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

Abstract 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.

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Longitudinal micro-computed tomography-derived biomarkers quantify non-resolving lung fibrosis in a silicosis mouse model

Scientific Reports ◽

10.1038/s41598-020-73056-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kaat Dekoster ◽

Tatjana Decaesteker ◽

Nathalie Berghen ◽

Sofie Van den Broucke ◽

Anne-Charlotte Jonckheere ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Lung Volume ◽

Lung Fibrosis ◽

Disease Onset ◽

Preclinical Studies ◽

Micro Computed Tomography ◽

Cross Sectional ◽

Total Lung Volume ◽

Imaging Results

Abstract In spite of many compounds identified as antifibrotic in preclinical studies, pulmonary fibrosis remains a life-threatening condition for which highly effective treatment is still lacking. Towards improving the success-rate of bench-to-bedside translation, we investigated in vivo µCT-derived biomarkers to repeatedly quantify experimental silica-induced pulmonary fibrosis and assessed clinically relevant readouts up to several months after silicosis induction. Mice were oropharyngeally instilled with crystalline silica or saline and longitudinally monitored with respiratory-gated-high-resolution µCT to evaluate disease onset and progress using scan-derived biomarkers. At weeks 1, 5, 9 and 15, we assessed lung function, inflammation and fibrosis in subsets of mice in a cross-sectional manner. Silica-instillation increased the non-aerated lung volume, corresponding to onset and progression of inflammatory and fibrotic processes not resolving with time. Moreover, total lung volume progressively increased with silicosis. The volume of healthy, aerated lung first dropped then increased, corresponding to an acute inflammatory response followed by recovery into lower elevated aerated lung volume. Imaging results were confirmed by a significantly decreased Tiffeneau index, increased neutrophilic inflammation, increased IL-13, MCP-1, MIP-2 and TNF-α concentration in bronchoalveolar lavage fluid, increased collagen content and fibrotic nodules. µCT-derived biomarkers enable longitudinal evaluation of early onset inflammation and non-resolving pulmonary fibrosis as well as lung volumes in a sensitive and non-invasive manner. This approach and model of non-resolving lung fibrosis provides quantitative assessment of disease progression and stabilization over weeks and months, essential towards evaluation of fibrotic disease burden and antifibrotic therapy evaluation in preclinical studies.

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Intraosteal Behavior of Porous Scaffolds: The mCT Raw-Data Analysis as a Tool for Better Understanding

Symmetry ◽

10.3390/sym11040532 ◽

2019 ◽

Vol 11 (4) ◽

pp. 532 ◽

Cited By ~ 1

Author(s):

Parrilla-Almansa ◽

González-Bermúdez ◽

Sánchez-Sánchez ◽

Meseguer-Olmo ◽

Martínez-Cáceres ◽

...

Keyword(s):

Computed Tomography ◽

Mathematical Analysis ◽

Demineralized Bone Matrix ◽

Bone Matrix ◽

Hounsfield Unit ◽

In Vivo Studies ◽

Porous Scaffolds ◽

Micro Computed Tomography ◽

Radiological Analysis

The aim of the study is to determine the existing correlation between high-resolution 3D imaging technique obtained through Micro Computed Tomography (mCT) and histological-histomorphometric images to determine in vivo bone osteogenic behavior of bioceramic scaffolds. A Ca-Si-P scaffold ceramic doped and non-doped (control) with a natural demineralized bone matrix (DBM) were implanted in rabbit tibias for 1, 3, and 5 months. A progressive disorganization and disintegration of scaffolds and bone neoformation occurs, from the periphery to the center of the implants, without any differences between histomorphometric and radiological analysis. However, significant differences (p < 0.05) between DMB-doped and non-doped materials where only detected through mathematical analysis of mCT. In this way, average attenuation coefficient for DMB-doped decreased from 0.99 ± 0.23 Hounsfield Unit (HU) (3 months) to 0.86 ± 0.32 HU (5 months). Average values for non-doped decreased from 0.86 ± 0.25 HU (3 months) to 0.66 ± 0.33 HU. Combination of radiological analysis and mathematical mCT seems to provide an adequate in vivo analysis of bone-implanted biomaterials after surgery, obtaining similar results to the one provided by histomorphometric analysis. Mathematical analysis of Computed Tomography (CT) would allow the conducting of long-term duration in vivo studies, without the need for animal sacrifice, and the subsequent reduction in variability.

