我们要感谢约书亚乙鲁宾博士慢病毒系统以及编制GL261 - Luc细胞有益的建议Mahil饶质粒厚礼。 我们感谢支持脑瘤研究（SSBTR），巴罗神经学基金会和华莱士基金会的慷慨支持的学生。 动物实验机构的动物护理和使用委员会的圣约瑟夫医院和医疗中心规定的准则和法规的规定执行。

1。细胞培养<ol><li> GL26细胞株是从癌症诊断和治疗（DCTD）国立癌症研究所（NCI），冯检基，MD司获得。为了促进肿瘤的生长速度GL261细胞的定量测量发光使用Lenti - X的HT包装组合（Clontech公司实验室公司）FUW Lentiphos HT系统（Clontech公司实验室，山景，加利福尼亚） GL质粒（JB鲁宾博士的实验室的一个厚礼）。细胞保持在贝科的改良Eagle培养基（DMEM），四环素10％胎牛血清（FCS; Clontech公司实验室公司）。 GL261细胞转染与基因编码使用的pGL4.51 [luc2 / CMV / NEO] luc2载体（Promega公司的公司，麦迪逊，威斯康星州）和FuGENE 6转染试剂（罗氏应用科学部，印第安纳波利斯，IN）指定下列条件制造商。 luc2基因密码子优化版本萤火虫luciferaSE，提供了显著高于标准luc基因的光输出。稳定转的DMEM含10％FCS和100微克/毫升Geneticin（G418，Invitrogen公司的公司，卡尔斯巴德，CA）的媒体选择和维护。 </li><li>植入前培养的细胞是由trypsinzation收获，洗一次在没有血清的DMEM悬浮无血清的DMEM浓度在1 × 10 7细胞/ ml。 </li></ol> 2。手术安装2 <ol><li>保持无菌的环境是整个手术，包括所有的手术器械，用品，手套，窗帘等。 </li><li>十周老C57BL/6-cBrd/cBrd/Cr（白化C57BL / 6）小鼠均购自国家癌症研究所的冯检基动物生产计划，并在平均体重20克（国家癌症研究所，冯检基，MD）。 </li><li>动物是由甲苯噻嗪（5毫克/公斤），氯胺酮（50毫克/千克）和丁丙诺啡（0.05毫克/千克）腹腔注射麻醉。要发送捏做是为了确保该动物是充分麻醉，手术前开始。动物的任何部分（即使轻微运动）是一个在减少麻醉水平的指示。动物立即给予额外的3.3毫克/千克甲苯噻嗪26.6毫克/公斤氯胺酮。使用一盏灯，无菌敷料覆盖身体，保持体温。 </li><li>一旦动物是正确麻醉的，他们放在立体井架气垫床（型号900小动物立体定位，大卫Kopf仪器）[图1]。 </li><li>阿克瓦泪眼科润滑油膏少量（1 / 4英寸）是应用在眼角膜。 </li><li>开放动物的口（动物的下巴周围放置你的食指和拇指）和滑动的立体框架中的缩进门牙的动物固定在立体框架。钳收紧，以确保安全耳机动物的头，日Ë头对准的目光都集中，确保但对动物的头施加过分的武力。 </li><li> O 2的软管录音动物的鼻孔附近。氧流量为0.5升/分钟。 </li><li>较低的背部和尾巴是录音小心，不要妥协呼吸的立体床。 </li><li>手术切口部位是剃光。 Povidine碘棉签棒是用来洗眼睛与碘的耳朵之间的区域之间的区域，确保碘不滴水成动物的眼睛。 </li><li>这些细胞植入手术前准备，并定期混合，以确保他们不解决。 </li><li> 10μL，50毫米世界精密仪器（WPI），萨拉索塔，佛罗里达州，与26计斜面针注射器装有细胞接种。如果细胞似乎是成群一起，可能需要重新载入注射器。 </li><li> 10μL注射器，然后放置到UMP3 - 1 UltraMicroPump微量进样器（WPI的，萨拉索塔，佛罗里达州）。 </li></ol> 3。颅内植入2 <ol><li>皮肤切口采用的是大小15手术刀刀片和齿镊。 10-15毫米的切口，纵向是从动物的眼睛之间，对动物的耳朵，暴露前囟门（在头骨的顶部矢状面和冠状缝合线交界处）移动。一定要正确识别前囟门，可以很容易地与鼻窦区前囟门[图2]这是远端混淆。 </li><li>动物是镇静剂后，它收到的0.25％（2.5毫克/毫升）布比卡因切口内注射。 </li><li>一个burrhole至前囟后0.1毫米和2.3毫米的中线右侧慢慢扭动16号1.5寸毫针手工，针小的压力而扭曲，直到头骨是渗透和大脑暴露。 </li><li>将注射器针头被移动到位置使用的Microdrive STEreotactic注射器架，直到它刚好触动大脑的表面。从这个位置，是先进的针入脑深度为3毫米的地方保持3分钟。 </li><li>针撤回.4毫米到2.6毫米以下的大脑表面的总深度，创造​​一个小口袋注入细胞。正是在这一点上，可选针在动物中的X射线图像，以确保妥善安置和深度。 </li><li>细胞悬液注入微量进样器与输液速度667 NL /分钟到2000年NL（2μL）量超过3分钟。 </li><li>针是左2分钟输液部位，以防止泄漏的地方。 </li><li>针慢慢地完全撤回。 </li><li> burrhole是充满骨蜡使用彭菲尔德剥离。 </li><li>使用4-0（1.5公制）薇乔缝合，确保没有大的差距是在皮肤上留下的切口缝合。