Name: high-frequency ultrasound imaging of mouse cervical lymph nodes
Uploaded: 2015-07-25T14:00:00.000+00:00
Duration: 10 min 2 s
Description: Watch this Scientific Journal Video about High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes at JoVE.com

这项工作是由来自西弗吉尼亚大学的玛丽·巴布兰多夫癌症中心多萝西·雷德福D.捐赠基金的支持。使用西弗吉尼亚大学动物模型和影像设备（AMIF）和显微成像设备（MIF）的（由玛丽·巴布兰多夫癌症研究中心和美国国立卫生研究院资助P20 RR16440支持，P30 RR032138 / GM103488和S10 RR026378）表示感谢。

在这个协议证明所有动物的程序进行了审查，并根据协议11-0412和14-0514批准西弗吉尼亚大学动物护理和使用委员会，并按照NIH指南中列出的照顾和使用的原则和程序进行动物。 1.动物的制备<ol><li>麻醉一个单一的鼠标使用3％异氟烷与1.5升/分钟100％的氧气混合感应室。除去从吸气室和地点的动物在预热至40℃之间，37-42℃（ 图2A）维护的成像平台上仰卧位置。由于缺乏响应脚趾捏麻醉确认。 </li><li>连接到麻醉系统在鼻锥内将鼠标吻。应用麻醉维持稳态镇静（1.5％异氟烷以1.5升/分钟100％的氧气混合）。 </li><li>敷眼润滑剂的每只眼睛以防止dryinG。应用电极凝胶到电极，并使用磁带的每个的四个爪固定到相应的电极上。电极焊盘将发送动物的心电图，以在成像系统，以允许监测心脏速率和呼吸速率。润滑和插入直肠温度探头进行连续监测体温。正常小鼠体温36.9℃。 1-2℃的变化是正常的，而在麻醉下。 </li><li>使用脱毛霜从鼠标的颈部取下毛皮。冲洗颈部用清水浸泡过的纱布去除毛发和多余的脱毛膏。任选地，使用的脱毛霜额外的应用以除去任何剩余的体毛（ 图2B）。 </li></ol> 2.识别和图像采集鼠颈淋巴结使用HFUS <ol><li>首先，涂上一层温暖的超声波凝胶的颈部区域缺乏皮草。使用凝胶的最佳图像自由主义的应用质量（ 图2C）。避免引入气泡在应用过程中的凝胶，它可以与超声成像造成干扰。 </li><li>调整成像平台20-30°使鼠标定位，微微隆起的头部。这个位置有助于确保最佳呼吸速率为鼠标。放置40兆赫传感器横向于所述安装系统和小心降低，直到换能器扫描头的前浸在超声凝胶（ 图2C）。 注意:一定不要把过多的压力到鼠标的脖子，因为它可能会导致不必要的呼吸窘迫。此外，它是有帮助的用于成像具有换能器和鼠标之间的凝胶的缓冲。 </li></ol>淋巴结B型成像与检测: <ol start="3"><li>利用计算机控制HFUS采集软件，调节亮度（B-）模式设置以下参数:增益22分贝，深度10.00毫米，宽14.08毫米。 注意:这些设置建议的出发点，可能需要略微调整为不同的应用程序之间的最佳图像采集。周围的高回声区域内观察正常宫颈淋巴结肿大为附近的皮肤表面椭圆形低回声结构。的患病淋巴结的外观可以模型之间变化。