The association between regional transcriptome profiles and lung volumes in response to mechanical ventilation and lung injury.

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.

Interaction between regional lung volumes and ventilator-induced lung injury in the normal and endotoxemic lung.

Both overdistension and atelectasis contribute to lung injury and mortality during mechanical ventilation. It has been proposed that combinations of tidal volume and end-expiratory lung volume exist that minimize lung injury linked to mechanical ventilation. The aim of this study was to examine this at the regional level in the healthy and endotoxemic lung. Adult female BALB/c mice were injected intraperitoneally with 10 mg/kg lipopolysaccharide (LPS) in saline or with saline alone. Four hours later, mice were mechanically ventilated for 2 h. Regional specific end-expiratory volume (sEEV) and tidal volume (sVt) were measured at baseline and after 2 h of ventilation using dynamic high-resolution four-dimensional computed tomography images. The regional expression of inflammatory genes was quantified by quantitative PCR. There was a heterogenous response in regional sEEV whereby endotoxemia increased gas trapping at end-expiration in some lung regions. Within the healthy group, there was a relationship between sEEV, sVt, and the expression of Tnfa, where high Vt in combination with high EEV or very low EEV was associated with an increase in gene expression. In endotoxemia there was an association between low sEEV, particularly when this was combined with moderate sVt, and high expression of IL6. Our data suggest that preexisting systemic inflammation modifies the relationship between regional lung volumes and inflammation and that although optimum EEV-Vt combinations to minimize injury exist, further studies are required to identify the critical inflammatory mediators to assess and the effect of different injury types on the response.

The Link between Regional Tidal Stretch and Lung Injury during Mechanical Ventilation.

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-1ß 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.

Imaging lung tissue oscillations using high-speed X-ray velocimetry.

This work utilized synchrotron imaging to achieve a regional assessment of the lung's response to imparted oscillations. The forced oscillation technique is increasingly being used in clinical and research settings for the measurement of lung function. During the forced oscillation technique, pressure oscillations are imparted to the lungs via the subjects' airway opening and the response is measured. This provides information about the mechanical properties of the airways and lung tissue. The quality of measurements is dependent upon the input signal penetrating uniformly throughout the lung. However, the penetration of these signals is not well understood. The development and use of a novel image-processing technique in conjunction with synchrotron-based imaging was able to regionally assess the lungs' response to input pressure oscillation signals in anaesthetized mice. The imaging-based technique was able to quantify both the power and distribution of lung tissue oscillations during forced oscillations of the lungs. It was observed that under forced oscillations the apices had limited lung tissue expansion relative to the base. This technique could be used to optimize input signals used for the forced oscillation technique or potentially as a diagnostic tool itself.

Mechanical ventilation decreases tidal volume heterogeneity but increases heterogeneity in end-expiratory volumes.

How the heterogeneous distribution of lung volumes changes in response to different mechanical ventilation (MV) strategies is unclear. Using our well-developed four-dimensional computed tomography (4DCT) high-resolution imaging technique, we aimed to assess the effect of different MV strategies on the distribution and heterogeneity of regional lung volumes. Healthy adult female BALB/c mice received either 2 h of "injurious" MV [n = 6, mechanical ventilation at high PIP with zero PEEP (HPZP)] with a peak inspiratory pressure (PIP) of 20 cmH2O and zero positive end-expiratory pressure (PEEP), or 2 h of "protective" MV [n = 8, mechanical ventilation at low PIP with PEEP (LPP)] with PIP = 12 cmH2O and PEEP = 2 cmH2O. 4DCT images were obtained at baseline (0 h) and after 2 h of MV. Tidal volume (Vt) and end-expiratory lung volume (EEV) were measured throughout the whole lung on a voxel-by-voxel basis. Heterogeneity of ventilation was determined by the coefficient of variation (COV) of Vt and EEV. Our data showed that MV had minimal impact on global Vt but decreased EEV in the HPZP group (P < 0.05). Both ventilation modes decreased the COV of Vt (39.4% for HPZP and 9.7% for LPP) but increased the COV in EEV (36.4% for HPZP and 29.2% for LPP). This was consistent with the redistribution index, which was significantly higher in the HVZP group than in the LPP group (P < 0.001). We concluded that regional assessment of the change in EEV showed different patterns in progression between LPP and HPZP strategies. Both ventilation strategies decreased heterogeneity in Vt after 2 h of MV but increased heterogeneity in EEV. Further work is required to determine the link between these effects and ventilator-induced lung injury.NEW & NOTEWORTHY Tidal volume heterogeneity decreases over time in response to mechanical ventilation, in contrast to end-expiratory volume heterogeneity which increases.

