Multiscale and multimodal imaging for three-dimensional vascular and histomorphological organ structure analysis of the pancreas.

Exocrine and endocrine pancreas are interconnected anatomically and functionally, with vasculature facilitating bidirectional communication. Our understanding of this network remains limited, largely due to two-dimensional histology and missing combination with three-dimensional imaging. In this study, a multiscale 3D-imaging process was used to analyze a porcine pancreas. Clinical computed tomography, digital volume tomography, micro-computed tomography and Synchrotron-based propagation-based imaging were applied consecutively. Fields of view correlated inversely with attainable resolution from a whole organism level down to capillary structures with a voxel edge length of 2.0 µm. Segmented vascular networks from 3D-imaging data were correlated with tissue sections stained by immunohistochemistry and revealed highly vascularized regions to be intra-islet capillaries of islets of Langerhans. Generated 3D-datasets allowed for three-dimensional qualitative and quantitative organ and vessel structure analysis. Beyond this study, the method shows potential for application across a wide range of patho-morphology analyses and might possibly provide microstructural blueprints for biotissue engineering.

In vivo low-dose phase-contrast CT for quantification of functional and anatomical alterations in lungs of an experimental allergic airway disease mouse model.

Introduction: Synchrotron-based propagation-based imaging (PBI) is ideally suited for lung imaging and has successfully been applied in a variety of in vivo small animal studies. Virtually all these experiments were tailored to achieve extremely high spatial resolution close to the alveolar level while delivering high x-ray doses that would not permit longitudinal studies. However, the main rationale for performing lung imaging studies in vivo in small animal models is the ability to follow disease progression or monitor treatment response in the same animal over time. Thus, an in vivo imaging strategy should ideally allow performing longitudinal studies. Methods: Here, we demonstrate our findings of using PBI-based planar and CT imaging with two different detectors-MÖNCH 0.3 direct conversion detector and a complementary metal-oxide-semiconductor (CMOS) detector (Photonics Science)-in an Ovalbumin induced experimental allergic airway disease mouse model in comparison with healthy controls. The mice were imaged free breathing under isoflurane anesthesia. Results: At x-ray dose levels below those once used by commercial small animal CT devices at similar spatial resolutions, we were able to resolve structural changes at a pixel size down to 25 µm and demonstrate the reduction in elastic recoil in the asthmatic mice in cinematic planar x-ray imaging with a frame rate of up to 100 fps. Discussion: Thus, we believe that our approach will permit longitudinal small animal lung disease studies, closely following the mice over longer time spans.

Characterization of transient and progressive pulmonary fibrosis by spatially correlated phase contrast microCT, classical histopathology and atomic force microscopy.

Pulmonary fibrosis (PF) is a severe and progressive condition in which the lung becomes scarred over time resulting in pulmonary function impairment. Classical histopathology remains an important tool for micro-structural tissue assessment in the diagnosis of PF. A novel workflow based on spatial correlated propagation-based phase-contrast micro computed tomography (PBI-microCT), atomic force microscopy (AFM) and histopathology was developed and applied to two different preclinical mouse models of PF - the commonly used and well characterized Bleomycin-induced PF and a novel mouse model for progressive PF caused by conditional Nedd4-2 KO. The aim was to integrate structural and mechanical features from hallmarks of fibrotic lung tissue remodeling. PBI-microCT was used to assess structural alteration in whole fixed and paraffin embedded lungs, allowing for identification of fibrotic foci within the 3D context of the entire organ and facilitating targeted microtome sectioning of planes of interest for subsequent histopathology. Subsequently, these sections of interest were subjected to AFM to assess changes in the local tissue stiffness of previously identified structures of interest. 3D whole organ analysis showed clear morphological differences in 3D tissue porosity between transient and progressive PF and control lungs. By integrating the results obtained from targeted AFM analysis, it was possible to discriminate between the Bleomycin model and the novel conditional Nedd4-2 KO model using agglomerative cluster analysis. As our workflow for 3D spatial correlation of PBI, targeted histopathology and subsequent AFM is tailored around the standard procedure of formalin-fixed paraffin-embedded (FFPE) tissue specimens, it may be a powerful tool for the comprehensive tissue assessment beyond the scope of PF and preclinical research.

