Effect of pneumolysin on rat brain ciliary function: comparison of brain slices with cultured ependymal cells

ABSTRACT This study compares two models for examining ependymal ciliary function: rat brain slices cut from the fourth ventricle and primary ependymal cells in culture. The cilia from both

preparations were very reproducible; each preparation had cilia beating at a constant frequency of between 38 and 44 Hz. With the brain slices, ciliary stasis occurred after 5 d in culture.

However, ependymal cells had fully functional cilia for up to 48 d in culture. The pneumococcal toxin, pneumolysin, caused a dose-dependent inhibition of cilia beat frequency within 15 min

in both models. There were no significant differences in the mean log 50% inhibitory concentration (pIC50) slice = 0.65 ± 0.05, equivalent to 4.4 hemolytic units (HU)/mL; cells = 0.57 ±

0.14, equivalent to 3.7 HU/mL. There were also no significant differences in the mean Hill slope factors for the curves (slice = 1.4 ± 0.05; cells = 1.6 ± 0.4). These data demonstrate that

both models can be used to examine the acute (15-min) effects of pneumolysin on cilia beat frequency. The main advantage of the primary ependymal culture model is that considerably more

cultured ependymal cells (∼70%) are available, compared with the number of ependymal cells on the brain slices (∼2%), thus reducing the number of animals used. A pure ependymal culture was

not achieved (∼30% of the cells were not ciliated). The increased survival time of the ependymal cells compared with the brain slices make cultured ependymal cells more useful for examining

long-term ciliary function, whereas brain slices may be more useful for examining the interactions between ependymal and other nearby cells. SIMILAR CONTENT BEING VIEWED BY OTHERS IIIG9

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access 07 August 2023 MAIN Despite the introduction of antibiotics and modern intensive care, a high morbidity and mortality rate is still associated with meningitis, particularly

pneumococcal meningitis (1). Although animal models have been used in the investigation of meningitis, there is still a need for other relevant models of the disease to enable dissection of

the mechanisms of pathogenesis. The ependymal epithelium is ciliated to varying degrees, depending on the ventricular location: for example, the cells lining the ventricular floor are more

ciliated than those lining the circumventricular organs such as the choroid plexus (2). A recent report has shown that ciliated ependymal cells may be adult neuronal stem cells from which

other neuronal cell phenotypes originate (3). In addition, the ependyma is believed to act as a filter relaying macromolecules to and from the CSF. However, the exact function of the

ependyma is unknown, as is its response to toxic insult. We have developed a system whereby brain slices are prepared with an intact ependymal lining. The ciliary beat frequency of the

ependymal cilia can be measured directly and continually. The advantage of such a technique is that ependymal cells are relevant, in that they are adjacent to the CSF, which is infected in

meningitis. Continual observation of ependymal ciliary movement enables us to assess the function and integrity of ependymal cells, thus allowing rapid screening for toxins. However, the use

of this tissue has disadvantages. The aim of this work, therefore, was to prepare a primary ependymal culture model that would allow the measurement of CBF. The advantage of such a method

would be that it would enable one to study only ependymal cells, without the presence of neuronal tissue associated with the brain slice model. In addition, the cells might survive over a

longer period of time, permitting extended time course experiments to be performed. A cell culture model may also yield more ependymal cells, hence reducing the number of animals needed for

the experiments. This study describes the development of a tissue culture model of the ependyma and its assessment, alongside the existing brain slice method, as a means of measuring

toxicity on CBF. The acute effects of pneumococcal toxin, pneumolysin, was used to compare the two experimental models. MATERIALS AND METHODS BRAIN SLICE. Wistar rats (14–17 d old) were

killed by cervical dislocation. Their cerebellum was removed and mounted on a vibratome under ice-cold M199 medium containing penicillin (100 IU/mL) and streptomycin (100 μg/mL). The brain

was sliced (250 μm) through the medulla oblongata and pons into the floor of the fourth ventricle so that the ciliated V-shaped floor was clear. The slices were mounted in 2 mL of prewarmed

M199 medium before use. CELL CULTURE. To grow the ependymal cells, we used an adaptation of the method by Wiebel _et al._ (4). Eight-well (25 × 35 mm) tissue culture trays were coated with

bovine fibronectin (75 μg/2 mL; ≅1 μg/cm2) and were incubated at 37°C in 5% CO2 for 2 h before use. Newborn (1- to 2-d-old) Wistar rats were killed by cervical dislocation, and their brains

were removed by careful dissection. The cerebellum was removed, as were small (3-mm) edge regions of the frontal cortex and the left and right cortical hemispheres. The remaining brain

regions (containing ependymal cells and ventricles) were mechanically dissociated by passing the tissue through an 18-gauge needle in 2 mL of tissue culture medium. The growth medium was

serum-free minimum essential medium (GIBCO Life Technologies, Paisley, Scotland) containing penicillin (100 IU/mL), streptomycin (100 μg/mL), Fungizone (2.5 μg/mL), bovine serum albumin (5

