这项工作是支持R01 AI32556（NIAID的国立卫生研究院）。这项工作也得到了支持路易斯安那疫苗中部和南部路易斯安那州路易斯安那州摄政理事会主办的传染病研究学会的部分。

1。阴道白色念珠菌接种<ol><li>接种前三天，同时抑制动物暴露腹部，注入100μL芝麻油含有0.1-0.5毫克β-雌二醇皮下注射小腹。前进针约5至10毫米，外侧的皮肤，以尽量减少注射部位的泄漏。 在这个模型中，由于接近生殖道的最佳皮下注射雌激素在小腹。有效剂量可能会有所不同小鼠品系，年龄或雌激素衍生物。在以往的研究中使用CBA - J（H -κ），C3H/HeN（H -κ），C57BL / 6（H - B），BALB / C（H - D），DBA / 2（ H 2 D），SJL 6-8周龄的小鼠（H - 2 S），0.1毫克μL发现有效证明阴道壁增厚，减少阴道粘液和增加上皮细胞脱落。以上，在此浓度的雌激素治疗的小鼠表现出一致的阴道念珠菌定植。接种其他菌株和年龄的小鼠，试验研究，建议，以确保修改后的条件下，雌激素的有效性，如果有必要增加雌激素的剂量。 雌激素的解决方案应准备新鲜，每次注射日。为了确保香油中的雌激素完整的溶解度，彻底的解决方案，使用一个旋涡混合器和热间歇混合，在37 ° C。在整个学习期间每周重复注射。 </li><li>为了准备接种，添加一个C 的 loopful从最近的亚文化编制成10毫升与0.1％葡萄糖补充Phytone蛋白胨肉汤（SDA），沙氏葡萄糖琼脂念珠菌blastoconidia 。在25 ° C在一个惊天水浴孵育肉汤培养18小时的固定相。 </li><li>孵化后，收集日发送到一个15毫升的锥形管和离心机在5分钟的800 XG的肉汤培养。用无菌PBS洗涤沉淀两次。 </li><li>枚举通过台盼蓝染色排除血球可行blastoconidia。调整细胞浓度为2.5 × 10 6 / ml的无菌PBS（或所需的接种浓度） 。 </li><li>为了稳定鼠标，用两个手指举行的尾巴基地，向上抬起臀部，使阴道口朝自己的方向（图1A）面临。它是理想的，如果鼠标被放置在平坦的磨碎的表面（如笼顶部），使鼠标可以提供对尾克制的阻力。 </li><li>吸取20μL（或所需的量不超过20μL）接种悬挂插入深约5毫米到阴道腔（图1B）枪头。完成这一步，尽快和尽可能轻轻减少鼠标窘​​迫。 </li></ol> 2。阴道灌洗<ol><li>以下安乐死（或麻醉），H旧的鼠标用两个手指，使阴道口变得露出尾巴的基地向下。 </li><li>通过引进与枪头反复抽吸和鼓动100μL无菌PBS灌洗阴道腔。枪头可能成为与细胞堵塞。如果发生这种情况，免除阻碍细胞，并在阴道内，继续与余下的PBS lavaging。收集到离心管灌洗液。 </li><li>另外，阴道灌洗，可与异氟醚吸入麻醉麻醉小鼠。对于这一点，暴露小鼠蒸发异氟醚，直到它们完全镇静剂（〜30秒）。向下按住老鼠尾巴的基地，并使用无菌PBS 50μL，轻轻灌洗阴道管腔。确保一个枪头，以避免恶劣的鼓动，在此过程中，尽量减少创伤阴道。镇静小鼠从麻醉中恢复接触周围空气的30秒内。 异氟醚可以使用异氟醚蒸发器和O 2（首选）或一个标准的降系统关闭蒸发器系统（同时服用镇静剂，以避免呼吸窘迫，需要密切监测动物）麻醉室蒸发。 对麻醉小鼠阴道灌洗液应在同一小鼠的纵向灌洗样本的首选方法。连续lavaging不影响评估随着时间的推移41真菌负担。 </li><li>湿安装的准备工作，转移到载玻片10μL灌洗液400 - 1000倍放大倍率光学显微镜观察。此外，灌洗液细胞成分可以染色研究细胞和核形态。涂抹的准备工作，转移到载玻片10μL灌洗液和使用一个枪头外壁轻轻蔓延。保持与CytoPrep固定液涂抹样品和标准的巴氏法染色（子宫颈抹片检查）。 400 ×放大倍率光学显微镜观察。 </li></ol> 3。阴道霉菌负担的量化<ol><li>在96圆底板，转移到一个顶行和该列分为以下5井（跌板）180μL无菌PBS灌洗液。 </li><li>使阴道灌洗液1:10系列稀释液20μL转移到旁边的列以及。调匀反复每次转让前的愿望。连续稀释至12灌洗样本（一个完整的横向排）可以同时进行，使用了12道移液器。 </li><li>从最低稀释，转移到沙氏葡萄糖琼脂（SDA），10μL的样品。可以进行多达36个样品的电镀1板使用方电网和可调间距多道移液器的Petri菜准备SDA。 </li><li>枚举孵化后的菌落形成单位（CFUs）在34 ° C F或48小时</li></ol> 4。阴道组织提取<ol><li>阴道灌洗过程，奠定了安乐死鼠标在它的后面，并用70％乙醇饱和腹股沟区。使用一对镊子，解除泌尿口向上，使阴道口变得暴露。 </li><li>一双弯钳插入阴道腔和宫颈定位。同时保持与镊子牢牢把握，提取子宫颈通过阴道腔（图2A - C的）。 </li><li>海关的阴道在阴道口的基地，然后取出宫颈从阴道手术剪。请记住，阴道组织繁难（阴道内的上皮端暴露向外）。组织可以是横向倒阴道保持原来的方向，或成片开使外侧切口。 切除阴道组织可用于1）淋巴细胞提取以下拼贴激酶消化（〜1 × 10 4 /鼠标）40，2）上皮细胞的分离以下dispase消化（〜5 × 10 4 /鼠标）28，3）冷冻或石蜡包埋的准备工作，为25个组织学分析。 </li></ol> 5。腰淋巴结切除术<ol><li>阴道灌洗过程，奠定了安乐死鼠标在它的后面，腹部用70％乙醇饱和。一个从小腹开始到胸部的外侧切口，暴露内部器官。使用双手一双镊子，向上移动肠子，使中央的血管变得可见。 </li><li>找到下腔静脉和腹主动脉。通常情况下，可确定一对腰淋巴结腹主动脉旁，之间大约一半位于肾和髂动脉的起源39。这些淋巴结，可以直观地区别于脂肪组织，弹性质感更轻，颜色更不透明相比，脂肪组织（图3）。这些淋巴结明显较大，在受感染的动物，比未接种动物。 </li><li>海关置于节点microforceps的淋巴结，然后轻轻地拉起周围组织分开。 </li></ol> 6。在单细胞悬液中分离淋巴细胞<ol><li>转移到无菌丝网屏幕置于无菌玻璃培养皿〜10毫升汉克斯&#39;平衡盐溶液（HBSS）（图4）菜内（约3 × 3厘米大小2）淋巴结肿大。 </li><li>倾向于轻微的培养皿中，按屏幕对用注射器柱塞头的淋巴结。确保打破所有淋巴结的节点，使细胞内容物通过屏幕传递节点的非细胞成分（即细胞膜，间质，脂肪），而留在屏幕上。 </li><li>使用相同的柱塞和注射器，吸吨他HBSS中含有细胞。屏幕〜5毫升的HBSS洗净，并收集到一个15毫升的锥形管，其余的液体。 </li><li>离心10分钟800 XG。在流体的顶部之前丢弃的液体用吸管吸任何脂肪沉积。与HBSS洗涤细胞沉淀三次。 1毫升的HBSS，并列举可行的细胞经台盼蓝染料排斥重悬沉淀。 </li></ol> 7。