Functional analysis and evaluation of respiratory cilia in healthy Chinese children

For this cross-sectional study, healthy Chinese school children aged less than 18 years and adult volunteers in Hong Kong (HK) were recruited. Subjects with a history of chronic respiratory or nasal disease, symptomatic upper respiratory tract infection (URTI) in the previous 6 weeks, those requiring long-term medication or known cigarette smokers were excluded [1]. Exposure to second hand smoking (SHS) at home was explored. Subjects were examined to exclude obvious nasal defects with an auroscope. The inferior turbinate of subjects was brushed with a 3 mm cytology brush (Conmed, product no. 149R) to obtain nasal epithelial cells, without topical anaesthetics. During the procedure, subjects were shown a video on a mobile phone as distraction. Nasal brushings were placed in medium 199 (pH 7.3) supplemented with streptomycin 50 μg/ml and penicillin 50 IU/ml (Gibco, UK).

Ciliated strips of epithelium were suspended in a chamber using a glass slide and cover slips. The slide was placed on a heated stage (37 °C) of a Leica light microscope mounted on an anti-vibration table. Specimens were examined using a × 100 interference contrast lens. Undisrupted ciliated strips of about 50 μm in length and devoid of mucus were studied. Beating ciliated edges from side-view profile (10 video chips), above-view profile (2 video chip) and towards the observer profile (1 video-chip), were recorded using a digital high speed video camera (IDT, model NX4) at a rate of 500 frames per second. The recorded side-view video sequences were projected onto a high resolution monitor and were played back at reduced frame rates or frame by frame to determine CBF. The ciliated edge was divided into 5 adjacent areas measuring 10 μm. Two measurements of CBF were recorded in each area, resulting in a total of 10 measurements for each edge. A maximum of 10 edges were analysed per subject. The number of frames for groups of beating cilia required to complete 10 cycles were recorded. CBF was converted using a calculation (CBF = 500/(number frames for 10 beats) × 10). The reproducibility for inter-observer (measured independently by two observers) and intra-observer (measured by the same observer after 2 days) CBF measurements were evaluated [1]. Normal ciliary beat pattern denoted a coordinated cilium beating in a forward and backward motion along the whole epithelial edge on side-view profile. Edges with dyskinetically beating cilia were counted and the percentage of these edges was calculated. Abnormal patterns, such as circular beating identified on above-view profile and towards the observer profile, were reported qualitatively.

Nasal brushing samples were fixed in 2% glutaraldehyde and processed through resin using standard techniques [1]. Ultrathin sections were cut at 100 nm, collected on 200 mesh thin bar copper grids, stained in 1% uranyl acetate and counterstained in Reynold’s lead phosphate.

The ultrastructural changes of the epithelium and cilia were assessed in a blinded fashion [7]. The distal and proximal regions of the axoneme were examined. Dynein arms were assessed in cross sections with clear structural features and an intact ciliary membrane. The number of ciliated cells, mucous cells, and dead cells of the epithelium were expressed as a percentage of the total number of cells examined. Disruption and damage to the tissue was quantified using a scoring system that assessed the degree of loss of cilia from ciliated cells, projection of cells from epithelial edge, cytoplasmic blebbing and mitochondrial damage with a summation epithelial integrity that incorporated all of these factors to assess the overall epithelial damage: 0 = no damage, 1 = minor, 2 = mild, 3 = moderate, 4 = major, 5 = severe damage [1]. Cilia in less than perfect cross-section but in which the microtubular arrangement could be recognised were recorded as being normal (9 + 2 microtubules) or defective. The results were recorded in batches of 100 counts, assembling as many such batches from a single section, and thus any pattern would be identified. Dynein arms were assessed in any high-quality cross-sections encountered during the recording of microtubular arrangements and documented as showing the presence of both arms, only an outer or inner arm, or neither arm. If the presence of an arm was equivocal, it was counted as being present [17].

