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A Comparison of Multiple Histotechnical and Morphological Methods for Measuring Hepatic Lipids in NASH Models
Author(s) -
Opsahl Alan C.,
Crowell Danielle M.,
Zhan Yutian,
Geoly Frank J.,
O'Neil Shawn P.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.546.2
Subject(s) - steatosis , pathology , steatohepatitis , cirrhosis , lipid droplet , fibrosis , oil red o , fatty liver , inflammation , sirius red , sinusoid , immunohistochemistry , staining , biology , medicine , disease , mesenchymal stem cell , adipogenesis
Nonalcoholic steatohepatitis (NASH) is a progressive syndrome in which fat (usually in the form of triglycerides) accumulates in the liver (steatosis), accompanied by inflammation and fibrosis in non‐alcoholic patients. Lipid accumulation occurs in intracellular vesicles in hepatocytes, typically producing a macrovesicular histologic pattern, and can lead to ballooning degeneration. The injury to hepatocytes can induce inflammation with subsequent fibrosis, and may ultimately lead to cirrhosis and/or hepatocellular carcinoma. A number of rodent models of NASH have been developed and quantification of hepatic lipids in liver sections is a common study endpoint due to the significant role that lipid accumulation plays in NASH pathogenesis. Several histotechnical and morphological methods for measuring hepatic lipid content have been developed. In this study, we used digital image analysis to compare three histological methods for measuring relative lipid burden in liver sections: 1) Oil Red O (ORO) histochemical stain, 2) liver vacuole area by morphometry (digital pathology), and 3) adipophilin expression by immunohistochemistry (IHC). Thirty C57 BL6/J mice were fed a high fat diet for 16 weeks to induce steatosis, followed by 7 weeks of high fat diet feeding in conjunction with daily oral dosing with vehicle or low or high doses of a proprietary compound. After the 23 week study period, livers were collected at necropsy and fixed in formalin before routine processing to paraffin (for IHC or morphology), or cryopreservation and snap‐freezing in OCT compound for cryosectioning (for ORO staining). Serial sections from the left lateral lobe of the liver were prepared from paraffin blocks for hematoxylin and eosin staining (vacuole morphometry) or IHC (for adipophilin), while the ORO stain was performed on cryosections from OCT blocks. Image analysis was performed to measure the area percent of the liver section that was occupied by ORO stain, DAB chromogen (adipophilin IHC) or lipid vacuoles, using Visiopharm image analysis software, and a non‐parametric statistical analysis was performed on the results. There were no significant differences among vehicle, low dose or high dose groups measured using any of the three endpoints for lipid quantification (Kruskal‐Wallis ANOVA); however, the relative patterns of median area percent for the three endpoints were identical among assays (median area percent for low dose compound was highest, and median area percent for high dose compound was lowest), suggesting that the three assays performed similarly, and that any of these assays is adequate for histotechnical measurement of hepatic lipids. Support or Funding Information Pfizer This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .