和结肠。</li>
	<li>用剪刀将氦部分分成两半。</li>
	<li>将近半部分放在RNA稳定解决方案中，以供以后的RNA定量。</li>
	<li>将解剖剂的一半放在 10% 中性缓冲形式中，用于幻灯片准备。</li>
</ol>3. 肠道损伤评分
<ol><li>部分石蜡嵌入组织到5 μm厚片，并安装在玻璃滑梯上。 注意:我们将标本发送到石蜡嵌入、分割和安装到幻灯片上的石蜡核心。</li>
	<li>根据标准程序去除幻灯片。</li>
	<li>根据标准程序，使用血氧林和异丙素的污渍部分。</li>
	<li>得分部分在3分尺度内肠损伤，如前所述42，43。<ol>
<li>使用光显微镜，评估两个单独的失明研究者在3点尺度上对全身肠道损伤进行评估，评估静脉的完整性和与地下室膜43 的分离（补充图1）。肠道损伤最好按放大20倍和数孔0.50进行评估。</li>
		<li>分配 0 分来描述正常的粘膜。</li>
		<li>分配一个分数1描述轻度损伤，其中包括亚皮鲁恩哈根的空间的发展，真空或副皮提升仅限于拉米纳丙丙或villi的提示。</li>
		<li>分配一个2分来描述严重的伤害，表示上皮提升和蒸发大于一半的维利，维利失真，或粘膜溃疡和拉米纳丙酸的解体。</li>
	</ol>
</li>
</ol>4. 帕内思和杯细胞的量化
<ol><li>去亲和后，组织部分的污渍滑动从步骤2.8与阿尔西亚蓝/周期酸希夫污渍，以表示杯和帕内思细胞，如先前描述的44，45根据以下步骤。 注:虽然阿尔西亚蓝/周期酸希夫污渍不是特定于帕内思或杯状细胞，在我们的经验，失明经验丰富的研究者有等效的细胞定量使用这种污渍相比，细胞靶向抗体，与显着较少的背景染色46。</li>
	<li>脱水、污渍和脱水滑梯如下。<ol>
<li>两次在二甲苯中浸入滑梯 10 分钟。 注意:二甲苯应用于烟气罩。</li>
		<li>用100%埃托冲洗。</li>
		<li>淹没幻灯片在100%EtOH 3分钟，然后在90%EtOH 3分钟，其次是70%EtOH 3分钟，最后淹没幻灯片在50%EtOH为3分钟。</li>
		<li>在自来水下清洗5分钟。 注意:将该部分远离自来水，以防止组织样本丢失。</li>
		<li>使用标准咖啡滤镜过滤阿尔西恩蓝色污渍溶液。</li>
		<li>污渍在阿尔西亚蓝色污渍中滑动 15 分钟，然后在自来水下清洗 2 分钟。</li>
		<li>在200mL的双蒸馏水中稀释1毫克周期酸。在此解决方案中浸入幻灯片 5 分钟。然后在自来水下洗1分钟。</li>
		<li>用希夫的试剂污渍 10 分钟。在自来水下清洗5分钟。</li>
		<li>用血氧林将滑梯染色1分钟，然后在自来水下清洗2分钟。</li>
		<li>将它们浸入酸醇（1 mL 盐酸混合在 99 mL 中，70% EtOH）中 1 分钟。</li>
		<li>浸入斯科特的自来水（自来水中NaHCO3 浓度0.1%）1分钟，然后在自来水下清洗1分钟。</li>
		<li>脱水滑梯。<ol>
<li>在 70% EtOH 中浸入每个幻灯片 10 次，然后在 90% EtOH 中浸 10 次，在 100% EtOH 中浸 10 次。</li>
			<li>在 100% EtOH 中浸入幻灯片 10 分钟，然后在新鲜的二甲苯中浸入两次，每次 3 分钟。</li>
		</ol>
</li>
		<li>在标本上放置一滴安装的介质，并在标本上放置盖子。</li>
	</ol>
</li>
	<li>杯细胞计数<ol>
<li>使用光显微镜，数数杯细胞（补充图2）。对于每一块肠道组织，计算杯细胞和500上皮细胞的数量，并表达杯细胞比作为每100个上皮细胞的比例。杯形细胞最好按放大20倍和数值光圈0.5计算。</li>
	</ol>
</li>
	<li>帕内思细胞计数<ol>
<li>使用光显微镜，数数帕内思细胞（补充图2）。对于每一块肠道组织，表达为每个肠道密码的Paneth细胞的比例。每片肠道组织数100个肠道密码。Paneth 细胞最好按 20x-60 倍的放大倍数和数值光圈 0.50-1.30 计算。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