代表性的成果: &gt; 4天接种小鼠阴道灌洗液细胞组分通常由念珠菌 ，上皮细胞和细胞浸润（图5）。 念珠菌湿装入镜， 可以直观地确定由菌丝的存在以及酵母（图5A） 。可染巴氏技术的研究，其中主要细胞是由三，形核确定中性粒细胞的上皮细胞和浸润的白细胞，阴道灌洗液涂片准备AR叶（图5B）。发现很少中性粒细胞，如果有的话，在未接种的小鼠（图5C）41 。 阴道霉菌负担的一个例子是如图6所示。在特定时间点收集的阴道灌洗液培养CFU枚举（图6a）。 念珠菌感染阴道定植/坚持周雌激素治疗的小鼠接种（图6B），而念珠菌不能建立在非雌激素治疗的小鼠接种（图6C）阴道定植。雌激素治疗的未接种的小鼠仍然念珠菌的负面整个时间（数据未显示）。此外，阴道灌洗，可以执行，也可以在不同的小鼠在各时间点或纵向相同的小鼠在麻醉状态下的一次。 腰淋巴结初级引流淋巴结，生殖道和阴道的挑战全身免疫反应，以评估最相关的网站。 ñOTE，这些淋巴结可能成为扩大接种小鼠，而他们通常出现在未接种的小鼠相当小。白血球细胞的回收率通常范围从8 × 10 5 /未接种的小鼠，以5 × 10 6 /接种鼠标。除了腰淋巴结，腹股沟，腘窝及肠系膜淋巴结也可使用。 <img alt="图1" src="/files/ftp_upload/3382/3382fig1.jpg" /> 图1。阴道念珠菌接种。 A）鼠标克制接种。鼠标放在一个铁丝笼插入和尾巴的基地举行，稍微向上抬起双腿，露出阴道口。用同样的手，因为它试图抵制尾巴克制，臀部的鼠标可以稳定。 b）介绍的接种到阴道腔。一个枪头轻轻地插入到阴道腔深约5毫米。然后敷悬挂接种。 图2。阴道组织提取。 AB）提取的子宫颈。宫颈是位于弯钳和向外通过阴道腔暴露。一旦出了阴道内，子宫颈是进一步拉向外充分暴露阴道。三）提取的阴道。阴道切除外阴用剪刀。分离后，取出从阴道内的子宫颈。 <img alt="图3" src="/files/ftp_upload/3382/3382fig3.jpg" /> 图3。鉴定腰淋巴结。之间周围器官/血管在骨盆附近的腰淋巴结的位置表示。一，腹主动脉。 B，膀胱。 C，髂总动脉。我肠子。 L时，肝。 R，直肠。 S，脾。 U，子宫。 <img alt="图4" src="/files/ftp_upload/3382/3382fig4.jpg" /> 图4。腰淋巴结丝网屏幕上的节点。淋巴结汇集到放置在培养皿中与HBSS屏幕。对用注射器推杆，获得单细胞悬液淋巴细胞的屏幕上按下淋巴结。 <img alt="图5" src="/files/ftp_upload/3382/3382fig5.jpg" /> 图5。接种小鼠阴道灌洗液的细胞组分。 a）湿安装和B）的巴氏涂片阴道灌洗样本的准备收集4天接种后和C）未接种小鼠。图像显示在1000 ×（A）或400 ×（B，C）的放大倍率。插入B中显示1000 ×阴道中性粒细胞的核形态。假丝酵母菌（Y）和菌丝（H）上皮细胞（EC）和neutrohils（N）表示。 <img alt="图6" src="/files/ftp_upload/3382/3382fig6.jpg" /> 图6检测离子阴道霉菌负担。一）代表白念珠菌菌落生长在SDA板。整齐（N）的6个不同的接种小鼠（上排）灌洗样品连续稀释和培养CFU枚举。二）在雌激素阴道霉菌负担的量化处理和C）非雌激素治疗的老鼠。 CFU/100μL接种小鼠肺泡灌洗液评估在指定的时间点。

