FIBROBLAST-SEEDED LUNG EXTRACELLULAR MATRIX (ECM)-DERIVED HYDROGELS AS AN IN VITRO MODEL FOR STROMAL BED IN IDIOPATHIC PULMONARY FIBROSIS (IPF)

Introduction: Idiopathic Pulmonary Fibrosis (IPF) is characterized by aberrant extracellular matrix (ECM) deposition and remodeling, which orchestrates cellular responses to the fibrotic microenvironment[1]. Decellularized lung ECM‐derived hydrogels resemble the mechanical properties[2] of native decellularized tissues, potentially providing a 3D model mimicking native cell‐ECM interactions. We aimed to characterize this 3D human lung microenvironment model, with respect to stiffness and viscoelastic properties, in the presence and absence of primary human lung fibroblasts.Materials & Methods: Lyophilized powders of decellularized IPF and control lung matrices (pool of 6 patients) were pepsin digested, and formed to hydrogels seeded with control primary lung fibroblasts (n = 4 donors), and cultured for 14 days. Stiffness and viscoelastic relaxation were measured by Low‐Load Compression Testing[2] (20% strain).Results: IPF hydrogels were stiffer than controls (1.84 ± 0.33 kPA vs 1.37 ± 0.35 kPA), and became even more stiff when cell‐seeded (1.91 ± 0.37 kPA) in contrast to controls which became softer (1.09 ± 0.27 kPA). Time to reach 100% viscoelastic relaxation was shorter in cell‐seeded compared to native hydrogels for both IPF (19.14 ± 3.17 vs 41.6 ± 37.66 seconds) and control (11.44 ± 6.55 vs 22.21 ± 19.59 seconds).Conclusion: The mechanical properties of the ECM hydrogels were modified by fibroblasts, while in turn the ECM microenvironment altered cellular responses. These data suggest that higher stiffnesses and altered relaxation patterns of the ECM could contribute to the fibrotic response in IPF by instructing the cells. Fibroblast‐seeded ECM‐derived hydrogels can provide more insight on cell‐ECM interactions in IPF.References[1] M. W. Parker et al., “Fibrotic extracellular matrix activates a profibrotic positive feedback loop,” J. Clin. Invest., vol. 124, no. 4, pp. 1622–1635, Apr. 2014.[2] R. H. J. De Hilster et al., “Human lung extracellular matrix hydrogels resemble the stiffness and viscoelasticity of native lung tissue,” Am. J. Physiol. ‐ Lung Cell. Mol. Physiol., vol. 318, no. 4, pp. L698–L704, Apr. 2020

Micrometer-resolution X-ray tomographic full-volume reconstruction of an intact post-mortem juvenile rat lung

In this article, we present an X-ray tomographic imaging method that is well suited for pulmonary disease studies in animal models to resolve the full pathway from gas intake to gas exchange. Current state-of-the-art synchrotron-based tomographic phase-contrast imaging methods allow for three-dimensional microscopic imaging data to be acquired non-destructively in scan times of the order of seconds with good soft tissue contrast. However, when studying multi-scale hierarchically structured objects, such as the mammalian lung, the overall sample size typically exceeds the field of view illuminated by the X-rays in a single scan and the necessity for achieving a high spatial resolution conflicts with the need to image the whole sample. Several image stitching and calibration techniques to achieve extended high-resolution fields of view have been reported, but those approaches tend to fail when imaging non-stable samples, thus precluding tomographic measurements of large biological samples, which are prone to degradation and motion during extended scan times. In this work, we demonstrate a full-volume three-dimensional reconstruction of an intact rat lung under immediate post-mortem conditions and at an isotropic voxel size of (2.75 µm)3. We present the methodology for collecting multiple local tomographies with 360° extended field of view scans followed by locally non-rigid volumetric stitching. Applied to the lung, it allows to resolve the entire pulmonary structure from the trachea down to the parenchyma in a single dataset. The complete dataset is available online (https://doi.org/10.16907/7eb141d3-11f1-47a6-9d0e-76f8832ed1b2)