胆管炎影响2\u20124%的期限和25\201230%的早产8，9。然而，胆管炎的影响可以延长很久的出生，因为它已被证明对胎儿和新生儿有显著的影响10，11，12，13，14，15，16。重要的是，胆管炎已被证明与NEC15，16的后续发展有关。虽然目前还不完全了解，NEC的发病机制可能涉及肠道不成熟、免疫系统调节不力、肠道炎症和细菌转移的组合，最终导致肠道坏死的最后一条常见途径19。然而，胆管炎与NEC随后发展之间的机械联系仍不清楚，以往的胆管炎动物模型不足以研究这种关系。为了解决这一知识差距，我们修改了通常使用的LPS诱发的胆囊炎和早产模型34，37，38，39，40，使新生儿出生和生存。在这样做，我们创造了一个模型，接近在胆管炎42看到的炎症状况，以研究胎儿接触母体炎症（FEMI）对随后的肠道发展的影响。
通过此协议，我们已经证明，这种使用 LPS 诱导的 FEMI 的胆管炎的穆林模型会导致短期和长期肠道损伤以及正常肠道发育的中断，最明显的是杯腔和 Paneth 细胞的下调节，这两种细胞都提供了肠道炎症的第一线先天免疫力。本模型显示的肠道损伤和组织细胞变化表明，模仿 NEC 中出现的损伤是一种有效的模型。这主要是因为Paneth细胞和杯状细胞的下降调节都与NEC的发病机理有关，组织损伤的模式与NEC41、42、49的人类病例相似。因此，这种LPS诱导的胆管炎FEMI模型是研究胆管炎和后来肠道损伤之间的机械联系的理想模型，特别是NEC的发展，以及胆管炎对发育中的微生物群的潜在影响，这在利用活细菌的现有模型中是不可能的。
在体内，胆管炎通常涉及上升的细菌感染，往往导致膜的早产破裂和胆管炎的临床表型，和动物模型的胆管炎存在，更准确地反映了这种病理生理学25，28，30，32。然而，由于我们的实验室研究肠道发育，包括发育中的微生物群，在胆管炎模型中存在活细菌会混淆微生物群分析。因此，现有的利用活细菌的胆管炎模型对于研究胆管炎与NEC后期发展的机械性联系是不切实际的。此外，LPS诱导的胆囊炎模型已经有效地模拟了产后白质和灰质脑损伤36，证明这种方法是模拟出生时接触胆囊炎的早产病的有效方法。
同样重要的是要注意，这种模型高度依赖于用于诱导FEMI的LPS剂量。本协议的主要关键步骤是在怀孕的水坝中引入FEMI，在内皮质激素注射LPS;因此，毫不奇怪，我们发现用于诱导FEMI的LPS剂量在这些实验的结果中极为重要。通过初步实验，使用了100微克/千克的LPS剂量，因为这种LPS剂量与孕产妇死亡率无关，导致约50%的新生儿存活率，以及41岁、42岁后代的肠道严重损伤。
LPS 与收费类受体 4 （TLR4） 复合物结合，导致细胞内信号蛋白、细胞因子生成和亲炎信号50的启动。收费类受体（TLR），包括TLR2、TLR3、TLR4、TLR7、TLR8和TLR9，对于诱导各种病原体和微生物（如病毒、细菌和真菌51）的炎症反应非常重要。有趣的是，TLR3的升级调节也被证明与新生儿穆林轮状病毒模型中增加的组织性肠道损伤和病毒脱落有关:此外，TLR3的淘汰改善了这些影响52。因此，虽然模型使用TLR4通路，但有理由认为，刺激其他TLR可能会得出类似的结果。
使用这种方法，我们已经能够证明，LPS注射在怀孕的水坝导致增加产妇，胎盘和胎儿炎症标记，同时节省羊水，以及诱导胎盘42的直接损害。有趣的是，这种方法没有显示子宫动脉阻力的改变。FEMI模型也对后代有重大影响，因为我们已经表明，暴露的幼崽有严重的肠道损伤42 通过IL-6依赖路径41， 并可能影响肠道的重要防御机制，如杯细胞和Paneth细胞41。这种损伤使新生儿越来越容易受到随后的LPS引起的肠道损伤和炎症41 的影响，这或许可以解释为什么患胆管炎的婴儿更容易患NEC。