<ol>
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没有利益冲突的声明。

念珠菌性阴道炎的实验小鼠模型已经建立，在过去的几十年历史念珠菌的黏膜宿主反应，以及用于研究测试抗真菌疗法3,4,11,13,16,17,19,21,24 ， 25,37。协议提出，这里纳入效率和劳动密集程度较低的方法，并出现念珠菌性阴道炎的最优化模型系统描述迄今为止。这些技术使真菌负担和收集阴道标本的快速定量分析。此外，以往的研究测试小鼠的几个haplotypic株（N = 13）和不同时间点接种后发现与 念珠菌 2,6,41变异相似的水平，在真菌的负担，并宿主反应阴道定植。因此，这种模式可以适应现有的协议小鼠品系或感染的持续时间没有限制。然而，应该认识到，阴道真菌负担的可变性高的动物给予相同的接种（图6B）之间。有证据支持这一变化的发生是由于不同程度的早期坚持阴道上皮41。因此，7-10小鼠/组建议用于统计目的。 C.不同菌株之间的阴道霉菌负担的变化也证明念珠菌 ，这表明，并非所有的 C株念珠菌有能力同样殖民鼠标阴道。例如，C念珠菌实验室3153A（株）在本议定书中使用已表现出更高的阴道定植比SC5314（临床分离），较高的接种（日志）须获得相当于阴道的真菌负担水平与3153A 27。事实上，一个充满变数的几个临床分离的阴道定植已记录 36 。因此，应德EN确定每个C.最佳接种念珠菌应变，以确保在小鼠一致定植。通常情况下，意味着从5 × 10 4到5 × 10 5 / 100μL灌洗液应该是一致的殖民检测的CFUs 。灌洗CFUs的量化评估阴道霉菌负担，是适合这种模式 ，念珠菌blastoconidia和菌丝通常不渗透超出了阴道上皮的表面层。我们以前的后灌洗阴道组织的组织学评价很少显示剩余 念珠菌 25,41 。然而，我们认识到，菌丝可miscounted随着他们的成长作为一个单一的殖民地的琼脂平板上，可能无法反映准确的真菌负担的可能性。 CFU枚举，在体内成像技术开发新的替代方法已成功地评估阴道真菌的负担，使用基因工程发光C.时白念珠菌 </EM&gt; 9,29。此外，该协议可以修改，诱导C 。 光滑定植在15,20糖尿病动物。迄今为止，其他非C。 白色念珠菌引起的阴道炎的小鼠模型尚未见报道。 雌激素的管理是这种模式的关键，发起强大的 阴道念珠菌6,14,23定植。此外香油悬浮液中的雌激素，可溶于水的雌二醇在PBS和皮内植入一个控释雌二醇颗粒为雌激素行政替代方法，并已受聘在女性生殖道感染10的其他小鼠模型注射皮下注射35。在这个模型中的高雌激素的要求可以由两个生理因素解释。其一是，提升的雌激素促进阴道上皮5,8,30分层。加厚从增加上皮细胞增殖的上皮都可能OW 念珠菌 ，阴道壁和随后的殖民统治获得更好地遵守。其次，已知有阴道上皮细胞糖原含量高。增加糖原沉积到30阴道壁组织中雌激素水平的结果。高架糖原在阴道的微环境， 从而可能使念珠菌得以蓬勃发展，提供额外的营养素。免疫前的分析表明，外源性雌激素没有影响细胞粘附分子表达 40 。外源性雌激素治疗的动物的一个缺点是，升高的雌激素也被称为促进阴道共生菌群30的过度生长。由于植物区系的组成可能会有所不同动物之间显著，这可能会增加一个变量共生生物体可能影响念珠菌的生长或调节宿主反应。在审议这一问题，该模型需要雌激素治疗控制（未接种）动物被列入所有的实验和分析并行。 在这种小鼠模型，可准确评估的抗真菌剂的疗效，由此处所述的协议，可提供在体内导致供临床使用的潜在疗法的发展的测试的关键。事实上，模型的稳健性提供了良好的临床疗效迹象。除了 ​​使用雌激素，在小鼠的阴道pH值中性促进菌丝，念珠菌致病性的形式在所有接种小鼠看到的生长。临床观察，一个强大的阴道发生中性粒细胞迁移而不影响真菌负担31,41的小鼠的一个子集。中性粒细胞的存在是突出妇女在对症阴道炎，似乎是平行的小鼠接种 12,41 。由于其他的临床症状（即，瘙痒，肿胀），无法精确衡量在小鼠，阴道中性粒细胞的评估作为简单而可靠的指标，在​​这个模型中的炎症（症状）。 如图5所示的液体灌洗阴道显微镜是在我们的实验室进行例行评估念珠菌定植和阴道炎症。此外，菌丝得分也可以被用来作为感染的措施。此后，灌洗液的细胞和可溶性组分可以被保存和未来的分析低温存档。值得注意的是，该协议的一个有利的功能是相同的小鼠在麻醉状态下的纵向，可以进行阴道灌洗。我们先前的研究证实，纵向评价，不影响阴道的真菌负担的评估。在感染的纵向分析所需的情况下，这种方法是特别有利的。 最后，我们已经开发出一种少侵入性切除术，阴道。这有效和快捷的的技术要求没有在切口腹部或任何内部器官，留下完整的生殖道的休息。这种模式的另一个有用的功能是缺乏免疫抑制剂的要求发起念珠菌定植。这一点尤为重要，因为维护自然宿主的免疫状态，是一个免疫学研究和宿主反应微生物的挑战的关键环节。因此，这些方法的组织和细胞，可应用于各种体外免疫检测。 总之，我们提供了几个重要的功能和阴道念珠菌病的实验模型的代表性成果。除了 ​​白色念珠菌引起的阴道炎，由其他病原体的女性下生殖道感染，包括淋球菌，阴道毛滴虫和单纯疱疹病毒进行了研究，利用小鼠模型，生物体intravaginally在激素治疗的小鼠 1 介绍 ，7,10,22,26,35,38。因此，涉及念珠菌性阴道炎的研究是有用的技术，可以应用于并有可能推进的方法来研究发病机制及主机女性下生殖道的其他传染病的免疫反应。

