7 Tesla MRI Liver Fat Quantification in Mice: Data Quality Assessment.

PURPOSE: The objective of this study was to evaluate the robustness of proton density fat fraction (PDFF) data determined by magnetic resonance imaging (MRI) and spectroscopy (MRS) via spatially resolved error estimation. MATERIALS AND METHODS: Using standard T2* relaxation time measurement protocols, in-vivo and ex-vivo MRI data with water and fat nominally in phase or out of phase relative to each other were acquired on a 7 T small animal scanner. Based on a total of 24 different echo times, PDFF maps were calculated in a magnitude-based approach. After identification of the decisive error-prone variables, pixel-wise error estimation was performed by simple propagation of uncertainty. The method was then used to evaluate PDFF data acquired for an explanted mouse liver and an in vivo mouse liver measurement. RESULTS: The determined error maps helped excluding measurement errors as cause of unexpected local PDFF variations in the explanted liver. For in vivo measurements, severe error maps gave rise to doubts in the acquired PDFF maps and triggered an in-depth analysis of possible causes, yielding abdominal movement or bladder filling as in vivo occurring reasons for the increased errors. CONCLUSION: The combination of pixel-wise acquisition of PDFF data and the corresponding error maps allows for a more specific, spatially resolved evaluation of the PDFF value reliability.

Limited potential of resolvin D1 in treatment of cholestatic liver fibrosis.

BACKGROUND: Several studies suggest a role for EPA- and DHA-derived pro-resolving mediators like resolvins in reversing metabolic and inflammatory disturbances seen in various chronic diseases. Here, we investigated the effects of resolvin D1 (RvD1) on bile duct ligation (BDL)-induced cholestatic liver injury. METHODS: Mice were treated daily with RvD1 or 0.1% ethanol (control) from the day of BDL until the final observation time points. Blood and liver tissue were collected 2, 5 and 14 days after BDL for different analyses. RESULTS: RvD1 treatment of mice had no impact on the extent of cholestatic liver injury upon BDL, neither in the acute phase nor in the progressive state of liver fibrosis. Although RvD1 treatment resulted in a significantly reduced activity of hepatic stellate cells as well as reduced deposition of extracellular matrix 2 days after BDL, mice were not protected from inflammation and further fibrosis progression. CONCLUSIONS: These data indicate that RvD1 has a limited therapeutic potential to treat cholestatic liver diseases, as it has no significant impact on regression of hepatic necroinflammation and fibrotic changes in bile duct-ligated mice.

Endogenously increased n-3 PUFA levels in fat-1 transgenic mice do not protect from non-alcoholic steatohepatitis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and fibrosis. Possible reasons for the NAFLD epidemic in industrialized countries are the high intake of pro-inflammatory n-6 polyunsaturated fatty acids (n-6 PUFAs) and low consumption of healthy n-3 PUFAs. Due to their anti-inflammatory properties, n-3 PUFAs may have the potential to alleviate chronic liver disease. Herein, we examined the therapeutic effect of increased n-3 PUFA tissue levels in fat-1 transgenic mice on progressive NASH. METHODS: Disease was induced in mice by streptozotocin and high fat diet (STZ/HFD) resulting in NASH. NAFLD in 6 and 8 weeks old wild type and fat-1 transgenic STZ/HFD treated mice was analyzed. Unlike all other mammals, fat-1 transgenic mice ubiquitously express an n-3 fatty acid desaturase, which converts n-6 to n-3 PUFAs, leading to increased n-3 and decreased n-6 PUFA tissue contents. RESULTS: Liver damage, NAFLD activity score (NAS), hepatic lipid accumulation and inflammation were significantly reduced in fat-1 transgenic STZ/HFD treated mice in the early (6 weeks) but not late (8 weeks) phase of NASH. Simultaneously, mRNA expression of genes involved in fatty acid uptake and storage (Cd36 and Plin3, respectively) was significantly down-regulated in 6 week old but not 8 week old fat-1 transgenic STZ/HFD treated mice. CONCLUSIONS: Endogenously elevated n-3 PUFA levels in fat-1 transgenic mice transiently delay the onset of STZ/HFD induced NASH but failed to efficiently protect from NASH development.

