We thank the Scientific Computing and Imaging Institute of the University of Utah for use of FluoRender software (R01GM09815). This work was supported by grants from the Glaucoma Research Foundation, Melsa M. and Frank Theodore Barr Foundation and the US National Institute of Health, (R01EY020878 and R01EY023621) to M.L.V., and (R01EY017182 and R01EY017950) to B.K.A.

在活体成像使用犹他州大学批准的机构动物护理和使用委员会协议无致病菌设施进行。 注意:此成像协议用于报告小鼠中视网膜小胶质细胞和浸润的单核细胞/巨噬细胞表达的fractalkine的受体基因座（CX3CR1）的控制下，绿色荧光蛋白（GFP）。 1. 在视网膜GFP +小胶质细胞的体内成像共聚焦扫描激光眼底镜检查（CSLO） <ol><li>转水控制的加热系统，建立稳定过程中，35〜37ºC鼠标之间的温度，并连接两个加热垫。 <ol><li>启动共焦扫描激光检眼镜（CSLO）系统（ 图2A）。打开CSLO程序，输入，将确定个人鼠标设置为2毫米（患者数据菜单）角膜曲率的信息。 </li></ol></li><li>准备IMA更改。安全地装配在其插槽干净55º宽视场物镜。通过确保加热垫铺上干净的纸准备眼底成像平台。使用夹子拼合垫和纸张，并保持他们从挡住物镜的移动。 </li><li>通过腹膜内注射1.3％2,2,2-三溴乙醇和0.8％的叔戊醇麻醉小鼠（250毫克/千克体重;0.5毫升/ 25克体重），使用30½ģ针装到1毫升的一次性注射器。返回鼠标笼子里，不停地在一个加热垫。 注:在麻醉注射与吸入的交付使鼠标成像和通畅的进入眼中，免费的鼻锥体和管更容易定位。 </li><li>一旦动物会不动，是没有响应于尾捏，诱导瞳孔扩大与托品和苯（各0.5％），通过定位动物俯卧和覆盖双眼用的液滴7分钟。 </li><li> 5分钟后，对花边鼠标俯卧在眼底平台的中心，适合隐形眼镜了双眼，将最小的压力。 注:隐形眼镜小且脆弱，但可以用手指小心处理，虽然镊子可以用来代替40。使用隐形眼镜是可选的，但是推荐，因为它最大限度地减少眼干涩和成像过程中保持角膜透明。 </li><li> 7分钟后，定位鼠标与右眼面对物镜和平行于物镜的轨道，保持无节制的平台（ 图2B）的边缘附近的动物。收集可比饱和度和取向的图像，不断维持从眼睛到物镜的距离，以及眼睛的对准的光路整个成像会话。剪贴长须与观察眼睛的干扰。 注:在接下来的8-10分钟，鼠标不移动或闪烁，并会呼吸，轻轻动ments，这是由CSLO眼睛跟踪技术（自动实时）模式，其调节基于眼底标具有高对比度的扫描头的位置进行跟踪。过去的那个时候，老鼠开始喘气和眨眼，使成像不切实际的。 </li><li>如果不使用隐形眼镜进行成像，应用PBS下降到双眼每隔2-3分钟，以防止它们干燥。 </li><li>收集的视网膜血管和视盘的眼底图像，聚焦在视网膜内层。 <ol><li>选择红外模式（IR）和调整设置到820纳米的激发，100％的激光功率，40-60％的敏感性（ 图2C）。 </li><li>工作在高速模式（12.6帧/秒），使用操纵杆来驱动眼底镜定位眼球。在低放大倍数，检查角膜和晶状体用于损伤或不透明，并且从研究眼睛排除具有缺陷或损伤，将影响成像的视网膜（ 图3A）。 </li><li>定位视盘区域（表面视神经乳头，ONH）通过使目标更接近眼球，然后居中在图像上，并通过拧操纵杆锁定这个位置。眼睛的眼底的这种对齐是关键，甚至获得整个图像聚焦和发射。 </li><li>可视化的视网膜的内面，以及视盘，使用位于在视网膜上作为参考焦平面（59和60 D）中，或更深（55 D）的玻璃体表面上的主要血管中的眼睛表示挖掘视神经光盘。最佳的重点应该在解决主要血管循环血细胞，用自己的管腔和墙壁明显不同。 </li><li>优化图像饱和度调节触摸屏控制面板上的旋钮，直到均匀照明的白色光晕在大多数视盘周围的55º眼底场的跨越（使用轻微过曝）。接着，降低饱和度，以优化对比度直到血细胞沿大血管运动可以清楚地分辨。如果震源深区坚持，不能因视网膜损伤，重新调整眼相机获得均匀明亮的图像，直到。这种调整是根本捕捉重现的图像集的位置和质量。 </li><li>收集中心视网膜的高分辨率红外图像（1.4-1.7毫米直径，这取决于年龄），平均实时30帧（4.7帧/秒;归一化），以提高信噪比（ 图2D ）。 </li></ol></li><li>立即，获得的GFP +小胶质细胞和/或局部视网膜和视盘的内面浸润的单核细胞的荧光图像。 注:视网膜的红外眼底图像的优化确定的GFP +细胞的荧光图像的分辨率，以及内层视网膜的程度跨越。未能集中在视网膜上，其可通过脉管系统的分辨率差被检测的内面，将导致在调暗的GFP +细胞视图（ 图3B）。未能调整IR凡城配给正确且均匀地影响荧光图像（FA），引入在GFP +细胞的强度和大小（ 图3C）人为的变化。 <ol><li> SWICH至荧光成像模式（FA）中的触摸屏面板，使用蓝色，488 nm激光激发（460-490毫微米阻挡滤波器集合），并采集设置（100％的激光功率和100-125％的灵敏度），其被保持常数所有小鼠（ 图2E）。 </li><li>收集单一的XY点，视网膜的二维荧光图像，并立即在同一位置拍摄眼底图像（100倍的扫描平均水平; 55°扫描角度为两个图像; 图2F）。 </li><li>通过在控制面板（ 图2G）选择"复合"和移动眼底镜左，右，平移整个鼻时间轴（ 图2H）视网膜捕获图像多。 注:扫描单XY-点图像后（100X s可以平均值），该软件自动实时平均值相同，新领域（外有绿色圆圈新的扫描过程中最佳的扫描，或当扫描不可行红色），和所有的针XY-位置。所得到的复合图像跨越1.5-1.7毫米的水平轴x 3-4毫米，在垂直轴（55°x120-124º扫描角）。 </li></ol></li><li>每只小鼠在15分钟内诱导麻醉，闪烁，运动前重新启动完成后成像。 </li><li>取下隐形眼镜，眼睛水合物用PBS和动物返回到其回暖笼子，检查行为，直到蠕动完全恢复。 </li><li>对于采用间歇CSLO成像会话在同一个体小鼠的纵向研究，重复成像不少于3天开以避免麻醉的蓄积毒性，以及角膜刺激。 </li></ol> 2.实时图像处理和分析<ol><li>连续的图像对齐: <ol><li>对齐眼底图像用血管和视盘作为​​路标的时间序列相同的眼球。在商业幻灯片放映演示程序，通过拖动每幅图像比前一个，直到他们视盘对准在XY-平面（顶部图像出现半透明的同时拖动），然后旋转顶部图像，直到所有打开的图像主要血管重叠下面（集中在船只最远从视盘）的图像上的血管。记录每个图像的旋转角度和保存这个时间序列以供将来参考，跟踪鼠标和眼睛的身份。 </li><li>通过将记录角度来校正在每个时间点在xy旋转，使用一个光栅图形编辑程序对准相应系列的单的xy点的荧光图像。此外，调整分辨率为300 dpi（不缩放）和背景（在RGB模式下，选择级别和品尝血管黑色的流明）。保存这些图片为TIF文件，加入"结盟"到他们的名字。 </li></ol></li><li>胶质细胞分割: <ol><li>以确定的GFP +细胞在中心视网膜，在FluoRender开放对应于最早的时间点/年龄的"对准"的TIF文件。缩放图像以适应屏幕，然后定义渲染视图（复合材料，正交，插值）及其属性（0.58伽玛，饱和度255点，255的亮度，195α和1.00色带所示），同时选择了RG渠道可见（隐藏双击它，在工作区框架B通道; 图4A）。 </li><li>选择单个细胞，阈红色通道（必须在工作空间框架中突出显示），通过增加低阈值，直到大部分细胞被屏蔽（白色）与最小的重叠和细胞过程（青色），同时保持高的阈值恒定（255）。介于100和170中的低阈值而变化，这取决于荧光强度（ 图4B &lt;/ STRONG&gt;）。 </li><li>保存这一观点与红色通道仅可见（捕获命令）作为一种新的TIF文件，添加"cellsegm"到原来的名称（ 图4C）。使用通用光栅图形编辑软件，它的反转灰阶调整分辨率为300 dpi的上面，并重新保存为RGB（ 图4D）。 注意:删除文件夹（项目）由FluoRender自动创建，并保存备查应用门槛。 </li></ol></li><li>小胶质细胞胞体分割和形态: <ol><li>以确定的GFP +细胞胞体为区域量化，使用图像分析软件以自动强度测量和阈值的功能，以及基于阈值的对象计数和测量。打开"cellsegm"TIF文件，并​​选择重调方法（样条）和红色通道（点击RGB红色标签）。通过取消选择"彩色视图通道"提高可视化（右键单击红色的RG乙标签），并选择"补色"（图像/调整图像）反转灰阶，看到了细胞的灰色/黑色在白色背景（ 图4E）。 </li><li>校准图像以1.4-1.7毫米取决于年龄，通过绘制一条水平线整个图像（校准，连结校准）。 </li><li>段个别细胞胞体通过施加强度阈值（ 图4B）。对于这一点，在红色的RGB通道的工作，并打开阈强度函数（对象计数;选择"计数更新"命令），以便跟踪二维参数的感兴趣区域（ROI），每个阈值区域表示各个胞体。 </li><li>限定在所述强度直方图的亮度临界值，通过选择255层次的最低50-60％，并通过目视评价阈颜色掩模（蓝色）和胞体周长（灰色）之间的重叠精炼最终阈值单个细胞（Figure 4E）。 </li><li>估计胞体掩模的精度来代表常染色体的尺寸由前测量区和后分割在10个细胞，并接受所选择的阈值范围内，如果测量相差低于10％（使用的二元工具栏和注释测量值）。 </li><li>手动识别常染色体的ROI包括多个细胞，并分离这些元件（消除或分开的ROI使用二元工具栏的控制，或在对象目录）而切换接通/断开的二值直接可视化的细胞（ 图4F）。 </li><li>分类小神经胶质细胞为感兴趣区与常染色体区域且小于50-60微米2以鉴别细胞胞体对应于激活的小胶质细胞和分别停用小胶质细胞，通过使用区域限制。 注意:每个细胞常染色体子集被标识与不同的伪彩色二进制层，并且可以进一步表征为其它形态参数（PERIME之三，圆等 ），以及离散的视网膜扇区（ 图4G）内量化。所分析的图像保存为一个文件ND2。 </li><li>验证过程和分支的复杂性在个别激活小神经胶质细胞，通过覆盖在面膜常染色体和单XY -点图像（对比度提高了50％; 图4H）。手动计数从细胞胞体直接延伸的处理或测量多边形包围心轴（使用二元工具栏和注释测量值）的直径。 注:活性细胞缺乏过程或承受几（&lt;4）和短（&lt;2×常染色体直径）的人，而相比之下，非活化细胞过程，其包括一个网状和广泛乔木（&gt; 3 - 10倍常染色体直径）周围的相对小胞体。 </li><li>手动识别细胞胞体小于&lt;20微米2和/或GFP表达低于检测极限，因此这是由在未被发现张力阈值。在使用的注解命令分类计数人工识别元素。 </li></ol></li></ol>

