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Background Computed Tomography (CT) ventilation imaging (CTVI) is an emerging ventilation imaging technique. CTVI implementations have been widely validated against alternative ventilation imaging techniques but have been limited to clinical research only. The first CTVI commercial product, CT LVAS (4DMedical, Melbourne, Australia), was recently released enabling its use in clinical practice. This study quantitatively compares ventilation images from CT LVAS and previously validated research CTVI algorithms to Galligas PET ventilation. Methods 16 patients with Galligas PET and paired inhale/exhale breath-hold CT images were taken from a publicly available dataset on The Cancer Imaging Archive. Ventilation images were produced using CT LVAS and two previously published algorithms: (1) utilising the Hounsfield Unit difference (CTVI_HU); and (2) utilising the Jacobian determinant (CTVI_Jac). CTVI images were compared to the reference standard Galligas PET using Bland-Altman analysis of lobar ventilation, voxel-wise Spearman correlation, and Dice similarity coefficient (DSC) of regions of interest representing the top 85% and 15% of ventilation function. Results Bland-Altman analysis showed overall bias of < 0.01% for all CTVI methods (95% confidence interval: ±7.4% for CT LVAS, ± 9.1% for CTVI_HU, ± 7.9% for CTVI_Jac). The mean Spearman correlation between CTVI and Galligas PET was 0.61 ± 0.14 ( p  < 0.01) for CT LVAS, 0.68 ± 0.10 ( p  < 0.01) for CTVI_HU, and 0.57 ± 0.15 ( p  < 0.01) for CTVI_Jac. The mean DSC for the top 85% was 0.91 ± 0.03 for CT LVAS, 0.92 ± 0.02 for CTVI_HU, and 0.91 ± 0.03 for CTVI_Jac, with the DSC for CTVI_HU significantly higher than the other two CTVI methods. The DSC for the top 15% was 0.47 ± 0.17 for CT LVAS, 0.53 ± 0.16 for CTVI_HU, and 0.47 ± 0.18 for CTVI_Jac. Conclusions In a comparison to Galligas PET ventilation imaging, CT LVAS performs similarly to previous CTVI methods. Bland-Altman analysis for quantification of lobar ventilation demonstrates negligible bias. Mean voxel-wise Spearman correlations are moderate to good. DSC of functionally thresholded lung regions are similar for all CTVI methods. These results warrant further investigation of CT LVAS as a readily available ventilation imaging tool in disease characterisation, lung health assessment, and surgical and targeted treatment planning. Trial registration Australian New Zealand Clinical Trials Registry (ANZCTR) registration number ACTRN12612000775819, registered on 23/07/2012.

We present detailed quantitative measurement analyses for flow in a spinner flask with spinning rates between 20 to 45 RPM, utilizing the optical velocimetry measurement technique of Particle Image Velocimetry (PIV). A partial section of the impeller was immersed in the working fluid to reduce the shear forces induced on the cells cultured on microcarriers. Higher rotational speeds improved the mixing effect in the medium at the expense of a higher shear environment. It was found that the mouse induced pluripotent stem (iPS) cells achieved the optimum number of cells over 7 days in 25 RPM suspension culture. This condition translates to 0.0984 Pa of maximum shear stress caused by the interaction of the fluid flow with the bottom surface. However, inverse cell growth was obtained at 28 RPM culture condition. Such a narrow margin demonstrated that mouse iPS cells cultured on microcarriers are very sensitive to mechanical forces. This study provides insight to biomechanical parameters, specifically the shear stress distribution, for a commercially available spinner flask over a wide range of Reynolds number.

Most measures of lung health independently characterise either global lung function or regional lung structure. The ability to measure airflow and lung function regionally would provide a more specific and physiologically focused means by which to assess and track lung disease in both pre-clinical and clinical settings. One approach for achieving regional lung function measurement is via phase contrast X-ray imaging (PCXI), which has been shown to provide highly sensitive, high-resolution images of the lungs and airways in small animals. The detailed images provided by PCXI allow the application of four-dimensional X-ray velocimetry (4DxV) to track lung tissue motion and provide quantitative information on regional lung function. However, until recently synchrotron facilities were required to produce the highly coherent, high-flux X-rays that are required to achieve lung PCXI at a high enough frame rate to capture lung motion. This paper presents the first translation of 4DxV technology from a synchrotron facility into a laboratory setting by using a liquid-metal jet microfocus X-ray source. This source can provide the coherence required for PCXI and enough X-ray flux to image the dynamics of lung tissue motion during the respiratory cycle, which enables production of images compatible with 4DxV analysis. We demonstrate the measurements that can be captured in vivo in live mice using this technique, including regional airflow and tissue expansion. These measurements can inform physiological and biomedical research studies in small animals and assist in the development of new respiratory treatments.

