Mouse Model of Metabolic Dysfunction-Associated Steatotic Liver Disease with Fibrosis

In our research, we focus on metabolic dysfunction-associated steatotic liver disease, or MASLD, and specifically on the mechanisms of lipid accumulation and fibrosis. Some of the recent developments are single cell and special transcriptomics, the standardization of animal models of MASLD, and the approval of semaglutide for the treatment of MASLD. There are many animal models of MASLD, but we found out that many did not induce liver fibrosis.

So, to address that point, we developed our mouse model of MASLD. Our mouse model induces MASLD with fibrosis by feeding hyperphagic mice a diet similar to the average American diet. This causes MASLD that is very similar to the disease in humans.

Our method will facilitate studies on the pathophysiology of MASLD and testing new therapies. To begin, obtain a paraffin-embedded liver tissue section on a slide. For de-paraffinization, submerge the slide in xylene or citrisolv for five to 15 minutes.

Then rehydrate the section serially in 100, 95, and 70%ethanol, followed by distilled water for five minutes in each. Then stain the section with Sirius Red and dehydrate the slide. Remove one slide at a time from the clearing solution and blot it on paper towel to remove excess liquid.

Add approximately 50 to 100 microliters of mounting medium to the section. Cover the section with a cover slip and gently press it down to remove air bubbles. Let the slide air dry.

Now, observe the prepared section under a light microscope with bright-field illumination using 4x to 20x objectives. Transfer approximately 500 to 1000 microliters of liver homogenate to 1.5-milliliter centrifuge tubes, and add trichloroacetic acid to reach a final concentration of 12%Vortex the tube briefly to mix the contents. Incubate the tube in an ice water bath for 30 minutes, and centrifuge it at 6, 000 g at four degrees Celsius for 10 minutes.

Then remove the supernatant with a pipette. Add one milliliter of 100%ice cold ethanol to wash the pellet and sonicate it to resuspend thoroughly. After centrifuging the suspension again, aspirate the supernatant.

Following the third wash, keep the tube open to air dry the pellet for 10 minutes or longer as necessary. Then add 800 microliters of 6 normal hydrochloric acid to the tube containing the protein pellet, and resuspend the pellet using a sonicator as demonstrated earlier. Transfer the suspension to a screw-capped glass tube or vial and tightly seal the cap.

Incubate the tube in an oven at 110 degrees Celsius for about 22 hours. If needed, transfer the contents from the glass vial to a centrifuge tube before spinning. Centrifuge the hydrolyzed sample at 16, 000 g for 10 minutes and aspirate the supernatant containing hydroxyproline into a 1.5-milliliter tube.

For hydroxyproline oxidation, add 450 microliters of chloramine solution to tubes with 50 microliters of sample and standards. Mix the contents in the tubes with a vortex, and incubate them at room temperature for 25 minutes. Then add 500 microliters of Ehrlich's reagent to each sample and standard.

After vortexing, incubate the samples at 65 degrees Celsius for 20 minutes in an incubator. After incubation, let the tubes cool to room temperature and observe the final color of the samples. Finally, measure the absorbance of samples and standards using a spectrophotometer at a wavelength of 550 nanometers.

Collagen staining of liver tissue with Picro Sirius Red, when observed with bright field illumination, showed collagen red with a light orange or yellow background. Collagen deposition corresponding to fibrosis was stained as well as collagen in the blood vessel walls, the hilum, and the liver capsule. The polarized light microscopy revealed distinct birefringent collagen fibers on a dark background, enhancing visualization of fibrosis.

Quantification of liver collagen positive area in agouti yellow mice fed with high fat and fructose diet, or HFFD, showed significantly higher fibrosis when compared to the wild type control mice fed with low fat and fructose diet. Quantification of hepatic hydroxyproline content revealed a significantly higher level in agouti yellow mice fed with HFFD diet compared to the control mice after 16 weeks on the diet, confirming increased collagen deposition in fibrotic livers.