Clustered gigantic stellate cells emerged in liver lobules of patients with alcoholic hepatitis/fibrosis and cirrhosis: focal augmentation of retinol storage as revealed by fluorescence microscopy

Abstract

Background Current concept maintains that liver injuries cause “activation” of hepatic stellate cells, the only cell type that normally store vitamin A. If continued, “activated” cells then lose this prime function, resulting in retinoid loss, and come to produce and secrete excessive collagens and extracellular matrix, while transforming into transitional cells and myofibroblast-like cells whose fibrogenicity accounts for the consequent fibrosis and cirrhosis. Materials and Methods Livers of nonalcoholic (n=2) and alcoholic (n=4) individuals obtained with ethical clearance from forensic autopsy were examined of vitamin A contents by 330-385 nm-fluorescence microscopy of 50-µm frozen sections; the same sections were subsequently stained by a newly modified OHA staining to visualize retinol-containing lipid droplets by oil red O counterstained with hematoxylin, and fibrous bundles by aniline blue. Morphovolumetry was assessed at 12-megapixel resolution. Results In healthy liver, spotty fluorescence of vitamin A-storing cells was glaring uniformly with and even distribution throughout. In alcoholic, upon a regular starry fluorescent background, huge vitamin A droplets formed a distinct cluster of 200-800 µm range found in centrilobular, intermediate, peripheral, or traversing multiple zones. Their intense autofluorescence signals lasted more than 5-10 times longer than the surrounding cells that usually faded within 5-20 seconds. Diameters measured 12-20 µm, comparing to 2-6 µm in the normal. Lobule perimeter was sharply demarcated by a narrow zone of diminished fluorescence. In cirrhotic, general fluorescence signals emitted from vitamin A-storing cell decreased, diminished, or disappeared. Clusters still remained but markedly sparse and reduced in size, sometimes single cell; nonfluorescent fibrous bands occupied most of the field. Under light microscopy, especially in alcoholic hepatitis/fibrosis, stellate cells were easily distinguished by the deep red stain of multilocular lipid droplets containing vitamin A; in the clusters, droplets were literally gigantic. Mononuclear infiltration, interrupted limiting plate, collagenous deposit upon hepatocyte deficit, fibrosed sinusoids, and CC-PP bridging fibrosis were identified fibrosis were identified. Quantification showed large droplets of 1,000~1,800 µm3, approximately 10-200 folds of the normal. Discussion Findings are clear that ethanol-injured livers expressed giant stellate cells characterized by individually massive store of vitamin A that occurred not vastly as in hypervitaminosis A but as a focal augmentation within part of the lobule. Increased vitamin A mobilization in stellate cells has been reported in diseased livers (Bromfenmajer et al., 1966; Cameron and Neuman, 1999) and CCI4 injury (Lukita-Atmadja and Subowo, 1993; Vollmar et al., 1998). We are tempted to think that quiescent cells store/mobilize less retinol than activated cells; the latter, with sustained in sult/inflammation, become “deactivated”, and undergo phenotypic transformation at the expense of total retinoid loss. (Supported by RBD Project #4703, and Toxicology Graduate Program).