薇乔缝合材料，解散S，因此缝线不切口愈合时，需要拆除。 </li><li>手术后的动物被放置在一个笼子里，下一个加热灯，是在所要求的高度暖笼的底部表面至30 ° C。当动物完全清醒（在笼子里的正常运动判断），他们返回到组房屋。手术后5天，口服布洛芬被添加到他们的饮用水。 100毫克的儿童布洛芬（100mg/5ml）被添加到一个标准的473毫升灭鼠一瓶水。动物每天观察和成像，体重每3天。两个星期的观察植入后增加至每天两次。动物安乐死时，显示健康状况下降，其中包括驼背的姿态，行动不便，可见体重下降（≥20％）的迹象。这些症状是肿瘤发布的回应，他们可重复出现约1天因肿瘤死亡前。 </li></ol> 在体内 4。 </EM生物发光成像3 <ol><li>启动[生活图片]软件。 </li><li> IVIS成像系统初始化按一下[初始化IVIS系统]按钮控制面板的底部右侧。 </li><li>选择[发光]在控制面板的顶部左侧的成像模式。 </li><li>要确定动力学的研究是十分必要的荧光素注射后的成像的最佳时间[图3]。这说明萤火虫荧光素酶; <ol class="lalpha"><li>注入加入10μl/克体重的D -荧光素萤火虫在PBS（15mg/ml;卡尺生命科学目录XR - 1001，或从另一家厂商的同类产品），到动物，如下所述。 </li><li>等待3分钟，然后放入气体麻醉室（2％异氟醚气体在O 2）麻醉鼠标。 </li><li>关闭气体麻醉室和开放的麻醉阀和真空IVIS多方面的。立即将镇静剂动物对温度控制的成像平台，确保鼠标的鼻孔是正确放置在气体麻醉气管。应采取的第一个图像的荧光素注射后约5分钟。 5动物可以IVIS光谱仪一次成像。如果少于5只动物进行成像，它可以插入未使用的歧管（S），异氟醚气体保护。 </li><li>继续拍摄图像，每3分钟，创造了长达一个小时的序列产生荧光素表达的动力学曲线。 <ol><li>在控制面板上点击[序列设置]按钮。 </li><li>序列编辑器中出现。 </li><li>在控制面板中，指定序列中的第一个发光图像的设置。 <ol class="lroman"><li>我们建议中等分级开始。 </li><li>我们还建议，以确定最佳的曝光时间的自动曝光。 </li><li>选择在序列编辑器[延迟]按钮ND指定一个彼此之间收购的3分钟的延迟时间。 </li></ol></li><li>在序列编辑器，单击[添加]。采集参数，然后添加到表中。 </li><li>步骤4重复序列中的每个图像。 </li></ol></li><li>一旦建立曲线，可通过绘制信号强度（强度）随时间变化的最佳成像时间。在图片动物体内光子最高时数得到最强，最准确的信号。 </li></ol></li><li>然而，早期的实验中，用荧光素注入腹腔（IP）IP注射偶尔导致间歇性的结果显示几乎没有在肿瘤的生物发光。我们推测，偶尔随机信号是由于交付的肠管或其他内脏器官的荧光素缺乏。因此，我们开始使用皮下（SC）荧光素注射，看到更大的成像重现性[图3]。 </li><li>第二十分钟皮下注射后，动物被放置在一个分庭在O 2 2％异氟醚气体，直到他们反应迟钝麻醉。 </li><li>麻醉动物（S）是移动成像室。眼药膏应用于动力学研究，因为成像的长度。它是不是需要其他成像过程，因为他们是在持续时间短。 </li><li>该图像是中等分级获得5分钟的曝光时间。也可用于自动采集选项。 </li><li>如果信号是饱和和/或在淡淡中等分级，分级或曝光时间可以调整。 </li><li>程序文件和后续的图像注释被保存在用户的计算机上的目录。 </li></ol> 5。三维成像3 <ol><li> [影像精灵]标签上按一下[序列设置]控制面板窗口。 </li><li>选择[生物发光成像向导开始屏幕上的成像模式和CLICK [下一步]。 </li><li>在“生物发光 - DLIT”成像向导窗口，选择[萤火虫]记者探头。选定的萤火虫源频谱发射/激发，会出现与被收购的六个相应的滤波器选择。 </li><li>最后的屏幕上会出现默认选择，其中包括自动曝光采集参数，并实地查看C.默认设置的工作很好，但是，这些设置，如有必要，可以修改。 </li><li>单击[下一步]和序列编辑器窗口将在20纳米宽过滤器（560纳米，580纳米，600nm处，620纳米，640纳米，和660nm）光谱的六个地区的序列填充。按[获取序列]。第一个过滤器（560纳米），将包括一个结构光模式，使用激光振建立表面形貌。 </li><li>在工具面板中选择[表面形貌]标签。在重建过程中创建的任何尖角，表面光滑，可应用到帐户。 “默认的低平滑建议。 </li><li>单击[创建]和断层扫描分析中会出现。画出一个包括整个动物的裁剪框，然后单击[下一步]。 </li><li>阈值工具，会出现一个紫色面具在选定的区域。口罩应被自动设置为与动物的照片。如果有必要，调整面膜的门槛，以便更恰当地适应动物的轮廓。 </li><li>单击[完成]和重建的网格会出现。重建，然后可以保存结果“选项卡中。 </li><li>创造动物的表面地形，进行[DLIT三维重建]下拉工具盘上。 </li><li> [分析]标签下，选择所有六个波长来进行重建。取消图像产生饱和像素或计数低于600。在[参数]标签保留为默认设置。 </li><li> [内容]标签下，应列出“肌肉”作为默认选择ř组织属性，“萤火虫”应该被列为源频谱​​。 </li><li>单击“分析”选项卡下的[重建]，和动物的表面三维重建和相应的信号源重建应该会出现[图4]。 </li><li>为了确定信号的位置和强度，选择工具面板的3D工具选项卡上的体素的按钮。 <ol><li>所有显示的体素周围画出一个正方形和总光通量测量显示音量“选项卡的底部。 </li><li> [地下中心]上按一下，以确定所选体素的信号的位置。冠状，矢状和transaxial片的动物会出现和使用的测量光标显示]从动物的表面体素中心的距离可以测量。 </li></ol></li><li>请参阅生活图像软件用户手册登记器官地图集和其他先进的功能的进一步信息。 </li></ol> 6。数据一nalysis 3 <ol><li>图像采集和保存后，按一下[浏览]按钮，选择文件访问程序文件。 </li><li>图像信息下可以找到[查看]→[图像信息]。 </li><li>可量化的利息率（ROI）投资回报率工具]按钮选择区域的信号强度。确保图像分析[光子]模式下，通过对图像的控制面板的顶部左上角的下拉列表中选择“光子”。 </li><li>从“类型”下拉列表中选择[测量投资回报率]按钮。请选择感兴趣的投资回报率的形状;选项包括圆，方，或电网。封面上所获得的图像的所有领域的强度。 </li><li>拖动ROI形状选择包含发光信号的地区的投资回报率的立场。 </li><li>信号强度的投资回报率的计算方法是点击[测量]按钮。 ROI标签显示的强度。投资回报率的可管理和保存USI吴的生活图像软件。 </li></ol> 7。代表性的成果： 植入的细胞检测手术当天使用IVIS谱细胞移植成功是显而易见的。 GL261 - luc和GL261 - luc2细胞可检测到，但是，luc2基因将提供更高水平的生物发光[图5]。植入后不久拍摄的图像可能有非特异性信号应为背景无视动物的爪子和鼻子。位于着床部位的信号是真实的，信号将随时间增加[图6]。在信号强度下降6天，重现性好，最有可能因肿瘤的损失采取一些植入的细胞的。肿瘤负荷的定量测量重复性好，他们应该稳步上升，动物，直到最终屈从于这种疾病。然而，生长曲线在很大程度上将取决于植入的细胞的状态，或不可避免的轻微VAR<html> iability在植入手术。我们的实验室已选出的形象植入动物每三天。 <img alt="图1" src="/files/ftp_upload/3403/3403fig1.jpg" /> 图1：后鼠标是正确麻醉的，它被放置在立体框架。小鼠的头部安全使用的口钳。 <img alt="图2" src="/files/ftp_upload/3403/3403fig2.jpg" /> 图2：打开后皮肤的主要解剖标志确定，包括包括前囟门，冠状面和矢状面的缝线。 burrhole是2.3毫米慢慢扭动的压力少量16号1.5寸毫针，直到头骨是渗透和大脑暴露前囟门右侧。 <img alt="图3" src="/files/ftp_upload/3403/3403fig3.jpg" /> 图3的皮下的动能比较（iles/ftp_upload/3403/3403fig3_1.jpg“ALT =”图3.1“/&gt;）与腹腔（ <img src="/files/ftp_upload/3403/3403fig3_2.jpg" alt="Figure3.2" />执行）荧光素注射证明皮下荧光素注射的效用，并确定图像下面的荧光素管理的最佳时机。三分钟后，荧光素被注入鼠标镇静剂，放置在IVIS光谱仪和成像每3分钟到一个小时，每6分钟后，产生的生物发光动力学曲线。这表明，皮下路线的荧光素管理腹腔注射优于在我们手中，这形象的动物的最佳时间是大约25分钟，GL261 - LUC细胞后，荧光素注射。 <img alt="图4" src="/files/ftp_upload/3403/3403fig4.jpg" /> 3维reconst多个视图图4。GL261 - luc2细胞颅内植入乱子共同注册用鼠标骨骼和大脑。 <img alt="图5" src="/files/ftp_upload/3403/3403fig5.jpg" /> 图5。GL261 - Luc细胞与GL261 - luc2细胞产生肿瘤获得的光子计数。结果是平均5只动物。 <img alt="图6" src="/files/ftp_upload/3403/3403fig6.jpg" /> 图6图GL261 - LUC在白化C57BL / 6小鼠的肿瘤细胞的生长。生物发光测定，每3天， 在体内光子计数与天植入后绘制。照片显示，在不同时间点的生物发光。着色是一个生物发光显示（像素力度），这是相对的肿瘤细胞数（彩条是右侧所示）。后死于疾病的动物大脑解剖和荧光素增加了局部获得前体内成像E所示图中的插图。