The authors have nothing to disclose.

神经变性疾病中在线监测的小胶质细胞数目和形态的激活需要使用的非侵入性的成像方法，使细胞的功能的详细可视化。成像后，小胶质细胞，必须分离（分段），用于形态测定分析通过使用多个阈值的步骤，以评估常染色体的大小和/或过程的复杂性作为读数为小胶质细胞活化。在这个协议中，我们描述了使用CSLO实时图像采集，和小胶质细胞活化的基于序列的细胞和胞体分割的定量分析方法。?...

小胶质细胞是自早期胚胎发育和整个成年期，专门驻留在中枢神经系统（CNS）神经免疫细胞。配备有受体的复杂剧目，小胶质细胞的活性和区域的异质性是由他们的双向相互作用调节与相邻的神经元，神经胶质细胞，血-脑屏障和渗透神经炎性细胞1,2-。小胶质细胞基础功能有助于生理保养和维修，因为他们品尝其领土的动态平衡3,4扰动。在中枢神经系统损伤或疾病，小胶质细胞是第一个响应者的神经信号，然后触发他们过渡到无功表型，被称为"激活小神经胶质细胞2,5-7。小胶质细胞活化涉及基因和蛋白的表达，其耦合到细胞胞体和过程调整大小和重塑6-9的一个复杂的循环。小胶质细胞的活化，以及细胞和再分配聚类，可伴随在细胞的数量（称为小神经胶质细胞）的本地整体增加。这可能是由于细胞增殖和自我更新，有或没有招募的血液衍生的单核细胞3,4,7,10-14。在范围广泛的年龄依赖性，慢性CNS疾病，持续小神经胶质细胞和小胶质细胞活化平行疾病进展15-19。小胶质细胞是如何影响神经退行性疾病仍不清楚，主要是因为他们玩，可能有疾病发生，发展多样化的贡献都神经保护和有害的作用。实时成像旨在了解的慢性CNS疾病监测了动物模型和人类的受损中枢神经系统的小神经胶质的行为，并证明了小胶质细胞的改变是可检测的开始在早期疾病阶段15-17,19,20。因此，关键是要开发的方法来检测和监测体内小胶质细胞活化。 无创DETE在脑小胶质细胞激活区域变化ction确立为在神经变性疾病的进展体内指示器的重要，使用分子成像或生物发光和正电子发射断层扫描或磁共振成像18,21,22。这些高度量化和非侵入性的分子和核成像方法检测胶质细胞增生与区域分辨率。或者，在CX3CR1 GFP / +小鼠的双光子共焦成像已经允许大脑胶质细胞的观察与细胞分辨率3,4,9,20,23-28。然而，这种方法限制了长期和反复观察慢性小胶质改建，由下式给出甚至微创脑成像程序29干扰它们的行为的潜在风险。或者，视网膜提供了直接的最佳条件， 在体内的可视化和小胶质细胞在其完整的CNS利基整个老化重复监控，急性损伤，并潜在期间慢性神经变性疾病。因此，最近的研究证明了视网膜小胶质细胞表达GFP的高分辨率成像的可行性，通过调整共焦激光扫描眼底镜（CSLO）图像实时CX3CR1 GFP / +小鼠。这已被用于跟踪在个体小鼠长达10周的GFP +细胞数每周改变以下急性诱发损伤或高眼压30-36。 我们已经扩展这种方法来进行长期的成像历时数月，并定量跟踪的基础上利用形态分析胞体大小小胶质细胞活化的变化。常染色体的大小被定义为小胶质细胞活化在实时成像研究使用皮质切片双光子激光共聚焦显微镜 CX3CR1-GFP +小胶质9 体内成像执行有用的指标。这些和其他的研究也证实常染色体大小和Iba1蛋白表达水平之间的相关性，的wh脑出血也随着活化9,37。因此，活化的小胶质细胞可在活体小鼠来鉴定，并且它们的数量和分配过程中的CNS健康和疾病随时间监测。 这个协议描述为CSLO实时图像采集和分析方法，监测小胶质细胞的数量，视网膜神经节细胞（RGC）变性（ 图1）中的分布和形态的活化。因此，本研究的用途:1）遗传性青光眼（DBA / 2J），该发生年龄依赖性视神经和视网膜神经变性和小鼠模型显示5和10个月38,39年龄之间显着的可变性疾病进展，2）每月CSLO 体内成像的GFP +细胞在视网膜长期可视化和杂CX3CR1 GFP / + DBA / 2J小鼠的年龄3-5个月，3）由分段和阈值实时成像分析以分离细胞胞体和测量无髓鞘视神经该IR区。施加这些策略在慢性青光眼的早期阶段，以评估视网膜小胶质细胞激活状态的动力学。

