Trauma and shock often severely affect the kidneys. This can lead to trauma-related acute kidney injury (TRAKI), which significantly increases the risk of adverse outcomes. To study the pathophysiology of TRAKI, we established a murine model of combined blunt thoracic trauma and pressure-controlled hemorrhage [trauma and hemorrhagic shock (THS)] that induces mild transient TRAKI. The mice displayed early and transiently increased plasma creatinine, urea, and neutrophil gelatinase-associated lipocalin and urine albumin, resolving 5 days after TRAKI induction. Morphological changes were only observed at the microscopic level, where proximal tubular cell damage and brush border loss were evident. We furthermore found kidney stress responses, for example, with induced heme oxygenase-1 expression in tubules. The upregulation of inflammatory mediators and kidney injury markers was followed by elevated leukocyte numbers, mainly consisting of monocytes/macrophages. Proteomic analyses revealed a distinct time course of intrarenal processes following trauma. Three-dimensional x-ray-based whole organ histology by contrast-enhanced microcomputed tomography showed significant impairment of capillary blood filling, particularly during the first day after THS, which was partly resolved by day 5. Our novel murine TRAKI model revealed previously unknown aspects of the complex temporal pathophysiologic response of the kidney along the nephron following trauma and hemorrhage, which may provide mechanistic starting points for future therapeutic approaches.NEW & NOTEWORTHY This study introduces a murine model of trauma-related acute kidney injury (TRAKI) via combined blunt thoracic trauma and hemorrhage, revealing transient kidney dysfunction despite normal morphology. Early damage to proximal tubular cells, inflammatory responses, and induction of stress markers like heme oxygenase-1 were observed. Proteomic analyses uncovered distinct intrarenal changes, whereas three-dimensional microcomputed tomography showed capillary blood supply impairment, resolving by day 5. These findings shed light on TRAKI's pathophysiology and may inform future therapeutic strategies.

The gold standard for studying biological soft tissues at the microscale (i.e., histology) is tissue sectioning with subsequent colorimetric or fluorescent staining and visual inspection under the microscope. When tissue integrity must be maintained for 3D histological assessment, contrast-enhanced microfocus X-ray computed tomography (CECT) is a promising solution, but there is still a lack of staining protocol optimization of contrast-enhancing staining agents (CESAs). Therefore, in this study, mouse auricles were incubated with Hafnium-substituted Wells-Dawson polyoxometalate, cationic iodinated contrast agent, or Lugol's iodine and were imaged with high-resolution CECT. Alignment with corresponding H&E-stained sections enabled the identification and segmentation of different tissue types. Contrast differences between tissue types were increased by washing the samples after staining or by combining CESAs. Finally, we proved that the latter could be used to quantitatively assess the 3D thickness distribution of the epidermis in the ears of a mouse model of psoriasis-like dermatitis. In conclusion, CECT and bright-field microscopy are complementary and not mutually exclusive techniques for the histological assessment of biological tissues. While bright-field microscopy gives detailed information about the cellular composition of tissues, CECT provides a better insight into the spatial interrelationship of tissues and is a powerful tool for performing 3D structural quantification.

Background. Failure to close the abdominal wall after intestinal transplantation (ITx) or multivisceral Tx remains a surgical challenge. An attractive method is the use of nonvascularized rectus fascia (NVRF) in which both layers of the donor abdominal rectus fascia are used as an inlay patch without vascular anastomosis. How this graft integrates over time remains unknown. The study aims to provide a multilevel analysis of the neovascularization and integration process of the NVRF. Methods. Three NVRF-Tx were performed after ITx. Clinical, radiological, histological, and immunological data were analyzed to get insights into the neovascularization and integration process of the NVRF. Moreover, cryogenic contrast-enhanced microfocus computed tomography (microCT) analysis was used for detailed reconstruction of the vasculature in and around the NVRF (3-dimensional histology). Results. Two men (31- and 51-y-old) and 1 woman (49-y-old) underwent 2 multivisceral Tx and 1 combined liver-ITx, respectively. A CT scan showed contrast enhancement around the fascia graft at 5 days post-Tx. At 6 weeks, newly formed blood vessels were visualized around the graft with Doppler ultrasound. Biopsies at 2 weeks post-Tx revealed inflammation around the NVRF and early fibrosis. At 6 months, classical 2-dimensional histological analysis of a biopsy confirmed integration of the fascia graft with strong fibrotic reaction without signs of rejection. A cryogenic contrast-enhanced microCT scan of the same biopsy revealed the presence of microvasculature, enveloping and penetrating the donor fascia. Conclusions. We showed clinical, histological, and microCT evidence of the neovascularization and integration process of the NVRF after Tx.

The kidney’s microstructure, which comprises a highly convoluted tubular and vascular network, can only be partially revealed using classical 2D histology. Considering that the kidney’s microstructure is closely related to its function and is often affected by pathologies, there is a need for powerful and high-resolution 3D imaging techniques to visualize the microstructure. Here, we present how cryogenic contrast-enhanced microCT (cryo-CECT) allowed 3D visualization of glomeruli, tubuli, and vasculature. By comparing different contrast-enhancing staining agents and freezing protocols, we found that the preferred sample preparation protocol was the combination of staining with 1:2 hafnium(IV)-substituted Wells-Dawson polyoxometalate and freezing by submersion in isopentane at −78°C. This optimized protocol showed to be highly sensitive, allowing to detect small pathology-induced microstructural changes in a mouse model of mild trauma-related acute kidney injury after thorax trauma and hemorrhagic shock. In summary, we demonstrated that cryo-CECT is an effective 3D histopathological tool that allows to enhance our understanding of kidney tissue microstructure and their related function.

Background Trauma and shock often severely affect the kidneys. This can lead to trauma-related acute kidney injury (TRAKI), which significantly increases the risk of adverse outcomes. Methods To study the pathophysiology of TRAKI, we developed a murine model of combined blunt thoracic trauma and pressure-controlled hemorrhage that induces mild transient TRAKI. Results The mice showed early and transient increased plasma creatinine, urea, NGAL, and urine albumin, resolving 5 days after TRAKI induction. Despite normal kidney morphology, significant damage to proximal tubular cells and a loss of the brush border was observed. This included kidney stress responses, e.g., with induced heme oxygenase-1 expression in tubules. The upregulation of inflammatory mediators and kidney injury markers was followed by elevated leukocyte numbers, mainly consisting of monocytes/macrophages. Proteomic analyses revealed a distinct time course of intrarenal processes after trauma. 3D x-ray-based whole-organ histology by contrast-enhanced microcomputed tomography showed significant impairment of capillary blood flow, especially during the first day post THS, which was partly resolved by day 5. Conclusions Our novel model of murine TRAKI has revealed previously unknown aspects of the complex temporal pathophysiological response of the kidney along the nephron after trauma and hemorrhage, which may provide mechanistic starting points for future therapeutic approaches. ### Competing Interest Statement The authors have declared no competing interest.

