Indocyanine-enhanced mouse model of bleomycin-induced lung fibrosis with hallmarks of progressive emphysema.

The development of new drugs for idiopathic pulmonary fibrosis strongly relies on preclinical experimentation, which requires the continuous improvement of animal models and integration with in vivo imaging data. Here, we investigated the lung distribution of bleomycin (BLM) associated with the indocyanine green (ICG) dye by fluorescence imaging. A long-lasting lung retention (up to 21 days) was observed upon oropharyngeal aspiration (OA) of either ICG or BLM + ICG, with significantly more severe pulmonary fibrosis, accompanied by the progressive appearance of emphysema-like features, uniquely associated with the latter combination. More severe and persistent lung fibrosis, together with a progressive air space enlargement uniquely associated with the BLM + ICG group, was confirmed by longitudinal micro-computed tomography (CT) and histological analyses. Multiple inflammation and fibrosis biomarkers were found to be increased in the bronchoalveolar lavage fluid of BLM- and BLM + ICG-treated animals, but with a clear trend toward a much stronger increase in the latter group. Similarly, in vitro assays performed on macrophage and epithelial cell lines revealed a significantly more marked cytotoxicity in the case of BLM + ICG-treated mice. Also unique to this group was the synergistic upregulation of apoptotic markers both in lung sections and cell lines. Although the exact mechanism underlying the more intense lung fibrosis phenotype with emphysema-like features induced by BLM + ICG remains to be elucidated, we believe that this combination treatment, whose overall effects more closely resemble the human disease, represents a valuable alternative model for studying fibrosis development and for the identification of new antifibrotic compounds.

A fully automated deep learning pipeline for micro-CT-imaging-based densitometry of lung fibrosis murine models.

Idiopathic pulmonary fibrosis, the archetype of pulmonary fibrosis (PF), is a chronic lung disease of a poor prognosis, characterized by progressively worsening of lung function. Although histology is still the gold standard for PF assessment in preclinical practice, histological data typically involve less than 1% of total lung volume and are not amenable to longitudinal studies. A miniaturized version of computed tomography (µCT) has been introduced to radiologically examine lung in preclinical murine models of PF. The linear relationship between X-ray attenuation and tissue density allows lung densitometry on total lung volume. However, the huge density changes caused by PF usually require manual segmentation by trained operators, limiting µCT deployment in preclinical routine. Deep learning approaches have achieved state-of-the-art performance in medical image segmentation. In this work, we propose a fully automated deep learning approach to segment right and left lung on µCT imaging and subsequently derive lung densitometry. Our pipeline first employs a convolutional network (CNN) for pre-processing at low-resolution and then a 2.5D CNN for higher-resolution segmentation, combining computational advantage of 2D and ability to address 3D spatial coherence without compromising accuracy. Finally, lungs are divided into compartments based on air content assessed by density. We validated this pipeline on 72 mice with different grades of PF, achieving a Dice score of 0.967 on test set. Our tests demonstrate that this automated tool allows for rapid and comprehensive analysis of µCT scans of PF murine models, thus laying the ground for its wider exploitation in preclinical settings.

The importance of routine quality control for reproducible pulmonary measurements by in vivo micro-CT.

Micro-computed tomography (CT) imaging provides densitometric and functional assessment of lung diseases in animal models, playing a key role either in understanding disease progression or in drug discovery studies. The generation of reliable and reproducible experimental data is strictly dependent on a system's stability. Quality controls (QC) are essential to monitor micro-CT performance but, although QC procedures are standardized and routinely employed in clinical practice, detailed guidelines for preclinical imaging are lacking. In this work, we propose a routine QC protocol for in vivo micro-CT, based on three commercial phantoms. To investigate the impact of a detected scanner drift on image post-processing, a retrospective analysis using twenty-two healthy mice was performed and lung density histograms used to compare the area under curve (AUC), the skewness and the kurtosis before and after the drift. As expected, statistically significant differences were found for all the selected parameters [AUC 532 ± 31 vs. 420 ± 38 (p < 0.001); skewness 2.3 ± 0.1 vs. 2.5 ± 0.1 (p < 0.001) and kurtosis 4.2 ± 0.3 vs. 5.1 ± 0.5 (p < 0.001)], confirming the importance of the designed QC procedure to obtain a reliable longitudinal quantification of disease progression and drug efficacy evaluation.

Alfaxalone and Dexmedetomidine as an Alternative to Gas Anesthesia for Micro-CT Lung Imaging in a Bleomycin-Induced Pulmonary Fibrosis Murine Model.

