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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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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Protokół

The protocols and use of experimental animals were approved by the institutional animal care and use committees at the University of Yamanashi and Waseda University. Animal care was performed in accordance with institutional guidelines.

1. CPEC Preparation

1. Prepare the following apparatuses and materials: a stereo microscope, preferably capable of transmitting illumination from the bottom; a pair of watchmaker forceps (Dumont #3 or #4), flame-sterilized, straight operating scissors, flame-sterilized; two sterile 10 cm plastic dishes containing 20 ml ice-cold Leibovitz L-15 medium; 35 mm glass-bottom dishes containing 2 ml RT Leibovitz L-15 medium; a 100 ml beaker containing 70% ethanol; neonatal mouse pups.
2. Briefly immerse a neonatal mouse in 70% ethanol and euthanize quickly by decapitation using the operating scissors.
3. Place the head immediately in ice-cold Leibovitz L-15 medium in the sterile 10 cm dish.
4. Remove the skin from the calvaria using the pair of watchmaker forceps, cut open the skull to expose the brain, and then cut the cranial nerves to isolate the whole brain.
5. Transfer the brain to a new dish containing ice-cold Leibovitz L-15 medium and observe under the stereo microscope, making sure that the brain is completely immersed in the medium.
6. Set the dorsal aspect of the brain facing up, and orient the brain so that the olfactory bulbs reside at the three o’clock position (for right-handed persons). Hold the brain gently with the forceps in the left hand.
7. Using the fine dissection forceps (Dumont #3 or #4) in the right hand, cut the corpus callosum and underneath the parenchyma connecting the cerebral hemispheres, along the longitudinal fissure of the cerebrum.
8. Gently push the cerebral hemispheres away to the lateral sides and expose the transverse cerebral fissure.
9. Separate the hemispheres by pinching out the parenchyma between the hemisphere and thalamus.
10. Gently pull out the lateral ventricular choroid plexus that is attached to the lateral side of the hippocampus by the lamina affixa.
11. Transfer the isolated choroid plexus to the 35 mm glass-bottom dish containing fresh Leibovitz L-15 medium, and overlay a weight (Materials List) gently to hold the tissue in place.

2. Live Imaging of CPEC Cilia

1. Confirm proper ultraviolet (UV) and inferred (IR) cut filter(s) to block light shorter than 400 nm and longer than 700 nm, and that neutral density (ND) filters (25% and 6%) are inserted in the light path of the inverted microscope.
2. Adjust the focus of the objective lens roughly by eye, and then adjust the condenser so that the center and focus to conform to the Köhler illumination. Insert an appropriate differential interference contrast (DIC) prism, a DIC element, as well as analyzer and polarizer elements in the light path to conform to the DIC optics.
3. Adjust the contrast of view by the DIC prism position so that the structure of the tissue surface is most recognizable. If all cilia of the target cells are motile, a clear view of motile cilia cannot be obtained by eye because of their movement.
4. Change the light path to the video camera, and remove the ND filters to increase the light power.
5. Use the camera in focusing mode to adjust the field of view and focus. During focusing, in which video images are displayed at real-time on the monitor, a clear view of motile cilia is not available.
6. Use the camera at 200 Hz with an exposure time of 0.1 msec for the desired period (seconds to minutes). After acquisition of the image stack, single frames will display clear ciliary structures. If ciliary edges are blurred, increase the frame rate or use a shorter exposure time.
7. Record the motion of CPEC cilia within 25–60 min after euthanasia in Leibovitz L-15 medium.

3. Analysis of Ciliary Motion

1. Manually track beating patterns of each cilium on the computer monitor. Mark ciliary tip positions in each frame with the mouse pointer, which are assembled for trajectory information of each cilium. Either analyze the trajectory information using the same software or export to other more general applications for further analysis. The efficiency of this analysis step is described in the Discussion.
2. Classify the trajectories into two modes of motion, back-and-forth or rotational, by eye.
3. Calculate the ciliary beating frequency (CBF) using the following formula: [CBF = (number of frames per second)/(average number of frames for a single beat)]¹⁴, which can be obtained from a ciliary tip motion diagram (Figure 3). Repeat this calculation for multiple ciliary beating cycles, because other cilia on the same cell can interfere with the motion of each cilium, resulting in irregularity.
4. To analyze the angular uniformity of the ciliary beating axes within a single cell, define the beating angle θ for each trajectory (Figure 4). For back-and-forth trajectories, fit the positions of the cilia tip to a straight line, and define θ as the angle the line makes with x-axis. For rotational trajectories, fit the positions to an ellipse, and define θ as the angle the major axis of the ellipse makes with x-axis. Details of the fitting are described in the Representative Results section.
5. For quantitative description of each trajectory, calculate generalized aspect ratio AR. Briefly, rotate the trajectory by -θ and define AR as the ratio between the widths of the distribution along x- and y-axes (Figure 4B). Details are shown in the Representative Results section, and the interpretation, significance, and the limitation of the parameter are described in the Discussion.