Introduction: Failure to close the abdominal wall after intestinal (ITx) and multiviseral transplantation (MvTx) remains a challenge, associated with increased morbidity and mortality. An attractive method is the use of non-vascularized rectus fascia (NVRF) in which both layers of the abdominal rectus fascia are used as an inlay patch without vascular anastomosis, with excellent short-term outcomes. The aim of our study is to provide an in-depth multi-level analysis (ranging from clinical, radiological, histological, contrast-enhanced microCT (CECT) to immunological evaluation) of the neovascularization and integration process of the NVRF based on three consecutive cases following ITx. Methods: Three patients underwent a NVRF transplantation in combination with an ITx between September 2019 and September 2022 at the Leuven Intestinal Failure and Transplant unit. A retrospective analysis was performed. Ethical approval for reporting was obtained (S67453). Results: The first patient was a 49-year old female who received a NVRF during combined liver-ITx and had an uneventful recovery. At 1 month, ultrasound doppler confirmed neovascularization of the graft. Five months later, at the time of continuity surgery, the donor fascia was macroscopically well integrated. H&E staining on biopsy confirmed the good integration of the graft with intense fibrotic reaction around the NVRF without rejection. CD31-staining showed neovascularisation on the interface with the native fascia. CECT analysis revealed the presence of microvasculature enveloping the donor fascia as well as penetrating the graft at the interface with the native fascia; Fig. 1a: sagittal view of CECT image, red arrow indicating blood vessels in the NVRF (blue line). Fig. 1b: 3-dimensial render of neovascularisation. The second patient was a 51-year old male who received a NVRF after a MvTx. Two weeks later, during a re-operation the fascia showed macroscopic neovascularization. Since the skin could not be closed, a VAC-system was placed on top of the fascia and secondary closure was obtained. The patient died six months post-transplant from a metastasized abdominal mesothelioma. The third patient was a 31-year old male who underwent MvTx. Eleven days post-transplant and after re-operation for intra-abdominal collections, primary closure could not be attained and a non-ABO-matched third party fascia was used to cover the defect. Six days after NVRF transplant, anti-A natural and immune antibodies were slightly increased suggesting the presence of de-novo specific antibodies against the third party fascia. Twelve days later, the patient died of an acute rupture of a mycotic aneurysm of the aorta tube. At re-intervention, the fascia looked macroscopically intact. Conclusion: We showed in this case series additional evidence of the neovascularization and integration, by fibrotic reaction, of donor NVRF after intestinal transplantation

Hypothesis: Characterizing the microstructure of an ice/surface interface and its effect on the icephobic behavior of surfaces remains a significant challenge. Introducing X-ray Computed Tomography (XCT) can provide unprecedented insights into the internal (porosity) and interfacial structures, i.e. wetting regime, between (super)hydrophobic surfaces and ice by visualizing these optically inaccessible regions. Experiments: Frozen droplets with controlled volume were deposited on top of metallic and polymeric substrates with different levels of wettability. Different modes of XCT (3D and 4D) were utilized to obtain information on the internal and interfacial structure of the ice/surface system. The results were supple-mented by conventional surface analysis techniques, including optical profilometry and contact angle measurements. Findings: Using XCT on ice/surface systems, the 3D and 4D (imaging with temporal resolution) structural information can be visualized. From these datasets, qualitative and quantitative results were obtained, not only for characterizing the interface but also for analyzing the entire droplet/surface system, e.g., measurement of porosity size, shape, and location. These results highlight the potential of XCT in the characterization of both droplets and substrates and proves that the technique can aid to develop hydrophobic surfaces for use as icephobic materials. CO 2023 Elsevier Inc. All rights reserved.

Biological tissues comprise a spatially complex structure, composition and organization at the microscale, named the microstructure. Given the close structure-function relationships in tissues, structural characterization is essential to fully understand the functioning of healthy and pathological tissues, as well as the impact of possible treatments. Here, we present a nondestructive imaging approach to perform quantitative 3D histo(patho)logy of biological tissues, termed Cryogenic Contrast-Enhanced MicroCT (cryo-CECT). By combining sample staining, using an X-ray contrast-enhancing staining agent, with freezing the sample at the optimal freezing rate, cryo-CECT enables 3D visualization and structural analysis of individual tissue constituents, such as muscle and collagen fibers. We applied cryo-CECT on murine hearts subjected to pressure overload following transverse aortic constriction surgery. Cryo-CECT allowed to analyze, in an unprecedented manner, the orientation and diameter of the individual muscle fibers in the entire heart, as well as the 3D localization of fibrotic regions within the myocardial layers. We foresee further applications of cryo-CECT in the optimization of tissue/food preservation and donor banking, showing that cryo-CECT also has clinical and industrial potential.