Micro-CT imaging could be considered a powerful non-invasive tool for accessing pulmonary fibrosis in mice. However, the choice of the anesthesia protocol plays a fundamental role to obtain robust and reproducible data, avoiding misinterpretations of the results. Inhaled anesthesia is commonly used for micro-CT lung imaging, but sometimes the standardization of the protocol may be challenging for routine activities in drug discovery. In this study we used micro-CT to evaluate the effects of two anesthetic protocols, consisting in Alfaxalone and Dexmedetomidine mixture, as injectable agents, and gaseous isoflurane, on vehicle and bleomycin-treated mice. No significant differences were highlighted between the protocols either for lung aeration degrees by micro-CT or histologic analyses in both the controls and bleomycin-treated groups. Our results support Alfaxalone and Dexmedetomidine mixture as a suitable and safe alternative compared to isoflurane for lung imaging. We also concluded that this injectable mixture may be applied for several imaging technologies and on different mice models.

Quantification of Lung Fibrosis in IPF-Like Mouse Model and Pharmacological Response to Treatment by Micro-Computed Tomography.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive degenerative lung disease leading to respiratory failure and death. Although anti-fibrotic drugs are now available for treating IPF, their clinical efficacy is limited and lung transplantation remains the only modality to prolong survival of IPF patients. Despite its limitations, the bleomycin (BLM) animal model remains the best characterized experimental tool for studying disease pathogenesis and assessing efficacy of novel potential drugs. In the present study, the effects of oropharyngeal (OA) and intratracheal (IT) administration of BLM were compared in C57BL/6 mice. The development of lung fibrosis was followed in vivo for 28 days after BLM administration by micro-computed tomography and ex vivo by histological analyses (bronchoalveolar lavage, histology in the left lung to stage fibrosis severity and hydroxyproline determination in the right lung). In a separate study, the antifibrotic effect of Nintedanib was investigated after oral administration (60 mg/kg for two weeks) in the OA BLM model. Lung fibrosis severity and duration after BLM OA and IT administration was comparable. However, a more homogeneous distribution of fibrotic lesions among lung lobes was apparent after OA administration. Quantification of fibrosis by micro-CT based on % of poorly aerated tissue revealed that this readout correlated significantly with the standard histological methods in the OA model. These findings were further confirmed in a second study in the OA model, evaluating Nintedanib anti-fibrotic effects. Indeed, compared to the BLM group, Nintedanib inhibited significantly the increase in % of poorly aerated areas (26%) and reduced ex vivo histological lesions and hydroxyproline levels by 49 and 41%, respectively. This study indicated that micro-computed tomography is a valuable in vivo technology for lung fibrosis quantification, which will be very helpful in the future to better evaluate new anti-fibrotic drug candidates.

In-vivo lung fibrosis staging in a bleomycin-mouse model: a new micro-CT guided densitometric approach.

Although increasing used in the preclinical testing of new anti-fibrotic drugs, a thorough validation of micro-computed tomography (CT) in pulmonary fibrosis models has not been performed. Moreover, no attempts have been made so far to define density thresholds to discriminate between aeration levels in lung parenchyma. In the present study, a histogram-based analysis was performed in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis by micro-CT, evaluating longitudinal density changes from 7 to 21 days after BLM challenge, a period representing the progression of fibrosis. Two discriminative densitometric indices (i.e. 40th and 70th percentiles) were extracted from Hounsfield Unit density distributions and selected for lung fibrosis staging. The strong correlation with histological findings (rSpearman = 0.76, p < 0.01) confirmed that variations in 70th percentile could reflect a pathological lung condition and estimate the effect of antifibrotic treatments. This index was therefore used to define lung aeration levels in mice distinguishing in hyper-inflated, normo-, hypo- and non-aerated pulmonary compartments. A retrospective analysis performed on a large cohort of mice confirmed the correlation between the proposed preclinical density thresholds and the histological outcomes (rSpearman = 0.6, p < 0.01), strengthening their suitability for tracking disease progression and evaluating antifibrotic drug candidates.

Maskless Arrayed Nanofiber Mats by Bipolar Pyroelectrospinning.

The numerous advantages of micro- and nanostructures produced by electrospinning (ES) have stimulated enormous interest in this technology with potential application in several fields. However, ES still has some limitations in controlling the geometrical arrangement of the fiber mats so that expensive and time-consuming technologies are usually employed for producing ordered geometries. Here we present a technique that we call "bipolar pyroelectrospinning" (b-PES) for generating ordered arrays of fiber mats in a direct manner by using the bipolar pyroelectric field produced by a periodically poled lithium niobate crystal (PPLN). The b-PES is free from expensive electrodes, nozzles, and masks because it makes use simply of the structured pyroelectric field produced by the PPLN crystal used as collector. The results show clearly the reliability of the technique in producing a wide variety of arrayed fiber mats that could find application in bioengineering or many other fields. Preliminary results of live cells patterning under controlled geometrical constraints is also reported and discussed in order to show potential exploitation as a scaffold in tissue engineering.

