Andreas Fouras

A shear gradient–dependent platelet aggregation mechanism drives thrombus formation

Platelet aggregation at sites of vascular injury is essential for hemostasis and arterial thrombosis. It has long been assumed that platelet aggregation and thrombus growth are initiated by soluble agonists generated at sites of vascular injury. By using high-resolution intravital imaging techniques and hydrodynamic analyses, we show that platelet aggregation is primarily driven by changes in blood flow parameters (rheology), with soluble agonists having a secondary role, stabilizing formed aggregates. We find that in response to vascular injury, thrombi initially develop through the progressive stabilization of discoid platelet aggregates. Analysis of blood flow dynamics revealed that discoid platelets preferentially adhere in low-shear zones at the downstream face of forming thrombi, with stabilization of aggregates dependent on the dynamic restructuring of membrane tethers. These findings provide insight into the prothrombotic effects of disturbed blood flow parameters and suggest a fundamental reinterpretation of the mechanisms driving platelet aggregation and thrombus growth.

Effect of Sustained Inflation Length on Establishing Functional Residual Capacity at Birth in Ventilated Premature Rabbits

The effect of inflation length on lung aeration pattern, tidal volumes, and functional residual capacity (FRC) immediately after birth was investigated. Preterm rabbits (28 d), randomized into four groups, received a 1-, 5-, 10-, or 20-s inflation (SI) followed by ventilation with 5 cm H2O end-expiratory pressure. Gas volumes were measured by plethysmography and uniformity of lung aeration by phase contrast x-ray imaging for 7 min. The first inspiratory volume significantly (p < 0.001) increased with inflation duration from a median (IQR) of 0.2 (0.1–3.1) mL/kg for 1-s inflation to 23.4 (19.3–30.4) mL/kg for 20-s SI. The lung was uniformly aerated, and the FRC and tidal volume fully recruited after 20-s SI. A 10-s SI caused a higher FRC (p < 0.05) at 7 min, and a 20-s SI caused a higher FRC (p < 0.05) at 20 s and 7 min than a 1- or 5-s SI. The mean (SD) time for 90% of the lung to aerate was 14.0 (4.1) s using 35 cm H2O peak inflation pressure. In these rabbits, 10- and 20-s SI increased the inspiratory volume and produced a greater FRC, and a 20-s SI uniformly aerated the lung before ventilation started.

Positive end-expiratory pressure enhances development of a functional residual capacity in preterm rabbits ventilated from birth

The factors regulating lung aeration and the initiation of pulmonary gas exchange at birth are largely unknown, particularly in infants born very preterm. As hydrostatic pressure gradients may play a role, we have examined the effect of a positive end-expiratory pressure (PEEP) on the spatial and temporal pattern of lung aeration in preterm rabbit pups mechanically ventilated from birth using simultaneous phase-contrast X-ray imaging and plethysmography. Preterm rabbit pups were delivered by caesarean section at 28 days of gestational age, anesthetized, intubated, and placed within a water-filled plethysmograph (head out). Pups were imaged as they were mechanically ventilated from birth with a PEEP of either 0 cmH(2)O or 5 cmH(2)O. The peak inflation pressure was held constant at 35 cmH(2)O. Without PEEP, gas only entered into the distal airways during inflation. The distal airways collapsed during expiration, and, as a result, the functional residual capacity (FRC) did not increase above the lungs anatomic dead space volume (2.5 +/- 0.8 ml/kg). In contrast, ventilation with 5-cmH(2)O PEEP gradually increased aeration of the distal airways, which did not collapse at end expiration. The FRC achieved in pups ventilated with PEEP (19.9 +/- 3.2 ml/kg) was significantly greater than in pups ventilated without PEEP (-2.3 +/- 3.5 ml/kg). PEEP greatly facilitates aeration of the distal airways and the accumulation of FRC and prevents distal airway collapse at end expiration in very preterm rabbit pups mechanically ventilated from birth.

Dynamic measures of regional lung air volume using phase contrast x-ray imaging

Phase contrast x-ray imaging can provide detailed images of lung morphology with sufficient spatial resolution to observe the terminal airways (alveoli). We demonstrate that quantitative functional and anatomical imaging of lung ventilation can be achieved in vivo using two-dimensional phase contrast x-ray images with high contrast and spatial resolution (<100 microm) in near real time. Changes in lung air volume as small as 25 microL were calculated from the images of term and preterm rabbit pup lungs (n = 28) using a single-image phase retrieval algorithm. Comparisons with plethysmography and computed tomography showed that the technique provided an accurate and robust method of measuring total lung air volumes. Furthermore, regional ventilation was measured by partitioning the phase contrast images, which revealed differences in aeration for different ventilation strategies.