Background Small airways disease is a feature of many respiratory conditions. Currently available methods of diagnosing small airways lack sensitivity and/or cannot evaluate spatial heterogeneity. New diagnostic strategies for diagnosing small airways disease are needed. Methods We determined the regional displacement of lung tissue calculated from fluoroscopic lung images acquired at multiple angles over sequential time points as a surrogate of ventilation. We applied this technique, which we call X-ray velocimetry (XV), to patients with chronic obstructive pulmonary disease (COPD) and impaired spirometry and veterans with deployment-related constrictive bronchiolitis (DR-CB) but preserved spirometry to determine XV-derived biomarkers specific for each condition. Results We identified disease- and stage-specific XV biomarkers for COPD patients that correlated with airflow obstruction on spirometry. Further, we identified a set of XV-derived biomarkers that could distinguish veterans with DR-CB from controls despite normal spirometry in most patients from both groups. Conclusions XV may provide a safe and widely-available strategy for diagnosing small airways disease while preserving spatial information. Future studies are required to validate the biomarkers described here in larger patient cohorts. Trial registration Not required for this study. However, participants enrolled at VUMC were enrolled under ClinicalTrials.gov study NCT04489758 (submitted 07/23/2020).

Rationale X-ray velocimetry (XV) has been utilized in preclinical models to assess lung motion and regional ventilation, though no studies have compared XV-derived physiologic parameters to measures derived through conventional means. Objectives To assess agreement between XV-analysis of fluoroscopic lung images and pitot tube flowmeter measures of ventilation. Methods XV- and pitot tube-derived ventilatory parameters were compared during tidal breathing and with bilevel-assisted breathing. Levels of agreement were assessed using the Bland-Altman analysis. Mixed models were used to characterize the association between XV- and pitot tube-derived values and optimize XV-derived values for higher ventilatory volumes. Measurements and main results Twenty-four healthy volunteers were assessed during tidal breathing and 11 were reassessed with increased minute ventilation with bilevel-assisted breathing. No clinically significant differences were observed between the two methods for respiratory rate (average Δ: 0.58; 95% limits of agreement: -1.55, 2.71) or duty cycle (average Δ: 0.02; 95% limits of agreement: 0.01, 0.03). Tidal volumes and flow rates measured using XV were lower than those measured using the pitot tube flowmeter, particularly at the higher volume ranges with bilevel-assisted breathing. Under these conditions, a mixed-model based adjustment was applied to the XV-derived values of tidal volume and flow rate to obtain closer agreement with the pitot tube-derived values. Conclusion Radiographically obtained measures of ventilation with XV demonstrate a high degree of correlation with parameters of ventilation. If the accuracy of XV were also confirmed for assessing the regional distribution of ventilation, it would provide information that goes beyond the scope of conventional pulmonary function tests or static radiographic assessments.

The aim of this study was to assess the association between regional tidal volume (Vt), regional functional residual capacity (FRC), and the expression of genes linked with ventilator-induced lung injury. Two groups of BALB/c mice (n = 8 per group) were ventilated for 2 hours using a protective or injurious ventilation strategy, with free-breathing mice used as control animals. Regional Vt and FRC of the ventilated mice was determined by analysis of high-resolution four-dimensional computed tomographic images taken at baseline and after 2 hours of ventilation and corrected for the volume of the region (i.e., specific [s]Vt and specific [s]FRC). RNA concentrations of 21 genes in 10 different lung regions were quantified using a quantitative PCR array. sFRC at baseline varied regionally, independent of ventilation strategy, whereas sVt varied regionally depending on ventilation strategy. The expression of IL-6 (P = 0.04), Ccl2 (P < 0.01), and Ang-2 (P < 0.05) was associated with sVt but not sFRC. The expression of seven other genes varied regionally (IL-1β and RAGE [receptor for advanced glycation end products]) or depended on ventilation strategy (Nfe2l2 [nuclear factor erythroid-derived 2 factor 2], c-fos, and Wnt1) or both (TNF-α and Cxcl2), but it was not associated with regional sFRC or sVt. These observations suggest that regional inflammatory responses to mechanical ventilation are driven primarily by tidal stretch.

