Organotypic liver culture in a fluid-air interface using slices of neonatal rat and adult human tissue - a model of fibrosis in vitro

Introduction: Fibrosis is the common end stage of most liver disease but there is no effective treatment currently available. We hypothesised that if viability of liver tissue slice culture could be improved, it should be possible to develop a model of liver fibrosis in vitro that could advance the development of antifibrotic therapy while at the same time reducing the need to use in vivo models. We have adapted a slice culture technique developed originally for organotypic culture of neural tissue to the liver.Methods: slices of neonatal rat or adult human liver, 100–400-?m thick, were cut and cultured on nitrocellulose inserts at the air/fluid interface for up to 28 days.Results: Hepatocytes expressed albumin by immunocytochemistry for up to 10 days and were viable for up to 21 days during which time new structures appeared, including cytokeratin 19 positive bile ductular structures and bands of smooth muscle actin positive stellate cells associated with new reticulin positive matrix. Smooth muscle actin expression by stellate cells could be pharmacologically inhibited by SDZ-RAD (everolimus).Discussion: In conclusion, we have successfully developed a novel model of liver culture, which may prove useful in both studies of the mechanisms of liver fibrosis and in developing therapeutic strategies

Nuclear factor-κB1 (p50) limits the inflammatory and fibrogenic responses to chronic injury

In this study we addressed the role of the nuclear factor (NF)-κB1/p50 subunit in chronic injury of the liver by determining the inflammatory and fibrotic responses of nfκb1-null mice in an experimental model that mimics chronic liver disease. Mice received repeated hepatic injuries throughout 12 weeks by intraperitoneal injection of the hepatotoxin carbon tetrachloride. In response nfκb1−/− mice developed more severe neutrophilic inflammation and fibrosis compared to nfκb1+/+ mice. This phenotype was associated with elevated hepatic expression of tumor necrosis factor (TNF)-α, which was localized to regions of the liver associated with inflammation and fibrosis. Hepatic stellate cells are important regulators of hepatic inflammatory and fibrogenic events but normally do not express TNF-α. Hepatic stellate cells derived from nfκb1−/− mice expressed TNF-α promoter activity, mRNA, and protein. By contrast the expression of other NF-κB-responsive genes (ICAM1 and interleukin-6) was similar between nfκb1−/− and nfκb1+/+ cells. We provide experimental evidence that the inappropriate expression of TNF-α by nfκb1−/− cells is because of lack of a p50-dependent histone deacetylase 1 (HDAC1)-mediated repression of TNF-α gene transcription. Taken together these data indicate that the p50 NF-κB subunit plays a critical protective role in the injured liver by limiting the expression of TNF-α and its recruitment of inflammatory cells

Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking

Background & Aims: Liver fibrosis and cirrhosis result from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs). Previously considered irreversible, we have studied a model of cirrhosis to determine the mechanisms mediating and limiting spontaneous recovery. Methods: A micronodular cirrhosis was induced in rats after 12 weeks of CCl4 intoxication. Livers were analyzed for evidence of matrix degradation, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) expression, stellate cell apoptosis, tissue transglutaminase (tTg) expression, and matrix cross-linking during spontaneous recovery of up to 366 days. Results: Over 366 days of recovery, micronodular cirrhosis underwent significant remodeling to a macronodular cirrhosis. Expression of collagen-1 and TIMP messenger RNA (mRNA) decreased significantly and active MMPs were shown in livers during remodeling of fibrosis. Resolution also was characterized by apoptosis of HSCs, predominantly at the margins of fibrotic septa. Residual septa, not remodeled at 366 days, were characterized by tTg-mediated cross-linking and relative hypocellularity. Conclusion: Recovery from comparatively advanced cirrhosis is possible and results in remodeling from a micronodular cirrhosis to a macronodular cirrhosis. We suggest resolution is limited by tTg-mediated matrix cross-linking and a failure of HSC apoptosis

Hepatic changes in the failing Fontan circulation

Background: The failing Fontan circulation is associated with hepatic impairment. The nature of this liver injury is poorly defined.Objective: To establish the gross and histological liver changes of patients with Fontan circulation relative to clinical, biochemical and haemodynamic findings.Methods: Patients were retrospectively assessed for extracardiac Fontan conversion between September 2003 and June 2005, according to an established clinical protocol. Twelve patients, mean age 24.6 (range 15.8–43.4) years were identified. The mean duration since the initial Fontan procedure was 14.1 (range 6.9–26.4) years.Results: Zonal enhancement of the liver (4/12) on CT was more common in patients with lower hepatic vein pressures (p = 0.007), and in those with absent cardiac cirrhosis on histological examination (p = 0.033). Gastro-oesophageal varices (4/12) were more common in patients with higher hepatic vein pressure (21 (6.3) vs 12.2 (2.2) mm Hg, p = 0.013) and associated with more advanced cirrhosis (p = 0.037). The extent of cirrhosis (7/12) was positively correlated with the hepatic vein pressure (r = 0.83, p = 0.003). A significant positive correlation was found between the Fontan duration and the degree of hepatic fibrosis (r = 0.75, p = 0.013), as well as presence of broad scars (r = 0.71, p = 0.021). Protein-losing enteropathy (5/12) occurred more frequently in patients with longer Fontan duration (11.7 (3.2) vs 17.9 (6.1) years, p = 0.038).Conclusions: Liver injury, which can be extensive in this patient group, is related to Fontan duration and hepatic vein pressures. CT scan assists non-invasive assessment. Cardiac cirrhosis with the risk of developing gastro-oesophageal varices and regenerative liver nodules, a precursor to hepatocellular carcinoma, is common in this patient group