Persistent mucus plugs in proximal airways are consequential for airflow limitation in asthma.

BACKGROUNDInformation about the size, airway location, and longitudinal behavior of mucus plugs in asthma is needed to understand their role in mechanisms of airflow obstruction and to rationally design muco-active treatments.METHODSCT lung scans from 57 patients with asthma were analyzed to quantify mucus plug size and airway location, and paired CT scans obtained 3 years apart were analyzed to determine plug behavior over time. Radiologist annotations of mucus plugs were incorporated in an image-processing pipeline to generate size and location information that was related to measures of airflow.RESULTSThe length distribution of 778 annotated mucus plugs was multimodal, and a 12 mm length defined short ("stubby", ≤12 mm) and long ("stringy", >12 mm) plug phenotypes. High mucus plug burden was disproportionately attributable to stringy mucus plugs. Mucus plugs localized predominantly to airway generations 6-9, and 47% of plugs in baseline scans persisted in the same airway for 3 years and fluctuated in length and volume. Mucus plugs in larger proximal generations had greater effects on spirometry measures than plugs in smaller distal generations, and a model of airflow that estimates the increased airway resistance attributable to plugs predicted a greater effect for proximal generations and more numerous mucus plugs.CONCLUSIONPersistent mucus plugs in proximal airway generations occur in asthma and demonstrate a stochastic process of formation and resolution over time. Proximal airway mucus plugs are consequential for airflow and are in locations amenable to treatment by inhaled muco-active drugs or bronchoscopy.TRIAL REGISTRATIONClinicaltrials.gov; NCT01718197, NCT01606826, NCT01750411, NCT01761058, NCT01761630, NCT01716494, and NCT01760915.FUNDINGAstraZeneca, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Sanofi-Genzyme-Regeneron, and TEVA provided financial support for study activities at the Coordinating and Clinical Centers beyond the third year of patient follow-up. These companies had no role in study design or data analysis, and the only restriction on the funds was that they be used to support the SARP initiative.

A Novel Air Trapping Segment Score Identifies Opposing Effects of Obesity and Eosinophilia on Air Trapping in Asthma.

Rationale: Density thresholds in computed tomography (CT) lung scans quantify air trapping (AT) at the whole-lung level but are not informative for AT in specific bronchopulmonary segments. Objectives: To apply a segment-based measure of AT in asthma to investigate the clinical determinants of AT in asthma. Methods: In each of 19 bronchopulmonary segments in CT lung scans from 199 patients with asthma, AT was categorized as present if lung attenuation was less than -856 Hounsfield units at expiration in ⩾15% of the lung area. The resulting AT segment score (0-19) was related to patient outcomes. Measurements and Main Results: AT varied at the lung segment level and tended to persist at the patient and lung segment levels over 3 years. Patients with widespread AT (⩾10 segments) had more severe asthma (P < 0.05). The mean (±SD) AT segment score in patients with a body mass index ⩾30 kg/m2 was lower than in patients with a body mass index <30 kg/m2 (3.5 ± 4.6 vs. 5.5 ± 6.3; P = 0.008), and the frequency of AT in lower lobe segments in obese patients was less than in upper and middle lobe segments (35% vs. 46%; P = 0.001). The AT segment score in patients with sputum eosinophils ⩾2% was higher than in patients without sputum eosinophilia (7.0 ± 6.1 vs. 3.3 ± 4.9; P < 0.0001). Lung segments with AT more frequently had airway mucus plugging than lung segments without AT (48% vs. 18%; P ⩽ 0.0001). Conclusions: In patients with asthma, air trapping is more severe in those with airway eosinophilia and mucus plugging, whereas those who are obese have less severe trapping because their lower lobe segments are spared.

Mucus Plugs Persist in Asthma, and Changes in Mucus Plugs Associate with Changes in Airflow over Time.

Rationale: Cross-sectional analysis of mucus plugs in computed tomography (CT) lung scans in the Severe Asthma Research Program (SARP)-3 showed a high mucus plug phenotype. Objectives: To determine if mucus plugs are a persistent asthma phenotype and if changes in mucus plugs over time associate with changes in lung function. Methods: In a longitudinal analysis of baseline and Year 3 CT lung scans in SARP-3 participants, radiologists generated mucus plug scores to assess mucus plug persistence over time. Changes in mucus plug score were analyzed in relation to changes in lung function and CT air trapping measures. Measurements and Main Results: In 164 participants, the mean (range) mucus plug score was similar at baseline and Year 3 (3.4 [0-20] vs. 3.8 [0-20]). Participants and bronchopulmonary segments with a baseline plug were more likely to have plugs at Year 3 than those without baseline plugs (risk ratio, 2.8; 95% confidence interval [CI], 2.0-4.1; P < 0.001; and risk ratio, 5.0; 95% CI, 4.5-5.6; P < 0.001, respectively). The change in mucus plug score from baseline to Year 3 was significantly negatively correlated with change in FEV1% predicted (rp = -0.35; P < 0.001) and with changes in CT air trapping measures (all P values < 0.05). Conclusions: Mucus plugs identify a persistent asthma phenotype, and susceptibility to mucus plugs occurs at the subject and the bronchopulmonary segment level. The association between change in mucus plug score and change in airflow over time supports a causal role for mucus plugs in mechanisms of airflow obstruction in asthma.