胆汁膜在哺乳动物怀孕中起着不可或缺的作用。它们包括具有多种功能的巧克力和氨骨。它们包围和保护胎儿，促进母体和胎儿隔间1之间的对羟基苯甲烷信号，并在胆汁膜内创建局部反馈回路，这可能参与启动隔膜1。目前对膜的了解表明，氨基安尼翁提供结构屏障功能，而胆汁提供免疫缓冲，主要保护发育中的胎儿免受母体免疫系统2。这些膜的炎症被称为胆囊炎。从历史上看，临床胆管炎的诊断是在孕产妇发烧加上一个或多个胎儿或产妇临床发现3，4后作出的。然而，虽然这个定义在临床上是有用的，但它缺乏精确性使得胆管炎的研究具有挑战性。2015年，为了澄清诊断结果，尤尼斯·肯尼迪·施莱佛国家儿童健康与人类发展研究所的专家小组研讨会将胆管炎定义为宫内炎症或感染，或两者兼有（三I）3。这一澄清是重要的，因为虽然微生物诱发感染是子宫/羊水炎症的一个重要原因，它发生的频率低于无菌子宫/羊水炎症5，6，7。总的来说，胆管炎仍然是一个严重的公共卫生问题，正如在2\u20124%的定期分娩和25\u201230%的早产8，9。
胆管炎对胎儿和新生儿有显著影响。文献记载，胆管炎与早产症许多发病率增加有关，包括支气管肺发育不良10、脑白质损伤11、心室出血12、早产视网膜病变13，以及疑似和确诊的早期新生儿败血症14、15。由于我们对不成熟的肠道的损伤和修复机制感兴趣，因此必须注意，胆管炎也与后来的坏死性肠球炎（NEC）15、16的发展有关。NEC是一种毁灭性的胃肠道疾病的早产儿，导致一个不良的调节主机反应炎症和随后的肠道坏死17。在美国，NEC每年影响超过4000名婴儿，其中多达三分之一的婴儿死于这种疾病18。NEC的发病机制可能涉及肠道不成熟、不成熟免疫系统调节不良、肠道炎症和细菌转移19的组合，最终导致肠道坏死的最后一条常见途径。重要的是，NEC的发病通常发生在出生后几周，并可能暴露于胆管炎，使得胆管炎与NEC随后的发展之间的机械联系变得模糊不清。胆管炎可能有助于NEC病理生理学的一个潜在机制是通过对母体免疫系统的调节，随后产生强烈的胎儿炎症反应，可能破坏正常的胎儿发育模式21，22，23。
啮齿动物和绵羊24、25、26、27、28、29、30、31、32等多种哺乳动物模型存在。然而，在胆管炎引起的胎儿接触产妇炎症（FEMI）之后，很少有关于肠道发育的数据。为了探讨FEMI与不成熟肠道损伤的后续发展之间的关系，我们采用了脂糖（LPS）诱导的FEMI模型。脂糖是克阴性细菌细胞外表面的主要成分，是多种真核物种（包括人类33）与生俱来的免疫系统的有力刺激剂。产妇LPS注射导致无菌炎症级联，没有活细菌的混淆作用，它是一个成熟的模式诱导早产34，以及急性胆管炎和胎儿炎症反应综合征（FIRS）的模型，这是最严重的形式的胆囊炎24，35。它也已被证明诱导大脑白质和灰质损伤的绵羊模型36和穆林模型37，38，39，40。然而，据我们所知，我们是第一个使用这种模型的胆管炎和FEMI来调查它对胃肠道发育的影响，过去出生，以及调查胆管炎和NEC41，42的后期发展之间的可能机械性联系。

