This work was supported by a Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (T.I.) and Grants-in-Aid for Scientific Research (C) from MEXT (S.T. and K.N).

该协议和使用实验动物被批准的机构动物护理和使用委员会在山梨和早稻田大学的大学。按照机构准则进行动物护理。 1. CPEC准备<ol><li>准备下列设备和材料:立体显微镜，最好是能够发送从底部照明;一对钟表匠钳（杜蒙＃3＃或4），火焰消毒，操作直剪，火焰消毒;疏导10厘米塑料盘含有20ml冰冷的Leibovitz L-15培养基; 35毫米玻璃底菜含2ml RT莱博维茨的L-15介质;的100毫升烧杯中含有70％的乙醇;新生小鼠的幼崽。 </li><li>简要地浸入一个新生小鼠在70％乙醇，并使用所述操作剪刀断头很快安乐死。 </li><li>在冰冷的Leibovitz L-15培养基，立即将头在无菌10厘米二SH。 </li><li>除去从使用一对钟表制造商镊子的颅盖的皮肤，剖开颅骨以暴露脑，然后切颅神经以分离全脑。 </li><li>脑转移到含有冰冷的Leibovitz L-15培养基的新菜和立体显微镜下观察，确保大脑完全浸入在介质中。 </li><li>设置大脑朝上背侧，并定向使大脑嗅球驻留在三点钟位置（右旋人）。与在左手镊子轻轻握住大脑。 </li><li>使用细镊子剥离（杜蒙＃3＃或4）在右手，切断胼胝体和实质下方连接大脑半球，沿大脑纵裂。 </li><li>轻轻大脑半球推到侧面和揭露横向脑裂。 </li><li>按捏出来个分离半球半球和丘脑两地电子实质。 </li><li>轻轻拉出附加到海马的侧面通过椎板affixa的侧脑室的脉络丛。 </li><li>转移孤立脉络丛含有新鲜莱博维茨L-15中型35毫米的玻璃底菜，并覆盖重量（材料清单）轻轻地握住组织到位。 </li></ol> 2.现场CPEC纤毛成像<ol><li>确认正确的紫外线（UV）和推断（IR）截止滤光器（S）以阻挡光线于400nm和大于700纳米，且该中性密度（ND）滤波器（25％和6％）短插入到光路倒置显微镜。 </li><li>调整物镜的焦点大致由眼睛，然后调整冷凝器，使得中心和聚焦，以符合科勒照明。插入一个适当的微分干涉对比（DIC）棱镜，DIC元件，以及在立分析器和偏振元件向右路径以符合DIC的光学。 </li><li>调整的视图由DIC棱镜位置的对比度，以使组织表面的结构是最容易识别的。如果目标小区的所有纤毛是能动的，不能由眼睛，因为它们的运动获得的运动纤毛的清晰视图。 </li><li>改变光路的摄像头，并删除ND滤镜增加了光功率。 </li><li>使用相机对焦模式调整的观点和重点领域。在重点，其中视频图像显示在监视器上，在实时性，运动纤毛的清晰视图不可用。 </li><li>使用相机以200Hz为0.1毫秒的所需时间（数秒至数分钟）的曝光时间。采集图像堆栈后，单框会显示清晰的纤毛结构。如果纤毛边缘模糊，提高帧速率或使用更短的曝光时间。 </li><li>在安乐死后，莱博维茨在25-60分钟记录CPEC纤毛的运动L-15培养基。 </li></ol>睫状运动的三分析<ol><li>手动跟踪每一个跳动的纤毛模式的电脑显示器上。在每一帧使用鼠标指针，这是组装每个纤毛的轨迹信息标记纤毛尖部位置。无论分析使用相同的软件或导出到用于进一步分析其他更普遍应用的轨迹信息。该分析步骤的效率在讨论中描述。 </li><li>分类轨迹为两种模式的运动，背面的往​​复或旋转，通过眼睛。 </li><li>使用下列公式计算睫状跳动频率（CBF）:[CBF =（每秒帧的数目）/（帧为单次搏动的平均数目）] 14，它可以从一个纤毛尖端运动图得到（ 图3 ）。