A tomographic microscopy-compatible Langendorff system for the dynamic structural characterization of the cardiac cycle

ntroduction: Cardiac architecture has been extensively investigated ex vivo using a broad spectrum of imaging techniques. Nevertheless, the heart is a dynamic system and the structural mechanisms governing the cardiac cycle can only be unveiled when investigating it as such. Methods: This work presents the customization of an isolated, perfused heart system compatible with synchrotron-based X-ray phase contrast imaging (X-PCI). Results: Thanks to the capabilities of the developed setup, it was possible to visualize a beating isolated, perfused rat heart for the very first time in 4D at an unprecedented 2.75 μm pixel size (10.6 μm spatial resolution), and 1 ms temporal resolution. Discussion: The customized setup allows high-spatial resolution studies of heart architecture along the cardiac cycle and has thus the potential to serve as a tool for the characterization of the structural dynamics of the heart, including the effects of drugs and other substances able to modify the cardiac cycle

Time Resolved in situ X-Ray Tomographic Microscopy Unraveling Dynamic Processes in Geologic Systems

X-ray tomographic microscopy is a well-established analysis technique in different fields of the Earth Sciences to access volumetric information of the internal microstructure of a large variety of opaque materials with high-spatial resolution and in a non-destructive manner. Synchrotron radiation, with its coherence and high flux, is required for pushing the temporal resolution into the second and sub-second regime and beyond, and therefore moving from the investigation of static samples to the study of fast dynamic processes as they happen in 3D. Over the past few years, several hardware and software developments at the TOMCAT beamline at the Swiss Light Source contributed to establishing its highly flexible and user-friendly fast tomography endstation, making a large variety of new dynamic in situ and operando investigations possible. Here we present an overview of the different devices, including an in-house developed detector, a new highly efficient macroscope and a programmable fast rotation stage. Their tight interplay and synchronization are key for lifting experimental design compromises and follow dynamic processes with high spatial and temporal resolution unfolding over prolonged periods of time, as often required by many applications. We showcase these new capabilities for the Earth Sciences community by presenting three different geological studies, which make use of different sample environments. With a tri-axial deformation rig, chemo-mechanical-hydraulic feedbacks between gypsum dehydration and halite deformation have been studied, while the spatio-temporal evolution of a solute plume has been investigated for the first time in 3D with a flow cell. A laser-based heating system available at the beamline provides access to the high temperatures required to address bubble growth and collapse as well as bubble-bubble interaction and coalescence in volcanological material. With the integration of a rheometer, information on bubble deformation could also be gained. In the near future, upgrades of most large-scale synchrotron radiation facilities to diffraction-limited storage rings will create new opportunities, for instance through sub-second tomographic imaging capabilities at sub-micron length scales

Real-time reconstruction and visualisation towards dynamic feedback control during time-resolved tomography experiments at TOMCAT

Tomographic X-ray microscopy beamlines at synchrotron light sources worldwide have pushed the achievable time-resolution for dynamic 3-dimensional structural investigations down to a fraction of a second, allowing the study of quickly evolving systems. The large data rates involved impose heavy demands on computational resources, making it difficult to readily process and interrogate the resulting volumes. The data acquisition is thus performed essentially blindly. Such a sequential process makes it hard to notice problems with the measurement protocol or sample conditions, potentially rendering the acquired data unusable, and it keeps the user from optimizing the experimental parameters of the imaging task at hand. We present an efficient approach to address this issue based on the real-time reconstruction, visualisation and on-the-fly an

Airspace Diameter Map-A Quantitative Measurement of All Pulmonary Airspaces to Characterize Structural Lung Diseases.

