Search

Background The roles of hypoxia and hypoxia inducible factor (HIF) during chronic kidney disease (CKD) are much debated. Interventional studies with HIF-α activation in rodents yielded contradictory results. The HIF pathway is regulated by prolyl and asparaginyl hydroxylases; while prolyl hydroxylase inhibition is a well-known method to stabilize HIF-α, little is known about the effect asparaginyl hydroxylase Factor Inhibiting HIF inhibiting (FIH). Methods We used a model of progressive proteinuric CKD and a model of obstructive nephropathy with unilateral fibrosis. In these models, we assessed hypoxia with pimonidazole and vascularization with three-dimensional micro-CT imaging. We analyzed a database of 217 CKD biopsies from stage 1 to 5 and we randomly collected 15 CKD biopsies from various severity degrees to assess FIH expression. Finally, we modulated FIH activity in vitro and in vivo using a pharmacologic approach, to assess its relevance in CKD. Results In our model of proteinuric CKD, we show that early CKD stages are not characterized by hypoxia or HIF activation. At late CKD stages, some areas of hypoxia are observed, but these are not colocalizing with fibrosis. In mice and in humans, we observed a downregulation of the HIF pathway, together with an increased FIH expression in CKD, according to its severity. Modulating FIH in vitro affects cellular metabolism, as described previously. In vivo, pharmacologic FIH inhibition increases the glomerular filtration rate of control and CKD animals and is associated with a reduced development of fibrosis. Conclusions The causative role of hypoxia and HIF activation in CKD progression is questioned. A pharmacological approach of FIH downregulation seem promising in proteinuric kidney disease.

Background Fixation and embedding of post mortem brain tissue is a pre-requisite for both gold-standard conventional histology and X-ray virtual histology. This process alters the morphology and density of the brain microanatomy. New method To quantify these changes, we employed synchrotron radiation-based hard X-ray tomography with 3 μm voxel length to visualize the same mouse brain after fixation in 4% formalin, immersion in ethanol solutions (50%, 70%, 80%, 90%, and 100%), xylene, and finally after embedding in a paraffin block. The volumetric data were non-rigidly registered to the initial formalin-fixed state to align the microanatomy within the entire mouse brain. Results Volumetric strain fields were used to characterize local shrinkage, which was found to depend on the anatomical region and distance to external surface. X-ray contrast was altered and enhanced by preparation-induced inter-tissue density changes. The preparation step can be selected to highlight specific anatomical features. For example, fiber tract contrast is amplified in 100% ethanol. Comparison with existing methods Our method provides volumetric strain fields, unlike approaches based on feature-to-feature or volume measurements. Volumetric strain fields are produced by non-rigid registration, which is less labor-intensive and observer-dependent than volume change measurements based on manual segmentations. X-ray microtomography provides spatial resolution at least an order of magnitude higher than magnetic resonance microscopy, allowing for analysis of morphology and density changes within the brain’s microanatomy. Conclusion Our approach belongs to three-dimensional virtual histology with isotropic micrometer spatial resolution and therefore complements atlases based on a combination of magnetic resonance microscopy and optical micrographs of serial histological sections.

Formalin fixation and paraffin embedding of post mortem tissue specimens is widely used for high-resolution neuroimaging with both conventional and X-ray virtual histology. Exchange of embedding solutions generates non-uniform brain shrinkage and changes relative tissue densities. We used synchrotron radiation-based X-ray micro computed tomography to visualize the embedding process for a single mouse brain. Non-rigid registration was employed to determine the volumetric strain fields and to track the X-ray absorption changes of corresponding features. This allows for a correction of the observed microanatomy to improve the anatomical context. Through embedding, the entire brain shrinks to around 40% of its volume in formalin. Shrinkage is non-uniform and varies over anatomical regions and the distance to external surfaces.

