这项工作是支持的补助金从德意志研究联合会（KA1270/3-1/2）"和BONFOR（O-112.0040）。

1。动物男C57BL6 / J小鼠的平均重量为25g，得到从Harlan Winkelmann的（Borchen，德国）。所有的实验都按照联邦法律有关保护动物的。实验动物管理的原则得到遵守。动物均保持在12小时光/暗周期，并提供使用市售的啮齿动物食物和自来水自由采食 。动物应该被安置在动物实验前至少7天到达后，所有实验都可以很容易地适应老鼠或其他物种，到大动物模型稍作修改。 2。手术过程<ol><li>麻醉诱导和维持与异氟醚（雅培，威斯巴登，德国）的氧气。发病后的麻醉地方的动物仰卧位和修复四肢用胶带（Leukosilk，拜尔斯道夫，德国汉堡）。 </li&gt; <li>剃须和外科消毒的腹壁中位数剖腹手术后为2厘米的长度上执行。沿白线通过切口进入腹腔。将两个无菌的拉钩，保持腹部开放。小心eventerate小肠向左侧，并把它放在潮湿的纯棉纱布。 </li><li>疏散湿润，无菌棉签涂抹（残废，德国诺因基兴）从幽门至盲肠，滚动涂抹的同时，整个小肠肠腔内容。注意:避免小肠壁或肠道血管病变出血。不加捻，以防止局部缺血或机械性肠梗阻和关闭使用两层连续的5/0聚酰胺不可吸收缝合线（爱惜康，Norderstedt，德国）的腹部切口的情况下，将肠道放回腹腔洞穴。 </li></ol> 3。术后护理<ol><li>后的程序将在一个加热灯，observi，下的动物纳克它，直到它从麻醉中恢复过来。把鼠标在它的笼子里，并允许获得水和食物的。由于阿片类药物的影响蠕动的财产，我们进行围手术期镇痛的饮用水中的非甾体抗炎药（如安乃近）。 </li></ol> 4。功能研究24小时后，IM <ol class="lalpha"><li> 胃肠道运输（GIT） <ol><li>与异氟醚氧22.5小时后，IM轻微麻醉小鼠。仔细的鼠标和超伸的头，用钝头镊子拔出舌头。填写1毫升的注射器，至少有100μL荧光标记的葡聚糖（FITC-葡聚糖，70,000 MW，Taufkirchen，德国，Sigma Aldrich公司），将它连接到动脉的导管（Vygon，旅游Ecouen，法国），并小心地将导管作为胃管进肚子里。 </li><li>简单地说注入100μLFITC-葡聚糖和拔出导管。让动物清醒，等待90分钟。在此期间，小鼠的食物或水。&lt;/ P&gt; <li>准备152毫升纺纱管，连续贴上标签。 </li><li>安乐死小鼠灌胃后90分钟，取出从胃到直肠的整个胃肠道。将肠道内到聚苯乙烯垫，避免拉伸。 CAVE:这是轻轻重要是清除肠道内，以避免液体FITC-葡聚糖转移到附近的段。开始使用剪辑的开始和结束时各分部可能会有所帮助。 </li><li>测量小肠和结肠全长全长。通过固定下来，以聚苯乙烯垫，套管，分为10个同等大小的段小肠分。的冒号分割成3段。您已经准备了15段（胃＃1），10个同样大小的小肠段（2-11），的盲肠（第12）和3个同样大小的结肠段（13-15）。 </li><li>断面肠道内的显着位置，用钳子抓住它，并刷新一次，用1.0毫升KHB到以前准备KHB containi的纳克2.0 ml微量离心管和涡大力，持续20秒。胃和盲肠直接放置和切在准备管。用1.0毫升KHB填充这些管子，从而漂洗从剪刀到管的管腔内容的痕迹。 </li><li>探针在12,000相对离心力离心5分钟，在室温下在台式离心机。在离心编制另外15个连续编号的1.5 ml离心管。 </li><li>上层清液转移到新的1.5 ml管和商店在黑暗中，在4°C，直到分析（下一步）。该探针可以保存数天，在4℃，无显着损失的FITC信号。对于较长的存储添加0.09％的钠，叠氮化物或储存在-18°C。 </li><li>吸移管100微升duplets在黑色96孔板中（Greiner公司生物之一，的Frickenhausen，德国）的上清液。用移液管100微升样品的KHB为空白。 </li><li>阅读在荧光板读取器（萨菲尔，Tecan公司，Crailsheim，德国），并量化fluorescencE在494纳米（吸收）/波长521纳米（排放）。从样品中减去空白值。 </li><li>计算的FITC-葡聚糖分布的几何中心（GC），这实际上是标记的重心的分布，由下式:GC =Σ（％的总荧光信号每段*段数）/ 100 </li></ol> 注:每个分部的荧光，其段数的百分比乘以肠道内的标记的分布的一个加权均值进行评估。通过标记的分布和行驶距离的影响这一点，但假定没有具体的基本分布。7计算出的GC值通常出现在组合的曲线图，展示了在胃肠道（ 图1A）的FITC葡聚糖分布。 </li><li> 结肠传输 <ol><li>老鼠应该是弱麻醉isoflur甲烷在氧气中的2％。注意:请确保动物会醒在40秒内。 </li><li>仔细检查通过插入的一个瘘探头（金属棒，直径为2 mm）进入结肠经肛门结肠通畅。 </li><li>使用瘘口的探头推进到2毫米的玻璃球3厘米的结肠，并立即拔出探头。将鼠标在一个透明的笼子里，并开始测量的时间后拉出的结肠瘘的探针。保持眼睛的鼠标和停止时的玻璃球后直接成为排出体外。 </li></ol></li></ol> 5。隔离ME标本组织化学分析<ol><li> Midjejunal段被切断从肠和沉浸在冷KHB在Sylgard的填充菜。 </li><li>肠系膜是固定的菜用0.2毫米的昆虫针（精细的科学工具，海德堡，德国）。 </li><li>剖开的部分沿其肠系膜边境和伸展〜120％，其长度和宽度。完成固定昆虫相反网站的肠系膜的引脚上。仔细冲洗Sylgard的菜，新鲜冰冷的KHB，从而冲洗所有管腔内容。 ，ME现在可以机械分离的粘膜，从肠系膜开始。在准备更换新鲜冷冻KHB几次。 </li><li>可以被分离的粘膜休克在液氮中冷冻，作进一步的分析，或丢弃。 </li><li>孤立我整个安装固定10分钟，在室温下以100％的乙醇。 清洗整个安装使用冷冻KHB三次。注意:也可以使用其他的固定剂（ 如 4％的甲醛，乙酸，丙酮）测试后，其与随后的分析的兼容性。 </li><li> ME整个安装准备进一步组织化学或immunhistochemical染色的: </li></ol><ol class="lalpha"><li> 组织化学:髓过氧化物酶（MPO）染色 浸润的中性粒细胞，可以检查以下MPO染色协议。 <ol><li>解决10毫克ĤANKER耶茨的试剂（Polyscience欧洲，Eppelheim，德国），在10毫升KHB，加入100μl的3％H 2 O 2。准备新鲜在使用前，不重用的解决方案。 </li><li> 10分钟，在室温下孵育整个安装。 </li><li>广泛KHB孵育10分钟用冷水冲洗标本。 </li><li>安装在水溶液中的安装介质（AQUATEK，默克公司，德国达姆施塔特）的标本，在光学显微镜下干燥后2小时分析幻灯片。 </li><li>计数的细胞数在5个随机选择的领域，作为MPO阳性细胞/ mm 2的计算。 </li></ol></li><li> 免疫组化染色: 因此MPO组织化学染色法，免疫组织化学染色的整个安装可以被执行。原则上，已建立的细胞培养或组织切片免疫组化染色协议，可以很容易地适应整个安装染色。然而，优化程序特定的反机构强烈推荐。 <ol><li>剪切成5×5毫米片标本，并在〜150微升 - 200微升抗体溶液在2.0毫升圆底离心管中进行染色。 </li><li>固定和阻塞的过程（ 即 3％BSA的PBS中，在室温下1小时），可以执行在"SYLGARD菜"。 </li><li>在PBS或其他缓冲液中洗涤步骤可以在12孔板中进行。 </li><li>仔细转移。标本染色管和洗涤板之间用锋利的镊子。 </li><li>与盖玻片在防褪色安装介质中最终的洗涤程序装载标本后，用荧光显微镜进行分析。 </li></ol></li></ol> 6。器官培养ME <ol><li>整个小肠被切除24小时后，IM放置在冷KHB含200 U / mL青霉素和100μg/ ml链霉素（KHB + P / S）。 </li><li>切肠切成3厘米长，脚各分部一个Sylgard的冰川SS菜。 </li><li>取出肠系膜细剪刀，滑的肠道织针。 </li><li>轻轻切开ME用锋利的镊子，并用湿润的棉签从下层剥离。粘膜保持的织针，并可以快速冷冻作进一步调查。 ME收集在寒冷的KHB + P / S。 </li><li>切孤立我带切成小块的2 - 4毫米的长度和孵化在冰冷的KHB + P / S为半小时，冲洗标本几次。 </li><li>转移约50-60毫克ME（至少一半ME 1小肠）在无菌的24孔板上，含有500微升的Dulbecco改良Eagle培养基（DMEM）。 </li><li>孵育在37℃和5％CO 2的额外的24小时。 </li><li>检查培养的细胞在显微镜下的细菌或真菌污染。 </li><li>收集上清，离心5分钟在1000 RCF和急速冷冻在液氮中。同时吸干的MUSC文件组织为30秒和重量上的干净的纸巾。 </li><li>可用于随后的分析释放的细胞因子（IL-6）的上清液通过ELISA或其他代谢产物（ 如亚硝酸盐），在上清液中通过遵循制造商的说明。 </li><li>每单位重量的ME组织标准化测得的浓度。 </li></ol> 7。代表性的成果<ol><li>功能的研究 最重要的参数，以评估严重程度的POI是考试的GIT 在体内 。 图1A说明了一个典型的分布FITC葡聚糖沿着胃肠道中未经处理的对照组小鼠24小时后，手术和小肠操纵。 "假手术"的动物表现出正常的GIT与计算几何中心（GC）在盲肠，IM导致GIT中的近端空肠一个显着延迟（ 图1B） 由测量球的排泄时间o分别集中在结肠的蠕动发2毫米的玻璃球。假手术组的小鼠显示IM的排泄时间:48 - 200秒，而导致长时间的排泄量之间的时间470 - 775秒（ 图1C）。 </li><li>形态学研究 要描述的炎症，内的ME表型的白细胞可以用组织化学或免疫组化分析。在图2A + B"假PMNs在低存在操作"观察动物（39±7个细胞/ mm 2）。 IM的小肠导致强烈的中性粒细胞浸润（660±86个/ mm 2）与假手术组小鼠相比， </li><li> ME器官培养 从细胞中释放出来的多种炎症介质是难以察觉，在组织裂解液。器官培养允许检测在培养上清液中释放介质。有代表性的调解员在POI是"NO"，这是在胃肠道中的主要抑制性神经递质。 无法检测到NO ME器官尽头TURE上清无操作控制动物（5±6μM/ 24小时/ mg组织）。在假手术（开腹手术）小鼠基础NO水平为53±36μM/ 24小时/ mg组织进行观察。缅因州文化小肠操纵（手术后24小时收获）小鼠产生显着更多的NO（2254±853μM/ 24小时/毫克组织）在ME器官培养24小时（ 图3）。 </li></ol><img alt="图1" src="/files/ftp_upload/4086/4086fig1.jpg" /> 图1。IM上的GIT的（A + B）和结肠转运的影响（C）GIT作为非吸收的荧光素标记的葡聚糖的百分比在15胃肠分部-胃（STO），小肠（SI 1-9测定），盲肠（CEC），冒号（公司）-90分钟口服后。 （A）胃肠道分布的异硫氰酸荧光素指数结肠传输测量的时间拉出瘘排泄的探头，直到2毫米的玻璃珠。转录后假手术或IM。假手术动物的标记位于盲肠或结肠近端与近端空肠位置，操纵动物相比。虚线表示计算GC。 （B）计算的GC表现出了长期的胃肠道转运时间后，IM。 （C）结肠转运时间与假手术动物表现出显着延迟在IM小鼠相比。 GC显示为15肠段的意思是（N = 5）。结肠传输时间显示意味着所有的个人价值组（n = 6）。 *** P &lt;0.001，学生的t检验。 <img alt="图2" src="/files/ftp_upload/4086/4086fig2.jpg" /> 图2。检测MPO阳性细胞在小肠ME（A）ME整个坐骑染色与瀚海耶茨试剂为检测MPO积极中性粒细胞24小时后剖腹手术（假）或IM程序。 （B）MPO阳性细胞定量分析每只小鼠在5个随机选择的领域。 N = 6个nimals每个组。 *** P &lt;0.001，学生的t检验。 <img alt="图3" src="/files/ftp_upload/4086/4086fig3.jpg" /> 图3的ME的培养物上清液中NO的产生。无操作控制的肌肉标本，假手术组和IM小鼠，术后24小时和24小时培养。未经处理的小鼠仅表现为非常低的基底产生NO。剖腹手术后，NO解放的增加而测量对照组和假手术组小鼠的显着差异。 24小时后，IM NO生产的未经处理或假手术组小鼠相比显着增加。例每组5只动物。 *** P &lt;0.001，1路ANOVA。

