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In mammals, ciliary transport aids the movement of gametes in the oviduct, sperm in the ductus efferentes of the testis, cerebrospinal fluid, and mucus and debris from the airways (1). Defective mucociliary transport is an important pathophysiologic feature in several human respiratory diseases, including cystic fibrosis, chronic bronchitis, and primary ciliary dyskinesia (2). Although the role of mucociliary transport in tubal infertility has not been clearly established, there are well-documented cases of the association of immotile cilia syndrome and infertility (3). Ciliated ependymal cells line the ventricular surface of the brain, the cerebral aqueducts, and the central canal of the spinal cord, separating the CSF from neuronal tissue. However, the role of ependymal cilia in the CNS is less well understood, and associated literature is sparse.

The movement of CSF close to the ventricular walls by ependymal cilia is in the predicted direction of CSF flow (4) and may serve to clear metabolites and toxins from the brain by improving the diffusion gradient between neuronal tissue and CSF. A role in host defense, keeping the surface of the brain and aqueducts clear from debris, has also been postulated.

This initial study was performed to obtain basic data on the effect of age on ependymal ciliary function and to compare respiratory and ependymal ciliary function. It is already known that the beat frequency of respiratory cilia is faster in newborn infants than in adults (5). Ependymal cilia are water propelling—as opposed to the mucus-propelling cilia of the respiratory tract—and may have different characteristics. Indeed, one previous study has suggested that ependymal cilia beat more quickly than respiratory cilia (6).

We have recently developed an ex vivo model that allows measurement of ependymal and respiratory ciliary beat frequency, using high-speed video analysis. This study was performed to add to our understanding of ependymal ciliary function. Our aims were to use this system to compare the ciliary beat frequency of ependymal and respiratory cilia and to determine whether ependymal cilia grow longer or change their beat frequency with age.

METHODS

Sample preparation.

The brains of 9- to 17-day-old Wistar rats were dissected after the animals had been killed. Brain slices were prepared from the floor of the 4th ventricle of these rats immediately after their death, and were mounted in a well containing 4 mL of medium 199 with Earl's salts (pH 7.4), plus penicillin 50 U/mL and streptomycin 50 μg/mL. Ciliary movement was observed by using a ×50 objective on an inverted microscope. Samples were enclosed in a purpose-designed environmental chamber that maintained the solution at 37°C and maintained the surrounding air at a humidity of 80% to minimize evaporation. Tracheal rings, 1 mm thick, were prepared by careful dissection from eight of the 11 adult rats whose ependymal ciliary frequency was measured. Tracheal rings were mounted and observed in a similar fashion to ependymal strips. It was not possible to obtain tracheal rings from 9- to 17-d-old rats that allowed measurement of ciliary beat frequency.

Measurement of ciliary beat frequency.

Beating cilia on ependymal strips were recorded by a high-speed video camera (Kodak EktaPro Motion Analyzer, model 1012) at a rate of 400 frames/sec. The camera allowed video sequences to be downloaded at reduced frame rates, permitting ciliary beat frequency to be determined directly by timing a given number of individual ciliary beat cycles. At each measurement time, ciliary beat frequency was measured from four different areas along each ependymal or tracheal edge.

Both respiratory and brain ciliary measurements were made after 30 min of incubation at 37°C.

We studied 32 ependymal edges from a total of 11 adult rats. Four readings of ciliary beat frequency were made on each edge (total number of readings, 128). Successful measurement of tracheal ciliary beat frequency was also obtained from eight of these rats (total number of readings, 29). Twenty-five 10- to 15-d-old rats had brain ciliary measurements (total number of readings, 100). A paired t test was used to compare adult brain and respiratory ciliary beat frequency.

Scanning electron microscopy measurement of cilial length.

The brain and tracheal samples were fixed in 2.5% phosphate-buffered glutaraldehyde and rinsed in fresh buffer before being post-fixed in 1% osmium tetroxide (OsO4). The rinsed samples were then dehydrated through graded ethanol and infiltrated and immersed in HMDS. The HMDS was then allowed to evaporate, reproducing the effect of critical point drying, allowing the tissue to dry to air, thus avoiding phase boundary damage. HMDS was chosen because critical point drying involves high rates of flow of liquid CO2 that would damage the thin and fragile brain samples. The dried samples were fixed to aluminum stubs and sputter-coated with gold before examination in the scanning electron microscope.

Ten fields from different cells bearing mature cilia were selected at random. Ten cilia from each field were measured using a computerized image analysis system. The brain and trachea from five different 9- to 15-d-old and four adult (4- to 6-month-old) rats were studied. The system was calibrated in microns by means of the scanning electron microscope's internal standard, and the calibration bar was marked upon each image.

Representative electron micrographs of respiratory and ependymal tissue are shown in Figures 1 and 2.

Figure 1
figure 1

Scanning electron micrograph of cilia lining the trachea of a 12-d-old Wistar rat. The 1-cm bar on the photograph is equivalent to a length of 2.1 μm.

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Figure 2
figure 2

Scanning electron micrograph of the ciliated ependymal lining of the floor of the 4th ventricle of a 12-d-old Wistar rat. The 1-cm bar on the photograph is equivalent to a length of 1.28 μm

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RESULTS

The mean ciliary beat frequencies of the brain ependymal edges and tracheal respiratory samples are shown graphically in Figure 3.

Figure 3
figure 3

Beat frequency measurements of ependymal and respiratory cilia. Each data point on this graph represents the mean beat frequency of four readings from an individual ependymal or respiratory ciliated edge. Respiratory cilia beat considerably more slowly than ependymal cilia.

