Evaluate Anti-Fibrotic Compounds in NASH Models
NASH is characterized by lobular inflammation, hepatocyte ballooning, and degeneration progressing to liver fibrosis. Left unchecked, NASH can progress to full blown cirrhosis and, in some instances, hepatocellular carcinoma. However, development of NASH is not universal among those affected by non-alcoholic fatty liver disease (NAFLD), and this is one of the factors that makes modeling NASH in animal models complex.
Animal models of NASH allow us to investigate the disease pathophysiology. While these experimental systems are not exact recapitulations of the human disease, they are useful for studying the pathways and processes that promote steatohepatitis and fibrosis.
Animal Models of NASH
Several animal models have been identified that faithfully replicate aspects of the disease in a reproducible manner. Developing NASH models involves the use of targeted gene knockouts or over expression, chemical manipulation, or feeding specialized diets (i.e., those deficient in essential nutrients or laden with calories from fat and carbohydrate).
Charles River has qualified several NASH models in both mice and rats. In each case, the animals develop a characteristic change in liver histopathology consistent with NASH. The following two diet-induced NASH models are useful to assess candidate agents.
- Feeding a choline deficient, defined amino acid (CDAA) diet to C57BL/6 mice
- Feeding a high fructose, high fat, cholesterol (HFHC) diet to ob/ob mice
We have also executed studies in short-term, chemically-induced insults resulting in hepatic fibrosis to assess pharmacodynamic assessment of target engagement.
Choline Deficient, Defined Amino Acid (CDAA) Mice
Defining the Onset of Fibrosis in a NASH Mouse Model
Objective of Time Course Study: Determine Onset of NASH in C57BL/6J Mice Fed CDA Diet
Example timeline for onset of fibrosis in a CDAA-diet induced C57BL/6 mouse NASH Model. Liver biopsies performed at baseline.
NASH Mouse Model Body Weight Data
Body weight: mice fed CDAA diet gain more weight than mice on chow diet.
CDAA Mouse Model: Serum Biomarkers
Serum biomarkers in mice fed CDAA diet compared to mice fed control diet.
Histology Score for the NASH Mouse Model
Histologic progression of NASH in mice fed CDAA diet. Control animals showed no disease at study termination.
Representative Micrographs of Livers from Mice Fed a Choline Deficient, Defined Amino Acid Diet
H&E and Picrosirius Red staining of liver after 18wks on CDAA diet. Arrow marks area of focal inflammation.
Utility Of The Liver Biopsy for the NASH Mouse Model
Liver biopsies demonstrate varying degrees of steatosis and fibrosis in animals prior to initiation of treatment.
Utility Of The Liver Biopsy for the NASH Mouse Model
6 Weeks Treatment with Pioglitazone or Return to Chow Diet (n=12/group)
Biopsy scores permit evaluation of response to treatment in individual animals.
High Fructose, High Fat, Cholesterol Diet in ob/ob Mice
The ob/ob Mouse Nash Model
Initial Study to Assess Efficacy of OCA
Example timeline for onset of fibrosis in a CDAA-diet induced C57BL/6 mouse NAFLD Model. Liver biopsies performed at baseline.
ob/ob Mouse Model Body Weight and Blood Glucose Data
Measures of body weight and blood glucose at several time points over the course of an 18-wk study.
ob/ob Mouse Model Biomarkers
Biomarker profile at several timepoints over the course of an 18-wk study
Baseline Pathology For ob/ob Mouse Model
The graphs above show the histology scores for ob/ob mouse model
Representative Micrographs of Livers from Rats Fed a Choline Deficient, High Fat Diet
H&E and Picrosirius Red staining of liver after 18wks on HFHC diet. Left: Extensive steatosis and lobular inflammation noted. Right: Picrosirius red decoration of fibrosis.
Pathology Data for ob/ob Mouse Model
Pathology scores for lobular inflammation and fibrosis increased after 24-weeks compared to 12-week timepoint.
Currently, the only treatment for NAFLD/NASH is lifestyle modification (diet and exercise). Pharmaceutical and biotech industries are actively pursuing the discovery and development of candidate therapies to address this unmet medical need. Need help selecting the best NASH model to evaluate your compound?
