FIGURE 2.

The initiation of NASH disease depends on resident liver macrophages. A, C57BL/6 mice fed CT or MCD diets were treated either with two injections of control liposomes (CT Lipo.) or clodronate-containing liposomes (clod. Lipo.). H&E-stained (a, d, g, and j) and Oil-Red O-stained (b, e, h, and k) tissue sections were examined under a bright field microscope at ×200 magnification. Liver sections were also stained with anti-F4/80 (green), anti-CD31 (red), and DAPI (blue), and examined under a apotome fluorescent microscope at ×200 magnification (c, f, i, and l). Arrows and arrowheads indicate ballooning hepatocytes and lipid inclusion, respectively. B, serum ALT was measured to assess liver injury. C, Oil-Red O staining revealed lipid accumulation in hepatocytes. Area of Oil-Red O-stained vesicles (μm²) was quantified using Zeiss axiovision software. Counting was done on 2–3 different views on each mouse/treatment group. D, macrophage depletion was assessed by the loss of AF488 anti-F4/80 fluorescence. The ratio of green pixel (AF488 anti-F4/80) was normalized to DAPI pixel (cell nucleus) on each tissue section and was measured by NIH ImageJ 64. *, p < 0.05 and ***, p < 0.0005 as determined by two-tailed t test.