μg/mL), insulin (5 μg/mL), transferrin (10 μg/mL), selenium (5 μg/mL), and from day 3 onward, thrombin (0.5I U/mL). Dissociated tissue from a single brain was seeded (500 μL/well) into

eight-well (25 × 35 mm) tissue culture trays, each well containing 2.5 mL of medium. The medium was replaced 3 d after seeding. The adherent ependymal cells were fed by the replacement of 2

mL of fresh medium two times a week. The ciliated ependymal cell colonies were identified at day 5, and experiments with pneumolysin were performed when cells were between 14 and 17 d old

and cell proliferation was optimal (4). CILIA BEAT FREQUENCY. To determine ciliary beat frequency, both the cells in culture and the brain slices were placed in a humidified (80–90%)

incubation (37°C) chamber and were observed via an inverted microscope system (Diphot, Nikon, UK). Tissue was allowed to equilibrate for 30 min before readings. Beating cilia were recorded

with a digital high-speed video camera (Kodak Ektapro Motion Analyzer, model 1012) at a rate of 400 frames/s, with a shutter speed of 1 in 2000. The camera allows video sequences to be

recorded and played back at reduced frame rates or frame by frame. Ciliary beat frequency can be determined by timing a given number of individual cilia beat cycles. Basal ciliary beat

frequency was measured at 30 min. Each time point represents the measurement of four individual cilia from each slice or each well. Calculation of CBF (Hz): 400 (number of frames/sec)/5

(frames elapsed for five ciliary beat cycles) × 5 (conversion per beat cycle). PNEUMOLYSIN PURIFICATION. Pneumolysin was purified as previously described (5), and was made up in M199. One

milliliter of pneumolysin solution was added to the tissue, plus a further 1 mL of medium. Repeated gentle pipetting mixed the medium, and the tissues were incubated for 15 min before final

measurement of CBF. ELECTRON MICROSCOPY. For scanning electron microscopy, the tissues were fixed in Sorensen's phosphate-buffered (pH 7.4) glutaraldehyde (4% wt/vol). After

postfixation in osmium tetroxide (1% wt/vol), samples were dehydrated through graded ethanols and immersed in hexamethyldisilazane. The hexamethyldisilazane evaporated, leaving dry tissue

with no phase boundary damage (damage caused to the tissue by pressure differences of the air/liquid interface). To visualize the most severe effects of pneumolysin on the ependyma, the

upper concentration (1 μg) was selected for electron microscopy. STATISTICS. All data presented are mean ± SEM of six independent experiments. Statistical analysis was performed as

appropriate. Individual curves were analyzed by GraphPad PRIZM (nonlinear regression, variable slope), and were tested by ANOVA. We compared the log 50% inhibitory concentration (pIC50) and

Hill slope factors, using unpaired Student's _t_ tests. RESULTS There was no significant (unpaired _t_ test, _p_ = 0.12) difference in the ciliary beat frequency between the two

ependymal models (slice = 38.5 ± 0.7 Hz; cells = 40.7 ± 0.9 Hz). Indeed, reproducibility of the cilia beat frequency (measured once daily for each tissue) with the two models was very good

for the first 3 days in culture (Fig. 1_A_). With regard to the brain slices, ciliary slowing had occurred after 2 days in culture, and complete ciliary stasis occurred on day 5. In

contrast, the cilia on the ependymal cells in culture maintained a beat frequency of 38–44 Hz for up to 48 d (Fig. 1_A_). The proportion of the cells in culture that were ciliated, _i.e._ of

ependymal origin, was approximately 70%. To examine the response of the cilia to a known bacterial toxin, we studied the acute effects of pneumolysin at a clinically relevant concentration.

The upper concentration of pneumolysin that we selected was 1 μg/mL (149 HU), which can be released from D39 wild-type bacteria at 2 × 107 colony-forming units/mL, a bacterial concentration

that is observed commonly in the CSF of patients with pneumococcal meningitis (6). Pneumolysin caused a dose-dependent inhibition of ependymal ciliary beat frequency in both models (Fig.

1_B_). There was no significant difference (unpaired _t_ test, _p_ = 0.58) in the log of the 50% inhibitory concentration (slice = 0.65 ± 0.05, which is equivalent to 4.4 HU/mL; cells = 0.57

± 0.14 equivalent to 3.7 HU/mL). The Hill slope factors were not significantly different (unpaired _t_ test, _p_ = 0.6) (slice = 1.4 ± 0.08; cells = 1.6 ± 0.4) for the two models. Scanning

electron microscopy revealed similar ependymal cell and ciliary structure in both brain slices (Fig. 2_A_) and cell culture (Fig. 2_B_). With the addition of pneumolysin [at 1 μg/mL (149 HU)

for 15 min] to brain slices, we observed widespread destruction of the epithelium. After treatment with pneumolysin, there were sparse and disrupted cilia, which seemed to be fused (Fig.