Frequently Asked Questions (FAQs) About NASH Models
What is NASH?
NASH, or nonalcoholic steatohepatitis, is an aggressive form of non-alcoholic fatty liver disease (NAFLD) characterized by excessive deposition of lipid, inflammation and fibrosis. NASH is on a continuum of liver diseases and is thought to be the tipping point for progression to cirrhosis and, in some cases, hepatocellular carcinoma. Estimates reveal that non-alcoholic fatty liver disease (NAFLD) affects ~25% of the world’s population. While considered benign, NAFLD can progress to non-alcoholic steatohepatitis (NASH) in a significant number of individuals.
How is NASH diagnosed?
Histopathologic assessment of a liver biopsy is the gold standard for confirming a NASH diagnosis. Elevation of liver enzymes and the presence of fatty liver on ultrasound may indicate the presence of NASH, but they are not specific. To date, there are no specific, sensitive, non-invasive imaging techniques that can reliably diagnose NASH.
Human liver biopsies are scored by a pathologist for steatosis, inflammation, hepatocyte ballooning, and fibrosis. Scoring is conducted using a semi-quantitative scale developed by Kleiner and co-workers. This system was developed for the expressed purpose of diagnosing and assessing changes in disease with therapy in humans.
How translatable are animal models of NASH to human disease?
Very few, if any, animal models faithfully mirror human disease. The liver pathology in rodent NASH models is no exception. The architecture of rodent livers is less well organized than that observed in human tissue. While hepatocyte ballooning is a key feature of human NASH, and some would say a qualification for diagnosis of NASH, this feature is quite rare in rodent models and many investigators do not score for this parameter. There is gathering opinion that the Kleiner scoring system developed to assess human disease does not provide the necessary granularity to evaluate candidate therapies tested in animal models. Charles River’s board-certified veterinary pathologists have developed a 6-point scale (0 – 5) to score steatosis, inflammation and fibrosis. Hepatocyte degeneration (as opposed to true ballooning) is infrequently observed and is scored as absent (0) or present (1).
What should I consider when selecting the most appropriate NASH model for my drug discovery and development program?
When assessing candidate therapies, your target and mechanism of action should drive your selection of an animal model of NASH. None of the rodent models faithfully reproduces the histopathology with the attendant metabolic context found in human disease. However, many of the pathways that have been implicated in human NASH also play a role in the etiology and pathogenesis of liver fibrosis in rodents. There are advantages and disadvantages to all the commonly used models, but their utility must be assessed in the context of the therapeutic target. Charles River scientists have a great deal of experience with a variety of mechanisms, therapeutic agents and classes, and approaches to addressing NASH, and can consult with you regarding your NASH program.
How can I reduce variability in my NASH study?
The development of NASH in any given animal study is a factor that can adversely affect the study outcome. Published estimates have reported as many as 30% of the animals on a diet-induced NASH study may develop mild disease or none at all. To address this issue, Charles River performs liver biopsies on our long term, diet-induced NASH models prior to enrolling animals in a study. This eliminates animals that have failed to develop disease and provides a way to rationally distribute the animals to the various treatment groups in such a way as to normalize the overall NAS scores. The biopsies also permit the evaluation of treatment effects in terms of response rates (improvement, stable disease, or disease progression).
What are some other endpoints I should add to my study?
Histopathologic evaluation of the liver is the primary endpoint in NASH studies. In addition to the semi-quantitative scores, Charles River’s pathology services can quantify the fibrosis area in liver sections by performing pixel counts of slides stained for connective tissue. Immunohistochemistry of collagens, α-SMA, galectin, and other markers is also available. Our Laboratory Sciences group can perform complete clinical chemistry profiles from serum samples. The Immunology and Biomarker groups provide data on a variety of analytes from circulating and tissue cell populations through customized ELISAs and multiplexed assays to document pharmacodynamic estimates of target engagement. Coupling these analyses with blood levels of the test article can provide the links between dose, exposure, and target engagement which should correlate with improvement in disease state.