A high-throughput 3D X-ray histology facility for biomedical research and preclinical applications.

Background: The University of Southampton, in collaboration with the University Hospital Southampton (UHS) NHS Foundation Trust and industrial partners, has been at the forefront of developing three-dimensional (3D) imaging workflows using X-ray microfocus computed tomography (μCT) -based technology. This article presents the outcomes of these endeavours and highlights the distinctive characteristics of a μCT facility tailored explicitly for 3D X-ray Histology, with a primary focus on applications in biomedical research and preclinical and clinical studies.

Methods: The UHS houses a unique 3D X-ray Histology (XRH) facility, offering a range of services to national and international clients.

3D visualization of trans-syncytial nanopores provides a pathway for paracellular diffusion across the human placental syncytiotrophoblast.

The placental syncytiotrophoblast, a syncytium without cell-cell junctions, is the primary barrier between the mother and the fetus. Despite no apparent anatomical pathway for paracellular diffusion of solutes across the syncytiotrophoblast, size-dependent paracellular diffusion is observed. Here we report data demonstrating that the syncytiotrophoblast is punctuated by -syncytial nanopores (TSNs).

Microvillous tip vesicles may be an origin of placental extracellular vesicles.

Introduction: Extracellular vesicles are now believed to be important mediators of placental-maternal communication. However, little is known about the formation of extracellular vesicles by human placenta. This study uses nanoscale three-dimensional imaging to investigate how and where placental extracellular vesicles form.

A High Fat "Western-style" Diet Induces AMD-Like Features in Wildtype Mice.

Scope: The intake of a "Western-style" diet rich in fats is linked with developing retinopathies including age-related macular degeneration (AMD). Wildtype mice are given a high fat diet (HFD) to determine how unhealthy foods can bring about retinal degeneration.

Methods And Results: Following weaning, female C57BL/6 mice are maintained on standard chow (7% kcal fat, n = 29) or a HFD (45% kcal fat, n = 27) for 12 months.

A novel model of nephrotic syndrome results from a point mutation in Lama5 and is modified by genetic background.

Nephrotic syndrome is characterized by severe proteinuria, hypoalbuminaemia, edema and hyperlipidaemia. Genetic studies of nephrotic syndrome have led to the identification of proteins playing a crucial role in slit diaphragm signaling, regulation of actin cytoskeleton dynamics and cell-matrix interactions. The laminin α5 chain is essential for embryonic development and, in association with laminin β2 and laminin γ1, is a major component of the glomerular basement membrane, a critical component of the glomerular filtration barrier.

Folding of the syncytiotrophoblast basal plasma membrane increases the surface area available for exchange in human placenta.

Introduction: The placental syncytiotrophoblast is the primary barrier between the mother and the fetus. To cross the placenta, nutrients and wastes must be transported across the apical microvillous and basal plasma membranes. While the syncytiotrophoblast basal plasma membrane is typically represented as relatively smooth, it has been shown to have invaginations that may increase its surface area.

Oligomeric Aβ Induces an AMD-Like Phenotype and Accumulates in Lysosomes to Impair RPE Function.

Alzheimer's disease-associated amyloid beta (Aβ) proteins accumulate in the outer retina with increasing age and in eyes of age-related macular degeneration (AMD) patients. To study Aβ-induced retinopathy, wild-type mice were injected with nanomolar human oligomeric Aβ, which recapitulate the Aβ burden reported in human donor eyes. In vitro studies investigated the cellular effects of Aβ in endothelial and retinal pigment epithelial (RPE) cells.

Pericytes on placental capillaries in terminal villi preferentially cover endothelial junctions in regions furthest away from the trophoblast.

Introduction: Pericytes are a common feature in the placental microvasculature but their roles are not well understood. Pericytes may provide physical or endocrine support for endothelium and in some tissues mediate vasoconstriction.

Methods: This study uses serial block-face scanning electron microscopy (SBFSEM) to generate three-dimensional (3D) reconstructions of placental pericytes of the terminal villi and transmission electron microscopy (TEM) to study pericyte endothelial cell interactions.