为了系统图像颈部区域内的所有淋巴结使用以下步骤: <ol><li>使用Y轴扫描颈部在颅尾鳍方式朝向胸部区域。使用X轴居中图像。 </li><li>确定主要景点:口腔，舌头和甲状腺（ 图3A，B和C，分别）;倾斜成像平台水平调节鼠标颈的腹面，使得颈部两侧，即使在B模式图像显示。倾侧量取决于每个单独小鼠的生理学。 </li></ol></l我&gt; <li>对整个颈部区域的三维扫描从口腔/舌区域甲状腺，以图淋巴结和关联的地标整个颈部。 <ol><li>找到舌头/口腔区域（ 图3A），并注意在Y尺度数值位置。 </li><li>找到甲状腺（ 图3C），并注意在Y尺度数值位置。 </li><li>计算（2.4.1）和（2.4.2）所获得的值之间的差，以确定被成像颈部区域的总长度在毫米。 </li><li>使用Y旋钮居中传感器上所确定的总长度的中点。 </li><li>按"3D"。进入总长度。为3D的步长，使用0.076毫米以获取图像系列堆栈为整个颈部区域。 </li></ol></li><li>一旦扫描完成后，选择脖子的右侧或左侧和中心的传感器感兴趣的个人淋巴结上，然后抬起40兆赫传感器关闭鼠标。删除40 MHz的换能器和更换50兆赫的Microscan换能器（也处于横向位置​​），以获得更高分辨率的图像。补充鼠标颈部超声凝胶，降低了50兆赫传感器到超声波凝胶。 </li></ol> 3D能量多普勒显像<ol start="6"><li>行为3D扫描与能量多普勒到驴的体积和个别颈部淋巴结如下血管: <ol><li>按下电源按钮系统键盘上获得权力和多普勒调整以下供电模式设置:PRF 4千赫，多普勒获得34分贝，2D增益调整30 dB的深度5.00毫米，宽4.73毫米。注:如前，这些设置是一个建议的出发点，并根据需要在各种型号的最佳图像采集可被修改。 </li><li>找到颅点最感兴趣的淋巴结，并注意在Y刻度的位置。 </li><li>找到相同的节点和票据的最尾端点在Y刻度的位置。 </li><li>计算距离差来确定淋巴结的总长度（见步骤2.4.3）。 </li><li>围绕所确定的总长度的中点的换能器，使用Y标尺位置。 </li><li>按"3D"，进入总淋巴结长度。使用0.051毫米为步长。 注意:由于该换能器到胸部区域的附近，在50兆赫传感器可能导致不稳定的多普勒图像由于检测正常呼吸运动。这可以通过使用"呼吸门控"选项中的"生理"标签下被淘汰。 </li><li>与周围的黄色框指定的区域由能量多普勒进行分析，并选择"3D扫描"，以获取图像选定的淋巴结。提高换能器关闭鼠标的，并将其移动到颈部的相对侧。降低换能器上的鼠标，重复上述对伊马的步骤在颈部的这一边戈淋巴结。 </li></ol></li><li>保存图像集进行后续分析。 </li></ol> 3.颈淋巴结活检<ol><li>选择用于活检所需淋巴结和保持HFUS成像的50兆赫传感器。选择了最大可见颈部淋巴结在小鼠颈部的每一侧。淋巴结肿大通常指示炎症反应，因此这样的节点是理想候选活检。 注意:我们已经发现这是很困难的，进行活组织切片检查的颈部淋巴结小于10 立方毫米。 </li><li>通过将1ml注射器与一个附加的27克，0.5英寸针头插入注射器座准备的针头和注射器活检。调整针保持器以定向针90°到鼠标颈部（ 图4A）。 </li><li>通过升高整个鼠标平台到针的水平制备。通过去除三维电机和切换到一个较高platf实现这一ORM，或者将合适的高度了坚实的对象下所提供的短期平台。使用塑料离心管架用于此目的。如果必要，可旋转平台180°，以位于所述颈部的侧相对的针头装置活检节点。 </li><li>通过选择"首选项"，然后通过选择"最大和扩展缓冲区"调整收购设置。放大的视场，以8.00毫米，宽9.73毫米的深度。使用屏幕按键转盘转动的导针。导针将预测在屏幕上的针的路径，并允许用户排队感兴趣的淋巴结中的正确位置活检。 </li><li>确保淋巴结由定心淋巴结中的中间或稍中心在屏幕（ 图4B）左侧恒定地保持在图。以获得该过程的整个电影回放，按系统键盘上预触发开始活检之前。 </li><li>调整针支架，直到针尖进入视野并接触皮肤（ 图4B）。推进针坚定，快速推穿刺皮肤。继续推进针，直到针尖也刺穿胶囊（ 图4C），并且是髓质（ 图4D）中可见。 </li><li>一旦针头被适当地定位在节点内，轻轻拉动针筒柱塞背面的200-300微升界线之间进行活检（ 图4D和4E）。需要注意的是活检材料通常不是注射器内可见。 </li><li>轻轻地取出从鼠标的脖子针。驱逐注射器内容到1.5 ml离心管。从注射器除去针，留在管中的针。收集1毫升活检培养基（从等分试样，分离了库存源）具有相同的注射器，然后重新连接针的注射器，同时保持在第针Ë管。 <ol><li>由排出所述活检介质插入管冲洗注射器和针头与活检介质。 注意:不要拉回到了活塞，而针被安装在切片后的任何一点。这减少了损失的活检材料的风险由于小样本大小。 </li></ol></li><li>确认通过组织学装置（ 图4F）淋巴结内容和通过另外的方法（组织化学，流式细胞术， 等 ）在适当分析。 </li><li>一旦活检完成后，关闭麻醉和删除直肠温度探头。从小鼠中取出多余的超声凝胶用纱布并从每个爪子取出磁带。 </li><li>从成像平台移开鼠标并返回到笼子。从活检部位出血极少可能会发生，但是这没有停止干预。监测恢复过程中鼠标，直到充分的活动重新开始。 </li></ol> 4.图像颈淋巴结分析<ol><lI&gt;在超声的软件，选择图像进行分析，然后导航到"图像处理"选项卡。选择"加载到3D"。 </li><li>在左上角选择"3D重建图像"，点击"显示单一窗口"按钮。使用变焦功能，如果需要的放大图像。切换"显示布局"，以仅在B模式，这从图中移除功率多普勒叠加查看图像。这使得更容易地看到在随后的3-D分析的淋巴结的边缘。滚动图像系列以定位淋巴结的开头。 </li><li>要外接淋巴结，定位到"3D设置"选项卡。选择"音量"，那么"开始"按钮旁边的"水货"。 </li><li>通过滚动周围绘制单个图像中感兴趣的区域轮廓。继续，直到包括整个淋巴结图像标记。选择"完成"完成分析。 </li><li>在图像的底部，3D体积和％血管将自动显示。 注:3D体积对应于淋巴结体积，以及百分之血管表示淋巴结阳性用于通过功率多普勒血流量的百分比。 </li><li>切换"显示布局"，以查看电源多普勒成像为B-模式图像上叠加。在表面视图观察感兴趣体积区域的净图。在标记的图像文件（TIF）格式或三维扫描图像导出为动画（.AVI）继续使用。 </li></ol>