重复此计算为多个纤毛打浆周期，因为在同一单元中的其他纤毛可与每个ciliu的运动干涉米，从而导致不​​均匀性。 </li><li>分析睫状单个细胞内跳动轴的角均匀性，定义为每条轨迹（ 图4）的打浆角度θ。对背的往复轨迹，适合纤毛尖的位置，以直线，并定义θ为线使得与x轴的角度。对于旋转轨迹，适合的位置，以椭圆形，并定义θ为椭圆的长轴使得与x轴的角度。配件的细节在代表结果部分所述。 </li><li>对于每一个轨迹的定量描述，计算广义长宽比AR。简言之，旋转轨迹由- θ和定义的AR作为沿x中的分布的宽度之间的比例-和y -axes（ 图4B）。细节显示在代表结果部分，和解释意义，并且参数的限制将在讨论中描述。 </li></ol> 4.样品制备SEM 注:SEM是评价以全面的方式纤毛上CPECs地位的重要手段。为了制作标本的SEM，一个标准程序之前报道15，使用了轻微的修改。 <ol><li>之前，从脑的解剖组织，在准备用聚乙烯帽5毫升的玻璃小瓶的固定液。固定剂由2％多聚甲醛，2.5％戊二醛（半Karnovsky氏溶液16）的0.1M磷酸盐缓冲液，pH 7.4中的。 </li><li>解剖出从脑组织如步骤1所述。 </li><li>简要地冲洗以在一个新的培养皿Hank氏平衡盐溶液（HBSS）中分离的组织，然后固定在玻璃小瓶中的固定剂的组织进行1小时，在室温下进行。使用一次性移液管和处理标本GEntly。漂洗在HBSS之后，将组织变得粘稠。 <ol><li>到试样转移到固定剂溶液，慢慢排出少量含有从移液管的组织作为液滴溶液，并加入到固定剂。 </li></ol></li><li>固定后，弃去固定剂和冲洗用磷酸盐缓冲液三次组织。 </li><li>浸入所述组织在10％蔗糖溶液洗出剩余的醛。以确保完全消除醛类，浸入样品溶液中的10分钟，然后用新鲜的10％蔗糖重复两次。这个步骤是重要的，以实现正确的后固定在随后的步骤。 </li><li>浸入所述组织中的1％四氧化锇在磷酸盐缓冲液进行30分钟的溶液中，然后放置在冰上后固定。判断osmification由样本的颜色的程度:当醛完全除去，所述样品是黑色的。 </li><li>双distille广泛洗净后固定的组织样本ð水数次。 </li><li>脱水通过浸入样品中的乙醇梯度浓度，通常为65％，75％，85％，95％，99％和100％，每10分钟。通过将分子筛到99.5％的乙醇从一个新购买的瓶子得到无水乙醇。用无水乙醇三次重复脱水。 </li><li>将脱水的样品成异戊酯，取代试剂为临界点干燥，10分钟。重复此步骤两次。该试剂迅速蒸发和样品会变得干燥，导致破坏表面张力。因此，不完全干燥的样品。 </li><li>乙酸异戊酯的最后交换后，除去大部分溶剂后，立即用铝箔包装的开口的玻璃小瓶中，并放置在干冰上的小瓶中。使用针或细镊子，使几个孔在箔覆盖小瓶的口，以使液态二氧化碳容易流动到在临界点干燥器小瓶。继续进行下一步骤尽快。 </li><li>在此步骤中，最小化的乙酸异戊酯的夹带进入干燥器的腔室，但不要让样品完全干燥临界点干燥前。此外，不要离开干冰的样本不必要长的时间，以避免霜冻对小瓶的形成。 </li><li>传输含有组织样品箔包裹的玻璃小瓶进入临界点干燥器，确保组织的表面结构保持完好，同时除去水中所含的组织。详细信息上操作的临界点干燥器可从制造商的说明来获得。 </li><li>仔细用牙签，以尽量减少机械损伤处理样品。所得干燥的组织样品是易碎。安装在使用离子溅射金属存根和外套钯金的样品。 </li></ol> 5.观察SEM <ol><li>通过SEM观察，并用数码相机记录影像配到扫描电子显微镜。 </li><li>数字图像数据传输到PC进行分析。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。本文旨在报告的详细方法，观察纤毛的蠕动孤立脉络丛组织。科学新奇已经报道在以往的研究1,8。