Evaluation of MR-safe bioptomes for MR-guided endomyocardial biopsy in minipigs: a potential radiation-free clinical approach.

BACKGROUND: Diagnostic accuracy of endomyocardial biopsy could improve if clinically safe magnetic resonance (MR)-compatible bioptomes were available. We explored two novel MR-compatible cardiac bioptomes for performance, safety, and clinical viability, employing in vivo minipig trials and phase-contrast synchrotron radiation computed microtomography (SRµCT). METHODS: Analysis of ex vivo obtained pig endomyocardial biopsies was performed using phase-contrast SRµCT and conventional two-dimensional histology. The technical performance was evaluated by measuring volume, inner and outer integrities, compression, and histological diagnostic value in 3 sets (6 per set) of biopsies for each experimental bioptome. The bioptomes were tested in vivo in 3 healthy minipigs per bioptome. The clinical feasibility was evaluated by procedural and cutting success as well as histological diagnostic value. RESULTS: The bioptome with the 'grind-grind' design achieved similar values to control in compression (p = 0.822), inner (p = 0.628), and outer (p = 0.507), integrities ex vivo. It showed a better performance in the in vivo real-time MRI setting demonstrating a higher cutting success (91.7%) than the 'grind-anvil' (86.2%) design. In both ex vivo and in vivo evaluations, the 'grind-grind' design displayed sufficient diagnostic value (83% and 95%). The 'grind-anvil' design showed adequate diagnostic value both ex vivo and in vivo (78% and 87.5%) but was not comparable to control according to the three-dimensional (3D) analysis. CONCLUSION: A novel MR-compatible bioptome was identified as plausible in a clinical setting. Additionally, SRµCT and subsequent 3D structural analysis could be valuable in the label-free investigation of myocardial tissue at a micrometer level. RELEVANCE STATEMENT: Implementation of MR-guided biopsy can improve animal studies on structural myocardial changes at any point in an experimental setup. With further improvements in guiding catheters, MR-guided biopsy, using the new bioptome, has a potential to increase quality and diagnostic accuracy in patients both with structural and inflammatory cardiomyopathies. KEY POINTS: • Novel MR-compatible bioptomes show promise for a clinical application. • SRµCT enabled detailed analysis of endomyocardial biopsies. • The bioptomes showed adequate in vivo performance without major complications.

Longitudinal x-ray based lung function measurement for monitoring Nintedanib treatment response in a mouse model of lung fibrosis.

Lung fibrosis (LF) is a chronic progressive, incurable, and debilitating condition of the lung, which is associated with different lung disease. Treatment options are still sparse. Nintedanib, an oral tyrosine kinase inhibitor, significantly slows the LF progression. However, there is a strong need of further research and the development of novel therapies. In this study, we used a correlative set-up that combines X-ray based lung function (XLF) with microCT and whole body plethysmography (WBP) for a comprehensive functional and structural evaluation of lung fibrosis (LF) as well as for monitoring response to orally administered Nintedanib in the mouse model of bleomycin induced LF. The decline in lung function as early as one week after intratracheal bleomycin instillation was reliably detected by XLF, revealing the lowest decay rate in the LF mice compared to healthy ones. Simultaneously performed microCT and WBP measurements corroborated XLF findings by exhibiting reduced lung volume [Formula: see text] and tidal volume [Formula: see text]. In LF mice XLF also revealed profound improvement in lung function one week after Nintedanib treatment. This positive response to Nintedanib therapy was further substantiated by microCT and WBP measurements which also showed significantly improved [Formula: see text] and [Formula: see text] in the Nintedanib treated mice. By comparing the XLF data to structural features assessing the extent of fibrosis obtained by ex-vivo high-resolution synchrotron radiation-based imaging and classical histology we demonstrate that: (1) a simple low dose x-ray measurement like XLF is sensitive enough to pick up treatment response, (2) Nintedanib treatment successfully improved lung function in a bleomycin induced LF mouse model and (3) differences between the fully restored lung function and the partially reduced fibrotic burden compared to healthy and untreated mice. The presented analysis pipeline underlines the importance of a combined functional and anatomical readout to reliably measure treatment response and could easily be adapted to other preclinical lung disease models.