To effectively diagnose, monitor and treat respiratory disease clinicians should be able to accurately assess the spatial distribution of airflow across the fine structure of lung. This capability would enable any decline or improvement in health to be located and measured, allowing improved treatment options to be designed. Current lung function assessment methods have many limitations, including the inability to accurately localise the origin of global changes within the lung. However, X-ray velocimetry (XV) has recently been demonstrated to be a sophisticated and non-invasive lung function measurement tool that is able to display the full dynamics of airflow throughout the lung over the natural breathing cycle. In this study we present two developments in XV analysis. Firstly, we show the ability of laboratory-based XV to detect the patchy nature of cystic fibrosis (CF)-like disease in β-ENaC mice. Secondly, we present a technique for numerical quantification of CF-like disease in mice that can delineate between two major modes of disease symptoms. We propose this analytical model as a simple, easy-to-interpret approach, and one capable of being readily applied to large quantities of data generated in XV imaging. Together these advances show the power of XV for assessing local airflow changes. We propose that XV should be considered as a novel lung function measurement tool for lung therapeutics development in small animal models, for CF and for other muco-obstructive diseases.

Background Lung inhomogeneity plays a pivotal role in the development of ventilator-induced lung injury (VILI), particularly in the context of pre-existing lung injury. The mechanisms that underlie this interaction are poorly understood. We aimed to elucidate the regional transcriptomic response to mechanical ventilation (MV), with or without pre-existing lung injury, and link this to the regional lung volume response to MV. Methods Adult female BALB/c mice were randomly assigned into one of four groups: Saline, MV, lipopolysaccharide (LPS) or LPS/MV. Lung volumes (tidal volume, Vt; end-expiratory volume, EEV) were measured at baseline or after 2 h of ventilation using four-dimensional computed tomography (4DCT). Regional lung tissue samples corresponding to specific imaging regions were analysed for the transcriptome response by RNA-Seq. Bioinformatics analyses were conducted and the regional expression of dysregulated gene clusters was then correlated with the lung volume response. Results MV in the absence of pre-existing lung injury was associated with regional variations in tidal stretch. The addition of LPS also caused regional increases in EEV. We identified 345, 141 and 184 region-specific differentially expressed genes in response to MV, LPS and LPS/MV, respectively. Amongst these candidate genes, up-regulation of genes related to immune responses were positively correlated with increased regional tidal stretch in the MV group, while dysregulation of genes associated with endothelial barrier related pathways were associated with increased regional EEV and Vt when MV was combined with LPS. Further protein–protein interaction analysis led to the identification of two protein clusters representing the PI3K/Akt and MEK/ERK signalling hubs which may explain the interaction between MV and LPS exposure. Conclusion The biological pathways associated with lung volume inhomogeneity during MV, and MV in the presence of pre-existing inflammation, differed. MV related tidal stretch induced up-regulation of immune response genes, while LPS combined with MV disrupted PI3K/Akt and MEK/ERK signalling.

Background: A sustained inflation (SI) facilitates lung aeration, but the most effective pressure and duration are unknown. We investigated the effect of gestational age (GA) and airway liquid volume on the required inflation pressure and SI duration. Methods: Rabbit kittens were delivered at 27, 29, and 30 d gestation, intubated and airway liquid was aspirated. Either no liquid (control) or 30 ml/kg of liquid was returned to the airways. Lung gas volumes were measured by plethysmography and phase-contrast X-ray-imaging. Starting at 22 cmH 2 O, airway pressure was increased until airflow commenced and pressure was then held constant. The SI was truncated when 20 ml/kg air had entered the lung and ventilation continued with intermittent positive pressure ventilation (iPPV). Results: Higher SI pressures and longer durations were required in 27-d kittens compared to 30-d kittens. During iPPV, 27-d kittens needed higher pressures and had lower functional residual capacity (FRC) compared to 30-d kittens. Adding lung liquid increased SI duration, reduced FRC, and increased resistance and pressures during iPPV in 29- and 30-d kittens. Conclusion: Immature kittens required higher starting pressures and longer SI durations to achieve a set inflation volume. Larger airway liquid volumes adversely affected lung function during iPPV in older but not young kittens.

In recent times, the study and use of induced pluripotent stem cells (iPSC) have become important in order to avoid the ethical issues surrounding the use of embryonic stem cells. Therapeutic, industrial and research based use of iPSC requires large quantities of cells generated in vitro. Mammalian cells, including pluripotent stem cells, have been expanded using 3D culture, however current limitations have not been overcome to allow a uniform, optimized platform for dynamic culture of pluripotent stem cells to be achieved. In the current work, we have expanded mouse iPSC in a spinner flask using Cytodex 3 microcarriers. We have looked at the effect of agitation on the microcarrier survival and optimized an agitation speed that supports bead suspension and iPS cell expansion without any bead breakage. Under the optimized conditions, the mouse iPSC were able to maintain their growth, pluripotency and differentiation capability. We demonstrate that microcarrier survival and iPS cell expansion in a spinner flask are reliant on a very narrow range of spin rates, highlighting the need for precise control of such set ups and the need for improved design of more robust systems.