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

Summary

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.

Abstract

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’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’s slices in a customized glass-bottom plate containing Dulbecco’s Modified Eagle’s Medium (DMEM)/High-Glucose at 37 °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.

Introduction

Cilia are sensory microtubule-based organelles that extend from the cell surface to the extracellular environment. Depending on their microtubule organization, cilia can be categorized into two types - “9+0” or “9+2”. Functionally, based on their motility, these can be classed as motile or non-motile cilia ¹. Primary cilia is a term commonly used to denote “9+0” non-motile cilia. These have nine parallel doublet microtubules (denoted by ‘9’) and a central pair of microtubules is absent within the central sheath (denoted by ‘0’). However, some “9+0” cilia, such as nodal cilia, which regulate embryo laterality are motile ². On the other hand, motile cilia are characterized, in addition to the nine parallel microtubule doublets, by an additional central pair of microtubule doublets and associated with dynein motor proteins to facilitate motility. In addition, some “9+2” cilia such as olfactory cilia are non-motile ³. Ependymal cells lining the brain ventricles and the central canal of the spinal cord are characterized by motile cilia that propel the cerebrospinal fluid (CSF) along the brain ventricles ⁴.

The overall goal of this method is to facilitate studying the motile cilia dynamics and structural abnormalities. The brain’s health and development heavily depend on efficient circulation of CSF within the brain ventricles. For instance, normal CSF flow and fluid balance require normal beating and functional ependymal cilia ^5,6, which in turn play critical roles in regulating the directional movement of neuronal cells and stem cell migration ⁷. As such, abnormal ependymal cilia function or structure can lead to abnormal CSF flow, which is associated with hydrocephalus, a medical condition in which there is an abnormal accumulation of CSF in the ventricles of the brain. This may consequently cause increased intracranial pressure and progressive enlargement of the head, convulsion, tunnel vision, and mental disability ⁸.

The advantages of this technique over existing methods is that it allowed for the first time to report three distinct ependymal cell types: I, II, and III, based on their unique ciliary beating frequency and beating angle. These ependymal cells are localized within certain regions in the brain ventricles. Furthermore, the effects of age and pharmacological agents such as alcohol and cilostazol on altering the ependymal cell types or their localizations can be demonstrated, which was not possible before this classification of ependymal cells. Cilostazol is an inhibitor of phosphodiesterase-3, an enzyme that metabolizes cAMP to AMP and it also regulates intracellular calcium ⁹. Using high-speed fluorescence imaging analysis allows imaging and quantifying of the unique intracellular calcium oscillation properties of the ependymal cells. For instance, both alcohol and cilostazol significantly altered the ependymal ciliary beating frequency as well as the intracellular calcium oscillations properties, which in turn, could lead to a change in the cerebrospinal fluid volume replacement by ependymal cilia ¹⁰. In summary, this technique was key to provide the first evidence of three distinct types of ependymal cells with different calcium oscillation properties.

In the following section, a detailed step-by-step overview of the procedure is provided, paying close attention to tissue preparation and handling.

Protocol

The procedures for animal use were approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Toledo in accordance with the guidelines of the Institutional Animal Care and Use Committee at the National Institutes of Health and the Guide for the Care and Use of Laboratory Animals.

1. Brain Extraction, Sectioning and Tissue Preparation

1. Sacrifice wild-type mouse strain C57BL/6 by deeply euthanizing with CO₂ asphyxiation for 5 min. Assure death by cervical dislocation.
2. Clean the mouse head with 70% ethanol.
3. Perform craniectomy using sterile scissors and forceps by first pulling the skin off, starting with the top of the head to expose the skull.
4. Then, when the skull is exposed, remove the skull by peeling the bone piece-by-piece, starting from the posterior side and moving toward the anterior side. Be cautious not to destroy the brain ventricles.
5. Collect the whole brain.
6. Place the brain in a 100 mm Petri dish containing DMEM/High-Glucose supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution containing 10,000 units/ml of penicillin and 10,000 µg/ml of streptomycin and pre-warmed to 37 °C.
7. Slice the brain on the median sagittal plane by hand with a sharp blade, and obtain the first 100-200 mm section from each half using a vibratome.
8. Rinse the brain tissue with pre-warmed 37 °C phosphate buffered saline (1x PBS) solution.
9. Immediately place the brain section in DMEM/High-Glucose media pre-warmed to 37 °C.

