Name: an il-8 transiently transgenized mouse model for the in vivo long-term monitoring of inflammatory responses
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这项工作得到意大利囊性纤维化基金会项目FFC＃18/2013，FFC＃29/2015和意大利囊性纤维变性联盟的支持，该联盟通过威尼托分公​​司 - 协奏曲Veneta Lotta控制Fibrosi Cistica Onlus。

描述的所有动物实验由维罗纳大学实验研究服务部门间动物实验室内动物福利委员会批准，并遵守欧洲指令2010/63 UE，意大利D.Lgs 26/2014和修订版"实验动物护理和使用指南"，华盛顿特区:国家科学院出版社，1996年。该方案和实验由美国国家卫生研究院（n 273/15）批准。动物可以自由进入标准的啮齿动物食物和软化的自来水，并且适应环境至少5天到当地的生殖条件（室温:20-24℃;相对湿度:40-70％;光暗周期:12小时）在任何治疗之前。 体内基因递送<ol><li>使用层流罩来制备体内递送试剂/核酸复合物。 </li><li>定义实验方案and参数遵循制造商的体内指示。 <ol><li>每只小鼠使用200μL复合物的总注射体积。 </li><li>从悬浮在无内毒素水中的40μgDNA开始;优化范围可达60μg。 注意:根据制造商的说明，注射体积中核酸的最终浓度不得超过0.5μg/μL。 </li><li>使用N / P比为6-8（每克核酸为0.12-0.16μL的递送试剂）。计算相应的输送试剂体积。 注意:复合物应该是阳离子的，用于有效的细胞进入。 N / P比定义为每个磷酸核酸（P）的体内递送试剂上的氮残基数（N），并表示复合物内离子平衡的量度。 </li></ol></li><li>在5％葡萄糖（最终浓度）中稀释计算量（参见步骤1.2.1）核酸使用10％葡萄糖储备溶液（提供）和无菌水。确保稀释体积是最终进样体积的一半。轻轻旋转或通过上下移动混合。 </li><li>使用10％葡萄糖储备溶液（提供）和无菌水将计算量（见步骤1.2.3）稀释为5％葡萄糖（终浓度）的一半注射体积。轻轻涡旋，旋转13,000 xg 15秒。 </li><li>将上述稀释的递送试剂一次性加入到稀释的核酸中。通过温和涡旋混合，并以13,000 xg的速度旋转15秒。 </li><li>在室温下将混合物从步骤1.5中孵育15分钟。 注意:从这一点开始，复合物在室温下稳定4小时，在4℃下储存多达7天。 </li><li>使用在室温下平衡的复合物进行尾静脉注射。将小鼠尾巴放入温水（50-53℃）30秒以允许静脉扩张。 <ol><li>将鼠标放在约束装置内。在20-30°角的尾静脉插入一个27至30号的针头，缓慢注射200μL。完成后，取下针头并向注射部位施加压力。 注意:注射期间尾部轻微的凸起表示定位不正确。如果发生这种情况，请取出针头并重复上一个位置附近的过程。 </li></ol></li><li>在静脉内注射后24和48 h，通过体内 BLI（见步骤2）显示基因表达。 </li></ol> 2. 体内 BLI 注意事项:事先准备，在DPBS中制备15 mg / mL D-荧光素的新鲜溶液，使用0.22μm过滤装置对其进行过滤灭菌，并将其储存在-20°C。 <ol><li>将小鼠放入透明有机玻璃麻醉室。确保异氟醚室已满。准备麻醉动物时，确保泵（left）和室（右）开关打开。将异氟烷表盘转换为2.5％的感应和2％的维护。 IVIS室内的动物在图像采集期间保持在2.5％异氟烷麻醉下。 </li><li>在小鼠完全麻醉后，在成像前15分钟通过腹膜内途径注射10mL / kg体重的D-萤光素溶液。 注意:应该对D荧光素进行动力学研究以确定给予D-荧光素后信号峰的时间。 </li><li>打开体内成像系统，并通过用一块黑色卡片衬垫来准备成像室（完成时丢弃）。根据需要放置鼻锥，以进行正确的麻醉（每只小鼠使用一个锥体）。 注意:将麻醉用于仪器的管是分开的，使得相同浓度的麻醉被引导到位于成像系统内部的麻醉歧管。 </li><li>从b转移小鼠（最多5个）牛到连接到成像系统中的歧管的鼻锥并且关闭门。图像采集持续5分钟。 </li><li>使用制造商的软件获取BLI，如下所示。 <ol><li>初始化软件在体内成像系统采集控制面板中，在Luminescent旁边加上一个复选标记。确认激励滤波器设置为阻塞，发射滤波器设置为打开。 </li><li>点击箭头:对于发光成像模式，选择5分钟的曝光时间，Binning 8和F / Stop 1;对于照相成像模式，选择Binning 4和F / stop 8。 </li><li>从"视野"下拉列表中，选择D，19厘米，主题高度为1.5厘米。准备好获取图像时单击"获取"。 </li></ol></li><li>图像采集完成后，将鼠标放回笼子中。 </li><li>使用制造商的软件量化从特定地区发射的光子。 <ol><li>点击<strong&gt;工具调色板中的ROI工具。在ROI工具中 ，从类型下拉列表中选择测量ROI 。 </li><li>点击Square图标，绘制一个平方的ROI，其尺寸适当，以覆盖一只动物的胸部。复制并粘贴每只动物的ROI，以获得具有相同尺寸的ROI。在"ROI工具"面板中，单击" 测量ROI"以获取ROI中总强度的度量。 </li><li>观察在会话期间在图像或序列中创建的所有ROI的ROI测量数据（每行一个ROI）。单击导出并选择文件将被保存的文件夹。 </li></ol></li></ol> 3.炎症刺激的老鼠挑战注意:在用促炎症刺激进行小鼠攻击之前，通过体内 BLI检查基线激活（参见步骤2）。至少7天必须在体内基因传递和摩尔之间通过挑战来让轻度和短暂的炎症消失。 <ol><li>准备用于气管内滴注的设备（参见图1和图2 ）。 <ol><li>将5 ml一次性注射器与弹簧（C，E），100μL注射器（B）和一次性量规（D）连接到三通旋塞阀（A）。将系统放在支架（H）上。将系统放在支架（H）上。 </li><li>将PE190微型医疗管（F）连接到一次性量规（D）和笔世纪（G）。 </li><li>向5 mL注射器中注入800μL空气，然后转动3路旋塞。 注意:通过在100μl注射器中吸出50μl向铜管填充铜绿假单胞菌培养物上清液。 </li></ol></li></ol><img alt="图1" class="xfigimg" src="/files/ftp_upload/55499/55499fig1.jpg" /> 图1:示意图代码气管内装置的单一组成部分。 （A） 3路旋塞阀， （B） 100μL汉密尔顿注射器， （C）一次性5 mL注射器， （D）一次性计量器， （E）一次性5 mL带弹簧的注射器， （F） PE190微型医用管， （G）笔世纪和（H）支持。 <a href="http://ecsource.jove.com/files/ftp_upload/55499/55499fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55499/55499fig2.jpg" /> 图2:组装的气管内装置的表示。 标识与图1相同。 <a href="http://ecsource.jove.com/files/ftp_upload/55499/55499fig2large.jpg" target="_blank">请点击</a>这里可以查看这个数字的较大版本。 <ol start="2"><li>使用设在2.5％异氟烷与氧气混合的异氟烷蒸发器室麻醉小鼠。 </li><li>监测动物，评估3-5分钟麻醉后的效果。 注意:要确认鼠标完全麻醉，请仔细监测以下标志:呼吸速度减慢，颈部捡起时手臂伸展不足，后肢刺激时缺乏反应。再等几分钟，再检查一下，如果不符合这些条件，则继续下一步。 </li><li>将麻醉的小鼠放在有机玻璃插管平台上，将其悬挂在门牙上，将其放置在电线上。 </li><li>用左手打开喉镜（右手研究人员），并抓住一双平头镊子。使用喉镜尖和镊子轻轻撬开口。 </li><li>拉舌头和h使用镊子将其老化到侧面。将喉镜刀片引导到嘴巴的后面。保持喉镜以90°的角度非常缓慢的按压直到气管的开口可见。握住喉镜就位。 </li><li>另一方面，将输送管连接到PE管的末端并将其插入气管。旋转三通阀以递送接种物。尽快将气管拔出气管。将鼠标悬挂几秒钟，让接种物吸入肺部。 注意:如果气管阻塞太久，小鼠会窒息死亡。 </li><li>从平台上移除鼠标。恢复时间可能会因应变而有所不同;仔细监测小鼠，确保动物在手术后30分钟内完全清醒。 </li><li>腹膜内注射150 mg / kg D-荧光素，并在气管内4,24和48 h后使用体内成像系统对肺进行成像滴注刺激。使用制造商的软件量化从特定地区发射的光子。 </li></ol>

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作者没有什么可以披露的。

在以前的工作11中 ，显示了bIL-8-Luc依赖性BLI和BAL标记之间的对比。它依赖于小鼠品系12的敏感程度差异。因此，bIL-8-Luc模型首次应用于不同的小鼠品系需要对BLI和更标准化的炎症标记物进行初步的炎症反应研究。 小鼠转染引起轻度肺部炎症和bIL-8-Luc的活化，这可以通过BLI在DNA注射后3〜4天可以检测到（ 图4 ）。 1周后完全消失<su...