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从大脑表面的细胞接种是注入后，创建一个0.4毫米的口袋的深度为2.6毫米。为了确保妥善安置针和深度的X射线，可以使用C型臂或类似的X射线图像加强设备，但是，这是可选的。手术并发症的可能出现如果动物是不正确镇静剂的，在这一点的动物细胞输注在移动。这可能会导致细胞混合或漏针道出血。细胞泄漏导致肿瘤细胞的异位的生长。它也是重要的不是可以做穿刺脑室如果burrhole协议4中所述的2.3毫米的内侧。注入2μL亚甲基蓝染料鼠标和解剖脑组织检查注入染料的位置，测试针和细胞传播的妥善安置。 在这个协议中，我们已经使用了体内成像系统和T IVIS频谱他生活图像软件（V 4.0），用该仪器使用设计。 （卡尺生命科学版）。任何可比在体内成像系统和图像分析工具可以用来获得类似的结果。这些系统提供了超过一些传统的磁共振成像（MRI），遵循的一个实验性的颅内肿瘤的生长优势。最明显的是2文书-动物MRI机器的相对成本昂贵得多，通常需要一个熟练的MRI技师的服务。最终用户可以通过在体内 ，如这里描述的成像。生物发光的数据是定量的，而MRI数据的定量是费时又有点不精确。此外，磁共振图像显示，除了对肿瘤细胞的水肿和炎症，并可能很难分开肿瘤的治疗效果。由于这些原因，获得准确增长肿瘤体积测量可以是一个挑战。生物发光需要ATP因此只有活肿瘤细胞有助于肿瘤的大小数据。尽管这样，也有一些与MRI的优势，如果一台机器可用细胞没有一个发光标记的MRI可视化标记。可视化瘤周水肿的能力可能会为一些实验性的协议的优势。的两种技术的优点是，使用一个，不排除使用其他功能，使数据可以得到对肿瘤的生长，以及从同一动物的瘤周围水肿和炎症的存在，当这两种技术可研究者。

