Morphological and injury analysis of bronchioles in saccular stage MV and control lungs after 24 h and 15 d.

<p>Light micrographs show cellular intraluminal debris in MV110+24 h lungs (A, B), intact epithelium of C110+24 h bronchiole (C) and bronchiole with denuded epithelium in MV110+24 h lung (D). The basement membrane perimeter of bronchioles was not different between all saccular stage MV and control lungs (E). Epithelial thickness of bronchioles was greater in MV110+24 h fetuses than in controls; however, it was lower in MV110+15 days fetuses relative to 15 days controls (F). The proportion of bronchioles that contained debris within the lumen was increased in MV110+24 h and MV110+15 d fetuses compared with age-matched controls (G). Values that do not share a common letter are significantly different from each other (P&lt;0.05, scale bar = 10 µm in A and B and 20 µm in C and D).</p

Oligonucleotide primer sequences.

<p>CTGF, Connective tissue growth factor; CYR-61, cysteine rich 61; EGR1, early growth response 1; HSPE1, heat shock 10 kDa protein; uPAR, urokinase plasminogen activator receptor; MT2a, metallothionein 2a; DLK-1, delta like homolog drosophila; F, forward primer; R, reverse primer.</p

Cell proliferation and myofibroblast density in saccular stage MV and control lungs after 24 h and 15 d.

<p>Immunohistochemical staining using Ki-67 antibody shows proliferating cells labelled brown in C110+24 h (A), MV110+24 h (B), C110+15 d (C) and MV110+15 d lungs (D). The proportion of proliferating cells in the gas-exchanging region was not different between MV lungs and there matched control groups (I). Myofibroblasts were detected using α-SMA antibody, which labelled the myofibroblasts brown as shown in C110+24 h (E), MV110+24 h (F), C110+15 d (G) and MV110+15 d lungs (H). Myofibroblasts were localised at developing septa. α-SMA was increased in MV110+24 h lungs, in comparison to controls (J, p&lt;0.05). Scale bar = 20 µm. Values that do not share a common letter are significantly different.</p

The mRNA levels of control and ventilated saccular lungs after 24 h, corrected for the level of housekeeping gene 18 S and expressed as fold change from the mean value in control fetuses ± SE.

<p>IL, interleukin, TNF-α tumor necrosis factor-α; CTGF, connective tissue growth factor; CYR61, cysteine-rich 61; EGR1, early growth response 1.</p

Injury analysis of saccular stage control and ventilated bronchioles after 24 h and 15 d.

<p>Mild injury: 45° bronchiole epithelium detached or absent; moderate: 45°–180° bronchiole epithelium detached or absent; severe: 180° bronchiole epithelium detached or absent. Injured data represent total no. of mild, moderate, and severely injured bronchioles. MV110+24 h lungs had a higher proportion of injured bronchioles relative to all other groups, of which most were classified as severely injured (p&lt;0.05).</p

Relative expression of potential repair gene mRNA in saccular and early alveolar stage lungs 24 h after MV.

<p>Metallothionein (A) and Urokinase Plasminogen Activator Receptor (B) mRNA expression was significantly increased in MV saccular and early alveolar stage lungs after 24 h when compared to controls (p&lt;0.05). Relative expression of Delta-Like Homolog Drosophila (C) and Heat Shock 10 kDa Protein (D) mRNA was not different between control and MV lungs at 24 h in the saccular or early-alveolar stage lung.</p

Lung morphometry, collagen and elastin density, percent tissue space and secondary septal crest density in saccular stage MV and control lungs after 24 h and 15 d.

<p>Light micrographs stained with hemotoxylin and eosin depicting lung morphology in C110+24 h (A), MV110+24 h (B), C110+15 d (C) and MV110+15 d (D) lung tissue. At 24 h after MV lung tissue showed signs of heterogeneous injury with regional hypercellularity and atelectasis (arrow, B). Tissue space fraction was increased in MV110+24 h lungs compared to controls (M, p&lt;0.05). Collagen fibres (black staining) are shown in C110 d+24 h (E), MV110+24 h (F), C110+15 d (G) and MV110+15 d (H) and elastin deposits (brown staining) in C110+24 h (I), MV110+24 h (J), C110+15 d (K) and MV110+15 d (L). Collagen fibres (brown staining) were not straight in MV110+24 h lungs (arrow, F), compared to controls at both ages and MV110+15 lungs (E,G,H). Collagen (N) and elastin density (O) was not different between MV lungs and their matched control group. Secondary septal crest density was reduced in MV110+24 h lungs (arrow, J) compared to those in C110+24 h group (I, P). Scale bar = 100 µm for A–D and 20 µm for E–L. Values that do not share a common letter are significantly different.</p

Method and apparatus for acquisition of high spatiotemporal resolution <i>in vivo</i> information.

<p>A) Experimental setup designed for the acquisition of synchrotron based phase contrast X-ray lung images with high spatial and temporal resolution (image modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048122#pone.0048122-Fouras3" target="_blank">[52]</a>). The set-up allows the acquisition of <i>in vivo</i> images with enhanced lung speckle, which provides detail necessary for a cross-correlation analysis. B) Cross-correlation analysis of images allows the determination of the displacement of lung tissue within each image interrogation region. The statistically most likely displacement for each respective window is determined as the displacement of lung tissue within that interrogation region. This is performed across the entire lung image, thus generating a vector field incorporating the entire lung.</p

Information determined from <i>in vivo</i> lung imaging and analysis technique.

<p>A) Average image of 10 phase contrast images of a rabbit pup taken with 2 ms exposures at a rate of 300 fps (1016×1016 pixels). The image shows the enhanced lung structure attained with this imaging technique. B) Vector map showing instantaneous lung motion between 2 subsequent images. A cross-correlation window size of 64×64 px and 75% window overlap was used, with only a quarter of the &gt;1000 vectors being shown for clarity. C) Map coloured according to the local lung tissue expansion at an instant in time, determined between 2 subsequent frames; red being largest and blue being smallest expansion values respectively.</p

Temporal measurement of lung activity via traditional methods and the X-ray velocimetry technique.

<p>A) Time plots showing the tidal volume delivered as measured with a flowmeter and X-ray velocimetry integrated expansion. The X-ray velocimetry integrated expansion was calibrated to volume according to the method of Fouras et. al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048122#pone.0048122-Fouras3" target="_blank">[52]</a>. The coefficient of determination between the flowmeter and the X-ray velocimetry volume was &gt;0.96. The animal was being ventilated at PEEP of 9 cmH2O, PIP of 21 cmH2O and a frequency of 1 Hz. B) X-ray velocimetry integrated expansion maps calculated from the end expiration to early inspiration (i), mid-inspiration (ii) and end-inspiration (iii). The X-ray velocimetry integrated expansion maps correlate to the time indicated by the (x) symbols on the X-ray velocimetry time plot in A). It can clearly be seen that as inspiration progresses a greater amount of lung tissue expansion has occurred and the expansion maps are able to show where the changes have occurred with high spatial resolution.</p