High-contrast, high-resolution imaging of biomedical specimens is indispensable for studying organ function and pathologies. Conventional histology, the gold standard for soft-tissue visualization, is limited by its anisotropic spatial resolution, elaborate sample preparation, and lack of quantitative image information. X-ray absorption or phase tomography have been identified as promising alternatives enabling non-destructive, distortion-free three-dimensional (3D) imaging. However, reaching sufficient contrast and resolution with a simple experimental procedure remains a major challenge. Here, we present a solution based on x-ray phase tomography through speckle-based imaging (SBI). We demonstrate on a mouse kidney that SBI delivers comprehensive 3D maps of hydrated, unstained soft tissue, revealing its microstructure and delivering quantitative tissue-density values at a density resolution of better than 2 m g / c m 3 and spatial resolution of better than 8 µm. We expect that SBI virtual histology will find widespread application in biomedicine and will open up new possibilities for research and histopathology.

Biological matter may change shape via water absorption or loss. For example, brain tissue shows non-uniform shrinkage during formalin fixation and paraffin embedding, which is the most common tissue preparation for conventional histological analysis. Local deformations can be analyzed with non-rigid registration of non-destructive three-dimensional imaging datasets. We utilized synchrotron radiation microtomography at the ANATOMIX beamline of Synchrotron SOLEIL to image a mouse brain with 3 micron voxel length after formalin fixation, immersion in ascending alcohol series and xylene, and after paraffin embedding. We created a pipeline for non- rigid registration to align the volumes and extract volumetric strain fields. In this way, we could visualize the swelling/shrinkage of anatomical features. This method avoids time-consuming segmentation of brain regions, however it is sensitive to the registration parameters. In this proceedings paper, we discuss the selection of registration parameters in order to generate plausible volumetric strain fields. This protocol can be deployed to any type of shape change of biological matter and allows for the quantification of the related processes.

Background Micrometer-resolution neuroimaging with gold-standard conventional histology requires tissue fixation and embedding. The exchange of solvents for the creation of sectionable paraffin blocks modifies tissue density and generates non-uniform brain shrinkage. New method We employed synchrotron radiation-based X-ray microtomography for slicing- and label-free virtual histology of the mouse brain at different stages of the standard preparation protocol from formalin fixation via ascending ethanol solutions and xylene to paraffin embedding. Segmentation of anatomical regions allowed us to quantify non-uniform tissue shrinkage. Global and local changes in X-ray absorption gave insight into contrast enhancement for virtual histology. Results The volume of the entire mouse brain was 60%, 56%, and 40% of that in formalin for, respectively, 100% ethanol, xylene, and paraffin. The volume changes of anatomical regions such as the hippocampus, anterior commissure, and ventricles differ from the global volume change. X-ray absorption of the full brain decreased, while local absorption differences increased, resulting in enhanced contrast for virtual histology. These trends were also observed with laboratory microtomography measurements. Comparison with existing methods Microtomography provided sub-10 μm spatial resolution with sufficient density resolution to resolve anatomical structures at each step of the embedding protocol. The spatial resolution of conventional computed tomography and magnetic resonance microscopy is an order of magnitude lower and both do not match the contrast of microtomography over the entire embedding protocol. Unlike feature-to-feature or total volume measurements, our approach allows for calculation of volume change based on segmentation. Conclusion We present isotropic micrometer-resolution imaging to quantify morphology and composition changes in a mouse brain during the standard histological preparation. The proposed method can be employed to identify the most appropriate embedding medium for anatomical feature visualization, to reveal the basis for the dramatic X-ray contrast enhancement observed in numerous embedded tissues, and to quantify morphological changes during tissue fixation and embedding.

A contrast agent for X-ray micro computed tomography (μCT), called XlinCA, that combines reliable perfusion and permanent retention and contrast properties, was developed for ex vivo imaging. The new imaging agent XlinCA features high molecular weight, low viscosity and a high iodine content.