<table border="1"><tbody><tr><td> 试剂名称或供应 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> GL261 - luc2 BioWare公司超</td><td>卡尺生命科学</td><td> GL261 - luc2 </td><td></td></tr><tr><td>贝科的改良Eagle培养基（DMEM） </td><td> Invitrogen公司</td><td> 10313039 </td><td></td></tr><tr><td> Geneticin（G418） </td><td> Gibco公司（Invitrogen公司） </td><td> 11811-023 </td><td></td></tr><tr><td>小牛血清（FCS） </td><td> Invitrogen公司</td><td> 26140079 </td><td></td></tr><tr><td> Phospate缓冲液（PBS） </td><td> Invitrogen公司</td><td> 70011044 </td><td></td></tr><tr><td> C57BL/6-cBrd/cBrd/Cr小鼠</td><td> NCI，冯检基</td><td></td><td></td></tr><tr><td>阿克瓦泪润滑油眼药膏</td><td> Akorn公司</td><td> 17478-062-35 </td><td></td></tr><tr><td> Ketaset（氯胺酮） </td><td>惠氏</td><td> 11570775 </td><td></td></tr><tr><td> Sedazine（甲苯噻嗪盐酸盐） </td><td>惠氏</td><td> 10031894 </td><td></td></tr><tr><td>小动物脑立体定位仪</td><td> Kopf仪器</td><td> 900 </td><td></td></tr><tr><td> UltraMicroPump与SYS - Micro4控制器</td><td>世界精密仪器</td><td> UMP3 - 1 </td><td>如果没有，它是可能的手动注入</td></tr><tr><td> 26计​​加入10μl注射器针头斜面</td><td>世界精密仪器</td><td> SGE010RNS </td><td></td></tr><tr><td>笛声钳</td><td>世界精密仪器</td><td> 500092 </td><td></td></tr><tr><td>彭菲尔德剥离</td><td>科德曼</td><td> 65-1015 </td><td></td></tr><tr><td> 16克1个半精密滑动针</td><td> Beckton Dickinson公司（BD） </td><td> 305198 </td><td></td></tr><tr><td>手术刀片手柄</td><td>屋宇署</td><td> 371030 </td><td></td></tr><tr><td>尺寸15刀片</td><td>屋宇署</td><td> 371315 </td><td></td></tr><tr><td> 4-0薇乔缝合</td><td>爱惜康</td><td> VCP496G </td><td></td></tr><tr><td>骨蜡</td><td> MEDLINE </td><td> DYNJBW25 </td><td></td></tr><tr><td> Povidine碘棉签棒</td><td> MEDLINE </td><td> MD93901 </td><td></td></tr><tr><td> D -荧光素钾盐</td><td>卡尺生命科学</td><td> 122796 </td><td></td></tr><tr><td> Forane（异氟醚） </td><td>巴克斯特</td><td> 1001936060 </td><td></td></tr><tr><td> OPMI Pentero显微镜</td><td>卡尔蔡司公司</td><td></td><td>任何手术显微镜就足够了</td></tr><tr><td>精诺真IVIS可选的麻醉系统的频谱</td><td>卡尺生命科学</td><td></td><td></td></tr></tbody></table>