Pyro-Electrification of Freestanding Polymer Sheets: A New Tool for Cation-Free Manipulation of Cell Adhesion in vitro.

Localized electric fields have become, in recent years, a source of inspiration to researchers and laboratories thanks to a huge amount of applications derived from it, including positioning of microparticles as building blocks for electrical, optical, and magnetic devices. The possibility of producing polymeric materials with surface charge thus opens new perspectives for applications where process simplicity and cost-effectiveness of flexible electronics are of fundamental importance. In particular, the influence of surface charges is widely studied and is a critical issue especially when new materials and functional technologies are introduced. Here, we report a voltage-free pyro-electrification (PE) process able to induce a permanent dipole orientation into polymer sheets under both mono- and bipolar distribution. The technique makes use of the pyroelectric effect for generating electric potentials on the order of kilovolts by an easy-to-accomplish thermal treatment of ferroelectric lithium niobate (LN) crystals. The PE allows us to avoid the expensive and time-consuming fabrication of high-power electrical circuits, as occurs in traditional generator-based techniques. Since the technique is fully compatible with spin-coating-based procedures, the pyro-electrified polymer sheets are easily peeled off the surface of the LN crystal after PE completion, thus providing highly stable and freestanding charged sheets. We show the reliability of the technique for different polymers and for different applications ranging from live cell patterning to biofilm formation tests for bacteria linked to food-processing environments.

Quick liquid packaging: Encasing water silhouettes by three-dimensional polymer membranes.

One of the most important substances on Earth is water. It is an essential medium for living microorganisms and for many technological and industrial processes. Confining water in an enclosed compartment without manipulating it or by using rigid containers can be very attractive, even more if the container is biocompatible and biodegradable. Here, we propose a water-based bottom-up approach for facile encasing of short-lived water silhouettes by a custom-made adaptive suit. A biocompatible polymer self-assembling with unprecedented degree of freedom over the water surface directly produces a thin membrane. The polymer film could be the external container of a liquid core or a free-standing layer with personalized design. The membranes produced have been characterized in terms of physical properties, morphology and proposed for various applications from nano- to macroscale. The process appears not to harm cells and microorganisms, opening the way to a breakthrough approach for organ-on-chip and lab-in-a-drop experiments.

Simple and Rapid Bioink Jet Printing for Multiscale Cell Adhesion Islands.

A simple and rapid process for multiscale printing of bioinks with dot widths ranging from hundreds of microns down to 0.5 µm is presented. The process makes use of spontaneous surface charges generated pyroelectrically that are able to draw little daughter droplets directly from the free meniscus of a mother drop through jetting ("p-jet"), thus avoiding time-consuming and expensive fabrication of microstructured nozzles. Multiscale can be easily achieved by modulating the parameters of the p-jet process. Here, it is shown that the p-jet allows us to print well-defined adhesion islands where NIH-3T3 fibroblasts are constrained to live into cluster configurations ranging from 20 down to single cell level. The proposed fabrication approach can be useful for high-throughput studies on cell adhesion, cytoskeleton organization, and stem cell differentiation.

Bipolar Patterning of Polymer Membranes by Pyroelectrification.

Polymer freestanding membranes with permanent bipolar patterns are fabricated by "pyroelectrification". The thermal stimulation of periodically poled lithium niobate (PPLN) crystals simultaneously generates the pyroelectric effect, the glass transition of the polymer, and therefore the periodic electric poling of the polymer. The reliability of these membranes is demonstrated for applications under both dry and wet conditions, including cell patterning.

Effects of Lithium Niobate Polarization on Cell Adhesion and Morphology.

Understanding how the interfacial effects influence cell adhesion and morphology is of fundamental interest for controlling function, growth, and movement of cells in vitro and in vivo. In particular, the influence of surface charges is well-known but still controversial, especially when new functional materials and methods are introduced. Here, the influence of the spontaneous polarization of ferroelectric lithium niobate (LN) on the adhesion properties of fibroblast cells is investigated. The spontaneous polarization of LN has one of the largest known magnitudes at room temperature (∼78 µC/cm(2)), and its orientation can be patterned easily by an external voltage, this motivating highly the investigation of its interaction with cells. Immunofluorescence and migration assays show strong evidence that the surface polarity regulates the adhesion functions, with enhanced spreading of the cytoskeleton on the negative face. The results suggest the potential of LN as a platform for investigating the role of charges on cellular processes, thus favoring new strategies in fabricating those biocompatible constructs used for tissue engineering. In fact, the orientation of the high-magnitude polarization can be patterned easily and, in combination with piezoelectric, pyroelectric, and photorefractive properties, may open the route to more sophisticated charge templates for modulating the cell response.