The generation of reactive oxygen species (ROS) plays an important role for the maintenance of cellular processes and functions in the body. However, the excessive generation of oxygen radicals under pathological conditions such as acute lung injury (ALI) and its most severe form acute respiratory distress syndrome (ARDS) leads to increased endothelial permeability. Within this hallmark of ALI and ARDS, vascular microvessels lose their junctional integrity and show increased myosin contractions that promote the migration of polymorphonuclear leukocytes (PMNs) and the transition of solutes and fluids in the alveolar lumen. These processes all have a redox component, and this chapter focuses on the role played by ROS during the development of ALI/ARDS. We discuss the origins of ROS within the cell, cellular defense mechanisms against oxidative damage, the role of ROS in the development of endothelial permeability, and potential therapies targeted at oxidative stress.

One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)-related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between -1,082 and -1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB-dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.

The purpose of this study was to evaluate the correlation of radiation dose with image quality in spiral CT. Seven clinical protocols were measured in six different radiological departments provided with four different types of high specification spiral CT scanners. Central and surface absorbed doses were measured in acrylic. The practical CT dose index (PCTDI) was calculated for seven clinical examination protocols and one standardized protocol using identical parameters on four different spiral CT scanners with a dedicated ionization chamber inserted into PMMA phantoms. For low contrast measurements, a cylindrical three-dimensional (3D) phantom (different sized spheres of defined contrast) was used. Image noise was measured with a cylindrical water phantom and high contrast resolution with a Perspex hole phantom. Image quality phantoms were scanned using the parameters of the clinical protocols. Images were randomized, blinded and read by six radiologists (one from each institution). PCTDI values for four different scanners varied up to a factor between 1.5 (centre) and 2.2 (surface) for the standardized protocol. A greater degree of variation was observed for seven clinical examination protocols of the six radiological departments. For example, PCTDI varied up to a factor between 1.7 (cerebrum protocol) and 8.3 (abdomen paediatric protocol). Low contrast resolution correlates closely with dose. An improvement in detection from 8 mm to 4 mm sized spheres needs approximately a ten-fold increase in dose. Noise shows a moderate correlation with PCTDI. High contrast resolution of clinical protocols is independent of PCTDI within a certain range. Differences in modern CT scanner technology seem to be of less importance for radiation exposure than selection of protocol parameters in different radiological institutes. Future discussion on guidelines regarding optimal (patient adapted) tube current for clinical protocols is desirable.

Biofilms – communities of microorganisms attached to surfaces – are a constant threat for long-term success in modern implantology. The application of laser scanning microscopy (LSM) has increased the knowledge about microscopic properties of biofilms, whereas a 3D imaging technique for the large scale visualization of bacterial growth and migration on curved and non-transparent surfaces is not realized so far.Towards this goal, we built a scanning laser optical tomography (SLOT) setup detecting scattered laser light to image biofilm on dental implant surfaces. SLOT enables the visualization of living biofilms in 3D by detecting the wavelength-dependent absorption of non-fluorescent stains like e.g. reduced triphenyltetrazolium chloride (TTC) accumulated within metabolically active bacterial cells. Thus, the presented system allows the large scale investigation of vital biofilm structure and in vitro development on cylindrical and non-transparent objects without the need for fluorescent vital staining. We suggest SLOT to be a valuable tool for the structural and volumetric investigation of biofilm formation on implants with sizes up to several millimeters.