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The adult respiratory cilia had a significantly (p= 0.003) lower mean ciliary beat frequency (mean 20.9 Hz: 95% confidence intervals 14–27) than adult ependymal cilia (mean 40.7 Hz: 95% confidence intervals 38.5–42.9). A two-sample t test was performed to compare adult and neonatal ependymal ciliary beat frequencies and cilial lengths. There was no significant difference between the ependymal ciliary beat frequencies (p= 0.19) of adult (mean 40.7: 95% confidence intervals 38.5–42.9) and 10- to 14-d-old rats (mean 38.8: 95% confidence intervals 37.1–40.6). Ependymal cilia (mean length ± SD: 8.2 ± 0.3 μm), measured by scanning electron microscopy, were significantly (p= 0.001) longer than respiratory cilia (mean length ± SD: 5.5 ± 0.6 μm) from the trachea of 9- to 15-d-old rats. Representative scanning electron micrographs are shown in Figure 1. The length of respiratory and ependymal cilia did not increase with age. Mean (± SD) adult respiratory and ependymal ciliary lengths were 5.6 ± 0.5 μm and 8.1 ± 0.2 μm, respectively, when measured by scanning electron microscopy.

DISCUSSION

The ependymal layer forms a barrier between the cerebrospinal fluid and neuronal tissue of the brain. Approximately 40 cilia, approximately 8 μm long, project from ciliated ependymal cells and beat, continuously, at up to 40 beats/sec (40 Hz). We found that brain ependymal cilia from young rats beat at a frequency similar to that of adult rats, and that no increase in cilial length occurred with growth from infancy to adulthood. However, we found that ependymal cilia beat at approximately twice the rate of cilia from the respiratory tract. Respiratory cilia for beat-frequency analysis were taken from adult rats because of technical difficulties in the preparation of cilia from the trachea of young rats. Using scanning electron microscopy, we subsequently demonstrated very poor ciliation of tracheas from 9- to 15-d-old rats. Measurements from the scanning electron micrographs revealed that respiratory cilia were significantly shorter than ependymal cilia. It is likely that the longer and faster-beating ependymal cilia are better suited to the rapid movement of cerebrospinal fluid.

The length of respiratory and brain cilia did not change from infancy to adulthood. Our findings that suggest caution must be exercised when extrapolating experimental results from studies on respiratory cilia to ependymal cilia

We have previously shown that respiratory cilia taken from human infants within 2 d of birth beat more quickly than those taken from healthy adults (5). Failure to find a similar difference in ependymal ciliary frequency between young and old rats may have resulted from sampling at a later stage, 10–15 d after birth. The mechanism whereby ependymal cilia beat at almost twice the frequency of respiratory cilia is being investigated.

Cathcart and Worthing (4) described the direction of the flow due to ependymal ciliary movement in the rat brain. The data they presented demonstrated conclusively that ciliary movements and ciliary currents are capable of circulating cells from the ventricular cavities toward the lateral apertures of Luschka and medial aperture of Magendie of the fourth ventricle at a very rapid rate. Large amounts of cellular debris were rapidly cleared from ependymal surfaces of blind areas. The direction taken by the ciliary currents in their specimens was the shortest distance to the next narrow opening in the system.

The needs for rapidly beating ependymal cilia are unclear. However, defective ciliary movement may be associated with pathologic states. There is strong evidence that defective ciliary movement in animals and humans (7) with primary ciliary dyskinesia may be primarily responsible for the occurrence of hydrocephalus. Afzelius (7) postulated that defective cilia were a primary cause of hydrocephalus and that high intraventricular pressure was its consequence. He examined the brains of seven patients with primary ciliary dyskinesia by CT scan. In three of these patients, the ventricular system and sulci were slightly enlarged. Subsequently, there have been a number of reports of hydrocephalus and primary ciliary dyskinesia (8–10).

Koto and colleagues (11, 12) reported that in WIC-Hyd rats, approximately 35% of the females developed slowly progressive and/or arrested hydrocephalus. Despite this, they grow up to maturity, are capable of reproduction, and seldom die of hydrocephalus. Approximately 34% of the males are affected by rapidly progressive hydrocephalus, all of which die. Approximately half of the males with hydrocephalus have total situs inversus viscera, and all cilia in affected male rats before and after development of hydrocephalus are immotile (13). A variety of movement disorders such as immobile, rotatory, and vibratory cilia were observed, in addition to normally beating cilia in affected female rats that did not develop severe hydrocephalus. The hydrocephalus developing in affected male and female WIC-Hyd rats appears to be secondary to a motility disorder of ependymal cilia, which is part of their primary ciliary dyskinesia.

Nakamura and Soto (14) infused Metavanadate, an inhibitor of ciliary movement, into the third ventricle of normal Sprague-Dawley rats for 1 wk. All of these rats developed hydrocephalus. This experiment supports the hypothesis that ciliary dyskinesia is primarily responsible for hydrocephalus occurring in the WIC-Hyd rats. From these studies, it would seem appropriate to consider the diagnosis of primary ciliary dyskinesia in children presenting with congenital hydrocephalus. Although hydrocephalus is uncommon in children with primary ciliary dyskinesia, clinicians should be aware of this association.

In summary, we have shown that ependymal cilia beat at approximately twice the rate of respiratory cilia and are also significantly longer. Caution must be observed when extrapolating results from experiments on respiratory cilia to ependymal cilia.