Johannes C. Schittny

Molecular architecture of basement membranes.

Basement membranes are specialized extracellular matrices with support, sieving, and cell regulatory functions. The molecular architectures of these matrices are created through specific binding interactions between unique glycoprotein and proteoglycan protomers. Type IV collagen chains, using NH2‐terminal, COOH‐terminal, and lateral association, form a covalently stabilized polygonal framework. Laminin, a four‐armed glycoprotein, self‐assembles through terminal‐domain interactions to form a second polymer network, Entactin/nidogen, a dumbbell‐shaped sulfated glycoprotein, binds laminin near its center and interacts with type IV collagen, bridging the two. A large heparan sulfate proteoglycan, important for charge‐dependent molecular sieving, is firmly anchored in the basement membrane and can bind itself through a core‐protein interaction to form dimers and oligomers and bind laminin and type IV collagen through its glycosaminoglycan chains. Heterogeneity of structure and function occur in different tissues, in development, and in response to different physiological needs. The molecular architecture of these matrices may be regulated during or after primary assembly through variations in compositions, isoform substitutions, and the modifying influence of exogenous macromolecules such as heparin and heparan sulfate.— Yurchenco, R D.; Schittny, J. C. Molecular architecture of basement membranes. FASEB J. 4: 1577‐1590; 1990.

Neonatal steroids induce a down-regulation of tenascin-C and elastin and cause a deceleration of the first phase and an acceleration of the second phase of lung alveolarization

Pre- and postnatal corticosteroids are often used in perinatal medicine to improve pulmonary function in preterm infants. To mimic this clinical situation, newborn rats were treated systemically with dexamethasone (Dex), 0.1–0.01xa0mg/kg/day on days P1–P4. We hypothesized that postnatal Dex may have an impact on alveolarization by interfering with extracellular matrix proteins and cellular differentiation. Morphological alterations were observed on 3D images obtained by high-resolution synchrotron radiation X-ray tomographic microscopy. Alveolarization was quantified stereologically by estimating the formation of new septa between days P4 and P60. The parenchymal expression of tenascin-C (TNC), smooth muscle actin (SMA), and elastin was measured by immunofluorescence and gene expression for TNC by qRT-PCR. After Dex treatment, the first phase of alveolarization was significantly delayed between days P6 and P10, whereas the second phase was accelerated. Elastin and SMA expressions were delayed by Dex treatment, whereas TNC expression was delayed and prolonged. A short course of neonatal steroids impairs the first phase of alveolarization, most likely by altering the TNC and elastin expression. Due to an overshooting catch-up during the second phase of alveolarization, the differences disappear when the animals reach adulthood.

Evolution of gene expression changes in newborn rats after mechanical ventilation with reversible intubation

Mechanical ventilation (MV) is life‐saving but potentially harmful for lungs of premature infants. So far, animal models dealt with the acute impact of MV on immature lungs, but less with its delayed effects. We used a newborn rodent model including non‐surgical and therefore reversible intubation with moderate ventilation and hypothesized that there might be distinct gene expression patterns after a ventilation‐free recovery period compared to acute effects directly after MV. Newborn rat pups were subjected to 8u2009hr of MV with 60% oxygen (O2), 24u2009hr after injection of lipopolysaccharide (LPS), intended to create a low inflammatory background as often recognized in preterm infants. Animals were separated in controls (CTRL), LPS injection (LPS), or full intervention with LPS and MV with 60% O2 (LPSu2009+u2009MVu2009+u2009O2). Lungs were recovered either directly following (T:0u2009hr) or 48u2009hr after MV (T:48u2009hr). Histologically, signs of ventilator‐induced lung injury (VILI) were observed in LPSu2009+u2009MVu2009+u2009O2 lungs at T:0u2009hr, while changes appeared similar to those known from patients with chronic lung disease (CLD) with fewer albeit larger gas exchange units, at T:48u2009hr. At T:0u2009hr, LPSu2009+u2009MVu2009+u2009O2 increased gene expression of pro‐inflammatory MIP‐2. In parallel anti‐inflammatory IL‐1Ra gene expression was increased in LPS and LPSu2009+u2009MVu2009+u2009O2 groups. At T:48u2009hr, pro‐ and anti‐inflammatory genes had returned to their basal expression. MMP‐2 gene expression was decreased in LPS and LPSu2009+u2009MVu2009+u2009O2 groups at T:0u2009hr, but no longer at T:48u2009hr. MMP‐9 gene expression levels were unchanged directly after MV. However, at T:48u2009hr, gene and protein expression increased in LPSu2009+u2009MVu2009+u2009O2 group. In conclusion, this study demonstrates the feasibility of delayed outcome measurements after a ventilation‐free period in newborn rats and may help to further understand the time‐course of molecular changes following MV. The differences obtained from the two time points could be interpreted as an initial transitory increase of inflammation and a delayed impact of the intervention on structure‐related genes. Pediatr Pulmonol. 2012; 47:1204–1214.

