Current approaches and future role of high content imaging in safety sciences and drug discovery

High content imaging combines automated microscopy with image analysis approaches to simultaneously quantify multiple phenotypic and/or functional parameters in biological systems. The technology has become an important tool in the fields of safety sciences and drug discovery, because it can be used for mode-of-action identification, determination of hazard potency and the discovery of toxicity targets and biomarkers. In contrast to conventional biochemical endpoints, high content imaging provides insight into the spatial distribution and dynamics of responses in biological systems. This allows the identification of signaling pathways underlying cell defense, adaptation, toxicity and death. Therefore high content imaging is considered a promising technology to address the challenges for the Toxicity testing in the 21st century approach. Currently high content imaging technologies are frequently applied in academia for mechanistic toxicity studies and in pharmaceutical industry for the ranking and selection of lead drug compounds or to identify/confirm mechanisms underlying effects observed in vivo. A recent workshop gathered scientists working on high content imaging in academia, pharmaceutical industry and regulatory bodies with the objective to compile the state-of-the-art of the technology in the different institutions. They defined technical and methodological gaps, addressed the need for quality control, suggested control compounds and acceptance criteria, highlighted cell sources and new readouts and discussed future requirements for regulatory implementation. This review summarizes the discussion, proposed solutions and recommendations of the specialists contributing to the workshop.JRC.I.5-Systems Toxicolog

Quantification of Pulmonary Fibrosis in a Bleomycin Mouse Model Using Automated Histological Image Analysis.

Current literature on pulmonary fibrosis induced in animal models highlights the need of an accurate, reliable and reproducible histological quantitative analysis. One of the major limits of histological scoring concerns the fact that it is observer-dependent and consequently subject to variability, which may preclude comparative studies between different laboratories. To achieve a reliable and observer-independent quantification of lung fibrosis we developed an automated software histological image analysis performed from digital image of entire lung sections. This automated analysis was compared to standard evaluation methods with regard to its validation as an end-point measure of fibrosis. Lung fibrosis was induced in mice by intratracheal administration of bleomycin (BLM) at 0.25, 0.5, 0.75 and 1 mg/kg. A detailed characterization of BLM-induced fibrosis was performed 14 days after BLM administration using lung function testing, micro-computed tomography and Ashcroft scoring analysis. Quantification of fibrosis by automated analysis was assessed based on pulmonary tissue density measured from thousands of micro-tiles processed from digital images of entire lung sections. Prior to analysis, large bronchi and vessels were manually excluded from the original images. Measurement of fibrosis has been expressed by two indexes: the mean pulmonary tissue density and the high pulmonary tissue density frequency. We showed that tissue density indexes gave access to a very accurate and reliable quantification of morphological changes induced by BLM even for the lowest concentration used (0.25 mg/kg). A reconstructed 2D-image of the entire lung section at high resolution (3.6 μm/pixel) has been performed from tissue density values allowing the visualization of their distribution throughout fibrotic and non-fibrotic regions. A significant correlation (p<0.0001) was found between automated analysis and the above standard evaluation methods. This correlation establishes automated analysis as a novel end-point measure of BLM-induced lung fibrosis in mice, which will be very valuable for future preclinical drug explorations

Distal lung epithelial progenitor cell function declines with age.

Tissue stem cell exhaustion is a key hallmark of aging, and in this study, we characterised its manifestation in the distal lung. We compared the lungs of 3- and 22-month old mice. We examined the gross morphological changes in these lungs, the density and function of epithelial progenitor populations and the epithelial gene expression profile. Bronchioles became smaller in their cross-sectional area and diameter. Using long-term EdU incorporation analysis and immunohistochemistry, we found that bronchiolar cell density remained stable with aging, but inferred rates of bronchiolar club progenitor cell self-renewal and differentiation were reduced, indicative of an overall slowdown in cellular turnover. Alveolar Type II progenitor cell density and self-renewal were maintained per unit tissue area with aging, but rates of inferred differentiation into Type I cells, and indeed overall density of Type I cells was reduced. Microarray analysis revealed age-related changes in multiple genes, including some with roles in proliferation and differentiation, and in IGF and TGFβ signalling pathways. By characterising how lung stem cell dynamics change with aging, this study will elucidate how they contribute to age-related loss of pulmonary function, and pathogenesis of common age-related pulmonary diseases

Automated computerized image analysis for the user-independent evaluation of disease severity in preclinical models of NAFLD/NASH.