Vector tomographic X-ray phase contrast velocimetry utilizing dynamic blood speckle.

We present a time-resolved tomographic reconstruction of the velocity field associated with pulsatile blood flow through a rotationally-symmetric stenotic vessel model. The in-vitro sample was imaged using propagation-based phase contrast with monochromated X-rays from a synchrotron undulator source, and a fast shutter-synchronized detector with high-resolution used to acquire frames of the resulting dynamic speckle pattern. Having used phase retrieval to decode the phase contrast from the speckle patterns, the resulting projected-density maps were analysed using the statistical correlation methods of particle image velocimetry (PIV). This yields the probability density functions of blood-cell displacement within the vessel. The axial velocity-field component of the rotationally-symmetric flow was reconstructed using an inverse-Abel transform. A modified inverse-Abel transform was used to reconstruct the radial component. This vector tomographic phase-retrieval velocimetry was performed over the full pumping cycle, to completely characterize the velocity field of the pulsatile blood flow in both space and time.

The proteomic response is linked to regional lung volumes in ventilator-induced lung injury.

It is unclear how acid-induced lung injury alters the regional lung volume response to mechanical ventilation (MV) and how this impacts protein expression. Using a mouse model, we investigated the separate and combined effects of acid aspiration and MV on regional lung volumes and how these were associated with the proteome. Adult BALB/c mice were divided into four groups: intratracheal saline, intratracheal acid, saline/MV, or acid/MV. Specific tidal volume (sVt) and specific end-expiratory volume (sEEV) were measured at baseline and after 2 h of ventilation using dynamic high-resolution four-dimensional computed tomography (4DCT) images. Lung tissue was dissected into 10 regions corresponding to the image segmentation for label-free quantitative proteomic analysis. Our data showed that acid aspiration significantly reduced sVt and caused further reductions in sVt and sEEV after 2 h of ventilation. Proteomic analysis revealed 42 dysregulated proteins in both Saline/MV and Acid/MV groups, and 37 differentially expressed proteins in the Acid/MV group. Mapping of the overlapping proteins showed significant enrichment of complement/coagulation cascades (CCC). Analysis of 37 unique proteins in the Acid/MV group identified six additional CCC proteins and seven downregulated proteins involved in the mitochondrial respiratory chain (MRC). Regional MRC protein levels were positively correlated with sEEV, while the CCC protein levels were negatively associated with sVt. Therefore, this study showed that tidal volume was associated with the expression of CCC proteins, while low end-expiratory lung volumes were associated with MRC protein expression, suggesting that tidal stretch and lung collapse activate different injury pathways.NEW & NOTEWORTHY This study provides novel insights into the regional response to mechanical ventilation in the setting of acid-induced lung injury and highlights the complex interaction between tidal stretch and low-end-expiratory lung volumes; both of which caused altered regulation of different injury pathways.

Quantification of muco-obstructive lung disease variability in mice via laboratory X-ray velocimetry.

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 ß-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.

Real-time in vivo imaging of regional lung function in a mouse model of cystic fibrosis on a laboratory X-ray source.

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.

Cardiogenic Airflow in the Lung Revealed Using Synchrotron-Based Dynamic Lung Imaging.

The beating heart is known to produce pressure and airflow oscillations in the lungs of mammals. This phenomenon is often disregarded as detailed measurement of its effects in the lung have hitherto not been possible. Previous studies have attempted to measure the effect of these oscillations on gas mixing. However, the results have proven inconclusive, due to the lack of a direct measurement tool capable of flow measurement throughout the entire bronchial tree. Here we present the first detailed measurement of cardiogenic oscillations, using synchrotron-based dynamic lung imaging of live mechanically ventilated mice. The results demonstrate large flow oscillations and pendelluft in the airways due to the mechanical action of the beating heart. Using a virtual tracer modelling analysis we show that cardiogenic oscillations produced up to 4 times increased gas mixing, but only in the absence of tidal ventilation. The results highlight the importance of considering this often-disregarded phenomenon when investigating lung function, particularly in situations where tidal ventilation is reduced or absent.

Application of a novel in vivo imaging approach to measure pulmonary vascular responses in mice.