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Radiographic and Micro—Computed Tomographic Imaging of Lipopolysaccharide-Mediated Bone Resorption

Annals of Otology Rhinology & Laryngology ◽

10.1177/000348940911800512 ◽

2009 ◽

Vol 118 (5) ◽

pp. 391-396 ◽

Cited By ~ 5

Author(s):

Robert Nason ◽

Dong H. Lee ◽

Jae Y. Jung ◽

Richard A. Chole

Keyword(s):

Bone Resorption ◽

Chronic Otitis Media ◽

Plain Radiography ◽

Micro Ct ◽

Micro Computed Tomography ◽

Gram Negative ◽

Successful Model ◽

Computed Tomographic ◽

Myeloid Differentiation Factor

Objectives: Chronic otitis media and cholesteatomas cause hearing loss as a result of bony erosion. This bone resorption is known to be more aggressive when cholesteatomas become infected. The most common organism isolated from both diseases is the gram-negative bacterium Pseudomonas aeruginosa. Lipopolysaccharide (LPS), a major virulence factor found in the gram-negative bacterial cell wall, is well known to incite inflammatory bone resorption. The mechanisms underlying this process, however, are poorly understood. In this study, we developed a mouse model of calvarial osteolysis in which resorption was reliably imaged by plain radiography and micro–computed tomography (micro-CT). Methods: A murine calvarial model was developed to study bone resorption induced by P aeruginosa LPS. Calvariae from wild-type and knockout mice used in this model were imaged by plain radiography and micro-CT. Results: A high degree of correlation between plain radiography and micro-CT was identified (R2 = 0.8554). Furthermore, maximal LPS-induced bone resorption required functioning toll-like receptor (TLR) 2, TLR4, and myeloid differentiation factor 88 (MyD88). Conclusions: We have developed a successful model of inflammatory osteolysis in which plain radiography can reliably delineate induced bone resorption. In vivo, we have shown that P aeruginosa LPS signals via TLR2, as well as TLR4 through MyD88.

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Characterization of Rotating Gantry Micro-CT Configuration for theIn VivoEvaluation of Murine Trabecular Bone

Microscopy and Microanalysis ◽

10.1017/s1431927613001396 ◽

2013 ◽

Vol 19 (4) ◽

pp. 907-913 ◽

Cited By ~ 3

Author(s):

Luke Arentsen ◽

Susanta Hui

Keyword(s):

Trabecular Bone ◽

Signal To Noise Ratio ◽

Hounsfield Unit ◽

Rate Of Change ◽

Physical Parameters ◽

Micro Ct ◽

Detector Geometry ◽

Ct System ◽

Filter Thickness

AbstractThe objective of this study is to determine the optimal physical parameters of a rotating gantry micro-CT system to assessin vivochanges to the trabecular bone of mice. Magnification, binning, peak kilovoltage, beam filtration, and tissue thickness are examined on a commercially available micro-CT system. The X-ray source and detector geometry provides 1.3×, 1.8×, or 3.3× magnification. Binning is examined from no binning to 2 to 4. Energy is varied from 40 to 80 kVp in 10 kVp increments and filter thickness is increased from no filtration to 1.5 mmAl in 0.5 mmAl increments. Mice are imaged at different magnifications and binning combinations to evaluate changes to image quality and microstructure estimation. Increasing magnification from 1.3× to 3.3× and lowering binning from 4 to 1 varies the spatial resolution from 2.5 to 11.8 lp/mm. Increasing the beam energy or filtration thickness decreases Hounsfield unit (HU) estimation, with a maximum rate of change being −286 HU/kVp for 80 kVp. Images for murine trabecular bone are blurred at effective pixel sizes above 60 μm. By comparing resolution, signal-to-noise ratio, and radiation dose, we find that a 3.3× magnification, binning of 2.80 kVp beam with a 0.5 mmAl filter comprises the optimal parameters to evaluate murine trabecular bone for this rotating gantry micro-CT.

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Inhibition of lncRNA PFRL prevents pulmonary fibrosis by disrupting the miR-26a/smad2 loop

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00434.2017 ◽

2018 ◽

Vol 315 (4) ◽

pp. L563-L575 ◽

Cited By ~ 11

Author(s):

Hua Jiang ◽

Yingzhun Chen ◽

Tong Yu ◽

Xiaoguang Zhao ◽

Huitong Shan ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Lung Fibrosis ◽

