Glucose oxidation drives trunk neural crest cell development and fate.

Bioenergetic metabolism is a key regulator of cellular function and signaling, but how it can instruct the behavior of cells and their fate during embryonic development remains largely unknown. Here, we investigated the role of glucose metabolism in the development of avian trunk neural crest cells (NCCs), a migratory stem cell population of the vertebrate embryo. We uncovered that trunk NCCs display glucose oxidation as a prominent metabolic phenotype, in contrast to what is seen for cranial NCCs, which instead rely on aerobic glycolysis. In addition, only one pathway downstream of glucose uptake is not sufficient for trunk NCC development. Indeed, glycolysis, mitochondrial respiration and the pentose phosphate pathway are all mobilized and integrated for the coordinated execution of diverse cellular programs, epithelial-to-mesenchymal transition, adhesion, locomotion, proliferation and differentiation, through regulation of specific gene expression. In the absence of glucose, the OXPHOS pathway fueled by pyruvate failed to promote trunk NCC adaptation to environmental stiffness, stemness maintenance and fate-decision making. These findings highlight the need for trunk NCCs to make the most of the glucose pathway potential to meet the high metabolic demands appropriate for their development.

Phase-contrast helium-3 MRI of aerosol deposition in human airways.

One of the key challenges in the study of health-related aerosols is predicting and monitoring sites of particle deposition in the respiratory tract. The potential health risks of ambient exposure to environmental or workplace aerosols and the beneficial effects of medical aerosols are strongly influenced by the site of aerosol deposition along the respiratory tract. Nuclear medicine is the only current modality that combines quantification and regional localization of aerosol deposition, and this technique remains limited by its spatial and temporal resolutions and by patient exposure to radiation. Recent work in MRI has shed light on techniques to quantify micro-sized magnetic particles in living bodies by the measurement of associated static magnetic field variations. With regard to lung MRI, hyperpolarized helium-3 may be used as a tracer gas to compensate for the lack of MR signal in the airways, so as to allow assessment of pulmonary function and morphology. The extrathoracic region of the human respiratory system plays a critical role in determining aerosol deposition patterns, as it acts as a filter upstream from the lungs. In the present work, aerosol deposition in a mouth-throat phantom was measured using helium-3 MRI and compared with single-photon emission computed tomography. By providing high sensitivity with high spatial and temporal resolutions, phase-contrast helium-3 MRI offers new insights for the study of particle transport and deposition.

Giant scaffolding protein AHNAK1 interacts with ß-dystroglycan and controls motility and mechanical properties of Schwann cells.

The profound morphofunctional changes that Schwann cells (SCs) undergo during their migration and elongation on axons, as well as during axon sorting, ensheathment, and myelination, require their close interaction with the surrounding laminin-rich basal lamina. In contrast to myelinating central nervous system glia, SCs strongly and constitutively express the giant scaffolding protein AHNAK1, localized essentially underneath the outer, abaxonal plasma membrane. Using electron microscopy, we show here that in the sciatic nerve of ahnak1(-) (/) (-) mice the ultrastructure of myelinated, and unmyelinated (Remak) fibers is affected. The major SC laminin receptor ß-dystroglycan co-immunoprecipitates with AHNAK1 shows reduced expression in ahnak1(-) (/) (-) SCs, and is no longer detectable in Cajal bands on myelinated fibers in ahnak1(-) (/) (-) sciatic nerve. Reduced migration velocity in a scratch wound assay of purified ahnak1(-) (/) (-) primary SCs cultured on a laminin substrate indicated a function of AHNAK1 in SC motility. This was corroborated by atomic force microscopy measurements, which revealed a greater mechanical rigidity of shaft and leading tip of ahnak1(-) (/) (-) SC processes. Internodal lengths of large fibers are decreased in ahnak1(-) (/) (-) sciatic nerve, and longitudinal extension of myelin segments is even more strongly reduced after acute knockdown of AHNAK1 in SCs of developing sciatic nerve. Together, our results suggest that by interfering in the cross-talk between the transmembrane form of the laminin receptor dystroglycan and F-actin, AHNAK1 influences the cytoskeleton organization of SCs, and thus plays a role in the regulation of their morphology and motility and lastly, the myelination process.

