We thank Maria Jepson, Dr. Paivi Jordan, and Dr. Linda Callahan at the University of Rochester Multiphoton Core for technical advice throughout the completion of this protocol. We also thank Dr. Changyong Feng for expert statistical advice, and Dr. Maiken Nedergaard at the University of Rochester Medical Center for the headplate design used in this paper. This work was supported in part by grants T32GM007356 and R01DA026325 from the National Institutes of Health (NIH); and by the University of Rochester Center for AIDS Research grant P30AI078498 (NIH).

罗切斯特大学委员会的动物资源学院批准在本文中进行的所有程序。 1.术前准备（和鼠标） <ol><li>所有必需的设备准备手术区。消毒预先用70％的乙醇的过程​​中使用的所有工具。或者，使用玻璃珠灭菌或高压灭菌消毒的工具。 </li><li>鼠标放置在异氟醚感应室，连接到一个异氟烷蒸发器。以1升/分钟的速率设置异氟烷水平至4％。 注意:用强力霉素（DOX）诱导的HIV-1的Tat转基因的星形胶质细胞特异性胶质纤维蛋白（GFAP）启动子（HIV TAT-Tg小鼠）驱动此报告的实验中，小鼠10被用来检测艾滋病病毒的效果-1诱导的神经炎症对脑血管结构，如前所述7,10。 </li><li>轻轻倾斜感应室，观察鼠标＃39; S翻正反射。一旦正位反射被抑制，异氟醚级别设置为2％和重定向从感应腔室到鼻锥的气流在手术台上。 </li><li>快速地从感应室向水注入加热垫移动鼠标。继续通过鼻锥申请异氟烷（1.5-2％）。根据个人老鼠容忍通过呼吸频率（RR）的监测评估，调整麻醉。 注:RR的抑郁症可能扰乱生理气体参数改变脑血流量和血管直径评估。试图保持每分钟55-65次呼吸之间RR。心脏速率（HR）也可以用于评估麻醉深度;让HR每分钟300-450次之间，以防止生理改变血管直径。通常情况下，麻醉就足够了1.5-2.0％异氟烷之间以1L /分钟的一个给定的鼠标。 </li><li>执行脚趾捏进一步检查麻醉深度。如果鼠标不响应，通讯ENCE的外科手术。 </li></ol> 2.制备薄头骨颅窗口<ol><li>应用人工泪液凝胶鼠标的眼睛。从用剪刀或电动剃须刀鼠标的头皮完全除去毛发。 </li><li>消毒头皮通过施加碘伏溶液;晾干。然后，在光学显微镜下，从小鼠的头皮去除皮肤，完全露出顶骨，尾椎额骨和前囟标记。 </li><li>为了干燥膜，便于拆卸应用少量的10％的氯化铁溶液到颅骨。与＃四十五分之五镊子轻轻刮了头骨中取出干燥的膜。 </li><li>围绕磁头板窗口涂抹一层薄薄的胶水。轻轻按压鼠标的头骨磁头板，保持感兴趣的区域，在窗口的中心。申请一滴牙科用粘固剂的给磁头板以聚合胶。 </li><li>涂抹薄薄的一层沿磁头板窗口的边缘胶以创建在其中容纳盐水的贮存器。一旦胶水已干，拧磁头板插入鼠标磁头板线束。 注意:在此过程中使用的鼠标磁头板线束是与金属基体和两个金属柱的结构。在每列有一个弹簧和一个蝶形螺母。的磁头板被放置在弹簧的顶部，并蝶形螺母被拧紧，直到头部是水平和不移动。 </li><li>从使用连接到微扭矩钻头设定在6000转钻头的磁头板窗口除去任何胶。停止每10-15秒，以防止小鼠头盖骨的过热，可能导致对血管结构损坏。 <ol><li>使用新的钻头，横向放置微扭矩钻头1.5毫米从中线（三分之一磁头板窗口的直径）。开始薄这个位置周围的头骨在4000转。将钻头轻轻划过头骨没有直接的下行压力。 </li><li>停火DRIL林志玲每隔10-15秒，以防止过热老鼠头盖骨。在此期间，利用压缩空气罐除去积累颅骨灰尘。 </li><li>使用RT生理盐水滴冷却头盖骨5-10秒。轻轻地取出所有流体用柔软的纸巾，继续颅骨变薄。 </li></ol></li><li>对于头骨的最终变薄，放置一个小体积的盐水中的磁头板容器和轻轻扫过牙齿microblade在感兴趣的区域，直到小血管都清晰可见。 </li></ol> 3.监测生理指标<ol><li>一旦颅骨完全减薄，从保持器拆下小鼠，并将其放置在它的后面。轻轻大盘回落两个后腿清楚地暴露大腿内侧。 </li><li>取下发过来两个大腿内侧，用剪刀或电动剃须刀。由覆盖用碘伏大腿消毒手术部位。 </li><li>捏紧内侧右大腿上方的股静脉皮肤并采用动脉＃5镊子。轻轻拉动皮肤向上，用剪刀剪下，并去除皮肤。 注:左大腿被保留在手术并发症（血管畸形，血管破裂）的事件。 </li><li>应用约3-5滴0.9％的盐水，以外科手术部位。这允许更大的放大血管，以及血管的保持湿润的部位，并防止干燥。通过解剖直言将用两个＃四十五分之五镊子股神经血管束分离动脉股静脉。 </li><li>放置两个，3厘米的片手术用缝合线的股动脉大约1cm相距下方。扭曲的缝线上顺时针创建血管止血带，这将有助于防止导管手术中失血过多。 </li><li>做一个小切口，用弹簧剪刀股动脉。放置导管中通过切口和预先动脉直到导管是牢固的动脉内。 </li><l我&gt;解开上缝合（止血带），以评估是否有泄漏和正确的导管放置。通过把两个结用缝线容器内围绕导管固定在股动脉导管。 </li><li>注入10微升肝素（100IU / ml）的通过导管，以防止血液凝固。然后，通过用4-0缝合线在一个简单的断续图案一起缝制皮肤手术关闭右大腿。 </li><li>注入50μl的尿烷（1.2毫克/克）腹腔内插入右下腹。五分钟后用另一个将50μl注射到左下腹，共100微升腹腔氨酯重复。慢慢降低的异氟烷水平至约0.25％，而伴随地重新检查小鼠麻醉的深度。 注意:使针浅沿后腹壁腹膜腔中，以使得它不穿孔肠道。这可以通过捏腹部腹直肌万亩完成scles远离内脏，然后注入。 </li><li>使用毛细管，收集动脉血从导管40微升样品。附加导管配备有生理盐水填充四通活栓和肝素灌封注射器的血压传感器。 </li><li>用胶带将血压换能器到手术装置，以防止意外取出导管。开始监视平均动脉压。 </li><li>插入血液填充毛细管成血液气体分析仪入口。一旦所有的血液已经被提取，从入口移除毛细管。在O 2和CO 2血气水平将出现在从血气分析仪打印纸。 </li></ol> 4.注射荧光染料<ol><li>捏上使用＃5镊子内侧左大腿皮肤。轻轻拉动皮肤向上，用剪刀剪下，并去除皮肤。准备葡聚糖结合红色荧光染料罗丹明（70 kDa）的（10毫克/千克溶解在盐水中）。 </li><li>定位股骨vein.Using 1ml注射器附有一个地下30针，得出适当的用于成像到注射器的130微升线荧光染料的预先制备溶液。通过染料完全拉入注射器，坚决轻弹注射器墙上拔下所有气泡。 <ol><li>弯曲的地下30针在通过按下它来对一个硬表面斜角朝上的大约30度角。填补了染料的针并丢弃任何剩余液体，留下了100微升样品。 </li></ol></li><li>染料的100微升样品注入慢慢进入股静脉。取出针后，应用稳定，轻轻压到注射部位，以制止任何出血。允许染料中循环5分钟。 注意:可替换地，可以使用右股静脉，而不是用于荧光染料注射，以防止进一步失血通过创造另一种外科部位。此外，如果动物是从手术部位出血不受控制，这可能是一个表明太多肝素被给冲洗导管。如果在10-15分钟无凝结和鼠标还在流血，应考虑重新启动实验，一个新的鼠标。 </li><li>关闭左大腿用4-0缝合线缝合在一起的皮肤，然后小心地翻转鼠标到它的肚子，然后将其放入鼠标磁头板线束。 </li></ol> 5. 体内双光子成像<ol><li>移动外科装置向双光子显微镜，确保连续保持麻醉的水平。放置一个小数量的0.9％盐水到磁头板容器和降低显微镜物镜，以便它进入与盐水接触。