Renal arteriovenous oxygen shunting has been proposed as a mechanism by which oxygen supplied to the kidney can bypass the renal parenchyma. Shunting could, therefore, play a crucial role in renal hypoxia and hyperoxia. In the absence of suitable quantitative experimental methods, computational modeling has been employed in recent years to estimate the extent and potential impact of oxygen shunting.Overestimation of the separation distance between arteries and veins was suggested to be responsible for previous findings that only negligible amounts of oxygen are shunted in the preglomerular vasculature. However, models considering the correct separation distance and wrapping of artery-vein pairs still showed shunting at negligible levels of less than 1% of total renal oxygen delivery. The effect of reverse CO2 shunting on the oxygen-hemoglobin dissociation curve was found to impair, rather than promote, preglomerular oxygen shunting.Oxygen is unlikely to be shunted along the preglomerular vasculature in sufficient quantities to affect renal oxygenation. There may be substantial shunting at the level of the postglomerular vasculature, but more extensive efforts in structural imaging and computational modeling are needed to quantify it reliably.

Background Lewy body disease (LBD) is the second most common cause of neurodegenerative dementia after Alzheimer’s disease and presents a challenge to healthcare services in an ageing society because current therapeutic approaches do not halt the disease’s progress. It has been suggested that aggregation of the protein α-synuclein plays a critical role in LBD. One pathway for aggregation involves the creation of low molecular weight α-synuclein oligomers, which have been observed to induce cell death. Precise location of Lewy bodies and their precursors could allow researchers to better relate their presence to clinical symptoms as well as infer their physiological effects. While conventional light microscopy of histological slices can visualize Lewy bodies and larger protein aggregates, it provides only two-dimensional information and diffraction-limited resolution prevents visualization of the smaller (approximately 50 nanometer) protein bundles. For this reason, we propose hard X-ray micro- and nano-tomography modalities for complementary, non-destructive, three-dimensional visualization of brain tissues taking advantage of developments at synchrotron radiation facilities. Methods As a proof-of-concept, we imaged paraffin-embedded fusiform gyrus tissue (provided by Newcastle Brain Tissue Resource) from a DLB case at the European Synchrotron Radiation Facility (ESRF Grenoble, France provided beamtime at ID19 from proposal MD-1055) using single-distance inline phase-contrast X-ray tomography with pixel size of 1.6 micrometers. A H&E stained histology slice of the same sample was also taken. Results A correspondence between the micro computed tomography and histology datasets could be made by tracking the orientation of vessels in the specimen. The micro computed tomography demonstrated as shown in figure (left) sufficient contrast and resolution for identification of the lower cortical layer as well as visualization of cells in three-dimensional manner, with confirmation from the annotated histology slice. Conclusions The present work serves as a proof-of-concept for the complementary use of phase contrast X-ray tomography and histology in the investigation of neurodegenerative diseases down to the cellular level. These results open the door for further nanometer resolution studies. Opens large image Nearby slices from phase-contrast microcomputed tomography (μ-CT) (left) and H&E stained histology (right) of diseased human brain tissue. The modalities can be used in complement, with the non-destructive μ-CT directing cutting planes for histological sectioning and extending histology into the third dimension.

Erythropoietin (Epo) is essential for erythropoiesis and is mainly produced by the fetal liver and the adult kidney following hypoxic stimulation. Epo regulation is commonly studied in hepatoma cell lines, but differences in Epo regulation between kidney and liver limit the understanding of Epo dysregulation in polycythaemia and anaemia. To overcome this limitation, we have generated a novel transgenic mouse model expressing Cre recombinase specifically in the active fraction of renal Epo-producing (REP) cells. Crossing with reporter mice confirmed the inducible and highly specific tagging of REP cells, located in the corticomedullary border region where there is a steep drop in oxygen bioavailability. A novel method was developed to selectively grow primary REP cells in culture and to generate immortalized clonal cell lines, called fibroblastoid atypical interstitial kidney (FAIK) cells. FAIK cells show very early hypoxia-inducible factor (HIF)-2α induction, which precedes Epo transcription. Epo induction in FAIK cells reverses rapidly despite ongoing hypoxia, suggesting a cell autonomous feedback mechanism. In contrast, HIF stabilizing drugs resulted in chronic Epo induction in FAIK cells. RNA sequencing of three FAIK cell lines derived from independent kidneys revealed a high degree of overlap and suggests that REP cells represent a unique cell type with properties of pericytes, fibroblasts, and neurons, known as telocytes. These novel cell lines may be helpful to investigate myofibroblast differentiation in chronic kidney disease and to elucidate the molecular mechanisms of HIF stabilizing drugs currently in phase III studies to treat anemia in end-stage kidney disease.