Cell mechanics of alveolar epithelial cells (AECs) and macrophages (AMs).

Cell mechanics provides an integrated view of many biological phenomena which are intimately related to cell structure and function. Because breathing constitutes a sustained motion synonymous with life, pulmonary cells are normally designed to support permanent cyclic stretch without breaking, while receiving mechanical cues from their environment. The authors study the mechanical responses of alveolar cells, namely epithelial cells and macrophages, exposed to well-controlled mechanical stress in order to understand pulmonary cell response and function. They discuss the principle, advantages and limits of a cytoskeleton-specific micromanipulation technique, magnetic bead twisting cytometry, potentially applicable in vivo. They also compare the pertinence of various models (e.g., rheological; power law) used to extract cell mechanical properties and discuss cell stress/strain hardening properties and cell dynamic response in relation to the structural tensegrity model. Overall, alveolar cells provide a pertinent model to study the biological processes governing cellular response to controlled stress or strain.

In vitro validation of computational fluid dynamic simulation in human proximal airways with hyperpolarized 3He magnetic resonance phase-contrast velocimetry.

Computational fluid dynamics (CFD) and magnetic resonance (MR) gas velocimetry were concurrently performed to study airflow in the same model of human proximal airways. Realistic in vivo-based human airway geometry was segmented from thoracic computed tomography. The three-dimensional numerical description of the airways was used for both generation of a physical airway model using rapid prototyping and mesh generation for CFD simulations. Steady laminar inspiratory experiments (Reynolds number Re = 770) were performed and velocity maps down to the fourth airway generation were extracted from a new velocity mapping technique based on MR velocimetry using hyperpolarized (3)He gas. Full two-dimensional maps of the velocity vector were measured within a few seconds. Numerical simulations were carried out with the experimental flow conditions, and the two sets of data were compared between the two modalities. Flow distributions agreed within 3%. Main and secondary flow velocity intensities were similar, as were velocity convective patterns. This work demonstrates that experimental and numerical gas velocity data can be obtained and compared in the same complex airway geometry. Experiments validated the simulation platform that integrates patient-specific airway reconstruction process from in vivo thoracic scans and velocity field calculation with CFD, hence allowing the results of this numerical tool to be used with confidence in potential clinical applications for lung characterization. Finally, this combined numerical and experimental approach of flow assessment in realistic in vivo-based human airway geometries confirmed the strong dependence of airway flow patterns on local and global geometrical factors, which could contribute to gas mixing.

Bench evaluation of flow limitation detection by automated continuous positive airway pressure devices.

STUDY OBJECTIVE: Automatic continuous positive airway pressure (CPAP) devices that adjust the pressure delivered to the patient are now available to treat sleep-disordered breathing. Sophisticated auto-CPAP devices can detect and correct flattened inspiratory flow contours (FIFCs) associated with subtle upper airway obstruction. However, evaluations of their performance are made difficult by differences across patients and devices. We performed a bench study of five commercially available auto-CPAP devices using a breath waveform simulator to evaluate sensitivity for detecting flattened inspiratory flow. DESIGN: Five degrees of FIFC were simulated. In addition, normal and abnormal flow contours from patients published in the literature were evaluated. MEASUREMENTS AND RESULTS: One device showed autotriggering leading to CPAP increases, and another device varied the CPAP level independently from the presence of an FIFC. The three remaining devices differed regarding the detection of FIFCs and the means used to increase CPAP. CONCLUSION: Based on the characteristics of each patient, physicians must choose among devices with different thresholds of FIFC detection and different pressure responses to detection. Therefore, physicians need details on the algorithms used in auto-CPAP devices. Manufacturers should supply detailed algorithms.

Nasal wall compliance in vasomotor rhinitis.