(1) Background: Stereological estimations significantly contributed to our understanding of lung anatomy and physiology. Taking stereology fully 3-dimensional facilitates the estimation of novel parameters. (2) Methods: We developed a protocol for the analysis of all airspaces of an entire lung. It includes (i) high-resolution synchrotron radiation-based X-ray tomographic microscopy, (ii) image segmentation using the free machine-learning tool Ilastik and ImageJ, and (iii) calculation of the airspace diameter distribution using a diameter map function. To evaluate the new pipeline, lungs from adult mice with cystic fibrosis (CF)-like lung disease (βENaC-transgenic mice) or mice with elastase-induced emphysema were compared to healthy controls. (3) Results: We were able to show the distribution of airspace diameters throughout the entire lung, as well as separately for the conducting airways and the gas exchange area. In the pathobiological context, we observed an irregular widening of parenchymal airspaces in mice with CF-like lung disease and elastase-induced emphysema. Comparable results were obtained when analyzing lungs imaged with μCT, sugges-ting that our pipeline is applicable to different kinds of imaging modalities. (4) Conclusions: We conclude that the airspace diameter map is well suited for a detailed analysis of unevenly distri-buted structural alterations in chronic muco-obstructive lung diseases such as cystic fibrosis and COPD

Transcriptomic characterization of culture-associated changes in murine and human precision-cut tissue slices

Our knowledge of complex pathological mechanisms underlying organ fibrosis is predominantly derived from animal studies. However, relevance of animal models for human disease is limited; therefore, an ex vivo model of human precision-cut tissue slices (PCTS) might become an indispensable tool in fibrosis research and drug development by bridging the animal-human translational gap. This study, presented as two parts, provides comprehensive characterization of the dynamic transcriptional changes in PCTS during culture by RNA sequencing. Part I investigates the differences in culture-induced responses in murine and human PCTS derived from healthy liver, kidney and gut. Part II delineates the molecular processes in cultured human PCTS generated from diseased liver, kidney and ileum. We demonstrated that culture was associated with extensive transcriptional changes and impacted PCTS in a universal way across the organs and two species by triggering an inflammatory response and fibrosis-related extracellular matrix (ECM) remodelling. All PCTS shared mRNA upregulation of IL-11 and ECM-degrading enzymes MMP3 and MMP10. Slice preparation and culturing activated numerous pathways across all PCTS, especially those involved in inflammation (IL-6, IL-8 and HMGB1 signalling) and tissue remodelling (osteoarthritis pathway and integrin signalling). Despite the converging effects of culture, PCTS display species-, organ- and pathology-specific differences in the regulation of genes and canonical pathways. The underlying pathology in human diseased PCTS endures and influences biological processes like cytokine release. Our study reinforces the use of PCTS as an ex vivo fibrosis model and supports future studies towards its validation as a preclinical tool for drug development

