Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Ferrini E

PROVIDER: S-EPMC7578219 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Ferrini Erica E Mecozzi Laura L Corsi Luisa L Ragionieri Luisa L Donofrio Gaetano G Stellari Franco Fabio FF

Frontiers in veterinary science 20201008

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 ...[more]

PMID: 33134367

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Project description:Background and aimsIdiopathic pulmonary fibrosis (IPF) is a fibrosing lung disease with unknown etiology, leading to cough and dyspnoea, which is also one of the most common sequelae affecting the quality of life of COVID-19 survivors. There is no cure for IPF patients. We aim to develop a reliable IPF animal model with quantification of fibrosis based on Micro-Computer Tomography (micro-CT) images for the new drug discovery, because different bleomycin administration routes, doses, and intervals are reported in the literature, and there is no quantitative assessment of pulmonary fibrosis based on micro-CT images in animal studies.MethodsWe compared three dosages (1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg) of intratracheal bleomycin administration and experiment intervals (14 and 21 days) in C57BL/6 mice by investigating survival rates, pulmonary histopathology, micro-CT, peripheral CD4+ & CD8+ cells, and cytokines. Moreover, a simple and reliable new method was developed for scoring fibrosis in live mice based on Micro-CT images by using Image J software, which transfers the dark sections in pulmonary Micro-CT images to light colors on a black background.ResultsThe levels of hydroxyproline, inflammation cytokine, fibrotic pathological changes, and collagen deposition in the lungs of mice were bleomycin dose-dependent and time-dependent as well as the body weight loss. Based on the above results, the mice model at 21 days after being given bleomycin at 1.25 mg/kg has optimal pulmonary fibrosis with a high survival rate and low toxicity. There is a significant decrease in the light area (gray value at 9.86 ± 0.72) in the BLM mice, indicating that a significant decrease in the alveolar air area was observed in BLM injured mice compared to normal groups (###p < 0.001), while the Pirfenidone administration increased the light area (gray value) to 21.71 ± 2.95 which is close to the value observed in the normal mice (gray value at 23.23 ± 1.66), which is consistent with the protein levels of Col1A1, and α-SMA. Notably, the standard deviations for the consecutive six images of each group indicate the precision of this developed quantitation method for the micro-CT image taken at the fifth rib of each mouse.ConclusionProvided a quantifying method for Micro-CT images in an optimal and repeatable pulmonary fibrosis mice model for exploring novel therapeutic interventions.

Project description:BackgroundBone marrow-derived fibrocytes reportedly play important roles in the pathogenesis of idiopathic pulmonary fibrosis. Pirfenidone is an anti-fibrotic agent; however, its effects on fibrocytes have not been investigated. The aim of this study was to investigate whether pirfenidone inhibits fibrocyte pool size in the lungs of bleomycin-treated mice.MethodsBleomycin (100 mg/kg) was infused with osmotic pumps into C57BL/6 mice, and pirfenidone (300 mg/kg/day) was orally administered daily for 2 wk. The lungs were removed, and single-cell suspensions were subjected to fluorescence-activated cell sorter (FACS) analysis to detect fibrocytes, which were defined as CD45 and collagen-I double-positive cells. Immunohistochemistry was performed on the lung specimens to quantify fibrocytes. Chemokines in the lung digests were measured with enzyme-linked immunosorbent assay. The effect of pirfenidone on alveolar macrophages was evaluated with bronchoalveolar lavage (BAL). In a therapeutic setting, pirfenidone administration was initiated 10 days after bleomycin treatment. For chemotaxis assay, lung fibrocytes were isolated with immunomagnetic selection (CD45-positive mesenchymal cells) after culture and allowed to migrate toward chemokines in the presence or absence of pirfenidone. Moreover, the effect of pirfenidone on the expression of chemokine receptors on fibrocytes was evaluated.ResultsPirfenidone significantly ameliorated bleomycin-induced pulmonary fibrosis as assessed with quantitative histology and collagen measurement. Fibrocyte pool size in bleomycin-treated mice lungs was attenuated from 26.5% to 13.7% by pirfenidone on FACS analysis. This outcome was also observed in a therapeutic setting. Immunohistochemistry revealed that fibrocytes were significantly decreased by pirfenidone administration compared with those in bleomycin-treated mice (P = 0.0097). Increased chemokine (CC motif) ligand-2 (CCL2) and CCL12 production in bleomycin-treated mouse lungs was significantly attenuated by pirfenidone (P = 0.0003 and P < 0.0001, respectively). Pirfenidone also attenuated macrophage counts stimulated by bleomycin in BAL fluid. Fibrocyte migration toward CCL2 and chemokine (CC motif) receptor-2 expression on fibrocytes was significantly inhibited by pirfenidone in vitro.ConclusionsPirfenidone attenuated the fibrocyte pool size in bleomycin-treated mouse lungs via attenuation of CCL2 and CCL12 production in vivo, and fibrocyte migration was inhibited by pirfenidone in vitro. Fibrocyte inhibition is considered a mechanism of anti-fibrotic action of pirfenidone.

Dataset Information

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

Publications

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

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