intracranial implantation with subsequent 3d in vivo bioluminescent imaging of murine gliomas

鼠脑胶质瘤261（GL261）是公认的体内模型系统概括人类胶质母细胞瘤（GBM）的许多功能。原是诱发细胞系成的C57BL / 6同基因小鼠品系1 3 - ​​甲基cholantrene颅内注射，因此，免疫主管C57BL / 6小鼠可以使用。虽然我们使用GL261，可用于以下协议的任何颅内的小鼠肿瘤模型的植入和监测。 GL261细胞工程，以稳定表达萤火虫荧光素酶（GL261 - LUC）。我们还创建了光明luc2基因表达的CMV启动子的稳定转染细胞系GL261 - luc2。 C57BL/6-cBrd/cBrd/Cr小鼠（C57BL / 6白化变种）来自国家癌症研究所，检，医师被用来消除黑皮肤和毛皮引起的光衰减。随着使用白化C57BL / 6小鼠体内成像使用在体内 IM IVIS频谱时效制度是从植入的一天（卡尺生命科学，霍普金顿，马）。 GL261 - luc和GL261 - luc2细胞株显示父母GL261细胞在体内的行为是相同的。一些共享的组织学特征在人类GBMS目前这种小鼠模型包括：肿瘤坏死，pseudopalisades，新生血管形成，侵袭，hypercellularity，炎症1。 之前被植入动物腹腔注射氯胺酮（50毫克/千克），甲苯噻嗪（5毫克/公斤）和丁丙诺啡（0.05毫克/千克），放置在一个立体的仪器和切口，麻醉是比手术刀颅中线。一个burrhole0.1毫米后，前囟门和2.3毫米中线的右侧。一针插入深度0.4mm至3mm和孤僻的2.6毫米深度。 GL261 - LUC或GL261 luc2细胞（10 7细胞/ ml）液注入超过3分钟的过程中。 burrhole被关闭bonewax和缝合切口。 立体定向植入发光细胞检测和肿瘤从着床之日起使用IVIS光谱仪的三维图像重建功能，可以分析。动物接受皮下注射的荧光素150微克/公斤体重前20分钟，影像。使用平均肿瘤生物发光随着时间的推移，肿瘤负荷是量化。荷瘤小鼠每天观察，评估发病率和被安乐死，当一个或存在下列症状：嗜睡，不能走动，弯腰驼背的姿势，没有新郎，在&gt; 10％的减肥导致厌食。肿瘤在所有的动物剖检可见一斑。

Watch this Scientific Journal Video about Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas at JoVE.com

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

颅内植入GL261细胞分化成C57BL / 6小鼠产生恶性胶质瘤，复述人类胶质母细胞瘤的许多特征。我们使用GL261细胞稳定表达荧光素酶，使我们能够使用<em&gt;在体内</em&gt;成像遵循肿瘤进展。手术和3D<em&gt;在体内</em&gt;成像演示。

随后的三维颅内植入在体内</em生物发光成像鼠脑胶质瘤

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

intracranial-implantation-with-subsequent-3d-vivo-bioluminescent

Research

JoVE Journal

Medicine

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

20.3K 次观看.  Barrow Neurological Institute of St. Joseph’s Hospital and Medical Center. 中文

视频: Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

发光法小鼠肝肿瘤模型

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

科研

医学

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

在体内的双基体生物光学成像

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

植入小鼠触发剪应力致动脉粥样硬化的颈袖

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

立体定向颅内植入和<em在体内</em生物发光成像的肿瘤异种移植的小鼠模型系统的多形性胶质母细胞瘤

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

颈动脉支架植入再狭窄的研究的小鼠模型

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

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

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

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

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

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

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

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

股动脉线损伤的小鼠模型的血管周围给药补丁植入

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...