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Various visualization modalities of the kidney vasculature and glomeruli. A: the reconstructed 3D stack of the aCT data set with the focus onto the vasculature of the kidney (bigger vessels are presented in yellow). B: the virtual section through the data set using another transfer function: visualization is focused on kidney tissue (bigger vessels are displayed blue; microvessels and glomeruli are seen as yellow structures; tissue is red). C and C: the visualization focused onto the glomeruli. C: a volume rendering of a virtual 500 m-thick slice, as indicated with the white box in the inset in the lower left corner of the image. The white frame (labeled C) indicates the site with the glomeruli, shown at higher magnification in C. D and D=: the advanced visualization option: aCT at a higher resolution (isotropic voxel size 0.59 m). D: the inset indicates the virtual section level displayed in D=. The 3D volume rendering of the microvasculature of glomerulus marked in D is displayed in D.
Source publication
In the last decades the contrast-enhanced microCT-imaging of whole animal kidney became increasingly important. The visualization was mainly limited to middle-sized vessels. Since modern desktop microCT-scanners provide the necessary detail resolution, we developed an approach for rapid visualization and consistent assessment of kidney vasculature...
Contexts in source publication
Context 1
... projection images were used for reconstruction using Recon Software of the micro-CT scanner manufacturer (Bruker microCT N.V.). The obtained 3D image stacks were then used to visualize the kidney and its vasculature by means of CTvox Software (Bruker microCT N.V.) or Imaris Software (Bitplane Scientific Solutions, Belfast, United Kingdom; Fig. 2). Application of threshold segmentation and different transfer functions of the mentioned software allowed the focusing on the following different aspects of the investigated kidney, for example: 1) only on the kidney (macro-) vasculature in 3D (Fig. 2A); 2) kidney tissue with embedded vascular structures with virtual sections looking ...
Context 2
... microCT N.V.) or Imaris Software (Bitplane Scientific Solutions, Belfast, United Kingdom; Fig. 2). Application of threshold segmentation and different transfer functions of the mentioned software allowed the focusing on the following different aspects of the investigated kidney, for example: 1) only on the kidney (macro-) vasculature in 3D (Fig. 2A); 2) kidney tissue with embedded vascular structures with virtual sections looking analog to the histological ones (Fig. 2B); or 3) on the distribution of the glomeruli at lower (Fig. 2C) and higher (Fig. 2C) magnifications. The obtained aCT data were used for the visualization of the vasculature and glomeruli (Fig. 1, step 3), as well ...
Context 3
... segmentation and different transfer functions of the mentioned software allowed the focusing on the following different aspects of the investigated kidney, for example: 1) only on the kidney (macro-) vasculature in 3D (Fig. 2A); 2) kidney tissue with embedded vascular structures with virtual sections looking analog to the histological ones (Fig. 2B); or 3) on the distribution of the glomeruli at lower (Fig. 2C) and higher (Fig. 2C) magnifications. The obtained aCT data were used for the visualization of the vasculature and glomeruli (Fig. 1, step 3), as well as quantitative and qualitative analysis (Fig. 1, steps 4a and b). They were also used for the definition of the potential ...
Context 4
... software allowed the focusing on the following different aspects of the investigated kidney, for example: 1) only on the kidney (macro-) vasculature in 3D (Fig. 2A); 2) kidney tissue with embedded vascular structures with virtual sections looking analog to the histological ones (Fig. 2B); or 3) on the distribution of the glomeruli at lower (Fig. 2C) and higher (Fig. 2C) magnifications. The obtained aCT data were used for the visualization of the vasculature and glomeruli (Fig. 1, step 3), as well as quantitative and qualitative analysis (Fig. 1, steps 4a and b). They were also used for the definition of the potential volumes of interest that should be further investigated in more detail (Fig. 1, step ...
Context 5
... of the vasculature and glomeruli (Fig. 1, step 3), as well as quantitative and qualitative analysis (Fig. 1, steps 4a and b). They were also used for the definition of the potential volumes of interest that should be further investigated in more detail (Fig. 1, step 5). The region of interest can later be rescanned at a higher resolution (Fig. 2D), providing further details on the microvasculature, including the one within a glomerulus (see Fig. 2D). ...
Context 6
... (Fig. 1, steps 4a and b). They were also used for the definition of the potential volumes of interest that should be further investigated in more detail (Fig. 1, step 5). The region of interest can later be rescanned at a higher resolution (Fig. 2D), providing further details on the microvasculature, including the one within a glomerulus (see Fig. 2D). ...
Context 7
... options. As already mentioned before ( Fig. 1), if needed, more advanced options are available. In particular, the correlative approach, as previously presented in the Fig. 3, which can be combined with the higher resolution scans of the volume of interest that remarkably increase the resolution of the capillaries within the glomerular structures (Fig. 2, D and ...
Citations
... Currently, alternative imaging modalities such as X-ray, CT, PET and MRI scan are unable to be used in space due to substantial limitations (e.g., limited space for large imaging structures, difficulties in interpretation due to microgravity). However, it is possible that the future development of a photocathode-based X-ray source may one day make this a possibility [101,246]. If X-ray imaging was possible, certain caveats would need to be taken into account for accurate interpretation. ...
The field of human space travel is in the midst of a dramatic revolution. Upcoming missions are looking to push the boundaries of space travel, with plans to travel for longer distances and durations than ever before. Both the National Aeronautics and Space Administration (NASA) and several commercial space companies (e.g., Blue Origin, SpaceX, Virgin Galactic) have already started the process of preparing for long-distance, long-duration space exploration and currently plan to explore inner solar planets (e.g., Mars) by the 2030s. With the emergence of space tourism, space travel has materialized as a potential new, exciting frontier of business, hospitality, medicine, and technology in the coming years. However, current evidence regarding human health in space is very limited, particularly pertaining to short-term and long-term space travel. This review synthesizes developments across the continuum of space health including prior studies and unpublished data from NASA related to each individual organ system, and medical screening prior to space travel. We categorized the extraterrestrial environment into exogenous (e.g., space radiation and microgravity) and endogenous processes (e.g., alteration of humans’ natural circadian rhythm and mental health due to confinement, isolation, immobilization, and lack of social interaction) and their various effects on human health. The aim of this review is to explore the potential health challenges associated with space travel and how they may be overcome in order to enable new paradigms for space health, as well as the use of emerging Artificial Intelligence based (AI) technology to propel future space health research.
... Accordingly, rarefaction of PTCs becomes a crucial limiting factor for the recovery of kidney tissue from injury 10,11 , and it correlates with the severity of kidney fibrosis in human patients and a variety of animal models [11][12][13][14][15][16] . Capillary rarefaction has been extensively studied by various, elaborate techniques 17,18 . ...
Functional and structural alterations of peritubular capillaries (PTCs) are a major determinant of chronic kidney disease (CKD). Using a software-based algorithm for semiautomatic segmentation and morphometric quantification, this study analyzes alterations of PTC shape associated with chronic tubulointerstitial injury in three mouse models and in human biopsies. In normal kidney tissue PTC shape was predominantly elongated, whereas the majority of PTCs associated with chronic tubulointerstitial injury had a rounder shape. This was reflected by significantly reduced PTC luminal area, perimeter and diameters as well as by significantly increased circularity and roundness. These morphological alterations were consistent in all mouse models and human kidney biopsies. The mean circularity of PTCs correlated significantly with categorized glomerular filtration rates and the degree of interstitial fibrosis and tubular atrophy (IFTA) and classified the presence of CKD or IFTA. 3D reconstruction of renal capillaries revealed not only a significant reduction, but more importantly a substantial simplification and reconfiguration of the renal microvasculature in mice with chronic tubulointerstitial injury. Computational modelling predicted that round PTCs can deliver oxygen more homogeneously to the surrounding tissue. Our findings indicate that alterations of PTC shape represent a common and uniform reaction to chronic tubulointerstitial injury independent of the underlying kidney disease.
... Traditional whole-kidney imaging methods, such as micro-CT imaging, have a resolution of several to tens of microns, which makes it difficult to correctly distinguish kidney microstructures smaller than 10 μm, such as capillaries [10,18]. In addition, the use of exogenous contrast agents in micro-CT imaging changes the perfusion status of the kidney, and incomplete perfusion of contrast agents may also lead to insufficient detection [10,52]. In contrast, optical imaging has a higher resolution than micro-CT. ...