这种方法的观点虽然此处所描述的技术并没有提供纤毛比以前公开的方法的更详细的分析，该技术的意义驻留在系统和成本效益，可以很容易地应用于筛选任何类型的纤毛运动离体的简单性。特别是，TI Workbench提供了一个简单和友好的用户界面，使研究人员观察和更容易地分析纤毛蠕动。有效的自动跟踪方法还没有被开发用于低对比度的物体，如在视频增强对比度的DIC未染色纤毛。虽然跟踪纤毛运动的方法是手工在该技术中，努力在睫状运动跟踪分析的量合适地降低与使用通用的软件包进行图像分析比较。 解剖样本为了准备êx体内CPEC样品，快速和温和的操作是必要的，以保持组织的生存力，这确保的纤毛体外的蠕动。因为污染红细胞模糊的观测领域，用磷酸盐缓冲盐水的解剖组织的温和但复杂的漂洗强烈建议。避免出血，必须小心，以避免撕裂后脉络膜动脉，可以被标识为附着到薄层affixa胶体丛的一个突出微红螺纹状结构。 显微镜倒置显微镜配备有DIC的光学和高功率传输光源是用于此方法是必不可少的。因为汞灯通常不能频繁接通和断开，在上述灯壳体的前方快门是必要的。手动或电动快门可以用于此目的。为了保护标本和观察者的眼睛FR嗡UV和IR光，滤光器是必需的，以允许仅可见光（400-700带通或紫外线和红外线截止滤光器的组合）。 ND滤镜也是必要的改变有所不同，根据情况，光功率:由眼监控，重点用摄像机，高速延时录制。的物镜具有高的数值孔径是必要的分辨率高。有盖玻片的顶部和因为组织的形态和厚度的焦点之间有一段距离。因此，水浸没透镜而不是油浸镜头优选为更好的图像质量。隔振还需要得到稳定的图像栈。空气自卸型表是用于这一目的是有用的，但在显微镜的通过插入网球以稳定的表也可以工作在显微镜基部下更简单的缓冲。 摄像机 CCD相机能够以LEAST 200帧/秒是必要跟踪纤毛的运动。在过去的十年中，CCD摄像机具有比标准视频速率（25-30赫兹）更快的帧速率变得可用低得多的成本。能够缩短暴露时间比帧间隔相机模型是有用的，以减少由于模糊到纤毛的运动，而不增加数据量。 0.1毫秒的曝光时间和一个5毫秒的帧间隔（200赫兹）是最低条件，以获得CPECs具有足够质量的睫状图像。 软件跟踪每个纤毛的运动的分析是耗时的，特别是当用于图像分析的通用软件包用于跟踪每个纤毛小费。减少的重复的任务，如鼠标单击显示器上的睫状尖端位置的步骤，选择一个菜单来记录点击位置，并点击一个按钮移动到下一个图像帧，结果在一个显著重duction的时间和精力。在这项研究中，一个特殊的常规加入到定制的TI工作台软件。在TI工作台软件的睫状跟踪模式，用户不断循环以下简单的两步操作:移动鼠标指针到睫状尖端和按箭头键在键盘上前进的框架，它不需要移动鼠标指针或用户的眼睛转向（向菜单和按钮在计算机屏幕上）从计算机屏幕上的睫状提示。