Automated evaluation of liver fibrosis in thioacetamide, carbon tetrachloride, and bile duct ligation rodent models using second-harmonic generation/two-photon excited fluorescence microscopy

Abstract

Animal models provide a useful platform for developing and testing new drugs to treat liver fibrosis. Accordingly, we developed a novel automated system to evaluate liver fibrosis in rodent models. This system uses second-harmonic generation (SHG)/two-photon excited fluorescence (TPEF) microscopy to assess a total of four mouse and rat models, using chemical treatment with either thioacetamide (TAA) or carbon tetrachloride (CCl), and a surgical method, bile duct ligation (BDL). The results obtained by the new technique were compared with that using Ishak fibrosis scores and two currently used quantitative methods for determining liver fibrosis: the collagen proportionate area (CPA) and measurement of hydroxyproline (HYP) content. We show that 11 shared morphological parameters faithfully recapitulate Ishak fibrosis scores in the models, with high area under the receiver operating characteristic (ROC) curve (AUC) performance. The AUC values of 11 shared parameters were greater than that of the CPA (TAA: 0.758-0.922 vs 0.752-0.908; BDL: 0.874-0.989 vs 0.678-0.966) in the TAA mice and BDL rat models and similar to that of the CPA in the TAA rat and CCl mouse models. Similarly, based on the trends in these parameters at different time points, 9, 10, 7, and 2 model-specific parameters were selected for the TAA rats, TAA mice, CCl mice, and BDL rats, respectively. These parameters identified differences among the time points in the four models, with high AUC accuracy, and the corresponding AUC values of these parameters were greater compared with those of the CPA in the TAA rat and mouse models (rats: 0.769-0.894 vs 0.64-0.799; mice: 0.87-0.93 vs 0.739-0.836) and similar to those of the CPA in the CCl mouse and BDL rat models. Similarly, the AUC values of 11 shared parameters and model-specific parameters were greater than those of HYP in the TAA rats, TAA mice, and CCl mouse models and were similar to those of HYP in the BDL rat models. The automated evaluation system, combined with 11 shared parameters and model-specific parameters, could specifically, accurately, and quantitatively stage liver fibrosis in animal models.

Serum and Hepatic Autofluorescence as a Real-Time Diagnostic Tool for Early Cholestasis Assessment

“…Hence, our results are consistent with the well-recognized ability of BDL to induce fibrosis [ 13 , 35 ], which is preceded by changes in the extracellular matrix, as we documented in 3-day BDL rats by matrix metalloproteinases activation [ 36 ]. The ability of AF to sense the deposition of the fibrillary precursors of collagen earlier than the detection of fibrosis by more conventional procedures [ 14 ] is also corroborated by our previous findings on the in vivo detection of the increase in the protein AF before collagen detection by conventional Sirius red staining in a methyl choline deficient (MCD) diet model of a fatty liver with oxidative stress [ 37 ].…”

“…For this reason, BDL has been recently used as a model of liver fibrosis to validate an automated system to improve the staging and monitoring of collagen deposition as a diagnostic support in experimental pharmacology. This system is based on the intrinsic optical properties of collagen, consisting of AF or anisotropy with the emission of light by a second-harmonic generation, to detect fibrosis in a label-free manner [ 14 ]. However, the various and complex bio-metabolic features of the liver involve additional, different endogenous biomolecules, able to fluoresce in the UV–visible interval when excited with light at a suitable wavelength.…”

INT-767 improves histopathological features in a diet-inducedob/obmouse model of biopsy-confirmed non-alcoholic steatohepatitis

“…To gain further insight into INT-767-induced histological improvements in ob/ob -NASH mice, morphometric features of fibrosis were measured using SHG/2-PE microscopy[ 22 ]. SHG-2PE is a label-free sensitive imaging method for the detection of tissue structural dissymmetry and for imaging and quantitative assessment of collagen fiber specifically with non-centrosymmetric structures like collagen 1 and 3 which are key contributors for hepatic fibrosis[ 30 ].…”

“…In addition, deparaffinized and unstained tissue sections of similar thickness where imaged by second harmonic generation (SHG, label free, specific to collagen 1 and 3, in transmission at 780 nm) and concurrently by 2-photon fluorescence excitation (2-PE at 780 nm, collected at 550 nm ± 88 nm to delineate the tissue structure). Both optical methods were performed on a mid-throughput, fully automated, nonlinear optical imaging system at 20 X objective and 0.39 μm resolution (Genesis 200 ® , HistoIndex, Singapore), as described previously[ 22 ]. Fat droplets appear as circular dark objects (no intrinsic fluorescence) and can thus be segregated from the parenchyma and quantified (droplet count, area, eccentricity, and size), Collagen fiber data (area, density, reticulation index) were expressed relative (%) to corresponding parenchymal area.…”

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“…In the research one of the important animal models on the cholestasis in the complete ligation of the simple BDL model 26 . Obstruction of bile duct and subsequent cholestasis that the concentration of bile acid is increased and toxins within the liver shown inactivation of kuffer cells and hepatic stellate 27 . Serological level of liver is classical marker of blocking cholestasis in clinical observation.…”