Multi-modal and multiscale imaging approaches reveal novel cardiovascular pathophysiology in Drosophila melanogaster.

Establishing connections between changes in linear DNA sequences and complex downstream mesoscopic pathology remains a major challenge in biology. Herein, we report a novel, multi-modal and multiscale imaging approach for comprehensive assessment of cardiovascular physiology in Drosophila melanogaster We employed high-speed angiography, optical coherence tomography (OCT) and confocal microscopy to reveal functional and structural abnormalities in the hdp2 mutant, pre-pupal heart tube and aorta relative to controls. hdp2 harbor a mutation in wupA, which encodes an ortholog of human troponin I (TNNI3). TNNI3 variants frequently engender cardiomyopathy. We demonstrate that the hdp2 aortic and cardiac muscle walls are disrupted and that shorter sarcomeres are associated with smaller, stiffer aortas, which consequently result in increased flow and pulse wave velocities. The mutant hearts also displayed diastolic and latent systolic dysfunction. We conclude that hdp2 pre-pupal hearts are exposed to increased afterload due to aortic hypoplasia. This may in turn contribute to diastolic and subtle systolic dysfunction via vascular-heart tube interaction, which describes the effect of the arterial loading system on cardiac function. Ultimately, the cardiovascular pathophysiology caused by a point mutation in a sarcomeric protein demonstrates that complex and dynamic micro- and mesoscopic phenotypes can be mechanistically explained in a gene sequence- and molecular-specific manner.

Visualization and quantification of injury to the ciliated epithelium using quantitative flow imaging and speckle variance optical coherence tomography.

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.

Ultrahigh-speed, phase-sensitive full-field interferometric confocal microscopy for quantitative microscale physiology.

We developed ultra-high-speed, phase-sensitive, full-field reflection interferometric confocal microscopy (FFICM) for the quantitative characterization of in vivo microscale biological motions and flows. We demonstrated 2D frame rates in excess of 1 kHz and pixel throughput rates up to 125 MHz. These fast FFICM frame rates were enabled by the use of a low spatial coherence, high-power laser source. Specifically, we used a dense vertical cavity surface emitting laser (VCSEL) array that synthesized low spatial coherence light through a large number of narrowband, mutually-incoherent emitters. Off-axis interferometry enabled single-shot acquisition of the complex-valued interferometric signal. We characterized the system performance (~2 µm lateral resolution, ~8 µm axial gating depth) with a well-known target. We also demonstrated the use of this highly parallelized confocal microscopy platform for visualization and quantification of cilia-driven surface flows and cilia beat frequency in an important animal model (Xenopus embryos) with >1 kHz frame rate. Such frame rates are needed to see large changes in local flow velocity over small distance (high shear flow), in this case, local flow around a single ciliated cell. More generally, our results are an important demonstration of low-spatial coherence, high-power lasers in high-performance, quantitative biomedical imaging.

Particle streak velocimetry-optical coherence tomography: a novel method for multidimensional imaging of microscale fluid flows.

We present a new OCT method for flow speed quantification and directional velocimetry: particle streak velocimetry-OCT (PSV-OCT). PSV-OCT generates two-dimensional, 2.5-vector component (vx ,|vy |,vz ) maps of microscale flow velocity fields. Knowledge of 2.5-vector components also enables the estimation of total flow speed. The enabling insight behind PSV-OCT is that tracer particles in sparsely-seeded fluid flow trace out streaks in (x,z,t)-space. The streak orientations in x-t and z-t yield vx and vz , respectively. The in-plane (x-z plane) residence time yields the out-of-plane speed |vy |. Vector component values are generated by fitting streaks to a model of image formation that incorporates equations of motion in 3D space. We demonstrate cross-sectional estimation of (vx ,|vy |,vz ) in two important animal models in ciliary biology: Xenopus embryos (tadpoles) and mouse trachea.

Erratum: Particle streak velocimetry-optical coherence tomography: a novel method for multidimensional imaging of microscale fluid flows: erratum.

[This corrects the article on p. 1590 in vol. 7.].

Improved velocimetry in optical coherence tomography using Bayesian analysis.

OCT is a popular cross-sectional microscale imaging modality in medicine and biology. While structural imaging using OCT is a mature technology in many respects, flow and motion estimation using OCT remains an intense area of research. In particular, there is keen interest in maximizing information extraction from the complex-valued OCT signal. Here, we introduce a Bayesian framework into the data workflow in OCT-based velocimetry. We demonstrate that using prior information in this Bayesian framework can significantly improve velocity estimate precision in a correlation-based, model-based framework for Doppler and transverse velocimetry. We show results in calibrated flow phantoms as well as in vivo in a Drosophila melanogaster (fruit fly) heart. Thus, our work improves upon the current approaches in terms of improved information extraction from the complex-valued OCT signal.