... The autofluorescence of the renal parenchyma and hydronephrosis observed in this study is mainly derived from FAD [24,52]. We have previously demonstrated that the cryo-MOST system can be used to observe the distribution of autofluorescence of two coenzymes in tissues, FAD and NADH, which are widely used to indicate metabolic status [28,29]. ...
Rationale: Mesoscopic visualization of the main anatomical structures of the whole kidney in vivo plays an important role in the pathological diagnosis and exploration of the etiology of hydronephrosis. However, traditional imaging methods cannot achieve whole-kidney imaging with micron resolution under conditions representing in vivo perfusion.
Methods: We used in vivo cryofixation (IVCF) to fix acute obstructive hydronephrosis (unilateral ureteral obstruction, UUO), chronic spontaneous hydronephrosis (db/db mice), and their control mouse kidneys for cryo-micro-optical sectioning tomography (cryo-MOST) autofluorescence imaging. We quantitatively assessed the kidney-wide pathological changes in the main anatomical structures, including hydronephrosis, renal subregions, arteries, veins, glomeruli, renal tubules, and peritubular functional capillaries.
Results: By comparison with microcomputed tomography imaging, we confirmed that IVCF can maintain the status of the kidney in vivo. Cryo-MOST autofluorescence imaging can display the main renal anatomical structures with a cellular resolution without contrast agents. The hydronephrosis volume reached 26.11 ± 6.00 mm³ and 13.01 ± 3.74 mm³ in 3 days after UUO and in 15-week-old db/db mouse kidneys, respectively. The volume of the cortex and inner stripe of the outer medulla (ISOM) increased while that of the inner medulla (IM) decreased in UUO mouse kidneys. Db/db mice also showed an increase in the volume of the cortex and ISOM volume but no atrophy in the IM. The diameter of the proximal convoluted tubule and proximal straight tubule increased in both UUO and db/db mouse kidneys, indicating that proximal tubules were damaged. However, some renal tubules showed abnormal central bulge highlighting in the UUO mice, but the morphology of renal tubules was normal in the db/db mice, suggesting differences in the pathology and severity of hydronephrosis between the two models. UUO mouse kidneys also showed vascular damage, including segmental artery and vein atrophy and arcuate vein dilation, and the density of peritubular functional capillaries in the cortex and IM was reduced by 37.2% and 49.5%, respectively, suggesting renal hypoxia. In contrast, db/db mouse kidneys showed a normal vascular morphology and peritubular functional capillary density. Finally, we found that the db/db mice displayed vesicoureteral reflux and bladder overactivity, which may be the cause of hydronephrosis formation.
Conclusions: We observed and compared main renal structural changes in hydronephrosis under conditions representing in vivo perfusion in UUO, db/db, and control mice through cryo-MOST autofluorescence imaging. The results indicate that cryo-MOST with IVCF can serve as a simple and powerful tool to quantitatively evaluate the in vivo pathological changes in three dimensions, especially the distribution of body fluids in the whole kidney. This method is potentially applicable to the three-dimensional visualization of other tissues, organs, and even the whole body, which may provide new insights into pathological changes in diseases.
... We then compared sULM to micro-CT considered as a gold standard for glomeruli observation. [24][25][26] Finally, we demonstrated that the observation of glomeruli by sULM is also possible on human transplanted kidneys. We provide materials, such as post-processing codes, to reproduce these experiments in hospitals equipped with conventional ultrasound scanners. ...
... The observation of glomeruli in this study is only partial compared to results found in the literature 25,26,30,[36][37][38][39][40][41] (Supplementary Table S4). On the one hand, this is due to a lack of acquisition time with the right concentration of microbubbles 42 (Supplementary Fig. S9). ...
Background:
Estimation of glomerular function is necessary to diagnose kidney diseases. However, the study of glomeruli in the clinic is currently done indirectly through urine and blood tests. A recent imaging technique called Ultrasound Localization Microscopy (ULM) has appeared. It is based on the ability to record continuous movements of individual microbubbles in the bloodstream. Although ULM improved the resolution of vascular imaging up to tenfold, the imaging of the smallest vessels had yet to be reported.
