Name: Mouse Model of Metabolic Dysfunction-Associated Steatotic Liver Disease with Fibrosis (Video) | JoVE Journal
Uploaded: 2025-07-18T13:30:00.000+00:00
Duration: 6 min 26 s
Description: Learn a mouse model of metabolic dysfunction-associated steatotic liver disease with fibrosis using hyperphagic mice fed a high-fat diet. This method enables studies of MASLD pathophysiology and therapeutic testing.

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5 minutes

This work was supported by National Institutes of Health grants K22CA178098, R03DK101863, and R15DK131627 and start-up funds from Brooklyn College (to JMC).

This protocol follows the animal care guidelines of Brooklyn College; IACUC protocol number 318.
1. Mouse model of MASLD with fibrosis
<ol>
	<li>Breed mice to produce Ay offspring. Set up breeding pairs with Ay mice (B6.Cg-Ay/J). Record the date of birth of the pups.</li>
	<li>Ensure that litters have similar numbers of pups to promote adequate and even feeding and growth. Aim to have five to six pups per litter, as it is the average litter size for C57BL/6J mice. Extra pups, preferentially those of the sex not needed, can be euthanized or fostered. 
	NOTE: Using Ay males and wild-type females (C57BL/6J Jax 0000664) may improve the efficiency of the breeding since wild-type C57BL/6J female mice are easy to obtain and may be better breeders than Ay female mice.</li>
	<li>Set up cages for experiments as described below.
	<ol>
		<li>Identify the Ay mice by their yellow coat color. Assign a unique identification to each mouse and tag or mark the mice. 
		NOTE: The agouti yellow (Ay) allele of the agouti gene confers a yellow coat color. The allele is semidominant in heterozygotes. Homozygosity causes embryonic lethality. Therefore, mice with yellow coat color are heterozygous for the Ay gene.</li>
		<li>Wean mice when they are 21 days old. Group together mice that will be housed in the same cage during the study. House three or four Ay mice per cage, according to expected body weight and institutional guidelines.</li>
		<li>Feed standard chow and water. House mice at standard temperature (20-23 &#176;C) and relative humidity (40% to 60%) with a 12 h light, 12 h dark cycle.</li>
	</ol>
	</li>
	<li>Feed the Ay mice the diet high in fat and fructose (High Fat and Fructose Diet, HFFD) starting at 8 weeks of age as described below.
	<ol>
		<li>Provide the Ay mice with the Western diet, which is high in fat and sucrose (Adjusted calories Western Diet Inotiv TD.88137) ad libitum.</li>
		<li>Prepare a solution containing fructose (23 g/L) and glucose (19 g/L) in drinking water. Sterilize the solution by filtration through a 0.2 &#181;m filter and store it in sterile bottles. Once opened, keep the solution refrigerated.</li>
		<li>Provide the mice with the solution containing fructose and glucose for drinking, in sterile drinking bottles. Replace the solution and bottles at least weekly, checking for growth of microorganisms.</li>
		<li>Use cage bedding with no nutritional value (e.g., wood chips).</li>
	</ol>
	</li>
	<li>Feed control mice a diet low in fat and fructose as described below. 
	NOTE: The type of control mice to use depends on the study design: If Ay-HFFD mice are used to evaluate the effect of drugs, the control mice can be Ay-HFFD mice treated with a placebo. If the model is used to study the effect of a genetic modification on the development of fibrosis, Ay-HFFD mice without the genetic modification can be used as controls. If controls without MASH are desired, follow the steps described here.
	<ol>
		<li>Use wild-type mice that are littermates of the Ay mice. They can be identified by their black coat color.</li>
		<li>Feed the mice a control diet (Low Fat Control Diet Inotiv TD.08485; referred to as Low Fat and Fructose Diet, LFFD) starting at the same age. Provide the mice with regular drinking water.</li>
	</ol>
	</li>
	<li>Monitor the development of obesity by weighing the mice weekly by placing the awake animals in a tared container on a laboratory balance and recording their weight.</li>
	<li>Ensure that the mice have sufficient food and drinking solution at all times. Monitor and record food and drinking solution consumption to estimate caloric intake, if desired.</li>
	<li>Collect tissues as described below.
