Behavioral Assessment of a Novel Hepatic Encephalopathy Model using CCl₄ and Manganese in Mice

Introduction and Objectives

Hepatic encephalopathy (HE), affecting around 40% of cirrhosis patients, impairs cognitive and motor functions. Developing HE experimental models is crucial for advancing our understanding of this condition. This study developed an HE models using intraperitoneal carbon tetrachloride (CCl₄) and manganese supplementation in mice, focusing on behavioral validation.

Materials and Patients

Two groups of male C57BL6 wild-type mice (8 mice per group), 10 weeks old, were used in this study. The first group (healthy controls) had access to standard food (Rodent Laboratory Chow* 5001, LabDiet, Richmond, IN, USA), and drinking water ad libitum and were euthanized at week

12. The second group (cirrhotic group) received the same diet but with 1 mg/ml of MnCl2 added to their drinking water. It was intraperitoneally injected twice a week with CCl4 for 12 weeks (1 ml/kg of body weight dissolved in olive oil for a final concentration of 30% in the first 5 weeks and 20% in the following 7 weeks). Behavioral tests, including the beam walking test and cylinder test, were conducted to assess motor coordination and motor asymmetry. Liver morphology changes were observed, and Hematoxylin-Eosin staining was used to determine inflammation. Data were analyzed using ANOVA for parametric data and the Kruskal-Wallis test for non-parametric data, with results presented as Mean ± SEM.

Results

Behavioral tests indicated signs of HE, such as gait abnormalities (tremor, rigidity), hind limb ataxia, and bristly hair. In the beam walking test, cirrhotic mice spent significantly longer to traverse the beam (P ≤ 0.05) and had a higher number of limb foot faults (P ≤ 0.001) compared to healthy mice. The cylinder test showed no significant difference in locomotor asymmetry. Morphological changes in the liver from healthy to cirrhotic were evident. Healthy livers had a smooth reddish-brown surface, regular shape, and firm texture. In contrast, cirrhotic livers appeared paler, with an irregular surface, and became harder and bumpy. Size alterations and the presence of leukocytic foci were also noted in cirrhotic livers.

Conclusions

The combination of CCl₄ and manganese successfully induced evidence of significant motor coordination impairments and distinct liver morphology changes, indicating a noticeable progress in developing the experimental model for HE.

Ethical statement: Technical specifications for the production, care, and use of laboratory animals followed the NOM-062-ZOO-1999. Additionally, guidelines from the animal facility of the University of Guadalajara and criteria outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health were adhered to.

Declaration of interests: None.

Funding: Program for Strengthening Institutes of the University Center of Health Sciences.

Fund to Basic Science and/or Frontier Science Modalities: Paradigms and Controversies in Science 2022. Project: “A paradigm shift in the therapeutic approach to obesity and metabolic syndrome in Mexicans” to JAB.