作者想雪莉谢和珍妮弗佩恩承认他们的专家技术援助。通过小儿泌尿外科学会和斯坦福大学的儿科研究基金的拨款，这项工作成为可能。

一，准备尿道导管<ol><li>对于每个鼠标膀胱和肾脏进行采样，5-6不锈钢珠（膀胱，1.6毫米大小0.9-2.0毫米混合）或氧化锆珠（肾，0.5毫米大小），分别放置在一个离心管管。高压灭菌的含珠管。 </li><li>管冷却至室温后，加入200微升无菌PBS，每个不锈钢含珠管和400微升无菌PBS每个氧化锆珠含管。 </li><li>我们准备由红，侯德群13所述的尿道导管。高压灭菌一双干净的平头镊子和剪刀（光圈或类似）的罚款，并让仪器冷却到环境温度。层流罩与70％的乙醇擦拭表面。在油烟机，切一个30厘米的聚乙烯管材（近似）段。 3毫升无菌注射器，无菌的地方无菌30号针头（1 / 2英寸长针）。用灭菌镊子拿起削减聚乙烯管材的一端滑入针管，直到它符合枢纽。切管，使约2厘米的延伸超出针尖。取出导管，注射器，并在无菌培养皿。 </li><li>要减少交叉污染的可能性，我们每个鼠标一个导管。所有导管后，他们发现培养皿中接触到至少30分钟的紫外线照射消毒。不要直视紫外线灯或灯被激活时，暴露在罩体的任何部分。紫外线照射后，导管可长期存储在培养皿中。我们建议用封口膜密封盘，以帮助保持无菌。 </li></ol>二。动物接种<ol><li>小鼠实验操作前至少一个星期到达，以避免应力引起的混杂因素。雌性小鼠的使用，因为雄性小鼠尿道是非常困难的导尿的。 CBA / J是一​​个普遍使用，尿路感染易感自交系小鼠品系。老鼠应使用8周和12岁之间，因为这是免疫成熟的窗口前衰老。在实验的第一天，浸成一小瓶冷冻细菌股票无菌接种环结束。刮了一个小接种适当的琼脂平板上，连胜了。一般而言，肾盂肾炎大肠杆菌大肠杆菌 （UPEC）和其他菌株可过夜卢里亚Bertani（LB）琼脂在37℃周围空气中的C。 UPEC菌株成长为坚实，黄白色半透明的菌落在LB琼脂。立即返回细菌股票冰柜。肾盂肾炎的菌株，可随着时间的推移变得不那么致命的反复，串行文化。因此，我们建议每一个实验裸奔新鲜琼脂平板。 </li><li>在实验的第2天，确认后适当的菌落形态，至少5毫升肉汤培养物中挑选单菌落琼脂平板和地方过夜。 LB培养基可用于大多数肾盂肾炎的菌株（37℃，周围空气中的200 RPM）。保持宽松的肉汤培养管的上限，允许曝气。 </li><li>在实验的第3天，第一肉汤培养和肉汤培养的10％再次过夜（一般为37 ° C和200 RPM），以提高I型菌毛的表达。串行肉汤培养有利于优化细菌uropathogenicity，因为I型菌毛已被证明是一个关键致病因子在体内 1尿路病原体。保持宽松的肉汤培养管的上限，允许曝气。 </li><li>在实验的第4天，从第3天测量的肉汤培养物的OD 600。如果尚未被测试的菌株，执行了至少7原木（用PBS进行稀释，LB琼脂上执行的文化，37 ° C孵育过夜）10倍连续稀释电镀确定外径 600和CFU之间的关系/毫升。 0.4-0.5外径600作为一个经验法则，通常对应于1 - 2X10 7 CFU每50微升。一旦外径600 CFU /毫升之间的关系已经确定，可以调整肉汤培养物的OD 600对应所需的50微升的PBS CFU每鼠。 10微升，而不是50，接种，减少回流到kidneys6导致了一些工作建议。然而，大多数出版物使用鼠标微升50％。一般来说，10 6 108 CFU /鼠标，但每个细菌/小鼠品系组合需要经验体内测试所需的CFU范围。 </li><li>为了帮助确保不作废，接种滴入后立即，老鼠应该被剥夺的水至少全身麻醉诱导前30分钟。另一个关键的动作是诱导scruffing小鼠麻醉诱导排尿前，轻轻地按较低的abdomEN。 2〜2.5％异氟醚是安全的诱导剂量为8-12星期岁的女CBA的小鼠，维持1.8-2％的异氟醚麻醉。 </li><li>一旦实现了麻醉，小鼠被放置在他们的头插在异氟醚含氧连接到一个头锥仰卧位。按摩小腹疏散膀胱中的尿液。如果膀胱是比较空的，这个演习可能不会产生尿液。下腹及会阴部，用70％乙醇浸泡。 </li><li> 1无菌40cc的注射器没有针头是用来绘制所需的细菌接种。下一步，无菌导尿管无菌注射器放在。多余的管被切断导管，用一把剪刀，只有1-2毫米的管道，以便保持远端针尖。然后，注射器是挖掘和柱塞，用注射器直立删除所有气泡郁闷。抑菌润滑dollop果冻是喷到无菌培养皿或无菌注射器包装内。导管的尖端（附注射器）浸在果冻。