Electric field stimulation of precision cut lung slices

Chronic airway diseases, such as bronchial asthma and chronic obstructive pulmonary disease (COPD), are the fourth-leading cause of death in developed countries and have a high personal, societal, and economical impact. Recurring inflammation is one characteristic of those diseases leading to airway remodelling and modulating the function of nerves resulting in excessive bronchoconstriction. The precision-cut lung slice (PCLS) technique is widely used in pulmonary pharmacology, but neurally-induced bronchoconstriction has not been studied yet. Hence, the aim of this study was to develop a system, which specifically activates nerves within PCLS of different species, allowing the investigation of neural function and its pharmacological interference throughout the whole lung - also in an allergic animal model. Electric field stimulation (EFS) is considered to activate nerve endings in tissue preparations. Therefore, a device was constructed in which the PCLS were faced by two platinum electrodes and which delivered serial rectangular electric stimuli. EFS-induced bronchoconstriction in PCLS was monitored by videomicroscopy. EFS of PCLS was first established for the rat. EFS conditions were systematically studied and stable repeated airway contractions were obtained at a frequency of 50 Hz, pulse duration of 1 ms, current of 200 mA and train width of 2.5 s. Magnesium antagonized airway contractions induced by EFS, but not by methacholine, indicating that they are neurally evoked. The EFS-triggered contractions were increased by the acetylcholine esterase inhibitor neostigmine and were highly reproducible, which allowed the study of muscarinic antagonists in consecutive EFS-trains. The decadic logarithmic inhibitory concentration of half-maximal effect (log IC50 M) of antagonists was determined in EFS of PCLS and revealed ipratropium (-10.0 ± 0.4) being more potent than atropine (-8.8 ± 0.4) and 4-DAMP (-8.4 ± 0.3). Furthermore, it was tested if thromboxane facilitates cholinergic bronchoconstriction by using the thromboxane prostanoid receptor antagonist SQ29548, but no effect was observed. In EFS of PCLS on different airway sizes, larger airways showed stronger response than smaller ones. In a model of allergic asthma, Brown-Norway rats were sensitized against ovalbumin (OVA). Besides haemorrhages in the lung also airway contractions in PCLS after OVA-provocation were found, pointing to a persisting inflammation and an acute allergic response, respectively. However, in PCLS from these rats no airway hyperresponsiveness was found in response to methacholine or EFS. Since airway responses are known to exhibit considerable species differences, the neural responses of PCLS prepared from mice, guinea pigs, sheep, marmosets and humans were examined. Peripheral neurons were activated either by EFS or additionally, in order to probe excitatory nonadrenergic noncholinergic (NANC) nerves, by capsaicin. Bronchoconstriction in response to EFS varied between species in magnitude ((2 ± 2)% in mouse to (61 ± 26)% in guinea pig) and sensitivity (half-maximal response in frequency-response curves: (0.5 ± 0.2) Hz in sheep to (23 ± 7) Hz in marmoset). As in rats, atropine antagonized the EFS-induced bronchoconstriction in PCLS from guinea pigs, sheep, marmosets and humans. Capsaicin caused bronchoconstriction only in human and guinea pig lungs. Transient receptor potential channel antagonists (SKF96365/Ruthenium red) inhibited airway contractions after EFS or capsaicin addition in guinea pigs. Only marmoset airways showed airway dilation after multiple EFS and this was blocked by propranolol and L-NAME. In conclusion, the findings within this study show that nerve endings remain intact in PCLS. The present method is useful to study neurogenic responses in airways of different size and pharmacological interventions are possible. In rat PCLS, lesser sensitivity to EFS with smaller airways is in contrast to the response of exogenously applied methacholine, which suggests that parasympathetic bronchoconstriction in the airways seems to be balanced by inversely arranged innervation and acetylcholine-responsiveness: rich innervations with low acetylcholine-responsiveness in large airways opposed to more sparse innervations with high sensitivity in smaller airways. In the rat model of allergic asthma, increased release of acetylcholine from postganglionic parasympathetic nerves does not take place and acetylcholine receptors and the postreceptor transduction mechanisms are not affected after allergen sensitization. As in rats, airways of guinea pigs, sheep, marmosets and humans do all receive cholinergic innervations. Moreover, distal guinea pig and human airways also receive excitatory NANC innervations. Marmoset airways even display relaxations by sympathetic and inhibitory NANC nerves. As guinea pig resembled the human response profile best, this model may be particularly suited for studies on the role of peripheral airway innervation and its involvement in human lung diseases

