Preparation of large biological samples for high-resolution, hierarchical, synchrotron phase-contrast tomography with multimodal imaging compatibility

Imaging across different scales is essential for understanding healthy organ morphology and pathophysiological changes. The macro- and microscale three-dimensional morphology of large samples, including intact human organs, is possible with X-ray microtomography (using laboratory or synchrotron sources). Preparation of large samples for high-resolution imaging, however, is challenging due to limitations such as sample shrinkage, insufficient contrast, movement of the sample and bubble formation during mounting or scanning. Here, we describe the preparation, stabilization, dehydration and mounting of large soft-tissue samples for X-ray microtomography. We detail the protocol applied to whole human organs and hierarchical phase-contrast tomography at the European Synchrotron Radiation Facility, yet it is applicable to a range of biological samples, including complete organisms. The protocol enhances the contrast when using X-ray imaging, while preventing sample motion during the scan, even with different sample orientations. Bubbles trapped during mounting and those formed during scanning (in the case of synchrotron X-ray imaging) are mitigated by multiple degassing steps. The sample preparation is also compatible with magnetic resonance imaging, computed tomography and histological observation. The sample preparation and mounting require 24-36 d for a large organ such as a whole human brain or heart. The preparation time varies depending on the composition, size and fragility of the tissue. Use of the protocol enables scanning of intact organs with a diameter of 150 mm with a local voxel size of 1 μm. The protocol requires users with expertise in handling human or animal organs, laboratory operation and X-ray imaging

An optimized non-destructive protocol for testing mechanical properties in decellularized rabbit trachea.

Successful tissue-engineered tracheal transplantation relies on the use of non-immunogenic constructs, which can vascularize rapidly, support epithelial growth, and retain mechanical properties to that of native trachea. Current strategies to assess mechanical properties fail to evaluate the trachea to its physiological limits, and lead to irreversible destruction of the construct. Our aim was to develop and evaluate a novel non-destructive method for biomechanical testing of tracheae in a rabbit decellularization model. To validate the performance of this method, we simultaneously analyzed quantitative and qualitative graft changes in response to decellularization, as well as in-vivo biocompatibility of implanted scaffolds. Rabbit tracheae underwent two, four and eight cycles of detergent-enzymatic decellularization. Biomechanical properties were analyzed by calculating luminal volume of progressively inflated and deflated tracheae with microCT. DNA, glycosaminoglycan and collagen contents were compared to native trachea. Scaffolds were prelaminated in vivo. Native, two- and four-cycle tracheae showed equal mechanical properties. Collapsibility of eight-cycle tracheae was significantly increased from -40 cmH2O (-3.9 kPa). Implantation of two- and four-cycle decellularized scaffolds resulted in favorable flap-ingrowth; eight-cycle tracheae showed inadequate integration. We showed a more limited detergent-enzymatic decellularization successfully removing non-cartilaginous immunogenic matter without compromising extracellular matrix content or mechanical stability. With progressive cycles of decellularization, important loss of functional integrity was detected upon mechanical testing and in-vivo implantation. This instability was not revealed by conventional quantitative nor qualitative architectural analyses. These experiments suggest that non-destructive, functional evaluation, e.g. by microCT, may serve as an important tool for mechanical screening of scaffolds before clinical implementation

The fatal trajectory of pulmonary COVID-19 is driven by lobular ischemia and fibrotic remodelling

BACKGROUND: COVID-19 is characterized by a heterogeneous clinical presentation, ranging from mild symptoms to severe courses of disease. 9-20% of hospitalized patients with severe lung disease die from COVID-19 and a substantial number of survivors develop long-COVID. Our objective was to provide comprehensive insights into the pathophysiology of severe COVID-19 and to identify liquid biomarkers for disease severity and therapy response. METHODS: We studied a total of 85 lungs (n = 31 COVID autopsy samples; n = 7 influenza A autopsy samples; n = 18 interstitial lung disease explants; n = 24 healthy controls) using the highest resolution Synchrotron radiation-based hierarchical phase-contrast tomography, scanning electron microscopy of microvascular corrosion casts, immunohistochemistry, matrix-assisted laser desorption ionization mass spectrometry imaging, and analysis of mRNA expression and biological pathways. Plasma samples from all disease groups were used for liquid biomarker determination using ELISA. The anatomic/molecular data were analyzed as a function of patients' hospitalization time. FINDINGS: The observed patchy/mosaic appearance of COVID-19 in conventional lung imaging resulted from microvascular occlusion and secondary lobular ischemia. The length of hospitalization was associated with increased intussusceptive angiogenesis. This was associated with enhanced angiogenic, and fibrotic gene expression demonstrated by molecular profiling and metabolomic analysis. Increased plasma fibrosis markers correlated with their pulmonary tissue transcript levels and predicted disease severity. Plasma analysis confirmed distinct fibrosis biomarkers (TSP2, GDF15, IGFBP7, Pro-C3) that predicted the fatal trajectory in COVID-19. INTERPRETATION: Pulmonary severe COVID-19 is a consequence of secondary lobular microischemia and fibrotic remodelling, resulting in a distinctive form of fibrotic interstitial lung disease that contributes to long-COVID. FUNDING: This project was made possible by a number of funders. The full list can be found within the Declaration of interests / Acknowledgements section at the end of the manuscript

Implications of the ISHLT 2005 and 2016 PGD Grading System.