Spatial correlation of 2D hard-tissue histology with 3D microCT scans through 3D printed phantoms.

Hard-tissue histology-the analysis of thin two-dimensional (2D) sections-is hampered by the opaque nature of most biological specimens, especially bone. Therefore, the cutting process cannot be assigned to regions of interest. In addition, the applied cutting-grinding method is characterized by significant material loss. As a result, relevant structures might be missed or destroyed, and 3D features can hardly be evaluated. Here, we present a novel workflow, based on conventual microCT scans of the specimen prior to the cutting process, to be used for the analysis of 3D structural features and for directing the sectioning process to the regions of interest. 3D printed fiducial markers, embedded together with the specimen in resin, are utilized to retrospectively register the obtained 2D histological images into the 3D anatomical context. This not only allows to identify the cutting position, but also enables the co-registration of the cell and extracellular matrix morphological analysis to local 3D information obtained from the microCT data. We have successfully applied our new approach to assess hard-tissue specimens of different species. After matching the predicted microCT cut plane with the histology image, we validated a high accuracy of the registration process by computing quality measures namely Jaccard and Dice similarity coefficients achieving an average score of 0.90 ± 0.04 and 0.95 ± 0.02, respectively. Thus, we believe that the novel, easy to implement correlative imaging approach holds great potential for improving the reliability and diagnostic power of classical hard-tissue histology.

RNF40 epigenetically modulates glycolysis to support the aggressiveness of basal-like breast cancer.

Triple-negative breast cancer (TNBC) is the most difficult breast cancer subtype to treat due to the lack of targeted therapies. Cancer stem cells (CSCs) are strongly enriched in TNBC lesions and are responsible for the rapid development of chemotherapy resistance and metastasis. Ubiquitin-based epigenetic circuits are heavily exploited by CSCs to regulate gene transcription and ultimately sustain their aggressive behavior. Therefore, therapeutic targeting of these ubiquitin-driven dependencies may reprogram the transcription of CSC and render them more sensitive to standard therapies. In this work, we identified the Ring Finger Protein 40 (RNF40) monoubiquitinating histone 2B at lysine 120 (H2Bub1) as an indispensable E3 ligase for sustaining the stem-cell-like features of the growing mammary gland. In addition, we found that the RNF40/H2Bub1-axis promotes the CSC properties and drug-tolerant state by supporting the glycolytic program and promoting pro-tumorigenic YAP1-signaling in TNBC. Collectively, this study unveils a novel tumor-supportive role of RNF40 and underpins its high therapeutic value to combat the malignant behavior of TNBC.

Bilateral dislocation of the hip joint and associated pathological changes in the ossa coxae and femora of a European roe deer (Capreolus capreolus).

We describe a bilateral craniodorsal dislocation of the hip joint in a free-ranging young roe buck and the associated pathological changes in the ossa coxae and femora of the animal. The highly symmetrical dislocation, which is considered to have developed secondary to hip dysplasia, caused the formation of two false acetabula that each consist of several, partially fused bone portions. The femora exhibit symmetrical outgrowths that extend from the greater trochanter along the intertrochanteric crest to the lesser trochanter. Formation of these outgrowths is attributed to abnormal traction at muscle attachment sites due to the displacement of the femora. On radiographic examination, both femora show signs of avascular necrosis in their head regions and of fatty marrow necrosis in their shafts, which is attributed to the damage of the arterial blood supply of the femora that was associated with the dislocation. The fact that, according to the hunter who shot the buck, the animal's locomotion was inconspicuous suggests that the false hip joints functioned quite well, thereby demonstrating a remarkable capacity of the musculoskeletal system for functional recovery.

Domestication effect of reduced brain size is reverted when mink become feral.