Methods:
We acquired ultrasound sequences from living humans and rats and then applied filters to divide the data set into slow-moving and fast-moving microbubbles. We performed a double tracking to highlight and characterize populations of microbubbles with singular behaviors. We decided to call this technique "sensing ULM" (sULM). We used post-mortem micro-CT for side-by-side confirmation in rats.
Findings:
In this study, we report the observation of microbubbles flowing in the glomeruli in living humans and rats. We present a set of analysis tools to extract quantitative information from individual microbubbles, such as remanence time or normalized distance.
Interpretation:
As glomeruli play a key role in kidney function, it would be possible that their observation yields a deeper understanding of the kidney. It could also be a tool to diagnose kidney diseases in patients. More generally, it will bring imaging capabilities closer to the functional units of organs, which is a key to understand most diseases, such as cancer, diabetes, or kidney failures.
Funding:
This study was funded by the European Research Council under the European Union Horizon H2020 program (ERC Consolidator grant agreement No 772786-ResolveStroke).
... The contrast agent µAngiofil has been prepared according to the manufacturer's recommendations (Fumedica AG, Switzerland). The perfusion of the mice was performed as previously described (Hlushchuk et al., 2020, Hlushchuk et al., 2018. Briefly, heparinized animals were deeply anaesthetized, the thorax and the peritoneal cavity were opened with scissors to expose the descending aorta. ...
... Thereafter, the corresponding (upper or lower) part of the body was perfused with µAngiofil at 1-1.5 ml/min using a syringe pump. The perfusion lasted until the organ of interest turned completely blue (Hlushchuk et al., 2019, Hlushchuk et al., 2018. In bones, it is not possible to visually monitor the described color change. ...
... If the sample is small, i.e. only a few millimeters in diameter, the voxel size may be as small as 1 µm or less enabling visualization of the microvasculature down to the capillary bed (Hlushchuk et al., 2019, Hlushchuk et al., 2020, Hlushchuk et al., 2018, Schaad et al., 2017. In case of large samples, like minipig hemimandible, the achievable voxel size is around 10 times larger (≥8 µm), and it is therefore, not possible to reach that level of resolution. ...
Angiogenesis is a physiological process essential for skeletal development and growth, for bone healing and regeneration. Various research areas, including implantology and tissue engineering, would benefit from improved three-dimensional (3D) imaging of the vasculature within bone tissue.
In the last decades, X-ray microtomography (microCT) has gained recognition as a non-destructive 3D imaging technique for bone morphology. The structural nature of skeletal tissue has rendered the direct 3D imaging of its vasculature extremely difficult. For the detection of the vessels, contrast or casting agents must be used. A major drawback of such an approach has been the limited contrast between the common perfusion agents and mineralized tissue, which makes their distinct segmentation problematic. The usually applied decalcification resolves this issue but makes simultaneous assessment of the intracortical bone microstructure and the vascular morphology impossible. Moreover, the problem of contrasting becomes compounded in the presence of a metal implant.
Herewith we introduce the micro Angiofil-enhanced microCT-based visualization of vasculature within bone tissue in various small and large animal models, with and without decalcification. This study documents simultaneous microvascular and bone imaging in murine tibia, murine bone metastatic model, pulp chamber, gingiva and periodontal ligaments. In a large animal model (minipig) we present the visualization and segmentation of different tissue types and vessels in the hemimandible containing several metal implants.
Herewith we provide for the first time a non-destructive 3D imaging approach of the vasculature within soft and hard tissues in the vicinity of metal implants in a large animal model.
... Recent work has demonstrated the gold standard for assessing peritubular capillary density is microangio-CT of the entire kidney. 22 Histologic measurements overestimate capillary density because of the artifacts from sample sectioning, mounting, and such. Such analysis was not possible in the archived tissues used in our study. ...
Background
Hypoxia is a key driver of fibrosis and is associated with capillary rarefaction in humans.
Objectives
Characterize capillary rarefaction in cats with chronic kidney disease (CKD).
Animals
Archival kidney tissue from 58 cats with CKD, 20 unaffected cats.