PURPOSE: In 2005, an ISHLT working group proposed a grading system for primary graft dysfunction (PGD) following lung transplantation (LTx), based on oxygenation (P/F) and chest X-ray. This system was revised in 2016. The aim of this study is to compare the impact of the 2016 revised versus the original 2005 grading system on PGD prevalence and early post-LTx outcome. METHODS: All patients receiving double LTx at our institution between 12/2016 and 01/2018 (n=75) were retrospectively studied. P/F ratios were calculated and X-rays were scored by two experts blinded for clinical information at time (T) points 0, 24, 48, and 72 hours. PGD grading was determined separately according to 2005 and 2016 PGD definition. When multiple P/F ratios were available, the lowest P/F was used. RESULTS: Results are visualized in figure 1. PGD distribution at T0, T24, T72 differed between 2005 and 2016 (p<0.001; p=0.005; p=0.05), but not at 48h (p=0.19). PGD-3 incidence at T0 (48%), T24 (29%), T48 (17%) was higher according to the 2005 score compared to 2016 score (31%, 25%, and 16%, respectively). In contrast, PGD-3 at T72 was lower according to the 2005 score (12%) compared to the 2016 score (16%). Time to extubation (p=0.93), ICU stay (p=0.33) and hospital stay (p=0.43) of patients with PGD-3 at any time (T0 - T72) hours did not differ according the grading system (figure 1, down). CONCLUSION: PGD grading differs between the original and the new PGD scoring system with a higher proportion of lower grades according to the 2016 definition at early time points. This might be explained by the more prominent role of chest radiography in the 2016 classification for PGD 0 regardless of the P/F ratio. On the other hand, according to the 2016 classification system, extubated patients are no longer automatically scored as PGD-0 or PGD-1, which might explain the higher proportion of PGD3 at T72. Early outcome was comparable between the two systems when patients suffered from PGD3 at any time point after transplantation. The impact of PGD grading on long-term outcome should be further investigated.status: publishe

The Value of Ex Situ CT Imaging of Donor Lungs Prior to Transplantation.

PURPOSE: Donor shortage remains a major obstacle associated with considerable mortality in patients waiting for lung transplantation (LTx). Objective donor lung assessment is crucial to obtain sufficient grafts of acceptable quality. We hypothesized that out of body chest CT may be an innovative way to assess grafts prior to LTx. Therefore, this study prospectively investigated the value of CT scan of donor lungs prior to LTx. METHODS: Between 12/2016 and 01/2018, all potential donor lungs were inspected in-situ, recovered and ex situ CT scanned during standard cold preservation. The transplant team and radiologist were blinded for the CT scan, which thus did not change the decision to transplant. CTs were scored for CT abnormalities and were compared between transplanted (Tx) and non-Tx grafts. In addition, CT of Tx grafts were compared between recipients developing PGD3 within 72 h ('PGD3') and recipients who did not ('No PGD3'). RESULTS: In total, 75 lungs were transplanted and 25 were not transplanted, of which 19 were declined for poor graft quality and 6 for extrapulmonary malignancies or logistics. CT abnormalities (CSL, GGO, EMPHY) differed between Tx and non-Tx grafts (Figure 1A). In lungs declined for poor graft quality (n=19), the reason for decline was confirmed in 13 lungs, however 4 lungs had only limited CT abnormalities present. In 2 out of 7 lungs declined at retrieval for clinical suspicion of emphysema, no emphysema was present on CT. In addition, CT abnormalities (CSL, RET/IST) of Tx grafts also significantly differed between recipients with PGD 3 and without PGD 3 (Fig 1B). CONCLUSION: Ex situ CT imaging of donor grafts during preservation is feasible and demonstrated significant differences between Tx and non-Tx grafts. Also grafts of patients with or without PGD3 differed in CT parameters. CT imaging could not confirm the reason for decline in 6 lungs, which might indicate an undisclosed graft potential and a role of CT imaging in donor assessment. The prognostic value of CT parameters on long-term outcomes remains elusive.status: publishe

Blood Eosinophilia Predicts Poor Outcome in Lung Transplant Recipients

PURPOSE: Eosinophils are associated with the onset of chronic respiratory diseases like asthma and COPD. In lung transplantation, patients with increased bronchoalveolar lavage eosinophils demonstrated a worse chronic lung allograft dysfunction (CLAD)-free survival and overall survival. We investigated the association between blood eosinophilia, graft survival and CLAD-free survival after lung transplantation. METHODS: A retrospective analysis was performed including all transplanted patients within our center between 2011 and 2016 (n=376). Blood eosinophils were measured as part of the routine clinical follow-up including all measurements prior to 01/06/2019. A ROC analysis was performed to define cut-offs for blood eosinophilia and patients were subsequently divided in those with high blood eosinophilia vs. those with lower eosinophilia based on the defined cut-off. All patients received oral steroids as part of their standard immunosuppressive drug regimen. RESULTS: ROC analysis revealed that the optimal threshold for blood eosinophilia is >7.85% (p=0.0026) for overall survival and >7.75% (p=0.001) for CLAD-free survival. Using a threshold of >8%, 112 patients had high blood eosinophilia and 264 had low eosinophilia (16 patients developed high eosinophilia after CLAD). Patients with blood eosinophilia >8% demonstrated worse graft survival (p=0.004) and CLAD-free survival (p=0.0076) compared to those with lower blood eosinophilia. Within the high eosinophilia group, 65 (58%) patients were diagnosed with CLAD, of which 42 (37.5%) patients had BOS and 23 (20.5%) had RAS. In the group with lower eosinophilia, 80 (30.3%) patients developed CLAD with 71 (26.9%) BOS and 9 (3.4%) RAS (p8%) demonstrate inferior graft survival and CLAD-free survival, specifically RAS. These findings require further research and may lead to the development of an easy to use, non-invasive marker for poor transplant outcome.status: publishe