Noninvasive imaging of the murine pulmonary vasculature is challenging due to the small size of the animal, limits of resolution of the imaging technology, terminal nature of the procedure, or the need for intravenous contrast. We report the application of laboratory-based high-speed, high-resolution x-ray imaging, and image analysis to detect quantitative changes in the pulmonary vascular tree over time in the same animal without the need for intravenous contrast. Using this approach, we detected an increased number of vessels in the pulmonary vascular tree of animals after 30 min of recovery from a brief exposure to inspired gas with 10% oxygen plus 5% carbon dioxide (mean ± standard deviation: 2193 ± 382 at baseline vs. 6177 ± 1171 at 30 min of recovery; P < 0.0001). In a separate set of animals, we showed that the total pulmonary blood volume increased (P = 0.0412) while median vascular diameter decreased from 0.20 mm (IQR: 0.15-0.28 mm) to 0.18 mm (IQR: 0.14-0.26 mm; P = 0.0436) over the respiratory cycle from end-expiration to end-inspiration. These findings suggest that the noninvasive, nonintravenous contrast imaging approach reported here can detect dynamic responses of the murine pulmonary vasculature and may be a useful tool in studying these responses in models of disease.

Technical Note: Contrast free angiography of the pulmonary vasculature in live mice using a laboratory x-ray source.

PURPOSE: In vivo imaging of the pulmonary vasculature in small animals is difficult yet highly desirable in order to allow study of the effects of a host of dynamic biological processes such as hypoxic pulmonary vasoconstriction. Here the authors present an approach for the quantification of changes in the vasculature. METHODS: A contrast free angiography technique is validated in silico through the use of computer-generated images and in vivo through microcomputed tomography (µCT) of live mice conducted using a laboratory-based x-ray source. Subsequent image processing on µCT data allowed for the quantification of the caliber of pulmonary vasculature without the need for external contrast agents. These measures were validated by comparing with quantitative contrast microangiography in the same mice. RESULTS: Quantification of arterial diameters from the method proposed in this study is validated against laboratory-based x-ray contrast microangiography. The authors find that there is a high degree of correlation (R = 0.91) between measures from microangiography and their contrast free method. CONCLUSIONS: A technique for quantification of murine pulmonary vasculature without the need for contrast is presented. As such, this technique could be applied for longitudinal studies of animals to study changes to vasculature without the risk of premature death in sensitive mouse models of disease. This approach may also be of value in the clinical setting.

Quantification of heterogeneity in lung disease with image-based pulmonary function testing.

Computed tomography (CT) and spirometry are the mainstays of clinical pulmonary assessment. Spirometry is effort dependent and only provides a single global measure that is insensitive for regional disease, and as such, poor for capturing the early onset of lung disease, especially patchy disease such as cystic fibrosis lung disease. CT sensitively measures change in structure associated with advanced lung disease. However, obstructions in the peripheral airways and early onset of lung stiffening are often difficult to detect. Furthermore, CT imaging poses a radiation risk, particularly for young children, and dose reduction tends to result in reduced resolution. Here, we apply a series of lung tissue motion analyses, to achieve regional pulmonary function assessment in ß-ENaC-overexpressing mice, a well-established model of lung disease. The expiratory time constants of regional airflows in the segmented airway tree were quantified as a measure of regional lung function. Our results showed marked heterogeneous lung function in ß-ENaC-Tg mice compared to wild-type littermate controls; identified locations of airway obstruction, and quantified regions of bimodal airway resistance demonstrating lung compensation. These results demonstrate the applicability of regional lung function derived from lung motion as an effective alternative respiratory diagnostic tool.

Imaging regional lung function: a critical tool for developing inhaled antimicrobial therapies.

Alterations in regional lung function due to respiratory infection have a significant effect on the deposition of inhaled treatments. This has consequences for treatment effectiveness and hence recovery of lung function. In order to advance our understanding of respiratory infection and inhaled treatment delivery, we must develop imaging techniques that can provide regional functional measurements of the lung. In this review, we explore the role of functional imaging for the assessment of respiratory infection and development of inhaled treatments. We describe established and emerging functional lung imaging methods. The effect of infection on lung function is described, and the link between regional disease, function, and inhaled treatments is discussed. The potential for lung function imaging to provide unique insights into the functional consequences of infection, and its treatment, is also discussed.

Synchrotron-based dynamic computed tomography of tissue motion for regional lung function measurement.