使用明收视目标定位感兴趣的领域</li><li>设置双光子激光激发到适当的荧光染料的波长。使用25XØ开始双光子成像BJECTIVE。 注意，在这些实验中，我们使用的缀合红色发光罗丹明染料被激发在780nm的波长的葡聚糖。甲三十六分之六百○七带通发射滤光片从染料检测荧光，以及一个带通二十零分之四百八发射器被用来检测动脉自发荧光</li><li>找到一个毛细血管床查看屏幕上，并放大使用光学变焦2.采集利用双光子成像软件毛细血管的图像这一领域。 </li><li>成像完成后，通过颈脱位随后断头牺牲鼠标。 </li></ol> 6. 离体双光子成像<ol><li>使用超等剪刀，使在头皮切口从interparietal骨骼鼠标的额骨。固定到皮肤用指针手指和拇指的头骨的侧面。 </li><li>将多余的精剪内侧interparietal骨下方切颅骨沿矢状​​缝。停割头颅约3mm后的前囟门标记。 注:切割颅骨，以防止切割时，大脑应用上行压力。 </li><li>使用＃5镊子，从大脑中分离颅骨和小心从大脑的表面去除任何脑膜。剩余的脑膜可无意中划破大脑;极端应注意避免这种情况。轻轻滑动大脑慢慢前进着下＃5镊子，直到大脑不受头骨。 注意:向下的压力，应使用以避免刺穿大脑。 </li><li>将大脑中的特定对小鼠脑切片工具。通过施加人工脑脊液滴在大脑洗大脑。 </li><li>从囟0除去大脑的2毫米冠状切面到前囟-2。放置冠状切面上含人工脑脊液与0前囟门凹陷载玻片（段大部分颅一部分）朝上。轻轻地覆盖在大脑SL冰，用玻璃盖玻片。 注意:不要按一次放置在大脑部分，因为这可能会变形的血管结构的玻璃。 </li><li>幻灯片转移到显微镜台上，放置一个小数量的0.9％盐水盖玻片上。降低显微镜物镜，直到它与盐水接触。定位使用明目标脑的中线。 </li><li>开始双光子成像和定位中线使用25倍的目标了。通过寻找在冠状切面的皮质表面纵裂做到这一点。放置在中线成象屏幕的右边缘和移动查看器屏幕上横向3个完整的帧（约1.5毫米，从中线）。 注意，在这些实验中，成像参数的那些相同，在步骤5.2中提到的音符。 </li><li>找到深度处的毛细血管都认为屏幕上几乎看不到。降低焦点的附加20的平面微米来确定Z堆叠的顶部。 </li><li>图像厚度设置为1微米。降低视图画面100微米，并调整激光功率在整个使得像素的小于1％的过饱和。 注意:与z堆栈图像从一系列的连续100 500x500x1微米图像编译。所述的Z堆叠的更准确的后处理的计算会较大。通常，试图收集的z堆叠100微米或更大。 </li><li>按XY重复图标其次是相邻的XY图标。一旦Z堆栈完成后，按系列完成图标，保存文件。 </li></ol> 7.数据处理<ol><li>在ImageJ的软件打开体内毛细血管的形象。手动设置基于从双光子软件获得的值的像素的距离的比率。 <ol><li>从工具菜单中的*直*图标。画从一个毛细血管壁延伸到OPPO线站点毛细血管壁，并记录由ImageJ的所提供的长度。 需要注意的是，可能有必要对放大图像，以便清楚地显现毛细管壁的边缘。 </li><li>在沿毛细管的不同位置重复步骤7.1.1，以及对多个毛细管的图像中。 </li></ol></li><li>打开Z堆栈文件到分析软件，如阿米拉。选择从子应用程序工具栏中的灯丝编辑器图标<ol><li>观察者厚度观察者设定为约20移动到顶部或在z堆栈底部。 </li><li> 选择跟踪图标，将光标移到加载的图像。上的毛细管在图2C描述的位置处的节点;段将相邻节点之间自动出现。毛细血管跟踪整个Z堆栈。 注意:这将是必要把节点端点和分支点之间，以便跟踪毛细管THRoughout的z栈。 </li><li>要删除这些节点，点击删除中间的图标。 注意:此可以创建必须手动通过使用选择单图标选择循环，然后选择移除选定图标被移除错误环段。如果环路继续跟踪期间出现在同一地区，它是可能的节点被放置在不同的毛细管。 </li><li>一旦跟踪完成后， 选择图形信息图标可打开包含自动提取血管参数的电子表格。可用的主视图屏幕上节点和段的数量。 </li></ol></li></ol>

