RenaSci

Non-alcoholic Steatohepatitis (NASH)

Hepatic Steatosis and Liver Function

 

Non-alcoholic fatty liver disease (NAFLD) is the excessive accumulation of lipid in the liver. Simple hepatic steatosis is a relatively benign condition and usually reversible. However, if lipids continue to accumulate and the liver becomes inflamed it can progress into non-alcoholic steatohepatitis (NASH). NASH is characterised by lobular inflammation and hepatocellular ballooning and is often associated with fibrosis (collagen deposition).  It can progress to cirrhosis, hepatocellular carcinoma and end-stage liver failure. Risk factors for NASH include obesity and diabetes. 

We offer a range of assays for evaluating the effects of novel drugs on liver lipids, liver function and liver pathology. 

 

Liver lipids


Total liver fat can be determined by chemical analysis (Soxhlet extraction) and assays can be performed on liver extracts to determine liver triglycerides, liver cholesterol, liver NEFA and liver glycogen levels. There is a good correlation between total liver fat determined by Soxhlet extraction and total liver triglycerides determined by enzyme assay.

Correlation between Total Liver Fat and Total Liver Triglycerides

We have used these assays to show that our dietary-induced obese mice and rats develop hepatic steatosis. Antiobesity agents such as the GLP-1 receptor agonist, liraglutide, significantly reduce liver lipids in dietary-induced obese animals.

 

Liver enzymes


Liver function can be assessed by measuring plasma levels of the liver enzymes, alanine aminotransferase ALT); aspartate aminotransferase (AST) and alkaline phosphatase (ALP), throughout and/or at the end of studies.

 

Liver pathology


At the end of studies, liver lobes can be collected and processed using standard histological staining procedures eg :-

 

     •     Haematoxylin and Eosin (H&E) staining to examine cell structures

     •     Oil Red O (ORO) staining for lipids and neutral triglycerides 

     •     Sirius Red (SR) staining for collagen deposition (fibrosis)


Liver sections can be sent for assessment by a fully-qualified pathologist (PathCelerate) using a light microscope and established graded scoring systems :-

 

     •    Steatosis scores (based on H&E staining; graded 0-3)

     •    Hepatocellular ballooning scores (based on H&E staining; graded 0-2)

     •    Lobular inflammation scores (based on H&E staining; graded 0-3)

 

The steatosis, hepatocellular ballooning and lobular inflammation scores are added together to form a non-alcoholic fatty liver disease (NAFLD) activity score (NAS; graded 0-8). .

 

Lipid deposition (lipid-postive vacuolation) can also be scored (based on ORO staining; graded 0-4).


The degree of fibrosis can be assessed by collagen deposition scores, which are based on SR staining and graded 0-4.  

 

Total collagen content in liver tissue can also be quantified as a direct measure of liver fibrosis. Total collagen content is determined using commercially available kits which measure levels of the amino acid, hydroxyproline, following acid hydrolysis of the tissue.

 

Liraglutide significantly reduced steatosis and hepatocellular ballooning scores in dietary-induced obese mice but not lobular inflammation, NAS or fibrosis scores. 

 

Mouse Models of NASH 

  

We have recently investigated the ability of choline deficient (CD) and methionine and choline deficient (MCD) diets to induce NASH in mice. The MCD diet has been employed for several years as an animal model of NASH. It produces the histological features of NASH but also marked weight-loss and reductions in plasma glucose and insulin. Thus, the MCD diet does not mirror the metabolic abnormalities observed in patients with NASH who are often obese and insulin resistant/diabetic. The CD diet was used as there is some evidence that it may produce less weight-loss than the MCD diet. The experiment included a control group of mice maintained on normal chow.  Pioglitazone was used as a standard as it has been shown to be of benefit in the treatment of NASH in man.

 

Mice were maintained on CD and MCD diets for 6 weeks. Plasma samples and livers were then taken for analysis.  Both diets significantly increased NAS and fibrosis (collagen deposition scores), liver lipids and plasma levels of the liver enzymes, ALT and AST, consistent with the development of NASH. These responses were, in general, inhibited by pioglitazone. 

NAS (NAFLD activity scores) and fibrosis (collagen deposition scores) in mice on CD and MCD diets

Liver Enzymes in CD and MCD Mouse Models of NASH

Liver Lipids in CD and MCD Mouse Models of NASH

 

The expression of genes associated with NASH (eg markers of fibrosis, inflammation and steatosis) were significantly increased in livers of mice maintained on CD and MCD diets compared to control mice on normal mouse chow. These reponses were also inhibited by pioglitazone.

 

These results demonstrate that mice on nutrient-deficient diets can be used as simple models to identify compounds with potential for the treatment of NASH. The CD diet had a clear advantage over the MCD diet as it produced much less weight-loss (see our Mouse Models of NASH Flyer).  

 

We have continued to validate the CD diet mouse model of NASH using the dual peroxisome proliferator activated receptor alpha/delta (PPARα/δ) agonist, elafibranor, which has produced promising results (the resolution of NASH without worsening of fibrosis) in Phase 2 clinical trials and is being developed for the treatment of NASH. Elafibranor reduced liver triglycerides and all of the histological features of NASH and fibrosis in mice on a CD diet confirming that this model can be used as an initital screen for anti-NASH activity. 

 

Please contact us for a copy of our NASH presentation or for further information about our assays for measuring liver lipids, plasma levels of liver enzymes, liver total collagen or for assessing liver pathology. 

 

All of these assays can be conducted on samples from external studies if required. 

 

Posters

 

Cheetham et al. 2017. Evaluation of elafibranor in the choline-deficient diet mouse model of NASH. NASH Summit Europe, Frankfurt, Germany, 10th-12th October 2017.