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Diet Induced NASH B6 | C57BL/6NTac Male Mice

  • Model #
  • Genotype
  • Nomenclature
  • NASH-B6-M
    C57BL/6NTac
  • NASHCONTROL-B6-M
    C57BL/6NTac

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.

NASH B6 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. They have significantly elevated ALT levels compared to controls, display robust steatosis, display hepatic inflammation including the presence of hepatic crown-like structures, have activated stellate cells, and develop consistent fibrosis by 26 weeks on diet (fibrosis score of 2 on a scale from 0-4 for most animals in one study, with bridging fibrosis observed only rarely and at late stages (1/10 mice at 53 weeks on diet)). More information is available in the phenotypic data tab below as well as in several webinar presentations: The Diet Induced NASH B6: A Translational NASH Model for Drug Discovery and Histopathological Characterization of the Diet Induced NASH B6.

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.

We recommend you review Policies, Recommendations, & Resources for Taconic's Diet Induced NASH Model prior to ordering. It includes important recommendations to support experimental success and animal welfare.

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.

Off the shelf availability permits researchers to start studies immediately, saving months of conditioning time and preserving vivarium space for active studies rather than conditioning cohorts.
Animal Diet: NASH-B6-M get D09100310i and NASHCONTROL-B6-M get NIH #31M Rodent Diet.

Tissue collection: Taconic can provide murine biospecimen collections on NASH and control animals as a separate service. Our Murine Biospecimen Services (MBS) provides general tissue and biospecimen collections which include blood, plasma, serum, liver, adipose tissue and many others. We offer various preservation methods including snap freezing, immersion in 10% neutral buffered formalin or immersion in culture media. Contact us for a complete list of available tissues and services.

Custom Diet Conditioning: Taconic also offers custom diet conditioning for any commercial or customer-provided strain as part of its Colony Management Solutions portfolio.

Color:

Black

Species:

Mouse

Initial Publication:

There is not yet a publication describing these mice, but multiple publications exist demonstrating applications using similar models. See other publications below.

Other Publications:
  • Trevaskis JL et al. Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice. Am J Physiol Gastrointest Liver Physiol 2012; 302: G762-G772. – original AMLN paper.
  • Kristiansen MN et al. Obese diet-induced mouse models of nonalcoholic steatohepatitis-tracking disease by liver biopsy. World J Hepatol. 2016 Jun 8;8(16):673-84. – overview of B6J (DIO) AMLN model at 26 week timepoint.
  • Hansen HH et al. Mouse models of nonalcoholic steatohepatitis in preclinical drug development. Drug Discov Today. 2017 Nov;22(11):1707-1718 - 2017 review paper with a table of compounds tested in the AMLN model.
  • Hernandez ED et al. Tropifexor‐mediated abrogation of steatohepatitis and fibrosis is associated with the antioxidative gene expression profile in rodents. Hepatology Communications 2019. – use of original AMLN model to evaluate FXR agonist therapeutic.
  • Oldham S et al. Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution. Journal of Visualized Experiments 2019, No. 146. – survival liver biopsy technique to assess NASH stage prior to study start.

Note that the data below was collected from animals which did not experience shipping. The stress of shipping can cause weight loss, which impacts the NASH phenotype. We recommend a pilot to assess the impact of transit to your facility and identify appropriate acclimation timelines.

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.
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.

NASH B6 have significantly elevated alanine aminotransferase (ALT) plasma levels.  2-way ANOVA Diet x Time: Only Diet is significant p<0.0001. Sidak's Multiple Comparisons Test. Not Longitudinal.
Figure 2: NASH B6 have significantly elevated alanine aminotransferase (ALT) plasma levels. 2-way ANOVA Diet x Time: Only Diet is significant p<0.0001. Sidak's Multiple Comparisons Test. Not Longitudinal.