软件跟踪睫状尖端位置，显示记录的轨迹，以帮助用户，并创建用于进一步分析纤毛运动信息的表。最通用的软件进行图像分析允许编程宏的这种重复的任务批处理。这样的自定义编程，以减少重复的步骤，强烈推荐。 轨迹分析在trajectori提取西文进行了使用了定制的TI工作台软件，如上所述。相对简单的分析和图像处理也进行了使用相同的软件。拟合分析，每个纤毛尖端的轨迹转移至伊戈尔Pro软件。其它先进的分析软件如MATLAB也可以用于此目的。 在目前的研究中，由于在分类的轨迹成背面的往复或旋转轨迹的困难，该轨迹是在以盲方式，这是由另一观察者确认，导致统一的分类分类眼。然而，更严格的数学措施进行分类的轨迹没有任何可能的随意性将是可取的。椭圆拟合的轨迹似乎是理想的，这可能量化为在这两个回的往复和旋转运动的拟合椭圆（）的长宽比椭圆的程度。然而，使用纵横比的分布以限定用于分类的阈值的尝试是无效的。经常失败回往复的轨迹拟合，参数经常没有收敛，而且收敛拟合显然虚幻。因此，椭圆拟合未获通过自动分类的轨迹或量化每一个轨迹的椭圆。嵌合进行提取的旋转运动技巧作为拟合椭圆的长轴的跳动方向。 在目前的研究中，AR提出量化，一个轨迹由一个理想的线性回的往复运动偏离的程度。的参数是分布宽度的比率在垂直于跳动方向沿的方向。的值应该为0时的轨迹是一条直线，和一个当该轨迹是一个圆，类似上述的椭圆的高宽比。在实践中，获得的AR值可以是通过转动轨迹相对容易，使得跳动方向平行于x轴（ 图4B）。这种量化会导致相当不同值的运动的两种模式，提示的潜在用途的参数不仅是量化每个轨迹的特性，而且要更严格的轨迹进行分类。但是，应该指出的是，跳动方向本身已经是明显的拟合过程在当前方法的两种模式的结果。 分析进一步的改进，如更先进的拟合算法，可以解决可能随意性的问题。 关于轨迹的椭圆率的一方面是，每个CPEC纤毛的打浆可以不发生在那是严格垂直于x的平面- ÿ </EM&gt;的观察平面。事实上，SEM成像的结果暗示，CPEC纤毛往往倾斜，并且倾斜可能导致明显的椭圆率或轨迹的各向异性。 为SEM准备在准备用于SEM观察，有几个重要的点，以确保采集的高质量的图像。第一，后固定由四氧化锇必须小心进行，包括预洗涤，用10％的蔗糖溶液。如果没有适当的固定由osmification过程，随后的步骤不能维持或恢复样品的条件，导致制剂的质量较低。因为醛固定剂是还原剂，检体中的任何剩余的醛抑制四氧化锇，强氧化性试剂的活性。为了确保消除多余的标本醛，仔细清洗用蔗糖溶液势在必行。在次优PO案件ST-固定，试样的颜色不改变从褐色至黑色。第二，四氧化锇的量必须至少为50倍大于该样品的厚度。否则，足以固定不能期待。第三，为了避免试样完全干燥，特别是在脱水过程，它采用高纯度的乙醇和乙酸异戊酯是重要的。标本干燥破坏精细的表面结构，如纤毛，导致较差的图像。