Nasal compliance is a measure related to the blood volume in the nasal mucosa. The objective of this study was to better understand the vascular response in vasomotor rhinitis by measuring nasal cross-sectional area and nasal compliance before and after mucosal decongestion in 10 patients with vasomotor rhinitis compared with 10 healthy subjects. Nasal compliance was inferred by measuring nasal area by acoustic rhinometry at pressures ranging from atmospheric pressure to a negative pressure of -10 cmH2O. Mucosal decongestion was obtained with one puff per nostril of 0.05% oxymetazoline. At atmospheric pressure, nasal cross-sectional areas were similar in the vasomotor rhinitis group and the healthy subject group. Mucosal decongestion did not induce any decrease of nasal compliance in patients with vasomotor rhinitis in contrast with healthy subjects. Our results support the hypothesis, already proposed, of an autonomic dysfunction based on a paradoxical response of the nasal mucosa in vasomotor rhinitis. Moreover, the clearly different behavior between healthy subjects and vasomotor rhinitis subjects suggests that nasal compliance measurement may therefore represent a potential line of research to develop a diagnostic tool for vasomotor rhinitis, which remains a diagnosis of exclusion.

Inspiratory flow in the nose: a model coupling flow and vasoerectile tissue distensibility.

We have developed a discrete multisegmental model describing the coupling between inspiratory flow and nasal wall distensibility. This model is composed of 14 individualized compliant elements, each with its own relationship between cross-sectional area and transmural pressure. Conceptually, this model is based on flow limitation induced by the narrowing of duct due to collapsing pressure. For a given inspiratory pressure and for a given compliance distribution, this model predicts the area profile and inspiratory flow. Acoustic rhinometry and posterior rhinomanometry were used to determine the initial geometric area and mechanical characteristics of each element. The proposed model, used under steady-state conditions, is able to simulate the pressure-flow relationship observed in vivo under normal conditions (4 subjects) and under pathological conditions (4 vasomotor rhinitis and 3 valve syndrome subjects). Our results suggest that nasal wall compliance is an essential parameter to understand the nasal inspiratory flow limitation phenomenon and the associated increase of resistance that is well known to physiologists. By predicting the functional pressure-flow relationship, this model could be a useful tool for the clinician to evaluate the potential effects of treatments.

Apical rigidity of an epithelial cell monolayer evaluated by magnetic twisting cytometry: ICAM-1 versus integrin linkages to F-actin structure.

Using Magnetic Twisting Cytometry (MTC) technique, we attempted to characterize in vitro the rigidity of the lining tissue covering the lung alveolar wall from its apical face. We purposely used a cellular model constituted by a monolayer of human alveolar epithelial cell (A549) over which microbeads, fixed to InterCellular Adhesion Molecule (ICAM-1), exert a controlled mechanical stress. ICAM-1 expression was induced by Tumor Necrosis Factor-alpha (TNF-alpha). Rigidity measurements, performed in the course of cytochalasin D depolymerization, reveal the force transmitter role of the transmembrane receptor ICAM-1 and demonstrate that ICAM-1 and F-actin linkages confers mechanical rigidity to the apical face of the epithelial cell monolayer resembling that provided by integrins. These results confirm the ability of MTC in identifying transmembrane mechanoreceptors in relation with F-actin. Molecular linkages between ICAM-1 and F-actin were observed by spatial visualisations of the structure after double staining of F-actin and anti ICAM-1 antibody through confocal microscopy.

Airflow modeling of steady inspiration in two realistic proximal airway trees reconstructed from human thoracic tomodensitometric images.

