Name: an intravital microscopy-based approach to assess intestinal permeability and epithelial cell shedding performance
Uploaded: 2020-12-03T13:00:00
Description: Watch this Scientific Journal Video about An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance at JoVE.com

导致这些结果的研究得到了《人民方案》（玛丽居里行动）根据欧洲联盟第七框架方案第302170号《REA赠款协定》（FP7/2007-2013）提供的资金;埃尔兰根-纽伦堡大学跨学科临床研究中心（ IZKF）;合作研究中心TRR241和德国研究理事会（DFG）的临床研究组KFO257;和 Dfg 。

以下议定书已获埃尔兰根（德国维尔茨堡雷吉隆·冯·翁特弗兰肯）相关地方当局批准。老鼠被安置在特定的无病原体条件下。
注：在IECs内抑制GGTase-中压的预膜抑制会导致 Pggt1bi+IEC 小鼠24肠道渗透性严重改变。因此，此鼠标模型用于演示该协议如何可用于研究肠道屏障缺陷。但是，此协议可用于研究任何其他鼠标行。
1. 手...

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虽然技术上具有挑战性，但基于病毒内显微镜的方法代表了一种独特的实验方法，可以实时可视化高动态细胞过程，例如细胞脱落性能。到目前为止，还没有其他的实验方法来可视化体内的细胞挤出。我们相信，该协议有助于描述不同的细胞过程在维持肠道平衡方面发挥作用。
利用病毒内显微镜，这里介绍的方法使活体动物的肠道上皮细胞脱落的实时可视化。因此，麻醉小?...

肠道是一个高度专业化的器官，具有严格调节的功能，能够产生冲突过程，即营养和防止有害发光物质。在人体与环境之间，肠道上皮作为物理和免疫屏障，有助于维持肠道1，2的粘膜平衡。上皮完整性的丧失和紧密结渗透性增加是众所周知的与炎症性肠病（IBD）3，4，5，6。3,4,5,6然后，上皮改变被认为是IBD慢性肠道炎症的原因和辅助放大器。因此，更好地了解IBD患者肠道的早期上皮变化，对于制定恢复上皮完整性的新战略，以便进行可靠的预测和随后预防IBD复发，将具有巨大的价值。
肠道上皮遵循一个复杂和严格监管的周转过程。从墓穴底部，从多能干细胞中提取的端点分化肠道上皮细胞（IECs）向上迁移至维卢斯尖端，在那里老化/受损的细胞被流变到流明7。分裂和细胞挤出之间的平衡能够维持肠道上皮细胞数，避免形成间隙和泄漏，以及上皮细胞的积累，可能导致细胞质量和肿瘤发生,8，9，10。,10尽管上皮细胞脱落在肠道上皮的生理更新中起着关键作用，但有关分子机制推动细胞在维卢斯尖端挤出的知识是有限的。因此，有必要进行基础研究，对上皮细胞脱落所涉及的分子事件序列进行精确描述。
肠道粘膜内不同细胞类型之间的复杂相互作用是了解调节上皮周转和肠道平衡的分子机制的关键。因此，在这种情况下，体内研究比体外和体外方法具有很高的优势。此外，实时成像技术允许描述控制特定现象的事件序列。在这方面，研究高动态过程需要使用优化的高分辨率技术直接观察组织。体内成像技术似乎作为独特的适当工具，用于研究上皮细胞在肠道脱落。
术语"病毒内显微镜"是指利用高分辨率成像技术（多光子或共生显微镜）直接可视化活体动物11内其原生环境内的细胞和组织的实验方法。它支持实时获取体内信息，高达单细胞分辨率，并且与静态或低分辨率方法相比具有明显的优势。内维图显微镜提供补充信息，并克服经典和/或高端技术的一些限制，如组织加工造成的伪影。相比之下，病毒内显微镜的主要局限性是，组织应直接暴露在显微镜下，在大多数情况下，这需要手术。虽然复杂的方法保持活力，并尽量减少图像组织（皮肤折叠室和成像窗口）的影响12，13，,13在大多数情况下，一个简单的皮肤切口执行组织外化（皮肤皮瓣）14。在过去十年中，这些方法为高度动态的过程提供了关键证据，这些进程以前是难以理解的。翻译上，实时成像提供了新的生物学见解干细胞和白细胞，以及癌症传播和转移形成13，16。13,16在临床背景下，内皮镜目前被利用作为癌症17和胃肠道疾病的诊断工具，如IBD18，19;,19而共体马赛克显微镜成为手术20期间的快速病理工具。一起，病毒内显微镜最近已成为生物医学研究和未来在临床应用的宝贵和通用的工具。
内维他微显微镜是在这里实现的肠道上皮泄漏的实时可视化和上皮细胞脱落事件的观察。肠道渗透性泄漏可以通过其他体内非侵入性技术进行识别，例如在血清21中对荧光示踪剂进行口头注射。然而，这种技术不允许直接观察脱落性能，也不允许分离准细胞和跨细胞渗透性。标准示踪剂实验和病毒内显微镜的结合代表了一种合适的方法：i） 识别肠道渗透性中的干扰，ii） 分离准细胞和跨细胞上皮渗透性。除了细胞脱落，体内显微镜与体内荧光标签相结合，使研究其他细胞和分子机制（例如，使用荧光报告小鼠22或在肠道粘膜23 内的其他细胞在细胞脱落过程中紧密结再分配或国际细胞与肠道粘膜23内的其他细胞之间的相互作用）。
此处介绍的方法表示对病毒内显微镜的适应，以便使用共和激光扫描显微镜 （CLSM） 实时观察肠道粘膜。因此，我们使用有条件的敲除小鼠GGTase（Geranylgeranyl转移酶）在肠道上皮细胞（IECs）在（Pggt1bi+IEC 小鼠），因为他们患有严重的肠道疾病和增加上皮渗透性24。介绍了小鼠的手术准备和肠道粘膜的染色，以及用于成像采集和采集后分析的适当设置。该协议可以使未来的研究有助于目前关于肠道上皮细胞脱落动力学和动力学的知识。此外，该协议可以作为各种适应的基础，以研究发生在肠道粘膜表面，甚至在其他组织的其他现象。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acriflavine hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>A8251</td>
			<td>1 mg/mL solution in PBS</td>
		</tr>
		<tr>
			<td>Deltaphase isothermal pad</td>
			<td>BrainTree</td>
			<td>B-DP-PAD</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Gemini Cautery System</td>
			<td>BrainTree</td>
			<td>B-GEM-5917</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ketamin</td>
			<td>WDT</td>
			<td>9089.01.00</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS X</td>
			<td>Leica</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>LSM microscope SP8</td>
			<td>Leica</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biochrom</td>
			<td>L182</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine B dextran</td>
			<td>Invitrogen</td>
			<td>D1824</td>
			<td>10,000 kDa MW; 2 mg/mL solution</td>
		</tr>
		<tr>
			<td>Standard forceps (Dumont SS)</td>
			<td>Fine Science Tools</td>
			<td>11203-23</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Straight fine scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma Aldrich</td>
			<td>T5648</td>
			<td>50 mg/mL in ethanol</td>
		</tr>
		<tr>
			<td>Xylazin</td>
			<td>Bayer</td>
			<td>1320422</td>
			<td></td>
		</tr>
	</tbody>
</table>