Imaging samples larger than the field of view: the SLS experience

Volumetric datasets with micrometer spatial and sub-second temporal resolutions are nowadays routinely acquired using synchrotron X-ray tomographic microscopy (SRXTM). Although SRXTM technology allows the examination of multiple samples with short scan times, many specimens are larger than the field-of-view (FOV) provided by the detector. The extension of the FOV in the direction perpendicular to the rotation axis remains non-trivial. We present a method that can efficiently increase the FOV merging volumetric datasets obtained by region-of interest tomographies in different 3D positions of the sample with a minimal amount of artefacts and with the ability to handle large amounts of data. The method has been successfully applied for the three-dimensional imaging of a small number of mouse lung acini of intact animals, where pixel sizes down to the micrometer range and short exposure times are required.

Gene expression profile in newborn rat lungs after two days of recovery of mechanical ventilation

Background:Preterm infants having immature lungs often require respiratory support, potentially leading to bronchopulmonary dysplasia (BPD). Conventional BPD rodent models based on mechanical ventilation (MV) present outcome measured at the end of the ventilation period. A reversible intubation and ventilation model in newborn rats recently allowed discovering that different sets of genes modified their expression related to time after MV. In a newborn rat model, the expression profile 48u2009h after MV was analyzed with gene arrays to detect potentially interesting candidates with an impact on BPD development.Methods:Rat pups were injected P4-5 with 2u2009mg/kg lipopolysaccharide (LPS). One day later, MV with 21 or 60% oxygen was applied during 6u2009h. Animals were sacrified 48u2009h after end of ventilation. Affymetrix gene arrays assessed the total gene expression profile in lung tissue.Results:In fully treated animals (LPS + MV + 60% O2) vs. controls, 271 genes changed expression significantly. All modified genes could be classified in six pathways: tissue remodeling/wound repair, immune system and inflammatory response, hematopoiesis, vasodilatation, and oxidative stress. Major alterations were found in the MMP and complement system.Conclusion:MMPs and complement factors play a central role in several of the pathways identified and may represent interesting targets for BPD treatment/prevention.Bronchopulmonary dysplasia (BPD) is a chronic lung disease occurring in ~30% of preterm infants born less than 30u2009wk of gestation (1). Its main risk factors include lung immaturity due to preterm delivery, mechanical ventilation (MV), oxygen toxicity, chorioamnionitis, and sepsis. The main feature is an arrest of alveolar and capillary formation (2). Models trying to decipher genes involved in the pathophysiology of BPD are mainly based on MV and oxygen application to young mammals with immature lungs of different species (3). In newborn rodent models, analyses of lung structure and gene and protein expression are performed for practical reasons directly at the end of MV (4,5,6). However, later appearing changes of gene expression might also have an impact on lung development and the evolution towards BPD and cannot be discovered by such models. Recently, we developed a newborn rat model of MV using an atraumatic (orotracheal) intubation technique that allows the weaning of the newborn animal off anesthesia and MV, the extubation to spontaneous breathing, and therefore allows the evaluation of effects of MV after a ventilation-free period of recovery (7). Indeed, applying this concept of atraumatic intubation by direct laryngoscopy, we recently were able to show significant differences between gene expression changes appearing directly after MV compared to those measured after a ventilation-free interval of 48u2009h. Immediately after MV, inflammation-related genes showed a transitory modified expression, while another set of more structurally related genes changed their expression only after a delay of 2 d (7). Lung structure, analyzed by conventional 2D histology and also by 3D reconstruction using synchrotron x-ray tomographic microscopy revealed, 48u2009h after end of MV, a reduced complexity of lung architecture compared to the nonventilated rat lungs, similar to the typical findings in BPD. To extend these observations about late gene expression modifications, we performed with a similar model a full gene expression profile of lung tissue 48u2009h after the end of MV with either room air or 60% oxygen. Essentially, we measured changes in the expression of genes related to the MMPs and complement system which played a role in many of the six identified mostly affected pathways.

Effective segmentation of fresh post-mortem murine lung parenchyma in phase contrast X-ray tomographic microscopy images

The acinus represents the functional unit of the mammalian lung. It is defined as the small tree of gas-exchanging airways, which is fed by the most distal purely conducting airway. Different hypotheses exist on how the fine structure of the acinus changes during ventilation and development. Since in classical 2-dimensional (2D) sections of the lung the borders of the acini are not detectable, every study of acini requires 3-dimensional (3D) datasets. As a basis for further studies of pulmonary acini we imaged rodent lungs as close to life as possible using phase contrast synchrotron radiation-based X-ray tomographic microscopy (SRXTM), and developed a protocol for the segmentation of the alveolar septa. The method is based on a combined multilevel filtering approach. Seeds are automatically defined for separate regions of tissue and airspace during each 2D filtering level and then given as input to a 3D random walk segmentation. Thus, the different types of artifacts present in the images are treated separately, taking into account the samples structural complexity. The proposed procedure yields high quality 3D segmentations of acinar microstructure that can be used for a reliable morphological analysis.