Cilia are hair-like projections on the surface of most vertebrate cells, which have attracted attention by medical researchers because of a class of diseases termed ciliopathies2&#8211;4. Despite the ubiquitous expression of the organelle, a wide variety of ciliary functions have been reported, including motility and biosensing. For example, motile cilia on the mucoepithelial surface transport mucus5 and epithelial debris to the outlet of tracts, thereby preventing disease by clearing the surface of epithelia. Moreover, during early developmental periods and embryonic stages, cilia regulate the proliferation of stem cells6, and are involved in the determination of left&#8211;right asymmetry of the vertebrate body7. 
Choroid plexus epithelial cells (CPECs) are derivatives of neuroepithelial cells that cover the surface of the choroid plexus tissue in the brain, which play important roles in maintaining homeostasis of the intracranial environment by production of cerebrospinal fluid (CSF). It has been previously demonstrated that CPECs have multiple non-motile cilia that regulate the production of CSF through G-protein-coupled receptors that are specifically concentrated on the cilia8. Although these cilia had been regarded as quiescent non-motile cilia, it was discovered that some CPEC cilia exhibit transient motility during the neonatal period1. This finding was quite important because it revealed that so-called non-motile cilia are not necessarily immotile from the beginning of development and might display transient motility during specific time windows, possibly in response to specific physiological demands and functions9. To precisely describe the motile nature of CPEC cilia, it is necessary to develop an ex vivo observation system that encompasses analysis of the kinetic profiles unique to CPEC cilia. 
With respect to motility, although several technical reports have described observations of the motile cilia of the tracheal epithelium5,10, motile single-cell flagella11, so-called conventional motile cilia12, and nodal cilia13, detailed analytical methods applicable to relatively undulated structures such as the choroid plexus have not been well documented so far. Moreover, a high time resolution is required to analyze the ciliary movement of CPECs, in which expensive high-speed cameras are indispensable. To circumvent this necessity and simplify monitoring the ciliary motility of various cell types, a low cost, high-speed camera has been introduced, and an easily accessible and convenient method to record the motility of motile cilia, especially to describe the speed and pattern of motion of each cilium, has been developed1. Moreover, original image analysis software &#8220;TI Workbench&#8221; has been used here to facilitate detailed analysis of motility. Collectively, this method provides a new concise strategy to analyze ciliary motion together with correlative scanning electron microscopy (SEM), which can be adopted in a wide range of cilium research.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Required reagents&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;for live cell imaging&lt;/strong&gt;</td></tr><tr><td>ethanol</td><td>Wako Chemicals</td><td>057-00456</td><td></td></tr><tr><td>Leibovitz L-15 medium</td><td>Life Technologies</td><td>11415-064</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;for SEM preparation&lt;/strong&gt;</td></tr><tr><td>ethanol</td><td>Wako Chemicals</td><td>057-00456</td><td></td></tr><tr><td>Hank&#039;s balanced salt solution</td><td>Life Technologies</td><td>14170112</td><td></td></tr><tr><td>paraformaldehyde&amp;nbsp;</td><td>Merck</td><td>1040051000</td><td></td></tr><tr><td>glutaraldehyde&amp;nbsp;</td><td>Nacalai tesque</td><td>17003-05</td><td></td></tr><tr><td>isoamyl acetate</td><td>Nacalai tesque</td><td>02710-95</td><td></td></tr><tr><td>Molecular Sieves 4A 1/8&amp;nbsp;</td><td>Wako Chemicals</td><td>130-08655</td><td>for preparation of anhydrous ethanol</td></tr><tr><td>phosphate buffer saline (PBS)</td><td>Sigma-Aldrich</td><td>D1408</td><td></td></tr><tr><td>phosphate buffer, 0.1 