Mucociliary Transport Deficiency and Disease Progression in Syrian Hamsters with SARS-CoV-2 Infection

Abstract

Substantial clinical evidence supports the notion that ciliary function in the airways plays an important role in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, consequent impaired mucociliary transport (MCT) remains unknown for the intact MCT apparatus from an in vivo model of disease. Using golden Syrian hamsters, a common animal model that recapitulates human COVID-19, we quantitatively followed the time course of physiological, virological, and pathological changes upon SARS-CoV-2 infection, as well as the deficiency of the MCT apparatus using micro-optical coherence tomography, a novel method to visualize and simultaneously quantitate multiple aspects of the functional microanatomy of intact airways. Corresponding to progressive weight loss up to 7 days post-infection (dpi), viral detection and histopathological analysis in both the trachea and lung revealed steadily descending infection from the upper airways, as the main target of viral invasion, to lower airways and parenchymal lung, which are likely injured through indirect mechanisms. SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 dpi, largely due to diminished motile ciliation coverage, but not airway surface liquid depth, periciliary liquid depth, or cilia beat frequency of residual motile cilia. Further analysis indicated that the fewer motile cilia combined with abnormal ciliary motion of residual cilia contributed to the delayed MCT. The time course of physiological, virological, and pathological progression suggest that functional deficits of the MCT apparatus predispose to COVID-19 pathogenesis by extending viral retention and may be a risk factor for secondary infection. As a consequence, therapies directed towards the MCT apparatus deserve further investigation as a treatment modality.

Figure 1.. Reduced body weight in hamsters through 7 days post-infection with SARS-CoV-2.

Golden Syrian hamsters were inoculated intranasally with 3×10⁵ plaque-forming units of SARS-CoV-2 or vehicle (mock), and body weight was monitored up to 7 days post-infection (dpi; N=8–25 for infected and N=4–12 for mock). P<0.0001 for time, infection, and interaction by two-way ANOVA; **P<0.01, ***P<0.001 and ****P<0.0001 by Šídák’s posthoc test.

Figure 2.. SARS-CoV-2 detection in hamsters through 7 days post-infection.

Golden Syrian hamsters were inoculated in Fig.1. Then samples were collected at 2, 4, and 7 dpi. Q-RT²-PCR vs. standard curve quantitated genomic (A) and subgenomic (B) viral titers for nasal brush, nasal wash, bronchial alveolar lavage fluid (BALF), oral swab, rectal swab, and serum. Viral titers in all types of samples from mock were under the detection limit, therefore, not shown. Subgenomic viral titers in serum, rectal swab, and most oral swabs were below quantitation limits by PCR, and are shown as 0. The dotted line indicates the limits of the PCR method. Square represents male and circle female. Values in log₁₀(copies/ml) were used for statistical analysis. For nasal brush, P<0.0001 and P=0.0161 for genomic and subgenomic titers, respectively, by one-way ANOVA with **P<0.01 (shown by a bracket) by following Tukey’s posthoc test. For nasal wash and BALF, **P<0.01 and ****P<0.0001 (shown by straight lines) by unpaired t-test. Representative images of the whole left lobe slices with SARS-CoV-2 detection by RNAscope® (C), with high power view of the airway (left) and parenchymal (right) at 2 dpi (D).

Figure 3.. Lung injury after SARS-CoV-2 infection in hamsters.

Golden Syrian hamsters were inoculated with SARS-CoV-2 as in Fig.1, then lung damage assessed by lung to body weight ratio (A), gross pathology (B), and histopathological analysis of the left lung, including representative hematoxylin and eosin (H&E) images (C-E) and quantitation by a blinded pathologist (F). (C) 100x (top) and 200x (bottom) total magnification of lungs from mock, 4dpi, and 7dpi displaying progression to interstitial pneumonia; 400x total magnification of (D) alveolus and alveolar interstitium displaying type II pneumocyte hyperplasia and mononuclear infiltrate and (E) a small-caliber artery displaying perivascular edema, hemorrhage, and intimal arteritis. Square is for males and circle for females. P<0.0001 (A and F) by one-way ANOVA, with *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 by Tukey’s post-hoc test.

