A mouse model of progressive lung fibrosis with cutaneous involvement induced by a combination of oropharyngeal and osmotic minipump bleomycin delivery.

Systemic sclerosis (SSc) with interstitial lung disease (SSc-ILD) lacks curative pharmacological treatments, thus necessitating effective animal models for candidate drug discovery. Existing bleomycin (BLM)-induced SSc-ILD mouse models feature spatially limited pulmonary fibrosis, spontaneously resolving after 28 days. Here, we present an alternative BLM administration approach in female C57BL/6 mice, combining oropharyngeal aspiration (OA) and subcutaneous mini-pump delivery (pump) of BLM to induce a sustained and more persistent fibrosis, while retaining stable skin fibrosis. A dose-finding study was performed with BLM administered as 10 µg (OA) +80 mg/kg (pump) (10 + 80), 10 + 100, and 15 + 100. Forty-two days after OA, micro-computed tomography (micro-CT) imaging and histomorphometric analyses showed that the 10 + 100 and 15 + 100 treatments induced significant alterations in lung micro-CT-derived readouts, Ashcroft score, and more severe fibrosis grades compared with saline controls. In addition, a marked reduction in hypodermal thickness was observed in the 15 + 100 group. A time-course characterization of the BLM 15 + 100 treatment at days 28, 35, and 42, including longitudinal micro-CT imaging, revealed progressing alterations in lung parameters. Lung histology highlighted a sustained fibrosis accompanied by a reduction in hypodermis thickness throughout the explored time-window, with a time-dependent increase in fibrotic biomarkers detected by immunofluorescence analysis. BLM-induced alterations were partly mitigated by Nintedanib treatment. Our optimized BLM delivery approach leads to extensive and persistent lung fibrotic lesions coupled with cutaneous fibrotic alterations: it thus represents a significant advance compared with current preclinical models of BLM-induced SSc-ILD.NEW & NOTEWORTHY This study introduces an innovative approach to enhance the overall performance of the mouse bleomycin (BLM)-induced model for systemic sclerosis with interstitial lung disease (SSc-ILD). By combining oropharyngeal aspiration and subcutaneous mini-pump delivery of BLM, our improved model leads to sustained lung fibrosis and stable skin fibrosis in female C57BL/6 mice. The optimized 15 + 100 treatment results in extensive and persistent lung fibrotic lesions and thus represents a significant improvement over existing preclinical models of BLM-induced SSc-ILD.

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 micro­CT deep learning approach for precision preclinical investigation of lung fibrosis progression and response to therapy.

Micro-computed tomography (µCT)-based imaging plays a key role in monitoring disease progression and response to candidate drugs in various animal models of human disease, but manual image processing is still highly time-consuming and prone to operator bias. Focusing on an established mouse model of bleomycin (BLM)-induced lung fibrosis we document, here, the ability of a fully automated deep-learning (DL)-based model to improve and speed-up lung segmentation and the precise measurement of morphological and functional biomarkers in both the whole lung and in individual lobes. µCT-DL whose results were overall highly consistent with those of more conventional, especially histological, analyses, allowed to cut down by approximately 45-fold the time required to analyze the entire dataset and to longitudinally follow fibrosis evolution and response to the human-use-approved drug Nintedanib, using both inspiratory and expiratory µCT. Particularly significant advantages of this µCT-DL approach, are: (i) its reduced experimental variability, due to the fact that each animal acts as its own control and the measured, operator bias-free biomarkers can be quantitatively compared across experiments; (ii) its ability to monitor longitudinally the spatial distribution of fibrotic lesions, thus eliminating potential confounding effects associated with the more severe fibrosis observed in the apical region of the left lung and the compensatory effects taking place in the right lung; (iii) the animal sparing afforded by its non-invasive nature and high reliability; and (iv) the fact that it can be integrated into different drug discovery pipelines with a substantial increase in both the speed and robustness of the evaluation of new candidate drugs. The µCT-DL approach thus lends itself as a powerful new tool for the precision preclinical monitoring of BLM-induced lung fibrosis and other disease models as well. Its ease of operation and use of standard imaging instrumentation make it easily transferable to other laboratories and to other experimental settings, including clinical diagnostic applications.

