Respiratory diseases are major causes of pediatric morbidity and mortality. These diseases are incompletely understood, which is a barrier to improving clinical care. Therefore, new mechanisms of disease need to be discovered. Optical imaging (e.g. optical coherence tomography [OCT]) will enable these discoveries since traditional imaging (e.g. x-ray, CT, MRI) cannot visualize structures smaller than ~1 mm. Microfluidic-scale cilia-driven fluid flow clears pathogen and allergen-containing mucus out of the lungs, yet we currently lack quantitative imaging technologies to characterize their flow performance. Moreover, while ciliary defects are traditionally considered a feature of rare but severe diseases (e.g. primary ciliary dyskinesia), development of biomechanical biomarkers extracted from quantitative flow imaging will allow us to test a paradigm-shifting hypothesis: intermediate defects in ciliary performance that are undetectable by current diagnostics are major modifiers of clinical severity in common respiratory diseases (e.g. asthma). Our research therefore has three aims. First, we will develop high-speed, cosine ambiguity-free Doppler OCT imaging systems. Cilia-driven fluid flow is three-dimensional in nature and not amenable to simplifying geometric assumptions such as parabolic flow profile. Traditional Doppler imaging suffers from cosine ambiguity that precludes the measurement of three-component flow velocities (v=vxi+vyj+vzk). We will develop a novel class of OCT interferometers that will enable three-dimensional, three-component flow imaging that will be demonstrated using the ciliated skin of Xenopus (tadpole) embryos, an important animal model in ciliary biology. Second, we will develop quantitative imaging assays of ciliary function that exploit cilia-driven microfluidic mixing. Taking a cue from work in biomimetic cilia, we have (a) demonstrated that ciliated biological surfaces can drive microfluidic mixing and (b) developed a novel microfluidic chip that uses a ciliated biological surface as a microfluidic "component." Building on these results, we will demonstrate that our microfluidic mixing-based assay can quantify biologically relevant perturbations to ciliary physiology including increased fluid viscosity and altered planar cell polarity. Third, we will demonstrate intermediate defects in Xenopus and mouse ciliary function using quantitative imaging. We will target two different classes of genes relevant in the performance of a ciliated surface in Xenopus embryos: ciliary molecular motors and notch signaling proteins (notch signaling controls the density of ciliated cells on the embryo skin). Given the importance of mouse models of pediatric respiratory disease, it is critical to demonstrate that our optical methods can be used to quantify the performance of mouse respiratory cilia. Moreover, this is an important step towards translating our diagnostic technologies to use in humans. We propose to use flow imaging to quantify performance modified by increased fluid viscosity (mechanical perturbation to decrease ciliary beat frequency) and increased extracellular ATP (pharmacological perturbation to increase ciliary beat frequency).
Respiratory diseases are very common in newborns and in children. These diseases are of significant public health interest because of their impact on pediatric morbidity and mortality. Our research will help us better understand the role of mucus-clearing cilia in pediatric respiratory disease and better diagnose clinically significant abnormalities in respiratory cilia.
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