Electric field stimulation of precision cut lung slices

Chronic airway diseases, such as bronchial asthma and chronic obstructive pulmonary disease (COPD), are the fourth-leading cause of death in developed countries and have a high personal, societal, and economical impact. Recurring inflammation is one characteristic of those diseases leading to airway remodelling and modulating the function of nerves resulting in excessive bronchoconstriction. The precision-cut lung slice (PCLS) technique is widely used in pulmonary pharmacology, but neurally-induced bronchoconstriction has not been studied yet. Hence, the aim of this study was to develop a system, which specifically activates nerves within PCLS of different species, allowing the investigation of neural function and its pharmacological interference throughout the whole lung - also in an allergic animal model. Electric field stimulation (EFS) is considered to activate nerve endings in tissue preparations. Therefore, a device was constructed in which the PCLS were faced by two platinum electrodes and which delivered serial rectangular electric stimuli. EFS-induced bronchoconstriction in PCLS was monitored by videomicroscopy. EFS of PCLS was first established for the rat. EFS conditions were systematically studied and stable repeated airway contractions were obtained at a frequency of 50 Hz, pulse duration of 1 ms, current of 200 mA and train width of 2.5 s. Magnesium antagonized airway contractions induced by EFS, but not by methacholine, indicating that they are neurally evoked. The EFS-triggered contractions were increased by the acetylcholine esterase inhibitor neostigmine and were highly reproducible, which allowed the study of muscarinic antagonists in consecutive EFS-trains. The decadic logarithmic inhibitory concentration of half-maximal effect (log IC50 M) of antagonists was determined in EFS of PCLS and revealed ipratropium (-10.0 ± 0.4) being more potent than atropine (-8.8 ± 0.4) and 4-DAMP (-8.4 ± 0.3). Furthermore, it was tested if thromboxane facilitates cholinergic bronchoconstriction by using the thromboxane prostanoid receptor antagonist SQ29548, but no effect was observed. In EFS of PCLS on different airway sizes, larger airways showed stronger response than smaller ones. In a model of allergic asthma, Brown-Norway rats were sensitized against ovalbumin (OVA). Besides haemorrhages in the lung also airway contractions in PCLS after OVA-provocation were found, pointing to a persisting inflammation and an acute allergic response, respectively. However, in PCLS from these rats no airway hyperresponsiveness was found in response to methacholine or EFS. Since airway responses are known to exhibit considerable species differences, the neural responses of PCLS prepared from mice, guinea pigs, sheep, marmosets and humans were examined. Peripheral neurons were activated either by EFS or additionally, in order to probe excitatory nonadrenergic noncholinergic (NANC) nerves, by capsaicin. Bronchoconstriction in response to EFS varied between species in magnitude ((2 ± 2)% in mouse to (61 ± 26)% in guinea pig) and sensitivity (half-maximal response in frequency-response curves: (0.5 ± 0.2) Hz in sheep to (23 ± 7) Hz in marmoset). As in rats, atropine antagonized the EFS-induced bronchoconstriction in PCLS from guinea pigs, sheep, marmosets and humans. Capsaicin caused bronchoconstriction only in human and guinea pig lungs. Transient receptor potential channel antagonists (SKF96365/Ruthenium red) inhibited airway contractions after EFS or capsaicin addition in guinea pigs. Only marmoset airways showed airway dilation after multiple EFS and this was blocked by propranolol and L-NAME. In conclusion, the findings within this study show that nerve endings remain intact in PCLS. The present method is useful to study neurogenic responses in airways of different size and pharmacological interventions are possible. In rat PCLS, lesser sensitivity to EFS with smaller airways is in contrast to the response of exogenously applied methacholine, which suggests that parasympathetic bronchoconstriction in the airways seems to be balanced by inversely arranged innervation and acetylcholine-responsiveness: rich innervations with low acetylcholine-responsiveness in large airways opposed to more sparse innervations with high sensitivity in smaller airways. In the rat model of allergic asthma, increased release of acetylcholine from postganglionic parasympathetic nerves does not take place and acetylcholine receptors and the postreceptor transduction mechanisms are not affected after allergen sensitization. As in rats, airways of guinea pigs, sheep, marmosets and humans do all receive cholinergic innervations. Moreover, distal guinea pig and human airways also receive excitatory NANC innervations. Marmoset airways even display relaxations by sympathetic and inhibitory NANC nerves. As guinea pig resembled the human response profile best, this model may be particularly suited for studies on the role of peripheral airway innervation and its involvement in human lung diseases