M</td><td></td><td></td><td>To make 100 ml, mix 19.0 ml of 0.1&amp;nbsp;M NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; and 81.0 ml of 0.1&amp;nbsp;M Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>monosodium phosphate (dihydrate)</td><td>Nacalai tesque</td><td>31718-15</td><td></td></tr><tr><td>disodium phosphate (anhydrous)</td><td>Nacalai tesque</td><td>31801-05</td><td></td></tr><tr><td>suclose</td><td>Nacalai tesque</td><td>30406-25</td><td></td></tr><tr><td>osmium tetroxide</td><td>Nisshin EM</td><td>300</td><td></td></tr><tr><td>dry ice</td><td></td><td></td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;Tools and materials for dissection&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;for both live imaging and SEM preparation&lt;/strong&gt;</td></tr><tr><td>stereo microscope</td><td>Olympus</td><td>SZX7</td><td></td></tr><tr><td>flat paper towel</td><td></td><td></td><td></td></tr><tr><td>&amp;Phi;10 cm plastic dish</td><td></td><td></td><td></td></tr><tr><td>100 ml beaker</td><td></td><td></td><td></td></tr><tr><td>straight operating scissors</td><td>Sansyo</td><td>S-2B</td><td></td></tr><tr><td>watchmaker forceps</td><td>Dumont</td><td>No.DU-3 or -4, INOX</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;for live cell imaging&lt;/strong&gt;</td></tr><tr><td>glass bottom dish</td><td>Matsunami Glass</td><td>D110300</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;for SEM preparation&lt;/strong&gt;</td></tr><tr><td>alminum foil</td><td></td><td></td><td></td></tr><tr><td>5 ml glass vial with a polyethylene cap</td><td>Nichiden Rika-Glass</td><td>PS-5A</td><td></td></tr><tr><td>transfer pipette</td><td>Samco Scientific</td><td>SM251-1S</td><td>for specimen tranfer</td></tr><tr><td>toothpick</td><td></td><td></td><td>for specimen transfer</td></tr><tr><td>ion sputter with gold-palladium</td><td>Hitachi</td><td>E-1030</td><td></td></tr><tr><td>critical point dryer</td><td>Hitachi</td><td>HCP-2</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;Microscopic equipment and materials&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;for live cell imaging&lt;/strong&gt;</td></tr><tr><td>inverted microscope</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>100 W mercury lump housing and power supply</td><td>Olympus</td><td>U-ULH and BH2-RFL-T3</td><td></td></tr><tr><td>100 W mercury lamp</td><td>Ushio</td><td>USH103D</td><td></td></tr><tr><td>DIC condenser, n.a. 0.55</td><td>Olympus</td><td>IX-LWUCD</td><td></td></tr><tr><td>electrrical shutter</td><td>Vincent Associates</td><td>VS35S22M1R3-24 and VMM-D1</td><td>manual shutter can be used.</td></tr><tr><td>band-pass filter (400-700&amp;nbsp;nm, &amp;Phi;45&amp;nbsp;mm)</td><td>Koshin Kagaku</td><td>C10-110621-1</td><td></td></tr><tr><td>ND filter (&amp;Phi;45 mm)</td><td>Olympus</td><td>45ND6, 45ND25</td><td>combination of 25% and 6% ND filters are used</td></tr><tr><td>objective lens (water immersion) with DIC element</td><td>Olympus</td><td>UApo 40XW/340, n.a., 1.15 with IX-DPAO40</td><td></td></tr><tr><td>high-speed video camera</td><td>Allied Vision Technologies</td><td>GE680</td><td>&amp;#8805;200 Hz frame rate and 1 msec expose time</td></tr><tr><td>image acquisition / analysis software</td><td>in-hous software</td><td>TI Workbench</td><td>capable of acquisition at high frame rates.</td></tr><tr><td>PC for camera control / analysis</td><td>Apple</td><td>Mac Pro</td><td></td></tr><tr><td>vibration isolation table</td><td>Meiritsu Seiki</td><td>AD0806</td><td></td></tr><tr><td>weight for tissue</td><td>Warner Instruments</td><td>slice anchor kits</td><td>It can be made with nylon mesh glued to a U-shape squashed &amp;Phi;0.5&amp;nbsp;mm platinum wire.</td></tr><tr><td>cover glass (alternative weight for tissue)</td><td>Matsunami</td><td>made-to-order</td><td>A cover glass can be used as a tissue weight.</td></tr><tr><td colspan="4">&lt;strong&gt;for SEM&lt;/strong&gt;</td></tr><tr><td>inverted microscope</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>scanning electron microscope</td><td>JEOL</td><td>JSM-6510</td><td></td></tr></tbody></table>