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The authors have nothing to disclose.

此处所描述的方法可用于分析脑微血管结构在大范围的实验模型/设置。对于这种方法的成功，有三个关键步骤，必须要掌握。首先，将薄颅骨窗不能损伤颅骨或底层大脑。这是很容易变薄时穿破头骨，或引起热诱导的血管渗漏。这可以通过摄像干扰作为荧光染料将泄漏到焦点的平面中，并掩盖了毛细血管。如果颅骨薄头骨制备过程中频频打破，它很可能是过大的下行压力造成的。握住微扭矩钻尽可能接近的毛刺允许更大的触摸控制，这将减少对头骨的下行压力。 第二，动脉插管应该尽可能快地，一旦动脉被切断发生。未能快速插入导管可导致失血过多，将COMPromise鼠标的生理完整性。难以插入导管通常是由于导管的相对大的尺寸相比股动脉。可能有必要以拉伸导管以减小其直径。此外，＃5钳提示可用于抓住并放大在动脉，以缓解在放置导管的所作的开口。 第三， 在体内的外科手术的持续时间应该被最小化，使得氨基甲酸酯麻醉，可尽快给药。在异氟烷麻醉可引起的脑血管13的血管舒张，从而倾斜毛细管直径的数据。 应该承认，阿米拉分析软件可以自动提取的手动追踪船只的半径;但是，使用此功能来分析从体外大脑Z堆栈图像时，该值是不准确的。这是因为，自动对焦之三除去脑，脑毛细血管不再加压，并可能成为形态变形。 体内毛细血管直径测量规避这个问题，因为毛细血管都在正常生理条件下加压。 还必须注意的是，这种方法也不是没有局限性。首先，显著培养掌握体内成像制剂是必需的。研究者必须学会有效地执行两项技术挑战性的技术（薄的头骨皮层的窗口和动脉插管）;在任一技术的错误会损害实验和数据无效。其次， 离体的z叠层的分析是时间密集型的。一个Z堆叠图像的骨架可长达2.5小时。此外，最好是，这种分析由有经验研究者完成，以确保骨架过程的稠度（这涉及仔细的手工TR血管acing）。 一旦该协议的所有方面都掌握了，得到的数据提供比较的更深入和血管生理参数的准确定量到显示毛细血管密度为每单位体积的毛细血管，或每视野毛细血管等传统使用的方法。 体内成像技术允许的其他参数的研究，包括，但不限于，红细胞流速，动脉扩张和红细胞磁通7,12，其也能够在纵向研究要被检查。此外，它可以很容易地调整和修改，以研究有关脑微血管结构研究的问题。这些研究可能包括定量毛细管形态在其他神经认知疾病的致病性的变化，或测量毛细血管密度与年龄有关的变化。因此，本文提出的方法是一个灵活和强大的工具的quantitat脑血管架构的香港专业教育学院的分析。

在病毒感染的急性期的人类免疫缺陷病毒-1（HIV-1）的侵入脑，和生产性感染都小胶质细胞和脑定居巨噬细胞，导致它们的活化 - 并且两个来自宿主的炎症介质和可溶性HIV-1的释放virotoxins如Tat和gp120的（ 在 1,2-综述）。因此，慢性神经炎状态成为中枢神经系统，这被认为是有助于HIV-1相关的神经认知障碍（手），3-5的发病机制建立。 HIV-1的Tat或小鼠的中枢神经系统内白细胞介素（IL）-17A的慢性过度表达已被证明导致微血管稀疏6,7。这就提出了慢性神经炎症可以通过对脑血管的影响造成的手发病的可能性。为了进一步探讨这个问题，我们已经制定的方法来量化脑血管STRUCTures的。 本文介绍的方法用于定量毛细管节点，毛细管段的数量，平均段长度，总段长度，平均毛细管直径，并通过一薄的颅骨皮质窗口使用毛细管网络的体内显像总毛细管体积（从先前描述的改性协议）8,9，以及离体成像的脑切片中，使用双光子显微镜。这种组合方法提供用于脑血管的参数的整体定量，由于体内薄颅骨皮质窗口允许大脑保护环境，同时毛细管网络在脑切片的离体成像能够重建完整，立体毛细管网络 - 然后可以使用市售软件定量。

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quantification of cerebral vascular architecture using two-photon microscopy in a mouse model of hiv-induced neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