<table><tbody><tr><td>DBA/2J and CX3CR1-GFP/+ mice</td><td>The Jackson Laboratory, Bar Harbor, ME</td><td>000671 and 00582</td><td>Mice are bred in house, introducing new breeders every 3 - 4 generations to prevent genetic drift.</td></tr><tr><td>30&amp;frac12; G needle and 1 ml tuberculin slip-tip syringe</td><td>BD, East Rutherford, NJ</td><td>305106 and 309659</td><td></td></tr><tr><td>2,2,2-tribromoethanol, tert-Amyl alcohol 99% and phosphate buffer saline tablets</td><td>Sigma-Aldrich, St. Louis, MO</td><td>T48402, 152463 and P4417</td><td>Avertin solution (pH 7.3, sterile filtered) must be freshly made solution or stored at 4 &amp;deg;C for up to 1 week.</td></tr><tr><td>Heat therapy T/Pump</td><td>Gaymar Industries, Orchard Park, NY</td><td>TP-650</td><td></td></tr><tr><td>Cotton-tipped applicators</td><td>Fisher Scientific, Pittsburgh, PA</td><td>23-400-100</td><td></td></tr><tr><td>Tropicamide 1%</td><td>Bausch &amp;amp; Lomb, Rochester, NY</td><td>NDC 24208-585-64</td><td></td></tr><tr><td>PMMA contact lenses, 1.70 base curve, 3.2 mm total diameter, 0.40 mm thick center</td><td>Cantor &amp;amp; Nissel Ltd., Northamptonshire, UK</td><td>G003709</td><td>Lenses are rinsed with sterile PBS and stored in polypropylene boxes.</td></tr><tr><td>HRA/Spectralis confocal scanning laser ophthalmoscope and Eye Explorer software</td><td>Heidelberg Engineering GmbH, Carlsbad, CA</td><td>Version 1.7.1.0</td><td></td></tr><tr><td>PowerPoint</td><td>Microsoft, Redmond, WA</td><td>Version 14.4.3</td><td></td></tr><tr><td>Adobe Photoshop</td><td>Adobe, San Jose, CA</td><td>Version CS3</td><td></td></tr><tr><td>FluoRender</td><td>Scientific Computing and Imaging Institute, University of Utah</td><td>Version 2.13</td><td>Freeware. &lt;a href=&quot;http://www.sci.utah.edu/software/13-software/127-fluorender.html&quot; target=&quot;_blank&quot;&gt;http://www.sci.utah.edu/software/13-software/127-fluorender.html&lt;/a&gt;</td></tr><tr><td>NIS-Elements C</td><td>Nikon, Melville, NY</td><td>Version 4.30.01</td><td></td></tr></tbody></table>

in vivo dynamics of retinal microglial activation during neurodegeneration: confocal ophthalmoscopic imaging and cell morphometry in mouse glaucoma

