作者感谢尼科Zantl，克劳斯普费弗和Bernhard Holzmann。这项研究是由德意志研究联合会，波恩坏哥德斯堡，德国（GRK - 840; VO 450/10-1）的支持。

1。鼠标的制备<ol><li>麻醉鼠标，通过腹腔注射麻醉液（见试剂表），并放置在仰卧位。 </li><li>鼠标的脚需要用胶布固定板，确保一个稳定的位置，在操作过程中的动物。 </li></ol> 2。操作<ol><li>以一个四套管，这是常见的静脉注射剂用于人类，而且其尖端的塑料管圆2毫米雕刻。我们使用三种不同大小的套管，以控制死亡率（BD Venflon 18GA（1,2 x45mm，80毫升/分钟）; 16GA（1,7 x45mm，180毫升/分钟）14GA（2,0 x45mm，270毫升/分钟））。 </li><li>彻底消毒腹部皮肤后，沿其中线约15mm切割。打开腹膜切开腹肌和腹膜，沿白线洞穴。 </li><li>确定盲肠极小心地拉出盲肠，回肠末端和升结肠腹部使用棉签。 </li><li> 15毫米从远端回盲瓣，刺破7 / 0缝合升结肠与墙。从而，肠道血管病变必须避免。缝合固定在结肠壁由两个手术节。 </li><li>现在穿刺升结肠近端从7 / 0缝线准备插管1-2毫米。小心地插入导管进入结肠的塑料管在畦上，直到浆膜水平。 </li><li> 7 / 0缝线周围套管的自由端和地点恰成畦塑料管的准备了两方面的结。 </li><li>现在，7 / 0缝线和针，通过结肠antimesenteric壁针。连续两个手术节必须执行，另外修复结肠壁塑料管。切割缝合结束。 </li><li>找回的一个小的套管铁部分，削减关闭关闭（1mm）的塑料管固定7 / 0缝线。 </li><li>现在，人们已经仔细牛奶大便从盲肠使用棉签对结肠支架的支架上，直到一个小技巧大便出现。 </li><li>把肠道进入腹膜洞穴，并通过腹腔注射0.5毫升生理盐水溶液performe液体复苏。 </li><li>关闭与连续缝合（4 / 0）腹膜。 </li><li> （4 / 0）奇异缝合关闭皮肤。 </li></ol> 3。术后护理<ol><li>把它在笼中的动物背部，其中应包含足够的食物和水。镇痛，一个强大的镇痛物质（我们使用丁丙诺啡）腹腔给药应定期进行。 </li><li>手术后的前两个天之内，一个人控制的动物，每6小时。 </li></ol> 4。深水CASP <ol><li>执行步骤1.1-2.4。 </li><li>不要刺破结肠。只要执行步骤2.6-2.8。 </li><li>执行步骤2.10-3.2。 </li></ol> 5。卡斯皮<ol><li>执行CASP使用一个14G套管以下步骤1.1-3.1 </li><li> 5H CASP后，再次经营的动物。准备和麻醉鼠标再次按照步骤1.1-1.2 </li><li>打开腹壁缝合。 </li><li>升结肠拉出插入支架。 </li><li>小心地切开缝合固定支架，并取出支架。 </li><li>在结肠单反相缝线（7 / 0）关闭的缺陷。 </li><li>把肠道进入腹腔，用10毫升生理盐水溶液冲洗后者的两倍。 </li><li>关闭与连续缝合（4 / 0）腹膜。 </li><li> （4 / 0）奇异缝合关闭皮肤。 </li><li>按照步骤3.1-3.2 </li></ol> 6。代表性的成果在操作几个小时，动物秀一开始败血症的临床体征。本病的典型症状是行动不便的，直立的外套，出汗，减少食物的摄入量，减肥和也减少了逃走的行为。一个严重的腹膜炎发展与连续的全身性感染的动物通常48小时内死亡。根据插入支架大小，可以产生明显的死亡率。 16G一个14G支架结果在100％的死亡率，使70％的死亡率，并可以通过使用一个18G支架（图1a）达到50％的死亡率。深水经营业绩在100％的成活率。通常情况下，所有的动物死亡inbetween后CASP的第一个48小时。因此，所有的动物，活在这个时间点可以被视为幸存者。不过，我们建议至少72小时的观察期发现罕见的“晚期死亡”后CASP。 卡斯皮结果取决于CASP后支架被删除时期间。干预后3h CASP的结果在45％的存活率。如果支架被删除后CASP，只有10％的5H动物生存的过程。支架拆除后死亡率100％（图1b）9H结果。深水卡斯皮导致100％的生存。卡斯皮设置，我们建议10天的观察期，因为通常是动物，死不迟于48小时后CASP。 腹部内脏24小时后CASP的宏观检查显示典型的腹膜炎的迹象：水肿，血管扩张，红斑肠壁，Peyer氏斑急剧划分，肠麻痹，自由流体，和化粪池的分泌（图2）。 全身感染细菌分析可以证明。 CASP 12小时后，大量的细菌金额可在腹腔灌洗检测，血，肝，肺，脾，和肾脏。细菌定性分析，揭示了典型的肠道细菌的全身性感染 ，像大肠杆菌大肠杆菌 ，类杆菌物种，肠球菌种，等（图3）。 CASP -诱导败血症表现在本地和亲和抗炎细胞因子和趋化因子的释放也全身。 12小时后CASP，TNF，IL -1β，IL - 6，IL - 10，MCP - 1和其他大量可以测量血液中，而且在悬浮器官，如肝，肺，脾，肾上清液用ELISA法（图4）。 建立在你的实验室，存活率，细菌学和细胞因子释放CASP时，应采取正确的性能CASP的控制参数。后来，这些参数可以被视为标准的读出所有的实验设置系统。 <img src="/files/ftp_upload/2299/2299fig1.jpg" alt="图1" /> 图1a：后CASP的生存。 CASP手术后的存活率取决于插入支架的直径。 14G CASP在100％的死亡率，死亡率在70％的16G CASP的结果，和18G CASP结果导致50％的死亡率。深水CASP与100％的生存。 N = 20/group 乙：卡斯皮手术依赖于支架拆除时的存活率。 14G CASP 3H支架拆除后，导致了55％的死亡率。 14G CASP的结果，在9H死亡率90％，而支架拆除后，在100％的死亡率14G CASP结果率在5小时后支架拆除。深水卡斯皮（5H）结果在100％的生存。 N = 10/group（深水卡斯皮;卡斯皮9H），N = 20/group（卡斯皮3H，5H卡斯皮）。 <img src="/files/ftp_upload/2299/2299fig2.jpg" alt="图2" /> 图2：CASP的腹部内脏后24小时 。腹壁被拆除后24小时，分别为16G CASP或假CASP。看起来生理假手术鼠标所在地。相比之下，水肿，充血，dilatated肠子，和Peyer氏斑的划分CASP的结果表现出严重的腹膜炎。 <img src="/files/ftp_upload/2299/2299fig3.jpg" alt="图3" /> 图3：细菌学 。 CASP导致严重感染的几个车厢，并在手术后12小时肠道细菌机关。菌落形成单位（CFU）每毫升，分别给予细菌数量。假手术组小鼠所采取的细菌培养是完全无菌的（未显示）。 CASP的16G，N = 5/group。 <img src="/files/ftp_upload/2299/2299fig4.jpg" alt="图4" /> 图4：细胞因子 。 CASP的触发全身免疫反应，一些细胞因子和趋化因子在血浆中检测到机关上清。模范的数字显示的促炎性细胞因子肿瘤坏死因子（TNF）和白细胞介素-6（IL - 6）以及主要是消炎的IL - 10。 12小时后CASP，可以检测到显着水平在血浆，肝，肺，脾。然而，IL - 10不能被检测到肺和脾后CASP。假手术不会导致任何细胞因子的分泌（未显示）。 CASP的16G，N = 5/group。