Pathologists use a semiquantitative scoring system (NAS or SAF score) to facilitate the reporting of disease severity and evolution. Similar scores are applied for the same purposes in rodents. Histological scores have inherent inter- and intra-observer variability and yield discrete and not continuous values. Here we performed an automatic numerical quantification of NASH features on liver sections in common preclinical NAFLD/NASH models. High-fat diet-fed foz/foz mice (Foz HF) or wild-type mice (WT HF) known to develop progressive NASH or an uncomplicated steatosis, respectively, and C57Bl6 mice fed a choline-deficient high-fat diet (CDAA) to induce steatohepatitis were analyzed at various time points. Automated software image analysis of steatosis, inflammation, and fibrosis was performed on digital images from entire liver sections. Data obtained were compared with the NAS score, biochemical quantification, and gene expression. As histologically assessed, WT HF mice had normal liver up to week 34 when they harbor mild steatosis with if any, little inflammation. Foz HF mice exhibited grade 2 steatosis as early as week 4, grade 3 steatosis at week 12 up to week 34; inflammation and ballooning increased gradually with time. Automated measurement of steatosis (macrovesicular steatosis area) revealed a strong correlation with steatosis scores (r = 0.89), micro-CT liver density, liver lipid content (r = 0.89), and gene expression of CD36 (r = 0.87). Automatic assessment of the number of F4/80-immunolabelled crown-like structures strongly correlated with conventional inflammatory scores (r = 0.79). In Foz HF mice, collagen deposition, evident at week 20 and progressing at week 34, was automatically quantified on picrosirius red-stained entire liver sections. The automated procedure also faithfully captured and quantitated macrovesicular steatosis, mixed inflammation, and pericellular fibrosis in CDAA-induced steatohepatitis. In conclusion, the automatic numerical analysis represents a promising quantitative method to rapidly monitor NAFLD activity with high-throughput in large preclinical studies and for accurate monitoring of disease evolution

Representative images of original and 2D-reconstructed lung sections for the quantification and the mapping of pulmonary tissue density by means of digital automatic analysis.

<p>Masson trichrome-stained images (A, E) and their respective 2D-reconstructed images (C, G) correspond to a lung section from saline control (A, C) and BLM-treated (0.75 mg/kg) lungs (E, G). Panels B, D, F, H show details from their original stained (B, F) and 2D-reconstructed (D, H) images at higher magnification. Lung tissue density was determined from thousands of micro-tiles crisscrossing entire lung sections. For mapping thousands tissue density values throughout lung section density values were graded in 20 classes of increasing values (I) and pseudocolours were assigned from blue (low density values) to yellow (high density values) according to their classification. Note that high density values (yellow) were restricted in alveolar parenchyma of BLM-treated lung (G, H) and located in fibrotic lesions evidenced in the respective original stained image (E, F). The frequency of tissue density (I) was determined from the classification of the whole unitary density values obtained in each lung section (A, E). HDFm index corresponds to the sum of the frequencies of the highest tissue density (classes 12 to 20) expressed in fibrotic foci. The mean tissue density (Dm) (J) was evaluated for each lung section from thousands of micro-tiles. Scale bars: 1 mm (A, C, E, G), 100 μm (B, D, F, H).</p

Representative micro-CT images of lung sections from saline control and bleomycin-treated mice.

<p>Transverse (top row) and corresponding coronal (bottom row) micro-CT images acquired at 14 days after saline (control) (A, B) and bleomycin administration at the concentration of 0.25 mg/kg (C, D), 0.50 mg/kg (E, F), 0.75 mg/kg (G, H) and 1mg/kg (I, J). Total lung volume is given as mean ± s.e.m. (n = 12/dosing group). **p<0.01, ***p<0.001 was considered statistically significant in comparison to the non bleomycin-treated control group.</p

Correlation of tissue density (Dm) and high frequency density (HDFm) indexes with Ashcroft score, micro-CT and lung function measurements in the bleomycin mice model.

<p>The agreement of the Dm and HDFm measurements with those obtained with Ashcroft scoring (A, B), micro-CT analysis (C, D), dynamic lung compliance (Cdyn) (E, F) and force vital capacity (FVC) (G, H) was evaluated using linear regression analysis. Data correspond to mean values per animal of saline control (n = 6) and BLM-treated (0.25, 0.50, 0.75, 1 mg/kg) (n = 12/dosing group) mice. r = Spearman correlation coefficient.</p

Distribution of tissue density frequency and determination of high tissue density frequency (HDFm) in saline control and bleomycin (BLM)-treated lungs.

<p>Lung tissue density was determined from several thousands of micro-tiles covering the whole lung sections of saline control (n = 6) and BLM-treated (n = 12/dosing group) mice. A: Tissue density values were graded in 20 classes of increasing values (mean ± s.e.m) and their frequency per class was expressed in percent (compared to the total number of density values). B: BLM administration induced a dose-dependent increase of HDFm which corresponded to the sum of the percentage of tissue densities from class 12 to 20. ****p<0.0001 was considered statistically significant in comparison to the non BLM-treated control group.</p

Representative original images of Masson trichrome stained lung sections and their corresponding automated 2D-reconstructed images according to their respective lung tissue density values.

<p>Original images (A, C, E, G, I) correspond to representative Masson trichrome stained lung sections from saline control (A) and BLM-treated lungs at 0.25 mg/kg (C), 0.50 mg/kg (E), 0.75 mg/kg (G) and 1 mg/kg (I). Reconstructed-2D lung sections of their respective original images were obtained by grading their tissue density values, obtained from thousands micro-tiles, in 20 classes of increasing values. Pseudocolours were then assigned to tissue densities according to their class from light blue (low density values) to yellow (high density values) (K). For each original image of saline control and BLM-treated lungs the value of their Ascroft score (mean ± s.e.m.) was indicated. Regarding their corresponding 2D-reconstructed images the value of their Dm (mean ± s.e.m.) and HDFm was indicated, respectively. Note that high density values visualized in 2D-reconstructed images were focalized in fibrotic regions evidenced in their corresponding Masson trichrome stained lung sections. Scale bars: 1 mm.</p