A typical consequence of breeding animal species for domestication is a reduction in relative brain size. When domesticated animals escape from captivity and establish feral populations, the larger brain of the wild phenotype is usually not regained. In the American mink (Neovison vison), we found an exception to this rule. We confirmed the previously described reduction in relative braincase size and volume compared to their wild North American ancestors in mink bred for their fur in Poland, in a dataset of 292 skulls. We then also found a significant regrowth of these measures in well-established feral populations in Poland. Closely related, small mustelids are known for seasonal reversible changes in skull and brain size. It seems that these small mustelids are able to regain the brain size, which is adaptive for living in the wild, and flexibly respond to selection accordingly.

Content aware image restoration improves spatiotemporal resolution in luminescence imaging.

Luminescent reporters are due to their intrinsically high signal-to-noise ratio a powerful labelling tool for microscopy and macroscopic in vivo imaging in biomedical research. However, luminescence signal detection requires longer exposure times than fluorescence imaging and is consequently less suited for applications requiring high temporal resolution or throughput. Here we demonstrate that content aware image restoration can drastically reduce the exposure time requirements in luminescence imaging, thus overcoming one of the major limitations of the technique.

High resolution propagation-based lung imaging at clinically relevant X-ray dose levels.

Absorption-based clinical computed tomography (CT) is the current imaging method of choice in the diagnosis of lung diseases. Many pulmonary diseases are affecting microscopic structures of the lung, such as terminal bronchi, alveolar spaces, sublobular blood vessels or the pulmonary interstitial tissue. As spatial resolution in CT is limited by the clinically acceptable applied X-ray dose, a comprehensive diagnosis of conditions such as interstitial lung disease, idiopathic pulmonary fibrosis or the characterization of small pulmonary nodules is limited and may require additional validation by invasive lung biopsies. Propagation-based imaging (PBI) is a phase sensitive X-ray imaging technique capable of reaching high spatial resolutions at relatively low applied radiation dose levels. In this publication, we present technical refinements of PBI for the characterization of different artificial lung pathologies, mimicking clinically relevant patterns in ventilated fresh porcine lungs in a human-scale chest phantom. The combination of a very large propagation distance of 10.7 m and a photon counting detector with [Formula: see text] pixel size enabled high resolution PBI CT with significantly improved dose efficiency, measured by thermoluminescence detectors. Image quality was directly compared with state-of-the-art clinical CT. PBI with increased propagation distance was found to provide improved image quality at the same or even lower X-ray dose levels than clinical CT. By combining PBI with iodine k-edge subtraction imaging we further demonstrate that, the high quality of the calculated iodine concentration maps might be a potential tool for the analysis of lung perfusion in great detail. Our results indicate PBI to be of great value for accurate diagnosis of lung disease in patients as it allows to depict pathological lesions non-invasively at high resolution in 3D. This will especially benefit patients at high risk of complications from invasive lung biopsies such as in the setting of suspected idiopathic pulmonary fibrosis (IPF).

Novel setup for rapid phase contrast CT imaging of heavy and bulky specimens.

This work introduces a novel setup for computed tomography of heavy and bulky specimens at the SYRMEP beamline of the Italian synchrotron Elettra. All the key features of the setup are described and the first application to off-center computed tomography scanning of a human chest phantom (approximately 45 kg) as well as the first results for vertical helical acquisitions are discussed.

Micro-CT evaluation of frozen and embalmed human cadavers on the effect of root canal preparation on microcrack formation in old dentin.

The aim of this study was to evaluate the existence of preoperative dentinal defects among differently preserved dentoalveolar bone-blocks (frozen vs. embalmed) and to investigate the effect of varying apical forces (low: <4 N, high: 4-8 N) during root canal preparation on microcrack formation using micro-computed tomography (micro-CT). Thirteen embalmed and seven frozen bone-blocks containing 1-3 single rooted teeth were collected. The teeth were evenly divided into three groups (n = 10): FLow (frozen, <4 N), ELow (embalmed, <4 N), EHigh (embalmed, 4-8 N). After working length determination all specimens were scanned preoperatively. Root canal preparation was performed using nickel-titanium instruments sizes 25/.06 and 40/.06 (F6 SkyTaper; Komet, Lemgo, Germany). A postoperative scan was performed and image stacks were co-registered. All cross-sectional images were screened to identify the presence of dentinal defects. The results were expressed as the percentage of teeth/slices presenting dentinal defects. The statistical analyses were performed with Kruskal-Wallis-Test and Mann-Whitney-U-Test (α = 5%). Embalmed specimens presented a significantly higher percentage of slices with preoperative microcracks (p<0.05) than frozen specimens. No significant difference between groups was observed regarding the induction of microcracks (p>0.05). Root canal preparation does not induce microcracks in dentoalveolar bone-blocks from donors of old age, irrespective of the preservation method and the apically directed forces.

