脉络丛上皮细胞的纤毛运动的观察<em&gt;离体</em

摘要

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.

摘要

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.

引言

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 ciliopathies^2–4. 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–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 epithelium^5,10, 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 “TI Workbench” 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.

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研究方案

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

1. 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。
2. 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。
3. 在冰冷的Leibovitz L-15培养基，立即将头在无菌10厘米二SH。
4. 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。
5. 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。
6. 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。
7. 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。
8. 轻轻大脑半球推到侧面和揭露横向脑裂。
9. 按捏出来个分离半球半球和丘脑两地电子实质。
10. 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。
11. 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。

2.现场CPEC纤毛成像

1. 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。
2. 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。
3. 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。
4. 改变光路的摄像头，并删除ND滤镜增加了光功率。
5. 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。
6. 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。
7. 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。

睫状运动的三分析

1. 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。
2. 分类轨迹为两种模式的运动，背面的往​​复或旋转，通过眼睛。
3. 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。
4. 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。
5. 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。

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。

1. 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。
2. 解剖出从脑组织如步骤1所述。
3. 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。
   1. 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。
4. 固定后，弃去固定剂和冲洗用磷酸盐缓冲液三次组织。
5. 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。
6. 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。
7. 双distille广泛洗净后固定的组织样本ð水数次。
8. 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。
9. 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。
10. 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。
11. 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。
12. 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。
13. 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。

5.观察SEM

1. 通过SEM观察，并用数码相机记录影像配到扫描电子显微镜。
2. 数字图像数据传输到PC进行分析。

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结果

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...

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讨论

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...

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材料

Name	Company	Catalog Number	Comments
Required reagents
for live cell imaging
ethanol	Wako Chemicals	057-00456
Leibovitz L-15 medium	Life Technologies	11415-064
for SEM preparation
ethanol	Wako Chemicals	057-00456
Hank's balanced salt solution	Life Technologies	14170112
paraformaldehyde	Merck	1040051000
glutaraldehyde	Nacalai tesque	17003-05
isoamyl acetate	Nacalai tesque	02710-95
Molecular Sieves 4A 1/8	Wako Chemicals	130-08655	for preparation of anhydrous ethanol
phosphate buffer saline (PBS)	Sigma-Aldrich	D1408
phosphate buffer, 0.1 M			To make 100 ml, mix 19.0 ml of 0.1 M NaH2PO4 and 81.0 ml of 0.1 M Na2HPO4
monosodium phosphate (dihydrate)	Nacalai tesque	31718-15
disodium phosphate (anhydrous)	Nacalai tesque	31801-05
suclose	Nacalai tesque	30406-25
osmium tetroxide	Nisshin EM	300
dry ice
[header]
Tools and materials for dissection
for both live imaging and SEM preparation
stereo microscope	Olympus	SZX7
flat paper towel
Φ10 cm plastic dish
100 ml beaker
straight operating scissors	Sansyo	S-2B
watchmaker forceps	Dumont	No.DU-3 or -4, INOX
for live cell imaging
glass bottom dish	Matsunami Glass	D110300
for SEM preparation
alminum foil
5 ml glass vial with a polyethylene cap	Nichiden Rika-Glass	PS-5A
transfer pipette	Samco Scientific	SM251-1S	for specimen tranfer
toothpick			for specimen transfer
ion sputter with gold-palladium	Hitachi	E-1030
critical point dryer	Hitachi	HCP-2
[header]
Microscopic equipment and materials
for live cell imaging
inverted microscope	Olympus	IX81
100 W mercury lump housing and power supply	Olympus	U-ULH and BH2-RFL-T3
100 W mercury lamp	Ushio	USH103D
DIC condenser, n.a. 0.55	Olympus	IX-LWUCD
electrrical shutter	Vincent Associates	VS35S22M1R3-24 and VMM-D1	manual shutter can be used.
band-pass filter (400-700 nm, Φ45 mm)	Koshin Kagaku	C10-110621-1
ND filter (Φ45 mm)	Olympus	45ND6, 45ND25	combination of 25% and 6% ND filters are used
objective lens (water immersion) with DIC element	Olympus	UApo 40XW/340, n.a., 1.15 with IX-DPAO40
high-speed video camera	Allied Vision Technologies	GE680	≥200 Hz frame rate and 1 msec expose time
image acquisition / analysis software	in-hous software	TI Workbench	capable of acquisition at high frame rates.
PC for camera control / analysis	Apple	Mac Pro
vibration isolation table	Meiritsu Seiki	AD0806
weight for tissue	Warner Instruments	slice anchor kits	It can be made with nylon mesh glued to a U-shape squashed Φ0.5 mm platinum wire.
cover glass (alternative weight for tissue)	Matsunami	made-to-order	A cover glass can be used as a tissue weight.
for SEM
inverted microscope	Olympus	IX81
scanning electron microscope	JEOL	JSM-6510