2_A,_ before and after pneumolysin). After the addition of pneumolysin (1 μg/mL: 149 HU for 15 min) to ependymal cells in culture, we observed that the cilia were stripped from the cell

surface. Unciliated ependymal cell debris remained in the place of heavily ciliated colonies (Fig. 2_B,_ before and after pneumolysin). DISCUSSION _Ex vivo_ rat brain slices had ciliated

ependymal cells that beat at 38–44 Hz for the first 3 d in culture. However, the brain slice loses approximately 65% of its ciliary function on day 4, and at day 5, no functional cilia were

evident. This is consistent with a study examining postmortem respiratory cilia, which showed that there was a dramatic reduction in CBF 7 days after death (7). The degree of ciliation in

the ependymal cells in culture was 70%, in agreement with results obtained by other workers (4). Ciliary function is a good indicator of ependymal viability, and CBF measurements can be made

quickly and used as an indicator of toxic potential. This technique will be useful for identifying and screening potential virulence factors active in diseases of the CSF. In addition,

destruction of the ciliary function may play a direct role in the disease process; thus, a determination of means to reverse this destruction could lead to clinical interventions. The effect

of pneumolysin on respiratory epithelial ciliary function has been studied previously (8). It was shown to cause rapid ciliary stasis when applied to nasal brush biopsy samples. We have

also shown that this is an effect common to the cilia lining the fourth ventricle of rat brain (9). In this study, the two dose-response curves for the inhibition of CBF in the two models

essentially overlapped, which demonstrates that the pneumolysin was equipotent. The steep slope factors for the dose-response curves may indicate positive cooperativity in the action of

pneumolysin; however, further experiments are required to confirm this. To visualize the most severe effects of pneumolysin on the ependyma, the upper concentration (1 μg) was selected for

electron microscopy. The toxic effects of pneumolysin (10, 11) on the ependymal models can be seen on the electron micrographs, which show that almost complete tissue destruction has been

caused by 1 μg of pneumolysin after 15 min in both models. It is clear from the data presented here that for long-term studies, the ciliated ependymal cells in culture represent a major

improvement over brain slices. In addition, ependymal cells in culture are advantageous in that the proportion of ciliated cells is increased over nonciliated cells when compared with brain

slices, thus reducing the number of animals used for experiments. However, the brain slice model is ideal for acute studies of ependymal ciliary function in which rapid measurements of CBF

are required, because the transverse section of ependyma can be visualized readily, without need for 14 d of culture. Brain slices would also be advantageous in experiments studying

neuronal-ependymal cell communication because of the presence of the underlying neuronal tissue. In conclusion, the brain slices were very useful for acute experiments with pneumolysin.

However, for longer-term low-dose recovery experiments, it is clear that ependymal cells in culture offer an improved alternative. The potency of pneumolysin-induced ciliary stasis remained

constant for both models tested. ABBREVIATIONS * CBF: ciliary beat frequency * CSF: cerebrospinal fluid * HMDS: hexamethyldisilazane * HU: hemolytic units REFERENCES * Quagliarello V, Scheld

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pathogenicity of _Streptococcus pneumonia_. _Trends Microbiol Sci_ 43: 103–106 Article  Google Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Child

Health, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, University of Leicester, Leicester, LE1 5WW, England Robert A Hirst, Andrew Rutman, Kulvinder Sikand & 

Christopher O'Callaghan * Department of Microbiology and Immunology Maurice Shock Medical Sciences Building, University of Leicester, Leicester, LE1 5WW, England Robert A Hirst & 

Peter W Andrew * Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ Timothy J Mitchell Authors * Robert A Hirst View

author publications You can also search for this author inPubMed Google Scholar * Andrew Rutman View author publications You can also search for this author inPubMed Google Scholar *

Kulvinder Sikand View author publications You can also search for this author inPubMed Google Scholar * Peter W Andrew View author publications You can also search for this author inPubMed 

Google Scholar * Timothy J Mitchell View author publications You can also search for this author inPubMed Google Scholar * Christopher O'Callaghan View author publications You can also

search for this author inPubMed Google Scholar ADDITIONAL INFORMATION This study was funded by a British United Providence Association (BUPA, UK) research grant to C.O'C. and P.W.A. and

approved by the University of Leicester. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Hirst, R., Rutman, A., Sikand, K. _et al._ Effect of

Pneumolysin on Rat Brain Ciliary Function: Comparison of Brain Slices with Cultured Ependymal Cells. _Pediatr Res_ 47, 381–384 (2000). https://doi.org/10.1203/00006450-200003000-00016

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