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.

DNA-PKcs modulates progenitor cell proliferation and fibroblast senescence in idiopathic pulmonary fibrosis.

BACKGROUND: Recent studies have highlighted the contribution of senescent mesenchymal and epithelial cells in Idiopathic Pulmonary Fibrosis (IPF), but little is known regarding the molecular mechanisms that regulate the accumulation of senescent cells in this disease. Therefore, we addressed the hypothesis that the loss of DNA repair mechanisms mediated by DNA protein kinase catalytic subunit (DNA-PKcs) in IPF, promoted the accumulation of mesenchymal progenitors and progeny, and the expression of senescent markers by these cell types. METHODS: Surgical lung biopsy samples and lung fibroblasts were obtained from patients exhibiting slowly, rapidly or unknown progressing IPF and lung samples lacking any evidence of fibrotic disease (i.e. normal; NL). The expression of DNA-Pkcs in lung tissue was assessed by quantitative immunohistochemical analysis. Chronic inhibition of DNA-PKcs kinase activity was mimicked using a highly specific small molecule inhibitor, Nu7441. Proteins involved in DNA repair (stage-specific embryonic antigen (SSEA)-4+ cells) were determined by quantitative Ingenuity Pathway Analysis of transcriptomic datasets (GSE103488). Lastly, the loss of DNA-PKc was modeled in a humanized model of pulmonary fibrosis in NSG SCID mice genetically deficient in PRKDC (the transcript for DNA-PKcs) and treated with Nu7441. RESULTS: DNA-PKcs expression was significantly reduced in IPF lung tissues. Chronic inhibition of DNA-PKcs by Nu7441 promoted the proliferation of SSEA4+ mesenchymal progenitor cells and a significant increase in the expression of senescence-associated markers in cultured lung fibroblasts. Importantly, mesenchymal progenitor cells and their fibroblast progeny derived from IPF patients showed a loss of transcripts encoding for DNA damage response and DNA repair components. Further, there was a significant reduction in transcripts encoding for PRKDC (the transcript for DNA-PKcs) in SSEA4+ mesenchymal progenitor cells from IPF patients compared with normal lung donors. In SCID mice lacking DNA-PKcs activity receiving IPF lung explant cells, treatment with Nu7441 promoted the expansion of progenitor cells, which was observed as a mass of SSEA4+ CgA+ expressing cells. CONCLUSIONS: Together, our results show that the loss of DNA-PKcs promotes the expansion of SSEA4+ mesenchymal progenitors, and the senescence of their mesenchymal progeny.

Lung hypoplasia in newborn rabbits with a diaphragmatic hernia affects pulmonary ventilation but not perfusion.

BackgroundA congenital diaphragmatic hernia (DH) can result in severe lung hypoplasia that increases the risk of morbidity and mortality after birth; however, little is known about the cardiorespiratory transition at birth.MethodsUsing phase-contrast X-ray imaging and angiography, we examined the cardiorespiratory transition at birth in rabbit kittens with DHs. Surgery was performed on pregnant New Zealand white rabbits (n=18) at 25 days' gestation to induce a left-sided DH. Kittens were delivered at 30 days' gestation, intubated, and ventilated to achieve a tidal volume (Vt) of 8 ml/kg in control and 4 ml/kg in DH kittens while they were imaged.ResultsFunctional residual capacity (FRC) recruitment and Vt in the hypoplastic left lung were markedly reduced, resulting in a disproportionate distribution of FRC into the right lung. Following lung aeration, relative pulmonary blood flow (PBF) increased equally in both lungs, and the increase in pulmonary venous return was similar in both control and DH kittens.ConclusionThese findings indicate that nonuniform lung hypoplasia caused by DH alters the distribution of ventilation away from hypoplastic and into normally grown lung regions. During transition, the increase in PBF and pulmonary venous return, which is vital for maintaining cardiac output, is not affected by lung hypoplasia.

Tracking extended mucociliary transport activity of individual deposited particles: longitudinal synchrotron X-ray imaging in live mice.

To assess potential therapies for respiratory diseases in which mucociliary transit (MCT) is impaired, such as cystic fibrosis and primary ciliary dyskinesia, a novel and non-invasive MCT quantification method has been developed in which the transit rate and behaviour of individual micrometre-sized deposited particles are measured in live mice using synchrotron phase-contrast X-ray imaging. Particle clearance by MCT is known to be a two-phase process that occurs over a period of minutes to days. Previous studies have assessed MCT in the fast-clearance phase, ∼20 min after marker particle dosing. The aim of this study was to non-invasively image changes in particle presence and MCT during the slow-clearance phase, and simultaneously determine whether repeat synchrotron X-ray imaging of mice was feasible over periods of 3, 9 and 25 h. All mice tolerated the repeat imaging procedure with no adverse effects. Quantitative image analysis revealed that the particle MCT rate and the number of particles present in the airway both decreased with time. This study successfully demonstrated for the first time that longitudinal synchrotron X-ray imaging studies are possible in live small animals, provided appropriate animal handling techniques are used and care is taken to reduce the delivered radiation dose.

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.

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.

Novel analysis of 4DCT imaging quantifies progressive increases in anatomic dead space during mechanical ventilation in mice.

Increased dead space is an important prognostic marker in early acute respiratory distress syndrome (ARDS) that correlates with mortality. The cause of increased dead space in ARDS has largely been attributed to increased alveolar dead space due to ventilation/perfusion mismatching and shunt. We sought to determine whether anatomic dead space also increases in response to mechanical ventilation. Mice received intratracheal lipopolysaccharide (LPS) or saline and mechanical ventilation (MV). Four-dimensional computed tomography (4DCT) scans were performed at onset of MV and after 5 h of MV. Detailed measurements of airway volumes and lung tidal volumes were performed using image analysis software. The forced oscillation technique was used to obtain measures of airway resistance, tissue damping, and tissue elastance. The ratio of airway volumes to total tidal volume increased significantly in response to 5 h of mechanical ventilation, regardless of LPS exposure, and airways demonstrated significant variation in volumes over the respiratory cycle. These findings were associated with an increase in tissue elastance (decreased lung compliance) but without changes in tidal volumes. Airway volumes increased over time with exposure to mechanical ventilation without a concomitant increase in tidal volumes. These findings suggest that anatomic dead space fraction increases progressively with exposure to positive pressure ventilation and may represent a pathological process.NEW & NOTEWORTHY We demonstrate that anatomic dead space ventilation increases significantly over time in mice in response to mechanical ventilation. The novel functional lung-imaging techniques applied here yield sensitive measures of airway volumes that may have wide applications.

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.

X-ray specks: low dose in vivo imaging of lung structure and function.

Respiratory health is directly linked to the structural and mechanical properties of the airways of the lungs. For studying respiratory development and pathology, the ability to quantitatively measure airway dimensions and changes in their size during respiration is highly desirable. Real-time imaging of the terminal airways with sufficient contrast and resolution during respiration is currently not possible. Herein we reveal a simple method for measuring lung airway dimensions in small animals during respiration from a single propagation-based phase contrast x-ray image, thereby requiring minimal radiation. This modality renders the lungs visible as a speckled intensity pattern. In the near-field regime, the size of the speckles is directly correlated with that of the dominant length scale of the airways. We demonstrate that Fourier space quantification of the speckle texture can be used to statistically measure regional airway dimensions at the alveolar scale, with measurement precision finer than the spatial resolution of the imaging system. Using this technique we discovered striking differences in developmental maturity in the lungs of rabbit kittens at birth.

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.