Tissue engineering provides a promising alternative therapy to the complex surgical reconstruction of auricular cartilage by using ear-shaped autologous costal cartilage. Bacterial nanocellulose (BNC) is proposed as a promising scaffold material for auricular cartilage reconstruction, as it exhibits excellent biocompatibility and secures tissue integration. Thus, this study evaluates a novel bilayer BNC scaffold for auricular cartilage tissue engineering. Bilayer BNC scaffolds, composed of a dense nanocellulose layer joined with a macroporous composite layer of nanocellulose and alginate, were seeded with human nasoseptaLchondrocytes (NC) and cultured in vitro for up to 6 weeks. To scale up for clinical translation, bilayer BNC scaffolds were seeded with a low number of freshly isolated (uncultured) human NCs combined with freshly isolated human mononuclear cells (MNC) from bone marrow in alginate and subcutaneously implanted in nude mice for 8 weeks. 3D morphometric analysis showed that bilayer BNC scaffolds have a porosity of 75% and mean pore size of 50 +/- 25 pm. Furthermore, endotoxin analysis and in vitro cytotoxicity testing revealed that the produced bilayer BNC scaffolds were non-pyrogenic (0.15 +/- 0.09 EU/ml) and non-cytotoxic (cell viability: 97.8 +/- 4.7%). This study demonstrates that bilayer BNC scaffolds offer a good mechanical stability and maintain a structural integrity while providing a porous architecture that supports cell ingrowth. Moreover, bilayer BNC scaffolds provide a suitable environment for culture-expanded NCs as well as a combination of freshly isolated NCs and MNCs to form cartilage in vitro and in vivo as demonstrated by immunohistochemistry, biochemical and biomechanical analyses. (C) 2014 Elsevier Ltd. All rights reserved.

Hard X-ray tomography with Paganin's widespread single-distance phase retrieval filter improves contrast-to-noise ratio (CNR) while reducing spatial resolution (SR). We demonstrate that a Gaussian filter provided larger CNR at high SR with interpretable density measurements for two medically relevant soft tissue samples. Paganin's filter produced larger CNR at low SR, though \emph{a priori} assumptions were generally false and image quality gains diminish for CNR $>1$. Therefore, simple absorption measurements of low-$Z$ specimens combined with Gaussian filtering can provide improved image quality and model-independent density measurements compared to single-distance phase retrieval., This article may be downloaded for personal use only. Any other use requires prior permission of the author and AIP Publishing. This article appeared in Applied Physics Letters 116, 023702 (2020), and may be found at https://aip.scitation.org/doi/10.1063/1.5133742

Malfunction of oxygen regulation in kidney and liver may lead to the pathogenesis of chronic diseases. The underlying mechanisms are poorly understood. In kidney, it is hypothesized that renal gas shunting from arteries to veins eliminates excess oxygen. Such shunting is highly dependent on the structure of the renal vascular network. The vascular tree has so far not been quantified under maintenance of its connectivity as three-dimensional imaging of the vessel tree down to the smallest capillaries, which in mouse model are smaller than 5 μm in diameter, is a challenging task. An established protocol uses corrosion casts and applies synchrotron radiation-based micro-computed tomography (SRμCT), which provides the desired spatial resolution with the necessary contrast. However, SRμCT is expensive and beamtime access is limited. We show here that measurements with a phoenix nanotomrm (General Electric, Wunstorf, Germany) can provide comparable results to those obtained with SRμCT, except for regions with small vessel structures, where the signal-to-noise level was significantly reduced. For this purpose the nanotom®m measurement was compared with its corresponding measurement acquired at the beamline P05 at PETRA III at DESY, Hamburg, Germany.

Biomedical Engineering / Biomedizinische Technik, 58 (S1), ISSN:0013-5585, ISSN:1862-278X