Early incisor lesions and Equine Odontoclastic Tooth Resorption and Hypercementosis: Reliability of radiographic findings.

BACKGROUND: In clinical practice, early diagnosis of Equine Odontoclastic Tooth Resorption and Hypercementosis (EOTRH) and other resorptive incisor diseases is difficult to achieve. The radiographic appearance of subtle pathological changes has not been described in detail and might be confused with age-related changes. OBJECTIVES: The study was performed to define typical radiographic signs of early incisor lesions and to evaluate the reliability of the radiographic findings. STUDY DESIGN: Descriptive and comparative study using post mortem clinical, radiographic, macroscopic and µCT examination. METHODS: The incisor region of 20 cadaveric horse heads, divided into three different age groups, was examined visually and by palpation. Intraoral radiographs were taken. After extraction, each incisor was macroscopically evaluated. Micro-computed tomography (µCT) scans were obtained. These scans were processed with Scry (v6.0, Kuchel & Sautter GbR) to obtain surface meshes which then were transferred to Meshlab (ISTI-CNR, version 2016.12). Attached tissues were virtually removed and surface curvature was computed to visualise and evaluate the quantity of unevenness (roughness) of the teeth's surface. Scoring systems for each diagnostic modality were developed. Scores were compared to describe and evaluate the radiographic appearance of early incisor lesions. RESULTS: The prevalence and severity of incisor lesions increased with age. Early, subtle lesions develop on the palatal/lingual side of incisors. While radiographically detected lesions were confirmed macroscopically and on the µCT scans, numerous teeth which were radiographically classified as healthy displayed lesions by macroscopic inspection (13.7%) and µCT analysis (58.1%). MAIN LIMITATIONS: Cadavers were studied and dental history was unknown. CONCLUSIONS: The detection of early and subtle incisor lesions indicating first signs of EOTRH on dorsoventral intraoral radiographs is limited due to the typical localisation of the lesions on the palatal/lingual side of the incisors.

Current Approaches for Image Fusion of Histological Data with Computed Tomography and Magnetic Resonance Imaging.

Classical analysis of biological samples requires the destruction of the tissue's integrity by cutting or grinding it down to thin slices for (Immuno)-histochemical staining and microscopic analysis. Despite high specificity, encoded in the stained 2D section of the whole tissue, the structural information, especially 3D information, is limited. Computed tomography (CT) or magnetic resonance imaging (MRI) scans performed prior to sectioning in combination with image registration algorithms provide an opportunity to regain access to morphological characteristics as well as to relate histological findings to the 3D structure of the local tissue environment. This review provides a summary of prevalent literature addressing the problem of multimodal coregistration of hard- and soft-tissue in microscopy and tomography. Grouped according to the complexity of the dimensions, including image-to-volume (2D ⟶ 3D), image-to-image (2D ⟶ 2D), and volume-to-volume (3D ⟶ 3D), selected currently applied approaches are investigated by comparing the method accuracy with respect to the limiting resolution of the tomography. Correlation of multimodal imaging could position itself as a useful tool allowing for precise histological diagnostic and allow the a priori planning of tissue extraction like biopsies.

Winter conditions, not resource availability alone, may drive reversible seasonal skull size changes in moles.