Cortical Tension Allocates the First Inner Cells of the Mammalian Embryo

Every cell in our body originates from the pluripotent inner mass of the embryo, yet it is unknown how biomechanical forces allocate inner cells in vivo. Here we discover subcellular heterogeneities in tensile forces, generated by actomyosin cortical networks, which drive apical constriction to position the first inner cells of living mouse embryos. Myosin II accumulates specifically around constricting cells, and its disruption dysregulates constriction and cell fate. Laser ablations of actomyosin networks reveal that constricting cells have higher cortical tension, generate tension anisotropies and morphological changes in adjacent regions of neighboring cells, and require their neighbors to coordinate their own changes in shape. Thus, tensile forces determine the first spatial segregation of cells during mammalian development. We propose that, unlike more cohesive tissues, the early embryo dissipates tensile forces required by constricting cells via their neighbors, thereby allowing confined cell repositioning without jeopardizing global architecture.

Inspiration regulates the rate and temporal pattern of lung liquid clearance and lung aeration at birth

At birth, the initiation of pulmonary gas exchange is dependent on air entry into the lungs, and recent evidence indicates that pressures generated by inspiration may be involved. We have used simultaneous plethysmography and phase-contrast X-ray imaging to investigate the contribution of inspiration and expiratory braking maneuvers (EBMs) to lung aeration and the formation of a functional residual capacity (FRC) after birth. Near-term rabbit pups (n = 26) were delivered by cesarean section, placed in a water plethysmograph, and imaged during the initiation of spontaneous breathing. Breath-by-breath changes in lung gas volumes were measured using plethysmography and visualized using phase-contrast X-ray imaging. Pups rapidly (1-5 breaths) generate a FRC (16.2 +/- 1.2 ml/kg) by inhaling a greater volume than they expire (by 2.9 +/- 0.4 ml.kg(-1).breath(-1) over the first 5 breaths). As a result, 94.8 +/- 1.4% of lung aeration occurred during inspiration over multiple breaths. The incidence of EBMs was rare early during lung aeration, with most (>80%) occurring after >80% of max FRC was achieved. Although EBMs were associated with an overall increase in FRC, 34.8 +/- 5.3% of EBMs were associated with a decrease in FRC. We conclude that lung aeration is predominantly achieved by inspiratory efforts and that EBMs help to maintain FRC following its formation.

The past, present, and future of x-ray technology for in vivo imaging of function and form

Scientists and clinicians have a keen interest in studying not just the structure of physiological systems, but their motion also, or more generally their form and function. This paper focuses on the technologies that underpin in vivo measurements of form and function of the human body for both research and medical treatment. A concise literature review of x-ray imaging, ultrasonography, magnetic resonance imaging, radionuclide imaging, laser Doppler velocimetry, and particle image velocimetry is presented. Additionally, a more detailed review of in vivo x-ray imaging is presented. Finally, two techniques, which the authors believe are representative of the present and future of in vivo x-ray imaging techniques, are presented.

Three-dimensional synchrotron x-ray particle image velocimetry

There is great potential to vastly improve biological flow measurement by using a combination of synchrotron imaging and the latest experimental flow measurement techniques. In the current paper, the three-dimensional velocity field within a cylindrical tube is measured using a combination of phase-contrast x-ray imaging and particle image velocimetry (PIV). We greatly refine the techniques previously used to undertake velocity measurements with a synchrotron light source, substantially enhancing accuracy. Furthermore a PIV correlation peak analysis is developed to allow three-dimensional measurement of the velocity field.

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.

IMAGING LUNG AERATION AND LUNG LIQUID CLEARANCE AT BIRTH USING PHASE CONTRAST X‐RAY IMAGING

1 The transition to extra‐uterine life at birth is critically dependent on airway liquid clearance to allow the entry of air and the onset of gaseous ventilation. We have used phase contrast X‐ray imaging to identify factors that regulate lung aeration at birth in spontaneously breathing term and mechanically ventilated preterm rabbit pups. 2 Phase contrast X‐ray imaging exploits the difference in refractive index between air and water to enhance image contrast, enabling the smallest air‐filled structures of the lung (alveoli; < 100 µm) to be resolved. Using this technique, the lungs become visible as they aerate, allowing the air–liquid interface to be observed as it moves distally during lung aeration. 3 Spontaneously breathing term rabbit pups rapidly aerate their lungs, with most fully recruiting their functional residual capacity (FRC) within the first few breaths. The increase in FRC occurs mainly during individual breaths, demonstrating that airway liquid clearance and lung aeration is closely associated with inspiration. We suggest that transpulmonary pressures generated by inspiration provide a hydrostatic pressure gradient for the movement of water out of the airways and into the surrounding lung tissue after birth. 4 In mechanically ventilated preterm pups, lung aeration is closely associated with lung inflation and a positive end‐expiratory pressure is required to generate and maintain FRC after birth. 5 In summary, phase contrast X‐ray imaging can image the air‐filled lung with high temporal and spatial resolution and is ideal for identifying factors that regulate lung aeration at birth in both spontaneously breathing term and mechanically ventilated preterm neonates.