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

Discovery of Sulforaphane as an Inducer of Ferroptosis in U-937 Leukemia Cells: Expanding Its Anticancer Potential

In recent years, natural compounds have emerged as inducers of non-canonical cell death. The isothiocyanate sulforaphane (SFN) is a well-known natural anticancer compound with remarkable pro-apoptotic activity. Its ability to promote non-apoptotic cell-death mechanisms remains poorly investigated. This work aimed to explore the capacity of SFN to induce non-apoptotic cell death modalities. SFN was tested on different acute myeloid leukemia cell lines. The mechanism of cell death was investigated using a multi-parametric approach including fluorescence microscopy, western blotting, and flow cytometry. SFN triggered different cell-death modalities in a dose-dependent manner. At 25 μM, SFN induced caspase-dependent apoptosis and at 50 μM ferroptosis was induced through depletion of glutathione (GSH), decreased GSH peroxidase 4 protein expression, and lipid peroxidation. In contrast, necroptosis was not involved in SFN-induced cell death, as demonstrated by the non-significant increase in phosphorylation of receptor-interacting protein kinase 3 and phosphorylation of the necroptotic effector mixed lineage kinase domain-like pseudokinase. Taken together, our results suggest that the antileukemic activity of SFN can be mediated via both ferroptotic and apoptotic cell death modalities

Nutraceuticals and Physical Activity as Antidepressants: The Central Role of the Gut Microbiota

Major depressive disorder (MDD) is a common mental illness. Evidence suggests that the gut microbiota play an essential role in regulating brain functions and the pathogenesis of neuropsychiatric diseases, including MDD. There are numerous mechanisms through which the gut microbiota and brain can exchange information in a continuous, bidirectional communication. Current research emphasizes the interexchange of signals influenced by the gut microbiota that are detected and transduced in information from the gut to the nervous system involving neural, endocrine, and inflammatory mechanisms, suggesting a relationship between oxidative stress and the pathophysiology of MDD via the hyperactivation of inflammatory responses. Potential sources of inflammation in the plasma and hippocampus of depressed individuals could stem from increases in intestinal permeability. Some nutraceuticals, such as specific probiotics, namely psychobiotics, polyphenols, carotenoids, butyrate, and prebiotics, have been demonstrated to exert an antidepressant activity, but most of them need to be metabolized and activated by gut microorganisms. By inducing changes in the gut microbiota composition, physical exercise might also exert a role in alleviating depression-like symptoms. The mutual relationships among nutraceuticals, exercise, and depression will be discussed, and the potential role of the gut microbiota as a therapeutic target to treat depression will be explored

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

A fully automated micro‑CT 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

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 þ 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 þ 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 þ 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 þ 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 þ 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.</p

Altered erythropoiesis and decreased number of erythrocytes in children with neuroblastoma

Neuroblastoma (NB) is a pediatric tumor presenting at diagnosis either as localized or metastatic disease, which mainly involves the bone marrow (BM). The physical occupancy of BM space by metastatic NB cells has been held responsible for impairment of BM function. Here, we investigated whether localized or metastatic NB may alter hematopoietic lineages' maturation and release of mature cells in the periphery, through gene expression profiling, analysis of BM smears, cell blood count and flow cytometry analysis. Gene ontology and disease-associated analysis of the genes significantly under-expressed in BM resident cells from children with localized and metastatic NB, as compared to healthy children, indicated anemia, blood group antigens, and heme and porphyrin biosynthesis as major functional annotation clusters. Accordingly, in children with NB there was a selective impairment of erythrocyte maturation at the ortho-chromic stage that resulted in reduced erythrocyte count in the periphery, regardless of the presence of metastatic cells in the BM. By considering all NB patients, low erythrocyte count at diagnosis associated with worse survival. Moreover, in the subset of metastatic patients, low erythrocyte count, hemoglobin and hematocrit and high red cell distribution width at follow-up also associated with worse outcome. These observations provide an alternative model to the tenet that infiltrating cells inhibit BM functions due to physical occupancy of space and may open a new area of research in NB to understand the mechanism(s) responsible for such selective impairment