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没有利益冲突的声明。

分析不同水平的IM（小肠膨出10 min比轻轻用的两个棉花喷头与IM的小肠进入盲肠内容的疏散的小肠进行检查）的比较研究显示手术操作和POI的程度之间的相关性。与其他组相比，IM显示出最高的白细胞数量在ME（PNMS，巨噬细胞和肥大细胞），导致长期和严重的POI 8。 在建立模型的IM，肠操作的规范运作和强度的关键步骤。触摸肠系膜在此过程中，必须严格避免，以防止?...

<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> Ethilon 5/0 </td><td>爱惜</td><td> 1666H </td></tr><tr><td>棉花喷头</td><td>残废</td><td> 71010 </td></tr><tr><td>动脉导管</td><td> Vygon </td><td> 115-798 </td></tr><tr><td> 96孔板（黑色） </td><td>格雷纳生物之一</td><td> 655096 </td></tr><tr><td>玻璃球（直径为2 mm） </td><td>武夫</td><td>可应要求提供</td></tr><tr><td>昆虫针</td><td>精细科学工具</td><td> 26000-25 </td></tr><tr><td>瀚海耶茨试剂</td><td> Polyscience </td><td> 560694 </td></tr><tr><td> AQUATEK </td><td>默克</td><td> HC109850 </td></tr><tr><td> DMEM </td><td>龙沙</td><td> BE12-604 F </td></tr><tr><td> FITC-葡聚糖（分子量70000） </td><td> Sigma Aldrich公司</td><td> 46945-500MG-F </td></tr></tbody></table>

functional assessment of intestinal motility and gut wall inflammation in rodents: analyses in a standardized model of intestinal manipulation

胃肠道的炎症，是一种常见的人类疾病的各种原因。在调查复杂的细胞和分子的肠道病理动物研究模型是至关重要的。虽然经常在许多炎症性疾病，最近的作品表现出兴趣的器官的粘膜，肌层（ME）是也是一个高度免疫功能的器官，港口密集的居民免疫网络。1,2这些作品内进行标准化肠道操纵模式（IM），导致炎症的肠壁，主要限于向ME。临床炎症导致长期的肠道运动功能障碍，术后肠梗阻（POI），这是一个频繁和不可避免的腹部手术后的并发症。炎症的特点是解放促炎症介质，如IL-6和IL-1β或抑制性神经递质像nitriÇ氧化氮（NO）。随后，免疫细胞的数量极大外渗到ME，中性粒细胞（PMN）和单核细胞为主的终于保持POI。2天为期，肠麻痹导致风险增加的愿望，细菌易位和感染并发症脓毒症和多器官功能衰竭，并导致较高的经济负担。 在这个手稿中，我们证明了IM标准化的模式，并在体内胃肠道过境评估（GIT）和结肠运输。此外，我们表现出的方​​法分离ME粘膜免疫分析，这是非常重要的区别，在这些具有高度的免疫活性肠壁车厢之间的炎症反应。所有的分析很容易转移到任何其他的研究模型，影响肠胃功能。

Watch this Scientific Journal Video about Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation at JoVE.com

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

术后肠梗阻（POI）是腹部外科手术的并发症导致的发病率和住院时间延长。由于缺乏预防或治疗策略加强研究是必要的。因此，我们建立了一个标准化的和可行的小鼠模型的病理生理POI调查和研究潜在的治疗方案。

在啮齿类动物的肠动力及胃肠壁的炎症分析功能评价一个标准化的模式肠道操作

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

functional-assessment-intestinal-motility-gut-wall-inflammation

Research

JoVE Journal

Medicine

22.0K 次观看.  University of Bonn. 术后肠梗阻（POI）是腹部外科手术的并发症导致的发病率和住院时间延长。由于缺乏预防或治疗策略加强研究是必要的。因此，我们建立了一个标准化的和可行的小鼠模型的病理生理POI调查和研究潜在的治疗方案。

视频: Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

鼠内镜<em&gt;体内</em&gt;发生和肠道的评价多模式成像伤口愈合和炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

科研

医学

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.

猪节段性小肠缺血模型中肠道干细胞的分离与培养

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ...

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 ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

使用化学活检进行移植质量评估

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

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

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

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 ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

通过将CBir1 TCR转基因CD4 + T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

小鼠肠间充质分离导致端细胞高产量

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

大鼠膝骨关节炎的标准化推拿手法

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

作者聚焦:了解大鼠推拿操作的机制 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

推拿操作以减少膝关节骨关节炎大鼠的炎症和软骨损失

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...