In calculating the sample size, in order to establish age-related references in children aged < 18 years with a local population size of around 1,000,000 [18], allowing 9% margin of error from the mean at 5% confidence level, 120 subjects were required. This sample size of 120 subjects would have a power of 80 and 5% level of significance (two-sided) to detect a 10% difference of mean CBF between the Chinese and European population [1]. Assuming 10% of sample insufficiency and loss during preparation, a total of 135 subjects were recruited. The mean and standard deviation (SD), 5th and 95th percentiles of CBF, the mean percentage, 5th and 95th percentiles of edges exhibiting dyskinetically beating cilia, cells with loss of cilia, cellular projections, cytoplasmic blebbing, mitochondrial damage and ultrastructural defects including microtubular, dynein arm or other defects of the 3 individual age groups and the whole group were calculated. A one way analysis of variance (ANOVA) was performed to detect a significant difference among individual groups. The mean CBF between children < 18 years and adults with and without SHS exposure in each individual age group and between subjects in the current study and the European study were compared using t-test. Inter-observer and intra-observer difference of CBF measurement were calculated by comparing the 95% confidence interval (CI).

We recruited 164 children (88 males, age range 2–17 years) and 50 adult volunteers from December 2015 to November 2016. We excluded 35 subjects because of known underlying diseases (11 children with allergic rhinitis [AR] with nasal congestion), URTI in previous 6 weeks (4 children with URTI alone and 11 also had underlying AR; 3 adults with URTI in previous 6 weeks alone and 2 also had underlying AR), inadequate samples (3 children) and being an active smoker (1 adult). Three of the 24 subjects with AR excluded were on intranasal medication. Samples from the remaining 135 children (67 males, age range 3–17 years) and 44 adults (25 males, age range 18–60 years) were analysed. The samples were processed for HSVM analysis and fixed for subsequent TEM with a mean of 5.5 h (range 1.5–11.5 h) from the time of nasal brushings [19]. Samples of 11 children aged 2–6 years and 10 children aged 13–17 years were processed at 9 to 11.5 h. Forty six children were exposed to SHS at home. Ten adults were exposed to SHS at home while 34 were not. The mean CBF and the percentage of dyskinetically beating edges for all subjects were summarized in Table 1. No significant difference was found in mean CBF (ANOVA, p = 0.542) and dyskinetically beating edges between the individual age groups (ANOVA, p = 0.212). The normal ciliary beat pattern from a subject showed coordinated cilia beating in a forward and backward motion along the whole epithelial edge (see Video 1) and the ciliated edges analysed that exhibited areas of dyskinetically beating cilia ranged from 18.0 to 24.2% (representative images of ciliated edge exhibited static cilia, see Video 2). There was also no significant difference in mean CBF between children with and without exposure to SHS for the 3 different age groups (ANOVA, p = 0.89 for children aged 2–6, p = 0.29 for children aged 7–12, p = 0.58 for children aged 13–17) but there was slightly higher mean CBF in adults with SHS exposure compared to adults without (ANOVA, p = 0.04) (Table 2). The mean CBF for children aged < 18 years was slightly higher than the adult group but it did not reach statistical significance [10.1 Hz (95% CI 9.8 to 10.4) versus 9.5 Hz (95% CI 8.9 to 10.0), ANOVA, p = 0.05]. Yet, it was lower than that of the European study [12.8 Hz (95% CI 12.3 to 13.3, ANOVA, p < 0.05)] [1]. There was no significant difference in mean CBF for samples processed within the plateau range of 3 to 9 h from collection [19] and those not within the plateau range for children aged < 18 years [10.52 Hz (95% CI 10.09 to 10.96) versus 10.50 Hz (95% CI 9.78 to 11.21), p = 0.969] and for adults [10.49 Hz (95% CI 9.81 to 11.16) versus 9.34 Hz (95% CI 8.44 to 10.24), p = 0.06].