	<ol>
		<li>Perform tissue collection after 16 weeks on the HFFD diet. Euthanize the mouse by carbon dioxide inhalation (or other methods approved by the institution&#39;s IACUC). 
		NOTE: After 16 weeks, Ay mice fed the HFFD diet usually have stage 1 fibrosis; prolonging the time will lead to greater fibrosis, progressing to stage 3 after 9 to 12 months.</li>
		<li>Weigh the mouse and record the weight19.</li>
		<li>Collect blood if needed. To do this, draw blood from the inferior vena cava with a 1 mL syringe and 27G needle after opening the abdomen and before dissecting the liver (or use any&#160;other method&#160;approved by the IACUC).</li>
		<li>Dissect the liver by opening the abdomen with a midline incision, holding the liver by its hilum with forceps, and cutting the ligaments that bind the liver to the diaphragm and retroperitoneum with scissors. Weigh the liver and record the weight.</li>
		<li>Rinse liver in ice-cold phosphate-buffered saline solution (PBS). Photograph the liver and &#160;include a ruler or other object as a size reference in the image. Place the liver in a Petri dish that is placed on ice.</li>
		<li>Dissect samples (approximately 100 to 150 mg per sample) of several lobes, including the median lobe, the left lobe, and the right lobe. Ensure samples have a thickness of no more than 4 mm to facilitate fixation. Place them in fixative solution (formalin 10% or formaldehyde 4%) for 24 h and process them for histopathological analysis (see below).</li>
		<li>Dissect additional samples of several lobes. Place them in tubes (1.5 or 2 mL), freeze them by submerging them in liquid nitrogen, and store them at -80 &#176;C. These samples will be used to measure hydroxyproline content.</li>
		<li>Collect samples of other organs if desired (adipose tissue, muscle, etc.)</li>
	</ol>
	</li>
</ol>
2. Collagen histochemical staining and morphometric quantification
NOTE: The steps that require the use of hazardous and/or volatile chemicals should be done in a fume hood. All slides should be processed in parallel using staining dishes and slide holders.
<ol>
	<li>Prepare liver sections as described below.
	<ol>
		<li>Fix liver tissue by keeping it in formalin (or formaldehyde) for 24 h at room temperature. Use a volume of fixative 20-fold larger than the tissue volume. Next, transfer tissues to 70% ethanol. The protocol can be paused at this point. 
		NOTE: Tissues can be placed in histology cassettes that are then placed in a container with fixative or, alternatively, in individual containers or tubes.</li>
		<li>Embed tissues in paraffin. This is usually done by a histopathology core facility or service.</li>
		<li>Obtain sections with a thickness of 0.5 &#181;m mounted on slides (non-charged slides are adequate). Allow sections to dry by exposing them to air at room temperature. A histopathology core facility or service usually does this. The protocol can be paused at this point.</li>
	</ol>
	</li>
	<li>Deparaffinize and rehydrate sections as described below.
	<ol>
		<li>Deparaffinize the sections by submerging them in two changes of deparaffinizing/clearing solution (xylene or citrisolv), for 5-15 min in each.</li>
		<li>Rehydrate by successively submerging slides in 100% ethanol, 95% ethanol, 70% ethanol, and water, for 5 min in each.</li>
	</ol>
	</li>
	<li>Stain sections with picro-sirius red as described below.
	<ol>
		<li>Submerge sections in a solution of Sirius red (direct red 80) 1 g/L in picric acid 1.3% for 1 h. Do not stain much longer, as direct red may stain structures other than collagen (e.g., nuclei). Add fast green (1 g/L) to the staining solution if counterstaining is desired26. Alternatively, stain nuclei with Weigert&#39;s hematoxylin27.</li>
		<li>Return picro-sirius red solution to a closed, dark glass or plastic container for storage. The solution can be reused multiple times (for at least 20 years27).</li>
	</ol>
	</li>
	<li>Submerge samples in ethanol (100%) for 5 min to dehydrate. Repeat with a second change of ethanol.</li>
	<li>Submerge in clearing solution (xylene or citrisolv) until they are individually removed for mounting.</li>
