Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution
April 16th, 2019
In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.
This survivable liver biopsy procedure in mice with non-alcoholic steatohepatitis, or NASH, allows both the exclusion of animals that don't have NASH and the accurate measurement of responses to interventions. This procedure closely mimics the clinical trial design for NASH studies that compare the post-versus pre-treatment pathology of the liver and allows a more accurate assessment of drug efficacy. Exposing the liver lobe can be challenging at first.
Practice exposing the tissue with your finger or a cotton-tipped applicator first to find the best fit for you. After confirming a lack of response to toe pinch, make a one-to two-centimeter, longitudinal abdominal skin incision immediately caudal to the xiphoid process and slightly to the left side of the anesthetized mouse. Use forceps and scissors to blunt dissect away the skin until the abdominal wall is exposed and the liver can be visualized underneath the muscle tissue.
Using a sterile scalpel blade, make a one-to two-centimeter incision in the abdominal wall to expose the left lateral lobe of the liver, and place a sterile cotton swab under the left lateral lobe to gently extract the portion to be exposed through the incision. Place a sterile saline-soaked piece of gauze under the exposed lobe, and use sterile scissors to excise a 30-to 50-milligram, wedge-shaped piece of tissue from the extracted liver. Cut a piece of absorbable gelatin compressed sponge similar in size to the wedge piece of liver, and use sterile forceps to insert the sponge into the cut portion of the liver.
Hold the sponge in place with the forceps until the gelatin adheres to the surface of the liver and hemostasis is achieved before placing the liver biopsy into an appropriate container for its downstream analysis. Gently return the liver to the abdominal cavity, taking care not to disturb the sponge, and, starting and ending with a full square knot, suture the abdominal wall with 5-0 coated absorbable sutures. Then, close the skin with seven-millimeter wound clips, confirming a complete closure after placement, and return the mouse to a clean cage on a heated surface with monitoring until full recovery.
In this representative experiment, 118 mice were biopsied after 29 weeks on a custom diet high in fat, fructose, and cholesterol to induce NASH. The level of fibrosis and steatosis were then scored for each biopsy. Mice were excluded from the study based on low or high fibrosis and abnormal weight loss or wound healing, and the remaining mice were treated with vehicle or the glucagon-like peptide-1 receptor agonist liraglutide for six weeks.
In the vehicle-treated groups, one mouse exhibited an increase in fibrosis and four mice demonstrated an increase in NASH activity score. Liraglutide treatment was associated with an overall improvement of fibrosis, with 17%of the treatment group improving and 83%remaining unchanged. Similarly, liraglutide treatment improved the overall NASH score, with 66%of the group exhibiting an improved NASH activity score, and 33%remained unchanged.
Additionally, liraglutide treatment improved inflammation, with 75%of the group having a lower inflammation score after treatment, while vehicle-treated animals experienced an increase in inflammation. Steatosis was not affected by agonist treatment, remaining unchanged in both groups before and after the intervention. Be careful not to overly damage the excised piece of liver to ensure that it can be appropriately analyzed for your needs.
This procedure can be used to mimic clinical trial designs by comparing pre-and post-treatment effects of new therapeutics on NASH livers.
Overview
This article presents a survivable liver biopsy procedure in mice with non-alcoholic steatohepatitis (NASH), facilitating the assessment of therapeutic agents. The method closely mimics clinical trial designs, allowing for accurate measurement of liver pathology pre- and post-treatment.
Key Study Components
Area of Science
- Neuroscience
- Biology
- Clinical Research
Background
- Non-alcoholic steatohepatitis (NASH) is a liver condition characterized by fat accumulation, inflammation, and fibrosis.
- Liver biopsies are crucial for assessing the severity of NASH and evaluating therapeutic interventions.
- This study aims to refine biopsy techniques in a preclinical model to better align with clinical trial methodologies.
- Understanding the effects of treatments like liraglutide on NASH is essential for developing effective therapies.
Purpose of Study
- To demonstrate a survivable liver biopsy technique in mice with NASH.
- To evaluate the efficacy of therapeutic agents in a manner that reflects clinical trial designs.