小胶质细胞，这是CNS驻地神经免疫细胞，改变其形态和大小响应中枢神经系统损伤，切换到激活状态具有不同的功能和基因表达谱。在健康，损伤和疾病胶质细胞活化的作用仍然不完全是由于他们的动态和复杂的调节反应的变化在他们的微环境的理解。因此，它是对非侵入性地监控关键和分析在完整生物体随时间的变化小胶质细胞活化。在体内胶质细胞活化的研究已经被推迟了技术限制，以跟踪小胶质细胞的行为没有改变中枢神经系统环境。这已经在慢性神经变性，在那里长期变化必须跟踪是特别具有挑战性的。视网膜，中枢神经系统器官适合于非侵入性的实时成像，提供了强大的系统，以可视化和在慢性病症表征小胶质细胞活化的动力学。 在体内成像的视网膜小胶质细胞，使用共聚焦检眼镜（CSLO）和CX3CR1 GFP / +报告小鼠，以可视化的小胶质细胞与细胞的分辨率的方法。此外，我们描述的方法来量化细胞活化和密度大细胞亚群每月改变（每视网膜200-300个细胞）。我们确认使用常染色体区域的作为用于在视网膜小胶质细胞活化的活跟踪一个有用的度量通过施加在体内图像的自动阈值基于形态测定分析。我们使用这些实时图像采集和分析的策略中的视网膜神经变性的慢性青光眼的小鼠模型的早期阶段，以监测小胶质细胞活化和小神经胶质细胞的动态变化。这种方法应该是有用，调查小胶质细胞在慢性中枢神经系统疾病，影响视网膜和视神经的神经元和轴突下降的贡献。

我们最近在体内的研究中使用这些实时图像采集和分析方法，以可视化，并在慢性青光眼和他们的关系的早期阶段跟踪动力学和视盘和视网膜小胶质细胞的变化模式来晚了神经退行性疾病59。在这里，我们示出了CSLO图像采集协议跨越大面积在个别年轻杂CX3CR1-GFP的DBA / 2J视网膜中央视网膜（ 图5）的可视化的小神经胶质细胞。基于高元分辨率，我们应用实时图像阈值和形?...

Watch this Scientific Journal Video about In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma at JoVE.co

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

小胶质细胞活化和小神经胶质细胞是关键应对慢性神经退行性病变。这里，我们提出在体内 ，视网膜CX3CR1-GFP +胶质细胞共聚焦检眼镜的长期可视化方法，以及用于阈和形态分析，以确定和量化它们的活化。我们监测期间与年龄有关的青光眼的早期阶段小胶质细胞的变化。

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

in-vivo-dynamics-retinal-microglial-activation-during

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

10.5K 次观看.  University of Utah. 小胶质细胞活化和小神经胶质细胞是关键应对慢性神经退行性病变。这里，我们提出在体内 ，视网膜CX3CR1-GFP +胶质细胞共聚焦检眼镜的长期可视化方法，以及用于阈和形态分析，以确定和量化它们的活化。我们监测期间与年龄有关的青光眼的早期阶段小胶质细胞的变化。 

视频: In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

激光诱导的慢性高眼压表征视觉缺陷的小鼠模型

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

科研

医学

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

免疫与感染

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

A磁条微珠缺血模型，以诱导小鼠高眼压，青光眼相关

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

青光眼诱导在程序<em&gt;在体内</em&gt;大鼠模型和全安装视网膜准备

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

神经科学

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

全鼠眼的消化，用于单核噬细胞的多参数流细胞学分析

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

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