在相同的实验测试，如果有多个细菌菌株正在小心，不要使用相同的果冻下降，润滑导管含有不同菌株。 </li><li>使用立体显微镜的放大倍数（0.8倍到5倍变焦范围），麻醉鼠标阴蒂是在非主导的手轻轻抓住一双细齿镊，并提出头侧揭露深粉红色的尿道口（图1）。注射器（附加导管）是掌握在占主导地位的手，握铅笔和导管尖端在从事尿道口在45度角。阴蒂是捉襟见肘，沿长轴注射器有效的“加载”到导管尿道。这一演习是必要的，因为鼠标尿道是多余的。应该很容易滑动导管进入膀胱（集线器，图2），可以轻轻飞旋，以帮助导航尿道多余的褶皱。一旦是hubbed导管，注射器，可降低，直到它与鼠标长轴平行。非主导的手可以放下镊子，以帮助稳定的立场是用来为主导的导管和注射器慢慢注入接种（至少5秒钟）。如果流体导管周围流动注射过程中，无论是膀胱全部或导管的阴道（只是尾椎尿道）。注射液，应立即停止，并仔细检查导管的位置。导管在尿道，如果给定的鼠标不应该被包括在实验中。另一方面，可以重新定位和错位的阴道的导管，重新尝试注射。注射后，导管和注射器慢慢地从鼠​​标撤回。 </li><li>为了减少早期接种后排尿，这可能撤离管理的细菌，从而导致变量的感染水平的可能性，一些研究人员在麻醉状态下保持30分钟后，经尿道前列腺电inoculation10的小鼠。手术后，小鼠恢复连续观测，以确认返回正常的呼吸方式和活动，在一个干净的变暖毯。一旦动物已经完全恢复抓取的能力，他们可能会返回到床上用品，含笼。 </li></ol>三。在受感染的组织测量的CFU <ol><li>各种鼠标UTI的出版物，报告从6小时到一个星期或更长的时间接种后的培养结果。需要凭经验确定每个细菌/小鼠品系组合的最佳时间点。尿scruffing无菌培养皿中的鼠标，获得文化。收集的尿液应保持在任何时候都冰。多的尝试，取得了很多时间，尿可能是必要的，因为鼠标排尿频繁，小批量（30-100微升每无效），并强调依赖。 </li><li>取得尿标本后，我们牺牲了使用异氟醚和颈椎脱位的小鼠。腹部是在无菌条件下打开。如果尿标本未成功获得早期，往往可以被吸出少量的尿液经膀胱壁使用30号针头和注射器。 </li><li>一个或两个肾脏取出，剁碎成至少4件（每件作品应不少于50微克）无菌培养皿中，并放入锆珠/ PBS含离心管放在冰上。膀胱取出，剁碎到至少4件，并放置到不锈钢珠/ PBS离心管放在冰上。组织/磁珠/ PBS离心管置于搅拌机均质机的子弹和组织匀浆使用“8”的速度设定为一分钟。这是常见的一些组织碎片后仍然同质化。只要50-100微升液体匀浆可吸管，固体碎片不一定是一个问题。如果同质化的时间是增加一些样品，关键是做所有组织，以保持同质化的一致性。我们利用一颗子弹搅拌机均质机，因为它避免了交叉污染的可能性，是较快速处理个别样品使用一个标准的均质之一，并没有明显的热量匀浆的样品。 </li><li>在微孔板应至少有两个或三个收集的尿液和组织匀浆稀释十倍。初步的实验中，它可能是审慎的做法，使至少7个10倍稀释。文化可达100微升尿，膀胱，和/或肾匀浆适当的琼脂。尿液标本可能是由于体积有限，可能有必要把其体积为100微升之前执行10倍稀释。如果这是必需的，初始稀释（前10倍稀释）将需要为细菌产量的最终计算的因素。我们宁可培养统一企业曙红亚甲蓝或麦康凯琼脂（在37 ° C孵育过夜），因为这些媒体是革兰氏阴性菌生物的选择性。 UPEC菌株麦康凯琼脂上生长，因为他们的能力发酵乳糖的中央脐，粉红色菌落。 </li><li>培养过夜后，每块板的菌落计数。对于连续稀释的样本，含有50-100个菌落的平板稀释被认为是最准确的计数。 “返回”计算，每组织CFU，每毫克组织，或每毫升。 </li></ol>四。代表性的成果<img src="/files/ftp_upload/2070/2070fig1.jpg" alt="图1" /> 图1。起重钳（照片上方超越）头侧的阴蒂与暴露尿道口（暗粉色组织） <img src="/files/ftp_upload/2070/2070fig2.jpg" alt="图2" /> 图2。尿道插管。双歧阴蒂，可以看到正上方的导管。 <img src="/files/ftp_upload/2070/2070fig3.jpg" alt="图3" /> 图3。膀胱CFU计数8周龄雌性CBA / J小鼠肾盂肾炎大肠杆菌经尿道感染后第2天大肠杆菌 。膀胱被同质化和麦康凯琼脂上连续稀释电镀CFU / ml的确定。钻石表明个别小鼠CFU /毫升，单杠表示位数CFU / ml为。