慢性肺部疾病，如哮喘，慢性阻塞性肺疾病（COPD），囊性纤维化（CF）和支气管扩张，其特征在于气道炎症。气道炎症的特征在于水肿，细胞浸润，T淋巴细胞和肥大细胞活化，气道分泌增加和胶原沉积过多。 CF是多系统疾病，其主要死因和发病原因是肺细菌感染与肺部恶化加重。肺功能的下降预测显着较差的结果1，2，3，4 。 呼吸道的炎症状态通常通过评估在炎症过程中引发的免疫标记来观察，所述免疫标记来源于下呼吸道和上呼吸道，例如痰液，其提供可变的resULTS。支气管镜也进行5 。鼠模型是研究以气道炎症为特征的疾病的发病机理和进展的有价值的工具，并且尚未确定有效的治疗或治疗方法。肺部感染和炎症的动物模型已被用于研究哮喘和宿主 - 病原体相互作用，包括模拟人类病症的化学物质的作用（ 例如，香烟烟雾暴露，LPS，弹性蛋白酶，卵白蛋白，poly I:C 等 ，作为以及上述的组合） 6 。炎症相关参数的测量需要牺牲动物，因为需要侵入性方法来测量诸如细菌负荷，肺中的细胞因子和收集的支气管肺泡灌洗液（BAL）液体等因素。此外，通常需要进行组织学检查。获得关于炎症反应动力学的信息的可能性需要使用numerous小鼠因此，在技术，道德，经济和运营基础上，无需牺牲动物就可以获得这种信息的技术是有价值的。 IL-8是炎症过程中的重要参与者，将白细胞募集到发炎组织。它代表了炎症途径激活研究的分子读数。 MIP-2和KC可以是小鼠中人IL-8的功能同源物。小鼠仅表达一种潜在的IL-8受体，即人CXCR2,7,8的同系物，但它们能够调节驱动报告基因的异源IL-8基因启动子。在观察到牛IL-8启动子/荧光素酶报告基因构建体可以在小鼠中反式激活之后，最近开发了肺炎症鼠模型。该功能允许利用生物发光成像（BLI）监测生活中的炎症反应小数9 。 该模型已经适应于研究由细菌外源产物（ 例如 LPS或细菌株释放的产物）或TNFα10,11引起的炎症。药物发现过程的重点是开发和优化可以治疗肺部疾病如CF，哮喘和COPD的新旧抗炎分子。这些新的化学实体必须在可以与特定临床表型相关联的动物模型中快速方便地进行测试，以便于智能临床试验的设计。