For ciliary beat pattern, 1 subject in the 13–17 year group had a mixed ciliary beat pattern with dyskinetic cilia, cilia with a normal pattern and cilia with a circular beat pattern when viewed from above on occasional ciliated cells (see Video 3). The mean CBF and TEM were normal. Normal beat pattern was observed in the ciliated epithelium from all other subjects (representative images of normal ciliary beat pattern from above and towards the observer view, see Video 4 and Video 5).

To establish a reference range, mean CBF was plotted against the age of each subject (Fig. 1a). A weak negative correlation was found between mean CBF and increasing age (r² = 0.021). As cilia were found to beat at different frequencies within each sample, we also plotted sample variation in CBF, the ciliated edges with the highest and lowest CBF against age. The highest mean CBF of edges ranged from 7.3 to 24.3 Hz (Fig. 1b) with 93% of subjects having a maximal CBF of > 10 Hz. The lowest mean CBF of edges ranged from 2.3 to 10.4 Hz (Fig. 1c) with 45% of subjects having a minimum CBF of > 6 Hz.

No significant difference was found for inter-observer and intra-observer measurements of CBF. The mean (SD) for inter-observer measurement was 0.57 (1.87) (95% CI − 0.77 to 1.9; range − 2 to 4) and intra-observer was 0.01 (1.4) (95% CI − 1.02 to 1.03; range − 3 to 3).

Some subjects had an inadequate sample for ultrastructural analysis as the tissue might have been lost during initial HSVM assessment and before the sample was processed for TEM [1]. Among 179 subjects analysed with CBF and beat pattern, 121 subjects had sufficient tissue for epithelial integrity measurements and 159 subjects had tissue processed for ultrastructure analysis. The mean of cross sections examined for each subject was 56 (range: 40–98). The percentages of different cell types observed in the ciliated epithelial strips are summarized in Table 3. There was no significant difference between the percentages of different cells types across age groups (ANOVA, p > 0.1). Ciliated cells formed about 50% of the cell population.

The integrity of ciliated epithelium was assessed by examining factors including loss of cilia, cellular extrusion, cytoplasmic blebbing, and mitochondrial damage in Fig. 2. Normal with a normal healthy mitochondrion (arrow, bar = 1 μm) in Fig. 2a. Loss of cilia, grade 3, and a cell with a damaged mitochondrion (arrow, bar = 1 μm) in Fig. 2b. Cellular extrusion, grade 2, (bar = 2 μm) in Fig. 2c. Cytoplasmic blebbing, grade 2 (arrow, bar = 2 μm) in Fig. 2d. Evidence of minor epithelial damage was observed and the analysis is summarised in Table 4. There was no significant difference of epithelial integrity score across age groups (ANOVA, p = 0.07). Normal nasal epithelium with an intact ciliated surface and minimal disruption (epithelial integrity score = 0, bar = 2 μm) is shown in Fig. 3a and abnormal epithelium with severely disrupted cell surface and marked loss of cilia (epithelial integrity score = 4, bar = 2 μm) in Fig. 3b.

Ultrastructural analysis is summarised in Table 5. Abnormal cilia were observed in some subjects. Microtubule defects including disarranged tubules (0.7%), extra-tubule including extra-single tubule and extra-microtubular pair (2.1%), 8 + gap (1.0%) and single tubule (5.5%); central microtubules defects including extra-inter tubule (0.3%) and central pair damage (1.2%); compound cilia (0.5%) and combination of defects (0.9%) were identified in all of the cilia counted in children aged < 18 years (Fig. 4 b-i). The commonest defect identified was single micro-tubule (Fig. 4e).

The mean microtubule defects in the 13–17 year group (14.4%) were significantly higher than those in the other age groups (8.3% in 2–6 year group, 5.3% in 7–12 year group; ANOVA, p < 0.01). Central microtubules defects, compound cilia and other microtubule defects referring to a combination of defects were found in less than 3% of all of the cilia counted in children aged < 18 years (Table 5). There was no significant difference between age groups for the central microtubules defects, compound cilia and other microtubule defects (ANOVA, p > 0.05).