Methods
Cross‐sectional study of paraffin‐embedded kidney tissue utilizing CD31 immunohistochemistry to highlight vascular structures. Consecutive high‐power fields from the cortex (10) and corticomedullary junction (5) were digitally photographed. An observer counted and colored the capillary area. Image analysis was used to determine the capillary number, average capillary size, and average percent capillary area in the cortex and corticomedullary junction. Histologic scoring was performed by a pathologist masked to clinical data.
Results
Percent capillary area (cortex) was significantly lower in CKD (median 3.2, range, 0.8‐5.6) compared to unaffected cats (4.4, 1.8‐7.0; P = <.001) and was negatively correlated with serum creatinine concentrations (r = −.36, P = .0013), glomerulosclerosis (r = −0.39, P = <.001), inflammation (r = −.30, P = .009), and fibrosis (r = −.30, P = .007). Capillary size (cortex) was significantly lower in CKD cats (2591 pixels, 1184‐7289) compared to unaffected cats (4523 pixels, 1801‐7618; P = <.001) and was negatively correlated with serum creatinine concentrations (r = −.40, P = <.001), glomerulosclerosis (r = −.44, P < .001), inflammation (r = −.42, P = <.001), and fibrosis (r = −.38, P = <.001).
Conclusions and Clinical Importance
Capillary rarefaction (decrease in capillary size and percent capillary area) is present in kidneys of cats with CKD and is positively correlated with renal dysfunction and histopathologic lesions.
... 8 The current gold standard in this application is therefore still vascular casting, a method wherein plastic resin mixtures are injected into the vasculature and left to solidify. Radiopacity is provided by additives such as lead chromate microparticles (Microfil, FlowTech Inc.) 9 or iodinated fatty acids (µAngiofil, Fumedica AG) 10 . As these plastic resin mixtures are hydrophobic, they do not pass the hydrated blood vessel walls or glomeruli, resulting in permanent retention within the vasculature. ...
Imaging of microvasculature is primarily performed with X-ray contrast agents, owing to the wide availability of absorption-contrast laboratory source microCT compared to phase contrast capable devices. Standard commercial contrast agents used in angiography are not suitable for high-resolution imaging ex vivo, however, as they are small molecular compounds capable of diffusing through blood vessel walls within minutes. Large nanoparticle-based blood pool contrast agents on the other hand exhibit problems with aggregation, resulting in clogging in the smallest blood vessels. Injection with solidifying plastic resins has, therefore, remained the gold standard for microvascular imaging, despite the considerable amount of training and optimization needed to properly perfuse the viscous compounds. Even with optimization, frequent gas and water inclusions commonly result in interrupted vessel segments. This lack of suitable compounds has led us to develop the polymeric, cross-linkable X-ray contrast agent XlinCA. As a water-soluble organic molecule, aggregation and inclusions are inherently avoided. High molecular weight allows it to be retained even in the highly fenestrated vasculature of the kidney filtration system. It can be covalently crosslinked using the same aldehydes used in tissue fixation protocols, leading to stable and permanent contrast. These properties allowed us to image whole mice and individual organs in 6 to 12-month-old C57BL/6J mice without requiring lengthy optimizations of injection rates and pressures, while at the same time achieving greatly improved filling of the vasculature compared to resin-based vascular casting. This work aims at illuminating the rationales, processes and challenges involved in creating this recently developed contrast agent.
... We chose to include the entire kidney in the μCT scan to match the SRUS images. Image comparisons can be made at a 5-10 μm scale using a smaller isometric voxel size in a selected smaller region of the kidney 24,26 . In Zhu et al. ...
... During the renal vascular flushing, the rat was euthanized by decapitation. Directly after flushing, 3 ml of μAngiofil contrast agent and hardener mixed according to the manufacturer's guidelines (Fumedica AG, Muri, Switzerland) was infused at 1 ml/ min 26,40 . The infusion continued until the entire surface of the kidney was blue, and a considerable amount of contrast had left the renal vein. ...