3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina.

The retinal pigment epithelium (RPE) is located between the neuroretina and the choroid, and plays a critical role in vision. RPE cells internalise outer segments (OS) from overlying photoreceptors in the daily photoreceptor renewal. Changes to RPE structure are linked with age and retinopathy, which has been described in the past by conventional 2D electron microscopy.

An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy.

Impaired cargo trafficking and the aggregation of intracellular macromolecules are key features of neurodegeneration, and a hallmark of aged as well as diseased retinal pigment epithelial (RPE) cells in the eye. Here, photoreceptor outer segments (POS), which are internalized daily by RPE cells, were modified by UV-irradiation to create oxidatively modified POS (OxPOS). Oxidative modification was quantified by a protein carbonyl content assay.

Serial block-face scanning electron microscopy reveals novel intercellular connections in human term placental microvasculature.

The placental microvasculature is a conduit for fetal blood allowing solute exchange between the mother and the fetus. Serial block-face scanning electron microscopy (SBF SEM) allows ultrastructure to be viewed in three dimensions and provides a new perspective on placental anatomy. This study used SBF SEM to study endothelial cells within the human placental microvasculature from uncomplicated pregnancies.

Revealing Sources of Variation for Reproducible Imaging of Protein Assemblies by Electron Microscopy.

Electron microscopy plays an important role in the analysis of functional nano-to-microstructures. Substrates and staining procedures present common sources of variation for the analysis. However, systematic investigations on the impact of these sources on data interpretation are lacking.

Persistent neuropathological effects 14 years following amyloid-β immunization in Alzheimer's disease.

We performed a 15-year post-mortem neuropathological follow-up of patients in the first trial of amyloid-β immunotherapy for Alzheimer's disease. Twenty-two participants of a clinical trial of active amyloid-β42 immunization (AN1792, Elan Pharmaceuticals) or placebo were studied. Comprehensive post-mortem neuropathological assessments were performed from 4 months to 15 years after the trial.

A lasered mouse model of retinal degeneration displays progressive outer retinal pathology providing insights into early geographic atrophy.

Early stages of geographic atrophy (GA) age-related macular degeneration is characterised by the demise of photoreceptors, which precedes the loss of underlying retinal pigment epithelial (RPE) cells. Sight-loss due to GA has no effective treatment; reflecting both the complexity of the disease and the lack of suitable animal models for testing potential therapies. We report the development and characterisation of a laser-induced mouse model with early GA-like pathology.

Oxidative Stress and Dysfunctional Intracellular Traffic Linked to an Unhealthy Diet Results in Impaired Cargo Transport in the Retinal Pigment Epithelium (RPE).

Scope: Oxidative stress and dysregulated intracellular trafficking are associated with an unhealthy diet which underlies pathology. Here, these effects on photoreceptor outer segment (POS) trafficking in the retinal pigment epithelium (RPE), a major pathway of disease underlying irreversible sight-loss, are studied.

Methods And Results: POS trafficking is studied in ARPE-19 cells using an algorithm-based quantification of confocal-immunofluorescence data supported by ultrastructural studies.

Serial block-face scanning electron microscopy of erythrocytes protruding through the human placental syncytiotrophoblast.

The syncytiotrophoblast forms a continuous barrier between the maternal and fetal circulations. Here we present a serial block-face scanning electron microscopy (SBFSEM) study, based on a single image stack, showing pooling of fetal blood underneath a region of stretched syncytiotrophoblast that has become detached from the basement membrane. Erythrocytes are protruding from discrete holes in the syncytiotrophoblast suggesting that, under specific circumstances, the syncytiotrophoblast may be permeable to fetal cells.

Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE.

The Retinal Pigment Epithelium (RPE) forms the primary site of pathology in several blinding retinopathies. RPE cultures are being continuously refined so that dynamic disease processes in this important monolayer can be faithfully studied outside the eye over longer periods. The RPE substrate, which mimics the supportive Bruch's membrane (BrM), plays a key role in determining how well in-vitro cultures recapitulate native RPE cells.