Detailed description of the flow field in human airways is highly important to better understand human breathing and provide a patient's customized diagnosis. An integrated numerical simulation platform is presently proposed in order to incorporate medical images into a numerical software to calculate flow field and to analyze it in terms of fluid dynamics. The platform was set up to compute steady inspiratory airflow in realistic human airways reconstructed from tomodensitometric medical images at resting breathing conditions. This morpho-functional simulation platform has been tested retrospectively with two CT-scanned patient airway morphological models: (i) a normal airway model (subject A) with no evidence of morphological alteration and (ii) a highly altered airway model (subject B) exhibiting a severe stenosis in the right main bronchus. First, various morphological aspects proper to each airway model are provided to show the performance and interest of the reconstruction method. Second, we describe the three-dimensional flow patterns associated to the global morphological features, which are mainly shared by the present realistic models and previous idealistic airway models. Finally, the flow characteristics associated to local morphological features specific to realistic airway models are discussed. The results demonstrate that the morpho-functional simulation platform is able to capture the main features of airway velocity patterns but also more specific airflow patterns which are related to customized patient morphological features such as laminar vortex formation. The present results suggest that the proposed airway functional imaging platform is adequate to provide most of functional information related to airflow and enable a patient to patient diagnosis.

Description and microbiology of endotracheal tube biofilm in mechanically ventilated subjects.

BACKGROUND: A biofilm is found on the inner side of endotracheal tubes (ETT) in mechanically ventilated patients, but its features and role in pneumonia remain unclear. METHODS: This prospective, observational, monocentric study included critically ill ventilated subjects. Measurement of the ETT inner volume was first performed before extubation using the acoustic reflection method. After extubation, the biofilm was studied by means of optical and atomic force microscopy. Bacteriological analysis was then performed and compared with clinical documentation. RESULTS: Twenty-four subjects were included. Duration of intubation lasted from 2 to 79 d (mean ± SD: 11 ± 15 d). The mean percentage of ETT volume loss evaluated in situ (n = 21) was 7.1% and was not linked with the duration of intubation. Analyses with atomic force microscopy (n = 6) showed a full coverage of the inner part of the tube with biofilm, even after saline rinse. Its thickness ranged from 0.8 to 5 µm. Bacteriological cultures of the biofilm (n = 22) often showed the same bacteria as in tracheal secretions, especially for pathogenic organisms. Pseudomonas aeruginosa and Candida albicans were among the most frequent microorganisms. In subjects who had experienced a successfully treated episode of ventilator-associated pneumonia (n = 5), the responsible bacteria were still present in the biofilm. CONCLUSIONS: ETT biofilm is always present in intubated patients whatever the duration of intubation and appears quickly after intubation. Even after soft rinse, a small but measurable part of biofilm remains always present, and seems strongly adherent to the ETT lumen. It contains potentially pathogenic bacteria for the lung.

Upper airway calibre and impedance in patients with Steinert's myotonic dystrophy.

Myotonic dystrophy (MD) can be responsible for increased inspiratory muscle loading, the origin of which is debated, with some authors incriminating distal lesions and others central abnormalities. Using a recent non-invasive method based on single transient pressure-wave reflection analysis, we measured central airway calibre from the mouth to the carina and respiratory impedance in a group of adults with MD, a group of patients with sleep apnoea syndrome (SAS) but no neuromuscular disease, and a group of normal controls. All participants were awake during the measurements. We found no reduction in central airway calibre in the patients with the adult form of MD, as compared to the normal controls. These data suggest that MD may be associated with peripheral airway obstruction related to alterations in the elastic properties of the lung.

Characterization of cytoskeleton mechanical properties and 3D-actin structure in twisted adherent epithelial cells.

Evaluation of the cytoskeleton mechanical properties requires specific micromanipulation techniques such as the magnetic twisting cytometry technique, in which microbeads are specifically linked to the cytoskeleton via transmembrane receptors. The aim of the study was to assess the structural relationship between the bead and the cytoskeleton structure. The spatial arrangement of the CSK network was therefore studied in fixed cells probed by beads and stained for F-actin by rhodamined phalloïdine. The spatial character of the actin CSK network, both in the bead neighborhood and at the cell scale, could then be studied for various degrees of fluorescent intensity from 3D-images of the actin structure, reconstructed from z-stack views obtained by confocal microscopy. Results show the feasibility of the staining/reconstruction technique which allows to reveal the three-dimensional organization of the cytoskeleton structure including an internal cytosolic structure with a high fluorescent F-actin intensity, and a sub-membranous cortical structure with a low fluorescent F-actin intensity.