Super-resolution ultrasound imaging (SRUS) enables in vivo microvascular imaging of deeper-lying tissues and organs, such as the kidneys or liver. The technique allows new insights into microvascular anatomy and physiology and the development of disease-related microvascular abnormalities. However, the microvascular anatomy is intricate and challenging to depict with the currently available imaging techniques, and validation of the microvascular structures of deeper-lying organs obtained with SRUS remains difficult. Our study aimed to directly compare the vascular anatomy in two in vivo 2D SRUS images of a Sprague–Dawley rat kidney with ex vivo μ CT of the same kidney. Co-registering the SRUS images to the μ CT volume revealed visually very similar vascular features of vessels ranging from ~ 100 to 1300 μm in diameter and illustrated a high level of vessel branching complexity captured in the 2D SRUS images. Additionally, it was shown that it is difficult to use μ CT data of a whole rat kidney specimen to validate the super-resolution capability of our ultrasound scans, i.e . , validating the actual microvasculature of the rat kidney. Lastly, by comparing the two imaging modalities, fundamental challenges for 2D SRUS were demonstrated, including the complexity of projecting a 3D vessel network into 2D. These challenges should be considered when interpreting clinical or preclinical SRUS data in future studies.
... This step would be unnecessary if the resolution of the µCT was high enough to clearly delineate the arterial walls and allow their morphometric analysis. The potential of the combination of stereology and µCT has been documented already (Vasilescu et al. 2013(Vasilescu et al. , 2020 and the use of contrast agents (Hlushchuk et al. 2018;Chadwick et al. 2021) will help to increase the potential of performing stereological analysis of the vascular tree on scans. In addition, other methods of X-ray imaging (such as synchrotron X-ray tomographic microscopy) reach better resolutions and have been used for lung morphometry already (Barré et al. 2014;Schittny 2018;Borisova et al. 2021). ...
The pulmonary vasculature consists of a large arterial and venous tree with a vast alveolar capillary network (ACN) in between. Both conducting blood vessels and the gas-exchanging capillaries are part of important human lung diseases, including bronchopulmonary dysplasia, pulmonary hypertension and chronic obstructive pulmonary disease. Morphological tools to investigate the different parts of the pulmonary vasculature quantitatively and in three dimensions are crucial for a better understanding of the contribution of the blood vessels to the pathophysiology and effects of lung diseases. In recent years, new stereological methods and imaging techniques have expanded the analytical tool box and therefore the conclusive power of morphological analyses of the pulmonary vasculature. Three of these developments are presented and discussed in this review article, namely (1) stereological quantification of the number of capillary loops, (2) serial block-face scanning electron microscopy of the ACN and (3) labeling of branching generations in light microscopic sections based on arterial tree segmentations of micro-computed tomography data sets of whole lungs. The implementation of these approaches in research work requires expertise in lung preparation, multimodal imaging at different scales, an advanced IT infrastructure and expertise in image analysis. However, they are expected to provide important data that cannot be obtained by previously existing methodology.
... These difficulties were similar for veins but less pronounced for airways. Possibly, the use of suitable contrast agents after perfusion fixation may help to enable the segmentation of smaller arteries as well (Hlushchuk et al. 2018;Faight et al. 2017;Jiménez et al. 2016). Furthermore, any increase in spatial resolution Fig. 5 Stereological counting procedure. ...
Various lung diseases, including pulmonary hypertension, chronic obstructive pulmonary disease or bronchopulmonary dysplasia, are associated with structural and architectural alterations of the pulmonary vasculature. The light microscopic (LM) analysis of the blood vessels is limited by the fact that it is impossible to identify which generation of the arterial tree an arterial profile within a LM microscopic section belongs to. Therefore, we established a workflow that allows for the generation-specific quantitative (stereological) analysis of pulmonary blood vessels. A whole left rabbit lung was fixed by vascular perfusion, embedded in glycol methacrylate and imaged by micro-computed tomography (µCT). The lung was then exhaustively sectioned and 20 consecutive sections were collected every 100 µm to obtain a systematic uniform random sample of the whole lung. The digital processing involved segmentation of the arterial tree, generation analysis, registration of LM sections with the µCT data as well as registration of the segmentation and the LM images. The present study demonstrates that it is feasible to identify arterial profiles according to their generation based on a generation-specific color code. Stereological analysis for the first three arterial generations of the monopodial branching of the vasculature included volume fraction, total volume, lumen-to-wall ratio and wall thickness for each arterial generation. In conclusion, the correlative image analysis of µCT and LM-based datasets is an innovative method to assess the pulmonary vasculature quantitatively.