During breathing, lung inflation is a dynamic process involving a balance of mechanical factors, including trans-pulmonary pressure gradients, tissue compliance and airway resistance. Current techniques lack the capacity for dynamic measurement of ventilation in vivo at sufficient spatial and temporal resolution to allow the spatio-temporal patterns of ventilation to be precisely defined. As a result, little is known of the regional dynamics of lung inflation, in either health or disease. Using fast synchrotron-based imaging (up to 60 frames s(-1)), we have combined dynamic computed tomography (CT) with cross-correlation velocimetry to measure regional time constants and expansion within the mammalian lung in vivo. Additionally, our new technique provides estimation of the airflow distribution throughout the bronchial tree during the ventilation cycle. Measurements of lung expansion and airflow in mice and rabbit pups are shown to agree with independent measures. The ability to measure lung function at a regional level will provide invaluable information for studies into normal and pathological lung dynamics, and may provide new pathways for diagnosis of regional lung diseases. Although proof-of-concept data were acquired on a synchrotron, the methodology developed potentially lends itself to clinical CT scanning and therefore offers translational research opportunities.

Functional lung imaging during HFV in preterm rabbits.

Although high frequency ventilation (HFV) is an effective mode of ventilation, there is limited information available in regard to lung dynamics during HFV. To improve the knowledge of lung function during HFV we have developed a novel lung imaging and analysis technique. The technique can determine complex lung motion information in vivo with a temporal resolution capable of observing HFV dynamics. Using high-speed synchrotron based phase contrast X-ray imaging and cross-correlation analysis, this method is capable of recording data in more than 60 independent regions across a preterm rabbit lung in excess of 300 frames per second (fps). This technique is utilised to determine regional intra-breath lung mechanics of preterm rabbit pups during HFV. Whilst ventilated at fixed pressures, each animal was ventilated at frequencies of 1, 3, 5 and 10 Hz. A 50% decrease in delivered tidal volume was measured at 10 Hz compared to 1 Hz, yet at the higher frequency a 500% increase in minute activity was measured. Additionally, HFV induced greater homogeneity of lung expansion activity suggesting this ventilation strategy potentially minimizes tissue damage and improves gas mixing. The development of this technique permits greater insight and further research into lung mechanics and may have implications for the improvement of ventilation strategies used to support severe pulmonary trauma and disease.

Altered lung motion is a sensitive indicator of regional lung disease.

Since lung diseases adversely affect airflow during breathing, they must also alter normal lung motion, which can be exploited to detect these diseases. However, standard imaging techniques such as CT and MRI imaging during breath-holds provide little or no information on lung motion and cannot detect diseases that cause subtle changes in lung structure. Phase-contrast X-ray imaging provides images of high contrast and spatial resolution with temporal resolutions that allow multiple images to be acquired throughout the respiratory cycle. Using X-ray phase-contrast imaging, coupled with velocimetry, we have measured lung tissue movement and determined velocity fields that define speed and direction of regional lung motion throughout a breath in normal Balb/c nude male mice and mice exposed to bleomycin. Regional maps of lung tissue motion reveal both the heterogeneity of normal lung motion, as well as abnormal motion induced by bleomycin treatment. Analysed histologically, bleomycin treatment caused pathological changes in lung structure that were heterogenous, occupying less than 12% of the lung at 6 days after treatment. Moreover, plethysmography failed to detect significant changes in compliance at either 36 h or 6 days after treatment. Detailed analysis of the vector fields demonstrated major differences (p < 0.001) in regional lung motion between control and bleomycin-treated mice at both 36 h and 6 days after treatment. The results of this study demonstrate that X-ray phase-contrast imaging, coupled with velocimetry, can detect early stage, subtle and non-uniform lung disease.

X-ray velocimetry and haemodynamic forces within a stenosed femoral model at physiological flow rates.

High resolution in vivo velocity measurements within the cardiovascular system are essential for accurate calculation of vessel wall shear stress, a highly influential factor for the progression of arterial disease. Unfortunately, currently available techniques for in vivo imaging are unable to provide the temporal resolution required for velocity measurement at physiological flow rates. Advances in technology and improvements in imaging systems are allowing a relatively new technique, X-ray velocimetry, to become a viable tool for such measurements. This study investigates the haemodynamics of pulsatile blood flow in an optically opaque in vitro model at physiological flow rates using X-ray velocimetry. The in vitro model, an asymmetric stenosis, is designed as a 3:1 femoral artery with the diameter and flow rate replicating vasculature of a mouse. Velocity measurements are obtained over multiple cycles of the periodic flow at high temporal and spatial resolution (1 ms and 29 µm, respectively) allowing accurate measurement of the velocity gradients and calculation of the wall shear stress. This study clearly illustrates the capability of in vitro X-ray velocimetry, suggesting it as a possible measurement technique for future in vivo vascular wall shear stress measurement.