Proteomic Fingerprint of Lung Fibrosis Progression and Response to Therapy in Bleomycin-Induced Mouse Model.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by the aberrant accumulation of extracellular matrix in the lungs. nintedanib is one of the two FDA-approved drugs for IPF treatment; however, the exact pathophysiological mechanisms of fibrosis progression and response to therapy are still poorly understood. In this work, the molecular fingerprint of fibrosis progression and response to nintedanib treatment have been investigated by mass spectrometry-based bottom-up proteomics in paraffin-embedded lung tissues from bleomycin-induced (BLM) pulmonary fibrosis mice. Our proteomics results unveiled that (i) samples clustered depending on the tissue fibrotic grade (mild, moderate, and severe) and not on the time course after BLM treatment; (ii) the dysregulation of different pathways involved in fibrosis progression such as the complement coagulation cascades, advanced glycation end products (AGEs) and their receptors (RAGEs) signaling, the extracellular matrix-receptor interaction, the regulation of actin cytoskeleton, and ribosomes; (iii) Coronin 1A (Coro1a) as the protein with the highest correlation when evaluating the progression of fibrosis, with an increased expression from mild to severe fibrosis; and (iv) a total of 10 differentially expressed proteins (padj-value ≤ 0.05 and Fold change ≤-1.5 or ≥1.5), whose abundance varied in the base of the severity of fibrosis (mild and moderate), were modulated by the antifibrotic treatment with nintedanib, reverting their trend. Notably, nintedanib significantly restored lactate dehydrogenase B (Ldhb) expression but not lactate dehydrogenase A (Ldha). Notwithstanding the need for further investigations to validate the roles of both Coro1a and Ldhb, our findings provide an extensive proteomic characterization with a strong relationship with histomorphometric measurements. These results unveil some biological processes in pulmonary fibrosis and drug-mediated fibrosis therapy.

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.

A new anesthesia protocol enabling longitudinal lung-function measurements in neonatal rabbits by micro-CT.

Micro-computed tomography (micro-CT) imaging is an emerging technology with many applications in small animals, for example, the study of pulmonary diseases, although clear guidelines and critical mass of evidence are still missing in the preclinical literature. The neonatal rabbit is a valuable model for studying pulmonary development. However, the longitudinal monitoring of lung function by micro-CT can be challenging. Distinctive datasets corresponding to the end-inspiration and end-expiration phases need to be generated and analyzed to derive lung-functional parameters. The quality of CT scans and the reliability of parameters obtained remain highly dependent on the anesthesia protocol used. Three different anesthetic protocols were tested. The combination of dexmedetomidine 0.25 mg/kg injected intraperitoneally followed by 1% isoflurane was found to facilitate CT imaging at 4 and 11 days after birth. Contrarily, isoflurane and ketamine-xylazine were found unsuitable and thus not investigated further. Total lung volumes significantly increased at day 11 compared with baseline in both respiratory phases, whereas lung tissue remained constant. As expected, functional residual capacity, air-to-tissue ratio, and minute ventilation were significantly increased at day 11 in each animal. Those parameters were correlated with inspiratory capacity, compliance, elastance, and resistance of both respiratory system and tissue component, as measured by flexiVent. Lung development was also evaluated by histomorphometric analyses. In conclusion, we have identified a safe and suitable anesthesia protocol for micro-CT imaging in neonatal rabbits. Moreover, the possibility to longitudinally measure lung function in the same subject dramatically reduced the intraexperimental variability.

A pilot study on Langerhans cells in keratoconus patients by in vivo confocal microscopy before and after corneal cross-linking and correlation with eye rubbing.

