Computational protocol: Antifibrotic Effects of the Dual CCR2/CCR5 Antagonist Cenicriviroc in Animal Models of Liver and Kidney Fibrosis

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Protocol publication

[…] Collagen deposition (the extent of fibrosis) was visualized in liver/kidney sections with picrosirius red staining. In the TAA model, collagen quantification was performed using computerized Life Science morphometry system (BIOQUANT, USA) on a total of 36 images per animal at 100x magnification (four picrosirius red-stained slides per animal, with nine images taken randomly per slide).In the NASH model, bright field images of picrosirius red-stained sections were captured around the central vein using a digital camera (DFC280; Leica, Germany) at 200x magnification; the ‘positive’ areas in five fields/sections were measured using ImageJ software (National Institutes of Health, USA). Perivascular areas were subtracted from the total positive areas for each field (modified fibrosis area). The NAS, a histological tool developed to assess disease severity in humans, was assessed in a blinded fashion and calculated according to Kleiner’s criteria on H&E-stained sections []. It is based on the semi-quantification of steatosis, lobular inflammation and hepatocellular ballooning.In the UUO model, ten images/depth/kidney were assessed in a blinded fashion using Axio Imager.A2 (Zeiss, USA) light microscopy (at 200x magnification to enable 60–70% sampling of renal cortical area) and quantified by a composite Collagen Volume Fraction (CVF [% total area imaged]) score expressed as the average positive stain across three anatomically distinct (200–250 μM apart) tissue sections, or depths, from the obstructed kidney. [...] Extracellular matrix protein content in tissue samples was measured by evaluating collagen type 1 and alpha-SMA protein expression in the TAA model and hydroxyproline content in the NASH and UUO models.In the TAA model, total protein was extracted from liver cells and expression levels of collagen type 1 and alpha-SMA were assessed by western blotting. Protein expression levels were normalized to the reference protein (glyceraldehyde-3-phosphate dehydrogenase [GAPDH] or calnexin).In the NASH model, frozen liver samples were subjected to an alkaline-acid hydrolysis, then centrifuged, and the supernatant collected. Hydroxyproline content was quantified against a hydroxyproline standard curve, with a BCA protein assay kit (Thermo Fisher Scientific, USA) used to normalize the calculated hydroxyproline values.In the UUO model, frozen renal cortical tissue biopsies were hydrolyzed and centrifuged, and the supernatant was analyzed by OD absorbance at 560 nM on a SpectraMax® 190 (Molecular Devices, USA). Standard curves for conversion of ODs to concentrations were generated using linear regression and sample concentrations were determined using SoftMax® Pro5 software (Molecular Devices, USA). […]

Pipeline specifications

Software tools ImageJ, SoftMax Pro
Application Microscopic phenotype analysis
Organisms Mus musculus, Rattus norvegicus, Homo sapiens
Diseases Fatty Liver, Alcoholic, Liver Cirrhosis, Peritonitis, Non-alcoholic Fatty Liver Disease
Chemicals Thioacetamide