Diet Induced NASH B6

Taconic is the first and only vendor to offer a diet-induced NASH model off the shelf

Taconic Biosciences maintains an inventory of C57BL/6NTac male mice conditioned on a modified Amylin liver NASH (AMLN) diet. Diet # D09100310i (source Research Diets) contains 40 kcal% fat, 20 kcal% fructose and 2% cholesterol and is an irradiated diet. C57BL/6NTac males are put on this diet at 6 weeks of age and housed at reduced density. Control males are housed in the same location, also at reduced density, and are fed NIH-31M diet. Control males can also be generated using a low-fat purified diet upon request.

Phenotypic characterization of the NASH B6 model is ongoing, but literature reports of B6 mice fed similar diets report that mice become obese, get fatty liver and develop liver inflammation and fibrosis after 26+ weeks on diet, with inter-animal variability observed for development of the inflammation and fibrosis phenotype.

NASH B6 mice in transit for an extended period of time (more than 48 hours by ground or any air transit) can lose 10-20% of their body weight and may require extended acclimation (two or more weeks). Weight loss induced by transit may delay disease phenotype relative to animals which have remained in the same facility for the entire conditioning period. Additionally, phenotypic penetrance of certain NASH phenotypes is incomplete, so animals conditioned on diet for the same length of time will vary in disease phenotype. Taconic recommends a pilot study to determine the appropriate number of mice to order for a specific study type.

Alopecia may occur in NASH mice and is thought to be a consequence of the metabolic disease phenotype. As long as the skin is normal and intact and no pruritus is observed, no treatment is required and the mice may be used in experiments.

NOTE: The NASH diet D09100310i may take several weeks to receive after ordering from Research Diets. Please ensure you understand timeline of diet receipt in your facility prior to ordering mice. Taconic cannot provide the NASH diet beyond the amount the mice are shipped with in the TTC for use in transit.

Animal Diet: NASH-B6-M get D09100310i and NASHCONTROL-B6-M get NIH #31M Rodent Diet.

Diet Conditioning: Taconic also offers diet conditioning as part of its Colony Management Solutions portfolio. Irradiated diets are approved for use in all MPF (TM) colony management barriers.

Benefits include:

• Lower cage costs
• Health testing is included
• Helps preserve the value of rederivation

Figure 1: Comparison between C57BL/6NTac mice placed on D09100310 diet (NASH B6NTac) or kept on chow diet (Control B6NTac) from 5 weeks of age. A) Liver weight as a percentage of total body weight. B) Liver hydroxyproline content. C) Liver triglycerides. D) Serum alanine aminotransferase levels (ALT). For each time point, n=8 for NASH B6NTac and n=4 for Control B6NTac. Different individual animals were used for each time point (i.e. data is not longitudinal by animal). * and ** indicate statistical significance between NASH and control animals. Two-way ANOVA with multiple comparisons, with p-value<0.05* or p-value<0.01** Data provided by an anonymous pharmaceutical company.

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Figure 2: Histopathology for C57BL/6NTac mice placed on D09100310 diet (NASH B6NTac) or kept on chow diet (Control B6NTac) from 5 weeks of age. Animals were on diet for 26 weeks (top set) or 38 weeks (bottom set). Picrosirius red (PSR) staining illustrates collagen I and III fibers, hematoxylin and eosin (H&E) staining illustrates steatosis. Two control and two NASH animals are shown for each time point, with PSR and H&E shown for the same individual animal. Different individual animals were used for each time point (i.e. data is not longitudinal by animal). Data provided by an anonymous pharmaceutical company.

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Necropsy results for NASH and chow control mice between 22-59 weeks of age

C57BL/6NTac mice either on NASH diet D09100310i or on control NIH-31M chow diet were necropsied at 22 (NASH only, no controls), 33, 41 or 59 weeks of age. With the exception of the 59 weeks of age group, NASH mice were selected as two full cagemate groups (10 total mice, 5 mice/cage). A portion of liver was collected for histopathology analysis. The remainder of the liver was dissected and any gross lesions or abnormalities were noted.

General NASH mouse findings: All mice were bright, alert and responsive. Fur coat condition was visibly oily and all mice had body condition score 5/5. Alopecia/barbering was evident in some mice, as were healed fight wounds. All animals had marked mesentary and subcutaneous adipose tissue. Liver in all mice was grossly pale to tan, smooth, and uniformly enlarged. All mice displayed moderate diffuse hepatomegaly with steatosis. For nearly all mice, no masses or lesions were observed on serosal or cut side of livers.

The following findings were noted: Male M671 (33 weeks old) was found to have an approximately 1.0 mm lesion, flat, circular, flush to the medial lobe, milky white in coloration, located on the lower right side of the medial lobe (Fig 3). In the 59 week old group, visible suspect lesions with fat-like deposit appearance measuring approximately 0.1 - 0.4 cm on serosal side of liver were found in M444, M445, M652, M653. Portions of M652 and M653 (Fig. 5) livers showing suspect lesions were fixed and sent for pathology analysis.

Pathology report for M652 and M653: There is diffuse marked lipidosis (microvesicular and macrovesicular) with multifocal neutrophil and mononuclear cell infiltrates (hepatosteatitis) and rare single cell necrosis/apoptosis. M655 showed a hepatic mass measuring approximately 0.8 cm, located between the left and caudal lobe tissues (Fig. 6); this was fixed and sent for pathology analysis.

Pathology report for M655: A large (roughly 8mm) hepatic adenoma compresses the adjacent liver parenchyma. In the remaining liver there is diffuse marked lipidosis (microvesicular and macrovesicular) with multifocal neutrophil and mononuclear cell infiltrates (hepatosteatitis) and rare single cell necrosis/apoptosis. No hepatocellular carcinoma was identified by histopathology (results pending on remaining 59 week old mice).



General chow control mouse findings: All mice were bright, alert and responsive. Fur coat appeared well groomed with mild oily appearance. Intermittent lighter colored tips on hairshafts of fur coat. Body condition score was 3-4 for controls at 33 and 41 weeks of age and 4 for controls at 59 weeks of age. All animals had adequate to plentiful mesentary and subcutaneous adipose tissue present. Liver appeared grossly dark pink with uniform color and texture, except in 2 males at 59 weeks for which liver appeared mildly pale with a mild icteric tinge in 2 of 5 males. No masses or lesions were observed on serosal or cut side of livers.

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Liver as % of Bodyweight

Figure 7

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Table 1: Raw bodyweight and liver weight data.