4. Sample Preparation for SEM

Note: SEM is an important method to evaluate the status of cilia on CPECs in a comprehensive manner. To prepare specimens for SEM, a standard procedure reported previously¹⁵ is employed with slight modifications.

1. Before dissecting the tissue from the brain, prepare the fixative in a 5 ml glass vial with a polyethylene cap. The fixative consists of 2% paraformaldehyde, 2.5% glutaraldehyde (half Karnovsky’s solution¹⁶) in 0.1 M phosphate buffer, pH 7.4.
2. Dissect out the tissue from the brain as described in step 1.
3. Briefly rinse the isolated tissue with Hank’s balanced salt solution (HBSS) in a new dish and then fix the tissue in the fixative in the glass vial for 1 hr at room temperature. Use disposable transfer pipettes and handle the specimens gently. After rinsing in HBSS, the tissue becomes sticky.
   1. To transfer the specimens into the fixative solution, slowly expel a small amount of solution containing the tissue from the transfer pipette as a droplet and add to the fixative.
4. After fixation, discard the fixative and rinse the tissue with phosphate buffer three times.
5. Immerse the tissue in a 10% sucrose solution to wash out the remaining aldehydes. To ensure complete elimination of aldehydes, immerse the samples in the solution for 10 min and then repeat twice with fresh 10% sucrose. This step is important to achieve proper post-fixation in subsequent steps.
6. Immerse the tissue in a solution of 1% osmium tetroxide in phosphate buffer for 30 min and then place on ice for post-fixation. Judge the degree of osmification by the sample color: when aldehydes are completely removed, the sample is black.
7. Wash the post-fixed tissue samples extensively with double distilled water several times.
8. Dehydrate the samples by immersion in graded concentrations of ethanol, usually 65%, 75%, 85%, 95%, 99%, and 100%, for 10 min each. Obtain anhydrous ethanol by placing molecular sieves into 99.5% ethanol from a newly purchased bottle. Repeat dehydration with anhydrous ethanol three times.
9. Place the dehydrated samples into isoamyl acetate, a substitution reagent for critical point drying, for 10 min. Repeat this step twice. This reagent evaporates rapidly and the sample can become dry, resulting in destruction by surface tension. Therefore, do not dry the sample completely.
10. After the final exchange of isoamyl acetate, remove most of the solvent, immediately wrap the open glass vial with aluminum foil, and place the vial on dry ice. Using a needle or fine forceps, make several holes in the foil covering the mouth of the vial, so that liquid carbon dioxide flows easily into the vial in the critical point dryer. Proceed to the next step as quickly as possible.
11. In this step, minimize the carryover of isoamyl acetate into the chamber of the dryer, but do not let the sample dry out completely before critical point drying. In addition, do not leave the sample on dry ice for an unnecessarily long time to avoid the formation of frost on the vial.
12. Transfer the foil-wrapped glass vials containing the tissue samples into the critical point dryer that ensures the surface structure of the tissue remains intact while removing water contained in the tissue. Detailed information on operating the critical point dryer can be obtained from the manufacturer’s instructions.
13. Handle the samples carefully using a toothpick to minimize mechanical damage. The resulting dried tissue samples are fragile. Mount the samples on metal stubs and coat with gold-palladium using an ion sputter.

5. Observation by SEM

1. Observe by SEM and record images with a digital camera equipped to scanning electron microscope.
2. Transfer digital image data to a PC for analysis.

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Wyniki

An overview of the workflow is shown in Figure 1, including images of the devices.

Live motion observations of CPECs

Movie 1 shows a movie of CPECs isolated from a perinatal mouse, and Movie 2 shows an expanded view of the images in Movie 1. It should be noted that individual ciliary tips are less clear in still images compared with those in movies. Figure 2 shows tracking of the mo...

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Dyskusje

Perspectives of this method

Although the technique described here does not provide a more detailed analysis of cilia than previously published methods, the significance of this technique resides in the simplicity of the system and cost effectiveness, which can be easily applied to screening any kind of ciliary motility ex vivo. In particular, TI Workbench provides a simple and user-friendly interface that enables researchers to observe and analyze ciliary motility more easily. Effecti...

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Materiały

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