The intratracheal instillation of bleomycin in mice induces early damage to alveolar epithelial cells and development of inflammation followed by fibrotic tissue changes and represents the most widely used model of pulmonary fibrosis to investigate human IPF. Histopathology is the gold standard for assessing lung fibrosis in rodents, however it precludes repeated and longitudinal measurements of disease progression and does not provide information on spatial and temporal distribution of tissue damage. Here we investigated the use of the Micro-CT technique to allow the evaluation of disease onset and progression at different time-points in the mouse bleomycin model of lung fibrosis. Micro-CT was throughout coupled with histological analysis for the validation of the imaging results. In bleomycin-instilled and control mice, airways and lung morphology changes were assessed and reconstructed at baseline, 7, 14 and 21 days post-treatment based on Micro-CT images. Ashcroft score, percentage of collagen content and percentage of alveolar air area were detected on lung slides processed by histology and subsequently compared with Micro-CT parameters. Extent (%) of fibrosis measured by Micro-CT correlated with Ashcroft score, the percentage of collagen content and the percentage of alveolar air area (r 2 = 0.91; 0.77; 0.94, respectively). Distal airway radius also correlated with the Ashcroft score, the collagen content and alveolar air area percentage (r 2 = 0.89; 0.78; 0.98, respectively). Micro-CT data were in good agreement with histological read-outs as micro-CT was able to quantify effectively and non-invasively disease progression longitudinally and to reduce the variability and number of animals used to assess the damage. This suggests that this technique is a powerful tool for understanding experimental pulmonary fibrosis and that its use could translate into a more efficient drug discovery process, also helping to fill the gap between preclinical setting and clinical practice.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease (ILD) whose cause and pathogenesis are not yet well understood. Until now, no animal model of lung fibrosis succeeds in recapitulating all IPF features, thus the use of different rodent models is essential for the evaluation and development of new effective pharmacological treatments. Recently, the alveolar epithelial dysfunction has been emphasized in the etiopathogenesis context of IPF. Remarkably, the role of an aberrant basaloid cell type, primarily found in humans and confirmed in mice, seems to be crucial in the establishment and progression of the disease/model. Our work aimed to characterize for the first time this cell population in a rat model of lung fibrosis induced by a double bleomycin (BLM) administration, demonstrating the translational value of the model and its potential use in the testing of effective new drugs. Methods: Rats received an intratracheal BLM administration at day 0 and 4. Animals were sacrificed 21 and 28 days post-BLM. The fibrosis evaluation was carried out through histological (Ashcroft score and automatic image analysis) and immunoenzymatic analysis. Immunofluorescence was used for the characterization of the aberrant basaloid cells markers. Results: Lung histology revealed an increase in severe grades of Ashcroft scores and areas of fibrosis, resulting in a rise of collagen deposition at both the analyzed time-points. Immunofluorescence staining indicated the presence of KRT8+ cells in bronchial epithelial cells from both controls (saline, SAL) and BLM-treated animals. Interesting, KRT8+ cells were found exclusively in the fibrotic parenchyma (confirmed by the alpha-smooth muscle actin (α-SMA) staining for myofibroblasts) of BLM-treated animals. Moreover, KRT8+ cells co-expressed markers as Prosurfactant protein C (Pro-SPC) and Vimentin, suggesting their intermediate state potentially originating from alveolar type II (AT2) cells, and participating to the abnormal epithelial–mesenchymal crosstalk. Conclusion: Previous preclinical studies demonstrated the presence of KRT8+ aberrant basaloid-like cells in murine models of lung fibrosis. This work investigated the same cell population in a different rodent (the rat) model of lung fibrosis triggered by a double administration of BLM. Our results provided a further confirmation that, in rats, the intratracheal administration of BLM induced the appearance of a population of cells compatible with the KRT8+ alveolar differentiation intermediate (ADI) cells, as described previously in the mouse. This piece of work enforces previous evidence and further support the use of a rat model of BLM resembling the alveolar epithelial dysfunction to evaluate new clinical candidates for development in IPF.

Nintedanib is an anti-fibrotic medication endowed with a multi-kinase inhibitor profile and approved for the treatment of Idiopathic Pulmonary Fibrosis (IPF). Nintedanib is believed to inhibit mainly Vascular Endothelial Growth Factor (VEGF), Platelet-Derived Growth Factor (PDGF), and Fibroblast Growth Factor (FGF) receptor kinases. The main objective was to identify potential tissue and/or circulating biomarkers to demonstrate Nintedanib’s target engagement and support its in vivo pharmacodynamic activity, consistent with its proposed mechanism(s) of action. In four independent experiments of bleomycin (BLM)-induced lung fibrosis model in rats, animals received Nintedanib (oral, 100 mg/kg/day) from day 7 post-BLM for 3 weeks. As expected, Nintedanib significantly reduced lung weight, the levels of lung fibrotic markers, and fibrotic areas. Moreover, Nintedanib-treated animals expressed lower levels of FGF2 in lung homogenates and higher plasma and lung levels of VEGF (≥3-fold, p < 0.05) compared to control animals. Lung proteomic analysis revealed the inhibition of receptor tyrosine kinases signaling in Nintedanib-treated animals. Circulating and lung levels of Nintedanib confirmed an optimal tissue distribution in the rat, consistent with the data reported for humans. Although VEGF ligand levels are elevated in the lungs of Nintedanib-treated animals, the VEGF signaling pathway remained functionally downregulated, strongly suggesting compensatory VEGF feedback delivery to its receptor blockade by Nintedanib. In summary, based on the present experimental findings in rats and supporting clinical preliminary evidence, increased VEGF levels can be reasonably considered an indicator of target engagement for Nintedanib and potentially for other VEGF modulators.

Idiopathic pulmonary fibrosis (IPF) remains a progressive and fatal disease despite major advances in antifibrotic therapy. Pirfenidone and nintedanib slow lung function decline, and the PDE4B inhibitor nerandomilast and treprostinil have recently shown promise as next-generation treatments. However, current therapies neither halt nor reverse disease progression, and tolerability issues often limit adherence. We compared the antifibrotic activity and plasma levels of nintedanib administered by either oral gavage or dietary supplementation in a rat model of pulmonary fibrosis. Male Sprague-Dawley rats received intratracheal bleomycin (1 U/kg) on days 0 and 4. From day 7 to day 28, animals were treated with nintedanib (100 mg/kg/day) by either oral gavage or medicated chow. Lung weight, fibrosis biomarkers (procollagen-I, metalloproteinase-7 or MMP7, WNT1-inducible signaling pathway protein or WISP-1), epithelial injury marker KL-6, target engagement biomarkers (Fibroblast Growth Factor 2 or FGF-2, Vascular Endothelial Growth Factor or VEGF), plasma drug levels, and histological fibrosis scores were evaluated. Both administration regimens significantly reduced procollagen-I, WISP-1 and KL-6, and histological fibrosis scores. Oral gavage produced approximately fourfold-higher peak plasma concentrations of nintedanib 30 min after dosing, whereas dietary administration resulted in lower, more stable plasma levels with reduced variability while resulting in comparable antifibrotic efficacy. In conclusion, nintedanib retained robust antifibrotic activity when administered via dietary supplementation despite lower, but continuous, active plasma concentrations. Collectively, these findings indicate that optimizing drug delivery and absorption kinetics may achieve sustained therapeutic efficacy while reducing unnecessary exposure to supratherapeutic concentrations, thereby potentially improving tolerability.