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所描述的协议允许可视化，并在使用非侵入HFUS成像鼠颈部淋巴结原位评价。利用B模式和功率多普勒成像可视颈部淋巴结形态，体积和淋巴结血流提供类似于用于颈椎病人节点在临床实践中表征的临床前小鼠模型系统的实验分析。监测通过细针活检颈部平面淋巴结的能力还提供了用于检测免疫细胞的改变，外细胞类型或细菌的过程中在小鼠口腔疾病诱导淋巴结病的存在下一种有用的技术。使用和低成本的具有HFUS相关联的容易允许的颈淋巴结状态在各种动物模型的快速筛选。 在这个协议中的一个关键步骤是最初的成功鉴定HFUS图像颈部淋巴结。我们的工厂有HFUS TRAN的分类sducers所描述的，所以我们用它们来获得最高质量的图像。然而，如果我们描述了换能器是不可用的，这是可能的，以适应使用其它换能器的成像。为了这个目的，调整图像的深度和宽度，以获得足够的图像是所有需要。这种图像的分辨率可能会发生变化，但它应该仍然有可能通过使用HFUS以获得高质量的图像。口腔和甲状腺的成像地标，将大大有助于定向用户所在的淋巴结被定位在适当的区域。特色椭圆形，低回声性质和位置表浅紧贴皮肤表面允许适当的颈部区域内快速识别验证颈淋巴结。虽然所有三个节点可以是在一个单一的成像平面（ 图3B）可见，通常为一个或两个节点成像期间捕获。换能器位置的细微调整可进行到裂呃不同成像平面可见，允许在颈部的单侧的所有节点的可视化。 虽然我们已经发现所描述的技术可靠识别颈部淋巴结，也有具体的限制，以在成像和活检技术。小鼠颈部淋巴结浅表性赋予过度的流动性时，即使轻微的压力被施加到通过所述换能器头在皮肤上。这可以通过缓慢应用传感器头放入超声波凝胶鼠标的脖子，直到里程碑意义的图像识别来抵消。淋巴结迁移率也可以用较高的分辨率（50兆赫）范围的换能器特别是当复杂的微细针活检。活检淋巴结居中图像通常被推出的视场由于刺穿覆盖皮肤和胶囊所需的活检针的力。这可以通过在淋巴结的偏离中心的定位来补救朝针进入的方向，对于淋巴结提供空间可以跨被推但仍活检期间保持在视野内。根据我们的经验，淋巴结"10 mm 3的非常难以活检，并且通过所述针中针前进经常推而不是穿透。因此，活检最好为淋巴结肿大其中该尺寸为保留"10 立方毫米，以确保在颈部区域内具有足够的节点目标的尺寸和稳定性。另外，活检材料可能不包含用于在需要较大的细胞数目（ 例如 ，流式细胞术）的程序足够的细胞数量。 HFUS已成功地用于可视原位HNSCC肿瘤25，并具有监测小鼠颈淋巴结转移口腔肿瘤24的潜力。除了 ​​超声，生物发光成像也被用于可视化在活体小鼠-26,27-原位口服肿瘤形成和颈淋巴结转移。作为一个alternative方法，生物发光成像具有能够直接量化肿瘤进展和转移负担随着时间的推移，在相同的动物在HFUS明显的优势。虽然不可否认有用，生物发光成像是无法衡量由许多可视化HFUS的参数，包括淋巴结形态，节点卷或血流。生物发光成像也需要专门的黑盒子成像过程中保持小鼠，使这种技术不适用于为适应细针活检。 此外，生物发光成像需要生产的肿瘤细胞的稳定表达荧光素酶，从而使该技术仅可在原位异种移植物的与荧光素酶转染的肿瘤细胞在免疫受损小鼠中，或与该限制萤光素酶表达诱导组织特异的转基因系统的情况下在特定肿瘤起源组织一个时空方式。相比之下，HFUS可以为used的与在这些模型中的生物发光图像结合，以及能够成像中的致癌物诱导口服肿瘤模型颈部淋巴结小鼠完整免疫系统28,29的。而HFUS可能更适合于口腔癌，可以从生物发光和HFUS成像系统中获得的组合信息最小鼠模型，其中肿瘤细胞表达的萤光素酶可以提供颈淋巴结受累比单独成像模态的一个更全面的了解。 识别并检测小鼠颈部淋巴结实时的能力允许在口腔疾病的大多数模型导致炎性淋巴结病，其中所述动物能保持在根据短期麻醉的倒置位置要使用这种技术。检测淋巴结转移或细菌感染，并在活的动物上淋巴结形态随之而来的影响提出了比传统方法显著的好处需要淋巴结从死亡的动物进行组织学处理来去除。结合HFUS细针穿刺活检可用于进行颈部淋巴结肿大，类似于在门诊进行，为监测疾病进展的口腔疾病最新的小鼠模型的改进方法，常规病理分析手段。