Figure 4.. Mucociliary dysfunction in hamster airways after SARS-CoV-2 infection.

Golden Syrian hamsters were inoculated in Fig.1 and following excision, tracheas were imaged by micro-optical coherence tomography (μOCT, A-G). Representative μOCT images from mock and 4 dpi (A) and M-mode projections of μOCT videos (B), ep: epithelium; mu: mucus. Mucociliary transport (MCT) rate (C), degree of active ciliation coverage (D), depths of airway surface liquid (ASL, E) and periciliary layer (PCL, F), and ciliary beat frequency (CBF, G) are shown. Square is for males and circle for females. P=0.0099, P<0.0001, P=0.0117, P=0.1106, P=0.7808 by one-way ANOVA for C-H, respectively; *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 by Tukey’s post-hoc test.

Figure 4.. Mucociliary dysfunction in hamster airways after SARS-CoV-2 infection.

Golden Syrian hamsters were inoculated in Fig.1 and following excision, tracheas were imaged by micro-optical coherence tomography (μOCT, A-G). Representative μOCT images from mock and 4 dpi (A) and M-mode projections of μOCT videos (B), ep: epithelium; mu: mucus. Mucociliary transport (MCT) rate (C), degree of active ciliation coverage (D), depths of airway surface liquid (ASL, E) and periciliary layer (PCL, F), and ciliary beat frequency (CBF, G) are shown. Square is for males and circle for females. P=0.0099, P<0.0001, P=0.0117, P=0.1106, P=0.7808 by one-way ANOVA for C-H, respectively; *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 by Tukey’s post-hoc test.

Figure 5.. Tracheal injury after SARS-CoV-2 infection in hamsters.

Golden Syrian hamsters were inoculated with SARS-CoV-2, then tracheas and lungs were processed for histopathological analysis. Representative H&E images (A) and quantitation by a blinded pathologist (B) are shown. Unstained slides were labeled with α tubulin by immunohistochemistry to specifically focus on ciliary injury. Representative images are shown of trachea (C), and quantitation of cilia coverage along the apical surface of the tracheal (D), bronchi (E), and bronchiolar (F) epithelia by a blinded investigator. Square is for males and circle for females. P<0.0001 (B and D) by one-way ANOVA, with *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 by Tukey’s post-hoc test.

Figure 5.. Tracheal injury after SARS-CoV-2 infection in hamsters.

Golden Syrian hamsters were inoculated with SARS-CoV-2, then tracheas and lungs were processed for histopathological analysis. Representative H&E images (A) and quantitation by a blinded pathologist (B) are shown. Unstained slides were labeled with α tubulin by immunohistochemistry to specifically focus on ciliary injury. Representative images are shown of trachea (C), and quantitation of cilia coverage along the apical surface of the tracheal (D), bronchi (E), and bronchiolar (F) epithelia by a blinded investigator. Square is for males and circle for females. P<0.0001 (B and D) by one-way ANOVA, with *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 by Tukey’s post-hoc test.

Figure 6.. Association of MCC functional parameters with delayed MCT in SARS-CoV-2 infection.

Linear correlation analysis of MCT with CBF (A); and PCL depth (B) in the presence and absence of SARS-CoV-2 infection. PCL: Periciliary liquid layer depth; MCT: Mucociliary transport rate; CBF: Ciliary beat frequency

Figure 7.. Abnormal ciliary beat frequency map and ciliary motion in SARS-CoV-2 infected hamsters.

Compared to mock controls (A), fewer cilia with intact and maintained ciliary beat frequency was evident in SARS-CoV-2 infected hamsters (A’). Two representative waveform analysis of detected cilia exhibited consistent amplitude and frequency in trachea from mock controls (B) compared to erratic amplitude and irregular beat patterns in residual motile cilia in trachea from SARS-CoV-2 infected hamsters (B’).