<ol>
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CASP是一个模式，满足了宝贵的脓毒症模型中假设的最标准的腹部败血症的多种微生物：CASP可以在小动物进行的，是高度重现性好，容易监测标准参数，提供了良好的读出来的可能性，并密切模拟临床环境腹部败血症。在比较研究中，我们可以证明，CASP的信息与早期和稳步增加全身感染和炎症（全身炎症反应综合征）弥漫性腹膜炎，而盲肠结扎穿孔（CLP）揭示了一个模型，腹腔脓肿形成持续的和次要（1）全身性炎症的迹象。卡斯皮技术提出了一种可能性来模拟连续的手术重点的卫生设施，这是人类最重要的治疗原则腹膜炎的临床过程。 CASP可以申请败血症或腹膜炎周围的所有问题，因此，用于处理与脓毒症研究的所有学科，免疫学，药理学，外科，重症监护医学，例如等有关败血症的不同科目的出版物，用CASP模型的选择下面给出（1-14）。 与CASP开始时，应注意一些的做法是必要的准确，快速地执行该操作。手术经验可能会有所帮助，但不是必需的。我们建议letality CASP的正确的性能参数的检测。一开始14G CASP作为一个大的支架比薄的更容易处理。一个训练有素的CASP的外科医生能够在10分钟内一个鼠标操作。任何实验开始之前，你应该确保你附近的那个时期。当你积累了一些经验与外科手术，你应该进行一次生存的动能，以检查您的技能。 14G CASP应导致100％的杀伤力。初学者在我们的实验室，以证明他们是与实验开始之前，他们能够产生100％的杀伤力与14G CASP。此外，细菌学和细胞因子的测量，应进行验证败血症。如果你有问题，在开始与CASP显微步骤，放大镜或手术显微镜的使用可能会有所帮助。但主要CASP可以很好地履行没有这些实用程序。再现我们的存活率，这是绝对必要的，相同的材料，尤其是支架（BD Venflon，见下文），用于。否则，结果大幅度变化是可能的。此外，我们建议可以有变化的结果而定，例如，外科医生的个性。然而，这样的因素的影响要小。 总之，CASP是一个可行的，简便，重现性和有价值的脓毒症模型，模仿密切腹部败血症的临床情况。因此，它是适合脓毒症研究的所有领域。