Quantitative Assessment of Cerebral Basement Membranes Using Electron Microscopy.

In this chapter we describe in detail the tissue processing techniques we employ for the study of cerebral tissue by transmission electron microscopy (TEM). In particular, we explain a technique that enables quantification of changes in cerebral basement membranes at the ultrastructural level. This is significant, as age related pathological conditions affecting the brain are often accompanied by ultrastructural changes in the cerebral vasculature.

Skin Microstructure is a Key Contributor to Its Friction Behaviour.

Due to its multifactorial nature, skin friction remains a multiphysics and multiscale phenomenon poorly understood despite its relevance for many biomedical and engineering applications (from superficial pressure ulcers, through shaving and cosmetics, to automotive safety and sports equipment). For example, it is unclear whether, and in which measure, the skin microscopic surface topography, internal microstructure and associated nonlinear mechanics can condition and modulate skin friction. This study addressed this question through the development of a parametric finite element contact homogenisation procedure which was used to study and quantify the effect of the skin microstructure on the skin frictional response.

The Alzheimer's-related amyloid beta peptide is internalised by R28 neuroretinal cells and disrupts the microtubule associated protein 2 (MAP-2).

Age-related Macular Degeneration (AMD) is a common, irreversible blinding condition that leads to the loss of central vision. AMD has a complex aetiology with both genetic as well as environmental risks factors, and share many similarities with Alzheimer's disease. Recent findings have contributed significantly to unravelling its genetic architecture that is yet to be matched by molecular insights.

Accuracy of diagnostic testing in primary ciliary dyskinesia.

Diagnosis of primary ciliary dyskinesia (PCD) lacks a "gold standard" test and is therefore based on combinations of tests including nasal nitric oxide (nNO), high-speed video microscopy analysis (HSVMA), genotyping and transmission electron microscopy (TEM). There are few published data on the accuracy of this approach.Using prospectively collected data from 654 consecutive patients referred for PCD diagnostics we calculated sensitivity and specificity for individual and combination testing strategies.

Exploring the diversity in Citrus fruit colouration to decipher the relationship between plastid ultrastructure and carotenoid composition.

Differentiation of new and characteristic plastid ultrastructures during ripening of citrus fruits in both peel and pulp appears to be strongly correlated with the content and complement of carotenoids. Most of the species of the Citrus genus display a wide range in fruit colouration due to differences in carotenoids; however, how this diversity is related and may contribute to plastid differentiation and ultrastructure is currently unknown. To that end, carotenoid profile and plastid ultrastructure were compared in peel and pulp of three sweet oranges: the ordinary orange-coloured Navel, rich in β,β-xanthophylls, the yellow Pinalate mutant with an elevated content of colourless carotenes and reduced β,β-xanthophylls, and the red-fleshed Cara Cara with high concentration of colourless carotenes and lycopene in the pulp; and two grapefruits: the white Marsh, with low carotenoid content, and the red Star Ruby, accumulating upstream carotenes and lycopene.

A mechanistic insight into the mechanical role of the stratum corneum during stretching and compression of the skin.

The study of skin biophysics has largely been driven by consumer goods, biomedical and cosmetic industries which aim to design products that efficiently interact with the skin and/or modify its biophysical properties for health or cosmetic benefits. The skin is a hierarchical biological structure featuring several layers with their own distinct geometry and mechanical properties. Up to now, no computational models of the skin have simultaneously accounted for these geometrical and material characteristics to study their complex biomechanical interactions under particular macroscopic deformation modes.

Fruit shading enhances peel color, carotenes accumulation and chromoplast differentiation in red grapefruit.

The distinctive color of red grapefruits is due to lycopene, an unusual carotene in citrus. It has been observed that red 'Star Ruby' (SR) grapefruits grown inside the tree canopy develop a more intense red coloration than those exposed to higher light intensities. To investigate the effect of light on SR peel pigmentation, fruit were bagged or exposed to normal photoperiodic conditions, and changes in carotenoids, expression of carotenoid biosynthetic genes and plastid ultrastructure in the peel were analyzed.