间质组织液淋巴引流是为传播在口腔颌面部1,2产生的感染性微生物和癌症的主要方法。颈部淋巴结临床评价是用来确定在口腔中是起源的疾病的存在或进展的共同诊断实践。这强调了收集颈部淋巴结肿大为有价值的解剖部位为口腔疾病的诊断3的重要性。一些专门的成像方法在临床上用于识别病变颈淋巴结肿大。这些包括正电子发射断层扫描（PET），计算机断层扫描（CT）和磁共振成像（MR）。而高度有价值的，这些方法都需要进行大量的患者准备，高度专业化的设备和/或化学输注进入循环，以启用或增强在成像过程。 超声成像（超声;美国）是用于一般即时通讯应用技术年龄颈部淋巴结肿大淋巴结病是由于感染或转移性参与4-6呈现。美国往往是结合PET-CT和磁共振成像提供病人淋巴结状况的全面表现，帮助确定手术切除7肿瘤分期和必要性。美国的非侵入性的性质也比其他成像模态，包括易用性，成本低，最小患者不适和制剂固有的优点。大多数颈淋巴结的表浅皮下位置允许我们引导微创细针穿刺活检增加精确度，提高诊断的准确性8。 商业高频（HF）美国提供内部解剖结构以30微米9具体的解决。使用的换能器范围从22-70兆赫，HFUS已容易地应用于各种实验啮齿类系统，以允许在体内的内部器官的实时成像。; HFUS已适应于肿瘤形成中常规亮度（B）中-mode可视化，以及与一些一般的和专业的对比度增强agents9。使用能量多普勒与HFUS提供监控内小鼠肿瘤的血流量，从而在活体小鼠血管10,11灌注进行全面评估的能力。 HFUS已被用来在主体腔内可视患病小鼠的淋巴结，表明该技术的平行效用临床实践。特别是，炎性和转移性内脏淋巴结改变在癌症窝藏乳房12,13，胰腺癌14，结肠15和肺16肿瘤，以及纤维状histocytomas 17，和获得积水18的老化小鼠模型的小鼠模型已观察。这些例子固化HFUS作为促进肿瘤诱发淋巴结在各种各样滚装的强大调查工具的值凹痕系统。 （头颈部鳞癌）21,22已经开发细菌感染19,20和头颈部鳞状细胞癌的几个模型来研究这些疾病在临床前设置。相反，人，小鼠含有淋巴调查从口腔组织（下颌骨，下颌骨颌下腺和肤浅的腮腺23）三种颈淋巴结。最近，我们报道了使用HFUS的映射这些淋巴结的位置和形态，监测头颈部鳞癌24的致癌物诱导小鼠模型的变化在淋巴结体积和血液流动。在这里，我们提供使用HFUS用于识别，成像和分析在活小鼠颈部淋巴结的详细协议。该协议还演示了使用HFUS进行的放大鼠标颈部淋巴结图像 - 引导下细针穿刺活检的可行性，使组织在监测T在颈部淋巴结含量的变化及病理学IME在相同的动物。这个协议是容易适用，以允许颈部淋巴结病理学从任何侵入性口腔疾病的小鼠得到的详细研究。