27 Weeks on Diet

Diet Induced NASH B6 27 Weeks on Diet

35 Weeks on Diet

Diet Induced NASH B6 35 Weeks on Diet

53 Weeks on Diet

Diet Induced NASH B6 53 Weeks on Diet
Figure 3: Summary of quantitative histology findings. T-Test Multiple Comparisons: Separate Analysis per Time, Unpaired. Significance level p<0.001

A

(A) Fibrosis score and (B) % fibrosis area. 2-Way ANOVA Time x Diet: Sidak's Multiple Comparisons. Significance level p<0.0001. Not Longitudinal. Fibrosis Score - Only diet is significant. % Fibrosis Area - Time vs Diet is significant and STRONG INTERACTION.

B

(A) Fibrosis score and (B) % fibrosis area. 2-Way ANOVA Time x Diet: Sidak's Multiple Comparisons. Significance level p<0.0001. Not Longitudinal. Fibrosis Score - Only diet is significant. % Fibrosis Area - Time vs Diet is significant and STRONG INTERACTION.
Figure 4: (A) Fibrosis score and (B) % fibrosis area. 2-Way ANOVA Time x Diet: Sidak's Multiple Comparisons. Significance level p<0.0001. Not Longitudinal. Fibrosis Score - Only diet is significant. % Fibrosis Area - Time vs Diet is significant and STRONG INTERACTION.

NASH B6 have hepatic crown-like structures (hCLS), a unique histological finding from NASH patients.  Hepatic crown-like structures (blue arrowheads) are macrophages surrounding hepatocytes with large lipid vacuoles. Macrophages also form solid inflammatory cell aggregates with other leukocytes (red arrowheads).
Figure 5: NASH B6 have hepatic crown-like structures (hCLS), a unique histological finding from NASH patients. Hepatic crown-like structures (blue arrowheads) are macrophages surrounding hepatocytes with large lipid vacuoles. Macrophages also form solid inflammatory cell aggregates with other leukocytes (red arrowheads).

Figure 6: Histopathology for C57BL/6NTac mice placed on D09100310 diet (NASH B6NTac) or kept on chow diet (Control B6NTac) from 6 weeks of age. Animals were on diet for 27, 35 or 53 weeks. Hematoxylin and eosin (H&E) staining illustrates morphological changes including steatosis and infiltration of immune cells. Activated HSCs were identified in liver sections by using a rabbit polyclonal antibody to alpha-smooth muscle actin (a-SMA) (1:500 dilution; ab5694; Abcam, Cambridge, MA) which was detected by an anti-rabbit IgG embedded polymer with embedded horseradish peroxidase and visualized with 3,3′-diaminobenzidine (DAB) chromogen (Agilent, Carpentaria, CA). Hepatocellular lipid droplets were identified by visualization of the lipid-binding protein adipophilin known to be expressed in steatotic livers. Sections of livers were probed using a rabbit polyclonal anti-adipophilin antibody (1:200 dilution; NB110-40877, Novus Biologics, Littleton, CO) which was detected by an anti-rabbit IgG embedded polymer with embedded horseradish peroxidase and visualized with 3,3′-diaminobenzidine (DAB) chromogen (Aligent, Carpentaria, CA). Picrosirius red (PSR) staining illustrates collagen I and III fibers and is used to measure fibrosis. The same individual NASH or control animal is shown for all four stains in a particular time point. Different individual animals were used for each time point (i.e. data is not longitudinal by animal). Histopathology services provided by IDEXX BioAnalytics.

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 7). 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. 9) livers showing suspect lesions were fixed and sent for pathology analysis. M655 showed a hepatic mass measuring approximately 0.8 cm, located between the left and caudal lobe tissues (Fig. 10); this was fixed and sent for pathology analysis.

Final histopathology report on 59 week old NASH mice found liver adenomas that compressed the adjacent parenchyma in 2 out of 10 mice (M655 and M444), foci of hepatocellular alteration and proliferation in 4 of 10 mice (M443, M444, M445, M652) and oval cell hyperplasia in all but 1 mouse (M770). An incidental finding observed in 1 of 10 mice (M655) was the presence of large aggregates of mononuclear cells including large and small lymphocytes, plasma cells and dendritic-like cells adjacent to central veins. These aggregates have features of lymphocyte neoplasia most consistent with follicular lymphoma (also described as follicular B-cell lymphoma), a common age-related neoplastic condition of C57BL/6 mice. No hepatocellular carcinoma was identified by histopathology in any of the NASH mice examined at 27, 35 or 53 weeks on diet (10/group).

Liver of C57BL/6NTac mouse on NASH diet D09100310i for 27 weeks displays moderate diffuse hepatomegaly with steatosis. This mouse (Male M671) displayed a flat lesion flush to the medial lobe.
Figure 7: Liver of C57BL/6NTac mouse
on NASH diet D09100310i for 27 weeks
displays moderate diffuse hepatomegaly
with steatosis. This mouse (Male M671)
displayed a flat lesion flush to
the medial lobe.
Liver of C57BL/6NTac mouse on NASH diet D09100310i for 35 weeks displays moderate diffuse hepatomegaly with steatosis.
Figure 8: Liver of C57BL/6NTac mouse on
NASH diet D09100310i for 35 weeks
displays moderate diffuse hepatomegaly
with steatosis.

Liver of C57BL/6NTac mouse on NASH diet D09100310i for 53 weeks displays moderate diffuse hepatomegaly with steatosis along with visible lesions with fat-like deposit appearance measuring approximately 0.1 - 0.4 cm on serosal side.
Figure 9: Liver of C57BL/6NTac mouse on
NASH diet D09100310i for 53 weeks
displays moderate diffuse hepatomegaly
with steatosis along with visible lesions
with fat-like deposit appearance
measuring approximately 0.1 - 0.4 cm
on serosal side.
Liver of C57BL/6NTac mouse on NASH diet D09100310i for 53 weeks displays moderate diffuse hepatomegaly with steatosis along with a hepatic mass measuring approximately 0.8 cm, located between the left and caudal lobe.
Figure 10: Liver of C57BL/6NTac mouse
on NASH diet D09100310i for 53
weeks displays moderate diffuse
hepatomegaly with steatosis along
with a hepatic mass measuring
approximately 0.8 cm, located between
the left and caudal lobe.

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.

The histopathology report for 59 week old control mice identified an incidental finding in 3 of 5 mice (M18, M19, M20): the presence of large aggregates of mononuclear cells including large and small lymphocytes, plasma cells and dendritic-like cells adjacent to central veins. These aggregates have features of lymphocyte neoplasia most consistent with follicular lymphoma (also described as follicular B-cell lymphoma), a common age-related neoplastic condition of C57BL/6 mice.

Liver as % of Bodyweight

Liver as % of Bodyweight
Figure 11

Table 1: Download raw data: bodyweight and liver weight.
Group Mouse ID Body Weight (g) Liver Weight (g)Liver as % of BW
NASH 22 wks old (16 wks on diet) H831 37.5 2.5 6.7%
NASH 22 wks old (16 wks on diet) H832 43.5 3.2 7.4%
NASH 22 wks old (16 wks on diet) H833 42.5 2.9 6.8%
NASH 22 wks old (16 wks on diet) H834 44.0 3.5 8.0%
NASH 22 wks old (16 wks on diet) H835 39.9 3.3 8.3%
NASH 22 wks old (16 wks on diet) H841 45.0 4.0 8.9%
NASH 22 wks old (16 wks on diet) H842 48.9 5.5 11.2%
NASH 22 wks old (16 wks on diet) H843 41.0 4.4 10.7%
NASH 22 wks old (16 wks on diet) H844 46.2 4.8 10.4%
NASH 22 wks old (16 wks on diet) H845 40.8 3.2 7.8%
     
NASH 33 wks old (27 wks on diet)M67146.83.697.9%
NASH 33 wks old (27 wks on diet)M67242.3 3.698.7%
NASH 33 wks old (27 wks on diet)M67344.43.968.9%
NASH 33 wks old (27 wks on diet)M67445.13.968.8%
NASH 33 wks old (27 wks on diet)M67544.14.139.4%
NASH 33 wks old (27 wks on diet)M67648.05.2310.9%
NASH 33 wks old (27 wks on diet)M67741.13.147.6%
NASH 33 wks old (27 wks on diet)M67845.24.5710.1%
NASH 33 wks old (27 wks on diet)M67943.93.668.3%
NASH 33 wks old (27 wks on diet)M68045.84.249.3%
Chow Control 33 wks oldM131.51.474.7%
Chow Control 33 wks oldM229.41.284.4%
Chow Control 33 wks oldM335.71.494.2%
Chow Control 33 wks oldM431.41.324.2%
Chow Control 33 wks oldM529.91.354.5%
Chow Control 33 wks oldM1135.31.64.5%
Chow Control 33 wks oldM1231.11.424.6%
Chow Control 33 wks oldM1340.81.84.4%
Chow Control 33 wks oldM1431.21.524.9%
Chow Control 33 wks oldM1530.61.444.7%
     
NASH 41 wks old (35 wks on diet)M68650.34.789.5%
NASH 41 wks old (35 wks on diet)M68741.43.799.2%
NASH 41 wks old (35 wks on diet)M68849.24.519.2%
NASH 41 wks old (35 wks on diet)M68945.16.2713.9%
NASH 41 wks old (35 wks on diet)M69044.86.4314.4%
NASH 41 wks old (35 wks on diet)M69147.64.49.2%
NASH 41 wks old (35 wks on diet)M69257.25.529.7%
NASH 41 wks old (35 wks on diet)M69347.34.068.6%
NASH 41 wks old (35 wks on diet)M69447.24.228.9%
NASH 41 wks old (35 wks on diet)M69549.94.158.3%
Chow Control 41 wks oldM644.72.124.7%
Chow Control 41 wks oldM736.71.734.7%
Chow Control 41 wks oldM831.61.364.3%
Chow Control 41 wks oldM932.51.444.4%
Chow Control 41 wks oldM1032.61.554.8%
     
NASH 59 wks of age (53 wks on diet)M77050.65.310.5%
NASH 59 wks of age (53 wks on diet)M43047.64.69.7%
NASH 59 wks of age (53 wks on diet)M43147.84.49.2%
NASH 59 wks of age (53 wks on diet)M44253.15.310.0%
NASH 59 wks of age (53 wks on diet)M44354.46.311.6%
NASH 59 wks of age (53 wks on diet)M44456.05.59.8%
NASH 59 wks of age (53 wks on diet)M44555.26.411.6%
NASH 59 wks of age (53 wks on diet)M65243.94.710.7%
NASH 59 wks of age (53 wks on diet)M65359.65.89.7%
NASH 59 wks of age (53 wks on diet)M65548.64.910.1%
Chow Control 59 wks of ageM1648.82.675.5%
Chow Control 59 wks of ageM1739.31.944.9%
Chow Control 59 wks of ageM1842.02.185.2%
Chow Control 59 wks of ageM1936.71.514.1%
Chow Control 59 wks of ageM2036.51.363.7%
Diet Induced NASH B6 mice are placed on a modified AMLN diet (D09100310i) starting at 6 weeks of age. Controls are fed NIH-31M. We are not able to accept NASH B6 orders by weight. Animals can lose weight in transit; for ground transit over 48 hours or air transit, mice may require extended acclimation time (2-4 weeks). We recommend acclimation time extends until mice have returned to within 5% of their pre-shipment bodyweight.

Customize this chart by clicking the legend elements, then explore download options by hovering your cursor over the down arrow to the right of the chart title.
Weeks of Age Weeks on Diet
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