62 - Microscopic Visualization of Lipid Droplets of Hepatic Stellate Cells: A Review

Poster Board Number: 62
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Abstract Body : Hepatic stellate cells (HSCs), the dominant fibrogenic cells residing in the space of Disse of the liver, display copious lipid droplets (LDs) that are distinct morphological markers of HSCs. In quiescent HSCs, LDs are composed of a core of predominantly retinyl esters (vitamin A) and triacylglycerols along with some cholesteryl esters and free fatty acids, all of which are surrounded by a layer of phospholipids. Embedded in the phospholipid layer are lipid-associated proteins perilipins 2, 3 and 5, which help control LD lipolysis. LDs are not just “great balls of fat;” rather, they are dynamic and metabolically active organelles, endowed with enzymes for synthesis and lipases for degradation of the lipids within LDs. Loss of LDs from HSCs is a hallmark of cell activation leading to enhanced fibrogenesis in liver injury. The ultrastructure of HSCs of humans and other mammalian species and fish have been described and reviewed. This report focuses on HSC LDs and reviews their morphology in livers of humans, baboons and rats as visualized by various techniques of microscopy. LDs are detected by blue-green autofluorescence for vitamin A at 328 nm excitation in conjunction with gold chloride or silver impregnation of liver tissue sections (25-µm thick). LDs are also readily discernable on plastic-embedded liver sections (1-µm thick) stained with toluidine blue, displaying a lightly greenish hue. Moreover, plastic sections offer a higher resolution than paraffin-embedded tissue sections do at the light microscopic level. Plastic-embedded specimens also allow for counting LDs in HSCs as well as for follow-up examination of LDs by electron microscopy. Nile red, Sudan III and oil red O stain LDs red while BODIPY 493/503 stains LDs green in frozen liver sections and in HSC cultures, as revealed by conventional light microscopy. The stains also impart fluorescence for detection by fluorescent microscopy and quantitative analysis by confocal microscopy. However, caution should be taken because these stains are non-specific neutral lipid stains. Moreover, Sudan III and oil red O staining can produce artificial fusion of LDs. Given that HSC LDs are coated with LD-associated perilipins 2, 3 and 5, immuno-cytochemical staining using specific antibodies against these perilipins provides a tool for microscopic detection of LDs in HSCs. Transmission electron microscopy remains the gold standard technique for visualization of HSC LDs. Electron microscopy distinguishes between two types of LDs. Type I LDs exhibit an electron dense matrix after fixation with osmium tetroxide. These droplets are limited by a unit membrane, generally ca. 1 µm in diameter, and are seen in association with acid phosphatase positive-lysosomes, which likely mediate digestion of LDs. In contrast, the matrix of type II LDs appears electron lucid, suggesting a different lipid composition than that of type I LDs. Also, type II LDs lack a membrane and appear to lie freely in the cytosol. In conclusion, the various techniques employed in microscopic visualization of LDs enable further elucidation of HSC LD dynamics in both healthy and injured livers.