Mechanical assessment by magnetocytometry of the cytosolic and cortical cytoskeletal compartments in adherent epithelial cells.

This study aims at quantifying the cellular mechanical properties based on a partitioning of the cytoskeleton in a cortical and a cytosolic compartments. The mechanical response of epithelial cells obtained by magnetocytometry - a micromanipulation technique which uses twisted ferromagnetic beads specifically linked to integrin receptors - was purposely analysed using a series of two Voigt bodies. Results showed that the cortical cytoskeleton has a faster response ( approximately 1 s) than the cytosolic compartment ( approximately 30 s). Moreover, the two cytoskeletal compartments have specific mechanical properties, i.e., the cortical (resp. cytosolic) cytoskeleton has a rigidity in the range: 49-85 Pa (resp.: 74-159 Pa) and a viscosity in the range 5-14 Pa.s (resp.: 593-1534 Pa.s), depending on the level of applied stress. Depolymerising actin-filaments strongly modified these values and especially those of the cytosolic compartment. The structural relevance of this two-compartment partitioning was supported by images of F-actin structure obtained on the same cells.

Frictional resistance sheds light on the multicomponent nature of nasal obstruction: a combined in vivo and computational fluid dynamics study.

Exploring nasal flow contributes to better understanding of pathophysiological functions of nasal cavities. We combined the rhinomanometry measurements of 11 patients and computational fluid dynamics (CFD) simulations in 3 nasal airway models to dissect the complex mechanisms that determine nasal flow obstruction: spatial complexity and pressure-dependent deformability of nasal airways. We quantified spatial complexity by calculating longitudinal variations of hydraulic diameter, perimeter and area of nasal cavities, and their impact on flow characteristics by examining the longitudinal variations of the kinetic energy coefficient and the kinetic to potential energy ratio. Airway distensibility variably affected in vivo pressure-flow relationships through the appearance of flow-limitation patterns characterized by maximum flow and/or flow plateau. We quantified deformability and spatial complexity effects on nasal airway resistance by normalizing all data with averaged reference parameters. The results show that discrepancies in nasal flow resistances reflect airway deformability and geometrical complexity, and thereby constitute a framework to better characterize nasal obstruction.

Evaluation of the trachea and intrathoracic airways by the acoustic reflection method in children with cystic fibrosis.

Tracheomalacia has been observed in older patients with cystic fibrosis (CF). The acoustic reflection method (ARM) allows a noninvasive calculation of the longitudinal cross-sectional area of the trachea (MTAv) and the airway resistance (Raw). ARM measurements were performed in 20 CF children and 20 controls during spontaneous breathing (SB), forced inspiration (FI), and forced expiration (FE). The mean MTAv value was comparable in the CF patients and the control subjects during SB, FI, and FE. The Raw was also comparable during SB and FI. However, the Raw during FE was higher in the CF patients than in the control subjects (7.9±2.3 vs. 5.0±1.5 cm H2O l(-1) s(-1), respectively, p<0.001). In the patients with CF, only the Raw during FE correlated with the predicted forced expiratory volume in 1s (R2=0.37, p=0.04). The tracheal cross-sectional area measured by the ARM is normal in children with CF but the increase in Raw during FE suggests an increase in intrathoracic airway distensibility.

Force control of endothelium permeability in mechanically stressed pulmonary micro-vascular endothelial cells.

Mechanical factors play a key role in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS) and Ventilator-Induced Lung Injury (VILI) as contributing to alveolo-capillary barrier dysfunction. This study aims at elucidating the role of the cytoskeleton (CSK) and cell-matrix adhesion system in the stressed endothelium and more precisely in the loss of integrity of the endothelial barrier. We purposely develop a cellular model made of a monolayer of confluent Human Pulmonary Microvascular Endothelial Cells (HPMVECs) whose cytoskeleton (CSK) is directly exposed to sustained cyclic mechanical stress for 1 and 2 h. We used RGD-coated ferromagnetic beads and measured permeability before and after stress application. We find that endothelial permeability increases in the stressed endothelium, hence reflecting a loss of integrity. Structural and mechanical results suggest that this endothelial barrier alteration would be due to physically-founded discrepancies in latero-basal reinforcement of adhesion sites in response to the global increase in CSK stiffness or centripetal intracellular forces. Basal reinforcement of adhesion is presently evidenced by the marked redistribution of αvß3 integrin with cluster formation in the stressed endothelium.