- To assess the impact of liraglutide on liver pathology in NASH models.
Methods Used
- Survivable liver biopsy procedure performed on mice with induced NASH.
- Histological analyses conducted on liver samples post-biopsy.
- Mice were treated with either vehicle or liraglutide for comparison.
- Assessment of fibrosis, inflammation, and NASH activity scores post-treatment.
Main Results
- Liraglutide treatment improved fibrosis in 17% of treated mice.
- 66% of liraglutide-treated mice exhibited improved NASH activity scores.
- 75% of the treatment group showed reduced inflammation scores.
- Steatosis levels remained unchanged across both treatment groups.
Conclusions
- The survivable liver biopsy technique is effective for evaluating NASH therapies.
- Liraglutide shows promise in improving liver pathology associated with NASH.
- This method enhances the relevance of preclinical studies to clinical trials.
Frequently Asked Questions
Executive Industry Relevance
This survivable liver biopsy procedure enables preclinical NASH studies to mirror clinical trial designs by establishing baseline fibrosis and tracking post-treatment changes, improving translational predictability. By excluding non-diseased animals and stratifying groups by baseline pathology, the method reduces biological noise and enhances confidence in target validation and lead identification efforts. This approach supports mechanistic de-risking and portfolio triage in NASH drug development programs.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables therapeutic hypothesis interrogation by confirming baseline NASH phenotype prior to intervention.
- Operational Value: Supports biological de-risking through exclusion of non-fibrotic mice and stratification by fibrosis severity.
- Predictive Value: Improves target confidence by aligning preclinical group allocation with clinical trial enrichment strategies.
Screening & Assay Development
- Scientific Value: Generates quantitative histopathological readouts (fibrosis, steatosis, inflammation) for assay standardization.
- Operational Value: Facilitates reproducible tissue sampling via standardized wedge biopsy and gelatin sponge hemostasis.
- Scalability: Enables platform reuse across multiple time points with 5-10 minute procedure duration per mouse.
Translational & Preclinical Research
- Translational Continuity: Mirrors clinical NASH trial design by comparing pre- and post-treatment liver pathology in the same animals.
- Mechanistic De-risking: Isolates pharmacological effects from baseline variability, improving interpretation of liraglutide’s impact on fibrosis and inflammation.
- Risk-Adjusted Advancement: Enables data-driven go/no-go decisions based on paired biopsy outcomes rather than terminal endpoints alone.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from target validation through preclinical efficacy testing by providing longitudinal, paired tissue assessments that inform biological activity and disease modification.
- Discovery Biology: Supports pathway clarification and target validation by confirming NASH phenotype before therapeutic challenge.
- Screening: Delivers standardized, quantitative tissue outputs that enable reliable compound evaluation across treatment groups.
- Analytics: Provides histopathological scoring (fibrosis, NASH activity, inflammation) as dependent variables for statistical comparison of pre- and post-treatment states.
- Translational Research: Bridges discovery and preclinical phases by replicating clinical biopsy timing and endpoints in a murine model.
- Enterprise Reuse: Establishes a reusable histopathological assessment capability for chronic liver disease models beyond single-study application.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target modulation by reducing confounding from baseline disease heterogeneity.
- Operational Value: Enhances reproducibility through standardized surgical technique, tissue handling, and histological scoring.
- Strategic Value: Improves go/no-go decision quality by enabling within-animal comparison of therapeutic response.
- Portfolio Impact: Supports risk-adjusted prioritization of NASH candidates based on fibrosis and activity score improvements.
Implementation Considerations
- Requires expertise in rodent surgical procedures and sterile tissue handling.
- Dependent on instrumentation for microsurgery, including forceps, scalpels, and wound closure tools.
- Necessitates cross-team standardization between surgery, histology, and pathology teams for consistent biopsy and scoring.
- Adaptation considerations include model-specific liver lobe accessibility and gelatin sponge sizing for hemostasis.
- Practical limitations include procedure survivability dependence on postoperative care and exclusion criteria based on wound healing or weight loss.