	<li>Remove one slide at a time from the clearing solution and remove excess liquid by blotting on paper towels. Add mounting medium (approximately 50 to 100 &#181;L). Cover the section with a coverslip and gently press the coverslip down to remove air bubbles. Let air dry. 
	NOTE: The protocol can be paused at this point.</li>
	<li>Observe sections using a light microscope with brightfield illumination and objectives 4x to 20x - collagen appears red over a light yellow-orange background. Image areas of interest with a color camera, according to the camera instructions.</li>
	<li>Perform imaging with polarized light microscopy as described below.
	<ol>
		<li>Engage and adjust the polarizer and analyzer filters orthogonally to ensure that collagen is visible and that the background signal is minimal. Collagen is visible as white, yellow, or orange on a dark background. In sections with little or no fibrosis, collagen in blood vessel walls will be visible. Keep settings, including illumination intensity, the same for all samples to be compared.</li>
		<li>Image using a monochrome camera or monochrome mode in a color camera. Use a 4x magnification objective. Take multiple images per section. Avoid areas near the hilum if they have large blood vessels. Use the same settings for all images to be compared.</li>
	</ol>
	</li>
	<li>Perform morphometric quantification using Photoshop (alternative a) as described below.
	<ol>
		<li>Quantify the white area in the images, which corresponds to collagen. In Photoshop, click Select &#62; Color range, then select Highlights, and adjust the parameters Fuzziness and Range to select pixels that are white, corresponding to collagen. Next, select Window &#62; Histogram &#62; Expanded View and record the number of pixels selected, shown next to the parameter Pixels. Use the same parameters for all images to be compared.</li>
		<li>Quantify the total area of the image. In Photoshop, click Select &#62; All, then record the total number of pixels as previously done.</li>
		<li>Calculate the percentage of the image area corresponding to collagen by dividing the number of white pixels by the number of total pixels. Values are usually expressed as area percentages.</li>
	</ol>
	</li>
	<li>Perform morphometric quantification using ImageJ (alternative b) as described below.
	<ol>
		<li>Select white areas in the images that correspond to collagen. Open the Threshold tool by clicking Image &#62; Adjust &#62; Threshold (or press Shift + T). Set the threshold range to ensure only collagen-stained regions (white areas) are highlighted in red (e.g., minimum threshold 64; maximum threshold: 255) and click Apply. This will binarize the image, converting the collagen region to black and the background to white.</li>
		<li>Measure the Total Image Area by clicking Analyze &#62; Set Measurements. Check Area and Display Label. Click OK. Click Analyze &#62; Measure (or press M on the keyboard). The Results window will display the total image area.</li>
		<li>To measure the collagen area and percentage, first, enable Limit to Threshold by clicking Analyze &#62; Set Measurements. Check Area, Area Fraction (ImageJ will calculate collagen % automatically), Limit to Threshold, and Display Label. Click OK. Then, click Analyze &#62; Measure (or press M). The Results window will show: Collagen Area (absolute pixel count) and Area Fraction (collagen% of the total image).</li>
		<li>Save the results by selecting Save As or copy them to the clipboard and paste them in a spreadsheet.</li>
	</ol>
	</li>
</ol>
3. Liver hydroxyproline quantification
NOTE: The steps that require the use of strong bases and acids should be conducted with appropriate personal protective equipment.
<ol>
	<li>Weigh liver samples prepared in step 1.8.7 and record weights. Place tissue aliquots in a tube appropriate for homogenization. Add deionized water using a water-to-tissue ratio of 900 &#181;L of water per 100 mg of tissue.</li>
	<li>Homogenize by bead beating or other method according to the manufacturer&#39;s instructions until the tissue is completely homogenized, as assessed by visual observation. The protocol can be paused at this point, and homogenates can be stored at -20 &#176;C.</li>
	<li>Perform protein precipitation as described below. The following steps are done at room temperature, except when a different temperature is specified.
	<ol>