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Narcotic fluid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Mix 0.5 mL Rompun with 4.0 mL Ketanest, and add 5.5 mL of saline solution (0.9%). 
For complete anesthesia, inject intraperitoneally 10 &mu;L/g body weight</td>
</tr>
<tr>
<td>Rompun (20 mg/ml Xylazine)</td>
<td>&nbsp;</td>
<td>Bayer AG 51368 Leverkusen, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ketanest (25mg/ml Esketamine)</td>
<td>&nbsp;</td>
<td>Pfizer Pharma GmbH, 10785 Berlin, Germany</td>
<td>647028001E</td>
<td>&nbsp;</td>
<tr>
<td>BD Venflon 18GA (1,2x45mm, 80ml/min)</td>
<td>&nbsp;</td>
<td>BD Biosciences, Franklin Lakes, NJ USA 07417</td>
<td>391457</td>
<td>&nbsp;</td>
<tr>
<td>BD Venflon 16GA (1,7x45mm, 180ml/min)</td>
<td>&nbsp;</td>
<td>BD Biosciences, Franklin Lakes, NJ USA 07417</td>
<td>391455</td>
<td>&nbsp;</td>
<tr>
<td>BD Venflon 14GA (2,0x45mm, 270ml/min)</td>
<td>&nbsp;</td>
<td>BD Biosciences, Franklin Lakes, NJ USA 07417</td>
<td>391456</td>
<td>&nbsp;</td>
<tr>
<td>Catgut Polyester 4/0 white, non absorbable</td>
<td>&nbsp;</td>
<td>Catgut GmbH, 08258 Markneukirchen, Germany</td>
<td>17218113</td>
<td>&nbsp;</td>
<tr>
<td>Mariderm black 7/0, non absorbable</td>
<td>&nbsp;</td>
<td>Catgut GmbH, 08258 Markneukirchen, Germany</td>
<td>18104900</td>
<td>&nbsp;</td>		</tbody>
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colon ascendens stent peritonitis (casp) - a standardized model for polymicrobial abdominal sepsis

败血症仍是一个长期存在的问题在世界各地的重症监护病房。了解败血症的复杂机制，是建立在这一领域的新的治疗方法的先决条件。因此，动物模型的要求是能够紧密地模仿人类疾病，也充分处理科学问题。科隆Ascendens支架腹膜炎（CASP）是一个高度标准化的模式，多种微生物在啮齿类动物的腹部败血症。在此模型中，一个小支架手术插入导致的肠道细菌进入腹膜腔的连续漏的小鼠或大鼠的升结肠。腹膜炎，全身菌血症，由肠道细菌感染的器官，和系统性，而​​且当地的一些亲和抗炎细胞因子的释放过程的结果。 CASP的杀伤力可以控制插入支架的直径。这个模型的一个变种，与所谓的干预（卡斯皮）CASP，提高机会，第二次手术，在临床实践中常见的程序，删除化粪池重点。 CASP是一个很容易学会的高度重复性的模式，密切模仿腹部败血症的临床过程。它导致就在几个科学领域，例如免疫，infectiology，或手术问题研究。

Watch this Scientific Journal Video about Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis at JoVE.com

Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

科隆Ascendens支架腹膜炎（CASP）是一个高度标准化的模式，多种微生物在啮齿类动物的腹部败血症。本文介绍的CASP的外科手术。 CASP模型及其变种，允许有关败血症的主题各种问题进行系统调查。

科隆Ascendens支架腹膜炎（CASP） - 标准化的模式为多种微生物腹部败血症

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for ...

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Research

JoVE Journal

Immunology and Infection

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

19.3K 次观看.  University of Greifswald. 科隆Ascendens支架腹膜炎（CASP）是一个高度标准化的模式，多种微生物在啮齿类动物的腹部败血症。本文介绍的CASP的外科手术。 CASP模型及其变种，允许有关败血症的主题各种问题进行系统调查。

视频: Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

盲肠结扎穿孔致脓毒症作为模型小鼠中自噬研究

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

科研

免疫与感染

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

新生儿多药脓毒症的对照小鼠模型

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

脓毒症诱导免疫抑制的切卡结扎和穿刺和鼻内感染双重模型的设计

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

腹部主动脉瘤小鼠模型中脉冲传播速度、失散性和应变的测量

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

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 ...

