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Sci Rep
2017 Nov 08;71:15115. doi: 10.1038/s41598-017-14670-9.
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Visualization and quantification of injury to the ciliated epithelium using quantitative flow imaging and speckle variance optical coherence tomography.
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Mucociliary flow is an important defense mechanism in the lung to remove inhaled pathogens and pollutants. Disruption of ciliary flow can lead to respiratory infections. Multiple factors, from drugs to disease can cause an alteration in ciliary flow. However, less attention has been given to injury of the ciliated epithelium. In this study, we show how optical coherence tomography (OCT) can be used to investigate injury to the ciliated epithelium in a multi-contrast setting. We used particle tracking velocimetry (PTV-OCT) to investigate the cilia-driven flow field and 3D speckle variance imaging to investigate size and extent of injury caused to the skin of Xenopus embryos. Two types of injuries are investigated, focal injury caused by mechanical damage and diffuse injury by a calcium chloride shock. We additionally investigate injury and regeneration of cilia to calcium chloride on ex vivo mouse trachea. This work describes how OCT can be used as a tool to investigate injury and regeneration in ciliated epithelium.
Figure 1.
Ex vivo tracheal explant culture. (a) Schematic of culture setup. (b) Photograph of petri dish housing a gelatin sponge upon which a trachea is placed. (c) Close-up photograph of trachea on gelatin sponge.
Figure 2. Imaging of ciliated Xenopus embryo skin before (a,c,e,g) and after focal injury (b,d,f,h). (a,b) Maximum intensity projection of a 100-frame video allows the visualization of tracer particles as particle streaks. The injury is visible as indentation in the embryos surface (marked with an arrow in b). (c,d) Vector flow field quantification of cilia-driven flow. The vector flow field is disrupted around location of injury (panel d). (e–h) 3D rendered speckle variance images visualize beating ciliary patches on embryo surface (panels e and f- view from top; panels g and h- view from side). The injury is visible as area with low speckle variance (white circle) in panel f and as indentation in panel h. The scale bar in panel a measures 0.5 mm.
Figure 3. Imaging of ciliated frog embryo skin before (a,c,e,g) and after CaCl2-shock (b,d,f,h). (a,b) Maximum intensity projection of a 100-frame video allows the visualization of flowing tracer particles as particle streaks. After application of CaCl2 flow is disrupted along the whole embryo surface and tracer particles appear stationary. (c,d) Vector flow field of cilia-driven flow. The vector field is non-existent after application of CaCl2 (panel d). (e,f) 3D rendered speckle variance imaging. After CaCl2-induced loss of cilia, the surface appears smooth with uniform grey intensity (panel f). The scale bar in panel a measures 0.5 mm.
Figure 4. (a) PTV-OCT results before and after application of CaCl2 to 6 Xenopus embryos. T1-T6 describes each individual animal. (b) Percentage decrease in flow speed after CaCl2 application. Error bars depict standard error. Wilcoxon signed rank test shows a significant decrease in flow speed (p = 0.03).
Figure 5. OCT Imaging of mouse trachea before (a,d,g), after CaCl2-shock (b,e,h) and after regeneration 8 days later (c,f,i). (a–c) Maximum intensity projection of a 200-frame video allows the visualization of flowing tracer particles as particle streaks. After application of CaCl2 flow is disrupted along the whole epithelium and tracer particles appear stationary. Flow is restored after 8 days of ex vivo culture. (d–f) Vector flow field of cilia-driven flow, gained by PTV analysis. The vector flow field shows very small and undirected movement of tracer beads after application of CaCl2 and complete restoration after regeneration. (g–i) 3D rendered speckle variance images visualize beating cilia as area of high image intensity. After CaCl2-induced loss of beating cilia, the surface appears spotted with an increase of low variance area h). After cilia restore, the surface appears smooth with high speckle variance again i). The scale bar in a) measures 0.5 mm.
Figure 6. PTV results before and after application of CaCl2 to 7 mouse tracheas and after regeneration 8 days later. P-values were calculated using the Wilcoxon signed rank test.
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