		<li>Transfer approximately 500 &#181;L to 1000 &#181;L of homogenate to 1.5 mL centrifuge tubes. Transfer the same volume for all samples.</li>
		<li>Add trichloroacetic acid for a final concentration of 12% (e.g., add 120 &#181;L of trichloroacetic acid to 880 &#181;L of homogenate)28. Vortex briefly to mix.</li>
		<li>Incubate samples in an ice/water bath for 30 min. Centrifuge at 6,000 x g at 4 &#176;C for 10 min. A precipitate containing the protein forms. Remove supernatant by aspiration. Keep tubes on ice.</li>
		<li>Add 1 mL of ice-cold ethanol (100%) to wash the pellet. Resuspend the pellet by sonication (or other method) and verify visually that the material is resuspended. Centrifuge at&#160;6,000 x g at 4 &#176;C for 10 min and remove supernatant by aspiration. Use sonication settings according to the equipment instructions (e.g., using a Branson SFX 150 sonifier with a 3.2 mm diameter microtip, an amplitude of 70% for 10 s is effective for most samples).&#160;Repeat washing 2x.</li>
		<li>Let the pellet air-dry for 10 min, or longer if necessary.</li>
	</ol>
	</li>
	<li>Perform protein hydrolysis as described below.
	<ol>
		<li>Add 800 &#181;L of 6 N hydrochloric acid to the tubes containing the protein precipitate. Resuspend the pellet by sonication (or other method), using the same settings as in step 3.3.4.</li>
		<li>Transfer protein suspension to glass tubes or vials with screw caps; close tightly. Incubate tubes in oven at 110 &#176;C for 22 &#177; 2 h29.</li>
		<li>Centrifuge tubes at 16,000 x g for 10 min; particulate will form a pellet. This usually requires transferring the hydrolysate from the glass vials to centrifuge tubes before centrifugation.</li>
		<li>Transfer the supernatant, which contains hydroxyproline, to 1.5 mL tubes. The protocol can be paused at this point. Hydrolysates can be stored at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare a standard curve for hydroxyproline concentration. For liver samples with fibrosis stage 1 to 3, using the amounts of tissue indicated in this protocol, standard concentrations up to 125 &#181;g/mL are adequate (e.g., serial dilutions of 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, and 0 &#181;g/mL). Prepare the standards by diluting hydroxyproline in 6 N hydrochloric acid.</li>
	<li>Transfer 40 &#181;L of each sample and standard to 1.5 mL labeled centrifuge tubes. Add 10 &#181;L of 10 N sodium hydroxide to each and vortex briefly to mix.</li>
	<li>Perform oxidation of hydroxyproline as described below.
	<ol>
		<li>Prepare acetate-citrate buffer containing acetic acid 1.2% v/v, citric acid 46 g/L, sodium acetate trihydrate 120 g/L, and sodium hydroxide 34 g/L in deionized water, and adjust buffer pH to 6.5.</li>
		<li>Prepare a solution of 12.7 g/L of chloramine T in water:n-propanol:acetate-citrate buffer (1:1:8 v:v:v). First, dissolve chloramine T in water by mixing, then add n-propanol, mix, and then add acetate-citrate buffer and mix.</li>
		<li>Add 450 &#181;L of chloramine solution to tubes with samples and standards. Mix by vortexing. Incubate at room temperature for 25 min30.</li>
	</ol>
	</li>
	<li>Prepare Ehrlich&#39;s reagent with 1 M 4-(Dimethylamino) benzaldehyde in perchloric acid:propanol 1:2 (v/v). Add 500 &#181;L of Ehrlich&#39;s reagent to samples and standards. Mix by vortexing.</li>
	<li>Incubate at 65 &#176;C for 20 min in a heating block, water bath, or incubator. When the incubation ends, let samples and standards cool to room temperature.</li>
	<li>Measure the absorbance of samples and standards at a wavelength of 550 nm using a spectrophotometer.</li>
	<li>Plot absorbance versus standard amounts and use a linear regression to make a standard curve. Use the parameters from the standard curve to calculate the concentration of hydroxyproline in the samples.</li>
	<li>Calculate the amount of hydroxyproline in the liver samples based on the total volume of the hydrolysate (step 3.4) and normalize by the mass of tissue corresponding to the volume of homogenate used for the protein precipitation (step 3.3.1). Express the results as ng of hydroxyproline per mg of tissue. Alternatively, the concentration of protein in homogenates can be measured and the results expressed as mass of hydroxyproline per mass of protein.</li>
</ol>