<table><tbody><tr><td>10% neutral buffered formalin</td><td>Sigma</td><td>HT501128</td><td></td></tr><tr><td>Alcian blue stain</td><td>Newcomer supply</td><td>1003A</td><td></td></tr><tr><td>C57Bl6/J mice</td><td>Jackson Laboratories</td><td>664</td><td></td></tr><tr><td>Ethanol</td><td>Decon labs</td><td>2701</td><td></td></tr><tr><td>HCl</td><td>Sigma</td><td>H1758</td><td></td></tr><tr><td>Hematoxylin stain</td><td>Leica</td><td>381562</td><td></td></tr><tr><td>LPS</td><td>Sigma</td><td>L2880</td><td></td></tr><tr><td>NaHCO3</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>Nikon Eclipse Ni-U Microscope</td><td>Nikon</td><td>2CE-MQVJ-1</td><td></td></tr><tr><td>Periodic Acid</td><td>ACROS</td><td>H5106</td><td>CAS# 10450-59-9</td></tr><tr><td>RNAlater</td><td>Thermofisher</td><td>Am7021</td><td></td></tr><tr><td>Schiff&#039;s reagent</td><td>Sigma</td><td>S5133</td><td></td></tr><tr><td>Secor Imager 2400</td><td>Meso Scale Discovery (MSD)</td><td></td><td></td></tr><tr><td>V-Plex Assay</td><td>Meso Scale Discovery (MSD)</td><td></td><td></td></tr><tr><td>Xylene</td><td>Sigma</td><td>534056</td><td></td></tr></tbody></table>

a murine model of fetal exposure to maternal inflammation to study the effects of acute chorioamnionitis on newborn intestinal development

胆管炎是早产的常见诱发剂，与早产的许多发病率有关，包括坏死性肠结肠炎（NEC）。然而，这两种情况之间的机械联系仍有待发现。我们采用了胆囊炎的穆林模型，该模型涉及脂糖（LPS）诱导胎儿接触母体炎症（FEMI）。FEMI的这种模型诱导无菌产妇、胎盘和胎儿炎症级联，这在许多临床胆管炎病例中也存在。虽然存在利用活细菌并更准确地模拟上升感染导致胆管炎的病理生理学的模型，但这些方法可能会对不成熟的肠道发育和相关发育的微生物群产生间接影响。使用此协议，我们已经证明，LPS 诱发的 FEMI 导致妊娠损失和早产的剂量依赖性增加，以及后代正常肠道发育的中断。此外，我们已经证明，FEMI显著增加后代的肠道损伤和血清细胞因子，同时减少杯状细胞和Paneth细胞，这两者都提供了肠道炎症的第一线先天免疫力。虽然类似的LPS诱导FEMI模型已经被用来模拟胆管炎和中枢神经系统随后的异常之间的关联，据我们所知，这个协议是第一个试图阐明胆管炎和后来肠道发育的扰动之间的机械联系，作为胆管炎和NEC之间的潜在联系。