Vascular cell hyperproliferation and metabolic reprogramming contribute to the pathophysiology of pulmonary arterial hypertension (PAH). An important cause of PAH in children with congenital heart disease (CHD) is increased pulmonary blood flow (PBF). To better characterize this disease course we studied early changes in pulmonary artery smooth muscle cell (PASMC) proliferation and metabolism using a unique ovine model of pulmonary overcirculation. Consistent with PAH in adults, PASMCs derived from 4-wk-old lambs exposed to increased PBF (shunt) exhibited increased rates of proliferation. While shunt PASMCs also exhibited significant decreases in mitochondrial oxygen consumption, membrane potential, and tricarboxylic acid (TCA) cycle function, suggesting a switch to Warburg metabolism as observed in advanced PAH in adults, they unexpectedly demonstrated decreased glycolytic lactate production, likely due to enhanced flux through the pentose phosphate pathway (PPP). This may be a response to the marked increase in NADPH oxidase (Nox) activity and decreased NADPH/NADP ratios observed in shunt PASMCs. Consistent with these findings, pharmacological inhibition of Nox activity preferentially slowed the growth of shunt PASMCs in vitro. Our results therefore indicate that PASMC hyperproliferation is observed early in the setting of pulmonary overcirculation and is accompanied by a unique metabolic profile that is independent of HIF-1α, PDHK1, or increased glycolytic flux. Our results also suggest that Nox inhibition may help prevent pulmonary overcirculation-induced PAH in children born with CHD.

The current study focuses on the use of scanning laser optical tomography (SLOT) in imaging of the mouse lung ex vivo. SLOT is a highly efficient fluorescence microscopy technique allowing rapid scanning of samples of a size of several millimeters, thus enabling volumetric visualization by using intrinsic contrast mechanisms of previously fixed lung lobes. Here, we demonstrate the imaging of airways, blood vessels, and parenchyma from whole, optically cleared mouse lung lobes with a resolution down to the level of single alveoli using absorption and autofluorescence scan modes. The internal structure of the lung can then be analyzed nondestructively and quantitatively in three-dimensional datasets in any preferred planar orientation. Moreover, the procedure preserves the microscopic structure of the lung and allows for subsequent correlative histologic studies. In summary, the current study has shown that SLOT is a valuable technique to study the internal structure of the mouse lung.

Correlative analysis requires examination of a specimen from macro to nano scale as well as applicability of analytical methods ranging from morphological to molecular. Accomplishing this with one and the same sample is laborious at best, due to deformation and biodegradation during measurements or intermediary preparation steps. Furthermore, data alignment using differing imaging techniques turns out to be a complex task, which considerably complicates the interconnection of results. We present correlative imaging of the accessory rat lung lobe by combining a modified Scanning Laser Optical Tomography (SLOT) setup with a specially developed sample preparation method (CRISTAL). CRISTAL is a resin-based embedding method that optically clears the specimen while allowing sectioning and preventing degradation. We applied and correlated SLOT with Multi Photon Microscopy, histological and immunofluorescence analysis as well as Transmission Electron Microscopy, all in the same sample. Thus, combining CRISTAL with SLOT enables the correlative utilization of a vast variety of imaging techniques.

Lysophosphatidic acid (LPA) signaling through one of its receptors, LPA 1 , contributes to both the development and the pathological remodeling after injury of many organs. Because we found previously that LPA-LPA 1 signaling contributes to pulmonary fibrosis, here we investigated whether this pathway is also involved in lung development. Quantitative assessment of lung architecture of LPA 1 -deficient knockout (KO) and wild-type (WT) mice at 3, 12, and 24 weeks of age using design-based stereology suggested the presence of an alveolarization defect in LPA 1 KO mice at 3 weeks, which persisted as alveolar numbers increased in WT mice into adulthood. Across the ages examined, the lungs of LPA 1 KO mice exhibited decreased alveolar numbers, septal tissue volumes, and surface areas, and increased volumes of the distal airspaces. Elastic fibers, critical to the development of alveolar septa, appeared less organized and condensed and more discontinuous in KO alveoli starting at P4. Tropoelastin messenger RNA expression was decreased in KO lungs, whereas expression of matrix metalloproteinases degrading elastic fibers was either decreased or unchanged. These results are consistent with the abnormal lung phenotype of LPA 1 KO mice, being attributable to reduced alveolar septal formation during development, rather than to increased septal destruction as occurs in the emphysema of chronic obstructive pulmonary disease. Peripheral septal fibroblasts and myofibroblasts, which direct septation in late alveolarization, demonstrated reduced production of tropoelastin and matrix metalloproteinases, and diminished LPA-induced migration, when isolated from LPA 1 KO mice. Taken together, our data suggest that LPA-LPA 1 signaling is critically required for septation during alveolarization.