Analysis of the pharynx and the trachea by the acoustic reflection method in children: a pilot study.

The acoustic reflection method (ARM) is based on the analysis of the reflection of acoustic waves. The aim of the study was to determine the feasibility of the ARM in healthy children, and the anthropometric parameters that were correlated with the minimum cross-sectional area (MCAv) of the upper airways. Fifty-nine children (age 2-18 years) participated to the study. The mean MCAv value were 1.47±0.15, 1.77±0.33, 1.80±0.23, 2.06±0.42, 2.19±0.37, and 2.22±0.29cm(2), in the 2-3, 4-6, 7-9, 10-12, 13-15, and 16-18 year age groups, respectively. The MCAv values correlated significantly with height, age, and weight, but in a stepwise multiple regression analysis, only height remained correlated with the MCAv (r=0.59, p<0.0001). The analysis of the upper airways by the ARM is feasible in children after 2 years of age and the MCAv values increase with height in childhood.

Sensitivity of alveolar macrophages to substrate mechanical and adhesive properties.

In order to understand the sensitivity of alveolar macrophages (AMs) to substrate properties, we have developed a new model of macrophages cultured on substrates of increasing Young's modulus: (i) a monolayer of alveolar epithelial cells representing the supple (approximately 0.1 kPa) physiological substrate, (ii) polyacrylamide gels with two concentrations of bis-acrylamide representing low and high intermediate stiffness (respectively 40 kPa and 160 kPa) and, (iii) a highly rigid surface of plastic or glass (respectively 3 MPa and 70 MPa), the two latter being or not functionalized with type I-collagen. The macrophage response was studied through their shape (characterized by 3D-reconstructions of F-actin structure) and their cytoskeletal stiffness (estimated by transient twisting of magnetic RGD-coated beads and corrected for actual bead immersion). Macrophage shape dramatically changed from rounded to flattened as substrate stiffness increased from soft ((i) and (ii)) to rigid (iii) substrates, indicating a net sensitivity of alveolar macrophages to substrate stiffness but without generating F-actin stress fibers. Macrophage stiffness was also increased by large substrate stiffness increase but this increase was not due to an increase in internal tension assessed by the negligible effect of a F-actin depolymerizing drug (cytochalasine D) on bead twisting. The mechanical sensitivity of AMs could be partly explained by an idealized numerical model describing how low cell height enhances the substrate-stiffness-dependence of the apparent (measured) AM stiffness. Altogether, these results suggest that macrophages are able to probe their physical environment but the mechanosensitive mechanism behind appears quite different from tissue cells, since it occurs at no significant cell-scale prestress, shape changes through minimal actin remodeling and finally an AMs stiffness not affected by the loss in F-actin integrity.

Phase-contrast velocimetry with hyperpolarized 3He for in vitro and in vivo characterization of airflow.

This paper describes a technique that combines radial MRI and phase contrast (PC) to map the velocities of hyperpolarized gases ((3)He) in respiratory airways. The method was evaluated on well known geometries (straight and U-shaped pipes) before it was applied in vivo. Dynamic 2D maps of the three velocity components were obtained from a 10-mm slice with an in-plane spatial resolution of 1.6 mm within 1 s. Integration of the in vitro through-plane velocity over the slice matched the input flow within a relative precision of 6.4%. As expected for the given Reynolds number, a parabolic velocity profile was obtained in the straight pipe. In the U-shaped pipe the three velocity components were measured and compared to a fluid-dynamics simulation so the precision was evaluated as fine as 0.025 m s(-1). The technique also demonstrated its ability to visualize vortices and localize characteristic points, such as the maximum velocity and vortex-center positions. Finally, in vivo feasibility was demonstrated in the human trachea during inhalation.