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<li>Brunt, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathology+of+fatty+liver+disease%5BTitle%5D)+AND+%22Mod+Pathol%22%5BJournal%5D)">Pathology of fatty liver disease.</a> Mod Pathol. 20 Suppl 1, 40(2007).</li>
<li>Ibrahim, S. H., Hirsova, P., Malhi, H., Gores, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+Models+of+Nonalcoholic+Steatohepatitis%3A+Eat%2C+Delete%2C+and+Inflame%5BTitle%5D)+AND+%22Dig+Dis+Sci%22%5BJournal%5D)">Animal Models of Nonalcoholic Steatohepatitis: Eat, Delete, and Inflame.</a> Dig Dis Sci. 61 (5), 1325-1336 (2016).</li>
<li>Caviglia, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA-21+and+Dicer+are+dispensable+for+hepatic+stellate+cell+activation+and+the+development+of+liver+fibrosis%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">MicroRNA-21 and Dicer are dispensable for hepatic stellate cell activation and the development of liver fibrosis.</a> Hepatology. 67 (6), 2414-2429 (2018).</li>
<li>Kiernan, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKiernan%2C+J+%22Histological+and+histochemical+methods%22">Histological and histochemical methods</a>. , Scion Publishing Ltd. (2015).</li>
<li>Rajalingam, D., Loftis, C., Xu, J. J., Kumar, T. K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trichloroacetic+acid-induced+protein+precipitation+involves+the+reversible+association+of+a+stable+partially+structured+intermediate%5BTitle%5D)+AND+%22Protein+Sci%22%5BJournal%5D)">Trichloroacetic acid-induced protein precipitation involves the reversible association of a stable partially structured intermediate.</a> Protein Sci. 18 (5), 980-993 (2009).</li>
<li>Davidson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrolysis+of+samples+for+amino+acid+analysis%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Hydrolysis of samples for amino acid analysis.</a> Methods Mol Biol. 211, 111-122 (2003).</li>
<li>Reddy, G. K., Enwemeka, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simplified+method+for+the+analysis+of+hydroxyproline+in+biological+tissues%5BTitle%5D)+AND+%22Clin+Biochem%22%5BJournal%5D)">A simplified method for the analysis of hydroxyproline in biological tissues.</a> Clin Biochem. 29 (3), 225-229 (1996).</li>
<li>Junqueira, L. C., Bignolas, G., Brentani, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Picrosirius+staining+plus+polarization+microscopy%2C+a+specific+method+for+collagen+detection+in+tissue+sections%5BTitle%5D)+AND+%22Histochem+J%22%5BJournal%5D)">Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections.</a> Histochem J. 11 (4), 447-455 (1979).</li>
<li>Mark, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contrasting+blood+pressure+effects+of+obesity+in+leptin-deficient+ob%2Fob+mice+and+agouti+yellow+obese+mice%5BTitle%5D)+AND+%22J+Hypertens%22%5BJournal%5D)">Contrasting blood pressure effects of obesity in leptin-deficient ob/ob mice and agouti yellow obese mice.</a> J Hypertens. 17 (12 Pt 2), 1949-1953 (1999).</li>
<li>Luo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+phenotype+and+adipose+and+liver+features+in+a+high-fat+Western+diet-induced+mouse+model+of+obesity-linked+NAFLD%5BTitle%5D)+AND+%22Am+J+Physiol+Endocrinol+Metab%22%5BJournal%5D)">Metabolic phenotype and adipose and liver features in a high-fat Western diet-induced mouse model of obesity-linked NAFLD.</a> Am J Physiol Endocrinol Metab. 310 (6), 418(2016).</li>
<li>Lonardo, A., Suzuki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sexual+Dimorphism+of+NAFLD+in+Adults.+Focus+on+Clinical+Aspects+and+Implications+for+Practice+and+Translational+Research%5BTitle%5D)+AND+%22J+Clin+Med%22%5BJournal%5D)">Sexual Dimorphism of NAFLD in Adults. Focus on Clinical Aspects and Implications for Practice and Translational Research.</a> J Clin Med. 9 (5), 1278(2020).</li>
<li>Lonardo, A., Ballestri, S., Mantovani, A., Targher, G., Bril, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endpoints+in+NASH+Clinical+Trials%3A+Are+We+Blind+in+One+Eye%5BTitle%5D)+AND+%22Metabolites%22%5BJournal%5D)">Endpoints in NASH Clinical Trials: Are We Blind in One Eye.</a> Metabolites. 14 (1), 40(2024).</li>
<li>Giles, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermoneutral+housing+exacerbates+nonalcoholic+fatty+liver+disease+in+mice+and+allows+for+sex-independent+disease+modeling%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Thermoneutral housing exacerbates nonalcoholic fatty liver disease in mice and allows for sex-independent disease modeling.</a> Nat Med. 23 (7), 829-838 (2017).</li>
<li>Hao, L., Khan, M. S. H., Zu, Y., Liu, J., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermoneutrality+Inhibits+Thermogenic+Markers+and+Exacerbates+Nonalcoholic+Fatty+Liver+Disease+in+Mice%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Thermoneutrality Inhibits Thermogenic Markers and Exacerbates Nonalcoholic Fatty Liver Disease in Mice.</a> Int J Mol Sci. 25 (15), 8482(2024).</li>
<li>Horakova, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermoneutral+housing+promotes+hepatic+steatosis+in+standard+diet-fed+C57BL%2F6N+mice%2C+with+a+less+pronounced+effect+on+NAFLD+progression+upon+high-fat+feeding%5BTitle%5D)+AND+%22Front+Endocrinol%22%5BJournal%5D)">Thermoneutral housing promotes hepatic steatosis in standard diet-fed C57BL/6N mice, with a less pronounced effect on NAFLD progression upon high-fat feeding.</a> Front Endocrinol. 14, 1205703(2023).</li>
<li>Suzuki, A., Diehl, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonalcoholic+Steatohepatitis%5BTitle%5D)+AND+%22Annu+Rev+Med%22%5BJournal%5D)">Nonalcoholic Steatohepatitis.</a> Annu Rev Med. 68, 85-98 (2017).</li>
<li>Arrese, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insights+into+Nonalcoholic+Fatty-Liver+Disease+Heterogeneity%5BTitle%5D)+AND+%22Semin+Liver+Dis%22%5BJournal%5D)">Insights into Nonalcoholic Fatty-Liver Disease Heterogeneity.</a> Semin Liver Dis. 41 (4), 421-434 (2021).</li>
<li>Wentworth, B. J., Caldwell, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pearls+and+pitfalls+in+nonalcoholic+fatty+liver+disease%3A+tricky+results+are+common%5BTitle%5D)+AND+%22Metab+Target+Organ+Damage%22%5BJournal%5D)">Pearls and pitfalls in nonalcoholic fatty liver disease: tricky results are common.</a> Metab Target Organ Damage. 1 (1), 2(2021).</li>
</ol>