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经尿道的细菌接种的小鼠泌尿道感染的感应，是一个历史悠久，但技术要求高的程序11。红和侯德群最近发表的鼠标尿路感染13经尿道前列腺电技术的一个很好的回顾。在本文中，我们试图进一步说明这个实验方法的更细点。 初学者可练习他们的技术，由执行中线剖腹探查术，暴露膀胱，注入了墨汁在PBS（1％）的稀溶液中，观察膀胱蓝色着色。我们发现，一?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Cryovial tubes, 2.0 ml polypropylene (no caps)</td>
<td>&nbsp;</td>
<td>E&amp;K Scientific</td>
<td>640201</td>
<td>&nbsp;</td>
<tr>
<td>Caps for cryovial tubes, with 'O' ring</td>
<td>&nbsp;</td>
<td>E&amp;K Scientific</td>
<td>440011-N</td>
<td>&nbsp;</td>
<tr>
<td>Bullet Blender</td>
<td>&nbsp;</td>
<td> Next Advance</td>
<td>BBX24</td>
<td>&nbsp;</td>
<tr>
<td>Zirconium Oxide Beads (0.5mm)</td>
<td>&nbsp;</td>
<td>Next Advance</td>
<td>ZROB05</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel Beads (1.6mm)</td>
<td>&nbsp;</td>
<td>Next Advance</td>
<td>SSB16</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel Bead Blend (0.9-2.0mm)</td>
<td>&nbsp;</td>
<td>Next Advance</td>
<td>SSB14B</td>
<td>&nbsp;</td>
<tr>
<td>CBA/J Mice, 8-12 week old female</td>
<td>&nbsp;</td>
<td>Jackson Laboratories</td>
<td>000656</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline (NaCl 137mM, KCl 2.7mM, Na2HPO4 4.3mM, KH2PO4 1.4mM)</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>EM-SX0420-13 (NaCl) EM-PX1405-1 (KCl) EM1.06585.0 (Na2HPO4) EM-5108-1 (KH2PO4)</td>
<td>&nbsp;</td>
<tr>
<td>LB agar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LB broth</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>1.10285.0500</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane (Aerrane)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>NDC 10019-773-60</td>
<td>&nbsp;</td>
<tr>
<td>Inoculating loops</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>22-363-601</td>
<td>&nbsp;</td>
<tr>
<td>Eosin-methylene blue (EMB) agar powder</td>
<td>&nbsp;</td>
<td>Himedia Laboratories</td>
<td>MM022-500G</td>
<td>&nbsp;</td>
<tr>
<td>MacConkey agar powder</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>212123</td>
<td>&nbsp;</td>
<tr>
<td>LB agar powder</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>244520</td>
<td>&nbsp;</td>
<tr>
<td>Intramedic non-radiopaque polyethylene tubing, PE10 [inner dimension 0.28 mm (0.011 inch); outer dimension 0.61 mm (0.024 inch)]</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>427401</td>
<td>&nbsp;</td>
<tr>
<td>30 gauge needles, 1/2 inch</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>305106</td>
<td>&nbsp;</td>
<tr>
<td>1 ml tuberculin Slip-Tip syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309602</td>
<td>&nbsp;</td>
<tr>
<td>3 ml Luer-Lock syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309585</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transurethral induction of mouse urinary tract infection