an intravital microscopy-based approach to assess intestinal permeability and epithelial cell shedding performance

使用共体成像对肠道进行病毒内显微镜，可以实时观察活体动物的上皮细胞脱落和屏障泄漏。因此，麻醉小鼠的肠道粘膜被局部染色，并染色有非特异性染色（acriflavine）和荧光示踪剂（rhodamine-B dextran），安装在盐水溶液冲洗板上，并使用共和显微镜直接成像。该技术可以补充其他非侵入性技术，以识别肠道渗透性泄漏，如口头管理的示踪剂的跨粘膜通道。除此之外，这里介绍的方法允许实时直接观察细胞脱落事件。与适当的荧光记者小鼠结合，这种方法适用于将光分入控制肠道上皮细胞挤出的细胞和分子机制，以及其他生物过程。过去几十年，使用病毒内显微镜进行有趣的研究有助于了解内皮渗透性、免疫细胞肠道定位、免疫上皮通信和灯具成分的入侵等。这里提出的协议不仅有助于增进对控制上皮细胞挤出的机制的理解，而且可以作为其他方法的发展的基础，这些方法可以用作工具，以可视化其他高动态细胞过程，即使在其他组织中。在技术限制中，特定组织的光学特性以及选定的成像技术和显微镜配置反过来将决定成像工作距离和采集图像的分辨率。

此处介绍的协议描述了一种基于病毒内显微镜的方法，用于可视化肠道上皮泄漏并实时观察肠道中的细胞脱落性能。简单地说，小鼠麻醉和提交手术准备，以暴露小肠粘膜的表面。然后，通过局部应用 acriflavine 来染色 IECs;而发光的罗达明B-dextran被用作示踪剂，以检测从流明到亚上皮空间的透气通道。因此，手术准备和麻醉小鼠被放置在安装在培养皿中的幻灯片上，并随着...

Watch this Scientific Journal Video about An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance at JoVE.com

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

利用病毒内显微镜，这里介绍的方法使活体动物的肠道上皮细胞脱落的实时可视化。因此，麻醉小鼠的局部染色肠道粘膜（acriflavine 和 rhodamineB-dextran）使用共和显微镜被成像为单细胞分辨率。

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. ...