在胚胎第15天接触FEMI会导致妊娠依赖剂量的丧失和早产的剂量依赖率（图1）42。在实验中，我们选择使用 100μg/kg 的 LPS 剂量来尽量减少妊娠损失和早产（早产期和宫内胎儿死亡之间 50% 的损失），同时使胎儿受到严重的炎症性侮辱。
使用这种方法，我们接下来检查了FEMI对后代后续伤害的影响。使用3点组织学量表测量全身肠道损伤，我们发现在出生（P0）和成年时（P56或8周生命）（图2）有重大损伤。需要注意的是，这种伤害发生在除了FEMI之外，对动物没有任何额外的刺激的情况下，这表明仅FEMI就扰乱了新生儿阴静脉肠道的正常平衡。由于小鼠出生时有一个相对不成熟的肠道，在生命的前4周继续发育47，48，这与早产儿谁也有不成熟的肠道有关。
为了进一步了解FEMI对肠道上皮的正常发育和不成熟肠道防御机制的影响，我们量化了与人类肠子相似的小肠道三分之一的胰腺细胞和抗菌肽产生胰腺细胞的数量。我们发现，与没有FEMI的动物相比，FEMI通过诱导高血糖细胞和帕内思细胞的丧失而扰乱了肠道上皮的正常组成（图3）。
为了研究FEMI对新生儿炎症反应的影响，使用ELISA与电化学发光，我们量化了各种血清炎症标记，其中包括IL-1+，IL-10，KC-GRO（相当于IL-8的穆林）和IL-6，从有和没有FEMI的幼崽血清（图4）。我们发现FEMI显著增加了P0所有细胞因子的炎症级联。晚年炎症级联（P7\u2012P56）因时间点和细胞因子而异。最有趣的是，对于 IL-6，FEMI 和虚假组的 P7\u2012P28 水平相似，但如果没有二次干预，P56 的 FEMI 组水平显著较高。这一点尤其重要，因为我们已经证明IL-6是FEMI模型中产后肠道损伤发展的关键细胞因子。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61464/61464fig01.jpg"> 图1:FEMI剂量对妊娠结果的影响。妊娠垃圾的存活率取决于剂量较高，导致妊娠损失率（A）和早产率（B）较高。图是经过弗里克等人的许可改编的。FEMI 使用 LPS 剂量为 100μg/kg，使幼崽在一周内存活 50%。每个数据点代表 n > 8 次怀孕和至少三个单独的实验。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61464/61464fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61464/61464fig02.jpg"> 图2:FEMI对小肠损伤模式的影响。肠道样本是在出生时、出生时、出生1周、生命2周和8周生命时从接触FEMI（100微克/千克LPS）或假控制的小鼠身上采集的。样本是由失明的调查人员42，43用3分的伤害评分。仅FEMI一项，没有进一步的侮辱，在出生时、生命1周和生命8周时就造成大量伤害。图已调整与弗里克等人的许可42。每个数据点代表一个 n > 10 只幼崽和至少 3 个怀孕水坝的至少三个单独实验。曼-惠特尼非参数T测试用于比较每个时间点的肠道损伤分数。星号表示p<0.05。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61464/61464fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61464/61464fig03.jpg"> 图3:FEMI在发育过程中诱发小肠中正常杯和泛血球细胞数量的变化。肠道样本是在出生时从接触FEMI（100微克/千克LPS）或假控制的小鼠身上采集的。样品沾染了阿尔西亚蓝/周期酸希夫污渍，以检测杯和帕内思细胞，这些是由失明的调查员量化的。与所有年龄段的假控制相比，来自FEMI动物的杯细胞和Paneth细胞都显示出一种趋势或显著减少。图经埃尔金等人许可改编。每个数据点代表一个 n > 10 只幼崽和至少三个单独的实验。错误条表示均值的标准误差。学生 T 测试用于比较每个时间点的杯和泛血球的数量。星号表示p<0.05。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61464/61464fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61464/61464fig04.jpg"> 图4:FEMI在P0出生后立即诱发全球所有细胞因子的新生儿炎症激增，在P56时出现IL-6的后期激增。血清细胞因子在 P0、P7、P14 和 P28 中使用 ELISA 进行量化，并根据制造商的说明进行电化学发光，板材读取为 620 nm。细胞因子值在雷达图中表示，所有细胞因子都绘制为最大值的百分比。与对照组相比，FEMI 组 P0 的所有细胞因子（IL-1+、IL-10、KC-GRO 和 IL-6）均显著增加（所有 p < 0.05）。与非参数化 Kruskal-Wallis 测试相比，与非参数化 Kruskal-Wallis 测试相比，与没有 FEMI（p < 0.05）相比，幼崽的 P56 的 IL-6 水平也重新上升，这是 FEMI 组中唯一显著升高的细胞因子，而此时的控制则处于后期。图经埃尔金等人许可改编。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61464/61464fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
补充图1:H&E染色肠组织肠道损伤评分。受伤分数由三分肠损伤评分表（0=正常，1=轻度伤害，2=严重伤害）根据维利蒸发程度、粘膜溃疡、拉米纳丙丙酸损伤以及维利体内出血（如前所述的43）确定。图经埃尔金等人许可改编。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61464/61464supfig01.jpg" target="_blank">请单击此处下载此图。</a>
补充图2:高血球和泛血球细胞的阿尔西亚蓝/PAS染色的代表外观。 阿尔西亚蓝/PAS染色肠道组织允许杯细胞清晰可视化， 存在于肠道维利（顶部面板标记为白色箭头，图像以20倍放大），和Paneth细胞，目前存在于利伯库恩的墓穴，位于肠道维利在拉米纳丙酸（底部面板标记为黄色箭头，图像拍摄于60倍放大）。 <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61464/61464supfig02.jpg" target="_blank">请单击此处下载此图。</a>