<table><tbody><tr><td>FMT 2500 Fluorescence Tomography System</td><td>Perkin Elmer Inc.</td><td>Experimental Builder</td><td></td></tr><tr><td>IVIS Lumina serie II Pre-clinical &lt;em&gt;In Vivo&lt;/em&gt;; Imaging System</td><td>Perkin Elmer Inc.</td><td>Experimental Builder</td><td></td></tr><tr><td>MMPsense 750 FAST</td><td>Perkin Elmer Inc.</td><td>NEV10001EX</td><td>Protect from light, store the probe at 4 &amp;deg;C</td></tr><tr><td>Female inbred BalbC</td><td>Harlan Laboratories Italy</td><td></td><td>Prior to use, animals were acclimatized for at least 5 days to the local vivarium conditions</td></tr><tr><td>bIL-8-Luc plasmid</td><td>Department of Medical Veterinary Science, University of Parma, Italy</td><td></td><td>Store the plasmid at -20 &amp;deg;C</td></tr><tr><td>pGL3basic vector</td><td>Promega</td><td>E1751</td><td>Store the vector at -20 &amp;deg;C</td></tr><tr><td>JetPEI DNA transfection reagent</td><td>Polyplus transfection</td><td>201B-001G</td><td>The DNA and JetPEI mix was formulated with a final N/P ratio of 7</td></tr><tr><td>D-luciferin potassium salt 1 g</td><td>Perkin Elmer Inc.</td><td>122796</td><td>Protect from light, store at -20 &amp;deg;C</td></tr><tr><td>Living Image software</td><td>Caliper Life Sciences,</td><td>Experimental Builder</td><td></td></tr><tr><td>Isoflurane</td><td>ESTEVE spa</td><td>571329.8</td><td>Do not inhale</td></tr><tr><td>Bio-Plex Cytokine Assay Kit</td><td>Bio-Rad Laboratories</td><td>M60-009RDPD</td><td>Store the unopened kit at 4 &amp;deg;C</td></tr><tr><td>Automated cell counter</td><td>Dasit XT 1800J</td><td>Experimental Builder</td><td></td></tr><tr><td>Penn-century model DP-4M Dry power insufflator</td><td>Penn-century</td><td>DPM-EXT</td><td></td></tr><tr><td>Gas anesthesia system XGI-8</td><td>Perkin Elmer Inc.</td><td>Experimental Builder</td><td></td></tr><tr><td>PE190 micro medical tubing</td><td>2biological instruments snc</td><td>BB31695-PE/8</td><td></td></tr><tr><td>Syringe without needle 5 mL</td><td>Terumo</td><td>SS*05SE1</td><td>Cut the boards of the piston by a scissors</td></tr><tr><td>Hamilton 0,10 mL (model 1710)</td><td>Gastight</td><td>81022</td><td></td></tr><tr><td>Discofix 3-way Stopcock</td><td>Braun</td><td>4095111</td><td></td></tr><tr><td>Syringe with needle 1 mL</td><td>Pic solution</td><td>3,071,260,300,320</td><td>Use without needle</td></tr><tr><td>Plastic feeding tubes 18ga x 50 mm</td><td>2biological instruments snc</td><td>FTP-18-50</td><td>Cut obliquely the tip&amp;nbsp;</td></tr></tbody></table>

an il-8 transiently transgenized mouse model for the in vivo long-term monitoring of inflammatory responses

气道炎症通常与细菌感染相关，代表肺部疾病的主要决定因素。各种因素的促炎能力的体内测定是具有挑战性的，并且需要终端程序，例如支气管肺泡灌洗和用于原位分析的肺的去除，排除了在同一只小鼠中的纵向可视化。在这里，通过在异源IL-8牛启动子的控制下，表达荧光素酶报告基因的瞬时转基因小鼠气管内滴注铜绿假单胞菌培养上清（SN）诱导肺部炎症。通过在滴注后2.5至48小时的时间范围内的体内生物发光图像（BLI）分析来监测肺中的荧光素酶表达。该过程可在2-3个月内重复多次，从而允许评估同一小鼠的炎症反应需要终止动物。这种方法允许实时监测在肺中起作用的促炎症因子和抗炎因子，并且似乎适合于功能和药理学研究。

将bIL-8-Luc瞬时转基因小鼠模型用于体内监测用含有分泌的毒力因子的浓缩细菌上清液（30x）攻击的小鼠的肺部炎症。诱导的炎症反应可通过体内成像检测，因为BLI信号的增加。尽管BLI信号在5和24小时之间达到最高峰，并且在48小时仍然是可检测的，但在滴注后2.5小时可清楚地检测出促炎活性。 <img alt="图3" class="xfig...

Watch this Scientific Journal Video about An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses at JoVE.com

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

这里描述的方法允许通过非侵入性生物发光成像（BLI）在小鼠肺中IL-8启动子依赖性炎症激活的可视化。荧光素酶报告基因构建体的交付时间可以将相同的动物多次进行BLI多至两个月。

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

an-il-8-transiently-transgenized-mouse-model-for-vivo-long-term

Research

JoVE Journal

Immunology and Infection

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

7.3K 次观看.  University of Verona. 这里描述的方法允许通过非侵入性生物发光成像（BLI）在小鼠肺中IL-8启动子依赖性炎症激活的可视化。荧光素酶报告基因构建体的交付时间可以将相同的动物多次进行BLI多至两个月。 

视频: An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

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

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

一个慢性双光子薄颅骨窗口技术在体内 Neuroinflammation模型小鼠小胶质细胞成像

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

神经科学

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

浸润性膀胱移行细胞癌中的完整的人MUC1转基因小鼠免疫诱导免疫治疗发展模型

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

医学

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

鼠标型号病原体引起的慢性炎症局部及全身站点

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

使用IL-1β启动子驱动的红色荧光蛋白记者小鼠中性粒细胞引发的活体成像

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

IL-22表达淋巴细胞使用记者小鼠的可视化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

IL-8瞬时转化的小鼠模型

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