The authors have no conflict of interest to disclose.

This protocol describes a mouse model of MASLD with liver fibrosis that replicates the disease in humans5,6. The mice develop liver steatosis, as shown by mixed micro- and macro-vesicular steatosis in histology and elevated hepatic triacylglycerol content19. The mice also develop obesity, hypertriglyceridemia, glucose intolerance, and have been reported to have hypertension, which, in combination with liver steatosis, correspond to the dia...

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common liver disease, with a prevalence of 31% to 40% in the US and 42% worldwide1,2,3,4. Patients with MASLD can develop metabolic dysfunction-associated steatohepatitis (MASH), previously named non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma5,6. MASLD increases the risk of hepatocellular carcinoma, and following the recent increase in prevalence of MASLD, it has become the most common liver disease in patients who develop hepatocellular carcinoma7,8. In MASLD, fibrosis is the primary predictor of progression to cirrhosis and of adverse clinical outcomes, including liver-related morbidity (hepatic decompensation, liver failure, or liver cancer) and liver-related mortality6,9,10,11. Accordingly, the Food and Drug Administration encourages the development of therapeutics focusing on MASH/NASH&#160;with fibrosis, as well as the use of appropriate animal models12.
In preclinical studies of drugs and in mechanistic studies of MASLD, mouse models are most commonly used13. Thousands of mouse models of MASLD have been described, suggesting a lack of consensus on an optimal model13,14. Establishment of optimal models faces several challenges. Although it is relatively easy to induce obesity and steatosis in mice by feeding them diets high in fat, liver fibrosis is more difficult to induce13,14,15,16. In MASLD models driven by obesogenic diets, fibrosis develops slowly17 Although this slow progression may better mimic the human disease18, it&#160;increases the duration and cost of animal studies. Diets with high cholesterol content (e.g., 2%) promote the development of fibrosis; however, they also decrease the endogenous synthesis of cholesterol, which is the opposite of the increase&#160;described in the human disease15,16. Some MASLD models use mutant or genetically modified mice; however, they may have caveats: ob/ob mice develop obesity, steatosis, and insulin resistance, but they lack leptin, which is a mediator of fibrosis; PTEN null mice develop steatosis but do not develop insulin resistance15,17. Therefore, mouse models of MASLD often require long study durations, diets with high cholesterol content, or genetic manipulations to develop significant fibrosis14. To address some of these problems, we have developed a mouse model in which the animals develop MASLD with fibrosis that progresses from pericellular/perisinusoidal (stage 1) to bridging fibrosis (stage 3)19.
The goal of this article is to describe this model of MASLD with liver fibrosis and two methods to quantify fibrosis: collagen histological staining by picro-sirius red and quantification of liver hydroxyproline content.
The rationale for developing this model was that the most useful animal models are those that best replicate the human disease, both in drivers of the disease and in alterations in histopathology and gene expression. Therefore, we used a diet that resembles the average diet in the US, including moderately high amounts of fat and fructose19,20. We fed this diet to mice carrying the Agouti yellow (Ay) mutation, which have been extensively used as models of obesity21. These mice ubiquitously express the agouti protein, an antagonist of the melanocortin receptors, including MC4R, causing hyperphagia and replicating the increased food intake that is common in human obesity21,22,23. This model recapitulates the alterations that define MASH, including steatosis, hepatocellular injury, inflammation, fibrosis, and metabolic dysfunction.
The main advantages of this model are the characteristics that make it similar to the human disease. The model uses a Western-type diet, which has been designed to resemble the typical US diet and has been shown to be the most effective diet in replicating the phenotype of human MASLD14,20. The mice develop liver histopathological alterations similar to those described in the livers of humans with MASH19,24. The mice develop metabolic dysfunction, which is an essential criterion for the diagnosis of MASLD5. The livers of mice with MASH show changes in gene expression that recapitulate those in humans with MASH/NASH19. A recent study compared 39 mouse models of MASLD for their similarity to the human disease. It included MC4R knock-out mice, which share the same&#160;mechanism of hyperphagia and received the same diet as the model that we describe14. That model ranked fifth (out of 39) for its similarity to human MASLD as assessed by phenotype, liver histopathology, and transcriptomics14.
Additional advantages of this model are: Ay mice are commercially available from the Jackson Laboratory, and are available in the C57BL/6J strain, which is commonly used in metabolic studies21,25; mutant mice are easily identifiable by their hair coat color; the model can be combined with models of gain- or loss-of-function to study the role of specific genes in the development of MASLD; and finally, the model does not use nutrient deficiencies (choline or methionine deficiency), unusual food components (cholic acid, ethionine),&#160;an unusually high content of specific nutrients (trans fats), or hepatotoxins (carbon tetrachloride), which are not common causes of MASLD and may not replicate the human disease13,14,17.
This method is appropriate for the investigation of mechanisms that contribute to the development and progression of MASLD as well as for the evaluation of interventions for its treatment or prevention. This model is particularly useful because the animals not only develop both steatohepatitis and metabolic dysfunction but also develop fibrosis that progresses to stage 3. The model may not be appropriate for the study of mechanisms that affect feeding behavior (e.g., GLP1R agonists), since these mice have hyperphagia.