observation of the ciliary movement of choroid plexus epithelial cells ex vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

工作流程的概述示于图1，包括设备的图像。 CPECs活运动观测 电影1示出了电影CPECs从围产期小鼠中分离的，和电影2示出了在电影1的图像的放大图。应当注意的是，个人睫状提示是在与那些在电影相比静像不太清楚。 图2示出了跟踪两个纤毛与不同模式的运动，背面的往复运动和旋转运动，从在视频1和2中所示的数据的运动。 图3示出了纤毛的运动轨迹的一个简单的分析，其中，所述相位差在两个坐标是显而易见的，在旋转运动。 CPEC纤毛尖轨迹分析德<html>尾的方法进行定量分析各轨迹示于图4与跳动的角度θ定义（A）和分析的代表性结果（B）的流程图的示意图。 定义为背面的往复轨迹跳动角度θ，适合睫状尖端的过程中多次循环的位置的直线Y =斧+ b和定义的跳动方向沿拟合线。定义角度跳动这里θ=反正切一个 。因为线往往拟合失败中提取旋转运动的情况下，方向，适合的旋转轨迹到椭圆形，并限定该方向与沿所述拟合椭圆的长轴。更精确地说，尖部位置（X，Y）中的多个打浆周期被安装到下面的功能。 1.JPG"WIDTH ="310"/&gt; 哪里 <img alt="公式（2）" fo:content-width="2in" src="/files/ftp_upload/52991/52991eq2.JPG" width="200" />椭圆拟合包括五个拟合参数:X 0，Y 0，A，B，θ。在x -和椭圆的中心的ÿ-spatial坐标是x 0和y 0分别。椭圆的长轴和短轴半径a和 b，分别。角度θ是角度，主要的轴与x轴，或打浆角度。这些参数的示意图示于图4A的右侧面板。嵌合是使用伊戈尔Pro软件通过限定上述的显函数。然后，击败角θ为所有纤毛的直方图观察d。在相同的细胞被绘制为圆形的直方图（ 图2中成田等人 8）。频率归一化到所分析睫状提示的数目。应当指出的是，每个纤毛被解释为具有两个θ值在圆形直方图， 例如 π/ 4和3π/ 4，导致方向的对称分布。 θ（ 图4B） -定义广义纵横比AR，睫状尖端的过程中多次循环的位置由旋转。根据旋转，既回的往复和旋转轨迹成为分布式大致平行于x轴，其中分布A和B的宽度，沿X轴和Y轴分别被定义为一半的的最小值和最大值之间的差。因此，这些参数是更相关的椭圆，a和 b的主要和次要的半径。AR被定义为与由AR = B / A A之间的比B。 脉络丛上皮细胞通过SEM观察 SEM可观察到的标本精细的表面结构。纤毛上CPECs确认存在整个发育时间过程。多纤毛上CPECs的出现是由胚胎天（E）13观察，不久后脉络丛发展开始于各地E11侧脑室。在这个阶段中，细胞是小的可变未成熟的外观，和睫状尖端指向各个方向。在E15，多个纤毛的刷毛束是建立在顶面的顶部，它们是从周围的微绒毛突出。在产后天（P）2，很多纤毛固定在弯曲的位置，反映了实时成像观察到的纤毛蠕动。在P14，大多数细胞具有多纤毛是不动的，以及微绒毛W¯¯<html>第i较细纹理与早期阶段进行比较。 图5显示CPECs的SEM图像与纤毛在这些阶段。基于图1和电影1所示的电影，很明显，运动纤毛通过实时成像容易识别。然而，这是难以通过DIC显微镜来区分非运动纤毛。因此，SEM是必不可少的理解纤毛的整体状态。 <img alt="图1" src="/files/ftp_upload/52991/52991fig1.jpg" /> 图1:工作流的概述 （ 一 ）立体声显微镜。 （二）倒置显微镜配备一个电荷耦合器件（CCD）相机。在分析过程中（C）电脑屏幕上。 （四）离子溅射。 （五 ）为样本SEM。 （六）扫描电子显微镜。 电影1。PLOAD / 52991 / Figure_2.mov"目标="_空白"&gt; CPECs的俯视图，从P2鼠标小狗在高速视频显微镜纤毛的电影。由于脉络丛组织的不规则表面，这种含有快照。无论是在聚焦和外的聚焦平面中的框的区域被扩大，在电影2比例尺所示:2微米。 电影2 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52991/Figure_3.mov" target="_blank">电影组织样品的电影1。扩大鉴于</a>纤毛跳动既表现出旋转或背的往复运动。比例尺:1微米。 <img alt="图4" src="/files/ftp_upload/52991/52991fig4.jpg" /> 图2:CPEC纤毛从电影2时间推移图象的轨迹的重构 （A） 的纤毛与背面的往复运动（上）和旋转运动（下）。 DIC<html>从电影数据图像呈现在50毫秒的时间间隔（1-8，比例尺:1微米）。目标睫状尖端的位置由箭头表示。 （B）中的轨迹（虚线）和在图像显示睫状提示（彩色圆圈）的位置重叠。 <img alt="图5" src="/files/ftp_upload/52991/52991fig5.jpg" /> 图3:两种模式从动画数据重构CPEC纤毛尖端运动的代表性睫状前端的动作，后面的往复运动（A）和旋转运动（B）中 ，在多个周期表示为轨迹（顶部，比例尺:0.5微米）。 x轴和前端位置的y坐标的时间进程作图，分别为（中间）。为了证明这两个坐标之间的相移，x坐标和y坐标是归一化到[1,1]和重叠（底部，红色和蓝色线条，分别）。 igure 6"SRC ="/文件/ ftp_upload / 52991 / 52991fig6.jpg"/&gt; 图4:示意图描述纤毛尖轨迹的分析背部和往复（左图）和旋转（右图）的轨迹跳动角度θ（A）的定义。描述拟合椭圆，A，B中，x 为0，且y 0参数式中，示出在右侧面板。 （B）的流程图的轨迹的拟合分析来定义打浆角度θ和AR。代表轨迹的拟合的实际结果显示在右侧面板。 <img alt="图7" src="/files/ftp_upload/52991/52991fig7.jpg" /> 图5:纤毛的SEM图像。 CPECs在E13，E15，P2和P14的代表性SEM显微照片。在时间过程中，CPECs尺寸增加，并且开发microviLLI和纤毛的顶面。纤毛的E13一簇尚未获得动力。在P2处，当纤毛与运动的比例达到最高点时，许多弯曲纤毛得到遵守。在P14，纤毛失去活力。比例尺:5微米。