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>雌性CBA / J小鼠</td><td>查尔斯河实验室</td><td> 01C38 </td><td> 5-6周龄</td></tr><tr><td> 白色念珠菌 （3153A） </td><td>国家征收的致病真菌，英国</td><td> NCPF3153 </td><td></td></tr><tr><td>芝麻油</td><td> Sigma - Aldrich公司</td><td> S3547 </td><td>不需要使用前必须预消毒</td></tr><tr><td> Β-雌二醇17戊酸</td><td> Sigma - Aldrich公司</td><td> E1631 </td><td> 0.1 - 0.5mg的香油</td></tr><tr><td> Phytone蛋白胨</td><td>碧迪</td><td> 211906 </td><td>补充0.1％葡萄糖</td></tr><tr><td>台盼蓝溶液</td><td> Sigma - Aldrich公司</td><td> T8154 </td><td></td></tr><tr><td>沙氏葡萄糖琼脂</td><td>流式细胞迪克inson </td><td> 211584 </td><td></td></tr><tr><td>型胶原酶IV </td><td> Sigma - Aldrich公司</td><td> C5138 </td><td> 0.25％ </td></tr><tr><td> Dispase </td><td> Invitrogen公司</td><td> 17105-041 </td><td> 1.7 U /毫升</td></tr><tr><td>丝网屏幕</td><td>雷公藤多甙</td><td> 060X060S0065W36T </td><td>第60号网，不锈钢</td></tr><tr><td>汉克斯"平衡盐溶液</td><td> Invitrogen公司</td><td> 24020-117 </td><td></td></tr><tr><td> CytoPrep固定液</td><td> Fisher Scientific则</td><td> 12-570-10 </td><td>保留涂抹幻灯片</td></tr><tr><td>巴氏染色EA - 65 </td><td> EMD化学品</td><td> 7054X - 85 </td><td></td></tr><tr><td>巴氏染色OG - 6 </td><td> EMD化学品</td><td> 7052X - 85 </td><td></td></tr><tr><td>哈里斯"明矾苏木</td><td> EMD化学品</td><td> 638A - 85 </td><td></td></tr><tr><td>异氟醚</td><td>百特医疗用品</td><td> NDC 10019-773-60 </td><td>异氟醚蒸发器，或在一个下拉系统中使用封闭麻醉室</td></tr></table>

