Hepatocellular ballooning in NASH - PubMed (original) (raw)

doi: 10.1016/j.jhep.2010.04.031. Epub 2010 Jun 25.

Yoshihiro Ikura, Daniela Dias, Kosuke Isomoto, Akito Yabu, Christopher Moskaluk, Patcharin Pramoonjago, Winsor Simmons, Harriet Scruggs, Nicholas Rosenbaum, Timothy Wilkinson, Patsy Toms, Curtis K Argo, Abdullah M S Al-Osaimi, Jan A Redick

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Hepatocellular ballooning in NASH

Stephen Caldwell et al. J Hepatol. 2010 Oct.

Abstract

Background & aims: Hepatocellular ballooning is a key finding in nonalcoholic steatohepatitis (NASH). It is conventionally defined by hemotoxylin and eosin (H&E) staining showing enlarged cells with rarefied cytoplasm and recently by changes in the cytoskeleton. Fat droplets are emerging as important organelles in cell metabolism. To address a possible relation between fat droplets and ballooning, we studied fat staining, H&E, and keratin 18 staining in human NASH.

Methods: Sequential staining and high resolution imaging were used to study freshly prepared cryo-sections from 10 patients with histologically confirmed steatohepatitis using oil red O for fat droplet identification, H&E to identify ballooning, and anti-K18 to confirm cytoskeletal changes. High resolution images were captured at each stage using the Aperio Scanscope. To provide ultrastructural correlation, glutaraldehyde-fixed specimens were studied using transmission electron microscopy (TEM) with serial sectioning for localization of ballooned cells by light microscopy and TEM in identical specimens.

Results: Serial staining consistently demonstrated that hepatocellular ballooning is associated with fat droplet accumulation evident by oil red O positivity and depletion of cytoplasmic keratin 18 with K-18 positive Mallory-Denk bodies (MDB). TEM confirmed the association between osmium stained fat droplets, MDB formation, and cellular enlargement and suggested droplet-associated dilation of the endoplasmic reticulum.

Conclusions: These results indicate a relationship between cellular ballooning, fat droplet accumulation, and cytoskeletal injury in NASH. We speculate that injury to multiple, organelles including fat droplets and endoplasmic reticulum, contribute to this characteristic finding.

Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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Figures

Fig. 1

Fig. 1. Patient series 1

(A) H&E (B) oil red O (C) anti-K 18. Note oil red O positive droplets and lack of CK-18 staining in two ballooned hepatocytes (solid arrows) compared to anti-K 18 positive neighboring cells. Open arrow indicates landmark red blood cell in central vein. (300X using the Aperio, 0.5 μm per pixel). Indicated magnifications are derived from the scale of the Aperio scanner (0.5 μm per pixel).

Fig. 2

Fig. 2. Patient series 2

(A) H&E, (B) oil red O, (C) anti-K 18. Solid arrow indicates a ballooned hepatocyte which contains numerous oil red O positive fat droplets (B) and diminished expression of keratin 18 (C) compared to neighboring cells. Note anti-K 18 immunostaining of Mallory-Denk body (arrowhead) in C. (300X using the Aperio, 0.5 μm per pixel). Indicated magnifications are derived from the scale of the Aperio scanner (0.5 μm per pixel).

Fig. 3

Fig. 3. Patient series 3

(A) H&E, (B) oil red O, (C) anti-K 18. Solid arrow indicates a ballooned hepatocyte. The arrowhead indicates a landmark red blood cell in a central vein. Note the dense fat droplet staining (open arrow) within a necrotic focus suggesting release of these structures with cell death. Indicated magnifications are derived from the scale of the Aperio scanner (0.5 μm per pixel).

Fig .4

Fig .4. Serial localization of ballooned cell for correlative light microscopy with toluidine blue and ultrastructure by TEM

(A) Toluidine blue staining of an osmium fixed specimen showing abundance of osmiophilic mediovesicular fat droplets (400X). Open arrow indicates a landmark red blood cell within the central vein. Asterisk indicates Mallory-Denk Body confirmed in the serially cut TEM shown in B. (B) Serially cut sample to examine by TEM (1000X) the same cell shown in A. Open arrow indicates landmark red blood cell within the central vein. TEM shows relationships between cellular enlargement, accumulation of fat droplets and Mallory-Denk body (asterisk). Inset (15000X) confirms the filamentous Mallory-Denk body (MDB). The solid arrow indicates landmark heterochromatin within the nucleus in reference to the inset.

Fig. 5

Fig. 5. Representative TEM series

(A-F) Ultrastructure of a ballooned hepatocyte showing the relationships between fat droplets, dilated endoplasmic reticulum and cytoskeletal injury evident as a perinuclear Mallory-Denk body. (up to 60,000X) Abbreviations: L-FD; large fat droplet, FD; fat droplet, Si; sinusoidal space, SoD; space of Disse, C; extracellular collagen bundle, N; nucleus, Nu; nucleolus, M; mitochondria, MDB; Mallory-Denk Body, dER; dilated endoplasmic reticulum. Arrows indicate continuity of the fat droplets with the endoplasmic reticulum (F).

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