Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

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.