protocols for vaginal inoculation and sample collection in the experimental mouse model of candida vaginitis

由念珠菌引起的外阴阴道念珠菌病（VVC），是一个较低的女性生殖道，在他们 18,32-34的生育年龄影响其他健康妇女中约75％的真菌感染。诱发因素包括生殖激素水平的抗生素使用，未经控制的糖尿病和干扰因怀孕，口服避孕药或激素替代疗法 33,34 。复发性VVC（RVVC），定义为三个或三个以上，每年发作，影响一个单独的5至8％的妇女没有诱发因素33。 的VVC的实验小鼠模型已经建立，并用于研究的发病机制和 粘膜念珠菌3,4,11,16,17,19,21,25,37宿主反应。这种模式也得到了测试体内 13,24潜在的抗真菌治疗。该模型要求的动物保持在一个国家选择pseudoestrusimal 念珠菌定植/感染6,14,23。在这种情况下，将已接种过的动物身上检测到的几个星期到几个月的阴道霉菌负担。过去的研究表明一个非常高的动物模型和人类感染的相对免疫和生理特性3,16,21之间的平行。差异，但是，包括念珠菌作为阴道正常菌群的缺乏和中性在小鼠的阴道pH值。 在这里，我们展示了一系列的鼠标阴道炎模型的主要方法，包括阴道接种，迅速收集阴道标本，评估阴道的真菌负担，细胞提取/隔离的组织筹备工作。其次是阴道灌洗液真菌的负担，及引流淋巴结白细胞产量的选民代表结果。随着使用麻醉剂，灌洗样本可以在多个时间点收集纵向评价相同的小鼠感染/定植。此外，这种模式要求没有启动感染的免疫抑制剂，使定义的主机条件下的免疫学研究。最后，模型和每种技术的介绍，这里可能引起的方法来研究降低女性生殖道（细菌，寄生虫，病毒）和各自的局部或全身的宿主防御的其他传染病。

Watch this Scientific Journal Video about Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis at JoVE.com

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

在评价中使用的关键技术<em&gt;念珠菌</em&gt;阴道炎在实验动物模型进行了描述。该方法将允许从阴道标本和淋巴细胞的快速收集，排水腰淋巴结。这些技术可能会引起女性下生殖道的其它疾病的小鼠模型。

实验小鼠模型阴道接种和样品采集的协议念珠菌阴道炎

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

protocols-for-vaginal-inoculation-sample-collection-experimental

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

32.9K 次观看.  Louisiana State University Health Sciences Center. 在评价中使用的关键技术念珠菌阴道炎在实验动物模型进行了描述。该方法将允许从阴道标本和淋巴细胞的快速收集，排水腰淋巴结。这些技术可能会引起女性下生殖道的其它疾病的小鼠模型。

视频: Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

实验转移和CTL过继转移免疫小鼠模型

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

科研

免疫与感染

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 ...

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 ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

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 ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

建立与艾滋病毒相关的肝脏发病机制的双人性化TK-NOG小鼠模型

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

在无脊椎动物病理学研究中发现血淋巴收集和接种转移到大黄微加分中

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

小鼠脚垫接种模型，研究病毒引起的神经炎反应

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

前体内分析研究胃 肠道的 念珠菌血脂磷

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...