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>

<ol><li>Kraft-Terry, S. D., Buch, S. J., Fox, H. S., Gendelman, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+coat+of+many+colors%3A+neuroimmune+crosstalk+in+human+immunodeficiency+virus+infection%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">A coat of many colors: neuroimmune crosstalk in human immunodeficiency virus infection.</a> Neuron. 64 (1), 133-145 (2009).</li>
<li>Ghafouri, M., Amini, S., Khalili, K., Sawaya, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-1+associated+dementia%3A+symptoms+and+causes%5BTitle%5D)+AND+%22Retrovirology%22%5BJournal%5D)">HIV-1 associated dementia: symptoms and causes.</a> Retrovirology. 3, 28(2006).</li>
<li>Antinori, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Updated+research+nosology+for+HIV-associated+neurocognitive+disorders%5BTitle%5D)+AND+%22Neurology%22%5BJournal%5D)">Updated research nosology for HIV-associated neurocognitive disorders.</a> Neurology. 69 (18), 1789-1799 (2007).</li>
<li>Clifford, D. B., Ances, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-associated+neurocognitive+disorder%5BTitle%5D)+AND+%22The+Lancet.+Infectious+diseases%22%5BJournal%5D)">HIV-associated neurocognitive disorder.</a> The Lancet. Infectious diseases. 13 (11), 976-986 (2013).</li>
<li>Lindl, K. A., Marks, D. R., Kolson, D. L., Jordan-Sciutto, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-associated+neurocognitive+disorder%3A+pathogenesis+and+therapeutic+opportunities%5BTitle%5D)+AND+%22Journal+of+neuroimmune+pharmacology+%3A+the+official+journal+of+the+Society+on+NeuroImmune+Pharmacology%22%5BJournal%5D)">HIV-associated neurocognitive disorder: pathogenesis and therapeutic opportunities.</a> Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology. 5 (3), 294-309 (2010).</li>
<li>Zimmermann, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CNS-targeted+production+of+IL-17A+induces+glial+activation%2C+microvascular+pathology+and+enhances+the+neuroinflammatory+response+to+systemic+endotoxemia%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">CNS-targeted production of IL-17A induces glial activation, microvascular pathology and enhances the neuroinflammatory response to systemic endotoxemia.</a> PloS one. 8 (2), e57307(2013).</li>
<li>Silva, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+central+nervous+system+expression+of+HIV-1+Tat+leads+to+accelerated+rarefaction+of+neocortical+capillaries+and+loss+of+red+blood+cell+velocity+heterogeneity%5BTitle%5D)+AND+%22Microcirculation%22%5BJournal%5D)">Chronic central nervous system expression of HIV-1 Tat leads to accelerated rarefaction of neocortical capillaries and loss of red blood cell velocity heterogeneity.</a> Microcirculation. 21 (7), 664-676 (2014).</li>
<li>Marker, D. F., Tremblay, M. E., Lu, S. M., Majewska, A. K., Gelbard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+thin-skull+window+technique+for+chronic+two-photon+in+vivo+imaging+of+murine+microglia+in+models+of+neuroinflammation%5BTitle%5D)+AND+%22Journal+of+visualized+experiments+%3A+JoVE%22%5BJournal%5D)">A thin-skull window technique for chronic two-photon in vivo imaging of murine microglia in models of neuroinflammation.</a> Journal of visualized experiments : JoVE. (43), (2010).</li>
<li>Kleinfeld, D., Mitra, P. P., Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluctuations+and+stimulus-induced+changes+in+blood+flow+observed+in+individual+capillaries+in+layers+2+through+4+of+rat+neocortex%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (26), 15741-15746 (1998).</li>
<li>Bruce-Keller, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphine+causes+rapid+increases+in+glial+activation+and+neuronal+injury+in+the+striatum+of+inducible+HIV-1+Tat+transgenic+mice%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Morphine causes rapid increases in glial activation and neuronal injury in the striatum of inducible HIV-1 Tat transgenic mice.</a> Glia. 56 (13), 1414-1427 (2008).</li>
<li>Zoumi, A., Yeh, A., Tromberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+cells+and+extracellular+matrix+in+vivo+by+using+second-harmonic+generation+and+two-photon+excited+fluorescence%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (17), 11014-11019 (2002).</li>
<li>Silva, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+hypercapnia+reveals+an+underlying+cerebrovascular+pathology+in+a+murine+model+for+HIV-1+associated+neuroinflammation%3A+role+of+NO-cGMP+signaling+and+normalization+by+inhibition+of+cyclic+nucleotide+phosphodiesterase-5%5BTitle%5D)+AND+%22Journal+of+neuroinflammation%22%5BJournal%5D)">Transient hypercapnia reveals an underlying cerebrovascular pathology in a murine model for HIV-1 associated neuroinflammation: role of NO-cGMP signaling and normalization by inhibition of cyclic nucleotide phosphodiesterase-5.</a> Journal of neuroinflammation. 9, 253(2012).</li>
<li>Farber, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Region-specific+and+agent-specific+dilation+of+intracerebral+microvessels+by+volatile+anesthetics+in+rat+brain+slices%5BTitle%5D)+AND+%22Anesthesiology%22%5BJournal%5D)">Region-specific and agent-specific dilation of intracerebral microvessels by volatile anesthetics in rat brain slices.</a> Anesthesiology. 87 (5), 1191-1198 (1997).</li>
</ol>

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...