Watch this Scientific Journal Video about A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development at JoVE.com

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

我们开发了一个胆管炎模型，以模拟胎儿接触产妇炎症（FEMI），而不会引起活体并发症，以检查FEMI对后代肠道发育的影响。这允许研究胆管炎后肠道损伤发展的机械原因。

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

a-murine-model-fetal-exposure-to-maternal-inflammation-to-study

Nanyang Technological University

Research

JoVE Journal

Medicine

3.7K 次观看.  University of Iowa. 我们开发了一个胆管炎模型，以模拟胎儿接触产妇炎症（FEMI），而不会引起活体并发症，以检查FEMI对后代肠道发育的影响。这允许研究胆管炎后肠道损伤发展的机械原因。

视频: A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

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 ...

科研

免疫与感染

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 ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

早产儿脑病建模使用产前缺氧缺血大鼠羊膜腔内毒素

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

医学

Probiotic Studies in Neonatal Mice Using Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

新生儿小鼠益生菌的研究

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

革兰阴性细菌败血症的新生儿成像模型

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus (GBS)

Group B Streptococcus (GBS) is one of the most common bacteria isolated during human pregnancy. It is a leading cause of placental infection/inflammation, termed chorioamnionitis. Chorioamnionitis exposes the developing fetus to a high risk of organ injuries, perinatal morbidity, and mortality, as well as life-long neurobehavioral impairments and other non-neurological developmental issues. The two most frequent subtypes of GBS isolates from maternal and fetal tissues are serotypes Ia (13%-23%) and III (25%-53%). Our lab has developed and characterized a rat model of GBS-induced chorioamnionitis to study subsequent impacts on the central nervous system of the developing fetus and to understand underlying mechanistic aspects. This article presents the design as well as uses of the preclinical rat model, which closely reproduces the hallmark of GBS-induced chorioamnionitis in humans. This article aims to help scientists reproduce the experimental design as well as to provide support through examples of troubleshooting. The present model may also contribute to potential discoveries through uncovering causes, mechanisms, and novel therapeutic avenues, which remain unsettled in many developmental impairments arising from chorioamnionitis. Furthermore, the use of this model may be extended to the studies of perinatal non-neurological common and severe morbidities affecting, for instance, the retina, bowel, lung, and kidney. The main interest of this research is in the field of GBS-induced fetal neurodevelopmental impairments such as cerebral palsy (CP), attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD). The rationale supporting this model is presented in this article, followed by procedures and results.