<table><tbody><tr><td>Vevo2100 High Resolution Micro-ultrasound Imaging System, with integrated software Version 1.6.0</td><td>VisualSonics, Toronto, Ontario, Canada</td><td>VS-11945</td><td></td></tr><tr><td>Power Dopper Mode and 3D Mode</td><td>VisualSonics, Toronto, Ontario, Canada</td><td>VS-11952; VS-11484</td><td></td></tr><tr><td>Vevo compact anesthesia system</td><td>VisualSonics, Toronto, Ontario, Canada</td><td></td><td></td></tr><tr><td>Vevo integrated rail system including 3D motor and micromanipulator for injections</td><td>VisualSonics, Toronto, Ontario, Canada</td><td>SA-11983</td><td></td></tr><tr><td>Thermasonic Gel Warmer</td><td>VisualSonics, Toronto, Ontario, Canada</td><td></td><td>Optional</td></tr><tr><td>Transducers &amp;ndash; MS-550D (Broadband frequency: 22 MHz - 55 MHz)</td><td>VisualSonics, Toronto, Ontario, Canada</td><td>VS-11960</td><td>Referred to as 40 MHz Transducer</td></tr><tr><td>Transducers &amp;ndash; MS-700 (Broadband frequency: 30 MHz - 70 MHz)</td><td>VisualSonics, Toronto, Ontario, Canada</td><td>VS-12026</td><td>Referred to as 50 MHz Transducer</td></tr><tr><td>Ophthalmic Ointment</td><td>Patterson Veterinary</td><td>07-888-1663</td><td></td></tr><tr><td>Electrode gel</td><td>Parker Laboratories</td><td>174-1525</td><td></td></tr><tr><td>Tape</td><td>Medical Arts Press</td><td>174-153000</td><td></td></tr><tr><td>Depilatory Cream</td><td>Carter Products</td><td></td><td></td></tr><tr><td>Cotton swabs</td><td>General Supply</td><td></td><td></td></tr><tr><td>Gauze</td><td>Fisher Scientific</td><td>22-037-907</td><td></td></tr><tr><td>Water</td><td>General Supply</td><td></td><td></td></tr><tr><td>Lubricating gel</td><td>Parker Laboratories</td><td>57-05</td><td></td></tr><tr><td>Ultrasound gel</td><td>Parker Laboratories</td><td>01-50</td><td></td></tr><tr><td>Microcentrifuge tube rack</td><td>General Supply</td><td></td><td>Used to raise mouse platform for optimal biopsy position</td></tr><tr><td>27 G &amp;frac12; inch needle with 1 ml syringe</td><td>Fisher Scientific</td><td>14-826-87</td><td></td></tr><tr><td>ThinPrep PreservCyt Solution</td><td>Hologic</td><td>70097-002</td><td>Refered to as biopsy media</td></tr><tr><td>Microcentrifuge tubes</td><td>General Supply</td><td></td><td></td></tr><tr><td>Thinprep 2000 processor</td><td>Cytyc, Marlborough, MA</td><td></td><td>Blue Filter</td></tr><tr><td>Olympus AX70 Provis Microscope</td><td>Olympus, Center Valley, PA</td><td></td><td></td></tr></tbody></table>