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。

<table><tbody><tr><td>Leica Microscope</td><td>Leica Inc.</td><td>MZ8</td><td></td></tr><tr><td>High Intensity Illuminator</td><td>Dolan-Jenner</td><td>180</td><td></td></tr><tr><td>Heating Pad</td><td>Stryker&amp;nbsp;</td><td>TP3E</td><td></td></tr><tr><td>T/PUMP</td><td>Gaymar Industries, Inc.</td><td>TP-500</td><td></td></tr><tr><td>TEC-4 Isoflurane Vaporizer</td><td>Datex Ohmeda</td><td>447</td><td></td></tr><tr><td>Artificial Tear Gel</td><td>Butler AHS</td><td>7312</td><td></td></tr><tr><td>Povidone-Iodine solution&amp;nbsp;</td><td>Aplicare</td><td>52380-1855-9</td><td></td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Dumot #5 Forceps</td><td>Fine Science Tools</td><td>11295-10</td><td></td></tr><tr><td>Dumont #5/45 Forceps</td><td>Fine Science Tools</td><td>11251-35</td><td></td></tr><tr><td>Ferric Chloride Solution</td><td>Ricca Chemical Company</td><td>3120-16</td><td></td></tr><tr><td>Loctite 454 Prism Instant Adhesive Gel</td><td>Henkel</td><td>45404</td><td></td></tr><tr><td>Dental Cement</td><td>Stoelting</td><td>51459</td><td></td></tr><tr><td>Microtoruqe II Handpiece Kit</td><td>Pearson Dental</td><td>R14-0002</td><td></td></tr><tr><td>005 Burr for Micro Drill</td><td>Fine Science Tools</td><td>19007-05</td><td></td></tr><tr><td>Norland Blade (Dental Microblade)</td><td>Salvin Dental</td><td>6900</td><td></td></tr><tr><td>Urethane</td><td>Sigma-Aldrich</td><td>U2500</td><td>Group 2B Carcinogen</td></tr><tr><td>Braided Suture</td><td>Ethicon</td><td>735G</td><td></td></tr><tr><td>Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15000-03</td><td></td></tr><tr><td>&amp;nbsp;Arterial Catheter</td><td>SAI Infusion Technologies</td><td>MAC-01</td><td>The end of the catheter was manually stretched out in order to decrease its diameter.&amp;nbsp;</td></tr><tr><td>Blood Pressure Moniter</td><td>World Precision Intruments</td><td>SYS-BP1</td><td></td></tr><tr><td>Blood Pressure Transducer and Cable</td><td>World Precision Intruments</td><td>BLPR2</td><td></td></tr><tr><td>RAPIDLab Blood Gas Analyzer&amp;nbsp;</td><td>Siemens&amp;nbsp;</td><td>248</td><td></td></tr><tr><td>40 &amp;mu;l Capillary Tube</td><td>VWR</td><td>15401-413</td><td></td></tr><tr><td>Texas Red-dextran (70,000 MW, 10 mg/kg dissolved in saline)</td><td>Invitrogen</td><td>D-1830</td><td></td></tr><tr><td>Adult Mouse Brain Slicer Matrix</td><td>Zivic Instruments</td><td>BSMAS001-1</td><td></td></tr><tr><td>Olympus Fluoview 1000 AOM-MPM Multiphoton Microscope</td><td>Olypmus</td><td>FV-1000 MPE</td><td></td></tr><tr><td>MaiTai HP DeepSee Ti:Sa laser</td><td>Spectra-Physics</td><td></td><td></td></tr><tr><td>ImageJ Software</td><td>National Institutes of Health (NIH)</td><td></td><td>Available at http://rsb.info.nih.gov/ij/download.html</td></tr><tr><td>Amira Software</td><td>Visage Imaging&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

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...

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

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

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

活体鼠脑微循环显微镜使用一个封闭的颅窗口

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

医学

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

纵<em&gt;在体内</em&gt;的Cerebrovasculature影像:相关性疾病CNS

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

使用磁共振成像实验性脑疟疾模型中的水肿发育​​和微血管病理学的体内追踪

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

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 ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

免疫与感染

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

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

摘要

探索更多视频

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

活体鼠脑微循环显微镜使用一个封闭的颅窗口

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

纵

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

使用磁共振成像实验性脑疟疾模型中的水肿发育和微血管病理学的体内追踪

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

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

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

体内小鼠海马体中小胶质细胞的慢性双光子成像

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

摘要

探索更多视频

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

活体鼠脑微循环显微镜使用一个封闭的颅窗口

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

纵

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

使用磁共振成像实验性脑疟疾模型中的水肿发育​​和微血管病理学的体内追踪

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

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

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

体内 小鼠海马体中小胶质细胞的慢性双光子成像

使用磁共振成像实验性脑疟疾模型中的水肿发育和微血管病理学的体内追踪

体内小鼠海马体中小胶质细胞的慢性双光子成像