PURPOSE: The aim of this study was to investigate the corneal microstructure and Langerhans cells using in vivo confocal microscopy in keratoconus patients before and after cross-linking, and to correlate the morphologic findings with clinical and patient-reported outcomes, including eye rubbing (ER) behavior. METHODS: Patients with progressive keratoconus undergoing iontophoresis-assisted epithelium-on cross-linking (I-CXL) were consecutively enrolled. In vivo confocal microscopy was performed before and 6 months after treatment. Patients were asked to quantify their ER behavior on a Visual Analogue Scale (VAS) and completed the Keratoconus Outcomes Research Questionnaire and the Ocular Surface Disease Index questionnaires at the same time points. Visual acuity, tear osmolarity, topography, aberrometry, and pachymetry of both eyes were assessed. RESULTS: Thirteen patients were included in this pilot study. Preoperatively, the mean Langerhans cells density was 35,615 cells per mm2, and the median morphology was 3. The mean ER VAS before treatment was 7,077 out of 10. The ER VAS showed significant positive correlations with both Langerhans cells density and morphology of the study eye. After treatment, a statistically significant reduction in ER VAS and in Langerhans cells variables was observed. The mean sub-basal plexus nerve density was comparable to pre-operative values 6 months after I-CXL. CONCLUSIONS: Based on this preliminary evidence, the presence of high density of mature Langerhans cells in the central cornea of keratoconus patients and its correlation with eye rubbing support the role of inflammation in keratoconus. The reduction in these markers after treatment may suggest a potential of CXL in moderating immune-related inflammation and eye rubbing in the medium term.

Multiphase micro-computed tomography reconstructions provide dynamic respiratory function in a mouse lung fibrosis model.

Micro-computed tomography derived functional biomarkers used in lung disease research can significantly complement end-stage histomorphometric measures while also allowing for longitudinal studies. However, no approach for visualizing lung dynamics across a full respiratory cycle has yet been described. Using bleomycin-induced lung fibrosis and the antifibrotic drug nintedanib as a test model, we implemented a four-dimensional (4D) micro-CT imaging approach consisting of 30 reconstructed volumes per respiratory cycle, coupled with deep-learning-assisted segmentation of lung volumes. 4D micro-CT provided an accurate description of inhalatory and exhalatory lung dynamics under resting conditions and revealed an inflammation-related obstructive pattern at day 7, followed by a restrictive pattern associated with fibrosis development at day 21. A milder restriction and fibrotic pathology resulted from nintedanib treatment. The similarity of 4D micro-CT data with those produced by diagnostic measurements, also points to its great potential as an exploratory tool for the discovery of clinically relevant therapeutic compounds.

Micro-CT-assisted identification of the optimal time-window for antifibrotic treatment in a bleomycin mouse model of long-lasting pulmonary fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a debilitating and fatal lung disease characterized by the excessive formation of scar tissue and decline of lung function. Despite extensive research, only two FDA-approved drugs exist for IPF, with limited efficacy and relevant side effects. Thus, there is an urgent need for new effective therapies, whose discovery strongly relies on IPF animal models. Despite some limitations, the Bleomycin (BLM)-induced lung fibrosis mouse model is widely used for antifibrotic drug discovery and for investigating disease pathogenesis. The initial acute inflammation triggered by BLM instillation and the spontaneous fibrosis resolution that occurs after 3 weeks are the major drawbacks of this system. In the present study, we applied micro-CT technology to a longer-lasting, triple BLM administration fibrosis mouse model to define the best time-window for Nintedanib (NINT) treatment. Two different treatment regimens were examined, with a daily NINT administration from day 7 to 28 (NINT 7-28), and from day 14 to 28 (NINT 14-28). For the first time, we automatically derived both morphological and functional readouts from longitudinal micro-CT. NINT 14-28 showed significant effects on morphological parameters after just 1 week of treatment, while no modulations of these biomarkers were observed during the preceding 7-14-days period, likely due to persistent inflammation. Micro-CT morphological data evaluated on day 28 were confirmed by lung histology and bronchoalveolar lavage fluid (BALF) cells; Once again, the NINT 7-21 regimen did not provide substantial benefits over the NINT 14-28. Interestingly, both NINT treatments failed to improve micro-CT-derived functional parameters. Altogether, our findings support the need for optimized protocols in preclinical studies to expedite the drug discovery process for antifibrotic agents. This study represents a significant advancement in pulmonary fibrosis animal modeling and antifibrotic treatment understanding, with the potential for improved translatability through the concurrent structural-functional analysis offered by longitudinal micro-CT.

Impact of corneal collagen cross-linking on vision-related quality of life measured with the keratoconus outcomes research questionnaire (KORQ) in patients with keratoconus.