<table><tbody><tr><td>4-(Dimethylamino)benzaldehyde</td><td>Sigma-Aldrich</td><td>D2004</td><td></td></tr><tr><td>Acetic acid</td><td>Various</td><td>NA</td><td></td></tr><tr><td>Adjusted calories diet Western diet</td><td>Inotiv</td><td>TD.88137</td><td>Diet containg fat (42 %kcal) and sucrose (34 %w/w). Other suppliers provide diets with the same composition</td></tr><tr><td>B6.Cg-Ay/J</td><td>The Jackson Laboratory</td><td>000021</td><td>Strain has the Agouti lethal yellow mutation and a C57BL6/J background. Currently, The Jackson Laboratory has the strain cryproserved.</td></tr><tr><td>C57BL/6J</td><td>The Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>Centrifuge</td><td>Various</td><td>NA</td><td>With rotor for 1.5 mL tubes, refrigerated, able to reach 16,000 xg.</td></tr><tr><td>Centrifuge</td><td>Various</td><td>NA</td><td></td></tr><tr><td>Chloramine-T hydrate</td><td>Sigma-Aldrich</td><td>857319</td><td></td></tr><tr><td>Citric acid</td><td>Various</td><td>NA</td><td></td></tr><tr><td>Direct Red 80</td><td>Sigma</td><td>365548-5G</td><td></td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>2701</td><td></td></tr><tr><td>Formalin solution, neutral buffered</td><td>Sigma</td><td>HT501128</td><td></td></tr><tr><td>Fructose</td><td>Sigma-Aldrich</td><td>F0127</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Histology processing equipment</td><td>NA</td><td>NA</td><td>Embedding and sectioning can be done in the investigator lab or external facility</td></tr><tr><td>Homogenizer</td><td>Various</td><td>NA</td><td>Bead beaters are efficient to process multiple samples. Other homogenization methods can be used</td></tr><tr><td>Hydrochloric acid</td><td>Various</td><td>NA</td><td></td></tr><tr><td>Hydroxyproline</td><td>Sigma</td><td>H5534</td><td></td></tr><tr><td>Low Fat Control Diet</td><td>Inotiv</td><td>TD.08485</td><td>Control diet, low in fat (13 %kcal) and sucrose (12 %w/w)</td></tr><tr><td>Microscope equiped for polarized light microscopy</td><td>Various</td><td>NA</td><td></td></tr><tr><td>N-propanol</td><td>Various</td><td>NA</td><td></td></tr><tr><td>Perchloric acid</td><td>Various</td><td>NA</td><td></td></tr><tr><td>Permount Mounting Medium</td><td>Fisher Scientific</td><td>SP15-100</td><td></td></tr><tr><td>Picric Acid, Saturated Aqueous Solution, Spectrum</td><td>Fisher</td><td>18612375</td><td></td></tr><tr><td>Sodium acetate trihydrate</td><td>Various</td><td>NA</td><td></td></tr><tr><td>Sodium hydroxyde</td><td>Various</td><td>NA</td><td></td></tr><tr><td>Sonicator</td><td>Various</td><td>NA</td><td>Sonicator is useful to resuspend pellets in step 2.5, but not absolutely necessary</td></tr><tr><td>Trichloroacetic acid</td><td>Various</td><td>NA</td><td></td></tr><tr><td>VWR Micro Cover Glasses, Rectangular</td><td>VWR&amp;nbsp;</td><td>48393-081</td><td></td></tr><tr><td>VWR Premium Superfrost Plus Microscope Slides | VWR</td><td>VWR&amp;nbsp;</td><td>48311-703</td><td></td></tr><tr><td>Water, deionized</td><td>Various</td><td>NA</td><td>Deionized water from water purification system or purchased from a supplier.</td></tr><tr><td>Xylenes</td><td>Fisher Scientific</td><td>X3F</td><td>Citrisolv Clearing Agent&amp;nbsp;(Fisher 22-143-975) is sold a an environmentally-friendly alternative</td></tr></tbody></table>