Feedback Loop ◽

Transforming Growth Factor ◽

Molecular Study ◽

Pulmonary Fibroblasts ◽

Proliferation And Differentiation ◽

Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease with increasing mortality and poor prognosis. The current understanding of the role of long noncoding RNAs (lncRNAs) in IPF remains limited. In the present study, we identified a lncRNA NONMMUT022554, designated pulmonary fibrosis-regulatory lncRNA (PFRL), with unknown functions and found that its levels were increased in fibrotic lung tissues of mice and pulmonary fibroblasts exposed to transforming growth factor (TGF)-β1. Furthermore, we found that enforced expression of PFRL induced fibroblast activation and collagen deposition, which could be mitigated by the overexpression of microRNA (miR)-26a. By contrast, the inhibition of PFRL could markedly alleviate the TGF-β1-induced upregulation of fibrotic markers and attenuate fibroblast proliferation and differentiation by regulating miR-26a. Meanwhile, our study confirmed that PFRL inhibited the expression and activity of miR-26a, which has been identified as an antifibrotic miRNA in our previous study. Interestingly, our molecular study further confirmed that Smad2 transcriptionally inhibits the expression of miR-26a and that the miR-26a/Smad2 feedback loop mediates the profibrotic effects of PFRL in lung fibrosis. More importantly, knockdown of PFRL ablated bleomycin-induced pulmonary fibrosis in vivo. Taken together, our findings indicate that lncRNA PFRL contributes to the progression of lung fibrosis by modulating the reciprocal repression between miR-26a and Smad2 and that this lncRNA may be a therapeutic target for IPF.

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Longitudinal in vivo micro-CT-based approach allows spatio-temporal characterization of fracture healing patterns and assessment of biomaterials in mouse femur defect models

10.1101/2020.10.02.324061 ◽

2020 ◽

Author(s):

Esther Wehrle ◽

Duncan C Tourolle né Betts ◽

Gisela A Kuhn ◽

Erica Floreani ◽

Malavika H Nambiar ◽

...

Keyword(s):

Bone Regeneration ◽

Healing Process ◽

Micro Ct ◽

Micro Computed Tomography ◽

Collagen Scaffolds ◽

Non Union ◽

Spatio Temporal ◽

Large Bone

AbstractThorough preclinical evaluation of novel biomaterials for treatment of large bone defects is essential prior to clinical application. Using in vivo micro-computed tomography (micro-CT) and mouse femoral defect models with different defect sizes, we were able to detect spatio-temporal healing patterns indicative of physiological and impaired healing in three defect sub-volumes and the adjacent cortex. The time-lapsed in vivo micro-CT-based approach was then applied to evaluate the bone regeneration potential of biomaterials using collagen and BMP-2 as test materials. Both collagen and BMP-2 treatment led to distinct changes in bone turnover in the different healing phases. Despite increased periosteal bone formation, 87.5% of the defects treated with collagen scaffolds resulted in non-unions. Additional BMP-2 application significantly accelerated the healing process and increased the union rate to 100%. This study further shows potential of time-lapsed in vivo micro-CT for capturing spatio-temporal deviations preceding non-union formation and how this can be prevented by application of biomaterials.This study therefore supports the application of longitudinal in vivo micro-CT for discrimination of normal and disturbed healing patterns and for the spatio-temporal characterization of the bone regeneration capacity of biomaterials.

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Multimodality Approach to Characterizing the Distinctive Hallmarks of Lung Fibrosis: A Mouse Model with Bleomycin and Indocyanine Green

10.21203/rs.3.rs-615697/v1 ◽

2021 ◽

Author(s):

Andrea Grandi ◽

Erica Ferrini ◽

Roberta Ciccimarra ◽

Martina Mambrini ◽

Laura Mecozzi ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Time Course ◽

Ex Vivo ◽

Imaging Techniques ◽

Lavage Fluid ◽

Pharmacological Therapy ◽

Micro Ct ◽

Oropharyngeal Aspiration ◽

Multimodality Approach

Abstract Background.Idiopathic Pulmonary Fibrosis is a progressive disease with short life expectancy and no disease-modifying pharmacological therapy. The continuous refinement of animal models and the integration of in-vivo imaging techniques is fundamental for the selection of new antifibrotic drugs.Indocyanine Green (ICG), a fluorescent dye, was administered by oropharyngeal aspiration (OA) to mice with Bleomycin (BLM) to map the lung exposure.Methods.Female mice C57bl/6 were treated via OA with BLM+ICG or ICG. Animals were imaged at 7, 14 and 21 days either with the fluorescent system or Micro-CT. At each time point subsets of mice were sampled for ex-vivo assessment. Histological assessment of fibrosis by Ashcroft score, airspace enlargements and mean linear intercept (MLI) were evaluated at 7, 14 and 21 days. Leukocytes and cytokines were measured in bronchoalveolar lavage fluid. Results.Fluorescence imaging revealed a persistent lung signal in both groups until 21 days. In BLM+ICG group, Micro-CT detected a marked increase in hypo- and non-aerated tissues throughout the study. At later time points hyper-inflated tissue was detected. Histology revealed high Ashcroft score throughout the time-course with a prominent increase in airspace size and MLI at day 21. ICG mice had healthy lungs.Conclusions.We showed that ICG can be used as a tracer to map the distribution of BLM in lungs. However, BLM+ICG produced unexpected severe lung changes different from pure BLM model, such as emphysema-like features which progressively worsened. The multimodalities approach warranted characterization of the distinctive features of this new pulmonary fibrosis model and provided fundamentals for in-vivo translation.

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The Importance of Routine Quality Control For Reproducible Pulmonary Measurements By in Vivo Micro-CT

10.21203/rs.3.rs-1194936/v1 ◽

2022 ◽

Author(s):

Martina Mambrini ◽

Laura Mecozzi ◽

Erica Ferrini ◽

Ludovica Leo ◽

Davide Bernardi ◽

...

Keyword(s):

Disease Progression ◽

Lung Diseases ◽

Drug Efficacy ◽

Micro Ct ◽

Micro Computed Tomography ◽

Efficacy Evaluation ◽

Lung Density ◽

Before And After ◽

The Impact

Abstract 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.

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Arctigenin Suppressed Epithelial-Mesenchymal Transition Through Wnt3a/β-Catenin Pathway in PQ-Induced Pulmonary Fibrosis

Frontiers in Pharmacology ◽

10.3389/fphar.2020.584098 ◽

2020 ◽

Vol 11 ◽

Author(s):

Fei Gao ◽

Yun Zhang ◽

Zhizhou Yang ◽

Mengmeng Wang ◽

Zhiyi Zhou ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Signaling Pathway ◽

Lung Fibrosis ◽

Epithelial Mesenchymal Transition ◽

A549 Cells ◽

Underlying Mechanism ◽

Alveolar Type ◽

Mesenchymal Transition

Arctigenin (ATG), a major bioactive substance of Fructus Arctii, counters renal fibrosis; however, whether it protects against paraquat (PQ)-induced lung fibrosis remains unknown. The present study was to determine the effect of ATG on PQ-induced lung fibrosis in a mouse model and the underlying mechanism. Firstly, we found that ATG suppressed PQ-induced pulmonary fibrosis by blocking the epithelial-mesenchymal transition (EMT). ATG reduced the expressions of Vimentin and α-SMA (lung fibrosis markers) induced by PQ and restored the expressions of E-cadherin and Occludin (two epithelial markers) in vivo and in vitro. Besides, the Wnt3a/β-catenin signaling pathway was significantly activated in PQ induced pulmonary fibrosis. Further analysis showed that pretreatment of ATG profoundly abrogated PQ-induced EMT-like phenotypes and behaviors in A549 cells. The Wnt3a/β-catenin signaling pathway was repressed by ATG treatment. The overexpression of Wnt3a could weaken the therapeutic effect of ATG in A549 cells. These findings suggested that ATG could serve as a new therapeutic candidate to inhibit or even reverse EMT-like changes in alveolar type II cells during PQ-induced lung fibrosis, and unraveled that the Wnt3a/β-catenin pathway might be a mechanistic tool for ATG to control pulmonary fibrosis.

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PEG-modified gadolinium nanoparticles as contrast agents for in vivo micro-CT

Scientific Reports ◽

10.1038/s41598-021-95716-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Charmainne Cruje ◽

P. Joy Dunmore-Buyze ◽

Eric Grolman ◽

David W. Holdsworth ◽

Elizabeth R. Gillies ◽

...

Keyword(s):

Contrast Agent ◽

Contrast Agents ◽

Soft Tissues ◽

Three Dimensional ◽

Blood Pool ◽

Micro Ct ◽

Micro Computed Tomography ◽

Poly Ethylene

AbstractVascular research is largely performed in rodents with the goal of developing treatments for human disease. Micro-computed tomography (micro-CT) provides non-destructive three-dimensional imaging that can be used to study the vasculature of rodents. However, to distinguish vasculature from other soft tissues, long-circulating contrast agents are required. In this study, we demonstrated that poly(ethylene glycol) (PEG)-coated gadolinium nanoparticles can be used as a vascular contrast agent in micro-CT. The coated particles could be lyophilized and then redispersed in an aqueous solution to achieve 100 mg/mL of gadolinium. After an intravenous injection of the contrast agent into mice, micro-CT scans showed blood pool contrast enhancements of at least 200 HU for 30 min. Imaging and quantitative analysis of gadolinium in tissues showed the presence of contrast agent in clearance organs including the liver and spleen and very low amounts in other organs. In vitro cell culture experiments, subcutaneous injections, and analysis of mouse body weight suggested that the agents exhibited low toxicity. Histological analysis of tissues 5 days after injection of the contrast agent showed cytotoxicity in the spleen, but no abnormalities were observed in the liver, lungs, kidneys, and bladder.

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