PURPOSE: To assess the impact of corneal collagen cross-linking on self-reported vision-related quality of life (VR-QoL) in keratoconus patients by means of the Keratoconus Outcomes Research Questionnaire (KORQ), a new disease-specific patient reported outcomes measures (PROMs) tool. METHODS: Patients with progressive keratoconus undergoing corneal collagen cross-linking were consecutively enrolled. Patients completed the KORQ before and after the treatment, at 1, 3 and 6 months. Data were collected regarding the visual acuity and the topographic, aberrometric and pachymetric parameters of both eyes. Patients were also asked to quantify their eye rubbing behavior on a Visual Analogue Scale (VAS) from 0 to 10, before and 6 months after treatment. The KORQ scores were associated with the visual acuity, topographic, aberrometric and pachymetric data by means of the Spearman correlation coefficient. RESULTS: The Activity Limitation (AL) subscale score was higher after surgery. Preoperatively, negative correlations were observed between the KORQ AL score and Best Corrected Visual Acuity (logMAR), maximum keratometry, flattest keratometry, steepest keratometry, symmetry index front and higher order aberrations (HOA) of the treatment eye. Postoperatively, we observed a shift toward the fellow eye, with stronger correlation of the KORQ scores with the fellow eye parameters. The Symptoms subscale score correlated with the aberrometric parameters of both eyes at various time points. Statistically significant correlations were observed between the change in KORQ scores and the change in aberrometric parameters. A statistically significant reduction in the eye rubbing behavior was detected (p < 0,0001). CONCLUSIONS: These results suggest that the corneal cross-linking treatment is effective both in improving the subjective perception of the disease by the patient and in stabilizing the objective indicators of disease progression. Keratometric, aberrometric and visual acuity values showed a significant impact on self-reported VR-QoL. Corneal cross-linking, by halting the worsening of these parameters, may bear a beneficial effect on VR-QoL.

Micro-CT-derived ventilation biomarkers for the longitudinal assessment of pathology and response to therapy in a mouse model of lung fibrosis.

Experimental in-vivo animal models are key tools to investigate the pathogenesis of lung disease and to discover new therapeutics. Histopathological and biochemical investigations of explanted lung tissue are currently considered the gold standard, but they provide space-localized information and are not amenable to longitudinal studies in individual animals. Here, we present an imaging procedure that uses micro-CT to extract morpho-functional indicators of lung pathology in a murine model of lung fibrosis. We quantified the decrease of lung ventilation and measured the antifibrotic effect of Nintedanib. A robust structure-function relationship was revealed by cumulative data correlating micro-CT with histomorphometric endpoints. The results highlight the potential of in-vivo micro-CT biomarkers as novel tools to monitor the progression of inflammatory and fibrotic lung disease and to shed light on the mechanism of action of candidate drugs. Our platform is also expected to streamline translation from preclinical studies to human patients.

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.

Pivotal role of micro-CT technology in setting up an optimized lung fibrosis mouse model for drug screening.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with no curative pharmacological treatment. The most used animal model of IPF for anti-fibrotic drug screening is bleomycin (BLM)-induced lung fibrosis. However, several issues have been reported: the balance among disease resolution, an appropriate time window for therapeutic intervention and animal welfare remain critical aspects yet to be fully elucidated. In this study, C57Bl/6 male mice were treated with BLM via oropharyngeal aspiration (OA) following either double or triple administration. The fibrosis progression was longitudinally assessed by micro-CT every 7 days for 4 weeks after BLM administration. Quantitative micro-CT measurements highlighted that triple BLM administration was the ideal dose regimen to provoke sustained lung fibrosis up to 28 days. These results were corroborated with lung histology and Bronchoalveolar Lavage Fluid cells. We have developed a mouse model with prolonged lung fibrosis enabling three weeks of a curative therapeutic window for the screening of putative anti-fibrotic drugs. Moreover, we have demonstrated the pivotal role of longitudinal micro-CT imaging in reducing the number of animals required per experiment in which each animal can be its own control. This approach permits a valuable decrease in costs and time to develop disease animal models.

The Italian version of the Keratoconus Outcomes Research Questionnaire (KORQ): Translation and validation of psychometric properties.