mouse model of metabolic dysfunction-associated steatotic liver disease with fibrosis

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

Liver fibrosis is the most important predictor of adverse outcomes in metabolic dysfunction-associated steatotic liver disease (MASLD). In pre-clinical studies of MASLD, mice are the most commonly used disease model. However, in those models, liver fibrosis is difficult to induce. This protocol describes a mouse model of MASLD in which hyperphagic Ay mice are fed a diet that is high in fat and fructose, which resembles the average diet in the US. The mice develop hepatic steatosis, injury, inflammation, and fibrosis. Fibrosis progresses from pericellular, stage 1 fibrosis after 16 weeks, to bridging, stage 3 fibrosis after 12 months. These mice also develop obesity, hypertriglyceridemia, glucose intolerance, and hyperinsulinemia. This disease model replicates the liver histopathology, the hepatic gene expression alterations, and the metabolic dysfunction of the human disease. This protocol includes two methods to quantify fibrosis: histological staining of collagen by picro-sirius red, and quantification of the liver content of hydroxyproline. These methods can be used for the study of MASLD pathophysiology and for preclinical studies of potential therapies.

Ay mice fed the HFFD gain more weight than wild-type mice fed the LFFD and develop obesity (Figure 1A). They gain weight more rapidly during the first 4 weeks of HFFD feeding, gaining approximately 3-4 g/week. After 8 weeks, they gain weight at a rate similar to control mice (approximately 0.6 g/week)19. After 16 weeks, the Ay mice fed the HFFD weigh approximately 45 g to 50 g (average of three separate experiments) and weigh approximately 40% to 80% more than control...

Mouse model of metabolic dysfunction-associated steatotic liver disease (MASLD) with metabolic dysfunction, hepatic gene expression changes, and liver histopathological alterations that resemble human MASLD, including fibrosis that progresses to advanced fibrosis stage 3. This model can be used in studies of MASLD pathophysiology and in pre-clinical studies of new therapies.

Liver fibrosis is the most important predictor of adverse outcomes in metabolic dysfunction-associated steatotic liver disease (MASLD). In pre-clinical ...

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.

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JoVE Journal

Medicine

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Cell Type-specific Gene Expression Profiling in the Mouse Liver

This protocol is intended to be a tool to study steatosis and the molecular, biochemical, cellular changes produced by the over exposure of hepatocytes to lipids in vitro.

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA treatments.

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Here, we present a robust method for in situ perfusion of the mouse liver to study the acute and direct regulation of liver metabolism without disturbing the hepatic architecture but in the absence of extra-hepatic factors.

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model

This article describes the use of oleic acid-induced HepG2 cells as a model for metabolic dysfunction-associated steatotic liver disease.

In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-Associated Steatotic Liver Disease

This protocol presents a comprehensive imaging pipeline to visualize and model hepatic stellate cells morphology in intact liver tissue. This approach combines fluorescent labeling of fibroblasts using mouse genetics, in situ liver perfusion-based tissue preparation, and a modified iDISCO tissue clearing method to achieve optical transparency.

Mapping Hepatic Stellate Cell Morphology in Mouse Models of Liver Fibrosis

Learn a mouse model of metabolic dysfunction-associated steatotic liver disease with fibrosis using hyperphagic mice fed a high-fat diet. This method enables studies of MASLD pathophysiology and therapeutic testing.

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

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