0 good macrophages, and the pink circle indicates a lipid droplet enclosed by macrophages with out discernible mitochondria or nuclear signal. (F) Intravital imaging of lipid droplets visualized by Bodipy; the yellow arrows indicate macrophages surrounding a lipid droplet. (See also Videos S3 and S4). Scale bars: 50 (A,B,E,F) and 200 (C).Cells 2021, ten,16 ofFigure four. Cell death throughout NASH progression. (A) TUNEL and Ki67 staining in liver sections of SD- (3 week) and WD-fed mice. (B) Liver enzyme activities (ALT and AST) inside the heart blood of mice fed a SD or WD. (C) Examples of ballooning (arrows) and Mallory enk bodies (arrowhead, MDB) in H E-stained liver tissue sections. (D) Visualization of ballooning and MDB by K18 immunostaining. (E,F) Representative image of Western blot with accompanying quantification from the necroptosis marker MLKL along with the apoptosis marker cleaved caspase-3 in livers of SD- and WD-fed mice over time. (G) Cleaved caspase3 immunostaining at unique time intervals after WD feeding; LPS: lipopolysaccharide. Information in B and F are means and regular error of 4 mice per time point. : p 0.05; : p 0.01; : p 0.001 in 12-LOX Inhibitor medchemexpress comparison to SD week 3, Dunnett’s a number of comparisons (B) or unpaired t (F) tests; data of individual mice are illustrated by dots; SD: common diet program; WD: Western diet regime. Scale bars: 50 (A,G) and 10 (C,D).Collectively, long-term feeding on WD led towards the progression from basic steatosis to NASH, which was characterized by inflammatory foci, the formation of lipogranulomas, necroptotic hepatocyte death, replacement proliferation, and late in the course of illness progression hepatocyte ballooning.Cells 2021, 10,17 of3.4. Ductular Reaction (DR) and Fibrosis Progression In human NASH, continuous hepatocyte death triggers a DR [42]. To study if DR also occurred within the present model, K19 immunostaining was performed. In SD-fed mice, K19 staining was only observed inside the bile ducts adjacent to the portal veins (Figure 5A; Figure S2). Nonetheless, in WD-fed mice, a progressive DR was evident, beginning at week 12 and rising more than time up to week 48 (Figure 5A,B). Development of DR was followed by elevated activities of alkaline phosphatase in the blood (Figure 5C). Whole slide scans demonstrated that the DR developed initially (weeks 128) in the periportal region, but later progressed towards the pericentral zone (Figure S8). Even though they’re believed to arise in order to α1β1 Formulation replenish lost hepatocytes as part of a reparative method [43], the functional significance of such DR is still not clear. Therefore, to investigate their function during NASH progression, we performed intravital imaging on the livers of WD-fed mice following tail vein injection with the green-fluorescent bile acid analogue CLF. Interestingly, CLF appeared inside the lumens of bile canaliculi and DR within a couple of minutes soon after intravenous injection (Figure 5D). This observation would fit to a mechanism, where hepatocytes secrete CLF into bile canaliculi from where it reached the DR.Figure five. Development of bile-draining ductular reaction throughout NAFLD progression. (A) Immunostaining of the cholangiocyte marker K19 in liver sections of mice on SD (3 week) or WD more than time. (B) Quantification with the K19 positive region. (C) ALP levels in blood of mice on SD or WD. (D) Intravital imaging just after intravenous injection on the bile acid analogue CLF (green). Yellow arrows indicate ductular structures. Data in B and C represent mean and normal errors of 3 mice per time poin