high-frequency ultrasound imaging of mouse cervical lymph nodes

高频超声（HFUS）被广泛地用作用于成像内部解剖结构在实验小动物系统的非侵入性方法。 HFUS具有检测结构小30微米的能力，已被用于可视化浅表淋巴结在啮齿类动物中的亮度（B）模式，一个属性。结合力与多普勒B超成像可以衡量淋巴结等器官内循环的血流量。而HFUS已用于淋巴结成像在多个小鼠模型系统中，一个详细的协议描述在小鼠HFUS成像和颈部淋巴结的鉴定尚未见报道。在这里，我们表明，HFUS可以适于检测和小鼠表征颈部淋巴结。合并的B模式和功率多普勒成像可用于检测的血流增加在免疫学上扩颈淋巴结。我们还描述了使用B型成像进行颈部淋巴结的细针活检无DES检索淋巴组织进行病理分析。最后，软件辅助步骤被描述以计算变化淋巴结体积和形象化以下图像重建在淋巴结形态学变化。在视觉上监测变化在颈部淋巴结生物学随时间的能力提供了一个简单而功能强大的技术在口腔疾病的临床前小鼠模型的非侵入性的监测颈淋巴结改变的。

用于成像和活检程序的整体示意图示于图1。在此过程中的关键步骤包括鼠标用于成像的适当的制备，鉴定颈部淋巴结的，正确制备和导电针活检，B的分析-mode和多普勒图像来测量体积和血管的使用的计算机软件的每个所选节点内的量。 小鼠颈部淋巴结HFUS成像需要施加并保持适当的麻醉整个摄像期间（ 图2A），以及完全去除毛发覆盖整个颈部区域（ 图2B）的。超声凝胶的自由施加到脱毛区域的步骤（ 图2C）期间保证了清晰HFUS信号。 颈部区域的HFUS成像是通过产生特征的超声图像宫颈解剖标志的可视化辅助图图3示出的关键器官的B模式（ 图3D），并在功率多普勒模式（ 图3E）的例子（ 图3A-C）中 ，颈部淋巴结。 在麻醉小鼠实时成像HFUS允许颈部淋巴结类似于在临床实践中进行的引导下细针穿刺活检。活检针和连接的收集注射器向控制微量设备的放置示于图4A中 。随后B模式超声图像显示理想针放置前活组织检查（ 图4B），针尖进入一个颈部淋巴结（ 图4C），和针位置活检（ 图4D）中。特写图像显示淋巴结（ 图4E）的髓质内针尖。活检组件通过离心涂片的处理揭示了丰富的淋巴样细胞团和相关的结缔组织，验证成功的淋巴结活检（ 图4F）。 计算为基础的HFUS图像的分析，可以了解有关淋巴结结构，体积和血管流量要获得详细信息。使用功率多普勒模式和3D体积测量，百分之血管（PV）可从图像系列包含整个节点（ 图5A）来计算。另外，三维成像允许虚拟淋巴结重建，揭示整体淋巴结地形（ 图5B）。 <img alt="图1" src="/files/ftp_upload/52718/52718fig1.jpg" /> 图1:参与小鼠诊断HFUS颈部淋巴结成像的步骤概述概略的关键步骤包括1:准备老鼠HFUS成像和获得含有三种小鼠颈部淋巴结颈部区域40和50兆赫分辨率图像。 2:细针影像引导下的颈部淋巴结和随后的病理切片分析<html>活检的材料。 3:计算机辅助图像分析和三维重建在B模式和多普勒得到确定血管流的各个淋巴结体积和百分比（％）淋巴结图像。 <img alt="图2" src="/files/ftp_upload/52718/52718fig2.jpg" /> 图2:高分辨率为颈部淋巴结评估和活检体内显微成像系统的概述 （ 一 ）HFUS系统被示为具有对颈淋巴结成像制备麻醉小鼠。还示出了微量（MI）和3D-马达阶段（3D MS）的辅助设备。 （ 二）特写视图HFUS成像头发颈部取出准备麻醉鼠标。 （ 三）同一鼠标代替50 MHz传感器在脖子上。注意用于促进颈部成像额外的超声凝胶。 718fig3.jpg"/&gt; 图3:在B模式和功率多普勒代表HFUS宫颈解剖学图像。 （A，B）在口腔的B模式图像，示出了口腔（BC）和舌部（T），通过成像最靠近鼻腔可视化。在颈部的各侧中的3中颈部淋巴结（标记男，下颌; SM，颌下; SP，浅表腮腺），如图（B）中显示为一组低回声结构在一个单成像平面。 （C）的甲状腺（钍），为可视化在上部胸部区域，出现固体，回声蝴蝶形结构。 （AC）显现具有40 MHz的换能器;比例尺= 1毫米。 （D，E）正常（D）的代表性图像和放大（E）的颈部淋巴结与B模式和功率多普勒（红色）的节点。虚线勾勒出个人的淋巴结。比例尺= 0.5毫米。 <img alt="图4" sRC ="/文件/ ftp_upload / 52718 / 52718fig4.jpg"/&gt; 图4:颈部淋巴结活检设置，成像和活检材料的离心涂片分析 （ 一 ）成像平台显示了微注射器和针头位置靠近颈部鼠标。宽的离心管架（橙色块）用于略微抬高平台，可实现合适的针放置，同时仍然允许在3D马达舞台空间。这种安排减少了花费的时间中除去电机阶段每只小鼠。采取全颈HFUS图像使用的50兆赫换能器颈部淋巴结活检的视频- （D B）。 （B）的 HFUS B模式图像显示位置到之前活检颈部侧的针。针尖略低于针导向的成像来表示针轨迹中重叠的位置（绿色虚线）的回声结构。淋巴结是在中心图片。比例尺= 1毫米。 （ 三）针刺进入淋巴结。颈淋巴结（D）活检。 （ 五）颈淋巴结活检缩放。比例尺= 0.5毫米。代表淋巴结活检证实的材料成功活检（F）细胞离心涂片分析。比例尺= 100微米。 <img alt="图5" src="/files/ftp_upload/52718/52718fig5.jpg" /> 图5:3D颈部淋巴结的图像的计算机分析一个淋巴结的（A） 的代表性屏幕截图使用计算机软件进行分析。该节点外接蓝色;分析结果表明，三维体积和百分比血管（PV）所示。 （B）3D分析后，同一节点的A面视图图像。呈现淋巴结基于在A截取测量整个体积。

Watch this Scientific Journal Video about High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes at JoVE.com

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

这个协议描述高频超声（HFUS）的应用为摄像小鼠颈部淋巴结。该技术优化了可视化和颈部淋巴结的形态，体积和血流量量化。颈部淋巴结和淋巴组织进行组织学评价的处理图像引导下穿刺活检还体现。

鼠标颈淋巴结高频超声成像

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

high-frequency-ultrasound-imaging-of-mouse-cervical-lymph-nodes

Research

JoVE Journal

Medicine

18.9K 次观看.  West Virginia University. 这个协议描述高频超声（HFUS）的应用为摄像小鼠颈部淋巴结。该技术优化了可视化和颈部淋巴结的形态，体积和血流量量化。颈部淋巴结和淋巴组织进行组织学评价的处理图像引导下穿刺活检还体现。 

视频: High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

夹层和小鼠的外周淋巴结的双光子成像

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

科研

生物学

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

小鼠浅表淋巴结手术

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

免疫与感染

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

小鼠腘窝淋巴结的活体成像

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

超高频超声成像分析黑色素瘤Braf/Pten基因工程小鼠模型中的淋巴结体积

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

癌症研究

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

用于分析基因工程小鼠模型中原位ATC肿瘤的高分辨率超声检查

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

小鼠中Pax8定向缺失肿瘤抑制因子 Arid1a 和 Pten 的子宫内膜癌进展的非侵入性超声评估

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

使用前 Vivo 共聚焦显微镜可视化淋巴节点结构和细胞定位

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Ultrafast Doppler Ultrasound for Hepatic Microvasculature Analysis in Mice

Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the microvasculature using Doppler ultrasound methods, imaging modalities employing the concept of ultrafast imaging are preferred. By increasing the frame rate of the ultrasound scanner to thousands of frames per second, it becomes possible to improve the sensitivity of the blood flow down to 2 mm/s and to obtain functional information about the microcirculation in comparison to a sensitivity of around 1 cm/s in conventional Doppler modes. While Ultrafast Doppler ultrasound (UFUS) imaging has become adopted in neuroscience, where it can capture brain activity through neurovascular coupling, it presents greater challenges when imaging the vasculature of abdominal organs due to larger motions linked to breathing. The liver, positioned anatomically under the diaphragm, is particularly susceptible to out-of-plane movement and oscillating respiratory motion. These artifacts not only adversely affect Doppler imaging but also complicate the anatomical analysis of vascular structures and the computation of vascular parameters. Here, we present a qualitative and quantitative imaging analysis of the hepatic vasculature in mice by UFUS. We identify major anatomical vascular structures and provide graphical illustrations of the hepatic macroscopical anatomy, comparing it to an in-depth anatomical assessment of the hepatic vasculature based on Doppler readouts. Additionally, we have developed a quantification protocol for robust measurements of hepatic blood volume of the microvasculature over time. To contemplate further research, qualitative vascular analysis provides a comprehensive overview and suggests a standardized terminology for researchers working with mouse models of liver disease. Furthermore, it offers the opportunity to apply ultrasound as a non-invasive complementary method to inspect hepatic vascular defects in vivo and measure functional microvascular alterations deep within the organ before unraveling blood vessel anomalies at the micron scale levels using ex vivo staining on tissue sections.

Ultrafast Doppler ultrasound (UFUS) with high spatial resolution (100 &#956;m) and sensitivity represents a suitable noninvasive imaging modality for obtaining a comprehensive qualitative overview of the hepatic vasculature. UFUS also facilitates quantitative measurements of the microvasculature, aiming to enhance our understanding of vascular disease mechanisms.

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the ...