Three-dimensional, three-vector-component velocimetry of cilia-driven fluid flow using correlation-based approaches in optical coherence tomography.

Microscale quantification of cilia-driven fluid flow is an emerging area in medical physiology, including pulmonary and central nervous system physiology. Cilia-driven fluid flow is most completely described by a three-dimensional, three-component (3D3C) vector field. Here, we generate 3D3C velocimetry measurements by synthesizing higher dimensional data from lower dimensional measurements obtained using two separate optical coherence tomography (OCT)-based approaches: digital particle image velocimetry (DPIV) and dynamic light scattering (DLS)-OCT. Building on previous work, we first demonstrate directional DLS-OCT for 1D2C velocimetry measurements in the sub-1 mm/s regime (sub-2.5 inch/minute regime) of cilia-driven fluid flow in Xenopus epithelium, an important animal model of the ciliated respiratory tract. We then extend our analysis toward 3D3C measurements in Xenopus using both DLS-OCT and DPIV. We demonstrate the use of DPIV-based approaches towards flow imaging of Xenopus cerebrospinal fluid and mouse trachea, two other important ciliary systems. Both of these flows typically fall in the sub-100 µm/s regime (sub-0.25 inch/minute regime). Lastly, we develop a framework for optimizing the signal-to-noise ratio of 3D3C flow velocity measurements synthesized from 2D2C measures in non-orthogonal planes. In all, 3D3C OCT-based velocimetry has the potential to comprehensively characterize the flow performance of biological ciliated surfaces.

Quantifying hyperoxia-mediated damage to mammalian respiratory cilia-driven fluid flow using particle tracking velocimetry optical coherence tomography.

Oxygen supplementation [hyperoxia, increased fraction of inspired oxygen (FiO 2 )] is an indispensable treatment in the intensive care unit for patients in respiratory failure. Like other treatments or drugs, hyperoxia has a risk-benefit profile that guides its clinical use. While hyperoxia is known to damage respiratory epithelium, it is unknown if damage can result in impaired capacity to generate cilia-driven fluid flow. Here, we demonstrate that quantifying cilia-driven fluid flow velocities in the sub-100 µm/s regime (sub-0.25 in./min regime) reveals hyperoxia-mediated damage to the capacity of ciliated respiratory mucosa to generate directional flow. Flow quantification was performed using particle tracking velocimetry optical coherence tomography (PTV-OCT) in ex vivo mouse trachea. The ability of PTV-OCT to detect biomedically relevant flow perturbations in the sub-100 µm/s regime was validated by quantifying temperature- and drug-mediated modulation of flow performance in ex vivo mouse trachea. Overall, PTV-OCT imaging of cilia-driven fluid flow in ex vivo mouse trachea is a powerful and straightforward approach for studying factors that modulate and damage mammalian respiratory ciliary physiology.

Quantitative optical coherence tomography imaging of intermediate flow defect phenotypes in ciliary physiology and pathophysiology.

Cilia-driven fluid flow is a critical yet poorly understood aspect of pulmonary physiology. Here, we demonstrate that optical coherence tomography-based particle tracking velocimetry can be used to quantify subtle variability in cilia-driven flow performance in Xenopus, an important animal model of ciliary biology. Changes in flow performance were quantified in the setting of normal development, as well as in response to three types of perturbations: mechanical (increased fluid viscosity), pharmacological (disrupted serotonin signaling), and genetic (diminished ciliary motor protein expression). Of note, we demonstrate decreased flow secondary to gene knockdown of kif3a, a protein involved in ciliogenesis, as well as a dose-response decrease in flow secondary to knockdown of dnah9, an important ciliary motor protein.

Microscale imaging of cilia-driven fluid flow.

Cilia-driven fluid flow is important for multiple processes in the body, including respiratory mucus clearance, gamete transport in the oviduct, right-left patterning in the embryonic node, and cerebrospinal fluid circulation. Multiple imaging techniques have been applied toward quantifying ciliary flow. Here, we review common velocimetry methods of quantifying fluid flow. We then discuss four important optical modalities, including light microscopy, epifluorescence, confocal microscopy, and optical coherence tomography, that have been used to investigate cilia-driven flow.

Resolving directional ambiguity in dynamic light scattering-based transverse motion velocimetry in optical coherence tomography.

Dynamic light scattering-based optical coherence tomography approaches have been successfully implemented to measure total transverse (xy) flow speed, but are unable to resolve directionality. We propose a method to extract directional velocity in the transverse plane by introducing a variable scan bias to our system. Our velocity estimation, which yields the directional velocity component along the scan axis, is also independent of any point-spread function calibration. By combining our approach with Doppler velocimetry, we show three-component velocimetry that is appropriately dependent on latitudinal and longitudinal angle.