A novel multi-parametric analysis of non-invasive methods to assess animal distress during chronic pancreatitis.

Ethical responsibility, legal requirements and the need to improve the quality of research create a growing interest in the welfare of laboratory animals. Judging the welfare of animals requires readout parameters, which are valid and sensitive as well as specific to assess distress after different interventions. In the present study, we evaluated the sensitivity and specificity of different non-invasive parameters (body weight change, faecal corticosterone metabolites concentration, burrowing and nesting activity) by receiver operating characteristic curves and judged the merit of a multi-parametric analysis by logistic regression. Chronic pancreatitis as well as laparotomy caused significant changes in all parameters. However, the accuracy of these parameters was different between the two animal models. In both animal models, the multi-parametric analysis relying on all the readout parameters had the highest accuracy when predicting distress. This multi-parametric analysis revealed that C57BL/6 mice during the course of chronic pancreatitis often experienced less distress than mice after laparotomy. Interestingly these data also suggest that distress does not steadily increase during chronic pancreatitis. In conclusion, combining these non-invasive methods for severity assessment represents a reliable approach to evaluate animal distress in models such as chronic pancreatitis.

n-3 PUFAs reduce tumor load and improve survival in a NASH-tumor mouse model.

BACKGROUND: With 9.1% of all cancer deaths, hepatocellular carcinoma is the second leading cause of cancer deaths worldwide. Due to the increasing prevalence of metabolic syndrome, nonalcoholic fatty liver disease (NAFLD) has evolved into a major risk factor for hepatocellular carcinoma development. Herein, we investigated whether a dietary n-3 polyunsaturated fatty acid (PUFA) supplementation improves the outcome of progressive NAFLD. METHODS: Feeding three high-fat diets, differing in n-3 and n-6 PUFA contents and ratios (n-3/n-6: 1:8, 1:1, 5:1), the impact of n-3 PUFAs and n-3/n-6 PUFA ratios on NAFLD-related liver fibrosis and tumorigenesis was analyzed in 12- and 20-week-old streptozotocin/high-fat diet (STZ/HFD)-treated mice. RESULTS: Feeding of n-3 PUFA-rich diets (1:1 and 5:1) resulted in increased hepatic n-3 PUFA content and n-3/n-6 PUFA ratio with decreased hepatic lipid accumulation. In 20-week-old mice, n-3 PUFA-rich diets alleviated tumor load significantly, with reduced liver/body weight index, tumor size, and tumor number. Finally, these effects were accompanied by a significant improvement of survival of these mice. CONCLUSIONS: Herein, we showed that increased n-3 PUFA content and n-3/n-6 PUFA ratios lead to improved survival and attenuated tumor progression in STZ/HFD-treated mice. Thus, n-3 PUFAs could be the basis for new therapeutic options against NAFLD-related tumorigenesis.

Repin1 deficiency in liver tissue alleviates NAFLD progression in mice.

There is an increasing prevalence of obesity and metabolic syndrome, which promote the development of non-alcoholic fatty liver disease (NAFLD), a disease that can evolve into cirrhosis and hepatocellular carcinoma. Repin1 loss was previously shown to have beneficial effects on lipid and glucose metabolism and obesity regulation. Herein, we characterized NAFLD in mice with hepatic deletion of Repin1 (LRep1-/-). For this purpose, liver disease was analysed in male LRep1-/- and wild-type mice treated with streptozotocin/high fat diet or a control diet over a period of 20 wks. Streptozotocin/high fat diet treated LRep1-/- mice showed a significant decrease in systemic and hepatic lipid accumulation, accompanied by diminished chronic inflammation and a subsequent reduction in liver injury. Remarkably, Repin1-deficient mice exhibited a lower tumour prevalence and tumour frequency, as well as a reduced liver weight/body weight index. A therapeutic approach using Repin1 siRNA in the early phase of NAFLD verified the observed beneficial effects of Repin1 deficiency. This study provides evidence that loss of Repin1 in the liver attenuates NAFLD progression, most likely by reducing fat accumulation and alleviating chronic tissue inflammation. Thus, modulating Repin1 expression may become a novel strategy and potential tool to inhibit NAFLD progression.