薄颅骨皮质窗口允许用于体内双光子皮质毛细血管成像（ 图1）。合适的区域，图像显示众多的，不同的毛细血管（ 图1A）。在同一视场，不存在动脉细胞壁自发荧光，以及可能还有其他的荧光信号，如胶原荧光，诱发二次谐波生成11（图1B）。 一旦脑切片的准备已完成，摄像区域约1.5mm处中线位于（图2A）。甲100μm的Z堆叠产生用于分析在阿米拉分析软件（图2B）的三维图像。毛细血管网，然后通过手动放置节点在开始和毛细管的端部，或者任何位置，一个毛细b被曝牧场到另一个（图2C）。这将产生一个完全镂空图像从其中形态学参数被自动萃取（ 图2D）。毛细管节点，段的数量，平均段长度，总段长度，和总体积的毛细管可以使用的小鼠的脑切片的双光子离体成像被提取（图2）。 图3示出了使用本文提出的方法获得的代表性结果。在该实验中，艾滋病毒神经炎症的转基因小鼠模型，使用10。生产艾滋病virotoxin Tat的诱导在康达+小鼠由强力霉素，注入食品12周。对照组由TAT-同窝喂食同样的食物。有任何测量的毛细管参数达+和TAT-小鼠之间无统计学差异显著。因此，12周暴露大脑的那朵起诉对HIV-1的Tat不足以引起脑微血管稀疏。与此相反，我们此前公布的数据显示，更多的长期（20周）的康达结果慢性中枢神经系统表达的脑血管7疏松。因此，此处提供的数据（图3）提供了在问候的康达诱导的血管重塑的小鼠的中枢神经系统内的动力学的有价值的新的信息。 <img alt="图1" src="/files/ftp_upload/53582/53582fig1.jpg" /> 图1.采集皮质毛细管直径:（A） 按照一个薄颅骨皮质窗口制备和静脉荧光染料注射，双光子显微镜被用于图像脑血管。在我们的实验中，我们使用缀合到红色发光罗丹明染料被激发在780nm的葡聚糖和荧光通过一个带通三十六分之六百零七发射滤波器可视化。 <strong&gt;的（B）的同一视场不能有通过二十○分之四百八十〇带通发射滤波器作为检测动脉自体荧光。二次谐波的产生可能诱发其他的荧光信号出现通过过滤器11。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53582/53582fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53582/53582fig2.jpg" /> 图2.代镂空毛细管网络的:（A）对小鼠进行静脉内注射一种荧光染料和处死。脑的2毫米冠状部分取前囟0和囟-2之间，并在从中线0前囟大约1.5毫米，进行成像。一个 示从C57BL / 6小鼠选择用于​​成像（黑盒子）皮质位置的代表图像。这个区域，索姆atosensory皮质，被选中，是因为我们先前已经显示脑血液失调可出现在康达+小鼠12的网站。（B）的毛细管网络的表征三维Z堆叠图像。该方法的（C）的示意图用于手动跟踪中船量化毛细血管。（D）镂空Z堆栈图像的形象代表。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53582/53582fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53582/53582fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53582/53582fig3.jpg" /> 图3.定量的TAT-小鼠脑血管结构与暴露于HIV的候选达virotoxin FO小鼠R 12周，小鼠用强力霉素（DOX）诱导的HIV-1的Tat转基因的星形胶质细胞特异性胶质纤维蛋白（GFAP）启动子（HIV TAT-Tg小鼠）驱动被用来研究对HIV-1引起的神经炎症的作用脑血管结构，如上所述7。这些小鼠（达+中，n = 3）暴露于达诱导的慢性（12周）的方案（即12周的DOX）。 TAT-小鼠（n = 4）用作年龄匹配的对照（这些小鼠对应的Tat +小鼠的非转基因同窝，并且因此不表达的HIV-1的Tat）。像达+小鼠中，TAT-老鼠也获得12周DOX暴露。在暴露于达12周（达+）的小鼠中，与那些未暴露于达（TAT-），有在节点（A）的数量没有统计学显著差异，段的数目（B），平均段长度（C）中，或总毛细管长度（D）中 ，毛细管直径（为达安+小鼠中，我们分析了14毛细血管6成像领域;为TAT-小鼠，我们分析了7毛细血管4摄像区域）（E），或总毛细管体积（F）。由于数据不是正态分布（由夏皮罗-威尔克试验确定），确切的Wilcoxon秩和检验来计算统计学意义（在这种情况下，确定为P &lt;0.05）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53582/53582fig3large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53582/53582fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation at JoVE.com

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

quantification-cerebral-vascular-architecture-using-two-photon

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.2K 次观看.  University of Rochester Medical Center. This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

视频: Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

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 ...

科研

神经科学

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

在皮肤中的单神经末梢体内双光子显微镜

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by&#160;local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

在 Vivo 三维双光子显微镜中,利用玻璃微移器研究本地 ATP 喷射的血管反应

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...