Seasonal changes in the environment can lead to astonishing adaptations. A few small mammals with exceptionally high metabolisms have evolved a particularly extreme strategy: they shrink before winter and regrow in spring, including changes of greater than 20% in skull and brain size. Whether this process is an adaptation to seasonal climates, resource availability or both remains unclear. We show that European moles (Talpa europaea) also decrease skull size in winter. As resources for closely related Iberian moles (Talpa occidentalis) are lowest in summer, we predicted they should shift the timing of size changes. Instead, they do not change size at all. We conclude that in moles, seasonal decrease and regrowth of skull size is an adaptation to winter climate and not to a changing resource landscape alone. We not only describe this phenomenon in yet another taxon, but take an important step towards a better understanding of this enigmatic cycle.

Simultaneous assessment of lung morphology and respiratory motion in retrospectively gated in-vivo microCT of free breathing anesthetized mice.

Retrospective gating (RG) is a well established technique in preclinical computed tomography (CT) to assess 3D morphology of the lung. In RG additional angular projections are recorded typically by performing multiple rotations. Consequently, the projections are sorted according to the expansion state of the chest and those sets are then reconstructed separately. Thus, the breathing motion artefacts are suppressed at a cost of strongly elevated X-ray dose levels. Here we propose to use the entire raw data to assess respiratory motion in addition to retrospectively gated 3D reconstruction that visualize anatomical structures of the lung. Using this RG based X-ray respiratory motion measurement approach, which will be referred to as RG based X-ray lung function measurement (rgXLF) on the example of the mdx mouse model of Duchenne muscle dystrophy (mdx) we accurately obtained both the 3D anatomical morphology of the lung and the thoracic bones as well as functional temporal parameters of the lung. Thus, rgXLF will remove the necessity for separate acquisition procedures by being able to reproduce comparable results to the previously established planar X-ray based lung function measurement approach in a single low dose CT scan.

A Preclinical Pilot Study on the Effects of Thermal Ablation on Lamb Growth Plates.

(1) Background: Thermal ablation has been demonstrated to affect the bone growth of osteoid osteoma in adolescents. Growth modulation due to thermal heat in children is conceivable, but has not yet been established. We used lamb extremities as a preclinical model to examine the effect of thermal ablation on growth plates in order to evaluate its potential for axial or longitudinal growth modulation in pediatric patients. (2) Methods: Thermal ablation was performed by electrocautery on eight different growth plates of the legs and distal radii of a stillborn lamb. After treatment, target hits and the physical extent of the growth plate lesions were monitored using micro-computed tomography (micro-CT) and histology. (3) Results: Lesions and their physical extent could be quantified in 75% of the treated extremities. The histological analysis revealed that the disruption of tissue was confined to a small area and the applied heat did not cause the entire growth plate to be disrupted or obviously damaged. (4) Conclusions: Thermal ablation by electrocautery is minimally invasive and can be used for targeted disruption of small areas in growth plates in the animal model. The results suggest that thermal ablation can be developed into a suitable method to influence epiphyseal growth in children.

Influence of moisture content of frozen and embalmed human cadavers for identification of dentinal microcracks using micro-computed tomography.

The aim of this study was to investigate the influence of moisture content in frozen and embalmed human cadavers on the detection of dentinal microcracks using micro-computed tomography (micro-CT). The group of embalmed specimens included three mandibular and two maxillary segments each containing one tooth. The group of frozen cadavers consisted of two frozen mandibular bone-blocks with two teeth and one mandibular segment containing one tooth. The final number of teeth for each preservation method was n = 5. All specimens were scanned with eight different moisture conditions: 48 h wet, 2 h dry, 48 h wet, 24 h dry, 48 h wet, 1 wk dry, 48 h wet, 1 wk dry. Micro-CT images were screened for the presence of dentinal microcracks. Statistical analysis was performed by nonparametric analysis of variance (α = 5%). Only few microcracks were observed in wet and in 2 h dried bone-blocks with no significant differences (p = 0.63 and p = 0.23, respectively). There was a significant and steady increase of microcracks within the groups of dried specimens as follows: 2 h dry < 24 h dry < first wk dry < second wk dry (all p < 0.008). Preservation method had no significant influence on the visibility of microcracks (p = 0.98). Identification of dentinal microcracks on micro-CT images is influenced by moisture content of cadaveric bone-blocks irrespective of the preservation method.