PURPOSE: To develop and validate the Italian version of the Keratoconus Outcomes Research Questionnaire (KORQ). METHODS: Cross-sectional validation study. Keratoconus patients with routine appointments at the outpatient clinic were consecutively enrolled in conjunction with a sample of non-keratoconus controls for comparison. The Italian translation from the original English version of the KORQ was administered to the patients together with the Italian version of the 25-item version of the National Eye Institute Visual Function Questionnaire (NEI VFQ-25). The reliability and validity of the Italian KORQ were assessed using standardized internationally accepted methods for cultural adaptation and validation of health-related quality of life measures. RESULTS: One hundred patients were deemed eligible and completed the questionnaires. The Cronbach's alpha coefficient for internal consistency ranged from 0.71 to 0.956 across the subscales. Spearman correlation coefficient and intraclass correlation coefficient of 0.98 showed excellent test-retest reliability. The control group had better scores on every subscale. Statistically significant correlations were found between the KORQ and analogous domain of the NEI VFQ-25 and with disease severity indicators, such as visual acuity, maximum keratometry, and steepest keratometry, thus demonstrating good construct and concurrent validity. CONCLUSION: The Italian version of the KORQ exhibited excellent internal consistency, test-retest reliability, validity, discriminatory power, and psychometric properties comparable with those of the original English version, and thus may be adopted as a powerful vision-targeted quality of life assessment tool for Italian keratoconus patients.

Persistency of Mesenchymal Stromal/Stem Cells in Lungs.

Mesenchymal stromal/stem cells (MSCs) are a fibroblast-like cell population with high regenerative potential that can be isolated from many different tissues. Several data suggest MSCs as a therapeutic tool capable of migrating to a site of injury and guide tissue regeneration mainly through their secretome. Pulmonary first-pass effect occurs during intravenous administration of MSCs, where 50 to 80% of the cells tend to localize in the lungs. This phenomenon has been exploited to study MSC potential therapeutic effects in several preclinical models of lung diseases. Data demonstrated that, regardless of the lung disease severity and the delivery route, MSCs were not able to survive longer than 24 h in the respiratory tract but still surprisingly determined a therapeutic effect. In this work, two different mouse bone marrow-derived mesenchymal stromal/stem cell (mBM-MSC) lines, stably transduced with a third-generation lentiviral vector expressing luciferase and green fluorescent protein reporter genes tracking MSCs in vivo biodistribution and persistency, have been generated. Cells within the engrafted lung were in vivo traced using the high-throughput bioluminescence imaging (BLI) technique, with no invasiveness on animal, minimizing biological variations and costs. In vivo BLI analysis allowed the detection and monitoring of the mBM-MSC clones up to 28 days after implantation independently from the delivery route. This longer persistency than previously observed (24 h) could have a strong impact in terms of pharmacokinetics and pharmacodynamics of MSCs as a therapeutic tool.

SSC-ILD mouse model induced by osmotic minipump delivered bleomycin: effect of Nintedanib.

Systemic sclerosis (SSc) is an autoimmune disease characterized by an excessive production and accumulation of collagen in the skin and internal organs often associated with interstitial lung disease (ILD). Its pathogenetic mechanisms are unknown and the lack of animal models mimicking the features of the human disease is creating a gap between the selection of anti-fibrotic drug candidates and effective therapies. In this work, we intended to pharmacologically validate a SSc-ILD model based on 1 week infusion of bleomycin (BLM) by osmotic minipumps in C57/BL6 mice, since it will serve as a tool for secondary drug screening. Nintedanib (NINT) has been used as a reference compound to investigate antifibrotic activity either for lung or skin fibrosis. Longitudinal Micro-CT analysis highlighted a significant slowdown in lung fibrosis progression after NINT treatment, which was confirmed by histology. However, no significant effect was observed on lung hydroxyproline content, inflammatory infiltrate and skin lipoatrophy. The modest pharmacological effect reported here could reflect the clinical outcome, highlighting the reliability of this model to better profile potential clinical drug candidates. The integrative approach presented herein, which combines longitudinal assessments with endpoint analyses, could be harnessed in drug discovery to generate more reliable, reproducible and robust readouts.

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.

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.