生长在琼脂利益肾盂肾炎的菌株。一般而言，肾盂肾炎<em&gt; E。大肠杆菌</em&gt;（统一企业）和其他菌株可过夜卢里亚Bertani（LB）琼脂在37 °环境空气中的C。 UPEC菌株的增长为黄白色半透明的菌落，LB琼脂。适当的菌落形态的确认，然后挑单菌落，在肉汤培养。 LB肉汤可用于大多数肾盂肾炎的菌株。可以采用双串口，一夜之间LB培养基培养，以提高I型菌毛，定义为肾盂肾炎细菌致病因子的表达。肉汤培养物稀释至所需浓度的磷酸盐缓冲液（PBS）。 8至12周龄雌性小鼠被放置在异氟醚麻醉和transurethrally用聚乙烯管材覆盖30个计注射器的细菌接种。典型的接种，必须凭经验确定每个细菌/小鼠品系的组合，是10<sup&gt; 6</sup&gt;到10<sup&gt; 8</sup&gt; CFU每鼠标在10至50微升的PBS。 （有一天至数周），尿液样本及膀胱和两个肾脏所需的感染期后收获。各器官剁碎，置于PBS中，并在一个蓝色的子弹匀浆器匀浆。在PBS系列稀释尿液和组织匀浆，并在适当的琼脂培养。翌日，菌落形成单位计数。

Watch this Scientific Journal Video about Transurethral Induction of Mouse Urinary Tract Infection at JoVE.com

Transurethral Induction of Mouse Urinary Tract Infection

此影片将展示transurethrally诱导小鼠尿路感染及造成的感染程度量化的方法。

经尿道诱导小鼠尿路感染

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

transurethral-induction-of-mouse-urinary-tract-infection

Research

JoVE Journal

Immunology and Infection

36.5K 次观看.  Stanford University. 此影片将展示transurethrally诱导小鼠尿路感染及造成的感染程度量化的方法。

视频: Transurethral Induction of Mouse Urinary Tract Infection

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

鼠标垂体免疫原的制备诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

科研

免疫与感染

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJL小鼠诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

小动物模型尿路感染的实验研究: 男性和女性的经尿道插管

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

从小鼠在原代肝细胞培养物的炎性反应的诱导

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Transurethral Instillation Procedure in Adult Male Mouse

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into the urinary bladder to either induce animal models of bladder pathologies or evaluate the effectiveness of intravesical treatments. Most rodent models of lower urinary tract (LUT) pathologies are induced in female mice due to ease of intravesical instillation of the substances via the female urethra. However, due to anatomical differences between the female and male LUT, transurethral instillation in a male mouse has been deemed a very challenging procedure, and it has not been previously described. In this manuscript, we provide a detailed description of how to prepare polyethylene (PE) tubing for subsequent insertion into the urethra of a male mouse. In addition, we discuss the ideal types of PE tubing to be used depending on the desired site of inoculation. Furthermore, we describe point by point how to prepare an animal for a successful transurethral instillation to avoid injury to the urethra and ensure the delivery of the solution to the desired location. The procedure is started by retracting the prepuce and the glans to expose the opening of the urethral meatus. Next, the glans are grasped by blunt non-crushing forceps to stabilize the penis and the PE tubing. The PE tubing is first inserted into the urethral meatus parallel to the animal body, then its angle is adjusted by tilting the catheter to maneuver it to follow the natural curvature of the urethra. This technique can be used to induced murine models of bladder pathologies and/or evaluate the effectiveness of intravesical treatments in male mice.

Transurethral instillation is a challenging procedure and has not been well described in the literature. The aim of this manuscript is to describe a technique for transurethral insertion of a catheter for intravesical delivery of liquids with active substances into the urinary bladder and/or prostate in adult male mice.

成年雄性小鼠经尿道灌注术

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

幽门螺杆菌感染与胃病理的小鼠模型

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

尿道感染小鼠模型产生的单细胞内细菌群落的分离, 用于下游单细胞分析

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

小鼠肺损伤的诱导,通过博霉素内切注射

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...