2. Live Imaging Configuration and Setup

1. Place the brain tissue sections in 30 mm glass-bottom culture dishes containing 1 ml of DMEM/High-Glucose media. Adjust the microscope’s enclosed chamber environment to 37 °C, 95%/5% O₂/CO₂ content (Figure 1).
2. Using a 60X objective oil immersion lens, collect the ependymal cells/cilia images by first placing an oil drop on the 60X objective lens and focusing on the cells with regular DIC transmitted light.
3. Then, follow the direction of the DMEM bubble movement as a guide to the location of the motile ependymal cilia as ciliary beatings create a kind of bubble movement in the area. Choose an area containing healthy cells with motile cilia in the brain’s lateral ventricle using the DIC filter. Once the ependymal cilia are found, adjust the light and focus to obtain a satisfactory image.
4. Set the live imaging parameters according to a specific purpose using Metamorph imaging software. In the present demonstration, acquire twenty four-bit images with the camera binning set to 1 x 1 combined with 60X objective and 5-10 msec exposure time.
5. Collect the DIC images by opening the microscope aperture to an optimal level in order to have a minimum exposure time. Observe live images stream to the camera to provide fast and immediate image acquisition without delay. Calculate the speed of cilia beating based on the requirement of the minimal exposure times to obtain sufficient image contrast.

3. Data Visualization and Analysis

1. Calculate the number of cilia beatings by counting the number of beatings in 1 min. Do this by decreasing the speed of the video and counting the number of beats using a cell counter or similar tool.
2. To calculate the frequency of beatings, multiply the exposure time at which the video is recorded by the number of the frames or time-lapse images acquired to get the number of sec. (Example: exposure time 5 msec 200 frames = 1,000 msec or 1 sec).
3. Calculate the number of beatings in one second to obtain the frequency which is expressed as the number of beating per one sec. Do this by dividing the number of cilia beatings over a one-second time interval (Example: cilia beats 50 times in a 200 frames video recorded at exposure time of 5 msec i.e. 5 msec x 200 frames = 1 sec; now divide 50 beats by 1 sec = 50 Hz).
4. Calculate the ciliary beating angle by evaluating the path taken by the ependymal cilia during both the power and recovery strokes. Perform this according to a previously described method, with minor modifications ¹¹. The precise movement of individual cilia is observed during the complete beat cycle.
5. On an acetate sheet placed over the monitor, draw a horizontal line along the ependymal edge and a vertical line through the midline position of the cilia at the start of the power stroke.
6. Plot the precise position of the cilium frame by frame as it moves forward during the power stroke. In a similar manner, plot the movement of the cilia during the recovery stroke.
7. Calculate the ciliary beating angle from the maximum deviation of the cilium from the midline during the power stroke as well as the recovery stroke.

4. Calcium Signal Recording

1. After slicing the brain, briefly rinse the brain slice with 1x PBS or Dulbecco’s PBS (pH 7.0). Prepare fresh Fluo-2 to avoid fluorescence quenching and to obtain good signal-to-noise ratio.
2. Prepare 1 mM stock solution of Fluo-2 solution in dimethylsulfoxide (DMSO), mix and vortex the solution for at least 5 min to ensure that Fluo-2 is homogenously dissolved in DMSO.
3. Dilute the Fluo-2 stock solution in 500 ml of DMEM/High-Glucose supplemented with 2% B27 pre-warmed to 37 °C to a final concentration of 20 mg/ml.
   NOTE: B-27 is an optimized serum-free supplement containing vitamin A, antioxidant cocktail and insulin used to support short- or long-term viability of hippocampal and other CNS neurons.
4. Immediately incubate the brain slice with 20 mg/ml Fluo-2 for 30 min at 37 °C in a glass-bottom plate.
5. To determine the optimal loading concentration of calcium fluorophore Fluo-2 and to avoid calcium dye cell toxicity, challenge cells with ATP, and check the cell viability by determining the time course and peak magnitude of calcium signals in response to ATP.
6. Record the video for calcium oscillation at a capture rate of 5 msec for a minimum of 1 sec (200 frames per sec), with excitation and emission wavelengths of 488 nm and 515 nm, respectively (Movie 2).
7. To distinguish the Fluo-2 calcium signal from autofluorescence or movement artifacts, ensure that the intensities emitted at 515 nm is monitored separately.
   NOTE: Fluo-2 is not the calcium fluoremetric suitable to quantify intracellular calcium. However, it is an excellent dynamic calcium dye to detect fast calcium changes, such as calcium oscillations. For more accurate calcium quantification, Fura-2 is recommended. However, the dynamic changes of Fura-2 are limited by its ratiometric nature of the dye.
8. Follow the formulas provided by the manufacturer to calculate the exact free intracellular calcium values. Example [Ca²⁺] = K_d × (R–R_min)/(R_max – R). Where K_d is the dissociation constant of the dye from the released calcium, R is the measured fluorescence at 488, and R_min and R_max are the fluorescence ratios at minimum and maximum ion concentration ¹².