Watch this Scientific Journal Video about Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo at JoVE.com

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

脉络丛上皮细胞的纤毛运动的观察<em&gt;离体</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

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Neuroscience

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

12.1K 次观看.  Waseda University. In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

视频: Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

科研

发育生物学

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

神经科学

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

在啮齿类动物呼吸道上皮纤毛运动功能的高分辨率成像的体外方法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

生物学

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

小鸡西里神经神经元的分离与培养

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in neuroscience. As it is becoming increasingly clear that this tiny structure plays a crucial role in health and disease of the central nervous system (CNS), it is of utmost importance to properly dissect the CP out of the brain ventricles in a way that allows downstream processing, ranging from functional to structural analysis. Here, isolation of the lateral and fourth brain ventricle mouse CP without the need for specialized tools or equipment is described. This isolation technique preserves the viability, function, and structure of cells within the CP. On account of its high vascularization, the CP can be visualized floating inside the ventricular cavities of the brain using a binocular microscope. However, transcardial perfusion required for downstream analysis can complicate the identification of the CP tissue. Depending on the further processing steps (e.g., RNA and protein analysis), this can be solved by visualizing the CP via transcardial perfusion with bromophenol blue. After isolation, the CP can be processed using several techniques, including RNA, protein, or single cell analysis, to gain further understanding on the function of this special brain structure. Here, scanning electron microscopy (SEM) on whole mount CP is used to get an overall view of the structure.

The choroid plexus (CP), an understudied tissue in neuroscience, plays a key role in health and disease of the central nervous system. This protocol describes a microdissection technique for isolating the CP and the use of scanning electron microscopy to obtain an overall view of its cellular structure.

小鼠脉络丛的显微切割和全安装扫描电子显微镜可视化

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

眼部微血管血管生成前维沃胆球状芽分析

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Choroid Sprouting Assay to Study Ocular Microvascular Proliferation Ex Vivo

Source: Tomita, Y., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/61677">An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis.</a> J. Vis. Exp. (2020)
This video demonstrates the development of an ex vivo model of choroidal sprouting to study ocular microvascular angiogenesis. The assay involves culturing mouse eye explants containing the choroid tissue attached to the sclera and retinal pigment epithelium under specific conditions that promote choroidal microvascular sprouting.

脉络膜发芽试验研究体外眼部微血管增殖

Source: Tomita, Y., et al. An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis. J. Vis. Exp. (2020)
This video demonstrates the ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

Isolating Immune Cells from Mouse Choroid Plexuses

Source: Dominguez-Belloso, et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63487">Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure of isolating immune cells from mouse choroid plexuses. This procedure is used in neuroimmunology research to understand how immune cells in the choroid plexuses impact brain health and various neurological diseases.

从小鼠脉络丛中分离免疫细胞

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 
1. ...

脉络丛上皮细胞的纤毛运动的观察

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