NASH Induction with CCl4
There are many ways to induce NAFLD/NASH for preclinical rodent modeling. Methods include dietary manipulation (employing high fat, high cholesterol, high fructose, methionine/choline deficient diets, or combinations of the above), chemotoxic agent induction, and acute liver injury models. The most widely used is the application of carbon tetrachloride (CCl4).
In the liver, CCl4 is metabolized by cytochrome P450 to two highly reactive radicals - trichloromethyl radical (CCl3) and oxygenation of CCl3. The CCl3 radical reacts with nucleic acids, proteins, and lipids, impairing key cellular processes. This results in altered lipid metabolism (fatty degeneration and steatosis) and lowered protein quantities.
This early phase of toxicity is mainly characterized by rising activities of liver speciﬁc enzymes and triglycerides. There’s a significant amount of hepatic fat accumulation (NASH) reducing hepatic function.
The oxygenation of CCl3 further initiates lipid peroxidation and the destruction of polyunsaturated fatty acids, which make up cell membranes. Consequently, membrane permeability in the mitochondria, endoplasmic reticulum, and plasma membrane is compromised. This leads to a strong inflammatory response which perpetuates chronic injury and promotes fibrosis.
Moreover, the interactions between CCl3 and DNA triggers mutations and eventually the formation of hepatocellular carcinoma (HCC).
Challenges with CCl4 NASH Induction
Despite CCl4 induction being one of the most widely used experimental models for toxin-induced liver fibrosis, several factors within the procedure can vary and impact results. These factors include, but are not limited to:
- Genetic background.
- Treatment duration.
- Administration routes.
- Hepatotoxin preparation.
Strain/Genetic Background Effect on NASH Induction
C57BL/6 inbred mice are the most frequently used strain for CCl4 NASH induction as they are easily accessible. Historically, however, this strain develops only intermediate liver ﬁbrosis. Studies have shown that BALB/c inbred mice are most sensitive to ﬁbrosis induction, whereas FVB/N mice are insensitive to CCl4.
Adjusting CCl4 Treatment Duration to Animal Models
The range in sensitivity to CCl4 toxicity, and impact on severity of ﬁbrogenesis, is due to the diverse genetic background seen across mouse strains. This varied sensitivity can be overcome by manipulating the frequency and duration of CCl4 treatment.
For example, in highly susceptible strains, four weeks of CCl4 application is usually suﬃcient for ﬁbrosis induction. In comparison, for ﬁbrosis-resistant strains the poor susceptibility can be partially compensated for by prolonged treatment of up to twelve weeks.
CCl4 Administration Route Affects Model Severity
The delivery route of the hepatotoxin also impacts the severity of the model. CCl4 delivery by oral or intraperitoneal (IP) route is common. Well-trained scientists should perform all procedures, as animal handling can induce a stress response that impacts results. Hepatoxins also need to be safely used.
Oral CCl4 delivery, either by gavage or oral feeding, can cause chemically-induced inﬂammation in the intestinal mucosa, while CCl4 delivery via IP is likely to be associated with abdominal adhesions and hepatic inﬂammation. Including appropriate controls for each induction method greatly benefits the interpretation of results.
Domenicali et al (2009) describe a novel method of CCl4-induced cirrhosis via inhalation intoxication. The study describes short cycles of CCl4 inhalation as an effective method to induce cirrhosis in mice.
Studies using inhaled CCl4 are typically performed for 11–15 weeks and aim towards the development of end stage liver cirrhosis with portal hypertension. The disadvantage to this using this method of delivery is the need for special equipment and trained experts as CCl4 vapors are toxic for humans.
Inducing NAFLD/NASH in a Dysmetabolic Background
The etiology in human liver fibrosis is very complex and many causative factors have been identified. Rodent models have become invaluable for understanding the pathogenesis of liver fibrosis, with the pharmacologically-induced toxicity in the CCl4 mouse model being practical and efficient. The model is great to use for studying pathways post liver injury, with the decline in liver function being pharmacologically induced.
However, models induced this way are lacking progression through a dysmetabolic environment, and pathways driving liver function decline are different from the pathways perturbed due to dysmetabolic consequences.
Metabolic disturbances such as insulin resistance, obesity, and dyslipidemia are prominent risk factors for the development of NAFLD and NASH. Therefore, it would be useful to recapitulate all factors of human disease simultaneously – both the liver injury/fibrosis and metabolic elements of NASH.
CCl4 NASH Induction in an Inherently Dysmetabolic Mouse Model
Multiple factors of human disease can be combined in one preclinical model if you start with the right strain. Using a polygenic, inherently dysmetabolic model e.g. MS-NASH (formerly FATZO) as your CCl4 induction starting point can combine severe fibrosis with dysmetabolism, diabetes, obesity, etc.
This type of translational model is suited to multiple study types, investigating both the pathogenic mechanisms of disease progression, as well as offering a human disease-relevant preclinical option for testing new approaches to NASH and antifibrotic therapies.
CCl4 NASH induction provides an efficient method for preclinical study of liver fibrosis using conventional rodent models. While these models feature the pathological features of NASH, they lack the disease dysmetabolic element. Inducing NASH with CCl4 in an inherently dysmetabolic strain provides a translational research model combining both severe fibrosis and dysmetabolism.