Liver-specific Repin1 deficiency impairs transient hepatic steatosis in liver regeneration.

Transient hepatic steatosis upon liver resection supposes functional relationships between lipid metabolism and liver regeneration. Repin1 has been suggested as candidate gene for obesity and dyslipidemia by regulating key genes of lipid metabolism and lipid storage. Herein, we characterized the regenerative potential of mice with a hepatic deletion of Repin1 (LRep1-/-) after partial hepatectomy (PH) in order to determine the functional significance of Repin1 in liver regeneration. Lipid dynamics and the regenerative response were analyzed at various time points after PH. Hepatic Repin1 deficiency causes a significantly decreased transient hepatic lipid accumulation. Defects in lipid uptake, as analyzed by decreased expression of the fatty acid transporter Cd36 and Fatp5, may contribute to attenuated and shifted lipid accumulation, accompanied by altered extent and chronological sequence of liver cell proliferation in LRep1-/- mice. In vitro steatosis experiments with primary hepatocytes also revealed attenuated lipid accumulation and occurrence of smaller lipid droplets in Repin1-deficient cells, while no direct effect on proliferation in HepG2 cells was observed. Based on these results, we propose that hepatocellular Repin1 might be of functional significance for early accumulation of lipids in hepatocytes after PH, facilitating efficient progression of liver regeneration.

Microcirculatory disturbances and cellular changes during progression of hepatic steatosis to liver tumors.

Non-alcoholic fatty liver disease is closely associated with metabolic syndrome and comprises a pathological spectrum of liver disease ranging from steatosis to steatohepatitis and can progress to fibrosis/cirrhosis and hepatocellular carcinoma. In 2013, a mouse model was described that mimics non-alcoholic fatty liver disease progression from steatohepatitis to tumors in a short time span and with high incidence. As microcirculatory disturbances play a crucial role in liver disease, the suitability of the steatosis-inflammation-tumor model for microcirculatory studies was assessed. Herein, we present a comprehensive view on morphological, microvascular, cellular, and functional aspects of non-alcoholic fatty liver disease progression in the steatosis-inflammation-tumor model using intravital microscopy, biochemical, and histological techniques. Mice develop steatohepatitis, mild fibrosis, and liver tumors at ages of 6, 12, and 20 weeks, respectively. Non-alcoholic fatty liver disease progression was accompanied by several general aspects of disease severity like increasing liver/body weight index, non-alcoholic fatty liver disease activity score, and hepatocellular apoptosis. Intravital microscopic analysis revealed significant changes in hepatic microcirculation with increasing structural alterations, elevated leukocyte adherence, and impaired nutritive perfusion. Non-alcoholic fatty liver disease was further characterized by a lower sinusoidal density with a striking rise at 20 weeks. The characteristic microcirculatory changes make the model a convenient tool for analysis of microcirculation during progression from steatosis to liver tumor. Impact statement Significant alterations of microcirculation contribute to progression of NAFLD, a chronic liver disease with increasing medical and socio-economic impact. Characterization of microcirculation in a NAFLD model reflecting all relevant stages of disease progression was still missing. Thus, we evaluated microcirculatory and cellular changes in a steatosis-inflammation-tumor model using in vivo microscopy. Analyses revealed increasing structural alterations, elevated leukocyte-endothelial interaction, and impaired nutritive perfusion. Thus, this model is suitable for further studies investigating therapeutic approaches targeting these progressive microcirculatory disturbances.