5. Immunofluorescence Microscopy

1. Fix the brain sections with phosphate buffered saline solution containing 4% paraformaldehyde (PFA) and 2% sucrose for 10 min. Alternatively, fix the whole brain with 4% PFA and then section into 50 mm sections using a cryostat.
2. Wash the tissue three times with 1x PBS for 5 min each time.
3. Incubate the brain slice with a solution of 0.1% Triton-X in 1x PBS for 5 min, then rinse three times with 1x PBS for 5 min each time.
4. Incubate the brain slice with mouse primary antibody, antiacetylated a-tubulin, used at a dilution of 1:5,000 in 10% FBS in 1x PBS for one hour at room temperature (RT) or overnight at 4 °C.
5. Wash the tissue three times with 1x PBS for 5 min each time.
6. Incubate the brain slice in secondary antibody, fluorescein anti-mouse IgG at a dilution of 1:500 in 10% FBS in 1x PBS solution for 1 hr at RT.
7. Before observation under a fluorescent microscope, counterstain the section with 4',6-diamidino-2-phenylindole (DAPI) for 5 min to stain the nucleus (or DNA) ¹³. To minimize photobleaching, image the sections immediately with minimum exposure time possible.

Results

Measuring ependymal cilia function in live mouse brain

The method described in this protocol is used to monitor ependymal cilia function and structure in the fresh tissue dissected from the mouse brain as well as to monitor and study cilia beating frequency. The steps followed to accomplish a complete experiment are depicted in a schematic flowchart (Figure 1). It is highly recommended that the experiment is conducted within a short time frame in order to keep the motile cilia...

Discussion

Described here is a protocol for the preparation of mouse brain tissue for both live-imaging and fluorescence microscopy that provides a rapid and sensitive close observation of the ependymal cilia within the brain ventricles. This technique is not restricted to the lateral ventricle only; it could be utilized to observe the cilia in other brain ventricles. This imaging technique provides a live stream that resembles the movement of the CSF by ciliary beating in an ex vivo setting. Moreover, it enables analysis ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/HIGH GLUCOSE	Cellgro Mediatech Inc.	10-013-CV
Fetal bovine serum (FBS)	Hyclone	SH30088-03
Penicillin/Streptomycin	Thermo Scientific	SV30010
Phosphate buffered saline	Thermo Scientific	SH30256-01
Paraformaldehyde	Electron Microscopy Sciences	15710-SP
Sucrose	Sigma-Aldrich	S-2395
Triton-X	Sigma-Aldrich	T9284
Fluo-2	TEF Labs	#0200
DMSO
B27	Gibco	17504044
VECTASHIELD HardSet Mounting Medium with DAPI	Vector Labs	H-1500
Anti-acetylated a-tubulin antibody	Sigma-Aldrich	T7451	clone 6-11B1
FITC Anti-mouse antibody	Vector Labs	FI-2000
Cell Culture plate	VWR Vista Vision	30-2041
Cover Slip (18 x 18)	VWR Vista Vision	16004.326
Vibratome	Leica Biosystems	Leica VT1200S
Cryostat	Leica Biosystems	Leica CM1860
Inverted Fluorescence Microscope	Nikon	Nikon TE2000	60X oil
Microscope cover glass 24 x 60 mm2	VWR Vista Vision	16004-312
Mounting Medium with DAPI	Vector Laboratories	H-1500
DAPI filter cube	Chroma Technology