<meta charset="utf8"></head><body>B 组链球菌 GBS 引发的绒毛膜羊膜炎临床前动物模型的剖腹产

The goal of this protocol is to describe a preclinical animal model of Group B Streptococcus (GBS)-induced chorioamnionitis. The study is designed to investigate mechanistic processes, potential causal links with developmental impairments, and finally to develop translational anti-inflammatory placento- and neuro-protective treatments.

B 组 链球菌 （GBS） 引发的绒毛膜羊膜炎临床前动物模型的剖腹产

Group B Streptococcus (GBS) is one of the most common bacteria isolated during human pregnancy. It is a leading cause of placental infection/inflammation, ...

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate across the gut invade the newborn's bloodstream, causing life-threatening bacteremia. The methodology presented here utilizes polarized intestinal epithelial cells grown on semipermeable inserts to assess the transcytosis of neonatal E. coli bacteremia isolates in vitro. This method uses the established T84 intestinal cell line that has the ability to grow to confluence and form tight junctions and desmosomes. After reaching confluence, mature T84 monolayers develop transepithelial resistance (TEER), which can be quantified using a voltmeter. The TEER values are inversely correlated with the paracellular permeability of extracellular components, including bacteria, across the intestinal monolayer. The transcellular passage of bacteria (transcytosis), on the other hand, does not necessarily alter the TEER measurements. In this model, bacterial passage across the intestinal monolayer is quantified for up to 6 h post-infection, and repeated measurements of TEER are made to monitor the paracellular permeability. In addition, this method facilitates the use of techniques such as immunostaining to study the structural changes in tight junctions and other cell-to-cell adhesion proteins during bacterial transcytosis across the polarized epithelium. The use of this model contributes to the characterization of the mechanisms by which neonatal E. coli transcytose across the intestinal epithelium to produce bacteremia.

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Escherichia coli&#160;causes sepsis in neonates who ingest the bacteria around the time of birth. The process involved in&#160;E. coli&#8217;s&#160;ability to travel from the enteric tract to the bloodstream is poorly understood. This&#160;in vitro&#160;model assesses the ability of&#160;E. coli&#160;strains to travel through the intestinal epithelial cells.

新生儿 大肠杆菌 分离菌血症肠道转吞作用的评估

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

一种新生儿BALB/c坏死性小肠结肠炎小鼠模型

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

摘要

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此视频中的章节

Neuropathogenic的非侵入型

诱导小鼠肠道炎症由效应CD4的过继转移

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

早产儿脑病建模使用产前缺氧缺血大鼠羊膜腔内毒素

新生儿小鼠益生菌的研究

革兰阴性细菌败血症的新生儿成像模型

B 组链球菌（GBS）引发的绒毛膜羊膜炎临床前动物模型的剖腹产

B 组链球菌（GBS）引发的绒毛膜羊膜炎临床前动物模型的剖腹产

新生儿大肠杆菌分离菌血症肠道转吞作用的评估

一种新生儿BALB/c坏死性小肠结肠炎小鼠模型

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

摘要

探索更多视频

此视频中的章节

Neuropathogenic的非侵入型

诱导小鼠肠道炎症由效应CD4的过继转移

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

早产儿脑病建模使用产前缺氧缺血大鼠羊膜腔内毒素

新生儿小鼠益生菌的研究

革兰阴性细菌败血症的新生儿成像模型

B 组 链球菌 （GBS） 引发的绒毛膜羊膜炎临床前动物模型的剖腹产

B 组 链球菌 （GBS） 引发的绒毛膜羊膜炎临床前动物模型的剖腹产

新生儿 大肠杆菌 分离菌血症肠道转吞作用的评估

一种新生儿BALB/c坏死性小肠结肠炎小鼠模型

B 组链球菌（GBS）引发的绒毛膜羊膜炎临床前动物模型的剖腹产

B 组链球菌（GBS）引发的绒毛膜羊膜炎临床前动物模型的剖腹产

新生儿大肠杆菌分离菌血症肠道转吞作用的评估