使用病毒内显微镜，该协议使肠道组织能够实时可视化，高达单细胞分辨率，并研究高动态过程，如细胞脱落。结合标准示踪剂实验，该协议允许识别体内肠道渗透性干扰，并区分半细胞和跨细胞渗透性。该协议可以用作开发其他病毒内显微镜方法的基础，以可视化各种组织内感兴趣的其他高动态细胞过程。这种方法的视觉演示对于了解手术准备步骤和定位活体动物，促进肠道粘膜表面的图像采集至关重要。在开始手术之前，打开共和激光扫描显微镜的底座和扫描仪盒，然后按开始打开计算机。启动图像采集软件并选择适当的配置。要设置适当的图像分辨率，请选择配置、硬件、分辨率、位深度和 12。在采集菜单下，为图像采集模式选择 XYZT，将目标设置为 20 或 40 倍。要设置顺序采集设置，请单击"查找"并在帧之间添加和选择。要配置序列 1，请打开可见的激光盒。将光电倍增管一设置为打开并定义发射波长。要配置序列 2，请打开可见的激光盒。将光电倍增管二设置为打开并定义发射波长。然后激活适当的激光器。在确认对手趾捏没有反应后，使用棉芽对麻醉小鼠的眼睛涂抹软膏，并在腹部左心室区域进行一厘米切口。使用钳子将肠道的三到五厘米段外化。在肠道部分的顶部和底部做两个小切口，并在肠道流明中引入玻璃微管。然后使用电烧打开外化肠道段纵向沿抗肠侧，并暴露粘膜。用盐水溶液短暂冲洗组织，以去除粪便。对于肠道粘膜表面的染色，在液滴中涂抹 100 微升每毫升 1 毫克的阿克里弗拉文溶液，让污渍在用 PBS 洗涤剩余溶液之前发展三分钟。接下来，在用PBS清洗之前，将每毫升2毫克的罗达明葡萄糖溶液中的每毫升2毫克的100微升涂抹在粘膜中，进行三分钟的孵育。然后将麻醉小鼠 supine 放在安装在用 37 摄氏度盐水冲洗的腔室的盖幻灯片上，必要时调整位置，然后将制备放在倒置显微镜阶段。将鼠标放置到舞台上后，立即打开光源并调整 XY 位置，直到照明轴聚焦在组织准备上。要选择视野，请选择相应的滤波数据集。打开快门，使用微轮聚焦肠道粘膜的表面。调整 XY 位置以定位可在视野中可视化多个 villi 的区域，并更改滤波块以验证罗达明二维克兰染色在该区域中是否可见。确定感兴趣区域后，开始图像采集并选择序列 1 以调整序列 1 的激光功率、增益和偏移。然后选择序列 2 并调整序列 2 的激光功率、增益和偏移。要定义 Z 堆栈范围，请打开 Z 堆栈放置菜单。使用 Z 访问控制聚焦粘膜表面，然后单击"开始"。关注信号仍可检测到的底限，然后单击结束。定义 Z 堆栈的数量，避免连续两个时间点的延时超过两分钟，并打开时间菜单以选择最小化和获取直到停止。要定义线平均值，请选择查找 1，线平均值 2，查找 2，线平均值 2。然后，要获取图像，请将格式设置为 1024 x 1024 像素，将速度设置为 400，然后单击"开始"。肠道上皮细胞特异性GGTase缺乏条件小鼠发展严重肠道病理学，小肠组织损伤评分增加，肠道上皮渗透性增加，也可以通过体内实验中的示踪剂使用口头施用的 FITC-dextran 来检测。细胞脱落事件可以识别为细胞从上皮单层进入流明，导致上皮天花板的暂时间隙，最终因相邻细胞之间的接触而闭合，即所谓的拉链效应。这些差距在控制和GGTase缺陷小鼠中观察到，尽管这些现象的频率在后者中较高。有趣的是，其他细胞似乎也吸收了脱克兰。这些所谓的可渗透细胞事件也主要发生在有条件的敲除小鼠中。可对细胞骨架相关和相关紧密结点蛋白进行免疫渗透，以确定有助于通过病毒内显微镜确定的潜在上皮渗透性中断的特定目标。该方法可用于分析肠道粘膜表面的其他现象，如内皮渗漏或肠道感染情况下的免疫上皮交流。

Research

JoVE Journal

Immunology and Infection

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

科研

免疫与感染

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Application of a Mouse Ligated PeyerÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

PRP作为一种新的方法，以防止感染的制备及<em在体外</em&gt;抗菌性能的PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒：三氯生的影响不使用有机溶剂的化学效应

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Development of an IFN-ÃƒÅ½Ã‚Â³ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

的IFN-γ酶联免疫斑点检测，以评估水痘 - 带状疱疹病毒特异性细胞免疫继脐血移植的发展

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对<i&gt;秒。肺炎</i&gt;<i&gt;小时。流感</i&gt;<i&gt; K。肺炎</i&gt;<em&gt;点。铜绿假单胞菌</em&gt;或<em&gt;一个。鲍曼不动杆菌</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

利用人类诱导的多能干细胞衍生的肠道有机体研究和改性上皮细胞对沙门氏菌和其他病原体的保护

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

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

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 ...