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve warming of the animal to promote vasodilation, which aids in both the identification of the blood vessels and positioning of the needle into the vessel lumen while securely restraining the animal. Although tail vein injections are common procedures in many protocols and are not considered highly technical if performed correctly, accurate and consistent injections are crucial to obtain reproducible results and minimize variability. Conventional methods for inducing vasodilation prior to tail vein injections generally depend on the use of a heat source such as a heat lamp, electrical/rechargeable heat pads, or pre-heated water at 37 &#176;C. Despite being readily accessible in a standard laboratory setting, these tools evidently suffer from poor/limited thermo-regulatory capacity. Similarly, although various forms of restraining devices are commercially available, they must be used carefully to avoid trauma to the animals. These limitations of the current methods create unnecessary variables in experiments or result in varying outcomes between experiments and/or laboratories.
In this article, we demonstrate an improved protocol using an innovative device that combines an independent, thermally regulated, warming device with an adjustable restraining unit into one system for efficient streamlined tail vein injection. The example we use is an intravenous model of fungal bloodstream infection that results in sepsis. The warming apparatus consists of a heat-reflective acrylic box installed with an adjustable automatic thermostat to maintain the internal temperature at a pre-set threshold. Likewise, the width and height of the cone restraining apparatus can be adjusted to safely accommodate various rodent sizes. With the advanced and versatile features of the device, the technique shown here could become a useful tool across a range of research areas involving rodent models that employ tail vein injections.

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

在穆林真菌败血症模型中用于高效尾静脉注射的当代加热/抑制装置

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...

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

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.
This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.
Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

沙 门氏菌 斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection ...

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous efforts to elucidate the pathophysiology of sepsis using various animal models; the mouse model of sepsis induced by cecal ligation and puncture (CLP) is widely used in laboratories. The three technical aspects that affect the severity and replicability of the CLP model are the percentage of cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. The rapid and specific diagnosis of sepsis is a crucial factor that affects the outcome. The gold standard for sepsis diagnosis is microbial culture; however, this process is time-consuming and sometimes inaccurate. The detection of sepsis-specific biomarkers is fast, but the existing biomarkers are unsatisfactory due to a short half-life, non-specificity, and insufficient sensitivity. Therefore, there is an urgent need for a reliable biomarker of sepsis in the early stages. Previous publications suggest that excessive neutrophil extracellular traps (NETs) occur in sepsis. Citrullinated histone H3 (CitH3), as a NET component, is elevated both in septic animals and patients, and the presence of CitH3 is a reliable diagnostic biomarker of sepsis. The present study aimed to describe a standardized mouse model of CLP-induced sepsis and establish a reliable blood biomarker of sepsis. Our work may contribute to the early and accurate diagnosis of sepsis in the future.

This protocol presents the operative details of cecal ligation and puncture (CLP) in a mouse model of sepsis. CLP is one of the most widely used techniques to create an animal model of sepsis. Therefore, a standardized CLP protocol is required for the attainment of reliable research results.

在盲肠结扎和穿刺诱导的脓毒症小鼠模型中评估可靠的生物标志物

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous ...

增强的 Intestine-on-chip 平台，用于免疫学洞察

Immunocompetent Intestine-on-Chip Model for Analyzing Gut Mucosal Immune Responses

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent model includes Human Umbilical Vein Endothelial Cells (HUVEC), Caco-2 intestinal epithelial cells, tissue-resident macrophages, and dendritic cells, which self-organize within the tissue, mirroring characteristics of the human intestinal mucosa. A unique aspect of this platform is its capacity to integrate circulating human primary immune cells, enhancing physiological relevance. The model is designed to investigate the intestinal immune system&#39;s response to bacterial and fungal colonization and infection. Due to its enlarged cavity size, the model offers diverse functional readouts such as permeation assays, cytokine release, and immune cell infiltration, and is compatible with immunofluorescence measurement of 3D structures formed by the epithelial cell layer. It hereby provides comprehensive insights into cell differentiation and function. The intestine-on-chip platform has demonstrated its potential in elucidating complex interactions between surrogates of a living microbiota and human host tissue within a microphysiological perfused biochip platform.

作者聚焦：为传染病研究开发免疫活性器官芯片模型

Our detailed protocol outlines the creation and use of the advanced intestine-on-chip model, which simulates human intestinal mucosa with 3D structures and various cell types, enabling in-depth analysis of immune responses and cellular functions in response to microbial colonization.

用于分析肠道粘膜免疫反应的免疫活性肠芯片模型

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent ...