Animal

Mouse iG-Std Model produces mild ASH (mASH) with severe fatty liver and limited mononuclear cell infiltration
The iG-Std model is prepared and provided by the Animal Core A mouse at the age of 2-3 months is aseptically operated under general anesthesia with ketamine and xylazine or isoflurane for implantation of a long-term gastrostomy catheter made of Tygon and silastic tubings with Dacron felt. The use of a swivel allows free movement of the mouse in a micro-isolator cage (1,2). The model can be given liquid diet with three varying content of polyunsaturated fat (corn oil): 8%Cal, 25%Cal, or 40%Cal. The most common diet used is high fat (40%Cal) diet which promotes the genesis of mild to moderate steatohepatitis when given together with alcohol. After one week of acclimatization period with infusion of a control diet, ethanol infusion is initiated at the initial ethanol dose of 22.7 g/kg/day which is incrementally increased to 33 g/kg/day (37.1%Cal) over a four weeks period. At the initial ethanol dose, total calories derived from a diet and ethanol is set at 568 Cal/kg/day and the caloric percentages of ethanol, dextrose, protein, and fat (corn oil) are 29%, 6%, 25%, and 40%, respectively. Vitamins, salts, and trace minerals are included at the recommended amounts by the Committee on Animal Nutrition of the National Research Council (Dyets Inc, PA). Vitamin and salt mix are included at the recommended amounts by the Committee on Animal Nutrition of the National Research Council (AIN-76A, 4.42 g/L and 15.4 g/L, respectively, Dyets Inc, PA). The attrition rate for the mouse iG-Std model for 4 wk feeding is 4.8+0.5% usually resulting from inadvertent technical errors, aggressive animals destroying the catheters, accidental over-intoxication or withdrawal.

Age-matched (8 weeks of age) male C57BL/6j mice are purchased from Jackson Laboratories (Bar Harbor, ME). The surgical procedure for intragastric catheter placement is performed as described for the iG-Std model. A high-fat liquid diet (40% calories from corn oil) is infused for 17 days with an incremental increase in caloric intake from 580Cal/kg/day to 986 Cal/kg/day (170%) to generate moderately obese mice (28% increase in body weight) before alcohol infusion commences. Alcohol dose is increased from 18g/kg/day to ~44g/kg/day over 4 weeks. In the end of the experiment, the overfed mice with or without alcohol feeding are moderately obese, with 34.6 + 9.4% heavier body weight than the controls (1).

1.  Xu J, Lai KK, Verlinsky A, Lugea A, French SW, Cooper MP, Ji C, Tsukamoto H. Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK. J.Hepatol. 2011 Sep;55(3):673-82. PMCID:PMC3094601

Hybrid HCFD+Alc Model produces chronic ASH (cASH) with fibrosis and macrophage inflammation

To best reproduce the common dietary condition among male Hispanic ALD patients, a high-risk population for ALD, the Core has incorporated into iG modeling, ad lib consumption of Western diet (1% w/w cholesterol, 20%Cal lard, 17% corn oil: HCFD, Dyets Inc #180529). For this hybrid feeding model, 8 wk old male C57Bl/6 mice are fed ad lib chow or HCFD for 2 wk prior to iG feeding for 8 wk of high fat diet (40%Cal from corn oil) plus ethanol (~27 g/kg/day) or isocaloric dextrose at 60% of total caloric intake. Mice continue to consume HCFD or chow ad lib for the remaining 40% calories. HCFD causes moderate obesity in both alcohol and control diet-fed mice compared to chow. Alcohol feeding at the low dose of 27g/kg/day, achieves 89+11 mg% of blood alcohol levels in chow-fed mice but 434+98 mg% in HCFD+Alc mice. Plasma ALT is not increased by HCFD alone, increased only 3-fold in Chow+Alc but 15-fold to 398+37 U/L in HCFD+Alc.  Liver histology shows no pathology by HCFD alone, moderate steatosis in Chow+Alc, but severe steatohepatitis with diffuse mononuclear cell inflammation in all HCFD+Alc mice.  Further, increased perisinusoidal, pericellular, and bridging fibrosis are noted in 62% of these mice, with increased Col1a1, aSma, Timp mRNA, hydroxyproline content, and aSMA immunostaining.

Publications with the model:

1. Kisseleva T, Cong M, Paik Y, Scholten D, Jiang C, Benner C, Iwaisako K, Moore-Morris T, Scott B, Tsukamoto H, Evans SM, Dillmann W, Glass CK, Brenner DA. Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis. Proc Natl Acad Sci U S A. 2012 Jun 12;109(24):9448-53.
2. Lazaro R, Wu R, Lee S, Zhu NL, Chen CL, French SW, Xu J, Machida K, Tsukamoto H. Osteopontin deficiency does not prevent but promotes alcoholic neutrophilic hepatitis in mice. Hepatology. 2014 Aug 18. doi: 10.1002/hep.27383

Hybrid HCFD+Alc+Binge Model produces alcohol-associated hepatitis  (AH) with neutrophil infiltration

One common feature of the history of patients with alcoholic hepatitis (AH) is binge drinking superimposing habitual chronic drinking. We have incorporated this binge drinking into the HCFD ad lib-alcohol iG hybrid feeding model (Hybrid HCFD+Alc).  For this modeling, alcohol iG infusion was withdrawn for 5~6 hr and a binge bolus dose (3.5~5g/kg) of ethanol was given weekly from the 2^nd week during the hybrid feeding.  By 8 weeks, approximately 50% of mice develop a varying degree of AH histologically characterized by infiltration of PMN around necrotic hepatocytes or lipogranuloma. This pathology is associated with 40-80 fold increases in myeloperoxidase (Mpo), Gro (Cxcl1), and osteopontin (Spp) mRNA in the liver.  Further microarray analysis reveals 8 of the top 10 upregulated genes in these livers are neutrophil-related, confirming the histological evidence of AH.  Further, clinical features of AH are also reproduced, including hypoalbuminemia, hyperbilirubinemia, and splenomegaly.

1. Lazaro R, Wu R, Lee S, Zhu NL, Chen CL, French SW, Xu J, Machida K, Tsukamoto H. Osteopontin deficiency does not prevent but promotes alcoholic neutrophilic hepatitis in mice. Hepatology. 2014 Aug 18. doi: 10.1002/hep.27383. [Epub ahead of print]
2. Khanova E, Wu R, Wang W, Yan R, Chen Y, French SW, Llorente C, Pan SQ, Yang Q, Li Y, Lazaro R, Ansong C, Smith RD, Bataller R, Morgan T, Schnabl B, Tsukamoto H. Pyroptosis by caspase11/4-gasdermin-D pathway in alcoholic hepatitis in mice and patients. Hepatology. 2018 May;67(5):1737-1753. doi: 10.1002/hep.29645. Epub 2018 Feb 27. PMID: 29108122; PMCID: PMC5906140.

Key features of transition and progression of liver pathology from mASH to cASH and AH in the mouse iG models are summarized in a figure below

Updated composite physiologic and pathologic data of these different versions of the iG model produced by the Core, are summarized below.

Detailed methods for the standard iG models (iG-Std) have been reported in a previous publication (1). The standard strain of rats we used for the iG model is specific pathogen free Wistar from Charles River and Jackson Laboratories. The model can be given liquid diet with three varying content of polyunsaturated fat (corn oil): 8%Cal, 25%Cal, or 40%Cal, with or without nutritional supplements (2-4).  The most common diet used is high fat (40%Cal) diet which promotes the genesis of mild to moderate steatohepatitis when given together with alcohol.  The composition of the standard high fat ethanol diet is: 40%Cal fat, 6%Cal dextrose, 24%Cal protein, and 35%Cal ethanol at the initial ethanol dose of 9 g/kg/day for the rat iG.  This dose is progressively increased to 13 g/kg/day (44%Cal) and 15.5 g/kg/day (48%Cal) at 4 and 9 wk respectively. Vitamins, salts, and trace minerals are included at the recommended amounts by the Committee on Animal Nutrition of the National Research Council (Dyets Inc, PA).

1. Tsukamoto H, Mkrtchyan H, Dynnyk A. Intragastric ethanol infusion model in rodents. L.E. Nagy (ed). Alcohol: Methods and Protocols. Humana Press, p33-48, 2008

2. Tsukamoto H, Horne W, Kamimura S, Niemelä O, Parkkila S, Ylä-Herttuala S, Brittenham GM. Experimental liver cirrhosis induced by alcohol and iron. J Clin Invest. 1995 Jul;96(1):620-30.

3. Tsukamoto H, French SW, Benson N, Delgado G, Rao GA, Larkin EC, Largman C.  Severe and progressive steatosis and focal necrosis in rat liver induced by continuous intragastric infusion of ethanol and low fat diet. Hepatology. 1985 Mar-Apr;5(2):224-32.

4.  Tsukamoto H, Towner SJ, Ciofalo LM, French SW. Ethanol-induced liver fibrosis in rats fed high fat diet. Hepatology. 1986 Sep-Oct;6(5):814-22.

Male B6 mice (8-10 wk old) are allowed free access to liquid diet purchased from Dyets, Inc. (Bethlehem, PA) containing ethanol (#710261 or #710260) or isocalorically substituted maltose dextrins (#710028 or #710027). The diet can contain either low (12.2%Cal, #710261 and #710028) or high (35%Cal, #710260 and #710027) level of fat. After adjusting to the control liquid diet for 2 days, mice are given a liquid diet containing 1% v/v ethanol (5.5%Cal) for 2 days, 2% (11%Cal) for 2 days, 4% (22%Cal) for 1 week, 5% (27%Cal) for 1 week, and finally 6% (32%Cal) for 1 week (1). For long-term feeding up to 12 months: the ethanol content in the liquid diet is reduced to 3.5% v/v (2).

For rats, the same diets can be given in the pair-feeding model with the highest alcohol concentration at 6.7%v/v.

1. Sebastian BM, Roychowdhury S, Tang H, Hillian AD, Feldstein AE, Stahl GL, Takahashi K, Nagy LE. Identification of a cytochrome P4502E1/Bid/C1q-dependent axis mediating inflammation in adipose tissue after chronic ethanol feeding to mice. J Biol Chem. 2011 Oct 14;286(41):35989-97.
2. Machida K, Tsukamoto H, Mkrtchyan H, Duan L, Dynnyk A, Liu HM, Asahina K, Govindarajan S, Ray R, Ou JH, Seki E, Deshaies R, Miyake K, Lai MM. Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog. Proc Natl Acad Sci U S A. 2009 Feb 3;106(5):1548-53.

Selected publications with the model:

1. Said HM, Mee L, Sekar VT, Ashokkumar B, Pandol SJ. Mechanism and regulation of folate uptake by pancreatic acinar cells: effect of chronic alcohol consumption. Am. J. Physiol Gastrointest. Liver Physiol 2010 Jun;298(6):G985-G993.
2. Chen CL, Tsukamoto H, Liu JC, Kashiwabara C, Feldman D, Sher L, Dooley S, French SW, Mishra L, Petrovic L, Jeong JH, Machida K. Reciprocal regulation by TLR4 and TGF-b in tumor-initiating stem-like cells. J Clin Invest. 2013 Jul 1;123(7):2832-49.
3. Zhong S, Machida K, Tsukamoto H, Johnson DL. Alcohol induces RNA polymerase III-dependent transcription through c-Jun by co-regulating TATA-binding protein (TBP) and Brf1 expression. J Biol Chem. 2011 Jan 28;286(4):2393-401.

Male or female B6 mice (8 wk old) are fed ad libitum for 2 weeks with a liquid diet containing two different levels of cholesterol and saturated fat: 2.32g/L cholesterol and 23.2g/L lard  (DYET#710142) or 46% reduced cholesterol and lard content (#710383).  The diet is then switched to those containing ethanol (#710362 or #710384) for ethanol-fed mice. Ethanol content is gradually increased from 1% (v/v) on day 1 to 4.35% (v/v) on day 12 until the end of feeding.  If additional alcohol binge is required, a weekly binge of alcohol is given from the second week of ethanol feeding, via a stomach tube at the initial dose of 3.5g/kg which is gradually increased to 4.5g/kg.  For control mice, isocaloric dextrose solution is given as a binge. This weekly binge induces heavy intoxication as evident by physical signs such as diminished motor coordination and immobilization, which dissipate as the animal recovers within 5 hours.  This Ad lib HCFD+Alc+Binge model induces significant hepatomegaly, increased plasma ALT (>200U/L), steatohepatitis with PMN infiltration in ~50% of the mice in 8 wk, plus activation of hepatic stellate cells, and pericellular and perisinusoidal liver fibrosis if treated for a prolonged duration (8-12 wk). These models can also be applied to long-term studies on liver and pancreatic oncogenesis.

Page A, Paoli PP, Hill SJ, Howarth R, Wu R, Kweon SM, French J, et al. Alcohol directly stimulates epigenetic modifications in hepatic stellate cells. J Hepatol 2015;62:388-397

The Core processes requests for services according to the following prioritization from the highest to lower: 1. Research projects and pilot projects funded by the center grant; 2. NIAAA-funded projects by center member investigators (not funded through the center); 3. NIAAA-funded projects by non-center investigators; 4. Projects by non-center investigators with other funding. The Animal Core plans to increase the maximal production to 750 rodents per year. Of this number, 33% will be allocated to research projects and 17% and 10% to pilot projects and the Core’s developmental projects without chargebacks (these costs for the category #1 are included in the Core budget). Services provided for projects in the categories #2~#4 which account for 40% of all Core services, will require payments for chargeback costs since these costs are not included in the Core’s budget.

The chargeback fees for different animal models are shown in tables below. Chargebacks for research projects pilot projects funded by the Center grant are waived to their annual use limits.  Differential charges are placed based on the service priority criteria. Concerning chargebacks, contact the center’s administrative office (323-442-3109).

Requests for services are made via e-mail to Dr. Tsukamoto (htsukamo@usc.edu) at least 3-4 months prior to the proposed commencement dates for the requested services. The Core staff and Tsukamoto schedule the requests according to the guideline above, and investigators are notified of the schedule. The Core orders animals from the vendors or initiates requesting an approval for animal transfer according to the schedule. During this scheduling process, the Core attempts to maximize the shared used of animals and tissues without compromising the integrity of research for each project. If such possibility can be considered, Dr. Tsukamoto, or the Core staff contacts PIs to address the feasibility of the sharing.

Revised chargebacks: February 6, 2014

*2 wks of pre-conditioning with Western diet prior to 8 wks of intragastric infusion of alcohol and liquid high fat diet (60% Cal) plus ad lib consumption of Western diet (40% Cal).

** Center members from institutions with a cost-share arrangement will be entitled to a 60% subsidy.

^Mice are house as a group of 3~5 per cage, while rats are individually housed.

Other Models

• Chargebacks for research project and pilot projects currently funded by the Center are fully waived.
• Differential surcharges are placed based on the service priority criteria

Another unique service provided by the Core is to facilitate access to tissues/samples collected from the models for different investigators.  This tissue sharing program encourages a collaborative climate and provides an invaluable resource for promoting research in an efficient and cost-effective manner.   In 2018-2022, a total of 460 specimens were shared by 15 center and non-center investigators, primarily tissues from the mouse iG models such as liver, pancreas, intestine, brain, and bone. Tissue sharing is facilitated by contacting the Animal Core personnel for pending experiments to be conducted by the Animal Core.

The Core offers services of measuring blood alcohol levels (BAL)) with no charges to the center research project and pilot project investigators and with the charge-back fee ($15/sample) for other investigators.  This assay requires only 10μl of plasma/serum using the Analox GM7 analyzer (Analox Instruments Ltd. Lunenburg, MA) available in the core.  This analyzer also performs assays for glucose, cholesterol, uric acid, urea, lactate pyruvate, ammonia, triglycerides besides alcohol, again requiring only 10μl per assay. For this alcohol assay, its reproducibility and inter-assay variability are routinely assessed by running the normal control (Data-Trol, Thermo Electron, Melbourne, Australia) or pooled, aliquoted and freezer banked plasma samples from alcohol-fed animals.  Our inter-assay variability for alcohol assay based on the most recent 20 assays is 2.9+0.8%. For a complete blood chemistry such as liver panel, the core sends out samples to Antech Diagnostics (Irvine, CA), per recommendation by the USC Department of Animal Resources.

Because of the Core’s commitment to serving center investigators from other local institutions as well as non-center investigators from other regions, the Animal Core Central Laboratory offers the functional space to perform their terminal experiments on models provided by the Core and collect samples. The Central Laboratory is located on the 4th floor of the Mudd bldg. of the USC Health Sciences campus, and the animals are housed in dedicated rat and mouse rooms on the ground floor of the same building.  The Laboratory is set up for anesthesia, terminal procedures such as infusion or perfusion, and sample collections; and the laboratory staff oversees, coordinates, and assists all procedures. The Laboratory is also equipped with low and high-speed centrifuges, a liquid nitrogen tank, and a refrigerator and freezers (-20oC and –80oC) for sample processing and storage.  Thus, PIs whose laboratories are not located on the USC campus can send laboratory personnel to the core laboratory for terminal procedures on specific dates of the core service completion.  If PI desires, the core personnel perform the routine terminal procedures on a charge-back basis ($35/hr/person).  The core also arranges a shipment of a dry ice sample package to PI’s laboratory via Federal Express.  Our routine procedure includes: 1) general anesthesia with ketamine and xylazine or isoflurane unless contraindicated; 2) weighing body weight; 3) collection of venous blood (~0.8ml) from inferior vena cava for subsequent separation and collection of plasma or serum; 4) collection of whole liver or/and pancreas which will be quickly placed into sterile PBS at 4oC; 5) weighing liver and pancreas; 6) Cryo-tissue preservation: tissue slices for fixation in 3% paraformaldehyde (freshly made) for 30 min~2hr followed by step-wise treatment with 15% and 30% sucrose and freezing with OTC in a Cryo-mold; 7) Fixation and paraffin embedding: a slice from each of left, middle and right liver lobes or pancreas from splenic, duodenal, and biliary portions are fixed in 3% paraformaldehyde for 2 hr and transferred to 80% ethanol for subsequent embedding; 8) Snap tissue freezing: tissues are divided and placed into 3 Eppendorf tubes (for mice) or 7 ml tubes (for rats) and snap-frozen in liquid nitrogen; 9) other tissues such as brain, fat,, lung, and intestine can be collected according to the investigators’ instructions.